Cohesin regulates tissue-specific expression by stabilizing highly occupied cis-regulatory modules

PubMed Central

Faure, Andre J.; Schmidt, Dominic; Watt, Stephen; Schwalie, Petra C.; Wilson, Michael D.; Xu, Huiling; Ramsay, Robert G.; Odom, Duncan T.; Flicek, Paul

2012-01-01

The cohesin protein complex contributes to transcriptional regulation in a CTCF-independent manner by colocalizing with master regulators at tissue-specific loci. The regulation of transcription involves the concerted action of multiple transcription factors (TFs) and cohesin's role in this context of combinatorial TF binding remains unexplored. To investigate cohesin-non-CTCF (CNC) binding events in vivo we mapped cohesin and CTCF, as well as a collection of tissue-specific and ubiquitous transcriptional regulators using ChIP-seq in primary mouse liver. We observe a positive correlation between the number of distinct TFs bound and the presence of CNC sites. In contrast to regions of the genome where cohesin and CTCF colocalize, CNC sites coincide with the binding of master regulators and enhancer-markers and are significantly associated with liver-specific expressed genes. We also show that cohesin presence partially explains the commonly observed discrepancy between TF motif score and ChIP signal. Evidence from these statistical analyses in wild-type cells, and comparisons to maps of TF binding in Rad21-cohesin haploinsufficient mouse liver, suggests that cohesin helps to stabilize large protein–DNA complexes. Finally, we observe that the presence of mirrored CTCF binding events at promoters and their nearby cohesin-bound enhancers is associated with elevated expression levels. PMID:22780989

Genetic Interactions Between the Meiosis-Specific Cohesin Components, STAG3, REC8, and RAD21L.

PubMed

Ward, Ayobami; Hopkins, Jessica; Mckay, Matthew; Murray, Steve; Jordan, Philip W

2016-06-01

Cohesin is an essential structural component of chromosomes that ensures accurate chromosome segregation during mitosis and meiosis. Previous studies have shown that there are cohesin complexes specific to meiosis, required to mediate homologous chromosome pairing, synapsis, recombination, and segregation. Meiosis-specific cohesin complexes consist of two structural maintenance of chromosomes proteins (SMC1α/SMC1β and SMC3), an α-kleisin protein (RAD21, RAD21L, or REC8), and a stromal antigen protein (STAG1, 2, or 3). STAG3 is exclusively expressed during meiosis, and is the predominant STAG protein component of cohesin complexes in primary spermatocytes from mouse, interacting directly with each α-kleisin subunit. REC8 and RAD21L are also meiosis-specific cohesin components. Stag3 mutant spermatocytes arrest in early prophase ("zygotene-like" stage), displaying failed homolog synapsis and persistent DNA damage, as a result of unstable loading of cohesin onto the chromosome axes. Interestingly, Rec8, Rad21L double mutants resulted in an earlier "leptotene-like" arrest, accompanied by complete absence of STAG3 loading. To assess genetic interactions between STAG3 and α-kleisin subunits RAD21L and REC8, our lab generated Stag3, Rad21L, and Stag3, Rec8 double knockout mice, and compared them to the Rec8, Rad21L double mutant. These double mutants are phenotypically distinct from one another, and more severe than each single knockout mutant with regards to chromosome axis formation, cohesin loading, and sister chromatid cohesion. The Stag3, Rad21L, and Stag3, Rec8 double mutants both progress further into prophase I than the Rec8, Rad21L double mutant. Our genetic analysis demonstrates that cohesins containing STAG3 and REC8 are the main complex required for centromeric cohesion, and RAD21L cohesins are required for normal clustering of pericentromeric heterochromatin. Furthermore, the STAG3/REC8 and STAG3/RAD21L cohesins are the primary cohesins required for axis formation. Copyright © 2016 Ward et al.

Building a foundation for structure-based cellulosome design for cellulosic ethanol: Insight into cohesin-dockerin complexation from computer simulation

PubMed Central

Xu, Jiancong; Crowley, Michael F; Smith, Jeremy C

2009-01-01

The organization and assembly of the cellulosome, an extracellular multienzyme complex produced by anaerobic bacteria, is mediated by the high-affinity interaction of cohesin domains from scaffolding proteins with dockerins of cellulosomal enzymes. We have performed molecular dynamics simulations and free energy calculations on both the wild type (WT) and D39N mutant of the C. thermocellum Type I cohesin-dockerin complex in aqueous solution. The D39N mutation has been experimentally demonstrated to disrupt cohesin-dockerin binding. The present MD simulations indicate that the substitution triggers significant protein flexibility and causes a major change of the hydrogen-bonding network in the recognition strips—the conserved loop regions previously proposed to be involved in binding—through electrostatic and salt-bridge interactions between β-strands 3 and 5 of the cohesin and α-helix 3 of the dockerin. The mutation-induced subtle disturbance in the local hydrogen-bond network is accompanied by conformational rearrangements of the protein side chains and bound water molecules. Additional free energy perturbation calculations of the D39N mutation provide differences in the cohesin-dockerin binding energy, thus offering a direct, quantitative comparison with experiments. The underlying molecular mechanism of cohesin-dockerin complexation is further investigated through the free energy profile, that is, potential of mean force (PMF) calculations of WT cohesin-dockerin complex. The PMF shows a high-free energy barrier against the dissociation and reveals a stepwise pattern involving both the central β-sheet interface and its adjacent solvent-exposed loop/turn regions clustered at both ends of the β-barrel structure. PMID:19384997

Structure of the Pds5-Scc1 Complex and Implications for Cohesin Function.

PubMed

Muir, Kyle W; Kschonsak, Marc; Li, Yan; Metz, Jutta; Haering, Christian H; Panne, Daniel

2016-03-08

Sister chromatid cohesion is a fundamental prerequisite to faithful genome segregation. Cohesion is precisely regulated by accessory factors that modulate the stability with which the cohesin complex embraces chromosomes. One of these factors, Pds5, engages cohesin through Scc1 and is both a facilitator of cohesion, and, conversely also mediates the release of cohesin from chromatin. We present here the crystal structure of a complex between budding yeast Pds5 and Scc1, thus elucidating the molecular basis of Pds5 function. Pds5 forms an elongated HEAT repeat that binds to Scc1 via a conserved surface patch. We demonstrate that the integrity of the Pds5-Scc1 interface is indispensable for the recruitment of Pds5 to cohesin, and that its abrogation results in loss of sister chromatid cohesion and cell viability. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The LSH/HELLS homolog Irc5 contributes to cohesin association with chromatin in yeast

PubMed Central

Bakowski, Tomasz; Maciaszczyk-Dziubinska, Ewa; Wysocki, Robert

2017-01-01

Abstract Accurate chromosome segregation is essential for every living cell as unequal distribution of chromosomes during cell division may result in genome instability that manifests in carcinogenesis and developmental disorders. Irc5 from Saccharomyces cerevisiae is a member of the conserved Snf2 family of ATP-dependent DNA translocases and its function is poorly understood. Here, we identify Irc5 as a novel interactor of the cohesin complex. Irc5 associates with Scc1 cohesin subunit and contributes to cohesin binding to chromatin. Disruption of IRC5 decreases cohesin levels at centromeres and chromosome arms, causing premature sister chromatid separation. Moreover, reduced cohesin occupancy at the rDNA region in cells lacking IRC5 leads to the loss of rDNA repeats. We also show that the translocase activity of Irc5 is required for its function in cohesion pathway. Finally, we demonstrate that in the absence of Irc5 both the level of chromatin-bound Scc2, a member of cohesin loading complex, and physical interaction between Scc1 and Scc2 are reduced. Our results suggest that Irc5 is an auxiliary factor that is involved in cohesin association with chromatin. PMID:28383696

Dalmatian: spotting the difference in cohesin protectors.

PubMed

Marston, Adele L

2017-06-01

The cohesin complex prevents separation of chromosomes following their duplication until the appropriate time during cell division. In vertebrates, establishment and maintenance of cohesin-dependent linkages depend on two distinct proteins, sororin and shugoshin. New findings published in The EMBO Journal show that in Drosophila , the function of both of these cohesin regulators is carried out by a single hybrid protein, Dalmatian. © 2017 The Author.

Scc2 regulates gene expression by recruiting cohesin to the chromosome as a transcriptional activator during yeast meiosis

PubMed Central

Lin, Weiqiang; Jin, Hui; Liu, Xiuwen; Hampton, Kristin; Yu, Hong-Guo

2011-01-01

To tether sister chromatids, a protein-loading complex, including Scc2, recruits cohesin to the chromosome at discrete loci. Cohesin facilitates the formation of a higher-order chromosome structure that could also influence gene expression. How cohesin directly regulates transcription remains to be further elucidated. We report that in budding yeast Scc2 is required for sister-chromatid cohesion during meiosis for two reasons. First, Scc2 is required for activating the expression of REC8, which encodes a meiosis-specific cohesin subunit; second, Scc2 is necessary for recruiting meiotic cohesin to the chromosome to generate sister-chromatid cohesion. Using a heterologous reporter assay, we have found that Scc2 increases the activity of its target promoters by recruiting cohesin to establish an upstream cohesin-associated region in a position-dependent manner. Rec8-associated meiotic cohesin is required for the full activation of the REC8 promoter, revealing that cohesin has a positive feedback on transcriptional regulation. Finally, we provide evidence that chromosomal binding of cohesin is sufficient for target-gene activation during meiosis. Our data support a noncanonical role for cohesin as a transcriptional activator during cell differentiation. PMID:21508318

The Chromosomal Association of the Smc5/6 Complex Depends on Cohesion and Predicts the Level of Sister Chromatid Entanglement

PubMed Central

Jeppsson, Kristian; Carlborg, Kristian K.; Nakato, Ryuichiro; Berta, Davide G.; Lilienthal, Ingrid; Kanno, Takaharu; Lindqvist, Arne; Brink, Maartje C.; Dantuma, Nico P.; Katou, Yuki; Shirahige, Katsuhiko; Sjögren, Camilla

2014-01-01

The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution. PMID:25329383

The LSH/HELLS homolog Irc5 contributes to cohesin association with chromatin in yeast.

PubMed

Litwin, Ireneusz; Bakowski, Tomasz; Maciaszczyk-Dziubinska, Ewa; Wysocki, Robert

2017-06-20

Accurate chromosome segregation is essential for every living cell as unequal distribution of chromosomes during cell division may result in genome instability that manifests in carcinogenesis and developmental disorders. Irc5 from Saccharomyces cerevisiae is a member of the conserved Snf2 family of ATP-dependent DNA translocases and its function is poorly understood. Here, we identify Irc5 as a novel interactor of the cohesin complex. Irc5 associates with Scc1 cohesin subunit and contributes to cohesin binding to chromatin. Disruption of IRC5 decreases cohesin levels at centromeres and chromosome arms, causing premature sister chromatid separation. Moreover, reduced cohesin occupancy at the rDNA region in cells lacking IRC5 leads to the loss of rDNA repeats. We also show that the translocase activity of Irc5 is required for its function in cohesion pathway. Finally, we demonstrate that in the absence of Irc5 both the level of chromatin-bound Scc2, a member of cohesin loading complex, and physical interaction between Scc1 and Scc2 are reduced. Our results suggest that Irc5 is an auxiliary factor that is involved in cohesin association with chromatin. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Meiosis-Specific Cohesin Component, Stag3 Is Essential for Maintaining Centromere Chromatid Cohesion, and Required for DNA Repair and Synapsis between Homologous Chromosomes

PubMed Central

Hopkins, Jessica; Bedigian, Rick; Oka, Kazuhiro; Overbeek, Paul; Murray, Steve; Jordan, Philip W.

2014-01-01

Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin complexes. Furthermore, our data suggests that STAG3 is required for structural changes of chromosomes that mediate chromosome pairing and synapsis, DNA repair and progression of meiosis. PMID:24992337

Meiosis-specific cohesin component, Stag3 is essential for maintaining centromere chromatid cohesion, and required for DNA repair and synapsis between homologous chromosomes.

PubMed

Hopkins, Jessica; Hwang, Grace; Jacob, Justin; Sapp, Nicklas; Bedigian, Rick; Oka, Kazuhiro; Overbeek, Paul; Murray, Steve; Jordan, Philip W

2014-07-01

Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin complexes. Furthermore, our data suggests that STAG3 is required for structural changes of chromosomes that mediate chromosome pairing and synapsis, DNA repair and progression of meiosis.

Sister acts: coordinating DNA replication and cohesion establishment

PubMed Central

Sherwood, Rebecca; Takahashi, Tatsuro S.; Jallepalli, Prasad V.

2010-01-01

The ring-shaped cohesin complex links sister chromatids and plays crucial roles in homologous recombination and mitotic chromosome segregation. In cycling cells, cohesin's ability to generate cohesive linkages is restricted to S phase and depends on loading and establishment factors that are intimately connected to DNA replication. Here we review how cohesin is regulated by the replication machinery, as well as recent evidence that cohesin itself influences how chromosomes are replicated. PMID:21159813

Many functions of the meiotic cohesin.

PubMed

Bardhan, Amit

2010-12-01

Sister chromatids are held together from the time of their formation in S phase until they segregate in anaphase by the cohesin complex. In meiosis of most organisms, the mitotic Mcd1/Scc1/Rad21 subunit of the cohesin complex is largely replaced by its paralog named Rec8. This article reviews the specialized functions of Rec8 that are crucial for diverse aspects of chromosome dynamics in meiosis, and presents some speculations relating to meiotic chromosome organization.

Long-Range Chromosome Interactions Mediated by Cohesin Shape Circadian Gene Expression

PubMed Central

Xu, Yichi; Guo, Weimin; Li, Ping; Zhang, Yan; Zhao, Meng; Fan, Zenghua; Zhao, Zhihu; Yan, Jun

2016-01-01

Mammalian circadian rhythm is established by the negative feedback loops consisting of a set of clock genes, which lead to the circadian expression of thousands of downstream genes in vivo. As genome-wide transcription is organized under the high-order chromosome structure, it is largely uncharted how circadian gene expression is influenced by chromosome architecture. We focus on the function of chromatin structure proteins cohesin as well as CTCF (CCCTC-binding factor) in circadian rhythm. Using circular chromosome conformation capture sequencing, we systematically examined the interacting loci of a Bmal1-bound super-enhancer upstream of a clock gene Nr1d1 in mouse liver. These interactions are largely stable in the circadian cycle and cohesin binding sites are enriched in the interactome. Global analysis showed that cohesin-CTCF co-binding sites tend to insulate the phases of circadian oscillating genes while cohesin-non-CTCF sites are associated with high circadian rhythmicity of transcription. A model integrating the effects of cohesin and CTCF markedly improved the mechanistic understanding of circadian gene expression. Further experiments in cohesin knockout cells demonstrated that cohesin is required at least in part for driving the circadian gene expression by facilitating the enhancer-promoter looping. This study provided a novel insight into the relationship between circadian transcriptome and the high-order chromosome structure. PMID:27135601

Meiosis in male Drosophila

PubMed Central

McKee, Bruce D.; Yan, Rihui; Tsai, Jui-He

2012-01-01

Meiosis entails sorting and separating both homologous and sister chromatids. The mechanisms for connecting sister chromatids and homologs during meiosis are highly conserved and include specialized forms of the cohesin complex and a tightly regulated homolog synapsis/recombination pathway designed to yield regular crossovers between homologous chromatids. Drosophila male meiosis is of special interest because it dispenses with large segments of the standard meiotic script, particularly recombination, synapsis and the associated structures. Instead, Drosophila relies on a unique protein complex composed of at least two novel proteins, SNM and MNM, to provide stable connections between homologs during meiosis I. Sister chromatid cohesion in Drosophila is mediated by cohesins, ring-shaped complexes that entrap sister chromatids. However, unlike other eukaryotes Drosophila does not rely on the highly conserved Rec8 cohesin in meiosis, but instead utilizes two novel cohesion proteins, ORD and SOLO, which interact with the SMC1/3 cohesin components in providing meiotic cohesion. PMID:23087836

Dual binding mode in cohesin-dockerin complexes as assessed through stretching studies

NASA Astrophysics Data System (ADS)

Wojciechowski, Michał; Cieplak, Marek

2016-10-01

A recent experimental study by Jobst et al. of stretching of a wild-type (WT) cohesin-dockerin complex has identified two kinds of the force-displacement patterns, with a single or double-peaked final rupture, which are termed "short" and "long" here. This duality has been interpreted as arising from the existence of two kinds of binding. Here, we analyze the separation of two cohesin-dockerin complexes of C. thermocellum theoretically. We use a coarse-grained structure-based model and the values of the pulling speeds are nearly experimental. In their native states, the two systems differ in the mutual binding orientations of the molecules in the complex. We demonstrate that the WT complex (PDB:1OHZ) unravels along two possible pathways that are qualitatively consistent with the presence of the short and long patterns observed experimentally. On the other hand, the mutated complex (PDB:2CCL) leads only to short trajectories. The short and long stretching pathways also appear in the cohesin-dockerin-Xmodule complex (PDB:4IU3, WT) of R. flavefaciens. Thus the duality in the stretching patterns need not be necessarily due to the duality in binding.

Cohesin Function in Cohesion, Condensation, and DNA Repair Is Regulated by Wpl1p via a Common Mechanism in Saccharomyces cerevisiae.

PubMed

Bloom, Michelle S; Koshland, Douglas; Guacci, Vincent

2018-01-01

Cohesin tethers DNA to mediate sister chromatid cohesion, chromosome condensation, and DNA repair. How the cell regulates cohesin to perform these distinct functions remains to be elucidated. One cohesin regulator, Wpl1p, was characterized in Saccharomyces cerevisiae as a promoter of efficient cohesion and an inhibitor of condensation. Wpl1p is also required for resistance to DNA-damaging agents. Here, we provide evidence that Wpl1p promotes the timely repair of DNA damage induced during S-phase. Previous studies have indicated that Wpl1p destabilizes cohesin's binding to DNA by modulating the interface between the cohesin subunits Mcd1p and Smc3p Our results suggest that Wpl1p likely modulates this interface to regulate all of cohesin's biological functions. Furthermore, we show that Wpl1p regulates cohesion and condensation through the formation of a functional complex with another cohesin-associated factor, Pds5p In contrast, Wpl1p regulates DNA repair independently of its interaction with Pds5p Together, these results suggest that Wpl1p regulates distinct biological functions of cohesin by Pds5p-dependent and -independent modulation of the Smc3p/Mcd1p interface. Copyright © 2018 by the Genetics Society of America.

Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA

PubMed Central

Yao, Hongjie; Brick, Kevin; Evrard, Yvonne; Xiao, Tiaojiang; Camerini-Otero, R. Daniel; Felsenfeld, Gary

2010-01-01

CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, although the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. Here we showed that the DEAD-box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF by binding to both, and is required for proper insulator function. PMID:20966046

Sisters Unbound Is Required for Meiotic Centromeric Cohesion in Drosophila melanogaster

PubMed Central

Krishnan, Badri; Thomas, Sharon E.; Yan, Rihui; Yamada, Hirotsugu; Zhulin, Igor B.; McKee, Bruce D.

2014-01-01

Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two structural maintenance of chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. We have identified a novel Drosophila gene called sisters unbound (sunn), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of non-disjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, our data show that SUNN is an essential meiosis-specific Drosophila cohesion protein. PMID:25194162

Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres

PubMed Central

Remeseiro, Silvia; Cuadrado, Ana; Carretero, María; Martínez, Paula; Drosopoulos, William C; Cañamero, Marta; Schildkraut, Carl L; Blasco, María A; Losada, Ana

2012-01-01

Cohesin is a protein complex originally identified for its role in sister chromatid cohesion, although increasing evidence portrays it also as a major organizer of interphase chromatin. Vertebrate cohesin consists of Smc1, Smc3, Rad21/Scc1 and either stromal antigen 1 (SA1) or SA2. To explore the functional specificity of these two versions of cohesin and their relevance for embryonic development and cancer, we generated a mouse model deficient for SA1. Complete ablation of SA1 results in embryonic lethality, while heterozygous animals have shorter lifespan and earlier onset of tumourigenesis. SA1-null mouse embryonic fibroblasts show decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromeric cohesion, which depends on cohesin-SA2. Instead, they arise from defective telomere replication, which requires cohesion mediated specifically by cohesin-SA1. We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis. PMID:22415365

Prophase pathway-dependent removal of cohesin from human chromosomes requires opening of the Smc3–Scc1 gate

PubMed Central

Buheitel, Johannes; Stemmann, Olaf

2013-01-01

Faithful transmission of chromosomes during eukaryotic cell division requires sister chromatids to be paired from their generation in S phase until their separation in M phase. Cohesion is mediated by the cohesin complex, whose Smc1, Smc3 and Scc1 subunits form a tripartite ring that entraps both DNA double strands. Whereas centromeric cohesin is removed in late metaphase by Scc1 cleavage, metazoan cohesin at chromosome arms is displaced already in prophase by proteolysis-independent signalling. Which of the three gates is triggered by the prophase pathway to open has remained enigmatic. Here, we show that displacement of human cohesin from early mitotic chromosomes requires dissociation of Smc3 from Scc1 but no opening of the other two gates. In contrast, loading of human cohesin onto chromatin in telophase occurs through the Smc1–Smc3 hinge. We propose that the use of differently regulated gates for loading and release facilitates unidirectionality of DNA's entry into and exit from the cohesin ring. PMID:23361318

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis.

PubMed

Wieczorek, Andrew S; Martin, Vincent J J

2012-12-15

The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli β-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli β-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study, chimeric protein scaffolds consisting of type 1 and type 2 cohesins anchored on the surface of L. lactis allowed for the controlled positioning of dockerin-fused reporter enzymes onto the scaffolds. By binding single enzymes or enzyme pairs to the scaffolds, our data also suggest that the size and relative positions of enzymes can affect the catalytic profiles of the resulting complexes. These insights will be of great value as we engineer more advanced scaffold-guided protein complexes to optimize biochemical pathways.

Principles of Chromosome Architecture Revealed by Hi-C.

PubMed

Eagen, Kyle P

2018-06-01

Chromosomes are folded and compacted in interphase nuclei, but the molecular basis of this folding is poorly understood. Chromosome conformation capture methods, such as Hi-C, combine chemical crosslinking of chromatin with fragmentation, DNA ligation, and high-throughput DNA sequencing to detect neighboring loci genome-wide. Hi-C has revealed the segregation of chromatin into active and inactive compartments and the folding of DNA into self-associating domains and loops. Depletion of CTCF, cohesin, or cohesin-associated proteins was recently shown to affect the majority of domains and loops in a manner that is consistent with a model of DNA folding through extrusion of chromatin loops. Compartmentation was not dependent on CTCF or cohesin. Hi-C contact maps represent the superimposition of CTCF/cohesin-dependent and -independent folding states. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cohesin organizes chromatin loops at DNA replication factories

PubMed Central

Guillou, Emmanuelle; Ibarra, Arkaitz; Coulon, Vincent; Casado-Vela, Juan; Rico, Daniel; Casal, Ignacio; Schwob, Etienne; Losada, Ana; Méndez, Juan

2010-01-01

Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication. PMID:21159821

Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module.

PubMed

Wojciechowski, Michał; Różycki, Bartosz; Huy, Pham Dinh Quoc; Li, Mai Suan; Bayer, Edward A; Cieplak, Marek

2018-03-22

The assembly of the polysaccharide degradating cellulosome machinery is mediated by tight binding between cohesin and dockerin domains. We have used an empirical model known as FoldX as well as molecular mechanics methods to determine the free energy of binding between a cohesin and a dockerin from Clostridium thermocellum in two possible modes that differ by an approximately 180° rotation. Our studies suggest that the full-length wild-type complex exhibits dual binding at room temperature, i.e., the two modes of binding have comparable probabilities at equilibrium. The ability to bind in the two modes persists at elevated temperatures. However, single-point mutations or truncations of terminal segments in the dockerin result in shifting the equilibrium towards one of the binding modes. Our molecular dynamics simulations of mechanical stretching of the full-length wild-type cohesin-dockerin complex indicate that each mode of binding leads to two kinds of stretching pathways, which may be mistakenly taken as evidence of dual binding.

Connected Gene Communities Underlie Transcriptional Changes in Cornelia de Lange Syndrome.

PubMed

Boudaoud, Imène; Fournier, Éric; Baguette, Audrey; Vallée, Maxime; Lamaze, Fabien C; Droit, Arnaud; Bilodeau, Steve

2017-09-01

Cornelia de Lange syndrome (CdLS) is a complex multisystem developmental disorder caused by mutations in cohesin subunits and regulators. While its precise molecular mechanisms are not well defined, they point toward a global deregulation of the transcriptional gene expression program. Cohesin is associated with the boundaries of chromosome domains and with enhancer and promoter regions connecting the three-dimensional genome organization with transcriptional regulation. Here, we show that connected gene communities, structures emerging from the interactions of noncoding regulatory elements and genes in the three-dimensional chromosomal space, provide a molecular explanation for the pathoetiology of CdLS associated with mutations in the cohesin-loading factor NIPBL and the cohesin subunit SMC1A NIPBL and cohesin are important constituents of connected gene communities that are centrally positioned at noncoding regulatory elements. Accordingly, genes deregulated in CdLS are positioned within reach of NIPBL- and cohesin-occupied regions through promoter-promoter interactions. Our findings suggest a dynamic model where NIPBL loads cohesin to connect genes in communities, offering an explanation for the gene expression deregulation in the CdLS. Copyright © 2017 by the Genetics Society of America.

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763

Insights into cellulosome assembly and dynamics: from dissection to reconstruction of the supramolecular enzyme complex.

PubMed

Smith, Steven P; Bayer, Edward A

2013-10-01

Cellulosomes are multi-enzyme complexes produced by anaerobic bacteria for the efficient deconstruction of plant cell wall polysaccharides. The assembly of enzymatic subunits onto a central non-catalytic scaffoldin subunit is mediated by a highly specific interaction between the enzyme-bearing dockerin modules and the resident cohesin modules of the scaffoldin, which affords their catalytic activities to work synergistically. The scaffoldin also imparts substrate-binding and bacterial-anchoring properties, the latter of which involves a second cohesin-dockerin interaction. Recent structure-function studies reveal an ever-growing array of unique and increasingly complex cohesin-dockerin complexes and cellulosomal enzymes with novel activities. A 'build' approach involving multimodular cellulosomal segments has provided a structural model of an organized yet conformationally dynamic supramolecular assembly with the potential to form higher order structures. Copyright © 2013. Published by Elsevier Ltd.

Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver

PubMed Central

2018-01-01

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity. PMID:29757144

Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver.

PubMed

Matthews, Bryan J; Waxman, David J

2018-05-14

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity. © 2018, Matthews et al.

Sister chromatid segregation in meiosis II

PubMed Central

Wassmann, Katja

2013-01-01

Meiotic divisions (meiosis I and II) are specialized cell divisions to generate haploid gametes. The first meiotic division with the separation of chromosomes is named reductional division. The second division, which takes place immediately after meiosis I without intervening S-phase, is equational, with the separation of sister chromatids, similar to mitosis. This meiotic segregation pattern requires the two-step removal of the cohesin complex holding sister chromatids together: cohesin is removed from chromosome arms that have been subjected to homologous recombination in meiosis I and from the centromere region in meiosis II. Cohesin in the centromere region is protected from removal in meiosis I, but this protection has to be removed—deprotected”—for sister chromatid segregation in meiosis II. Whereas the mechanisms of cohesin protection are quite well understood, the mechanisms of deprotection have been largely unknown until recently. In this review I summarize our current knowledge on cohesin deprotection. PMID:23574717

Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii

PubMed Central

Ze, Xiaolei; Ben David, Yonit; Laverde-Gomez, Jenny A.; Dassa, Bareket; Sheridan, Paul O.; Duncan, Sylvia H.; Louis, Petra; Henrissat, Bernard; Juge, Nathalie; Koropatkin, Nicole M.; Bayer, Edward A.

2015-01-01

ABSTRACT Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energy from dietary starches that escape digestion by host enzymes via its exceptional activity against particulate “resistant” starches. Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family (GH13). We found that amylase activity in R. bromii is expressed constitutively, with the activity seen during growth with fructose as an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptides were detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted to carry dockerin modules, with one, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediate the formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerins from R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymes Amy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wall-anchoring protein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylases Amy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, as its dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides the first example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an “amylosome.” PMID:26419877

Structural maintenance of chromosome complexes differentially compact mitotic chromosomes according to genomic context

PubMed Central

Schalbetter, S. A.; Goloborodko, A.; Fudenberg, G.; Belton, J.-M.; Miles, C.; Yu, M.; Dekker, J.; Mirny, L.; Baxter, J.

2017-01-01

Structural Maintenance of Chromosomes (SMC) protein complexes are key determinants of chromosome conformation. Using Hi-C and polymer modeling, we study how cohesin and condensin, two deeply conserved SMC complexes, organize chromosomes in the budding yeast Saccharomyces cerevisiae. The canonical role of cohesin is to co-align sister chromatids whilst condensin generally compacts mitotic chromosomes. We find strikingly different roles for the two complexes in budding yeast mitosis. First, cohesin is responsible for compacting mitotic chromosome arms, independently of sister chromatid cohesion. Polymer simulations demonstrate this role can be fully accounted for through cis-looping of chromatin. Second, condensin is generally dispensable for compaction along chromosome arms. Instead it plays a targeted role compacting the rDNA proximal regions and promoting resolution of peri-centromeric regions. Our results argue that the conserved mechanism of SMC complexes is to form chromatin loops and that distinct SMC-dependent looping activities are selectively deployed to appropriately compact chromosomes. PMID:28825700

Solution conformation of a cohesin module and its scaffoldin linker from a prototypical cellulosome.

PubMed

Galera-Prat, Albert; Pantoja-Uceda, David; Laurents, Douglas V; Carrión-Vázquez, Mariano

2018-04-15

Bacterial cellulases are drawing increased attention as a means to obtain plentiful chemical feedstocks and fuels from renewable lignocellulosic biomass sources. Certain bacteria deploy a large extracellular multi-protein complex, called the cellulosome, to degrade cellulose. Scaffoldin, a key non-catalytic cellulosome component, is a large protein containing a cellulose-specific carbohydrate-binding module and several cohesin modules which bind and organize the hydrolytic enzymes. Despite the importance of the structure and protein/protein interactions of the cohesin module in the cellulosome, its structure in solution has remained unknown to date. Here, we report the backbone 1 H, 13 C and 15 N NMR assignments of the Cohesin module 5 from the highly stable and active cellulosome from Clostridium thermocellum. These data reveal that this module adopts a tightly packed, well folded and rigid structure in solution. Furthermore, since in scaffoldin, the cohesin modules are connected by linkers we have also characterized the conformation of a representative linker segment using NMR spectroscopy. Analysis of its chemical shift values revealed that this linker is rather stiff and tends to adopt extended conformations. This suggests that the scaffoldin linkers act to minimize interactions between cohesin modules. These results pave the way towards solution studies on cohesin/dockerin's fascinating dual-binding mode. Copyright © 2018 Elsevier Inc. All rights reserved.

Cohesin Function in Cohesion, Condensation, and DNA Repair Is Regulated by Wpl1p via a Common Mechanism in Saccharomyces cerevisiae

PubMed Central

Bloom, Michelle S.; Koshland, Douglas; Guacci, Vincent

2018-01-01

Cohesin tethers DNA to mediate sister chromatid cohesion, chromosome condensation, and DNA repair. How the cell regulates cohesin to perform these distinct functions remains to be elucidated. One cohesin regulator, Wpl1p, was characterized in Saccharomyces cerevisiae as a promoter of efficient cohesion and an inhibitor of condensation. Wpl1p is also required for resistance to DNA-damaging agents. Here, we provide evidence that Wpl1p promotes the timely repair of DNA damage induced during S-phase. Previous studies have indicated that Wpl1p destabilizes cohesin’s binding to DNA by modulating the interface between the cohesin subunits Mcd1p and Smc3p. Our results suggest that Wpl1p likely modulates this interface to regulate all of cohesin’s biological functions. Furthermore, we show that Wpl1p regulates cohesion and condensation through the formation of a functional complex with another cohesin-associated factor, Pds5p. In contrast, Wpl1p regulates DNA repair independently of its interaction with Pds5p. Together, these results suggest that Wpl1p regulates distinct biological functions of cohesin by Pds5p-dependent and -independent modulation of the Smc3p/Mcd1p interface. PMID:29158426

LDB1-mediated enhancer looping can be established independent of mediator and cohesin.

PubMed

Krivega, Ivan; Dean, Ann

2017-08-21

Mechanistic studies in erythroid cells indicate that LDB1, as part of a GATA1/TAL1/LMO2 complex, brings erythroid-expressed genes into proximity with enhancers for transcription activation. The role of co-activators in establishing this long-range interaction is poorly understood. Here we tested the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and cohesin to establishment of locus control region (LCR)/β-globin proximity. CRISPR/Cas9 editing of the β-globin promoter to eliminate the RNA Pol II PIC by deleting the TATA-box resulted in loss of transcription, but enhancer-promoter interaction was unaffected. Additional deletion of the promoter GATA1 site eliminated LDB1 complex and mediator occupancy and resulted in loss of LCR/β-globin proximity. To separate the roles of LDB1 and mediator in LCR looping, we expressed a looping-competent but transcription-activation deficient form of LDB1 in LDB1 knock down cells: LCR/β-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the β-globin promoter forced LCR loop formation in the absence of mediator or cohesin occupancy. Moreover, ENCODE data and our chromatin immunoprecipitation results indicate that cohesin is almost completely absent from validated and predicted LDB1-regulated erythroid enhancer-gene pairs. Thus, lineage specific factors largely mediate enhancer-promoter looping in erythroid cells independent of mediator and cohesin. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

Cohesin and related coiled-coil domain-containing complexes physically and functionally connect the dots across the genome

PubMed Central

Poon, Betty P.K

2011-01-01

Interactions between genetic regions located across the genome maintain its three-dimensional organization and function. Recent studies point to key roles for a set of coiled-coil domain-containing complexes (cohibin, cohesin, condensin and monopolin) and related factors in the regulation of DNA-DNA connections across the genome. These connections are critical to replication, recombination, gene expression as well as chromosome segregation. PMID:21822055

Cohesin Can Remain Associated with Chromosomes during DNA Replication.

PubMed

Rhodes, James D P; Haarhuis, Judith H I; Grimm, Jonathan B; Rowland, Benjamin D; Lavis, Luke D; Nasmyth, Kim A

2017-09-19

To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We developed a non-fluorescent HaloTag ligand to discriminate the fluorescence recovery signal from labeling of newly synthesized Halo-tagged Scc1 protein (pulse-chase or pcFRAP). In cells where cohesin turnover is inactivated by deletion of WAPL, Scc1 can remain associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to un-replicated DNA. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Cleavage of cohesin rings coordinates the separation of centrioles and chromatids.

PubMed

Schöckel, Laura; Möckel, Martin; Mayer, Bernd; Boos, Dominik; Stemmann, Olaf

2011-07-10

Cohesin pairs sister chromatids by forming a tripartite Scc1-Smc1-Smc3 ring around them. In mitosis, cohesin is removed from chromosome arms by the phosphorylation-dependent prophase pathway. Centromeric cohesin is protected by shugoshin 1 and protein phosphatase 2A (Sgo1-PP2A) and opened only in anaphase by separase-dependent cleavage of Scc1 (refs 4-6). Following chromosome segregation, centrioles loosen their tight orthogonal arrangement, which licenses later centrosome duplication in S phase. Although a role of separase in centriole disengagement has been reported, the molecular details of this process remain enigmatic. Here, we identify cohesin as a centriole-engagement factor. Both premature sister-chromatid separation and centriole disengagement are induced by ectopic activation of separase or depletion of Sgo1. These unscheduled events are suppressed by expression of non-cleavable Scc1 or inhibition of the prophase pathway. When endogenous Scc1 is replaced by artificially cleavable Scc1, the corresponding site-specific protease triggers centriole disengagement. Separation of centrioles can alternatively be induced by ectopic cleavage of an engineered Smc3. Thus, the chromosome and centrosome cycles exhibit extensive parallels and are coordinated with each other by dual use of the cohesin ring complex.

RAD21L, a novel cohesin subunit implicated in linking homologous chromosomes in mammalian meiosis

PubMed Central

Lee, Jibak

2011-01-01

Cohesins are multi-subunit protein complexes that regulate sister chromatid cohesion during mitosis and meiosis. Here we identified a novel kleisin subunit of cohesins, RAD21L, which is conserved among vertebrates. In mice, RAD21L is expressed exclusively in early meiosis: it apparently replaces RAD21 in premeiotic S phase, becomes detectable on the axial elements in leptotene, and stays on the axial/lateral elements until mid pachytene. RAD21L then disappears, and is replaced with RAD21. This behavior of RAD21L is unique and distinct from that of REC8, another meiosis-specific kleisin subunit. Remarkably, the disappearance of RAD21L at mid pachytene correlates with the completion of DNA double-strand break repair and the formation of crossovers as judged by colabeling with molecular markers, γ-H2AX, MSH4, and MLH1. RAD21L associates with SMC3, STAG3, and either SMC1α or SMC1β. Our results suggest that cohesin complexes containing RAD21L may be involved in synapsis initiation and crossover recombination between homologous chromosomes. PMID:21242291

RAD21L, a novel cohesin subunit implicated in linking homologous chromosomes in mammalian meiosis.

PubMed

Lee, Jibak; Hirano, Tatsuya

2011-01-24

Cohesins are multi-subunit protein complexes that regulate sister chromatid cohesion during mitosis and meiosis. Here we identified a novel kleisin subunit of cohesins, RAD21L, which is conserved among vertebrates. In mice, RAD21L is expressed exclusively in early meiosis: it apparently replaces RAD21 in premeiotic S phase, becomes detectable on the axial elements in leptotene, and stays on the axial/lateral elements until mid pachytene. RAD21L then disappears, and is replaced with RAD21. This behavior of RAD21L is unique and distinct from that of REC8, another meiosis-specific kleisin subunit. Remarkably, the disappearance of RAD21L at mid pachytene correlates with the completion of DNA double-strand break repair and the formation of crossovers as judged by colabeling with molecular markers, γ-H2AX, MSH4, and MLH1. RAD21L associates with SMC3, STAG3, and either SMC1α or SMC1β. Our results suggest that cohesin complexes containing RAD21L may be involved in synapsis initiation and crossover recombination between homologous chromosomes.

Mps1 kinase-dependent Sgo2 centromere localisation mediates cohesin protection in mouse oocyte meiosis I.

PubMed

El Yakoubi, Warif; Buffin, Eulalie; Cladière, Damien; Gryaznova, Yulia; Berenguer, Inés; Touati, Sandra A; Gómez, Rocío; Suja, José A; van Deursen, Jan M; Wassmann, Katja

2017-09-25

A key feature of meiosis is the step-wise removal of cohesin, the protein complex holding sister chromatids together, first from arms in meiosis I and then from the centromere region in meiosis II. Centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage, in order to maintain sister chromatids together until their separation in meiosis II. Failures in step-wise cohesin removal result in aneuploid gametes, preventing the generation of healthy embryos. Here, we report that kinase activities of Bub1 and Mps1 are required for Sgo2 localisation to the centromere region. Mps1 inhibitor-treated oocytes are defective in centromeric cohesin protection, whereas oocytes devoid of Bub1 kinase activity, which cannot phosphorylate H2A at T121, are not perturbed in cohesin protection as long as Mps1 is functional. Mps1 and Bub1 kinase activities localise Sgo2 in meiosis I preferentially to the centromere and pericentromere respectively, indicating that Sgo2 at the centromere is required for protection.In meiosis I centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage ensuring that sister chromatids are kept together until their separation in meiosis II. Here the authors demonstrate that Bub1 and Mps1 kinase activities are required for Sgo2 localisation to the centromere region.

Lesson from the stoichiometry determination of the cohesin complex: a short protease mediated elution increases the recovery from cross-linked antibody-conjugated beads.

PubMed

Holzmann, Johann; Fuchs, Johannes; Pichler, Peter; Peters, Jan-Michael; Mechtler, Karl

2011-02-04

Affinity purification of proteins using antibodies coupled to beads and subsequent mass spectrometric analysis has become a standard technique for the identification of protein complexes. With the recent transfer of the isotope dilution mass spectrometry principle (IDMS) to the field of proteomics, quantitative analyses-such as the stoichiometry determination of protein complexes-have become achievable. Traditionally proteins were eluted from antibody-conjugated beads using glycine at low pH or using diluted acids such as HCl, TFA, or FA, but elution was often found to be incomplete. Using the cohesin complex and the anaphase promoting complex/cyclosome (APC/C) as examples, we show that a short 15-60 min predigestion with a protease such as LysC (modified on-bead digest termed protease elution) increases the elution efficiency 2- to 3-fold compared to standard acid elution protocols. While longer incubation periods-as performed in standard on-bead digestion-led to partial proteolysis of the cross-linked antibodies, no or only insignificant cleavage was observed after 15-60 min protease mediated elution. Using the protease elution method, we successfully determined the stoichiometry of the cohesin complex by absolute quantification of the four core subunits using LC-SRM analysis and 19 reference peptides generated with the EtEP strategy. Protease elution was 3-fold more efficient compared to HCl elution, but measurements using both elution techniques are in agreement with a 1:1:1:1 stoichiometry. Furthermore, using isoform specific reference peptides, we determined the exact STAG1:STAG2 stoichiometry within the population of cohesin complexes. In summary, we show that the protease elution protocol increases the recovery from affinity beads and is compatible with quantitative measurements such as the stoichiometry determination of protein complexes.

Determination of binding affinity upon mutation for type I dockerin-cohesin complexes from Clostridium thermocellum and Clostridium cellulolyticum using deep sequencing.

PubMed

Kowalsky, Caitlin A; Whitehead, Timothy A

2016-12-01

The comprehensive sequence determinants of binding affinity for type I cohesin toward dockerin from Clostridium thermocellum and Clostridium cellulolyticum was evaluated using deep mutational scanning coupled to yeast surface display. We measured the relative binding affinity to dockerin for 2970 and 2778 single point mutants of C. thermocellum and C. cellulolyticum, respectively, representing over 96% of all possible single point mutants. The interface ΔΔG for each variant was reconstructed from sequencing counts and compared with the three independent experimental methods. This reconstruction results in a narrow dynamic range of -0.8-0.5 kcal/mol. The computational software packages FoldX and Rosetta were used to predict mutations that disrupt binding by more than 0.4 kcal/mol. The area under the curve of receiver operator curves was 0.82 for FoldX and 0.77 for Rosetta, showing reasonable agreements between predictions and experimental results. Destabilizing mutations to core and rim positions were predicted with higher accuracy than support positions. This benchmark dataset may be useful for developing new computational prediction tools for the prediction of the mutational effect on binding affinities for protein-protein interactions. Experimental considerations to improve precision and range of the reconstruction method are discussed. Proteins 2016; 84:1914-1928. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The Opposing Actions of Arabidopsis CHROMOSOME TRANSMISSION FIDELITY7 and WINGS APART-LIKE1 and 2 Differ in Mitotic and Meiotic Cells

PubMed Central

Mitra, Sayantan; Yang, Xiaohui

2016-01-01

Sister chromatid cohesion, which is mediated by the cohesin complex, is essential for the proper segregation of chromosomes during mitosis and meiosis. Stable binding of cohesin with chromosomes is regulated in part by the opposing actions of CTF7 (CHROMOSOME TRANSMISSION FIDELITY7) and WAPL (WINGS APART-LIKE). In this study, we characterized the interaction between Arabidopsis thaliana CTF7 and WAPL by conducting a detailed analysis of wapl1-1 wapl2 ctf7 plants. ctf7 plants exhibit major defects in vegetative growth and development and are completely sterile. Inactivation of WAPL restores normal growth, mitosis, and some fertility to ctf7 plants. This shows that the CTF7/WAPL cohesin system is not essential for mitosis in vegetative cells and suggests that plants may contain a second mechanism to regulate mitotic cohesin. WAPL inactivation restores cohesin binding and suppresses ctf7-associated meiotic cohesion defects, demonstrating that WAPL and CTF7 function as antagonists to regulate meiotic sister chromatid cohesion. The ctf7 mutation only had a minor effect on wapl-associated defects in chromosome condensation and centromere association. These results demonstrate that WAPL has additional roles that are independent of its role in regulating chromatin-bound cohesin. PMID:26813623

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hinshaw, Stephen M.; Makrantoni, Vasso; Kerr, Alastair

The cohesin ring holds newly replicated sister chromatids together until their separation at anaphase. Initiation of sister chromatid cohesion depends on a separate complex, Scc2NIPBL/Scc4Mau2 (Scc2/4), which loads cohesin onto DNA and determines its localization across the genome. Proper cohesin loading is essential for cell division, and partial defects cause chromosome missegregation and aberrant transcriptional regulation, leading to severe developmental defects in multicellular organisms. We present here a crystal structure showing the interaction between Scc2 and Scc4. Scc4 is a TPR array that envelops an extended Scc2 peptide. Using budding yeast, we demonstrate that a conserved patch on the surfacemore » of Scc4 is required to recruit Scc2/4 to centromeres and to build pericentromeric cohesion. These findings reveal the role of Scc4 in determining the localization of cohesin loading and establish a molecular basis for Scc2/4 recruitment to centromeres.« less

Superresolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact Caenorhabditis elegans tissue

PubMed Central

Köhler, Simone; Wojcik, Michal; Dernburg, Abby F.

2017-01-01

When cells enter meiosis, their chromosomes reorganize as linear arrays of chromatin loops anchored to a central axis. Meiotic chromosome axes form a platform for the assembly of the synaptonemal complex (SC) and play central roles in other meiotic processes, including homologous pairing, recombination, and chromosome segregation. However, little is known about the 3D organization of components within the axes, which include cohesin complexes and additional meiosis-specific proteins. Here, we investigate the molecular organization of meiotic chromosome axes in Caenorhabditis elegans through STORM (stochastic optical reconstruction microscopy) and PALM (photo-activated localization microscopy) superresolution imaging of intact germ-line tissue. By tagging one axis protein (HIM-3) with a photoconvertible fluorescent protein, we established a spatial reference for other components, which were localized using antibodies against epitope tags inserted by CRISPR/Cas9 genome editing. Using 3D averaging, we determined the position of all known components within synapsed chromosome axes to high spatial precision in three dimensions. We find that meiosis-specific HORMA domain proteins span a gap between cohesin complexes and the central region of the SC, consistent with their essential roles in SC assembly. Our data further suggest that the two different meiotic cohesin complexes are distinctly arranged within the axes: Although cohesin complexes containing the kleisin REC-8 protrude above and below the plane defined by the SC, complexes containing COH-3 or -4 kleisins form a central core, which may physically separate sister chromatids. This organization may help to explain the role of the chromosome axes in promoting interhomolog repair of meiotic double-strand breaks by inhibiting intersister repair. PMID:28559338

Casein Kinase 1 and Phosphorylation of Cohesin Subunit Rec11 (SA3) Promote Meiotic Recombination through Linear Element Formation.

PubMed

Phadnis, Naina; Cipak, Lubos; Polakova, Silvia; Hyppa, Randy W; Cipakova, Ingrid; Anrather, Dorothea; Karvaiova, Lucia; Mechtler, Karl; Smith, Gerald R; Gregan, Juraj

2015-05-01

Proper meiotic chromosome segregation, essential for sexual reproduction, requires timely formation and removal of sister chromatid cohesion and crossing-over between homologs. Early in meiosis cohesins hold sisters together and also promote formation of DNA double-strand breaks, obligate precursors to crossovers. Later, cohesin cleavage allows chromosome segregation. We show that in fission yeast redundant casein kinase 1 homologs, Hhp1 and Hhp2, previously shown to regulate segregation via phosphorylation of the Rec8 cohesin subunit, are also required for high-level meiotic DNA breakage and recombination. Unexpectedly, these kinases also mediate phosphorylation of a different meiosis-specific cohesin subunit Rec11. This phosphorylation in turn leads to loading of linear element proteins Rec10 and Rec27, related to synaptonemal complex proteins of other species, and thereby promotes DNA breakage and recombination. Our results provide novel insights into the regulation of chromosomal features required for crossing-over and successful reproduction. The mammalian functional homolog of Rec11 (STAG3) is also phosphorylated during meiosis and appears to be required for fertility, indicating wide conservation of the meiotic events reported here.

Polo kinase Cdc5 is a central regulator of meiosis I

PubMed Central

Attner, Michelle A.; Miller, Matthew P.; Ee, Ly-sha; Elkin, Sheryl K.; Amon, Angelika

2013-01-01

During meiosis, two consecutive rounds of chromosome segregation yield four haploid gametes from one diploid cell. The Polo kinase Cdc5 is required for meiotic progression, but how Cdc5 coordinates multiple cell-cycle events during meiosis I is not understood. Here we show that CDC5-dependent phosphorylation of Rec8, a subunit of the cohesin complex that links sister chromatids, is required for efficient cohesin removal from chromosome arms, which is a prerequisite for meiosis I chromosome segregation. CDC5 also establishes conditions for centromeric cohesin removal during meiosis II by promoting the degradation of Spo13, a protein that protects centromeric cohesin during meiosis I. Despite CDC5’s central role in meiosis I, the protein kinase is dispensable during meiosis II and does not even phosphorylate its meiosis I targets during the second meiotic division. We conclude that Cdc5 has evolved into a master regulator of the unique meiosis I chromosome segregation pattern. PMID:23918381

Chl1 DNA helicase and Scc2 function in chromosome condensation through cohesin deposition.

PubMed

Shen, Donglai; Skibbens, Robert V

2017-01-01

Chl1 DNA helicase promotes sister chromatid cohesion and associates with both the cohesion establishment acetyltransferase Eco1/Ctf7 and the DNA polymerase processivity factor PCNA that supports Eco1/Ctf7 function. Mutation in CHL1 results in precocious sister chromatid separation and cell aneuploidy, defects that arise through reduced levels of chromatin-bound cohesins which normally tether together sister chromatids (trans tethering). Mutation of Chl1 family members (BACH1/BRIP/FANCJ and DDX11/ChlR1) also exhibit genotoxic sensitivities, consistent with a role for Chl1 in trans tethering which is required for efficient DNA repair. Chl1 promotes the recruitment of Scc2 to DNA which is required for cohesin deposition onto DNA. There is limited evidence, however, that Scc2 also directs the deposition onto DNA of condensins which promote tethering in cis (intramolecular DNA links). Here, we test the ability of Chl1 to promote cis tethering and the role of both Chl1 and Scc2 to promote condensin recruitment to DNA. The results reveal that chl1 mutant cells exhibit significant condensation defects both within the rDNA locus and genome-wide. Importantly, chl1 mutant cell condensation defects do not result from reduced chromatin binding of condensin, but instead through reduced chromatin binding of cohesin. We tested scc2-4 mutant cells and similarly found no evidence of reduced condensin recruitment to chromatin. Consistent with a role for Scc2 specifically in cohesin deposition, scc2-4 mutant cell condensation defects are irreversible. We thus term Chl1 a novel regulator of both chromatin condensation and sister chromatid cohesion through cohesin-based mechanisms. These results reveal an exciting interface between DNA structure and the highly conserved cohesin complex.

Chl1 DNA helicase and Scc2 function in chromosome condensation through cohesin deposition

PubMed Central

Shen, Donglai

2017-01-01

Chl1 DNA helicase promotes sister chromatid cohesion and associates with both the cohesion establishment acetyltransferase Eco1/Ctf7 and the DNA polymerase processivity factor PCNA that supports Eco1/Ctf7 function. Mutation in CHL1 results in precocious sister chromatid separation and cell aneuploidy, defects that arise through reduced levels of chromatin-bound cohesins which normally tether together sister chromatids (trans tethering). Mutation of Chl1 family members (BACH1/BRIP/FANCJ and DDX11/ChlR1) also exhibit genotoxic sensitivities, consistent with a role for Chl1 in trans tethering which is required for efficient DNA repair. Chl1 promotes the recruitment of Scc2 to DNA which is required for cohesin deposition onto DNA. There is limited evidence, however, that Scc2 also directs the deposition onto DNA of condensins which promote tethering in cis (intramolecular DNA links). Here, we test the ability of Chl1 to promote cis tethering and the role of both Chl1 and Scc2 to promote condensin recruitment to DNA. The results reveal that chl1 mutant cells exhibit significant condensation defects both within the rDNA locus and genome-wide. Importantly, chl1 mutant cell condensation defects do not result from reduced chromatin binding of condensin, but instead through reduced chromatin binding of cohesin. We tested scc2-4 mutant cells and similarly found no evidence of reduced condensin recruitment to chromatin. Consistent with a role for Scc2 specifically in cohesin deposition, scc2-4 mutant cell condensation defects are irreversible. We thus term Chl1 a novel regulator of both chromatin condensation and sister chromatid cohesion through cohesin-based mechanisms. These results reveal an exciting interface between DNA structure and the highly conserved cohesin complex. PMID:29186203

Preliminary X-ray characterization of a novel type of anchoring cohesin from the cellulosome of Ruminococcus flavefaciens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alber, Orly; Noach, Ilit; Lamed, Raphael

2008-02-01

The cloning, expression, purification, crystallization and preliminary X-ray characterization of a novel class of cohesin module (type III) from the R. flavefaciens ScaE anchoring scaffoldin are described. Ruminococcus flavefaciens is an anaerobic bacterium that resides in the gastrointestinal tract of ruminants. It produces a highly organized multi-enzyme cellulosome complex that plays a key role in the degradation of plant cell walls. ScaE is one of the critical structural components of its cellulosome that serves to anchor the complex to the cell wall. The seleno-l-methionine-labelled derivative of the ScaE cohesin module has been cloned, expressed, purified and crystallized. The crystals belongmore » to space group C2, with unit-cell parameters a = 155.6, b = 69.3, c = 93.0 Å, β = 123.4°, and contain four molecules in the asymmetric unit. Diffraction data were phased to 1.95 Å using the anomalous signal from the Se atoms.« less

Inactivation of the budding yeast cohesin loader Scc2 alters gene expression both globally and in response to a single DNA double strand break

PubMed Central

Lindgren, Emma; Hägg, Sara; Giordano, Fosco; Björkegren, Johan; Ström, Lena

2014-01-01

Genome integrity is fundamental for cell survival and cell cycle progression. Important mechanisms for keeping the genome intact are proper sister chromatid segregation, correct gene regulation and efficient repair of damaged DNA. Cohesin and its DNA loader, the Scc2/4 complex have been implicated in all these cellular actions. The gene regulation role has been described in several organisms. In yeast it has been suggested that the proteins in the cohesin network would effect transcription based on its role as insulator. More recently, data are emerging indicating direct roles for gene regulation also in yeast. Here we extend these studies by investigating whether the cohesin loader Scc2 is involved in regulation of gene expression. We performed global gene expression profiling in the absence and presence of DNA damage, in wild type and Scc2 deficient G2/M arrested cells, when it is known that Scc2 is important for DNA double strand break repair and formation of damage induced cohesion. We found that not only the DNA damage specific transcriptional response is distorted after inactivation of Scc2 but also the overall transcription profile. Interestingly, these alterations did not correlate with changes in cohesin binding. PMID:25483075

A parts list for fungal cellulosomes revealed by comparative genomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haitjema, Charles H.; Gilmore, Sean P.; Henske, John K.

Cellulosomes are large, multi-protein complexes that tether plant biomass degrading enzymes together for improved hydrolysis1. These complexes were first described in anaerobic bacteria where species specific dockerin domains mediate assembly of enzymes onto complementary cohesin motifs interspersed within non-catalytic protein scaffolds1. The versatile protein assembly mechanism conferred by the bacterial cohesin-dockerin interaction is now a standard design principle for synthetic protein-scale pathways2,3. For decades, analogous structures have been reported in the early branching anaerobic fungi, which are known to assemble by sequence divergent non-catalytic dockerin domains (NCDD)4. However, the enzyme components, modular assembly mechanism, and functional role of fungal cellulosomesmore » remain unknown5,6. Here, we describe the comprehensive set of proteins critical to fungal cellulosome assembly, including novel, conserved scaffolding proteins unique to the Neocallimastigomycota. High quality genomes of the anaerobic fungi Anaeromyces robustus, Neocallimastix californiae and Piromyces finnis were assembled with long-read, single molecule technology to overcome their repeat-richness and extremely low GC content. Genomic analysis coupled with proteomic validation revealed an average 320 NCDD-containing proteins per fungal strain that were overwhelmingly carbohydrate active enzymes (CAZymes), with 95 large fungal scaffoldins identified across 4 genera that contain a conserved amino acid sequence repeat that binds to NCDDs. Fungal dockerin and scaffoldin domains have no similarity to their bacterial counterparts, yet several catalytic domains originated via horizontal gene transfer with gut bacteria. Though many catalytic domains are shared with bacteria, the biocatalytic activity of anaerobic fungi is expanded by the inclusion of GH3, GH6, and GH45 enzymes in the enzyme complexes. Collectively, these findings suggest that the fungal cellulosome is an evolutionarily chimeric structure – an independently evolved fungal complex that co-opted useful activities from bacterial neighbors within the gut microbiome.« less

Crystal Structure of the Cohesin Gatekeeper Pds5 and in Complex with Kleisin Scc1.

PubMed

Lee, Byung-Gil; Roig, Maurici B; Jansma, Marijke; Petela, Naomi; Metson, Jean; Nasmyth, Kim; Löwe, Jan

2016-03-08

Sister chromatid cohesion is mediated by cohesin, whose Smc1, Smc3, and kleisin (Scc1) subunits form a ring structure that entraps sister DNAs. The ring is opened either by separase, which cleaves Scc1 during anaphase, or by a releasing activity involving Wapl, Scc3, and Pds5, which bind to Scc1 and open its interface with Smc3. We present crystal structures of Pds5 from the yeast L. thermotolerans in the presence and absence of the conserved Scc1 region that interacts with Pds5. Scc1 binds along the spine of the Pds5 HEAT repeat fold and is wedged between the spine and C-terminal hook of Pds5. We have isolated mutants that confirm the observed binding mode of Scc1 and verified their effect on cohesin by immunoprecipitation and calibrated ChIP-seq. The Pds5 structure also reveals architectural similarities to Scc3, the other large HEAT repeat protein of cohesin and, most likely, Scc2. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Structure and function of the PP2A-shugoshin interaction

PubMed Central

Xu, Zheng; Cetin, Bulent; Anger, Martin; Cho, Uhn Soo; Helmhart, Wolfgang; Nasmyth, Kim; Xu, Wenqing

2009-01-01

SUMMARY Accurate chromosome segregation during mitosis and meiosis depends on shugoshin proteins that prevent precocious dissociation of cohesin from centromeres. Shugoshins associate with PP2A, which is thought to de-phosphorylate cohesin and thereby prevent cleavage by separase during meiosis I. A crystal structure of a complex between a fragment of human Sgo1 and an AB’C PP2A holoenzyme reveals that Sgo1 forms a homodimeric parallel coiled-coil that docks simultaneously onto PP2A’s C and B’ subunits. Sgo1 homo-dimerization is a pre-requisite for PP2A binding. While hSgo1 interacts only with the AB’C holoenzymes, its relative Sgo2 interacts with all PP2A forms and may thus lead to dephosphorylation of distinct substrates. Mutant shugoshin proteins defective in the binding of PP2A cannot protect centromeric cohesin from separase during meiosis I or support the spindle assembly checkpoint in yeast. Finally, we provide evidence that PP2A’s recruitment to chromosomes may be sufficient to protect cohesin from separase in mammalian oocytes. PMID:19716788

Polycomb purification by in vivo biotinylation tagging reveals cohesin and Trithorax group proteins as interaction partners

PubMed Central

Strübbe, Gero; Popp, Christian; Schmidt, Alexander; Pauli, Andrea; Ringrose, Leonie; Beisel, Christian; Paro, Renato

2011-01-01

The maintenance of specific gene expression patterns during cellular proliferation is crucial for the identity of every cell type and the development of tissues in multicellular organisms. Such a cellular memory function is conveyed by the complex interplay of the Polycomb and Trithorax groups of proteins (PcG/TrxG). These proteins exert their function at the level of chromatin by establishing and maintaining repressed (PcG) and active (TrxG) chromatin domains. Past studies indicated that a core PcG protein complex is potentially associated with cell type or even cell stage-specific sets of accessory proteins. In order to better understand the dynamic aspects underlying PcG composition and function we have established an inducible version of the biotinylation tagging approach to purify Polycomb and associated factors from Drosophila embryos. This system enabled fast and efficient isolation of Polycomb containing complexes under near physiological conditions, thereby preserving substoichiometric interactions. Novel interacting proteins were identified by highly sensitive mass spectrometric analysis. We found many TrxG related proteins, suggesting a previously unrecognized extent of molecular interaction of the two counteracting chromatin regulatory protein groups. Furthermore, our analysis revealed an association of PcG protein complexes with the cohesin complex and showed that Polycomb-dependent silencing of a transgenic reporter depends on cohesin function. PMID:21415365

Splitting the chromosome: cutting the ties that bind sister chromatids.

PubMed

Nasmyth, K; Peters, J M; Uhlmann, F

2000-05-26

In eukaryotic cells, sister DNA molecules remain physically connected from their production at S phase until their separation during anaphase. This cohesion is essential for the separation of sister chromatids to opposite poles of the cell at mitosis. It also permits chromosome segregation to take place long after duplication has been completed. Recent work has identified a multisubunit complex called cohesin that is essential for connecting sisters. Proteolytic cleavage of one of cohesin's subunits may trigger sister separation at the onset of anaphase.

Separase Is Required for Homolog and Sister Disjunction during Drosophila melanogaster Male Meiosis, but Not for Biorientation of Sister Centromeres.

PubMed

Blattner, Ariane C; Chaurasia, Soumya; McKee, Bruce D; Lehner, Christian F

2016-04-01

Spatially controlled release of sister chromatid cohesion during progression through the meiotic divisions is of paramount importance for error-free chromosome segregation during meiosis. Cohesion is mediated by the cohesin protein complex and cleavage of one of its subunits by the endoprotease separase removes cohesin first from chromosome arms during exit from meiosis I and later from the pericentromeric region during exit from meiosis II. At the onset of the meiotic divisions, cohesin has also been proposed to be present within the centromeric region for the unification of sister centromeres into a single functional entity, allowing bipolar orientation of paired homologs within the meiosis I spindle. Separase-mediated removal of centromeric cohesin during exit from meiosis I might explain sister centromere individualization which is essential for subsequent biorientation of sister centromeres during meiosis II. To characterize a potential involvement of separase in sister centromere individualization before meiosis II, we have studied meiosis in Drosophila melanogaster males where homologs are not paired in the canonical manner. Meiosis does not include meiotic recombination and synaptonemal complex formation in these males. Instead, an alternative homolog conjunction system keeps homologous chromosomes in pairs. Using independent strategies for spermatocyte-specific depletion of separase complex subunits in combination with time-lapse imaging, we demonstrate that separase is required for the inactivation of this alternative conjunction at anaphase I onset. Mutations that abolish alternative homolog conjunction therefore result in random segregation of univalents during meiosis I also after separase depletion. Interestingly, these univalents become bioriented during meiosis II, suggesting that sister centromere individualization before meiosis II does not require separase.

Separase Is Required for Homolog and Sister Disjunction during Drosophila melanogaster Male Meiosis, but Not for Biorientation of Sister Centromeres

PubMed Central

Blattner, Ariane C.; McKee, Bruce D.; Lehner, Christian F.

2016-01-01

Spatially controlled release of sister chromatid cohesion during progression through the meiotic divisions is of paramount importance for error-free chromosome segregation during meiosis. Cohesion is mediated by the cohesin protein complex and cleavage of one of its subunits by the endoprotease separase removes cohesin first from chromosome arms during exit from meiosis I and later from the pericentromeric region during exit from meiosis II. At the onset of the meiotic divisions, cohesin has also been proposed to be present within the centromeric region for the unification of sister centromeres into a single functional entity, allowing bipolar orientation of paired homologs within the meiosis I spindle. Separase-mediated removal of centromeric cohesin during exit from meiosis I might explain sister centromere individualization which is essential for subsequent biorientation of sister centromeres during meiosis II. To characterize a potential involvement of separase in sister centromere individualization before meiosis II, we have studied meiosis in Drosophila melanogaster males where homologs are not paired in the canonical manner. Meiosis does not include meiotic recombination and synaptonemal complex formation in these males. Instead, an alternative homolog conjunction system keeps homologous chromosomes in pairs. Using independent strategies for spermatocyte-specific depletion of separase complex subunits in combination with time-lapse imaging, we demonstrate that separase is required for the inactivation of this alternative conjunction at anaphase I onset. Mutations that abolish alternative homolog conjunction therefore result in random segregation of univalents during meiosis I also after separase depletion. Interestingly, these univalents become bioriented during meiosis II, suggesting that sister centromere individualization before meiosis II does not require separase. PMID:27120695

Osmotic mechanism of the loop extrusion process

NASA Astrophysics Data System (ADS)

Yamamoto, Tetsuya; Schiessel, Helmut

2017-09-01

The loop extrusion theory assumes that protein factors, such as cohesin rings, act as molecular motors that extrude chromatin loops. However, recent single molecule experiments have shown that cohesin does not show motor activity. To predict the physical mechanism involved in loop extrusion, we here theoretically analyze the dynamics of cohesin rings on a loop, where a cohesin loader is in the middle and unloaders at the ends. Cohesin monomers bind to the loader rather frequently and cohesin dimers bind to this site only occasionally. Our theory predicts that a cohesin dimer extrudes loops by the osmotic pressure of cohesin monomers on the chromatin fiber between the two connected rings. With this mechanism, the frequency of the interactions between chromatin segments depends on the loading and unloading rates of dimers at the corresponding sites.

The expanding universe of cohesin functions: a new genome stability caretaker involved in human disease and cancer.

PubMed

Mannini, Linda; Menga, Stefania; Musio, Antonio

2010-06-01

Cohesin is responsible for sister chromatid cohesion, ensuring the correct chromosome segregation. Beyond this role, cohesin and regulatory cohesin genes seem to play a role in preserving genome stability and gene transcription regulation. DNA damage is thought to be a major culprit for many human diseases, including cancer. Our present knowledge of the molecular basis underlying genome instability is extremely limited. Mutations in cohesin genes cause human diseases such as Cornelia de Lange syndrome and Roberts syndrome/SC phocomelia, and all the cell lines derived from affected patients show genome instability. Cohesin mutations have also been identified in colorectal cancer. Here, we will discuss the human disorders caused by alterations of cohesin function, with emphasis on the emerging role of cohesin as a genome stability caretaker.

An inherited immunoglobulin class-switch recombination deficiency associated with a defect in the INO80 chromatin remodeling complex

PubMed Central

Kracker, Sven; Di Virgilio, Michela; Schwartzentruber, Jeremy; Cuenin, Cyrille; Forveille, Monique; Deau, Marie-Céline; McBride, Kevin M.; Majewski, Jacek; Gazumyan, Anna; Seneviratne, Suranjith; Grimbacher, Bodo; Kutukculer, Necil; Herceg, Zdenko; Cavazzana, Marina; Jabado, Nada; Nussenzweig, Michel C.; Fischer, Alain; Durandy, Anne

2015-01-01

Background Immunoglobulin class-switch recombination defects (CSR-D) are rare primary immunodeficiencies characterized by impaired production of switched immunoglobulin isotypes and normal or elevated IgM levels. They are caused by impaired T:B cooperation or intrinsic B cell defects. However, many immunoglobulin CSR-Ds are still undefined at the molecular level. Objective This study's objective was to delineate new causes of immunoglobulin CSR-Ds and thus gain further insights into the process of immunoglobulin class-switch recombination (CSR). Methods Exome sequencing in 2 immunoglobulin CSR-D patients identified variations in the INO80 gene. Functional experiments were performed to assess the function of INO80 on immunoglobulin CSR. Results We identified recessive, nonsynonymous coding variations in the INO80 gene in 2 patients affected by defective immunoglobulin CSR. Expression of wild-type INO80 in patients' fibroblastic cells corrected their hypersensitivity to high doses of γ-irradiation. In murine CH12-F3 cells, the INO80 complex accumulates at Sα and Eμ regions of the IgH locus, and downregulation of INO80 as well as its partners Reptin and Pontin impaired CSR. In addition, Reptin and Pontin were shown to interact with activation-induced cytidine deaminase. Finally, an abnormal separation of sister chromatids was observed upon INO80 downregulation in CH12-F3 cells, pinpointing its role in cohesin activity. Conclusion INO80 deficiency appears to be associated with defective immunoglobulin CSR. We propose that the INO80 complex modulates cohesin function that may be required during immunoglobulin switch region synapsis. PMID:25312759

Methods for Discovery of Novel Cellulosomal Cellulases Using Genomics and Biochemical Tools.

PubMed

Ben-David, Yonit; Dassa, Bareket; Bensoussan, Lizi; Bayer, Edward A; Moraïs, Sarah

2018-01-01

Cell wall degradation by cellulases is extensively explored owing to its potential contribution to biofuel production. The cellulosome is an extracellular multienzyme complex that can degrade the plant cell wall very efficiently, and cellulosomal enzymes are therefore of great interest. The cellulosomal cellulases are defined as enzymes that contain a dockerin module, which can interact with a cohesin module contained in multiple copies in a noncatalytic protein, termed scaffoldin. The assembly of the cellulosomal cellulases into the cellulosomal complex occurs via specific protein-protein interactions. Cellulosome systems have been described initially only in several anaerobic cellulolytic bacteria. However, owing to ongoing genome sequencing and metagenomic projects, the discovery of novel cellulosome-producing bacteria and the description of their cellulosomal genes have dramatically increased in the recent years. In this chapter, methods for discovery of novel cellulosomal cellulases from a DNA sequence by bioinformatics and biochemical tools are described. Their biochemical characterization is also described, including both the enzymatic activity of the putative cellulases and their assembly into mature designer cellulosomes.

Near-complete genome sequence of the cellulolytic Bacterium Bacteroides ( Pseudobacteroides) cellulosolvens ATCC 35603

DOE PAGES

Dassa, Bareket; Utturkar, Sagar M.; Hurt, Richard A.; ...

2015-09-24

We report the single-contig genome sequence of the anaerobic, mesophilic, cellulolytic bacterium, Bacteroides cellulosolvens. The bacterium produces a particularly elaborate cellulosome system, whereas the types of cohesin-dockerin interactions are opposite of other known cellulosome systems: cell-surface attachment is thus mediated via type-I interactions whereas enzymes are integrated via type-II interactions.

Clinical, developmental and molecular update on Cornelia de Lange syndrome and the cohesin complex: abstracts from the 2014 Scientific and Educational Symposium.

PubMed

Kline, Antonie D; Calof, Anne L; Lander, Arthur D; Gerton, Jennifer L; Krantz, Ian D; Dorsett, Dale; Deardorff, Matthew A; Blagowidow, Natalie; Yokomori, Kyoko; Shirahige, Katsuhiko; Santos, Rosaysela; Woodman, Julie; Megee, Paul C; O'Connor, Julia T; Egense, Alena; Noon, Sarah; Belote, Maurice; Goodban, Marjorie T; Hansen, Blake D; Timmons, Jenni Glad; Musio, Antonio; Ishman, Stacey L; Bryan, Yvon; Wu, Yaning; Bettini, Laura R; Mehta, Devanshi; Zakari, Musinu; Mills, Jason A; Srivastava, Siddharth; Haaland, Richard E

2015-06-01

Cornelia de Lange Syndrome (CdLS) is the most common example of disorders of the cohesin complex, or cohesinopathies. There are a myriad of clinical issues facing individuals with CdLS, particularly in the neurodevelopmental system, which also have implications for the parents and caretakers, involved professionals, therapists, and schools. Basic research in developmental and cell biology on cohesin is showing significant progress, with improved understanding of the mechanisms and the possibility of potential therapeutics. The following abstracts are presentations from the 6th Cornelia de Lange Syndrome Scientific and Educational Symposium, which took place on June 25-26, 2014, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting in Costa Mesa, CA. The Research Committee of the CdLS Foundation organizes the meeting, reviews and accepts abstracts, and subsequently disseminates the information to the families through members of the Clinical Advisory Board. In addition to the scientific and clinical discussions, there were educationally focused talks related to practical aspects of behavior and development. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD. © 2015 Wiley Periodicals, Inc.

An inherited immunoglobulin class-switch recombination deficiency associated with a defect in the INO80 chromatin remodeling complex.

PubMed

Kracker, Sven; Di Virgilio, Michela; Schwartzentruber, Jeremy; Cuenin, Cyrille; Forveille, Monique; Deau, Marie-Céline; McBride, Kevin M; Majewski, Jacek; Gazumyan, Anna; Seneviratne, Suranjith; Grimbacher, Bodo; Kutukculer, Necil; Herceg, Zdenko; Cavazzana, Marina; Jabado, Nada; Nussenzweig, Michel C; Fischer, Alain; Durandy, Anne

2015-04-01

Immunoglobulin class-switch recombination defects (CSR-D) are rare primary immunodeficiencies characterized by impaired production of switched immunoglobulin isotypes and normal or elevated IgM levels. They are caused by impaired T:B cooperation or intrinsic B cell defects. However, many immunoglobulin CSR-Ds are still undefined at the molecular level. This study's objective was to delineate new causes of immunoglobulin CSR-Ds and thus gain further insights into the process of immunoglobulin class-switch recombination (CSR). Exome sequencing in 2 immunoglobulin CSR-D patients identified variations in the INO80 gene. Functional experiments were performed to assess the function of INO80 on immunoglobulin CSR. We identified recessive, nonsynonymous coding variations in the INO80 gene in 2 patients affected by defective immunoglobulin CSR. Expression of wild-type INO80 in patients' fibroblastic cells corrected their hypersensitivity to high doses of γ-irradiation. In murine CH12-F3 cells, the INO80 complex accumulates at Sα and Eμ regions of the IgH locus, and downregulation of INO80 as well as its partners Reptin and Pontin impaired CSR. In addition, Reptin and Pontin were shown to interact with activation-induced cytidine deaminase. Finally, an abnormal separation of sister chromatids was observed upon INO80 downregulation in CH12-F3 cells, pinpointing its role in cohesin activity. INO80 deficiency appears to be associated with defective immunoglobulin CSR. We propose that the INO80 complex modulates cohesin function that may be required during immunoglobulin switch region synapsis. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast.

PubMed

Jonak, Katarzyna; Zagoriy, Ievgeniia; Oz, Tugce; Graf, Peter; Rojas, Julie; Mengoli, Valentina; Zachariae, Wolfgang

2017-06-18

Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to "deprotect" Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/C Cdc20 removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes.

APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast

PubMed Central

Jonak, Katarzyna; Oz, Tugce; Graf, Peter; Rojas, Julie; Mengoli, Valentina; Zachariae, Wolfgang

2017-01-01

ABSTRACT Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to “deprotect” Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/CCdc20 removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes. PMID:28514186

The Aurora kinase Ipl1 maintains the centromeric localization of PP2A to protect cohesin during meiosis.

PubMed

Yu, Hong-Guo; Koshland, Douglas

2007-03-26

Homologue segregation during the first meiotic division requires the proper spatial regulation of sister chromatid cohesion and its dissolution along chromosome arms, but its protection at centromeric regions. This protection requires the conserved MEI-S332/Sgo1 proteins that localize to centromeric regions and also recruit the PP2A phosphatase by binding its regulatory subunit, Rts1. Centromeric Rts1/PP2A then locally prevents cohesion dissolution possibly by dephosphorylating the protein complex cohesin. We show that Aurora B kinase in Saccharomyces cerevisiae (Ipl1) is also essential for the protection of meiotic centromeric cohesion. Coupled with a previous study in Drosophila melanogaster, this meiotic function of Aurora B kinase appears to be conserved among eukaryotes. Furthermore, we show that Sgo1 recruits Ipl1 to centromeric regions. In the absence of Ipl1, Rts1 can initially bind to centromeric regions but disappears from these regions after anaphase I onset. We suggest that centromeric Ipl1 ensures the continued centromeric presence of active Rts1/PP2A, which in turn locally protects cohesin and cohesion.

Independent mechanisms recruit the cohesin loader protein NIPBL to sites of DNA damage.

PubMed

Bot, Christopher; Pfeiffer, Annika; Giordano, Fosco; Manjeera, Dharani E; Dantuma, Nico P; Ström, Lena

2017-03-15

NIPBL is required to load the cohesin complex on to DNA. While the canonical role of cohesin is to couple replicated sister chromatids together until the onset of mitosis, it also promotes tolerance to DNA damage. Here, we show that NIPBL is recruited to DNA damage throughout the cell cycle via independent mechanisms, influenced by type of damage. First, the heterochromatin protein HP1γ (also known as CBX3) recruits NIPBL to DNA double-strand breaks (DSBs) through the corresponding HP1-binding motif within the N-terminus. By contrast, the C-terminal HEAT repeat domain is unable to recruit NIPBL to DSBs but independently targets NIPBL to laser microirradiation-induced DNA damage. Each mechanism is dependent on the RNF8 and RNF168 ubiquitylation pathway, while the recruitment of the HEAT repeat domain requires further ATM or ATR activity. Thus, NIPBL has evolved a sophisticated response to damaged DNA that is influenced by the form of damage, suggesting a highly dynamic role for NIPBL in maintaining genomic stability. © 2017. Published by The Company of Biologists Ltd.

Releasing the cohesin ring: A rigid scaffold model for opening the DNA exit gate by Pds5 and Wapl.

PubMed

Ouyang, Zhuqing; Yu, Hongtao

2017-04-01

The ring-shaped ATPase machine, cohesin, regulates sister chromatid cohesion, transcription, and DNA repair by topologically entrapping DNA. Here, we propose a rigid scaffold model to explain how the cohesin regulators Pds5 and Wapl release cohesin from chromosomes. Recent studies have established the Smc3-Scc1 interface as the DNA exit gate of cohesin, revealed a requirement for ATP hydrolysis in ring opening, suggested regulation of the cohesin ATPase activity by DNA and Smc3 acetylation, and provided insights into how Pds5 and Wapl open this exit gate. We hypothesize that Pds5, Wapl, and SA1/2 form a rigid scaffold that docks on Scc1 and anchors the N-terminal domain of Scc1 (Scc1N) to the Smc1 ATPase head. Relative movements between the Smc1-3 ATPase heads driven by ATP and Wapl disrupt the Smc3-Scc1 interface. Pds5 binds the dissociated Scc1N and prolongs this open state of cohesin, releasing DNA. We review the evidence supporting this model and suggest experiments that can further test its key principles. © 2017 WILEY Periodicals, Inc.

Cornelia de Lange syndrome and molecular implications of the cohesin complex: Abstracts from the 7th biennial scientific and educational symposium 2016

PubMed Central

Kline, Antonie D.; Krantz, Ian D.; Deardorff, Matthew A.; Shirahige, Katsuhiko; Dorsett, Dale; Gerton, Jennifer L.; Wu, Meng; Mehta, Devanshi; Mills, Jason A.; Carrico, Cheri S.; Noon, Sarah; Herrera, Pamela S.; Horsfield, Julia A.; Bettale, Chiara; Morgan, Jeremy; Huisman, Sylvia A.; Moss, Jo; McCleery, Joseph; Grados, Marco; Hansen, Blake D.; Srivastava, Siddharth; Taylor-Snell, Emily; Kerr, Lynne M.; Katz, Olivia; Calof, Anne L.; Musio, Antonio; Egense, Alena; Haaland, Richard E.

2017-01-01

Cornelia de Lange Syndrome (CdLS) is due to mutations in the genes for the structural and regulatory proteins that make up the cohesin complex, and is considered a cohesinopathy disorder or, more recently, a transcriptomopathy. New phenotypes have been recognized in this expanding field. There are multiple clinical issues facing individuals with all forms of CdLS, particularly in the neurodevelopmental system, but also gastrointestinal, cardiac, and musculoskeletal. Aspects of developmental and cell biology have found common endpoints in the biology of the cohesin complex, with improved understanding of the mechanisms, easier diagnostic tests, and the possibility of potential therapeutics, all major clinical implications for the individual with CdLS. The following abstracts are the presentations from the 7th Cornelia de Lange Syndrome Scientific and Educational Symposium, June 22–23, 2016, in Orlando, FL, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting. In addition to the scientific and clinical discussions, there were talks related to practical aspects of behavior including autism, transitions, communication, access to medical care, and databases. At the end of the symposium, a panel was held, which included several parents, affected individuals and genetic counselors, and discussed the greatest challenges in life and how this information can assist in guiding future research. The Research Committee of the CdLS Foundation organizes this meeting, reviews, and accepts abstracts, and subsequently disseminates the information to the families through members of the Clinical Advisory Board and publications. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD. PMID:28190301

Formation of chromosomal domains in interphase by loop extrusion

NASA Astrophysics Data System (ADS)

Fudenberg, Geoffrey

While genomes are often considered as one-dimensional sequences, interphase chromosomes are organized in three dimensions with an essential role for regulating gene expression. Recent studies have shown that Topologically Associating Domains (TADs) are fundamental structural and functional building blocks of human interphase chromosomes. Despite observations that architectural proteins, including CTCF, demarcate and maintain the borders of TADs, the mechanisms underlying TAD formation remain unknown. Here we propose that loop extrusion underlies the formation TADs. In this process, cis-acting loop-extruding factors, likely cohesins, form progressively larger loops, but stall at TAD boundaries due to interactions with boundary proteins, including CTCF. This process dynamically forms loops of various sizes within but not between TADs. Using polymer simulations, we find that loop extrusion can produce TADs as determined by our analyses of the highest-resolution experimental data. Moreover, we find that loop extrusion can explain many diverse experimental observations, including: the preferential orientation of CTCF motifs and enrichments of architectural proteins at TAD boundaries; TAD boundary deletion experiments; and experiments with knockdown or depletion of CTCF, cohesin, and cohesin-loading factors. Together, the emerging picture from our work is that TADs are formed by rapidly associating, growing, and dissociating loops, presenting a clear framework for understanding interphase chromosomal organization.

Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

NASA Astrophysics Data System (ADS)

Liu, Fang

Globally, there is growing appreciation for developing a sustainable economy that uses eco-efficient bio-processes. Biotechnology provides an increasing range of tools for industry to help reduce cost and improve environmental performance. Inspired by the naturally evolved machineries of protein scaffolds and their binding ligands, synthetic protein scaffolds were engineered based on cohesin-dockerin interactions and metal chelating peptides to tackle the challenges and make improvements in three specific areas: (1) protein purification, (2) biofuel cells, and (3) nanomaterial synthesis. The first objective was to develop efficient and cost-effective non-chromatographic purification processes to purify recombinant proteins in an effort to meet the dramatically growing market of protein drugs. In our design, the target protein was genetically fused with a dockerin domain from Clostridium thermocellum and direct purification and recovery was achieved using thermo-responsive elastin-like polypeptide (ELP) scaffold containing the cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by another cycle of thermal precipitation. The purification cost can be further reduced by regenerating and recycling the ELP-cohesin capturing scaffolds. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated dockerin with the calcium-coordinating function impaired was used in replace of the original full length dockerin domain. The truncated dockerin domain maintained its functionality as an effective affinity tag, and efficient EDTA mediated dissociation of the bound dockerin-intein tag was also realized. The regenerated ELP capturing scaffold was reused for additional purification cycles without any decrease in efficiency. The second objective was to assemble biocatalysts for biofuel cells. Three beta-NAD dependent dehydrogenases, alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FALDH) and formate dehydrogenase (FDH), were site-specifically co-localized onto the scaffolds displayed on the yeast surface based on the high-affinity interactions between three orthogonal cohesin/dockerin pairs. The assembled multi-enzyme cascades, which can completely convert methanol to CO2, showed improved production yield compared with that of the non-complexed enzyme mixture, indicating efficient substrate channeling among the three enzymes. This strategy can be easily extended to other complex cascade reactions for enzymatic fuel cell applications. To further explore the role of biotechnology toward environmental sustainability, Escherichia coli was engineered to express phytochelatin synthase, which converted glutathione into the metal-binding peptide phytochelatin (PC). PCs served as peptide scaffolds and mediated synthesis of CdS nanocrystals. This approach may be generalized to guide the in vitro self-assembly of a wide range of nanocrystals with different compositions and sizes.

Whole-exome sequencing reveals the spectrum of gene mutations and the clonal evolution patterns in paediatric acute myeloid leukaemia.

PubMed

Shiba, Norio; Yoshida, Kenichi; Shiraishi, Yuichi; Okuno, Yusuke; Yamato, Genki; Hara, Yusuke; Nagata, Yasunobu; Chiba, Kenichi; Tanaka, Hiroko; Terui, Kiminori; Kato, Motohiro; Park, Myoung-Ja; Ohki, Kentaro; Shimada, Akira; Takita, Junko; Tomizawa, Daisuke; Kudo, Kazuko; Arakawa, Hirokazu; Adachi, Souichi; Taga, Takashi; Tawa, Akio; Ito, Etsuro; Horibe, Keizo; Sanada, Masashi; Miyano, Satoru; Ogawa, Seishi; Hayashi, Yasuhide

2016-11-01

Acute myeloid leukaemia (AML) is a molecularly and clinically heterogeneous disease. Targeted sequencing efforts have identified several mutations with diagnostic and prognostic values in KIT, NPM1, CEBPA and FLT3 in both adult and paediatric AML. In addition, massively parallel sequencing enabled the discovery of recurrent mutations (i.e. IDH1/2 and DNMT3A) in adult AML. In this study, whole-exome sequencing (WES) of 22 paediatric AML patients revealed mutations in components of the cohesin complex (RAD21 and SMC3), BCORL1 and ASXL2 in addition to previously known gene mutations. We also revealed intratumoural heterogeneities in many patients, implicating multiple clonal evolution events in the development of AML. Furthermore, targeted deep sequencing in 182 paediatric AML patients identified three major categories of recurrently mutated genes: cohesion complex genes [STAG2, RAD21 and SMC3 in 17 patients (8·3%)], epigenetic regulators [ASXL1/ASXL2 in 17 patients (8·3%), BCOR/BCORL1 in 7 patients (3·4%)] and signalling molecules. We also performed WES in four patients with relapsed AML. Relapsed AML evolved from one of the subclones at the initial phase and was accompanied by many additional mutations, including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process causing leukaemia recurrence. © 2016 John Wiley & Sons Ltd.

Cornelia de Lange syndrome and molecular implications of the cohesin complex: Abstracts from the 7th biennial scientific and educational symposium 2016.

PubMed

Kline, Antonie D; Krantz, Ian D; Deardorff, Matthew A; Shirahige, Katsuhiko; Dorsett, Dale; Gerton, Jennifer L; Wu, Meng; Mehta, Devanshi; Mills, Jason A; Carrico, Cheri S; Noon, Sarah; Herrera, Pamela S; Horsfield, Julia A; Bettale, Chiara; Morgan, Jeremy; Huisman, Sylvia A; Moss, Jo; McCleery, Joseph; Grados, Marco; Hansen, Blake D; Srivastava, Siddharth; Taylor-Snell, Emily; Kerr, Lynne M; Katz, Olivia; Calof, Anne L; Musio, Antonio; Egense, Alena; Haaland, Richard E

2017-05-01

Cornelia de Lange Syndrome (CdLS) is due to mutations in the genes for the structural and regulatory proteins that make up the cohesin complex, and is considered a cohesinopathy disorder or, more recently, a transcriptomopathy. New phenotypes have been recognized in this expanding field. There are multiple clinical issues facing individuals with all forms of CdLS, particularly in the neurodevelopmental system, but also gastrointestinal, cardiac, and musculoskeletal. Aspects of developmental and cell biology have found common endpoints in the biology of the cohesin complex, with improved understanding of the mechanisms, easier diagnostic tests, and the possibility of potential therapeutics, all major clinical implications for the individual with CdLS. The following abstracts are the presentations from the 7th Cornelia de Lange Syndrome Scientific and Educational Symposium, June 22-23, 2016, in Orlando, FL, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting. In addition to the scientific and clinical discussions, there were talks related to practical aspects of behavior including autism, transitions, communication, access to medical care, and databases. At the end of the symposium, a panel was held, which included several parents, affected individuals and genetic counselors, and discussed the greatest challenges in life and how this information can assist in guiding future research. The Research Committee of the CdLS Foundation organizes this meeting, reviews, and accepts abstracts, and subsequently disseminates the information to the families through members of the Clinical Advisory Board and publications. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD. © 2017 Wiley Periodicals, Inc.

Is cohesin required for spindle-pole-body/centrosome cohesion?

PubMed Central

Jin, Hui; Avey, Martin

2012-01-01

Centrosomes are microtubule-organizing centers that nucleate spindle microtubules during cell division. In budding yeast, the centrosome, often referred to as the spindle pole body, shares structural components with the centriole, the central core of the animal centrosome. The parental centrosome is duplicated when DNA replication takes place. Like sister chromatids tethered together by cohesin, duplicated centrosomes are linked and then separate to form the bipolar spindle necessary for chromosome segregation. Recent studies have shown that cohesin is also localized to the animal centrosome and is perhaps directly involved in engaging paired centrioles. Here we discuss the potential role of cohesin in mediating spindle-pole-body cohesion in the context of yeast meiosis. We propose that the coordination of chromosome segregation with centrosome cohesion and duplication is mediated by the antagonistic interaction between the Aurora kinase and the Polo kinase and that the role of cohesin in centrosome regulation appears to be indirect in budding yeast. PMID:22482005

Sub-kb Hi-C in D. melanogaster reveals conserved characteristics of TADs between insect and mammalian cells.

PubMed

Wang, Qi; Sun, Qiu; Czajkowsky, Daniel M; Shao, Zhifeng

2018-01-15

Topologically associating domains (TADs) are fundamental elements of the eukaryotic genomic structure. However, recent studies suggest that the insulating complexes, CTCF/cohesin, present at TAD borders in mammals are absent from those in Drosophila melanogaster, raising the possibility that border elements are not conserved among metazoans. Using in situ Hi-C with sub-kb resolution, here we show that the D. melanogaster genome is almost completely partitioned into >4000 TADs, nearly sevenfold more than previously identified. The overwhelming majority of these TADs are demarcated by the insulator complexes, BEAF-32/CP190, or BEAF-32/Chromator, indicating that these proteins may play an analogous role in flies as that of CTCF/cohesin in mammals. Moreover, extended regions previously thought to be unstructured are shown to consist of small contiguous TADs, a property also observed in mammals upon re-examination. Altogether, our work demonstrates that fundamental features associated with the higher-order folding of the genome are conserved from insects to mammals.

ROCC, a conserved region in cohesin's Mcd1 subunit, is essential for the proper regulation of the maintenance of cohesion and establishment of condensation

PubMed Central

Eng, Thomas; Guacci, Vincent; Koshland, Doug

2014-01-01

Cohesin helps orchestrate higher-order chromosome structure, thereby promoting sister chromatid cohesion, chromosome condensation, DNA repair, and transcriptional regulation. To elucidate how cohesin facilitates these diverse processes, we mutagenized Mcd1p, the kleisin regulatory subunit of budding yeast cohesin. In the linker region of Mcd1p, we identified a novel evolutionarily conserved 10–amino acid cluster, termed the regulation of cohesion and condensation (ROCC) box. We show that ROCC promotes cohesion maintenance by protecting a second activity of cohesin that is distinct from its stable binding to chromosomes. The existence of this second activity is incompatible with the simple embrace mechanism of cohesion. In addition, we show that the ROCC box is required for the establishment of condensation. We provide evidence that ROCC controls cohesion maintenance and condensation establishment through differential functional interactions with Pds5p and Wpl1p. PMID:24966169

Diverse Developmental Disorders from The One Ring: Distinct Molecular Pathways Underlie the Cohesinopathies

PubMed Central

Horsfield, Julia A.; Print, Cristin G.; Mönnich, Maren

2012-01-01

The multi-subunit protein complex, cohesin, is responsible for sister chromatid cohesion during cell division. The interaction of cohesin with DNA is controlled by a number of additional regulatory proteins. Mutations in cohesin, or its regulators, cause a spectrum of human developmental syndromes known as the “cohesinopathies.” Cohesinopathy disorders include Cornelia de Lange Syndrome and Roberts Syndrome. The discovery of novel roles for chromatid cohesion proteins in regulating gene expression led to the idea that cohesinopathies are caused by dysregulation of multiple genes downstream of mutations in cohesion proteins. Consistent with this idea, Drosophila, mouse, and zebrafish cohesinopathy models all show altered expression of developmental genes. However, there appears to be incomplete overlap among dysregulated genes downstream of mutations in different components of the cohesion apparatus. This is surprising because mutations in all cohesion proteins would be predicted to affect cohesin’s roles in cell division and gene expression in similar ways. Here we review the differences and similarities between genetic pathways downstream of components of the cohesion apparatus, and discuss how such differences might arise, and contribute to the spectrum of cohesinopathy disorders. We propose that mutations in different elements of the cohesion apparatus have distinct developmental outcomes that can be explained by sometimes subtly different molecular effects. PMID:22988450

Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy.

PubMed

Jobst, Markus A; Milles, Lukas F; Schoeler, Constantin; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Gaub, Hermann E; Nash, Michael A

2015-10-31

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicted dual binding modes across multiple bacterial species, our approach opens up new possibilities for understanding assembly and catalytic properties of a broad range of multi-enzyme complexes.

Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction.

PubMed

Bule, Pedro; Pires, Virgínia M R; Alves, Victor D; Carvalho, Ana Luísa; Prates, José A M; Ferreira, Luís M A; Smith, Steven P; Gilbert, Harry J; Noach, Ilit; Bayer, Edward A; Najmudin, Shabir; Fontes, Carlos M G A

2018-05-03

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ortiz, Rosario, E-mail: r_oh@ciencias.unam.mx; Kouznetsova, Anna, E-mail: Anna.Kouznetsova@ki.se; Echeverría-Martínez, Olga M., E-mail: omem@ciencias.unam.mx

The synaptonemal complex (SC) is a proteinaceous structure that holds the homologous chromosomes in close proximity while they exchange genetic material in a process known as meiotic recombination. This meiotic recombination leads to genetic variability in sexually reproducing organisms. The ultrastructure of the SC is studied by electron microscopy and it is observed as a tripartite structure. Two lateral elements (LE) separated by a central region (CR) confer its classical tripartite organization. The LEs are the anchoring platform for the replicated homologous chromosomes to properly exchange genetic material with one another. An accurate assembly of the LE is indispensable formore » the proper completion of meiosis. Ultrastructural studies suggested that the LE is organized as a multilayered unit. However, no validation of this model has been previously provided. In this ultrastructural study, by using mice with different genetic backgrounds that affect the LE width, we provide further evidence that support a multilayered organization of the LE. Additionally, we provide data suggesting additional roles of the different cohesin complex components in the structure of the LEs of the SC. - Highlights: • The lateral element of the synaptonemal complex is a multilayered structure. • The width of the lateral element in synaptonemal complex-null mice is different. • Two cohesin complex cores plus one axial element form a wild-type lateral element. • The layers of the lateral element can be analyzed in different null mice models.« less

Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia

PubMed Central

2013-01-01

BACKGROUND Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear. METHODS We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis. RESULTS AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcription-factor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumor-suppressor genes (16%), DNA-methylation–related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories. CONCLUSIONS We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.) PMID:23634996

Mechanical Stability of a High-Affinity Toxin Anchor from the Pathogen Clostridium perfringens.

PubMed

Milles, Lukas F; Bayer, Edward A; Nash, Michael A; Gaub, Hermann E

2017-04-20

The opportunistic pathogen Clostridium perfringens assembles its toxins and carbohydrate-active enzymes by the high-affinity cohesin-dockerin (Coh-Doc) interaction. Coh-Doc interactions characterized previously have shown considerable resilience toward mechanical stress. Here, we aimed to determine the mechanics of this interaction from C. perfringens in the context of a pathogen. Using atomic force microscopy based single-molecule force spectroscopy (AFM-SMFS) we probed the mechanical properties of the interaction of a dockerin from the μ-toxin with the GH84C X82 cohesin domain of C. perfringens. Most probable complex rupture forces were found to be approximately 60 pN and an estimate of the binding potential width was performed. The dockerin was expressed with its adjacent FIVAR (found in various architectures) domain, whose mechanostability we determined to be very similar to the complex. Additionally, fast refolding of this domain was observed. The Coh-Doc interaction from C. perfringens is the mechanically weakest observed to date. Our results establish the relevant force range of toxin assembly mechanics in pathogenic Clostridia.

Coarse-Grained MD Simulations and Protein-Protein Interactions: The Cohesin-Dockerin System.

PubMed

Hall, Benjamin A; Sansom, Mark S P

2009-09-08

Coarse-grained molecular dynamics (CG-MD) may be applied as part of a multiscale modeling approach to protein-protein interactions. The cohesin-dockerin interaction provides a valuable test system for evaluation of the use of CG-MD, as structural (X-ray) data indicate a dual binding mode for the cohesin-dockerin pair. CG-MD simulations (of 5 μs duration) of the association of cohesin and dockerin identify two distinct binding modes, which resemble those observed in X-ray structures. For each binding mode, ca. 80% of interfacial residues are predicted correctly. Furthermore, each of the binding modes identified by CG-MD is conformationally stable when converted to an atomistic model and used as the basis of a conventional atomistic MD simulation of duration 20 ns.

Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy

PubMed Central

Jobst, Markus A; Milles, Lukas F; Schoeler, Constantin; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Gaub, Hermann E; Nash, Michael A

2015-01-01

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicteddual binding modes across multiple bacterial species, our approach opens up newpossibilities for understanding assembly and catalytic properties of a broadrange of multi-enzyme complexes. DOI: http://dx.doi.org/10.7554/eLife.10319.001 PMID:26519733

A Dual-Color Reporter Assay of Cohesin-Mediated Gene Regulation in Budding Yeast Meiosis.

PubMed

Fan, Jinbo; Jin, Hui; Yu, Hong-Guo

2017-01-01

In this chapter, we describe a quantitative fluorescence-based assay of gene expression using the ratio of the reporter green fluorescence protein (GFP) to the internal red fluorescence protein (RFP) control. With this dual-color heterologous reporter assay, we have revealed cohesin-regulated genes and discovered a cis-acting DNA element, the Ty1-LTR, which interacts with cohesin and regulates gene expression during yeast meiosis. The method described here provides an effective cytological approach for quantitative analysis of global gene expression in budding yeast meiosis.

Comprehensive mutational profiling of core binding factor acute myeloid leukemia

PubMed Central

Duployez, Nicolas; Marceau-Renaut, Alice; Boissel, Nicolas; Petit, Arnaud; Bucci, Maxime; Geffroy, Sandrine; Lapillonne, Hélène; Renneville, Aline; Ragu, Christine; Figeac, Martin; Celli-Lebras, Karine; Lacombe, Catherine; Micol, Jean-Baptiste; Abdel-Wahab, Omar; Cornillet, Pascale; Ifrah, Norbert; Dombret, Hervé; Leverger, Guy; Jourdan, Eric

2016-01-01

Acute myeloid leukemia (AML) with t(8;21) or inv(16) have been recognized as unique entities within AML and are usually reported together as core binding factor AML (CBF-AML). However, there is considerable clinical and biological heterogeneity within this group of diseases, and relapse incidence reaches up to 40%. Moreover, translocations involving CBFs are not sufficient to induce AML on its own and the full spectrum of mutations coexisting with CBF translocations has not been elucidated. To address these issues, we performed extensive mutational analysis by high-throughput sequencing in 215 patients with CBF-AML enrolled in the Phase 3 Trial of Systematic Versus Response-adapted Timed-Sequential Induction in Patients With Core Binding Factor Acute Myeloid Leukemia and Treating Patients with Childhood Acute Myeloid Leukemia with Interleukin-2 trials (age, 1-60 years). Mutations in genes activating tyrosine kinase signaling (including KIT, N/KRAS, and FLT3) were frequent in both subtypes of CBF-AML. In contrast, mutations in genes that regulate chromatin conformation or encode members of the cohesin complex were observed with high frequencies in t(8;21) AML (42% and 18%, respectively), whereas they were nearly absent in inv(16) AML. High KIT mutant allele ratios defined a group of t(8;21) AML patients with poor prognosis, whereas high N/KRAS mutant allele ratios were associated with the lack of KIT or FLT3 mutations and a favorable outcome. In addition, mutations in epigenetic modifying or cohesin genes were associated with a poor prognosis in patients with tyrosine kinase pathway mutations, suggesting synergic cooperation between these events. These data suggest that diverse cooperating mutations may influence CBF-AML pathophysiology as well as clinical behavior and point to potential unique pathogenesis of t(8;21) vs inv(16) AML. PMID:26980726

Architectural protein subclasses shape 3-D organization of genomes during lineage commitment

PubMed Central

Phillips-Cremins, Jennifer E.; Sauria, Michael E. G.; Sanyal, Amartya; Gerasimova, Tatiana I.; Lajoie, Bryan R.; Bell, Joshua S. K.; Ong, Chin-Tong; Hookway, Tracy A.; Guo, Changying; Sun, Yuhua; Bland, Michael J.; Wagstaff, William; Dalton, Stephen; McDevitt, Todd C.; Sen, Ranjan; Dekker, Job; Taylor, James; Corces, Victor G.

2013-01-01

Summary Understanding the topological configurations of chromatin may reveal valuable insights into how the genome and epigenome act in concert to control cell fate during development. Here we generate high-resolution architecture maps across seven genomic loci in embryonic stem cells and neural progenitor cells. We observe a hierarchy of 3-D interactions that undergo marked reorganization at the sub-Mb scale during differentiation. Distinct combinations of CTCF, Mediator, and cohesin show widespread enrichment in looping interactions at different length scales. CTCF/cohesin anchor long-range constitutive interactions that form the topological basis for invariant sub-domains. Conversely, Mediator/cohesin together with pioneer factors bridge shortrange enhancer-promoter interactions within and between larger sub-domains. Knockdown of Smc1 or Med12 in ES cells results in disruption of spatial architecture and down-regulation of genes found in cohesin-mediated interactions. We conclude that cell type-specific chromatin organization occurs at the sub-Mb scale and that architectural proteins shape the genome in hierarchical length scales. PMID:23706625

The Energetics and Physiological Impact of Cohesin Extrusion.

PubMed

Vian, Laura; Pękowska, Aleksandra; Rao, Suhas S P; Kieffer-Kwon, Kyong-Rim; Jung, Seolkyoung; Baranello, Laura; Huang, Su-Chen; El Khattabi, Laila; Dose, Marei; Pruett, Nathanael; Sanborn, Adrian L; Canela, Andres; Maman, Yaakov; Oksanen, Anna; Resch, Wolfgang; Li, Xingwang; Lee, Byoungkoo; Kovalchuk, Alexander L; Tang, Zhonghui; Nelson, Steevenson; Di Pierro, Michele; Cheng, Ryan R; Machol, Ido; St Hilaire, Brian Glenn; Durand, Neva C; Shamim, Muhammad S; Stamenova, Elena K; Onuchic, José N; Ruan, Yijun; Nussenzweig, Andre; Levens, David; Aiden, Erez Lieberman; Casellas, Rafael

2018-05-17

Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development. Copyright © 2018 Elsevier Inc. All rights reserved.

Two independent modes of chromatin organization revealed by cohesin removal.

PubMed

Schwarzer, Wibke; Abdennur, Nezar; Goloborodko, Anton; Pekowska, Aleksandra; Fudenberg, Geoffrey; Loe-Mie, Yann; Fonseca, Nuno A; Huber, Wolfgang; H Haering, Christian; Mirny, Leonid; Spitz, Francois

2017-11-02

Imaging and chromosome conformation capture studies have revealed several layers of chromosome organization, including segregation into megabase-sized active and inactive compartments, and partitioning into sub-megabase domains (TADs). It remains unclear, however, how these layers of organization form, interact with one another and influence genome function. Here we show that deletion of the cohesin-loading factor Nipbl in mouse liver leads to a marked reorganization of chromosomal folding. TADs and associated Hi-C peaks vanish globally, even in the absence of transcriptional changes. By contrast, compartmental segregation is preserved and even reinforced. Strikingly, the disappearance of TADs unmasks a finer compartment structure that accurately reflects the underlying epigenetic landscape. These observations demonstrate that the three-dimensional organization of the genome results from the interplay of two independent mechanisms: cohesin-independent segregation of the genome into fine-scale compartments, defined by chromatin state; and cohesin-dependent formation of TADs, possibly by loop extrusion, which helps to guide distant enhancers to their target genes.

Casein Kinase 1 Coordinates Cohesin Cleavage, Gametogenesis, and Exit from M Phase in Meiosis II.

PubMed

Argüello-Miranda, Orlando; Zagoriy, Ievgeniia; Mengoli, Valentina; Rojas, Julie; Jonak, Katarzyna; Oz, Tugce; Graf, Peter; Zachariae, Wolfgang

2017-01-09

Meiosis consists of DNA replication followed by two consecutive nuclear divisions and gametogenesis or spore formation. While meiosis I has been studied extensively, less is known about the regulation of meiosis II. Here we show that Hrr25, the conserved casein kinase 1δ of budding yeast, links three mutually independent key processes of meiosis II. First, Hrr25 induces nuclear division by priming centromeric cohesin for cleavage by separase. Hrr25 simultaneously phosphorylates Rec8, the cleavable subunit of cohesin, and removes from centromeres the cohesin protector composed of shugoshin and the phosphatase PP2A. Second, Hrr25 initiates the sporulation program by inducing the synthesis of membranes that engulf the emerging nuclei at anaphase II. Third, Hrr25 mediates exit from meiosis II by activating pathways that trigger the destruction of M-phase-promoting kinases. Thus, Hrr25 synchronizes formation of the single-copy genome with gamete differentiation and termination of meiosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiplex CRISPR/Cas9-Based Genome Editing in Human Hematopoietic Stem Cells Models Clonal Hematopoiesis and Myeloid Neoplasia.

PubMed

Tothova, Zuzana; Krill-Burger, John M; Popova, Katerina D; Landers, Catherine C; Sievers, Quinlan L; Yudovich, David; Belizaire, Roger; Aster, Jon C; Morgan, Elizabeth A; Tsherniak, Aviad; Ebert, Benjamin L

2017-10-05

Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34 + human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents. Copyright © 2017 Elsevier Inc. All rights reserved.

Meiosis and Maternal Aging: Insights from Aneuploid Oocytes and Trisomy Births

PubMed Central

Herbert, Mary; Kalleas, Dimitrios; Cooney, Daniel; Lamb, Mahdi; Lister, Lisa

2015-01-01

In most organisms, genome haploidization requires reciprocal DNA exchanges (crossovers) between replicated parental homologs to form bivalent chromosomes. These are resolved to their four constituent chromatids during two meiotic divisions. In female mammals, bivalents are formed during fetal life and remain intact until shortly before ovulation. Extending this period beyond ∼35 years greatly increases the risk of aneuploidy in human oocytes, resulting in a dramatic increase in infertility, miscarriage, and birth defects, most notably trisomy 21. Bivalent chromosomes are stabilized by cohesion between sister chromatids, which is mediated by the cohesin complex. In mouse oocytes, cohesin becomes depleted from chromosomes during female aging. Consistent with this, premature loss of centromeric cohesion is a major source of aneuploidy in oocytes from older women. Here, we propose a mechanistic framework to reconcile data from genetic studies on human trisomy and oocytes with recent advances in our understanding of the molecular mechanisms of chromosome segregation during meiosis in model organisms. PMID:25833844

Stimulation of mTORC1 with L-leucine Rescues Defects Associated with Roberts Syndrome

PubMed Central

Xu, Baoshan; Lee, Kenneth K.; Zhang, Lily; Gerton, Jennifer L.

2013-01-01

Roberts syndrome (RBS) is a human disease characterized by defects in limb and craniofacial development and growth and mental retardation. RBS is caused by mutations in ESCO2, a gene which encodes an acetyltransferase for the cohesin complex. While the essential role of the cohesin complex in chromosome segregation has been well characterized, it plays additional roles in DNA damage repair, chromosome condensation, and gene expression. The developmental phenotypes of Roberts syndrome and other cohesinopathies suggest that gene expression is impaired during embryogenesis. It was previously reported that ribosomal RNA production and protein translation were impaired in immortalized RBS cells. It was speculated that cohesin binding at the rDNA was important for nucleolar form and function. We have explored the hypothesis that reduced ribosome function contributes to RBS in zebrafish models and human cells. Two key pathways that sense cellular stress are the p53 and mTOR pathways. We report that mTOR signaling is inhibited in human RBS cells based on the reduced phosphorylation of the downstream effectors S6K1, S6 and 4EBP1, and this correlates with p53 activation. Nucleoli, the sites of ribosome production, are highly fragmented in RBS cells. We tested the effect of inhibiting p53 or stimulating mTOR in RBS cells. The rescue provided by mTOR activation was more significant, with activation rescuing both cell division and cell death. To study this cohesinopathy in a whole animal model we used ESCO2-mutant and morphant zebrafish embryos, which have developmental defects mimicking RBS. Consistent with RBS patient cells, the ESCO2 mutant embryos show p53 activation and inhibition of the TOR pathway. Stimulation of the TOR pathway with L-leucine rescued many developmental defects of ESCO2-mutant embryos. Our data support the idea that RBS can be attributed in part to defects in ribosome biogenesis, and stimulation of the TOR pathway has therapeutic potential. PMID:24098154

Stimulation of mTORC1 with L-leucine rescues defects associated with Roberts syndrome.

PubMed

Xu, Baoshan; Lee, Kenneth K; Zhang, Lily; Gerton, Jennifer L

2013-01-01

Roberts syndrome (RBS) is a human disease characterized by defects in limb and craniofacial development and growth and mental retardation. RBS is caused by mutations in ESCO2, a gene which encodes an acetyltransferase for the cohesin complex. While the essential role of the cohesin complex in chromosome segregation has been well characterized, it plays additional roles in DNA damage repair, chromosome condensation, and gene expression. The developmental phenotypes of Roberts syndrome and other cohesinopathies suggest that gene expression is impaired during embryogenesis. It was previously reported that ribosomal RNA production and protein translation were impaired in immortalized RBS cells. It was speculated that cohesin binding at the rDNA was important for nucleolar form and function. We have explored the hypothesis that reduced ribosome function contributes to RBS in zebrafish models and human cells. Two key pathways that sense cellular stress are the p53 and mTOR pathways. We report that mTOR signaling is inhibited in human RBS cells based on the reduced phosphorylation of the downstream effectors S6K1, S6 and 4EBP1, and this correlates with p53 activation. Nucleoli, the sites of ribosome production, are highly fragmented in RBS cells. We tested the effect of inhibiting p53 or stimulating mTOR in RBS cells. The rescue provided by mTOR activation was more significant, with activation rescuing both cell division and cell death. To study this cohesinopathy in a whole animal model we used ESCO2-mutant and morphant zebrafish embryos, which have developmental defects mimicking RBS. Consistent with RBS patient cells, the ESCO2 mutant embryos show p53 activation and inhibition of the TOR pathway. Stimulation of the TOR pathway with L-leucine rescued many developmental defects of ESCO2-mutant embryos. Our data support the idea that RBS can be attributed in part to defects in ribosome biogenesis, and stimulation of the TOR pathway has therapeutic potential.

The length but not the sequence of peptide linker modules exerts the primary influence on the conformations of protein domains in cellulosome multi-enzyme complexes.

PubMed

Różycki, Bartosz; Cazade, Pierre-André; O'Mahony, Shane; Thompson, Damien; Cieplak, Marek

2017-08-16

Cellulosomes are large multi-protein catalysts produced by various anaerobic microorganisms to efficiently degrade plant cell-wall polysaccharides down into simple sugars. X-ray and physicochemical structural characterisations show that cellulosomes are composed of numerous protein domains that are connected by unstructured polypeptide segments, yet the properties and possible roles of these 'linker' peptides are largely unknown. We have performed coarse-grained and all-atom molecular dynamics computer simulations of a number of cellulosomal linkers of different lengths and compositions. Our data demonstrates that the effective stiffness of the linker peptides, as quantified by the equilibrium fluctuations in the end-to-end distances, depends primarily on the length of the linker and less so on the specific amino acid sequence. The presence of excluded volume - provided by the domains that are connected - dampens the motion of the linker residues and reduces the effective stiffness of the linkers. Simultaneously, the presence of the linkers alters the conformations of the protein domains that are connected. We demonstrate that short, stiff linkers induce significant rearrangements in the folded domains of the mini-cellulosome composed of endoglucanase Cel8A in complex with scaffoldin ScafT (Cel8A-ScafT) of Clostridium thermocellum as well as in a two-cohesin system derived from the scaffoldin ScaB of Acetivibrio cellulolyticus. We give experimentally testable predictions on structural changes in protein domains that depend on the length of linkers.

[Identification of novel pathogenic gene mutations in pediatric acute myeloid leukemia by whole-exome resequencing].

PubMed

Shiba, Norio

2015-12-01

A new class of gene mutations, identified in the pathogenesis of adult acute myeloid leukemia (AML), includes DNMT3A, IDH1/2, TET2 and EZH2. However, these mutations are rare in pediatric AML cases, indicating that pathogeneses differ between adult and pediatric forms of AML. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across entire genomes or proteincoding sequences. In order to reveal a complete registry of gene mutations, we performed whole exome resequencing of paired tumor-normal specimens from 19 pediatric AML cases using Illumina HiSeq 2000. In total, 80 somatic mutations or 4.2 mutations per sample were identified. Many of the recurrent mutations identified in this study involved previously reported targets in AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1 and EZH2. On the other hand, several genes were newly identified in the current study, including BCORL1 and major cohesin components such as SMC3 and RAD21. Whole exome resequencing revealed a complex array of gene mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that could be discriminated from its adult counterpart, in terms of the spectrum of gene mutations.

Unique organization and unprecedented diversity of the Bacteroides (Pseudobacteroides) cellulosolvens cellulosome system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhivin, Olga; Dassa, Bareket; Moraïs, Sarah

The organization of the B. cellulosolvens cellulosome is unique compared to previously described cellulosome systems. In contrast to all other known cellulosomes, the cohesin types are reversed for all scaffoldins i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. Many of the type II dockerin-bearing ORFs include X60 modules, which are known to stabilize type II cohesin–dockerin interactions. In the present work, we focused on revealing the architectural arrangement of cellulosome structure in this bacterium by examining numerous interactions between the various cohesin and dockerin modules.more » In total, we cloned and expressed 43 representative cohesins and 27 dockerins. The results revealed various possible architectures of cell-anchored and cell-free cellulosomes, which serve to assemble distinctive cellulosome types via three distinct cohesin–dockerin specificities: type I, type II, and a novel-type designated R (distinct from type III interactions, predominant in ruminococcal cellulosomes). The results of this study provide novel insight into the architecture and function of the most intricate and extensive cellulosomal system known today, thereby extending significantly our overall knowledge base of cellulosome systems and their components. The robust cellulosome system of B. cellulosolvens, with its unique binding specificities and reversal of cohesin–dockerin types, has served to amend our view of the cellulosome paradigm. Revealing new cellulosomal interactions and arrangements is critical for designing high-efficiency artificial cellulosomes for conversion of plant-derived cellulosic biomass towards improved production of biofuels.« less

Unique organization and unprecedented diversity of the Bacteroides (Pseudobacteroides) cellulosolvens cellulosome system

DOE PAGES

Zhivin, Olga; Dassa, Bareket; Moraïs, Sarah; ...

2017-09-07

The organization of the B. cellulosolvens cellulosome is unique compared to previously described cellulosome systems. In contrast to all other known cellulosomes, the cohesin types are reversed for all scaffoldins i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. Many of the type II dockerin-bearing ORFs include X60 modules, which are known to stabilize type II cohesin–dockerin interactions. In the present work, we focused on revealing the architectural arrangement of cellulosome structure in this bacterium by examining numerous interactions between the various cohesin and dockerin modules.more » In total, we cloned and expressed 43 representative cohesins and 27 dockerins. The results revealed various possible architectures of cell-anchored and cell-free cellulosomes, which serve to assemble distinctive cellulosome types via three distinct cohesin–dockerin specificities: type I, type II, and a novel-type designated R (distinct from type III interactions, predominant in ruminococcal cellulosomes). The results of this study provide novel insight into the architecture and function of the most intricate and extensive cellulosomal system known today, thereby extending significantly our overall knowledge base of cellulosome systems and their components. The robust cellulosome system of B. cellulosolvens, with its unique binding specificities and reversal of cohesin–dockerin types, has served to amend our view of the cellulosome paradigm. Revealing new cellulosomal interactions and arrangements is critical for designing high-efficiency artificial cellulosomes for conversion of plant-derived cellulosic biomass towards improved production of biofuels.« less

Physical Association of Saccharomyces cerevisiae Polo-like Kinase Cdc5 with Chromosomal Cohesin Facilitates DNA Damage Response.

PubMed

Pakchuen, Sujiraporn; Ishibashi, Mai; Takakusagi, Emi; Shirahige, Katsuhiko; Sutani, Takashi

2016-08-12

At the onset of anaphase, a protease called separase breaks the link between sister chromatids by cleaving the cohesin subunit Scc1. This irreversible step in the cell cycle is promoted by degradation of the separase inhibitor, securin, and polo-like kinase (Plk) 1-dependent phosphorylation of the Scc1 subunit. Plk could recognize substrates through interaction between its phosphopeptide interaction domain, the polo-box domain, and a phosphorylated priming site in the substrate, which has been generated by a priming kinase beforehand. However, the physiological relevance of this targeting mechanism remains to be addressed for many of the Plk1 substrates. Here, we show that budding yeast Plk1, Cdc5, is pre-deposited onto cohesin engaged in cohesion on chromosome arms in G2/M phase cells. The Cdc5-cohesin association is mediated by direct interaction between the polo-box domain of Cdc5 and Scc1 phosphorylated at multiple sites in its middle region. Alanine substitutions of the possible priming phosphorylation sites (scc1-15A) impair Cdc5 association with chromosomal cohesin, but they make only a moderate impact on mitotic cell growth even in securin-deleted cells (pds1Δ), where Scc1 phosphorylation by Cdc5 is indispensable. The same scc1-15A pds1Δ double mutant, however, exhibits marked sensitivity to the DNA-damaging agent phleomycin, suggesting that the priming phosphorylation of Scc1 poses an additional layer of regulation that enables yeast cells to adapt to genotoxic environments. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Comprehensive mutational profiling of core binding factor acute myeloid leukemia.

PubMed

Duployez, Nicolas; Marceau-Renaut, Alice; Boissel, Nicolas; Petit, Arnaud; Bucci, Maxime; Geffroy, Sandrine; Lapillonne, Hélène; Renneville, Aline; Ragu, Christine; Figeac, Martin; Celli-Lebras, Karine; Lacombe, Catherine; Micol, Jean-Baptiste; Abdel-Wahab, Omar; Cornillet, Pascale; Ifrah, Norbert; Dombret, Hervé; Leverger, Guy; Jourdan, Eric; Preudhomme, Claude

2016-05-19

Acute myeloid leukemia (AML) with t(8;21) or inv(16) have been recognized as unique entities within AML and are usually reported together as core binding factor AML (CBF-AML). However, there is considerable clinical and biological heterogeneity within this group of diseases, and relapse incidence reaches up to 40%. Moreover, translocations involving CBFs are not sufficient to induce AML on its own and the full spectrum of mutations coexisting with CBF translocations has not been elucidated. To address these issues, we performed extensive mutational analysis by high-throughput sequencing in 215 patients with CBF-AML enrolled in the Phase 3 Trial of Systematic Versus Response-adapted Timed-Sequential Induction in Patients With Core Binding Factor Acute Myeloid Leukemia and Treating Patients with Childhood Acute Myeloid Leukemia with Interleukin-2 trials (age, 1-60 years). Mutations in genes activating tyrosine kinase signaling (including KIT, N/KRAS, and FLT3) were frequent in both subtypes of CBF-AML. In contrast, mutations in genes that regulate chromatin conformation or encode members of the cohesin complex were observed with high frequencies in t(8;21) AML (42% and 18%, respectively), whereas they were nearly absent in inv(16) AML. High KIT mutant allele ratios defined a group of t(8;21) AML patients with poor prognosis, whereas high N/KRAS mutant allele ratios were associated with the lack of KIT or FLT3 mutations and a favorable outcome. In addition, mutations in epigenetic modifying or cohesin genes were associated with a poor prognosis in patients with tyrosine kinase pathway mutations, suggesting synergic cooperation between these events. These data suggest that diverse cooperating mutations may influence CBF-AML pathophysiology as well as clinical behavior and point to potential unique pathogenesis of t(8;21) vs inv(16) AML. © 2016 by The American Society of Hematology.

l-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome

PubMed Central

Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E.; Gerton, Jennifer L.

2015-01-01

Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS. PMID:25378554

L-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome.

PubMed

Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E; Gerton, Jennifer L

2015-03-15

Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS. © The Author 2014. Published by Oxford University Press.

Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells

PubMed Central

Hindriksen, Sanne; Bramer, Arne J.; Truong, My Anh; Vromans, Martijn J. M.; Post, Jasmin B.; Verlaan-Klink, Ingrid; Snippert, Hugo J.; Lens, Susanne M. A.

2017-01-01

The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B. PMID:28640891

Coordination of tRNA transcription with export at nuclear pore complexes in budding yeast.

PubMed

Chen, Miao; Gartenberg, Marc R

2014-05-01

tRNAs are encoded by RNA polymerase III-transcribed genes that reside at seemingly random intervals along the chromosomes of budding yeast. Existing evidence suggests that the genes congregate together at the nucleolus and/or centromeres. In this study, we re-examined spatial and temporal aspects of tRNA gene (tDNA) expression. We show that tDNA transcription fluctuates during cell cycle progression. In M phase, when tRNA synthesis peaks, tDNAs localize at nuclear pore complexes (NPCs). Docking of a tDNA requires the DNA sequence of the contacted gene, nucleoporins Nup60 and Nup2, and cohesin. Characterization of mutants that block NPC localization revealed that docking is a consequence of elevated tDNA transcription. NPC-tDNA contact falters in the absence of the principal exportin of nascent tRNA, Los1, and genetic assays indicate that gating of tDNAs at NPCs favors cytoplasmic accumulation of functional tRNA. Collectively, the data suggest that tDNAs associate with NPCs to coordinate RNA polymerase III transcription with the nuclear export of pre-tRNA. The M-phase specificity of NPC contact reflects a regulatory mechanism that may have evolved, in part, to avoid collisions between DNA replication forks and transcribing RNA polymerase III machinery at NPCs.

Coordination of tRNA transcription with export at nuclear pore complexes in budding yeast

PubMed Central

Chen, Miao; Gartenberg, Marc R.

2014-01-01

tRNAs are encoded by RNA polymerase III-transcribed genes that reside at seemingly random intervals along the chromosomes of budding yeast. Existing evidence suggests that the genes congregate together at the nucleolus and/or centromeres. In this study, we re-examined spatial and temporal aspects of tRNA gene (tDNA) expression. We show that tDNA transcription fluctuates during cell cycle progression. In M phase, when tRNA synthesis peaks, tDNAs localize at nuclear pore complexes (NPCs). Docking of a tDNA requires the DNA sequence of the contacted gene, nucleoporins Nup60 and Nup2, and cohesin. Characterization of mutants that block NPC localization revealed that docking is a consequence of elevated tDNA transcription. NPC–tDNA contact falters in the absence of the principal exportin of nascent tRNA, Los1, and genetic assays indicate that gating of tDNAs at NPCs favors cytoplasmic accumulation of functional tRNA. Collectively, the data suggest that tDNAs associate with NPCs to coordinate RNA polymerase III transcription with the nuclear export of pre-tRNA. The M-phase specificity of NPC contact reflects a regulatory mechanism that may have evolved, in part, to avoid collisions between DNA replication forks and transcribing RNA polymerase III machinery at NPCs. PMID:24788517

Casein kinase 1 (α, δ and ϵ) localize at the spindle poles, but may not be essential for mammalian oocyte meiotic progression

PubMed Central

Qi, Shu-Tao; Wang, Zhen-Bo; Huang, Lin; Liang, Li-Feng; Xian, Ye-Xing; Ouyang, Ying-Chun; Hou, Yi; Sun, Qing-Yuan; Wang, Wei-Hua

2015-01-01

CK1 (casein kinase 1) is a family of serine/threonine protein kinase that is ubiquitously expressed in eukaryotic organism. CK1 members are involved in the regulation of many cellular processes. Particularly, CK1 was reported to phosphorylate Rec8 subunits of cohesin complex and regulate chromosome segregation in meiosis in budding yeast and fission yeast.1-3 Here we investigated the expression, subcellular localization and potential functions of CK1α, CK1δ and CK1ϵ during mouse oocyte meiotic maturation. We found that CK1α, CK1δ and CK1ϵ all concentrated at the spindle poles and co-localized with γ-tubulin in oocytes at both metaphase I (MI) and metaphase II (MII) stages. However, depletion of CK1 by RNAi or overexpression of wild type or kinase-dead CK1 showed no effects on either spindle organization or chromosome segregation during oocyte meiotic maturation. Thus, CK1 is not the kinase that phosphorylates Rec8 cohesin in mammalian oocytes, and CK1 may not be essential for spindle organization and meiotic progression although they localize at spindle poles. PMID:25927854

RNA polymerase III transcription - regulated by chromatin structure and regulator of nuclear chromatin organization.

PubMed

Pascali, Chiara; Teichmann, Martin

2013-01-01

RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its transcription factors have been implicated in the organization of nuclear chromatin in several organisms. In yeast, the ability of the Pol III transcription system to contribute to nuclear organization seems to be dependent on direct interactions of Pol III genes and/or its transcription factors TFIIIC and TFIIIB with the structural maintenance of chromatin (SMC) protein-containing complexes cohesin and condensin. In human cells, Pol III genes and transcription factors have also been shown to colocalize with cohesin and the transcription regulator and genome organizer CCCTC-binding factor (CTCF). Furthermore, chromosomal sites have been identified in yeast and humans that are bound by partial Pol III machineries (extra TFIIIC sites - ETC; chromosome organizing clamps - COC). These ETCs/COC as well as Pol III genes possess the ability to act as boundary elements that restrict spreading of heterochromatin.

Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells

PubMed Central

Eid, Rita; Demattei, Marie-Véronique; Episkopou, Harikleia; Augé-Gouillou, Corinne; Decottignies, Anabelle; Grandin, Nathalie

2015-01-01

Mutations in ATRX (alpha thalassemia/mental retardation syndrome X-linked), a chromatin-remodeling protein, are associated with the telomerase-independent ALT (alternative lengthening of telomeres) pathway of telomere maintenance in several types of cancer, including human gliomas. In telomerase-positive glioma cells, we found by immunofluorescence that ATRX localized not far from the chromosome ends but not exactly at the telomere termini. Chromatin immunoprecipitation (ChIP) experiments confirmed a subtelomeric localization for ATRX, yet short hairpin RNA (shRNA)-mediated genetic inactivation of ATRX failed to trigger the ALT pathway. Cohesin has been recently shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked diminution in the amount of cohesin in subtelomeric regions of telomerase-positive glioma cells. Inactivation of ATRX also led to diminution in the amount of TERRAs, noncoding RNAs resulting from transcription of telomeric DNA, as well as to a decrease in RNA polymerase II (RNAP II) levels at the telomeres. Our data suggest that ATRX might establish functional interactions with cohesin on telomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be relevant to the triggering of the ALT pathway in cancer cells that exhibit genetic inactivation of ATRX. PMID:26055325

Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells.

PubMed

Eid, Rita; Demattei, Marie-Véronique; Episkopou, Harikleia; Augé-Gouillou, Corinne; Decottignies, Anabelle; Grandin, Nathalie; Charbonneau, Michel

2015-08-01

Mutations in ATRX (alpha thalassemia/mental retardation syndrome X-linked), a chromatin-remodeling protein, are associated with the telomerase-independent ALT (alternative lengthening of telomeres) pathway of telomere maintenance in several types of cancer, including human gliomas. In telomerase-positive glioma cells, we found by immunofluorescence that ATRX localized not far from the chromosome ends but not exactly at the telomere termini. Chromatin immunoprecipitation (ChIP) experiments confirmed a subtelomeric localization for ATRX, yet short hairpin RNA (shRNA)-mediated genetic inactivation of ATRX failed to trigger the ALT pathway. Cohesin has been recently shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked diminution in the amount of cohesin in subtelomeric regions of telomerase-positive glioma cells. Inactivation of ATRX also led to diminution in the amount of TERRAs, noncoding RNAs resulting from transcription of telomeric DNA, as well as to a decrease in RNA polymerase II (RNAP II) levels at the telomeres. Our data suggest that ATRX might establish functional interactions with cohesin on telomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be relevant to the triggering of the ALT pathway in cancer cells that exhibit genetic inactivation of ATRX. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

CTCF and Cohesin in Genome Folding and Transcriptional Gene Regulation.

PubMed

Merkenschlager, Matthias; Nora, Elphège P

2016-08-31

Genome function, replication, integrity, and propagation rely on the dynamic structural organization of chromosomes during the cell cycle. Genome folding in interphase provides regulatory segmentation for appropriate transcriptional control, facilitates ordered genome replication, and contributes to genome integrity by limiting illegitimate recombination. Here, we review recent high-resolution chromosome conformation capture and functional studies that have informed models of the spatial and regulatory compartmentalization of mammalian genomes, and discuss mechanistic models for how CTCF and cohesin control the functional architecture of mammalian chromosomes.

Cornelia de Lange Syndrome: NIPBL haploinsufficiency downregulates canonical Wnt pathway in zebrafish embryos and patients fibroblasts.

PubMed

Pistocchi, A; Fazio, G; Cereda, A; Ferrari, L; Bettini, L R; Messina, G; Cotelli, F; Biondi, A; Selicorni, A; Massa, V

2013-10-17

Cornelia de Lange Syndrome is a severe genetic disorder characterized by malformations affecting multiple systems, with a common feature of severe mental retardation. Genetic variants within four genes (NIPBL (Nipped-B-like), SMC1A, SMC3, and HDAC8) are believed to be responsible for the majority of cases; all these genes encode proteins that are part of the 'cohesin complex'. Cohesins exhibit two temporally separated major roles in cells: one controlling the cell cycle and the other involved in regulating the gene expression. The present study focuses on the role of the zebrafish nipblb paralog during neural development, examining its expression in the central nervous system, and analyzing the consequences of nipblb loss of function. Neural development was impaired by the knockdown of nipblb in zebrafish. nipblb-loss-of-function embryos presented with increased apoptosis in the developing neural tissues, downregulation of canonical Wnt pathway genes, and subsequent decreased Cyclin D1 (Ccnd1) levels. Importantly, the same pattern of canonical WNT pathway and CCND1 downregulation was observed in NIPBL-mutated patient-specific fibroblasts. Finally, chemical activation of the pathway in nipblb-loss-of-function embryos rescued the adverse phenotype and restored the physiological levels of cell death.

Cornelia de Lange syndrome: further delineation of phenotype, cohesin biology and educational focus, 5th Biennial Scientific and Educational Symposium abstracts.

PubMed

Kline, Antonie D; Calof, Anne L; Schaaf, Cheri A; Krantz, Ian D; Jyonouchi, Soma; Yokomori, Kyoko; Gauze, Maria; Carrico, Cheri S; Woodman, Julie; Gerton, Jennifer L; Vega, Hugo; Levin, Alex V; Shirahige, Katsuhiko; Champion, Michele; Goodban, Marjorie T; O'Connor, Julia T; Pipan, Mary; Horsfield, Julia; Deardorff, Matthew A; Ishman, Stacey L; Dorsett, Dale

2014-06-01

Cornelia de Lange syndrome (CdLS) is the prototype for the cohesinopathy disorders that have mutations in genes associated with the cohesin subunit in all cells. Roberts syndrome is the next most common cohesinopathy. In addition to the developmental implications of cohesin biology, there is much translational and basic research, with progress towards potential treatment for these conditions. Clinically, there are many issues in CdLS faced by the individual, parents and caretakers, professionals, and schools. The following abstracts are presentations from the 5th Cornelia de Lange Syndrome Scientific and Educational Symposium on June 20-21, 2012, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting, Lincolnshire, IL. The research committee of the CdLS Foundation organizes the meeting, reviews and accepts abstracts and subsequently disseminates the information to the families. In addition to the basic science and clinical discussions, there were educationally-focused talks related to practical aspects of management at home and in school. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD. © 2014 Wiley Periodicals, Inc.

Combing Chromosomal DNA Mediated by the SMC Complex: Structure and Mechanisms.

PubMed

Kamada, Katsuhiko; Barillà, Daniela

2018-02-01

Genome maintenance requires various nucleoid-associated factors in prokaryotes. Among them, the SMC (Structural Maintenance of Chromosomes) protein has been thought to play a static role in the organization and segregation of the chromosome during cell division. However, recent studies have shown that the bacterial SMC is required to align left and right arms of the emerging chromosome and that the protein dynamically travels from origin to Ter region. A rod form of the SMC complex mediates DNA bridging and has been recognized as a machinery responsible for DNA loop extrusion, like eukaryotic condensin or cohesin complexes, which act as chromosome organizers. Attention is now turning to how the prototype of the complex is loaded on the entry site and translocated on chromosomal DNA, explaining its overall conformational changes at atomic levels. Here, we review and highlight recent findings concerning the prokaryotic SMC complex and discuss possible mechanisms from the viewpoint of protein architecture. © 2017 The Authors. BioEssays Published by WILEY Periodicals, Inc.

Function of YY1 in Long-Distance DNA Interactions

PubMed Central

Atchison, Michael L.

2014-01-01

During B cell development, long-distance DNA interactions are needed for V(D)J somatic rearrangement of the immunoglobulin (Ig) loci to produce functional Ig genes, and for class switch recombination (CSR) needed for antibody maturation. The tissue-specificity and developmental timing of these mechanisms is a subject of active investigation. A small number of factors are implicated in controlling Ig locus long-distance interactions including Pax5, Yin Yang 1 (YY1), EZH2, IKAROS, CTCF, cohesin, and condensin proteins. Here we will focus on the role of YY1 in controlling these mechanisms. YY1 is a multifunctional transcription factor involved in transcriptional activation and repression, X chromosome inactivation, Polycomb Group (PcG) protein DNA recruitment, and recruitment of proteins required for epigenetic modifications (acetylation, deacetylation, methylation, ubiquitination, sumoylation, etc.). YY1 conditional knock-out indicated that YY1 is required for B cell development, at least in part, by controlling long-distance DNA interactions at the immunoglobulin heavy chain and Igκ loci. Our recent data show that YY1 is also required for CSR. The mechanisms implicated in YY1 control of long-distance DNA interactions include controlling non-coding antisense RNA transcripts, recruitment of PcG proteins to DNA, and interaction with complexes involved in long-distance DNA interactions including the cohesin and condensin complexes. Though common rearrangement mechanisms operate at all Ig loci, their distinct temporal activation along with the ubiquitous nature of YY1 poses challenges for determining the specific mechanisms of YY1 function in these processes, and their regulation at the tissue-specific and B cell stage-specific level. The large numbers of post-translational modifications that control YY1 functions are possible candidates for regulation. PMID:24575094

ASXL2 mutations are frequently found in pediatric AML patients with t(8;21)/ RUNX1-RUNX1T1 and associated with a better prognosis.

PubMed

Yamato, Genki; Shiba, Norio; Yoshida, Kenichi; Shiraishi, Yuichi; Hara, Yusuke; Ohki, Kentaro; Okubo, Jun; Okuno, Haruna; Chiba, Kenichi; Tanaka, Hiroko; Kinoshita, Akitoshi; Moritake, Hiroshi; Kiyokawa, Nobutaka; Tomizawa, Daisuke; Park, Myoung-Ja; Sotomatsu, Manabu; Taga, Takashi; Adachi, Souichi; Tawa, Akio; Horibe, Keizo; Arakawa, Hirokazu; Miyano, Satoru; Ogawa, Seishi; Hayashi, Yasuhide

2017-05-01

ASXL2 is an epigenetic regulator involved in polycomb repressive complex regulation or recruitment. Clinical features of pediatric acute myeloid leukemia (AML) patients with ASXL2 mutations remain unclear. Thus, we investigated frequencies of ASXL1 and ASXL2 mutations, clinical features of patients with these mutations, correlations of these mutations with other genetic alterations including BCOR/BCORL1 and cohesin complex component genes, and prognostic impact of these mutations in 369 pediatric patients with de novo AML (0-17 years). We identified 9 (2.4%) ASXL1 and 17 (4.6%) ASXL2 mutations in 25 patients. These mutations were more common in patients with t(8;21)(q22;q22)/RUNX1-RUNX1T1 (ASXL1, 6/9, 67%, P = 0.02; ASXL2, 10/17, 59%, P = 0.01). Among these 25 patients, 4 (27%) of 15 patients with t(8;21) and 6 (60%) of 10 patients without t(8;21) relapsed. However, most patients with relapse were rescued using stem cell transplantation irrespective of t(8;21). The overall survival (OS) and event-free survival (EFS) rates showed no differences among pediatric AML patients with t(8;21) and ASXL1 or ASXL2 mutations and ASXL wild-type (5-year OS, 75% vs. 100% vs. 91% and 5-year EFS, 67% vs. 80% vs. 67%). In 106 patients with t(8;21) AML, the coexistence of mutations in tyrosine kinase pathways and chromatin modifiers and/or cohesin complex component genes had no effect on prognosis. These results suggest that ASXL1 and ASXL2 mutations play key roles as cooperating mutations that induce leukemogenesis, particularly in pediatric AML patients with t(8;21), and these mutations might be associated with a better prognosis than that reported previously. © 2017 Wiley Periodicals, Inc.

Recombination protein Tid1p controls resolution of cohesin-dependent linkages in meiosis in Saccharomyces cerevisiae

PubMed Central

Kateneva, Anna V.; Konovchenko, Anton A.; Guacci, Vincent; Dresser, Michael E.

2005-01-01

Sister chromatid cohesion and interhomologue recombination are coordinated to promote the segregation of homologous chromosomes instead of sister chromatids at the first meiotic division. During meiotic prophase in Saccharomyces cerevisiae, the meiosis-specific cohesin Rec8p localizes along chromosome axes and mediates most of the cohesion. The mitotic cohesin Mcd1p/Scc1p localizes to discrete spots along chromosome arms, and its function is not clear. In cells lacking Tid1p, which is a member of the SWI2/SNF2 family of helicase-like proteins that are involved in chromatin remodeling, Mcd1p and Rec8p persist abnormally through both meiotic divisions, and chromosome segregation fails in the majority of cells. Genetic results indicate that the primary defect in these cells is a failure to resolve Mcd1p-mediated connections. Tid1p interacts with recombination enzymes Dmc1p and Rad51p and has an established role in recombination repair. We propose that Tid1p remodels Mcd1p-mediated cohesion early in meiotic prophase to facilitate interhomologue recombination and the subsequent segregation of homologous chromosomes. PMID:16230461

The kinetochore prevents centromere-proximal crossover recombination during meiosis

PubMed Central

Vincenten, Nadine; Kuhl, Lisa-Marie; Lam, Isabel; Oke, Ashwini; Kerr, Alastair RW; Hochwagen, Andreas; Fung, Jennifer; Keeney, Scott; Vader, Gerben; Marston, Adèle L

2015-01-01

During meiosis, crossover recombination is essential to link homologous chromosomes and drive faithful chromosome segregation. Crossover recombination is non-random across the genome, and centromere-proximal crossovers are associated with an increased risk of aneuploidy, including Trisomy 21 in humans. Here, we identify the conserved Ctf19/CCAN kinetochore sub-complex as a major factor that minimizes potentially deleterious centromere-proximal crossovers in budding yeast. We uncover multi-layered suppression of pericentromeric recombination by the Ctf19 complex, operating across distinct chromosomal distances. The Ctf19 complex prevents meiotic DNA break formation, the initiating event of recombination, proximal to the centromere. The Ctf19 complex independently drives the enrichment of cohesin throughout the broader pericentromere to suppress crossovers, but not DNA breaks. This non-canonical role of the kinetochore in defining a chromosome domain that is refractory to crossovers adds a new layer of functionality by which the kinetochore prevents the incidence of chromosome segregation errors that generate aneuploid gametes. DOI: http://dx.doi.org/10.7554/eLife.10850.001 PMID:26653857

Architectural roles of multiple chromatin insulators at the human apolipoprotein gene cluster

PubMed Central

Mishiro, Tsuyoshi; Ishihara, Ko; Hino, Shinjiro; Tsutsumi, Shuichi; Aburatani, Hiroyuki; Shirahige, Katsuhiko; Kinoshita, Yoshikazu; Nakao, Mitsuyoshi

2009-01-01

Long-range regulatory elements and higher-order chromatin structure coordinate the expression of multiple genes in cluster, and CTCF/cohesin-mediated chromatin insulator may be a key in this regulation. The human apolipoprotein (APO) A1/C3/A4/A5 gene region, whose alterations increase the risk of dyslipidemia and atherosclerosis, is partitioned at least by three CTCF-enriched sites and three cohesin protein RAD21-enriched sites (two overlap with the CTCF sites), resulting in the formation of two transcribed chromatin loops by interactions between insulators. The C3 enhancer and APOC3/A4/A5 promoters reside in the same loop, where the APOC3/A4 promoters are pointed towards the C3 enhancer, whereas the APOA1 promoter is present in the different loop. The depletion of either CTCF or RAD21 disrupts the chromatin loop structure, together with significant changes in the APO expression and the localization of transcription factor hepatocyte nuclear factor (HNF)-4α and transcriptionally active form of RNA polymerase II at the APO promoters. Thus, CTCF/cohesin-mediated insulators maintain the chromatin loop formation and the localization of transcriptional apparatus at the promoters, suggesting an essential role of chromatin insulation in controlling the expression of clustered genes. PMID:19322193

The Electrosome: A Surface-Displayed Enzymatic Cascade in a Biofuel Cell’s Anode and a High-Density Surface-Displayed Biocathodic Enzyme

PubMed Central

Szczupak, Alon; Aizik, Dror; Moraïs, Sarah; Vazana, Yael; Barak, Yoav; Bayer, Edward A.; Alfonta, Lital

2017-01-01

The limitation of surface-display systems in biofuel cells to a single redox enzyme is a major drawback of hybrid biofuel cells, resulting in a low copy-number of enzymes per yeast cell and a limitation in displaying enzymatic cascades. Here we present the electrosome, a novel surface-display system based on the specific interaction between the cellulosomal scaffoldin protein and a cascade of redox enzymes that allows multiple electron-release by fuel oxidation. The electrosome is composed of two compartments: (i) a hybrid anode, which consists of dockerin-containing enzymes attached specifically to cohesin sites in the scaffoldin to assemble an ethanol oxidation cascade, and (ii) a hybrid cathode, which consists of a dockerin-containing oxygen-reducing enzyme attached in multiple copies to the cohesin-bearing scaffoldin. Each of the two compartments was designed, displayed, and tested separately. The new hybrid cell compartments displayed enhanced performance over traditional biofuel cells; in the anode, the cascade of ethanol oxidation demonstrated higher performance than a cell with just a single enzyme. In the cathode, a higher copy number per yeast cell of the oxygen-reducing enzyme copper oxidase has reduced the effect of competitive inhibition resulting from yeast oxygen consumption. This work paves the way for the assembly of more complex cascades using different enzymes and larger scaffoldins to further improve the performance of hybrid cells. PMID:28644390

Genome Organization Drives Chromosome Fragility.

PubMed

Canela, Andres; Maman, Yaakov; Jung, Seolkyoung; Wong, Nancy; Callen, Elsa; Day, Amanda; Kieffer-Kwon, Kyong-Rim; Pekowska, Aleksandra; Zhang, Hongliang; Rao, Suhas S P; Huang, Su-Chen; Mckinnon, Peter J; Aplan, Peter D; Pommier, Yves; Aiden, Erez Lieberman; Casellas, Rafael; Nussenzweig, André

2017-07-27

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT. Published by Elsevier Inc.

Association of mutations with morphological dysplasia in de novo acute myeloid leukemia without 2016 WHO Classification-defined cytogenetic abnormalities

PubMed Central

Weinberg, Olga K.; Gibson, Christopher J.; Blonquist, Traci M.; Neuberg, Donna; Pozdnyakova, Olga; Kuo, Frank; Ebert, Benjamin L.; Hasserjian, Robert P.

2018-01-01

Despite improvements in our understanding of the molecular basis of acute myeloid leukemia (AML), the association between genetic mutations with morphological dysplasia remains unclear. In this study, we evaluated and scored dysplasia in bone marrow (BM) specimens from 168 patients with de novo AML; none of these patients had cytogenetic abnormalities according to the 2016 World Health Organization Classification. We then performed targeted sequencing of diagnostic BM aspirates for recurrent mutations associated with myeloid malignancies. We found that cohesin pathway mutations [q (FDR-adjusted P)=0.046] were associated with a higher degree of megakaryocytic dysplasia and STAG2 mutations were marginally associated with greater myeloid lineage dysplasia (q=0.052). Frequent megakaryocytes with separated nuclear lobes were more commonly seen among cases with cohesin pathway mutations (q=0.010) and specifically in those with STAG2 mutations (q=0.010), as well as NPM1 mutations (q=0.022 when considering the presence of any vs. no megakaryocytes with separated nuclear lobes). RAS pathway mutations (q=0.006) and FLT3-ITD (q=0.006) were significantly more frequent in cases without evaluable erythroid cells. In univariate analysis of the 153 patients treated with induction chemotherapy, NPM1 mutations were associated with longer event-free survival (EFS) (P=0.042), while RUNX1 (P=0.042), NF1 (P=0.040), frequent micromegakaryocytes (P=0.018) and presence of a subclone (P=0.002) were associated with shorter EFS. In multivariable modeling, NPM1 was associated with longer EFS, while presence of a subclone and frequent micromegakaryocytes remained significantly associated with shorter EFS. PMID:29326119

Interdependence of the rad50 hook and globular domain functions.

PubMed

Hohl, Marcel; Kochańczyk, Tomasz; Tous, Cristina; Aguilera, Andrés; Krężel, Artur; Petrini, John H J

2015-02-05

Rad50 contains a conserved Zn(2+) coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here, we focused on rad50 mutations flanking the Zn(2+)-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double-strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end joining, and DNA double-strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled-coil and globular ATPase domains, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions. Copyright © 2015 Elsevier Inc. All rights reserved.

Diversity and strain specificity of plant cell wall degrading enzymes revealed by the draft genome of Ruminococcus flavefaciens FD-1.

PubMed

Berg Miller, Margret E; Antonopoulos, Dionysios A; Rincon, Marco T; Band, Mark; Bari, Albert; Akraiko, Tatsiana; Hernandez, Alvaro; Thimmapuram, Jyothi; Henrissat, Bernard; Coutinho, Pedro M; Borovok, Ilya; Jindou, Sadanari; Lamed, Raphael; Flint, Harry J; Bayer, Edward A; White, Bryan A

2009-08-14

Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components.

Esco2 regulates cx43 expression during skeletal regeneration in the zebrafish fin.

PubMed

Banerji, Rajeswari; Eble, Diane M; Iovine, M Kathryn; Skibbens, Robert V

2016-01-01

Roberts syndrome (RBS) is a rare genetic disorder characterized by craniofacial abnormalities, limb malformation, and often severe mental retardation. RBS arises from mutations in ESCO2 that encodes an acetyltransferase and modifies the cohesin subunit SMC3. Mutations in SCC2/NIPBL (encodes a cohesin loader), SMC3 or other cohesin genes (SMC1, RAD21/MCD1) give rise to a related developmental malady termed Cornelia de Lange syndrome (CdLS). RBS and CdLS exhibit overlapping phenotypes, but RBS is thought to arise through mitotic failure and limited progenitor cell proliferation while CdLS arises through transcriptional dysregulation. Here, we use the zebrafish regenerating fin model to test the mechanism through which RBS-type phenotypes arise. esco2 is up-regulated during fin regeneration and specifically within the blastema. esco2 knockdown adversely affects both tissue and bone growth in regenerating fins-consistent with a role in skeletal morphogenesis. esco2-knockdown significantly diminishes cx43/gja1 expression which encodes the gap junction connexin subunit required for cell-cell communication. cx43 mutations cause the short fin (sof(b123) ) phenotype in zebrafish and oculodentodigital dysplasia (ODDD) in humans. Importantly, miR-133-dependent cx43 overexpression rescues esco2-dependent growth defects. These results conceptually link ODDD to cohesinopathies and provide evidence that ESCO2 may play a transcriptional role critical for human development. © 2015 Wiley Periodicals, Inc.

Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae

PubMed Central

Tong, Kevin; Skibbens, Robert V.

2015-01-01

Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures—processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated. PMID:25986377

Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae.

PubMed

Tong, Kevin; Skibbens, Robert V

2015-06-02

Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures-processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated.

Regional centromeres in the yeast Candida lusitaniae lack pericentromeric heterochromatin

PubMed Central

Kapoor, Shivali; Zhu, Lisha; Froyd, Cara; Liu, Tao; Rusche, Laura N.

2015-01-01

Point centromeres are specified by a short consensus sequence that seeds kinetochore formation, whereas regional centromeres lack a conserved sequence and instead are epigenetically inherited. Regional centromeres are generally flanked by heterochromatin that ensures high levels of cohesin and promotes faithful chromosome segregation. However, it is not known whether regional centromeres require pericentromeric heterochromatin. In the yeast Candida lusitaniae, we identified a distinct type of regional centromere that lacks pericentromeric heterochromatin. Centromere locations were determined by ChIP-sequencing of two key centromere proteins, Cse4 and Mif2, and are consistent with bioinformatic predictions. The centromeric DNA sequence was unique for each chromosome and spanned 4–4.5 kbp, consistent with regional epigenetically inherited centromeres. However, unlike other regional centromeres, there was no evidence of pericentromeric heterochromatin in C. lusitaniae. In particular, flanking genes were expressed at a similar level to the rest of the genome, and a URA3 reporter inserted adjacent to a centromere was not repressed. In addition, regions flanking the centromeric core were not associated with hypoacetylated histones or a sirtuin deacetylase that generates heterochromatin in other yeast. Interestingly, the centromeric chromatin had a distinct pattern of histone modifications, being enriched for methylated H3K79 and H3R2 but lacking methylation of H3K4, which is found at other regional centromeres. Thus, not all regional centromeres require flanking heterochromatin. PMID:26371315

Meiotic Cohesin SMC1β Provides Prophase I Centromeric Cohesion and Is Required for Multiple Synapsis-Associated Functions

PubMed Central

Biswas, Uddipta; Wetzker, Cornelia; Lange, Julian; Christodoulou, Eleni G.; Seifert, Michael; Beyer, Andreas; Jessberger, Rolf

2013-01-01

Cohesin subunit SMC1β is specific and essential for meiosis. Previous studies showed functions of SMC1β in determining the axis-loop structure of synaptonemal complexes (SCs), in providing sister chromatid cohesion (SCC) in metaphase I and thereafter, in protecting telomere structure, and in synapsis. However, several central questions remained unanswered and concern roles of SMC1β in SCC and synapsis and processes related to these two processes. Here we show that SMC1β substantially supports prophase I SCC at centromeres but not along chromosome arms. Arm cohesion and some of centromeric cohesion in prophase I are provided by non-phosphorylated SMC1α. Besides supporting synapsis of autosomes, SMC1β is also required for synapsis and silencing of sex chromosomes. In absence of SMC1β, the silencing factor γH2AX remains associated with asynapsed autosomes and fails to localize to sex chromosomes. Microarray expression studies revealed up-regulated sex chromosome genes and many down-regulated autosomal genes. SMC1β is further required for non-homologous chromosome associations observed in absence of SPO11 and thus of programmed double-strand breaks. These breaks are properly generated in Smc1β−/− spermatocytes, but their repair is delayed on asynapsed chromosomes. SMC1α alone cannot support non-homologous associations. Together with previous knowledge, three main functions of SMC1β have emerged, which have multiple consequences for spermatocyte biology: generation of the loop-axis architecture of SCs, homologous and non-homologous synapsis, and SCC starting in early prophase I. PMID:24385917

Resolving complex chromosome structures during meiosis: versatile deployment of Smc5/6.

PubMed

Verver, Dideke E; Hwang, Grace H; Jordan, Philip W; Hamer, Geert

2016-03-01

The Smc5/6 complex, along with cohesin and condensin, is a member of the structural maintenance of chromosome (SMC) family, large ring-like protein complexes that are essential for chromatin structure and function. Thanks to numerous studies of the mitotic cell cycle, Smc5/6 has been implicated to have roles in homologous recombination, restart of stalled replication forks, maintenance of ribosomal DNA (rDNA) and heterochromatin, telomerase-independent telomere elongation, and regulation of chromosome topology. The nature of these functions implies that the Smc5/6 complex also contributes to the profound chromatin changes, including meiotic recombination, that characterize meiosis. Only recently, studies in diverse model organisms have focused on the potential meiotic roles of the Smc5/6 complex. Indeed, Smc5/6 appears to be essential for meiotic recombination. However, due to both the complexity of the process of meiosis and the versatility of the Smc5/6 complex, many additional meiotic functions have been described. In this review, we provide a clear overview of the multiple functions found so far for the Smc5/6 complex in meiosis. Additionally, we compare these meiotic functions with the known mitotic functions in an attempt to find a common denominator and thereby create clarity in the field of Smc5/6 research.

SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes

PubMed Central

Hwang, Grace; Sun, Fengyun; Eppig, John J.; Handel, Mary Ann

2017-01-01

SMC complexes include three major classes: cohesin, condensin and SMC5/6. However, the localization pattern and genetic requirements for the SMC5/6 complex during mammalian oogenesis have not previously been examined. In mouse oocytes, the SMC5/6 complex is enriched at the pericentromeric heterochromatin, and also localizes along chromosome arms during meiosis. The infertility phenotypes of females with a Zp3-Cre-driven conditional knockout (cKO) of Smc5 demonstrated that maternally expressed SMC5 protein is essential for early embryogenesis. Interestingly, protein levels of SMC5/6 complex components in oocytes decline as wild-type females age. When SMC5/6 complexes were completely absent in oocytes during meiotic resumption, homologous chromosomes failed to segregate accurately during meiosis I. Despite what appears to be an inability to resolve concatenation between chromosomes during meiosis, localization of topoisomerase IIα to bivalents was not affected; however, localization of condensin along the chromosome axes was perturbed. Taken together, these data demonstrate that the SMC5/6 complex is essential for the formation of segregation-competent bivalents during meiosis I, and findings suggest that age-dependent depletion of the SMC5/6 complex in oocytes could contribute to increased incidence of oocyte aneuploidy and spontaneous abortion in aging females. PMID:28302748

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

PubMed Central

2013-01-01

Background Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture. Results Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins. Conclusions The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production. PMID:24341331

SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes.

PubMed

Hwang, Grace; Sun, Fengyun; O'Brien, Marilyn; Eppig, John J; Handel, Mary Ann; Jordan, Philip W

2017-05-01

SMC complexes include three major classes: cohesin, condensin and SMC5/6. However, the localization pattern and genetic requirements for the SMC5/6 complex during mammalian oogenesis have not previously been examined. In mouse oocytes, the SMC5/6 complex is enriched at the pericentromeric heterochromatin, and also localizes along chromosome arms during meiosis. The infertility phenotypes of females with a Zp3-Cre -driven conditional knockout (cKO) of Smc5 demonstrated that maternally expressed SMC5 protein is essential for early embryogenesis. Interestingly, protein levels of SMC5/6 complex components in oocytes decline as wild-type females age. When SMC5/6 complexes were completely absent in oocytes during meiotic resumption, homologous chromosomes failed to segregate accurately during meiosis I. Despite what appears to be an inability to resolve concatenation between chromosomes during meiosis, localization of topoisomerase IIα to bivalents was not affected; however, localization of condensin along the chromosome axes was perturbed. Taken together, these data demonstrate that the SMC5/6 complex is essential for the formation of segregation-competent bivalents during meiosis I, and findings suggest that age-dependent depletion of the SMC5/6 complex in oocytes could contribute to increased incidence of oocyte aneuploidy and spontaneous abortion in aging females. © 2017. Published by The Company of Biologists Ltd.

Diversity and Strain Specificity of Plant Cell Wall Degrading Enzymes Revealed by the Draft Genome of Ruminococcus flavefaciens FD-1

PubMed Central

Berg Miller, Margret E.; Antonopoulos, Dionysios A.; Rincon, Marco T.; Band, Mark; Bari, Albert; Akraiko, Tatsiana; Hernandez, Alvaro; Thimmapuram, Jyothi; Henrissat, Bernard; Coutinho, Pedro M.; Borovok, Ilya; Jindou, Sadanari; Lamed, Raphael; Flint, Harry J.; Bayer, Edward A.; White, Bryan A.

2009-01-01

Background Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. Methodology/Principal Findings The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. Conclusions/Significance The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components. PMID:19680555

Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: advances and applications.

PubMed

Hyeon, Jeong Eun; Jeon, Sang Duck; Han, Sung Ok

2013-11-01

The cellulosome is one of nature's most elegant and elaborate nanomachines and a key biological and biotechnological macromolecule that can be used as a multi-functional protein complex tool. Each protein module in the cellulosome system is potentially useful in an advanced biotechnology application. The high-affinity interactions between the cohesin and dockerin domains can be used in protein-based biosensors to improve both sensitivity and selectivity. The scaffolding protein includes a carbohydrate-binding module (CBM) that attaches strongly to cellulose substrates and facilitates the purification of proteins fused with the dockerin module through a one-step CBM purification method. Although the surface layer homology (SLH) domain of CbpA is not present in other strains, replacement of the cell surface anchoring domain allows a foreign protein to be displayed on the surface of other strains. The development of a hydrolysis enzyme complex is a useful strategy for consolidated bioprocessing (CBP), enabling microorganisms with biomass hydrolysis activity. Thus, the development of various configurations of multi-functional protein complexes for use as tools in whole-cell biocatalyst systems has drawn considerable attention as an attractive strategy for bioprocess applications. This review provides a detailed summary of the current achievements in Clostridium-derived multi-functional complex development and the impact of these complexes in various areas of biotechnology. Copyright © 2013 Elsevier Inc. All rights reserved.

Redirection of pyruvate flux toward desired metabolic pathways through substrate channeling between pyruvate kinase and pyruvate-converting enzymes in Saccharomyces cerevisiae.

PubMed

Kim, Sujin; Bae, Sang-Jeong; Hahn, Ji-Sook

2016-04-07

Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates. Here, we investigated the effect of substrate channeling on the flux partitioning at a metabolic branch point, focusing on pyruvate metabolism in Saccharomyces cerevisiae. As a platform strain for the channeling of pyruvate flux, PYK1-Coh-Myc strain was constructed in which PYK1 gene encoding pyruvate kinase is tagged with cohesin domain. By using high-affinity cohesin-dockerin interaction, the pyruvate-forming enzyme Pyk1 was tethered to heterologous pyruvate-converting enzymes, lactate dehydrogenase and α-acetolactate synthase, to produce lactic acid and 2,3-butanediol, respectively. Pyruvate flux was successfully redirected toward desired pathways, with a concomitant decrease in ethanol production even without genetic attenuation of the ethanol-producing pathway. This pyruvate channeling strategy led to an improvement of 2,3-butanediol production by 38%, while showing a limitation in improving lactic acid production due to a reduced activity of lactate dehydrogenase by dockerin tagging.

CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function

PubMed Central

Guo, Ya; Xu, Quan; Canzio, Daniele; Shou, Jia; Li, Jinhuan; Gorkin, David U.; Jung, Inkyung; Wu, Haiyang; Zhai, Yanan; Tang, Yuanxiao; Lu, Yichao; Wu, Yonghu; Jia, Zhilian; Li, Wei; Zhang, Michael Q.; Ren, Bing; Krainer, Adrian R.; Maniatis, Tom; Wu, Qiang

2015-01-01

SUMMARY CTCF/cohesin play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and β-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters, and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. The findings reveal how 3D chromosome architecture can be encoded by genome sequence. PMID:26276636

Structural Basis of Clostridium perfringens Toxin Complex Formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adams,J.; Gregg, K.; Bayer, E.

2008-01-01

The virulent properties of the common human and livestock pathogen Clostridium perfringens are attributable to a formidable battery of toxins. Among these are a number of large and highly modular carbohydrate-active enzymes, including the {mu}-toxin and sialidases, whose catalytic properties are consistent with degradation of the mucosal layer of the human gut, glycosaminoglycans, and other cellular glycans found throughout the body. The conservation of noncatalytic ancillary modules among these enzymes suggests they make significant contributions to the overall functionality of the toxins. Here, we describe the structural basis of an ultra-tight interaction (Ka = 1.44 x 1011 M-1) between themore » X82 and dockerin modules, which are found throughout numerous C. perfringens carbohydrate-active enzymes. Extensive hydrogen-bonding and van der Waals contacts between the X82 and dockerin modules give rise to the observed high affinity. The {mu}-toxin dockerin module in this complex is positioned {approx}180 relative to the orientation of the dockerin modules on the cohesin module surface within cellulolytic complexes. These observations represent a unique property of these clostridial toxins whereby they can associate into large, noncovalent multitoxin complexes that allow potentiation of the activities of the individual toxins by combining complementary toxin specificities.« less

Manipulation of Karyotype in Caenorhabditis elegans Reveals Multiple Inputs Driving Pairwise Chromosome Synapsis During Meiosis

PubMed Central

Roelens, Baptiste; Schvarzstein, Mara; Villeneuve, Anne M.

2015-01-01

Meiotic chromosome segregation requires pairwise association between homologs, stabilized by the synaptonemal complex (SC). Here, we investigate factors contributing to pairwise synapsis by investigating meiosis in polyploid worms. We devised a strategy, based on transient inhibition of cohesin function, to generate polyploid derivatives of virtually any Caenorhabditis elegans strain. We exploited this strategy to investigate the contribution of recombination to pairwise synapsis in tetraploid and triploid worms. In otherwise wild-type polyploids, chromosomes first sort into homolog groups, then multipartner interactions mature into exclusive pairwise associations. Pairwise synapsis associations still form in recombination-deficient tetraploids, confirming a propensity for synapsis to occur in a strictly pairwise manner. However, the transition from multipartner to pairwise association was perturbed in recombination-deficient triploids, implying a role for recombination in promoting this transition when three partners compete for synapsis. To evaluate the basis of synapsis partner preference, we generated polyploid worms heterozygous for normal sequence and rearranged chromosomes sharing the same pairing center (PC). Tetraploid worms had no detectable preference for identical partners, indicating that PC-adjacent homology drives partner choice in this context. In contrast, triploid worms exhibited a clear preference for identical partners, indicating that homology outside the PC region can influence partner choice. Together, our findings, suggest a two-phase model for C. elegans synapsis: an early phase, in which initial synapsis interactions are driven primarily by recombination-independent assessment of homology near PCs and by a propensity for pairwise SC assembly, and a later phase in which mature synaptic interactions are promoted by recombination. PMID:26500263

Pan-Cellulosomics of Mesophilic Clostridia: Variations on a Theme.

PubMed

Dassa, Bareket; Borovok, Ilya; Lombard, Vincent; Henrissat, Bernard; Lamed, Raphael; Bayer, Edward A; Moraïs, Sarah

2017-11-18

The bacterial cellulosome is an extracellular, multi-enzyme machinery, which efficiently depolymerizes plant biomass by degrading plant cell wall polysaccharides. Several cellulolytic bacteria have evolved various elaborate modular architectures of active cellulosomes. We present here a genome-wide analysis of a dozen mesophilic clostridia species, including both well-studied and yet-undescribed cellulosome-producing bacteria. We first report here, the presence of cellulosomal elements, thus expanding our knowledge regarding the prevalence of the cellulosomal paradigm in nature. We explored the genomic organization of key cellulosome components by comparing the cellulosomal gene clusters in each bacterial species, and the conserved sequence features of the specific cellulosomal modules (cohesins and dockerins), on the background of their phylogenetic relationship. Additionally, we performed comparative analyses of the species-specific repertoire of carbohydrate-degrading enzymes for each of the clostridial species, and classified each cellulosomal enzyme into a specific CAZy family, thus indicating their putative enzymatic activity (e.g., cellulases, hemicellulases, and pectinases). Our work provides, for this large group of bacteria, a broad overview of the blueprints of their multi-component cellulosomal complexes. The high similarity of their scaffoldin clusters and dockerin-based recognition residues suggests a common ancestor, and/or extensive horizontal gene transfer, and potential cross-species recognition. In addition, the sporadic spatial organization of the numerous dockerin-containing genes in several of the genomes, suggests the importance of the cellulosome paradigm in the given bacterial species. The information gained in this work may be utilized directly or developed further by genetically engineering and optimizing designer cellulosome systems for enhanced biotechnological biomass deconstruction and biofuel production.

The selfish yeast plasmid uses the nuclear motor Kip1p but not Cin8p for its localization and equal segregation.

PubMed

Cui, Hong; Ghosh, Santanu K; Jayaram, Makkuni

2009-04-20

The 2 micron plasmid of Saccharomyces cerevisiae uses the Kip1 motor, but not the functionally redundant Cin8 motor, for its precise nuclear localization and equal segregation. The timing and lifetime of Kip1p association with the plasmid partitioning locus STB are consistent with Kip1p being an authentic component of the plasmid partitioning complex. Kip1-STB association is not blocked by disassembling the mitotic spindle. Lack of Kip1p disrupts recruitment of the cohesin complex at STB and cohesion of replicated plasmid molecules. Colocalization of a 2 micron reporter plasmid with Kip1p in close proximity to the spindle pole body is reminiscent of that of a CEN reporter plasmid. Absence of Kip1p displaces the plasmid from this nuclear address, where it has the potential to tether to a chromosome or poach chromosome segregation factors. Exploiting Kip1p, which is subsidiary to Cin8p for chromosome segregation, to direct itself to a "partitioning center" represents yet another facet of the benign parasitism of the yeast plasmid.

Probing Protein Structure in Vivo with FRET

PubMed Central

Davis, Trisha; Muller, Eric

2012-01-01

Fluorescence resonance energy transfer (FRET) is widely used to construct probes for cellular activities and to complement two-hybrid results that predict protein-protein interactions. The Yeast Resource Center promotes an underutilized potential of FRET as an in vivo tool to position proteins within low resolution structures derived from electron microscopy. The success of this approach using widefield microscopy depends upon the choice of filter sets, standardized image acquisition, a robust metric and controls matched to the structure under investigation. A comparison of various CFP and YFP filter combinations from Chroma and Semrock demonstrated the strength of the Chroma filters when coupled with our FRET metric, termed FretR. Coupling CFP and YFP to a selection of proteins of known structure allowed us to create a standard curve of FretR versus distance. How well other FRET metrics conform was also evaluated. Finally FretR was linked to an approximation of the efficiency of energy transfer. Together this feature set has allowed us to contribute to our understanding of the organization of the yeast spindle pole body, cohesin complex and gamma-tubulin complex.

RPA Mediates Recruitment of MRX to Forks and Double-Strand Breaks to Hold Sister Chromatids Together.

PubMed

Seeber, Andrew; Hegnauer, Anna Maria; Hustedt, Nicole; Deshpande, Ishan; Poli, Jérôme; Eglinger, Jan; Pasero, Philippe; Gut, Heinz; Shinohara, Miki; Hopfner, Karl-Peter; Shimada, Kenji; Gasser, Susan M

2016-12-01

The Mre11-Rad50-Xrs2 (MRX) complex is related to SMC complexes that form rings capable of holding two distinct DNA strands together. MRX functions at stalled replication forks and double-strand breaks (DSBs). A mutation in the N-terminal OB fold of the 70 kDa subunit of yeast replication protein A, rfa1-t11, abrogates MRX recruitment to both types of DNA damage. The rfa1 mutation is functionally epistatic with loss of any of the MRX subunits for survival of replication fork stress or DSB recovery, although it does not compromise end-resection. High-resolution imaging shows that either the rfa1-t11 or the rad50Δ mutation lets stalled replication forks collapse and allows the separation not only of opposing ends but of sister chromatids at breaks. Given that cohesin loss does not provoke visible sister separation as long as the RPA-MRX contacts are intact, we conclude that MRX also serves as a structural linchpin holding sister chromatids together at breaks. Copyright © 2016 Elsevier Inc. All rights reserved.

Hotspots of aberrant enhancer activity punctuate the colorectal cancer epigenome

PubMed Central

Cohen, Andrea J.; Saiakhova, Alina; Corradin, Olivia; Luppino, Jennifer M.; Lovrenert, Katreya; Bartels, Cynthia F.; Morrow, James J.; Mack, Stephen C.; Dhillon, Gursimran; Beard, Lydia; Myeroff, Lois; Kalady, Matthew F.; Willis, Joseph; Bradner, James E.; Keri, Ruth A.; Berger, Nathan A.; Pruett-Miller, Shondra M.; Markowitz, Sanford D.; Scacheri, Peter C.

2017-01-01

In addition to mutations in genes, aberrant enhancer element activity at non-coding regions of the genome is a key driver of tumorigenesis. Here, we perform epigenomic enhancer profiling of a cohort of more than forty genetically diverse human colorectal cancer (CRC) specimens. Using normal colonic crypt epithelium as a comparator, we identify enhancers with recurrently gained or lost activity across CRC specimens. Of the enhancers highly recurrently activated in CRC, most are constituents of super enhancers, are occupied by AP-1 and cohesin complex members, and originate from primed chromatin. Many activate known oncogenes, and CRC growth can be mitigated through pharmacologic inhibition or genome editing of these loci. Nearly half of all GWAS CRC risk loci co-localize to recurrently activated enhancers. These findings indicate that the CRC epigenome is defined by highly recurrent epigenetic alterations at enhancers which activate a common, aberrant transcriptional programme critical for CRC growth and survival. PMID:28169291

In situ analysis of DNA damage response and repair using laser microirradiation.

PubMed

Kim, Jong-Soo; Heale, Jason T; Zeng, Weihua; Kong, Xiangduo; Krasieva, Tatiana B; Ball, Alexander R; Yokomori, Kyoko

2007-01-01

A proper response to DNA damage is critical for the maintenance of genome integrity. However, it is difficult to study the in vivo kinetics and factor requirements of the damage recognition process in mammalian cells. In order to address how the cell reacts to DNA damage, we utilized a second harmonic (532 nm) pulsed Nd:YAG laser to induce highly concentrated damage in a small area in interphase cell nuclei and cytologically analyzed both protein recruitment and modification. Our results revealed for the first time the sequential recruitment of factors involved in two major DNA double-strand break (DSB) repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR), and the cell cycle-specific recruitment of the sister chromatid cohesion complex cohesin to the damage site. In this chapter, the strategy developed to study the DNA damage response using the 532-nm Nd:YAG laser will be summarized.

Sgol2 provides a regulatory platform that coordinates essential cell cycle processes during meiosis I in oocytes

PubMed Central

Rattani, Ahmed; Wolna, Magda; Ploquin, Mickael; Helmhart, Wolfgang; Morrone, Seamus; Mayer, Bernd; Godwin, Jonathan; Xu, Wenqing; Stemmann, Olaf; Pendas, Alberto; Nasmyth, Kim

2013-01-01

Accurate chromosome segregation depends on coordination between cohesion resolution and kinetochore-microtubule interactions (K-fibers), a process regulated by the spindle assembly checkpoint (SAC). How these diverse processes are coordinated remains unclear. We show that in mammalian oocytes Shugoshin-like protein 2 (Sgol2) in addition to protecting cohesin, plays an important role in turning off the SAC, in promoting the congression and bi-orientation of bivalents on meiosis I spindles, in facilitating formation of K-fibers and in limiting bivalent stretching. Sgol2’s ability to protect cohesin depends on its interaction with PP2A, as is its ability to silence the SAC, with the latter being mediated by direct binding to Mad2. In contrast, its effect on bivalent stretching and K-fiber formation is independent of PP2A and mediated by recruitment of MCAK and inhibition of Aurora C kinase activity respectively. By virtue of its multiple interactions, Sgol2 links many of the processes essential for faithful chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.01133.001 PMID:24192037

SA1 and TRF1 synergistically bind to telomeric DNA and promote DNA-DNA pairing

NASA Astrophysics Data System (ADS)

Wang, Hong; Lin, Jiangguo; Countryman, Preston; Pan, Hai; Parminder Kaur Team; Robert Riehn Team; Patricia Opresko Team; Jane Tao Team; Susan Smith Team

Impaired telomere cohesion leads to increased aneuploidy and early onset of tumorigenesis. Cohesion is thought to occur through the entrapment of two DNA strands within tripartite cohesin ring(s), along with a fourth subunit (SA1/SA2). Surprisingly, cohesion rings are not essential for telomere cohesion, which instead requires SA1 and shelterin proteins including TRF1. However, neither this unique cohesion mechanism at telomeres or DNA-binding properties of SA1 is understood. Here, using single-molecule fluorescence imaging of quantum dot-labeled proteins on DNA we discover that while SA1 diffuses across multiple telomeric and non-telomeric regions, the diffusion mediated through its N-terminal domain is slower at telomeric regions. However, addition of TRF1 traps SA1 within telomeric regions, which form longer DNA-DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy. Together, these experimental results and coarse-grained molecular dynamics simulations suggest that TRF1 and SA1 synergistically interact with DNA to support telomere cohesion without cohesin rings.

SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster.

PubMed

Yan, Rihui; Thomas, Sharon E; Tsai, Jui-He; Yamada, Yukihiro; McKee, Bruce D

2010-02-08

Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis.

Higher Order Chromatin Modulator Cohesin SA1 Is an Early Biomarker for Colon Carcinogenesis: Race-Specific Implications.

PubMed

Wali, Ramesh K; Momi, Navneet; Dela Cruz, Mart; Calderwood, Audrey H; Stypula-Cyrus, Yolanda; Almassalha, Luay; Chhaparia, Anuj; Weber, Christopher R; Radosevich, Andrew; Tiwari, Ashish K; Latif, Bilal; Backman, Vadim; Roy, Hemant K

2016-11-01

Alterations in high order chromatin, with concomitant modulation in gene expression, are one of the earliest events in the development of colorectal cancer. Cohesins are a family of proteins that modulate high-order chromatin, although the role in colorectal cancer remains incompletely understood. We, therefore, assessed the role of cohesin SA1 in colorectal cancer biology and as a biomarker focusing in particular on the increased incidence/mortality of colorectal cancer among African-Americans. Immunohistochemistry on tissue arrays revealed dramatically decreased SA1 expression in both adenomas (62%; P = 0.001) and adenocarcinomas (75%; P = 0.0001). RT-PCR performed in endoscopically normal rectal biopsies (n = 78) revealed a profound decrease in SA1 expression in adenoma-harboring patients (field carcinogenesis) compared with those who were neoplasia-free (47%; P = 0.03). From a racial perspective, colorectal cancer tissues from Caucasians had 56% higher SA1 expression than in African-Americans. This was mirrored in field carcinogenesis where healthy Caucasians expressed more SA1 at baseline compared with matched African-American subjects (73%; P = 0.003). However, as a biomarker for colorectal cancer risk, the diagnostic performance as assessed by area under ROC curve was greater in African-Americans (AUROC = 0.724) than in Caucasians (AUROC = 0.585). From a biologic perspective, SA1 modulation of high-order chromatin was demonstrated with both biophotonic (nanocytology) and chromatin accessibility [micrococcal nuclease (MNase)] assays in SA1-knockdown HT29 colorectal cancer cells. The functional consequences were underscored by increased proliferation (WST-1; P = 0.0002, colony formation; P = 0.001) in the SA1-knockdown HT29 cells. These results provide the first evidence indicating a tumor suppressor role of SA1 in early colon carcinogenesis and as a risk stratification biomarker giving potential insights into biologic basis of racial disparities in colorectal cancer. Cancer Prev Res; 9(11); 844-54. ©2016 AACR. ©2016 American Association for Cancer Research.

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance

PubMed Central

Payne, Felicity; Colnaghi, Rita; Rocha, Nuno; Seth, Asha; Harris, Julie; Carpenter, Gillian; Bottomley, William E.; Wheeler, Eleanor; Wong, Stephen; Saudek, Vladimir; Savage, David; O’Rahilly, Stephen; Carel, Jean-Claude; Barroso, Inês; O’Driscoll, Mark; Semple, Robert

2014-01-01

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5–6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase–deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress. PMID:25105364

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance.

PubMed

Payne, Felicity; Colnaghi, Rita; Rocha, Nuno; Seth, Asha; Harris, Julie; Carpenter, Gillian; Bottomley, William E; Wheeler, Eleanor; Wong, Stephen; Saudek, Vladimir; Savage, David; O'Rahilly, Stephen; Carel, Jean-Claude; Barroso, Inês; O'Driscoll, Mark; Semple, Robert

2014-09-01

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5-6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase-deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress.

A potential source for cellulolytic enzyme discovery and environmental aspects revealed through metagenomics of Brazilian mangroves

PubMed Central

2013-01-01

The mangroves are among the most productive and biologically important environments. The possible presence of cellulolytic enzymes and microorganisms useful for biomass degradation as well as taxonomic and functional aspects of two Brazilian mangroves were evaluated using cultivation and metagenomic approaches. From a total of 296 microorganisms with visual differences in colony morphology and growth (including bacteria, yeast and filamentous fungus), 179 (60.5%) and 117 (39.5%) were isolated from the Rio de Janeiro (RJ) and Bahia (BA) samples, respectively. RJ metagenome showed the higher number of microbial isolates, which is consistent with its most conserved state and higher diversity. The metagenomic sequencing data showed similar predominant bacterial phyla in the BA and RJ mangroves with an abundance of Proteobacteria (57.8% and 44.6%), Firmicutes (11% and 12.3%) and Actinobacteria (8.4% and 7.5%). A higher number of enzymes involved in the degradation of polycyclic aromatic compounds were found in the BA mangrove. Specific sequences involved in the cellulolytic degradation, belonging to cellulases, hemicellulases, carbohydrate binding domains, dockerins and cohesins were identified, and it was possible to isolate cultivable fungi and bacteria related to biomass decomposition and with potential applications for the production of biofuels. These results showed that the mangroves possess all fundamental molecular tools required for building the cellulosome, which is required for the efficient degradation of cellulose material and sugar release. PMID:24160319

Characterization of the cellulosomal scaffolding protein CbpC from Clostridium cellulovorans 743B.

PubMed

Nakajima, Daichi; Shibata, Toshiyuki; Tanaka, Reiji; Kuroda, Kouichi; Ueda, Mitsuyoshi; Miyake, Hideo

2017-10-01

Clostridium cellulovorans 743B, an anaerobic and mesophilic bacterium, produces an extracellular enzyme complex called the cellulosome on the cell surface. Recently, we have reported the whole genome sequence of C. cellulovorans, which revealed that a total of 4 cellulosomal scaffolding proteins: CbpA, HbpA, CbpB, and CbpC were encoded in the C. cellulovorans genome. In particular, cbpC encoded a 429-residue polypeptide that includes a carbohydrate-binding module (CBM), an S-layer homology module, and a cohesin. CbpC was also detected in the culture supernatant of C. cellulovorans. Genomic DNA coding for CbpC was subcloned into a pET-22b+ vector in order to express and produce the recombinant protein in Escherichia coli BL21(DE3). Measurement of CbpC adsorption to crystalline cellulose indicated a dissociation constant of 0.60 μM, which is a similar to that of CBM from CbpA. We also subcloned the region encoding xylanase B (XynB) with the dockerin from C. cellulovorans and analyzed the interaction between XynB and CbpC by GST pull-down assay. It was observed that GST-CbpC assembles with XynB to form a minimal cellulosome. The activity of XynB against rice straw tended to be increased in the presence of CbpC. These results showed a synergistic effect on rice straw as a representative cellulosic biomass through the formation of a minimal cellulosome containing XynB bound to CbpC. Thus, our findings provide a foundation for the development of cellulosic biomass saccharification using a minimal cellulosome. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The active enhancer network operated by liganded RXR supports angiogenic activity in macrophages

PubMed Central

Daniel, Bence; Hah, Nasun; Horvath, Attila; Czimmerer, Zsolt; Poliska, Szilard; Gyuris, Tibor; Keirsse, Jiri; Gysemans, Conny; Van Ginderachter, Jo A.; Balint, Balint L.; Evans, Ronald M.; Barta, Endre; Nagy, Laszlo

2014-01-01

RXR signaling is predicted to have a major impact in macrophages, but neither the biological consequence nor the genomic basis of its ligand activation is known. Comprehensive genome-wide studies were carried out to map liganded RXR-mediated transcriptional changes, active binding sites, and cistromic interactions in the context of the macrophage genome architecture. The macrophage RXR cistrome has 5200 genomic binding sites, which are not impacted by ligand. Active enhancers are characterized by PU.1 binding, an increase of enhancer RNA, and P300 recruitment. Using these features, 387 liganded RXR-bound enhancers were linked to 226 genes, which predominantly reside in CTCF/cohesin-limited functional domains. These findings were molecularly validated using chromosome conformation capture (3C) and 3C combined with sequencing (3C-seq), and we show that selected long-range enhancers communicate with promoters via stable or RXR-induced loops and that some of the enhancers interact with each other, forming an interchromosomal network. A set of angiogenic genes, including Vegfa, has liganded RXR-controlled enhancers and provides the macrophage with a novel inducible program. PMID:25030696

Successful Growth Hormone Therapy in Cornelia de Lange Syndrome.

PubMed

de Graaf, Michael; Kant, Sarina G; Wit, Jan Maarten; Willem Redeker, Egbert Johan; Eduard Santen, Gijs Willem; Henriëtta Verkerk, Annemieke Johanna Maria; Uitterlinden, André Gerardus; Losekoot, Monique; Oostdijk, Wilma

2017-12-15

Cornelia de Lange syndrome (CdLS) is a both clinically and genetically heterogeneous syndrome. In its classical form, it is characterised by distinctive facial features, intra-uterine growth retardation, short stature, developmental delay, and anomalies in multiple organ systems. NIPBL, SMC1A, SMC3, RAD21 and HDAC8, all involved in the cohesin pathway, have been identified to cause CdLS. Growth hormone (GH) secretion has been reported as normal, and to our knowledge, there are no reports on the effect of recombinant human GH treatment in CdLS patients. We present a patient born small for gestational age with persistent severe growth retardation [height -3.4 standard deviation score (SDS)] and mild dysmorphic features, who was treated with GH from 4.3 years of age onward and was diagnosed 6 years later with CdLS using whole-exome sequencing. Treatment led to a height gain of 1.6 SDS over 8 years. Treatment was interrupted shortly due to high serum insulin-like growth factor-1 serum values. In conclusion, GH therapy may be effective and safe for short children with CdLS.

CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription.

PubMed

Tang, Zhonghui; Luo, Oscar Junhong; Li, Xingwang; Zheng, Meizhen; Zhu, Jacqueline Jufen; Szalaj, Przemyslaw; Trzaskoma, Pawel; Magalska, Adriana; Wlodarczyk, Jakub; Ruszczycki, Blazej; Michalski, Paul; Piecuch, Emaly; Wang, Ping; Wang, Danjuan; Tian, Simon Zhongyuan; Penrad-Mobayed, May; Sachs, Laurent M; Ruan, Xiaoan; Wei, Chia-Lin; Liu, Edison T; Wilczynski, Grzegorz M; Plewczynski, Dariusz; Li, Guoliang; Ruan, Yijun

2015-12-17

Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Successful Growth Hormone Therapy in Cornelia de Lange Syndrome

PubMed Central

de Graaf, Michael; Kant, Sarina G; Wit, Jan Maarten; Redeker, Egbert Johan Willem; Santen, Gijs Willem Eduard; Verkerk, Annemieke Johanna Maria Henriëtta; Uitterlinden, André Gerardus; Losekoot, Monique; Oostdijk, Wilma

2017-01-01

Cornelia de Lange syndrome (CdLS) is a both clinically and genetically heterogeneous syndrome. In its classical form, it is characterised by distinctive facial features, intra-uterine growth retardation, short stature, developmental delay, and anomalies in multiple organ systems. NIPBL, SMC1A, SMC3, RAD21 and HDAC8, all involved in the cohesin pathway, have been identified to cause CdLS. Growth hormone (GH) secretion has been reported as normal, and to our knowledge, there are no reports on the effect of recombinant human GH treatment in CdLS patients. We present a patient born small for gestational age with persistent severe growth retardation [height -3.4 standard deviation score (SDS)] and mild dysmorphic features, who was treated with GH from 4.3 years of age onward and was diagnosed 6 years later with CdLS using whole-exome sequencing. Treatment led to a height gain of 1.6 SDS over 8 years. Treatment was interrupted shortly due to high serum insulin-like growth factor-1 serum values. In conclusion, GH therapy may be effective and safe for short children with CdLS. PMID:28588001

The selfish yeast plasmid uses the nuclear motor Kip1p but not Cin8p for its localization and equal segregation

PubMed Central

Cui, Hong; Ghosh, Santanu K.

2009-01-01

The 2 micron plasmid of Saccharomyces cerevisiae uses the Kip1 motor, but not the functionally redundant Cin8 motor, for its precise nuclear localization and equal segregation. The timing and lifetime of Kip1p association with the plasmid partitioning locus STB are consistent with Kip1p being an authentic component of the plasmid partitioning complex. Kip1–STB association is not blocked by disassembling the mitotic spindle. Lack of Kip1p disrupts recruitment of the cohesin complex at STB and cohesion of replicated plasmid molecules. Colocalization of a 2 micron reporter plasmid with Kip1p in close proximity to the spindle pole body is reminiscent of that of a CEN reporter plasmid. Absence of Kip1p displaces the plasmid from this nuclear address, where it has the potential to tether to a chromosome or poach chromosome segregation factors. Exploiting Kip1p, which is subsidiary to Cin8p for chromosome segregation, to direct itself to a “partitioning center” represents yet another facet of the benign parasitism of the yeast plasmid. PMID:19364922

Mad2, Bub3, and Mps1 regulate chromosome segregation and mitotic synchrony in Giardia intestinalis, a binucleate protist lacking an anaphase-promoting complex

PubMed Central

Vicente, Juan-Jesus; Cande, W. Zacheus

2014-01-01

The binucleate pathogen Giardia intestinalis is a highly divergent eukaryote with a semiopen mitosis, lacking an anaphase-promoting complex/cyclosome (APC/C) and many of the mitotic checkpoint complex (MCC) proteins. However, Giardia has some MCC components (Bub3, Mad2, and Mps1) and proteins from the cohesin system (Smc1 and Smc3). Mad2 localizes to the cytoplasm, but Bub3 and Mps1 are either located on chromosomes or in the cytoplasm, depending on the cell cycle stage. Depletion of Bub3, Mad2, or Mps1 resulted in a lowered mitotic index, errors in chromosome segregation (including lagging chromosomes), and abnormalities in spindle morphology. During interphase, MCC knockdown cells have an abnormal number of nuclei, either one nucleus usually on the left-hand side of the cell or two nuclei with one mislocalized. These results suggest that the minimal set of MCC proteins in Giardia play a major role in regulating many aspects of mitosis, including chromosome segregation, coordination of mitosis between the two nuclei, and subsequent nuclear positioning. The critical importance of MCC proteins in an organism that lacks their canonical target, the APC/C, suggests a broader role for these proteins and hints at new pathways to be discovered. PMID:25057014

Elucidate the Mechanism of Telomere Maintenance in STAG2 Mutated Tumor Cells

DTIC Science & Technology

2017-12-01

recent analysis identified the cohesin subunit STAG2 as one of twelve genes mutated in four or more tumor types including melanoma, pancreatic...conferences, seminars, study groups , and individual study. Include participation in conferences, workshops, and seminars not listed under major...only 12 genes found to be significantly mutated in four or more cancer types (18). Approximately 85% of STAG2 mutations are truncating and often result

Statistical radii associated with amino acids to determine the contact map: fixing the structure of a type I cohesin domain in the Clostridium thermocellum cellulosome

NASA Astrophysics Data System (ADS)

Chwastyk, Mateusz; Poma Bernaola, Adolfo; Cieplak, Marek

2015-07-01

We propose to improve and simplify protein refinement procedures through consideration of which pairs of amino acid residues should form native contacts. We first consider 11 330 proteins from the CATH database to determine statistical distributions of contacts associated with a given type of amino acid. The distributions are set across the distances between the α-C atoms that are in contact. Based on this data, we determine typical radii of effective spheres that can be placed on the α-C atoms in order to reconstruct the distribution of the contact lengths. This is done by checking for overlaps with enlarged van der Waals spheres associated with heavy atoms on other amino acids. The resulting contacts can be used to identify non-native contacts that may arise during the time evolution of structure-based models. Here, the radii are used to guide reconstruction of nine missing side chains in a type I cohesin domain with the Protein Data Bank code 1AOH. We first identify the likely missing contacts and then sculpt the corresponding side chains by standard refinement tools to achieve consistency with the expected contact map. One ambiguity in refinement is resolved by determining all-atom conformational energies.

Extrusion without a motor: a new take on the loop extrusion model of genome organization

PubMed Central

Johnson, J.; Michieletto, D.; Morozov, A. N.; Nicodemi, M.; Cook, P. R.; Marenduzzo, D.

2018-01-01

ABSTRACT Chromatin loop extrusion is a popular model for the formation of CTCF loops and topological domains. Recent HiC data have revealed a strong bias in favour of a particular arrangement of the CTCF binding motifs that stabilize loops, and extrusion is the only model to date which can explain this. However, the model requires a motor to generate the loops, and although cohesin is a strong candidate for the extruding factor, a suitable motor protein (or a motor activity in cohesin itself) has yet to be found. Here we explore a new hypothesis: that there is no motor, and thermal motion within the nucleus drives extrusion. Using theoretical modelling and computer simulations we ask whether such diffusive extrusion could feasibly generate loops. Our simulations uncover an interesting ratchet effect (where an osmotic pressure promotes loop growth), and suggest, by comparison to recent in vitro and in vivo measurements, that diffusive extrusion can in principle generate loops of the size observed in the data. Extra View on : C. A. Brackley, J. Johnson, D. Michieletto, A. N. Morozov, M. Nicodemi, P. R. Cook, and D. Marenduzzo “Non-equilibrium chromosome looping via molecular slip-links”, Physical Review Letters 119 138101 (2017) PMID:29300120

Arabidopsis MZT1 homologs GIP1 and GIP2 are essential for centromere architecture.

PubMed

Batzenschlager, Morgane; Lermontova, Inna; Schubert, Veit; Fuchs, Jörg; Berr, Alexandre; Koini, Maria A; Houlné, Guy; Herzog, Etienne; Rutten, Twan; Alioua, Abdelmalek; Fransz, Paul; Schmit, Anne-Catherine; Chabouté, Marie-Edith

2015-07-14

Centromeres play a pivotal role in maintaining genome integrity by facilitating the recruitment of kinetochore and sister-chromatid cohesion proteins, both required for correct chromosome segregation. Centromeres are epigenetically specified by the presence of the histone H3 variant (CENH3). In this study, we investigate the role of the highly conserved γ-tubulin complex protein 3-interacting proteins (GIPs) in Arabidopsis centromere regulation. We show that GIPs form a complex with CENH3 in cycling cells. GIP depletion in the gip1gip2 knockdown mutant leads to a decreased CENH3 level at centromeres, despite a higher level of Mis18BP1/KNL2 present at both centromeric and ectopic sites. We thus postulate that GIPs are required to ensure CENH3 deposition and/or maintenance at centromeres. In addition, the recruitment at the centromere of other proteins such as the CENP-C kinetochore component and the cohesin subunit SMC3 is impaired in gip1gip2. These defects in centromere architecture result in aneuploidy due to severely altered centromeric cohesion. Altogether, we ascribe a central function to GIPs for the proper recruitment and/or stabilization of centromeric proteins essential in the specification of the centromere identity, as well as for centromeric cohesion in somatic cells.

STAG2 promotes error correction in mitosis by regulating kinetochore-microtubule attachments.

PubMed

Kleyman, Marianna; Kabeche, Lilian; Compton, Duane A

2014-10-01

Mutations in the STAG2 gene are present in ∼20% of tumors from different tissues of origin. STAG2 encodes a subunit of the cohesin complex, and tumors with loss-of-function mutations are usually aneuploid and display elevated frequencies of lagging chromosomes during anaphase. Lagging chromosomes are a hallmark of chromosomal instability (CIN) arising from persistent errors in kinetochore-microtubule (kMT) attachment. To determine whether the loss of STAG2 increases the rate of formation of kMT attachment errors or decreases the rate of their correction, we examined mitosis in STAG2-deficient cells. STAG2 depletion does not impair bipolar spindle formation or delay mitotic progression. Instead, loss of STAG2 permits excessive centromere stretch along with hyperstabilization of kMT attachments. STAG2-deficient cells display mislocalization of Bub1 kinase, Bub3 and the chromosome passenger complex. Importantly, strategically destabilizing kMT attachments in tumor cells harboring STAG2 mutations by overexpression of the microtubule-destabilizing enzymes MCAK (also known as KIF2C) and Kif2B decreased the rate of lagging chromosomes and reduced the rate of chromosome missegregation. These data demonstrate that STAG2 promotes the correction of kMT attachment errors to ensure faithful chromosome segregation during mitosis. © 2014. Published by The Company of Biologists Ltd.

Mutations in chromatin regulators functionally link Cornelia de Lange syndrome and clinically overlapping phenotypes.

PubMed

Parenti, Ilaria; Teresa-Rodrigo, María E; Pozojevic, Jelena; Ruiz Gil, Sara; Bader, Ingrid; Braunholz, Diana; Bramswig, Nuria C; Gervasini, Cristina; Larizza, Lidia; Pfeiffer, Lutz; Ozkinay, Ferda; Ramos, Feliciano; Reiz, Benedikt; Rittinger, Olaf; Strom, Tim M; Watrin, Erwan; Wendt, Kerstin; Wieczorek, Dagmar; Wollnik, Bernd; Baquero-Montoya, Carolina; Pié, Juan; Deardorff, Matthew A; Gillessen-Kaesbach, Gabriele; Kaiser, Frank J

2017-03-01

The coordinated tissue-specific regulation of gene expression is essential for the proper development of all organisms. Mutations in multiple transcriptional regulators cause a group of neurodevelopmental disorders termed "transcriptomopathies" that share core phenotypical features including growth retardation, developmental delay, intellectual disability and facial dysmorphism. Cornelia de Lange syndrome (CdLS) belongs to this class of disorders and is caused by mutations in different subunits or regulators of the cohesin complex. Herein, we report on the clinical and molecular characterization of seven patients with features overlapping with CdLS who were found to carry mutations in chromatin regulators previously associated to other neurodevelopmental disorders that are frequently considered in the differential diagnosis of CdLS. The identified mutations affect the methyltransferase-encoding genes KMT2A and SETD5 and different subunits of the SWI/SNF chromatin-remodeling complex. Complementary to this, a patient with Coffin-Siris syndrome was found to carry a missense substitution in NIPBL. Our findings indicate that mutations in a variety of chromatin-associated factors result in overlapping clinical phenotypes, underscoring the genetic heterogeneity that should be considered when assessing the clinical and molecular diagnosis of neurodevelopmental syndromes. It is clear that emerging molecular mechanisms of chromatin dysregulation are central to understanding the pathogenesis of these clinically overlapping genetic disorders.

Role of Hypomethylating Agents in the Treatment of Bone Marrow Failure

DTIC Science & Technology

2016-10-01

functional studies, as proposed in Aim 2, to find that cells with cohesin gene mutations are sensitized to hypomethylating agents. We used CRISPR /Cas9...screen loss of function mutations in MDS for response to azacitidine. We used CRISPR /Cas9 genome engineering of primary human hematopoietic stem and...investigate whether sites of altered methylation occur at hydroxymethylated loci. We generated isogenic TF-1 cell line clones using CRISPR -Cas9

The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis

PubMed Central

Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J. Julian; Gartner, Anton

2016-01-01

Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis. PMID:27010650

The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis.

PubMed

Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J Julian; Gartner, Anton

2016-03-01

Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis.

Location of RAD51-like protein during meiotic prophase in Eimeria tenella.

PubMed

Del Cacho, Emilio; Gallego, Margarita; Pagés, Marc; Barbero, José Luís; Monteagudo, Luís; Sánchez-Acedo, Caridad

2011-05-31

This study focuses on reporting events in Eimeria tenella oocysts from early to late prophase I in terms of RAD51 protein in association with the synaptonemal complex formed between homologous chromosomes. The aim of the study was the sequential localization of RAD51 protein, which is involved in the repair of double-strand breaks (DSBs) on the eimerian chromosomes as they synapse and desynapse. Structural Maintenance of Chromosome protein SMC3, which plays a role in synaptonemal complex formation, was labeled to identify initiation and progress of chromosome synapsis and desynapsis in parallel with the appearance and disappearance of RAD51 foci. Antibodies directed against RAD51 and cohesin subunit SMC3 proteins were labeled with either fluorescence or colloidal gold to visualize RAD51 protein foci and synaptonemal complexes. RAD51 protein localization during prophase I was studied on meiotic chromosomes spreads obtained from oocysts at different points in time after the start of sporulation. The present findings showed that foci detected with the antibody directed against RAD51 protein first appeared at the pre-leptotene stage before homologous chromosomes began pairing. Subsequently, the foci were detected in association with the lateral elements at the precise sites where synapsis were in progress. These findings lead us to suggest that in E. tenella, homologous chromosome pairing was a DSB-dependent mechanism and reinforced the participation of RAD51 protein in meiotic homology search, alignment and pairing of chromosomes. Copyright © 2010 Elsevier B.V. All rights reserved.

Securin is a target of the UV response pathway in mammalian cells.

PubMed

Romero, Francisco; Gil-Bernabé, Ana M; Sáez, Carmen; Japón, Miguel A; Pintor-Toro, José A; Tortolero, María

2004-04-01

All eukaryotic cells possess elaborate mechanisms to protect genome integrity and ensure survival after DNA damage, ceasing proliferation and granting time for DNA repair. Securin is an inhibitory protein that is bound to a protease called Separase to inhibit sister chromatid separation until the onset of anaphase. At the metaphase-to-anaphase transition, Securin is degraded by the anaphase-promoting complex or cyclosome, and Separase contributes to the release of cohesins from the chromosome, allowing for the segregation of sister chromatids to opposite spindle poles. Here we provide evidence that human Securin (hSecurin) has a novel role in cell cycle arrest after exposure to UV light or ionizing radiation. In fact, irradiation downregulated the level of hSecurin protein, accelerating its degradation via the proteasome and reducing hSecurin mRNA translation, but the presence of hSecurin is necessary for cell proliferation arrest following UV treatment. Moreover, an alteration of UV-induced hSecurin downregulation could lead directly to the accumulation of DNA damage and the subsequent development of malignant tumors.

Securin Is a Target of the UV Response Pathway in Mammalian Cells†

PubMed Central

Romero, Francisco; Gil-Bernabé, Ana M.; Sáez, Carmen; Japón, Miguel A.; Pintor-Toro, José A.; Tortolero, María

2004-01-01

All eukaryotic cells possess elaborate mechanisms to protect genome integrity and ensure survival after DNA damage, ceasing proliferation and granting time for DNA repair. Securin is an inhibitory protein that is bound to a protease called Separase to inhibit sister chromatid separation until the onset of anaphase. At the metaphase-to-anaphase transition, Securin is degraded by the anaphase-promoting complex or cyclosome, and Separase contributes to the release of cohesins from the chromosome, allowing for the segregation of sister chromatids to opposite spindle poles. Here we provide evidence that human Securin (hSecurin) has a novel role in cell cycle arrest after exposure to UV light or ionizing radiation. In fact, irradiation downregulated the level of hSecurin protein, accelerating its degradation via the proteasome and reducing hSecurin mRNA translation, but the presence of hSecurin is necessary for cell proliferation arrest following UV treatment. Moreover, an alteration of UV-induced hSecurin downregulation could lead directly to the accumulation of DNA damage and the subsequent development of malignant tumors. PMID:15024062

Blood-Based Detection of Radiation Exposure in Humans Based on Novel Phospho-Smc1 ELISA

PubMed Central

Ivey, Richard G.; Moore, Heather D.; Voytovich, Uliana J.; Thienes, Cortlandt P.; Lorentzen, Travis D.; Pogosova-Agadjanyan, Era L.; Frayo, Shani; Izaguirre, Venissa K.; Lundberg, Sally J.; Hedin, Lacey; Badiozamani, Kas Ray; Hoofnagle, Andrew N.; Stirewalt, Derek L.; Wang, Pei; Georges, George E.; Gopal, Ajay K.; Paulovich, Amanda G.

2011-01-01

The structural maintenance of chromosome 1 (Smc1) protein is a member of the highly conserved cohesin complex and is involved in sister chromatid cohesion. In response to ionizing radiation, Smc1 is phosphorylated at two sites, Ser-957 and Ser-966, and these phosphorylation events are dependent on the ATM protein kinase. In this study, we describe the generation of two novel ELISAs for quantifying phospho-Smc1Ser-957 and phospho-Smc1Ser-966. Using these novel assays, we quantify the kinetic and biodosimetric responses of human cells of hematological origin, including immortalized cells, as well as both quiescent and cycling primary human PBMC. Additionally, we demonstrate a robust in vivo response for phospho-Smc1Ser-957 and phospho-Smc1Ser-966 in lymphocytes of human patients after therapeutic exposure to ionizing radiation, including total-body irradiation, partial-body irradiation, and internal exposure to 131I. These assays are useful for quantifying the DNA damage response in experimental systems and potentially for the identification of individuals exposed to radiation after a radiological incident. PMID:21388270

From genes to protein mechanics on a chip.

PubMed

Otten, Marcus; Ott, Wolfgang; Jobst, Markus A; Milles, Lukas F; Verdorfer, Tobias; Pippig, Diana A; Nash, Michael A; Gaub, Hermann E

2014-11-01

Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever. The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

Faithful anaphase is ensured by Mis4, a sister chromatid cohesion molecule required in S phase and not destroyed in G1 phase

PubMed Central

Furuya, Kanji; Takahashi, Kohta; Yanagida, Mitsuhiro

1998-01-01

The loss of sister chromatid cohesion triggers anaphase spindle movement. The budding yeast Mcd1/Scc1 protein, called cohesin, is required for associating chromatids, and proteins homologous to it exist in a variety of eukaryotes. Mcd1/Scc1 is removed from chromosomes in anaphase and degrades in G1. We show that the fission yeast protein, Mis4, which is required for equal sister chromatid separation in anaphase is a different chromatid cohesion molecule that behaves independent of cohesin and is conserved from yeast to human. Its inactivation in G1 results in cell lethality in S phase and subsequent premature sister chromatid separation. Inactivation in G2 leads to cell death in subsequent metaphase–anaphase progression but missegregation occurs only in the next round of mitosis. Mis4 is not essential for condensation, nor does it degrade in G1. Rather, it associates with chromosomes in a punctate fashion throughout the cell cycle. mis4 mutants are hypersensitive to hydroxyurea (HU) and UV irradiation but retain the ability to restrain cell cycle progression when damaged or sustaining a block to replication. The mis4 mutation results in synthetic lethality with a DNA ligase mutant. Mis4 may form a stable link between chromatids in S phase that is split rather than removed in anaphase. PMID:9808627

Elevated intracellular pH appears in aged oocytes and causes oocyte aneuploidy associated with the loss of cohesion in mice

PubMed Central

Cheng, Jin-Mei; Li, Jian; Tang, Ji-Xin; Chen, Su-Ren; Deng, Shou-Long; Jin, Cheng; Zhang, Yan; Wang, Xiu-Xia; Zhou, Chen-Xi; Liu, Yi-Xun

2016-01-01

ABSTRACT Increases in the aneuploidy rate caused by the deterioration of cohesion with increasing maternal age have been well documented. However, the molecular mechanism for the loss of cohesion in aged oocytes remains unknown. In this study, we found that intracellular pH (pHi) was elevated in aged oocytes, which might disturb the structure of the cohesin ring to induce aneuploidy. We observed for the first time that full-grown germinal vesicle (GV) oocytes displayed an increase in pHi with advancing age in CD1 mice. Furthermore, during the in vitro oocyte maturation process, the pHi was maintained at a high level, up to ∼7.6, in 12-month-old mice. Normal pHi is necessary to maintain protein localization and function. Thus, we put forward a hypothesis that the elevated oocyte pHi might be related to the loss of cohesion and the increased aneuploidy in aged mice. Through the in vitro alkalinization treatment of young oocytes, we observed that the increased pHi caused an increase in the aneuploidy rate and the sister inter-kinetochore (iKT) distance associated with the strength of cohesion and caused a decline in the cohesin subunit SMC3 protein level. Young oocytes with elevated pHi exhibited substantially the increase in chromosome misalignment. PMID:27472084

The role of meiotic cohesin REC8 in chromosome segregation in gamma irradiation-induced endopolyploid tumour cells.

PubMed

Erenpreisa, Jekaterina; Cragg, Mark S; Salmina, Kristine; Hausmann, Michael; Scherthan, Harry

2009-09-10

Escape from mitotic catastrophe and generation of endopolyploid tumour cells (ETCs) represents a potential survival strategy of tumour cells in response to genotoxic treatments. ETCs that resume the mitotic cell cycle have reduced ploidy and are often resistant to these treatments. In search for a mechanism for genome reduction, we previously observed that ETCs express meiotic proteins among which REC8 (a meiotic cohesin component) is of particular interest, since it favours reductional cell division in meiosis. In the present investigation, we induced endopolyploidy in p53-dysfunctional human tumour cell lines (Namalwa, WI-L2-NS, HeLa) by gamma irradiation, and analysed the sub-cellular localisation of REC8 in the resulting ETCs. We observed by RT-PCR and Western blot that REC8 is constitutively expressed in these tumour cells, along with SGOL1 and SGOL2, and that REC8 becomes modified after irradiation. REC8 localised to paired sister centromeres in ETCs, the former co-segregating to opposite poles. Furthermore, REC8 localised to the centrosome of interphase ETCs and to the astral poles in anaphase cells where it colocalised with the microtubule-associated protein NuMA. Altogether, our observations indicate that radiation-induced ETCs express features of meiotic cell divisions and that these may facilitate chromosome segregation and genome reduction.

TAD-free analysis of architectural proteins and insulators.

PubMed

Mourad, Raphaël; Cuvier, Olivier

2018-03-16

The three-dimensional (3D) organization of the genome is intimately related to numerous key biological functions including gene expression and DNA replication regulations. The mechanisms by which molecular drivers functionally organize the 3D genome, such as topologically associating domains (TADs), remain to be explored. Current approaches consist in assessing the enrichments or influences of proteins at TAD borders. Here, we propose a TAD-free model to directly estimate the blocking effects of architectural proteins, insulators and DNA motifs on long-range contacts, making the model intuitive and biologically meaningful. In addition, the model allows analyzing the whole Hi-C information content (2D information) instead of only focusing on TAD borders (1D information). The model outperforms multiple logistic regression at TAD borders in terms of parameter estimation accuracy and is validated by enhancer-blocking assays. In Drosophila, the results support the insulating role of simple sequence repeats and suggest that the blocking effects depend on the number of repeats. Motif analysis uncovered the roles of the transcriptional factors pannier and tramtrack in blocking long-range contacts. In human, the results suggest that the blocking effects of the well-known architectural proteins CTCF, cohesin and ZNF143 depend on the distance between loci, where each protein may participate at different scales of the 3D chromatin organization.

Improved transcription and translation with L-leucine stimulation of mTORC1 in Roberts syndrome.

PubMed

Xu, Baoshan; Gogol, Madelaine; Gaudenz, Karin; Gerton, Jennifer L

2016-01-05

Roberts syndrome (RBS) is a human developmental disorder caused by mutations in the cohesin acetyltransferase ESCO2. We previously reported that mTORC1 signaling was depressed and overall translation was reduced in RBS cells and zebrafish models for RBS. Treatment of RBS cells and zebrafish RBS models with L-leucine partially rescued mTOR function and protein synthesis, correlating with increased cell division and improved development. In this study, we use RBS cells to model mTORC1 repression and analyze transcription and translation with ribosome profiling to determine gene-level effects of L-leucine. L-leucine treatment partially rescued translational efficiency of ribosomal subunits, translation initiation factors, snoRNA production, and mitochondrial function in RBS cells, consistent with these processes being mTORC1 controlled. In contrast, other genes are differentially expressed independent of L-leucine treatment, including imprinted genes such as H19 and GTL2, miRNAs regulated by GTL2, HOX genes, and genes in nucleolar associated domains. Our study distinguishes between gene expression changes in RBS cells that are TOR dependent and those that are independent. Some of the TOR independent gene expression changes likely reflect the architectural role of cohesin in chromatin looping and gene expression. This study reveals the dramatic rescue effects of L-leucine stimulation of mTORC1 in RBS cells and supports that normal gene expression and translation requires ESCO2 function.

Cornelia de Lange syndrome: Congenital heart disease in 149 patients.

PubMed

Ayerza Casas, Ariadna; Puisac Uriol, Beatriz; Teresa Rodrigo, María Esperanza; Hernández Marcos, María; Ramos Fuentes, Feliciano J; Pie Juste, Juan

2017-10-11

Cornelia de Lange syndrome (CdLS) is produced by mutations in genes that encode regulatory or structural proteins of the cohesin complex. Congenital heart disease (CHD) is not a major criterion of the disease, but it affects many individuals. The objective of this study was to study the incidence and type of CHD in patients with CdLS. Cardiological findings were evaluated in 149 patients with CdLS and their possible relationship with clinical and genetic variables. A percentage of 34.9 had CHD (septal defects 50%, pulmonary stenosis 27%, aortic coarctation 9.6%). The presence of CHD was related with neonatal hospitalisation (P=.04), hearing loss (P=.002), mortality (P=.09) and lower hyperactivity (P=.02), it being more frequent in HDAC8+ patients (60%), followed by NIPBL+ (33%) and SMC1A+ (28.5%). While septal defects predominate in NIPBL+, pulmonary stenosis is more common in HDAC8+. Patients with CdLS have a high incidence of CHD, which varies according to the affected gene, the most frequent findings being septal defects and pulmonary stenosis. Perform a cardiologic study in all these patients is suggested. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

Bone marrow fibrosis in myelodysplastic syndromes: a prospective evaluation including mutational analysis

PubMed Central

Ramos, Fernando; Robledo, Cristina; Izquierdo-García, Francisco Miguel; Suárez-Vilela, Dimas; Benito, Rocío; Fuertes, Marta; Insunza, Andrés; Barragán, Eva; del Rey, Mónica; de Morales, José María García-Ruiz; Tormo, Mar; Salido, Eduardo; Zamora, Lurdes; Pedro, Carmen; Sánchez-del-Real, Javier; Díez-Campelo, María; del Cañizo, Consuelo; Sanz, Guillermo F.; Hernández-Rivas, Jesús María

2016-01-01

The biological and molecular events that underlie bone marrow fibrosis in patients with myelodysplastic syndromes are poorly understood, and its prognostic role in the era of the Revised International Prognostic Scoring System (IPSS-R) is not yet fully determined. We have evaluated the clinical and biological events that underlie bone marrow fibrotic changes, as well as its prognostic role, in a well-characterized prospective patient cohort (n=77) of primary MDS patients. The degree of marrow fibrosis was linked to parameters of erythropoietic failure, marrow cellularity, p53 protein accumulation, WT1 gene expression, and serum levels of CXCL9 and CXCL10, but not to other covariates including the IPSS-R score. The presence of bone marrow fibrosis grade 2 or higher was associated with the presence of mutations in cohesin complex genes (31.5% vs. 5.4%, p=0.006). By contrast, mutations in CALR, JAK2, PDGFRA, PDGFRB, and TP53 were very rare. Survival analysis showed that marrow fibrosis grade 2 or higher was a relevant significant predictor for of overall survival, and independent of age, performance status, and IPSS-R score in multivariate analysis. PMID:27127180

Distinct TERB1 Domains Regulate Different Protein Interactions in Meiotic Telomere Movement.

PubMed

Zhang, Jingjing; Tu, Zhaowei; Watanabe, Yoshinori; Shibuya, Hiroki

2017-11-14

Meiotic telomeres attach to the nuclear envelope (NE) and drive the chromosome movement required for the pairing of homologous chromosomes. The meiosis-specific telomere proteins TERB1, TERB2, and MAJIN are required to regulate these events, but their assembly processes are largely unknown. Here, we developed a germ-cell-specific knockout mouse of the canonical telomere-binding protein TRF1 and revealed an essential role for TRF1 in directing the assembly of TERB1-TERB2-MAJIN. Further, we identified a TERB2 binding (T2B) domain in TERB1 that is dispensable for the TRF1-TERB1 interaction but is essential for the subsequent TERB1-TERB2 interaction and therefore for telomere attachment to the NE. Meanwhile, cohesin recruitment at telomeres, which is required for efficient telomere movement, is mediated by the MYB-like domain of TERB1, but not by TERB2-MAJIN. Our results reveal distinct protein interactions through various domains of TERB1, which enable the sequential assembly of the meiotic telomere complex for their movements. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

From Genes to Protein Mechanics on a Chip

PubMed Central

Milles, Lukas F.; Verdorfer, Tobias; Pippig, Diana A.; Nash, Michael A.; Gaub, Hermann E.

2014-01-01

Single-molecule force spectroscopy enables mechanical testing of individual proteins, however low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip protein expression and measurement of single-molecule mechanical properties. We constructed microarrays of proteins covalently attached to a chip surface, and found that a single cohesin-modified cantilever that bound to the terminal dockerin-tag of each protein remained stable over thousands of pulling cycles. The ability to synthesize and mechanically probe protein libraries presents new opportunities for high-throughput mechanical phenotyping. PMID:25194847

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription

PubMed Central

Stefanovsky, Victor Y.; Tremblay, Michel G.; Lindsay, Helen; Robinson, Mark D.

2017-01-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes. PMID:28715449

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

PubMed

Herdman, Chelsea; Mars, Jean-Clement; Stefanovsky, Victor Y; Tremblay, Michel G; Sabourin-Felix, Marianne; Lindsay, Helen; Robinson, Mark D; Moss, Tom

2017-07-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.

PyContact: Rapid, Customizable, and Visual Analysis of Noncovalent Interactions in MD Simulations.

PubMed

Scheurer, Maximilian; Rodenkirch, Peter; Siggel, Marc; Bernardi, Rafael C; Schulten, Klaus; Tajkhorshid, Emad; Rudack, Till

2018-02-06

Molecular dynamics (MD) simulations have become ubiquitous in all areas of life sciences. The size and model complexity of MD simulations are rapidly growing along with increasing computing power and improved algorithms. This growth has led to the production of a large amount of simulation data that need to be filtered for relevant information to address specific biomedical and biochemical questions. One of the most relevant molecular properties that can be investigated by all-atom MD simulations is the time-dependent evolution of the complex noncovalent interaction networks governing such fundamental aspects as molecular recognition, binding strength, and mechanical and structural stability. Extracting, evaluating, and visualizing noncovalent interactions is a key task in the daily work of structural biologists. We have developed PyContact, an easy-to-use, highly flexible, and intuitive graphical user interface-based application, designed to provide a toolkit to investigate biomolecular interactions in MD trajectories. PyContact is designed to facilitate this task by enabling identification of relevant noncovalent interactions in a comprehensible manner. The implementation of PyContact as a standalone application enables rapid analysis and data visualization without any additional programming requirements, and also preserves full in-program customization and extension capabilities for advanced users. The statistical analysis representation is interactively combined with full mapping of the results on the molecular system through the synergistic connection between PyContact and VMD. We showcase the capabilities and scientific significance of PyContact by analyzing and visualizing in great detail the noncovalent interactions underlying the ion permeation pathway of the human P2X 3 receptor. As a second application, we examine the protein-protein interaction network of the mechanically ultrastable cohesin-dockering complex. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Functional analysis of sporophytic transcripts repressed by the female gametophyte in the ovule of Arabidopsis thaliana.

PubMed

Armenta-Medina, Alma; Huanca-Mamani, Wilson; Sanchez-León, Nidia; Rodríguez-Arévalo, Isaac; Vielle-Calzada, Jean-Philippe

2013-01-01

To investigate the genetic and molecular regulation that the female gametophyte could exert over neighboring sporophytic regions of the ovule, we performed a quantitative comparison of global expression in wild-type and nozzle/sporocyteless (spl) ovules of Arabidopsis thaliana (Arabidopsis), using Massively Parallel Signature Sequencing (MPSS). This comparison resulted in 1517 genes showing at least 3-fold increased expression in ovules lacking a female gametophyte, including those encoding 89 transcription factors, 50 kinases, 25 proteins containing a RNA-recognition motif (RRM), and 20 WD40 repeat proteins. We confirmed that eleven of these genes are either preferentially expressed or exclusive of spl ovules lacking a female gametophyte as compared to wild-type, and showed that six are also upregulated in determinant infertile1 (dif1), a meiotic mutant affected in a REC8-like cohesin that is also devoided of female gametophytes. The sporophytic misexpression of IOREMPTE, a WD40/transducin repeat gene that is preferentially expressed in the L1 layer of spl ovules, caused the arrest of female gametogenesis after differentiation of a functional megaspore. Our results show that in Arabidopsis, the sporophytic-gametophytic cross talk includes a negative regulation of the female gametophyte over specific genes that are detrimental for its growth and development, demonstrating its potential to exert a repressive control over neighboring regions in the ovule.

Sequence Complexity of Chromosome 3 in Caenorhabditis elegans

PubMed Central

Pierro, Gaetano

2012-01-01

The nucleotide sequences complexity in chromosome 3 of Caenorhabditis elegans (C. elegans) is studied. The complexity of these sequences is compared with some random sequences. Moreover, by using some parameters related to complexity such as fractal dimension and frequency, indicator matrix is given a first classification of sequences of C. elegans. In particular, the sequences with highest and lowest fractal value are singled out. It is shown that the intrinsic nature of the low fractal dimension sequences has many common features with the random sequences. PMID:22919380

Not all transmembrane helices are born equal: Towards the extension of the sequence homology concept to membrane proteins

PubMed Central

2011-01-01

Background Sequence homology considerations widely used to transfer functional annotation to uncharacterized protein sequences require special precautions in the case of non-globular sequence segments including membrane-spanning stretches composed of non-polar residues. Simple, quantitative criteria are desirable for identifying transmembrane helices (TMs) that must be included into or should be excluded from start sequence segments in similarity searches aimed at finding distant homologues. Results We found that there are two types of TMs in membrane-associated proteins. On the one hand, there are so-called simple TMs with elevated hydrophobicity, low sequence complexity and extraordinary enrichment in long aliphatic residues. They merely serve as membrane-anchoring device. In contrast, so-called complex TMs have lower hydrophobicity, higher sequence complexity and some functional residues. These TMs have additional roles besides membrane anchoring such as intra-membrane complex formation, ligand binding or a catalytic role. Simple and complex TMs can occur both in single- and multi-membrane-spanning proteins essentially in any type of topology. Whereas simple TMs have the potential to confuse searches for sequence homologues and to generate unrelated hits with seemingly convincing statistical significance, complex TMs contain essential evolutionary information. Conclusion For extending the homology concept onto membrane proteins, we provide a necessary quantitative criterion to distinguish simple TMs (and a sufficient criterion for complex TMs) in query sequences prior to their usage in homology searches based on assessment of hydrophobicity and sequence complexity of the TM sequence segments. Reviewers This article was reviewed by Shamil Sunyaev, L. Aravind and Arcady Mushegian. PMID:22024092

Method and apparatus for enhanced sequencing of complex molecules using surface-induced dissociation in conjunction with mass spectrometric analysis

DOEpatents

Laskin, Julia [Richland, WA; Futrell, Jean H [Richland, WA

2008-04-29

The invention relates to a method and apparatus for enhanced sequencing of complex molecules using surface-induced dissociation (SID) in conjunction with mass spectrometric analysis. Results demonstrate formation of a wide distribution of structure-specific fragments having wide sequence coverage useful for sequencing and identifying the complex molecules.

A single mutation in Securin induces chromosomal instability and enhances cell invasion.

PubMed

Mora-Santos, Mar; Castilla, Carolina; Herrero-Ruiz, Joaquín; Giráldez, Servando; Limón-Mortés, M Cristina; Sáez, Carmen; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

2013-01-01

Pituitary tumour transforming gene (pttg1) encodes Securin, a protein involved in the inhibition of sister chromatid separation binding to Separase until the onset of anaphase. Separase is a cysteine-protease that degrades cohesin to segregate the sister chromatids to opposite poles of the cell. The amount of Securin is strongly regulated because it should allow Separase activation when it is degraded by the anaphase promoting complex/cyclosome, should arrest the cell cycle after DNA damage, when it is degraded through SKP1-CUL1-βTrCP ubiquitin ligase, and its overexpression induces tumour formation and correlates with metastasis in multiple tumours. Securin is a phosphoprotein that contains 32 potentially phosphorylatable residues. We mutated and analysed most of them, and found a single mutant, hSecT60A, that showed enhanced oncogenic properties. Our fluorescence activated cell sorting analysis, fluorescence in situ hybridisation assays, tumour cell migration and invasion experiments and gene expression by microarrays analysis clearly involved hSecT60A in chromosomal instability and cell invasion. These results show, for the first time, that a single mutation in pttg1 is sufficient to trigger the oncogenic properties of Securin. The finding of this point mutation in patients might be used as an effective strategy for early detection of cancer. Copyright © 2012 Elsevier Ltd. All rights reserved.

Meiotic double-strand breaks at the interface of chromosome movement, chromosome remodeling, and reductional division

PubMed Central

Storlazzi, Aurora; Tessé, Sophie; Gargano, Silvana; James, Françoise; Kleckner, Nancy; Zickler, Denise

2003-01-01

Chromosomal processes related to formation and function of meiotic chiasmata have been analyzed in Sordaria macrospora. Double-strand breaks (DSBs), programmed or γ-rays-induced, are found to promote four major events beyond recombination and accompanying synaptonemal complex formation: (1) juxtaposition of homologs from long-distance interactions to close presynaptic coalignment at midleptotene; (2) structural destabilization of chromosomes at leptotene/zygotene, including sister axis separation and fracturing, as revealed in a mutant altered in the conserved, axis-associated cohesin-related protein Spo76/Pds5p; (3) exit from the bouquet stage, with accompanying global chromosome movements, at zygotene/pachytene (bouquet stage exit is further found to be a cell-wide regulatory transition and DSB transesterase Spo11p is suggested to have a new noncatalytic role in this transition); (4) normal occurrence of both meiotic divisions, including normal sister separation. Functional interactions between DSBs and the spo76-1 mutation suggest that Spo76/Pds5p opposes local destabilization of axes at developing chiasma sites and raise the possibility of a regulatory mechanism that directly monitors the presence of chiasmata at metaphase I. Local chromosome remodeling at DSB sites appears to trigger an entire cascade of chromosome movements, morphogenetic changes, and regulatory effects that are superimposed upon a foundation of DSB-independent processes. PMID:14563680

Do Exogenous DNA Double-Strand Breaks Change Incomplete Synapsis and Chiasma Localization in the Grasshopper Stethophyma grossum?

PubMed Central

2016-01-01

Meiotic recombination occurs as a programmed event that initiates by the formation of DNA double-strand breaks (DSBs) that give rise to the formation of crossovers that are observed as chiasmata. Chiasmata are essential for the accurate chromosome segregation and the generation of new combinations of parental alleles. Some treatments that provoke exogenous DSBs also lead to alterations in the recombination pattern of some species in which full homologous synapsis is achieved at pachytene. We have carried out a similar approach in males of the grasshopper Stethophyma grossum, whose homologues show incomplete synapsis and proximal chiasma localization. After irradiating males with γ rays we have studied the distribution of both the histone variant γ-H2AX and the recombinase RAD51. These proteins are cytological markers of DSBs at early prophase I. We have inferred synaptonemal complex (SC) formation via identification of SMC3 and RAD 21 cohesin subunits. Whereas thick and thin SMC3 filaments would correspond to synapsed and unsynapsed regions, the presence of RAD21 is only restricted to synapsed regions. Results show that irradiated spermatocytes maintain restricted synapsis between homologues. However, the frequency and distribution of chiasmata in metaphase I bivalents is slightly changed and quadrivalents were also observed. These results could be related to the singular nuclear polarization displayed by the spermatocytes of this species. PMID:28005992

Sequence Complexity of Amyloidogenic Regions in Intrinsically Disordered Human Proteins

PubMed Central

Das, Swagata; Pal, Uttam; Das, Supriya; Bagga, Khyati; Roy, Anupam; Mrigwani, Arpita; Maiti, Nakul C.

2014-01-01

An amyloidogenic region (AR) in a protein sequence plays a significant role in protein aggregation and amyloid formation. We have investigated the sequence complexity of AR that is present in intrinsically disordered human proteins. More than 80% human proteins in the disordered protein databases (DisProt+IDEAL) contained one or more ARs. With decrease of protein disorder, AR content in the protein sequence was decreased. A probability density distribution analysis and discrete analysis of AR sequences showed that ∼8% residue in a protein sequence was in AR and the region was in average 8 residues long. The residues in the AR were high in sequence complexity and it seldom overlapped with low complexity regions (LCR), which was largely abundant in disorder proteins. The sequences in the AR showed mixed conformational adaptability towards α-helix, β-sheet/strand and coil conformations. PMID:24594841

An efficient approach to BAC based assembly of complex genomes.

PubMed

Visendi, Paul; Berkman, Paul J; Hayashi, Satomi; Golicz, Agnieszka A; Bayer, Philipp E; Ruperao, Pradeep; Hurgobin, Bhavna; Montenegro, Juan; Chan, Chon-Kit Kenneth; Staňková, Helena; Batley, Jacqueline; Šimková, Hana; Doležel, Jaroslav; Edwards, David

2016-01-01

There has been an exponential growth in the number of genome sequencing projects since the introduction of next generation DNA sequencing technologies. Genome projects have increasingly involved assembly of whole genome data which produces inferior assemblies compared to traditional Sanger sequencing of genomic fragments cloned into bacterial artificial chromosomes (BACs). While whole genome shotgun sequencing using next generation sequencing (NGS) is relatively fast and inexpensive, this method is extremely challenging for highly complex genomes, where polyploidy or high repeat content confounds accurate assembly, or where a highly accurate 'gold' reference is required. Several attempts have been made to improve genome sequencing approaches by incorporating NGS methods, to variable success. We present the application of a novel BAC sequencing approach which combines indexed pools of BACs, Illumina paired read sequencing, a sequence assembler specifically designed for complex BAC assembly, and a custom bioinformatics pipeline. We demonstrate this method by sequencing and assembling BAC cloned fragments from bread wheat and sugarcane genomes. We demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes.

ComplexContact: a web server for inter-protein contact prediction using deep learning.

PubMed

Zeng, Hong; Wang, Sheng; Zhou, Tianming; Zhao, Feifeng; Li, Xiufeng; Wu, Qing; Xu, Jinbo

2018-05-22

ComplexContact (http://raptorx2.uchicago.edu/ComplexContact/) is a web server for sequence-based interfacial residue-residue contact prediction of a putative protein complex. Interfacial residue-residue contacts are critical for understanding how proteins form complex and interact at residue level. When receiving a pair of protein sequences, ComplexContact first searches for their sequence homologs and builds two paired multiple sequence alignments (MSA), then it applies co-evolution analysis and a CASP-winning deep learning (DL) method to predict interfacial contacts from paired MSAs and visualizes the prediction as an image. The DL method was originally developed for intra-protein contact prediction and performed the best in CASP12. Our large-scale experimental test further shows that ComplexContact greatly outperforms pure co-evolution methods for inter-protein contact prediction, regardless of the species.

Study of base pair mutations in proline-rich homeodomain (PRH)-DNA complexes using molecular dynamics.

PubMed

Jalili, Seifollah; Karami, Leila; Schofield, Jeremy

2013-06-01

Proline-rich homeodomain (PRH) is a regulatory protein controlling transcription and gene expression processes by binding to the specific sequence of DNA, especially to the sequence 5'-TAATNN-3'. The impact of base pair mutations on the binding between the PRH protein and DNA is investigated using molecular dynamics and free energy simulations to identify DNA sequences that form stable complexes with PRH. Three 20-ns molecular dynamics simulations (PRH-TAATTG, PRH-TAATTA and PRH-TAATGG complexes) in explicit solvent water were performed to investigate three complexes structurally. Structural analysis shows that the native TAATTG sequence forms a complex that is more stable than complexes with base pair mutations. It is also observed that upon mutation, the number and occupancy of the direct and water-mediated hydrogen bonds decrease. Free energy calculations performed with the thermodynamic integration method predict relative binding free energies of 0.64 and 2 kcal/mol for GC to AT and TA to GC mutations, respectively, suggesting that among the three DNA sequences, the PRH-TAATTG complex is more stable than the two mutated complexes. In addition, it is demonstrated that the stability of the PRH-TAATTA complex is greater than that of the PRH-TAATGG complex.

Detection and isolation of nucleic acid sequences using competitive hybridization probes

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

1997-01-01

A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided.

Detection and isolation of nucleic acid sequences using competitive hybridization probes

DOEpatents

Lucas, J.N.; Straume, T.; Bogen, K.T.

1997-04-01

A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided. 7 figs.

Encoding and choice in the task span paradigm.

PubMed

Reiman, Kaitlin M; Weaver, Starla M; Arrington, Catherine M

2015-03-01

Cognitive control during sequences of planned behaviors requires both plan-level processes such as generating, maintaining, and monitoring the plan, as well as task-level processes such as selecting, establishing and implementing specific task sets. The task span paradigm (Logan in J Exp Psychol Gen 133:218-236, 2004) combines two common cognitive control paradigms, task switching and working memory span, to investigate the integration of plan-level and task-level processes during control of sequential behavior. The current study expands past task span research to include measures of encoding processes and choice behavior with volitional sequence generation, using the standard task span as well as a novel voluntary task span paradigm. In two experiments, we consider how sequence complexity, defined separately for plan-level and task-level complexity, influences sequence encoding (Experiment 1), sequence choice (Experiment 2), sequence memory, and task performance of planned sequences of action. Results indicate that participants were sensitive to sequence complexity, but that different aspects of behavior are most strongly influenced by different types of complexity. Hierarchical complexity at the plan level best predicts voluntary sequence generation and memory; while switch frequency at the task level best predicts encoding of externally defined sequences and task performance. Furthermore, performance RTs were similar for externally and internally defined plans, whereas memory was improved for internally defined sequences. Finally, participants demonstrated a significant sequence choice bias in the voluntary task span. Consistent with past research on choice behavior, volitional selection of plans was markedly influenced by both the ease of memory and performance.

BAC-pool 454-sequencing: A rapid and efficient approach to sequence complex tetraploid cotton genomes

USDA-ARS?s Scientific Manuscript database

New and emerging next generation sequencing technologies have been promising in reducing sequencing costs, but not significantly for complex polyploid plant genomes such as cotton. Large and highly repetitive genome of G. hirsutum (~2.5GB) is less amenable and cost-intensive with traditional BAC-by...

Tapping the promise of genomics in species with complex, nonmodel genomes.

PubMed

Hirsch, Candice N; Buell, C Robin

2013-01-01

Genomics is enabling a renaissance in all disciplines of plant biology. However, many plant genomes are complex and remain recalcitrant to current genomic technologies. The complexities of these nonmodel plant genomes are attributable to gene and genome duplication, heterozygosity, ploidy, and/or repetitive sequences. Methods are available to simplify the genome and reduce these barriers, including inbreeding and genome reduction, making these species amenable to current sequencing and assembly methods. Some, but not all, of the complexities in nonmodel genomes can be bypassed by sequencing the transcriptome rather than the genome. Additionally, comparative genomics approaches, which leverage phylogenetic relatedness, can aid in the interpretation of complex genomes. Although there are limitations in accessing complex nonmodel plant genomes using current sequencing technologies, genome manipulation and resourceful analyses can allow access to even the most recalcitrant plant genomes.

Complexity: an internet resource for analysis of DNA sequence complexity

PubMed Central

Orlov, Y. L.; Potapov, V. N.

2004-01-01

The search for DNA regions with low complexity is one of the pivotal tasks of modern structural analysis of complete genomes. The low complexity may be preconditioned by strong inequality in nucleotide content (biased composition), by tandem or dispersed repeats or by palindrome-hairpin structures, as well as by a combination of all these factors. Several numerical measures of textual complexity, including combinatorial and linguistic ones, together with complexity estimation using a modified Lempel–Ziv algorithm, have been implemented in a software tool called ‘Complexity’ (http://wwwmgs.bionet.nsc.ru/mgs/programs/low_complexity/). The software enables a user to search for low-complexity regions in long sequences, e.g. complete bacterial genomes or eukaryotic chromosomes. In addition, it estimates the complexity of groups of aligned sequences. PMID:15215465

Improving performance of DS-CDMA systems using chaotic complex Bernoulli spreading codes

NASA Astrophysics Data System (ADS)

Farzan Sabahi, Mohammad; Dehghanfard, Ali

2014-12-01

The most important goal of spreading spectrum communication system is to protect communication signals against interference and exploitation of information by unintended listeners. In fact, low probability of detection and low probability of intercept are two important parameters to increase the performance of the system. In Direct Sequence Code Division Multiple Access (DS-CDMA) systems, these properties are achieved by multiplying the data information in spreading sequences. Chaotic sequences, with their particular properties, have numerous applications in constructing spreading codes. Using one-dimensional Bernoulli chaotic sequence as spreading code is proposed in literature previously. The main feature of this sequence is its negative auto-correlation at lag of 1, which with proper design, leads to increase in efficiency of the communication system based on these codes. On the other hand, employing the complex chaotic sequences as spreading sequence also has been discussed in several papers. In this paper, use of two-dimensional Bernoulli chaotic sequences is proposed as spreading codes. The performance of a multi-user synchronous and asynchronous DS-CDMA system will be evaluated by applying these sequences under Additive White Gaussian Noise (AWGN) and fading channel. Simulation results indicate improvement of the performance in comparison with conventional spreading codes like Gold codes as well as similar complex chaotic spreading sequences. Similar to one-dimensional Bernoulli chaotic sequences, the proposed sequences also have negative auto-correlation. Besides, construction of complex sequences with lower average cross-correlation is possible with the proposed method.

Regulation of Centromere Localization of the Drosophila Shugoshin MEI-S332 and Sister-Chromatid Cohesion in Meiosis

PubMed Central

Nogueira, Cristina; Kashevsky, Helena; Pinto, Belinda; Clarke, Astrid; Orr-Weaver, Terry L.

2014-01-01

The Shugoshin (Sgo) protein family helps to ensure proper chromosome segregation by protecting cohesion at the centromere by preventing cleavage of the cohesin complex. Some Sgo proteins also influence other aspects of kinetochore-microtubule attachments. Although many Sgo members require Aurora B kinase to localize to the centromere, factors controlling delocalization are poorly understood and diverse. Moreover, it is not clear how Sgo function is inactivated and whether this is distinct from delocalization. We investigated these questions in Drosophila melanogaster, an organism with superb chromosome cytology to monitor Sgo localization and quantitative assays to test its function in sister-chromatid segregation in meiosis. Previous research showed that in mitosis in cell culture, phosphorylation of the Drosophila Sgo, MEI-S332, by Aurora B promotes centromere localization, whereas Polo phosphorylation promotes delocalization. These studies also suggested that MEI-S332 can be inactivated independently of delocalization, a conclusion supported here by localization and function studies in meiosis. Phosphoresistant and phosphomimetic mutants for the Aurora B and Polo phosphorylation sites were examined for effects on MEI-S332 localization and chromosome segregation in meiosis. Strikingly, MEI-S332 with a phosphomimetic mutation in the Aurora B phosphorylation site prematurely dissociates from the centromeres in meiosis I. Despite the absence of MEI-S332 on meiosis II centromeres in male meiosis, sister chromatids segregate normally, demonstrating that detectable levels of this Sgo are not essential for chromosome congression, kinetochore biorientation, or spindle assembly. PMID:25081981

Not just gene expression: 3D implications of chromatin modifications during sexual plant reproduction.

PubMed

Dukowic-Schulze, Stefanie; Liu, Chang; Chen, Changbin

2018-01-01

DNA methylation and histone modifications are epigenetic changes on a DNA molecule that alter the three-dimensional (3D) structure locally as well as globally, impacting chromatin looping and packaging on a larger scale. Epigenetic marks thus inform higher-order chromosome organization and placement in the nucleus. Conventional epigenetic marks are joined by chromatin modifiers like cohesins, condensins and membrane-anchoring complexes to support particularly 3D chromosome organization. The most popular consequences of epigenetic modifications are gene expression changes, but chromatin modifications have implications beyond this, particularly in actively dividing cells and during sexual reproduction. In this opinion paper, we will focus on epigenetic mechanisms and chromatin modifications during meiosis as part of plant sexual reproduction where 3D management of chromosomes and re-organization of chromatin are defining features and prime tasks in reproductive cells, not limited to modulating gene expression. Meiotic chromosome organization, pairing and synapsis of homologous chromosomes as well as distribution of meiotic double-strand breaks and resulting crossovers are presumably highly influenced by epigenetic mechanisms. Special mobile small RNAs have been described in anthers, where these so-called phasiRNAs seem to direct DNA methylation in meiotic cells. Intriguingly, many of the mentioned developmental processes make use of epigenetic changes and small RNAs in a manner other than gene expression changes. Widening our approaches and opening our mind to thinking three-dimensionally regarding epigenetics in plant development holds high promise for new discoveries and could give us a boost for further knowledge.

Method for identifying and quantifying nucleic acid sequence aberrations

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

1998-01-01

A method for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe.

Method for identifying and quantifying nucleic acid sequence aberrations

DOEpatents

Lucas, J.N.; Straume, T.; Bogen, K.T.

1998-07-21

A method is disclosed for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe. 11 figs.

Assessing randomness and complexity in human motion trajectories through analysis of symbolic sequences

PubMed Central

Peng, Zhen; Genewein, Tim; Braun, Daniel A.

2014-01-01

Complexity is a hallmark of intelligent behavior consisting both of regular patterns and random variation. To quantitatively assess the complexity and randomness of human motion, we designed a motor task in which we translated subjects' motion trajectories into strings of symbol sequences. In the first part of the experiment participants were asked to perform self-paced movements to create repetitive patterns, copy pre-specified letter sequences, and generate random movements. To investigate whether the degree of randomness can be manipulated, in the second part of the experiment participants were asked to perform unpredictable movements in the context of a pursuit game, where they received feedback from an online Bayesian predictor guessing their next move. We analyzed symbol sequences representing subjects' motion trajectories with five common complexity measures: predictability, compressibility, approximate entropy, Lempel-Ziv complexity, as well as effective measure complexity. We found that subjects' self-created patterns were the most complex, followed by drawing movements of letters and self-paced random motion. We also found that participants could change the randomness of their behavior depending on context and feedback. Our results suggest that humans can adjust both complexity and regularity in different movement types and contexts and that this can be assessed with information-theoretic measures of the symbolic sequences generated from movement trajectories. PMID:24744716

Compositional segmentation and complexity measurement in stock indices

NASA Astrophysics Data System (ADS)

Wang, Haifeng; Shang, Pengjian; Xia, Jianan

2016-01-01

In this paper, we introduce a complexity measure based on the entropic segmentation called sequence compositional complexity (SCC) into the analysis of financial time series. SCC was first used to deal directly with the complex heterogeneity in nonstationary DNA sequences. We already know that SCC was found to be higher in sequences with long-range correlation than those with low long-range correlation, especially in the DNA sequences. Now, we introduce this method into financial index data, subsequently, we find that the values of SCC of some mature stock indices, such as S & P 500 (simplified with S & P in the following) and HSI, are likely to be lower than the SCC value of Chinese index data (such as SSE). What is more, we find that, if we classify the indices with the method of SCC, the financial market of Hong Kong has more similarities with mature foreign markets than Chinese ones. So we believe that a good correspondence is found between the SCC of the index sequence and the complexity of the market involved.

Kit for detecting nucleic acid sequences using competitive hybridization probes

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

2001-01-01

A kit is provided for detecting a target nucleic acid sequence in a sample, the kit comprising: a first hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the first hybridization probe including a first complexing agent for forming a binding pair with a second complexing agent; and a second hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the first hybridization probe does not selectively hybridize, the second hybridization probe including a detectable marker; a third hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the third hybridization probe including the same detectable marker as the second hybridization probe; and a fourth hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the third hybridization probe does not selectively hybridize, the fourth hybridization probe including the first complexing agent for forming a binding pair with the second complexing agent; wherein the first and second hybridization probes are capable of simultaneously hybridizing to the target sequence and the third and fourth hybridization probes are capable of simultaneously hybridizing to the target sequence, the detectable marker is not present on the first or fourth hybridization probes and the first, second, third, and fourth hybridization probes each include a competitive nucleic acid sequence which is sufficiently complementary to a third portion of the target sequence that the competitive sequences of the first, second, third, and fourth hybridization probes compete with each other to hybridize to the third portion of the target sequence.

Mining sequence variations in representative polyploid sugarcane germplasm accessions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xiping; Song, Jian; You, Qian

Sugarcane (Saccharum spp.) is one of the most important economic crops because of its high sugar production and biofuel potential. Due to the high polyploid level and complex genome of sugarcane, it has been a huge challenge to investigate genomic sequence variations, which are critical for identifying alleles contributing to important agronomic traits. In order to mine the genetic variations in sugarcane, genotyping by sequencing (GBS), was used to genotype 14 representative Saccharum complex accessions. GBS is a method to generate a large number of markers, enabled by next generation sequencing (NGS) and the genome complexity reduction using restriction enzymes.more » To use GBS for high throughput genotyping highly polyploid sugarcane, the GBS analysis pipelines in 14 Saccharum complex accessions were established by evaluating different alignment methods, sequence variants callers, and sequence depth for single nucleotide polymorphism (SNP) filtering. By using the established pipeline, a total of 76,251 non-redundant SNPs, 5642 InDels, 6380 presence/absence variants (PAVs), and 826 copy number variations (CNVs) were detected among the 14 accessions. In addition, non-reference based universal network enabled analysis kit and Stacks de novo called 34,353 and 109,043 SNPs, respectively. In the 14 accessions, the percentages of single dose SNPs ranged from 38.3% to 62.3% with an average of 49.6%, much more than the portions of multiple dosage SNPs. Concordantly called SNPs were used to evaluate the phylogenetic relationship among the 14 accessions. The results showed that the divergence time between the Erianthus genus and the Saccharum genus was more than 10 million years ago (MYA). The Saccharum species separated from their common ancestors ranging from 0.19 to 1.65 MYA. The GBS pipelines including the reference sequences, alignment methods, sequence variant callers, and sequence depth were recommended and discussed for the Saccharum complex and other related species. A large number of sequence variations were discovered in the Saccharum complex, including SNPs, InDels, PAVs, and CNVs. Genome-wide SNPs were further used to illustrate sequence features of polyploid species and demonstrated the divergence of different species in the Saccharum complex. The results of this study showed that GBS was an effective NGS-based method to discover genomic sequence variations in highly polyploid and heterozygous species.« less

Mining sequence variations in representative polyploid sugarcane germplasm accessions

DOE PAGES

Yang, Xiping; Song, Jian; You, Qian; ...

2017-08-09

Sugarcane (Saccharum spp.) is one of the most important economic crops because of its high sugar production and biofuel potential. Due to the high polyploid level and complex genome of sugarcane, it has been a huge challenge to investigate genomic sequence variations, which are critical for identifying alleles contributing to important agronomic traits. In order to mine the genetic variations in sugarcane, genotyping by sequencing (GBS), was used to genotype 14 representative Saccharum complex accessions. GBS is a method to generate a large number of markers, enabled by next generation sequencing (NGS) and the genome complexity reduction using restriction enzymes.more » To use GBS for high throughput genotyping highly polyploid sugarcane, the GBS analysis pipelines in 14 Saccharum complex accessions were established by evaluating different alignment methods, sequence variants callers, and sequence depth for single nucleotide polymorphism (SNP) filtering. By using the established pipeline, a total of 76,251 non-redundant SNPs, 5642 InDels, 6380 presence/absence variants (PAVs), and 826 copy number variations (CNVs) were detected among the 14 accessions. In addition, non-reference based universal network enabled analysis kit and Stacks de novo called 34,353 and 109,043 SNPs, respectively. In the 14 accessions, the percentages of single dose SNPs ranged from 38.3% to 62.3% with an average of 49.6%, much more than the portions of multiple dosage SNPs. Concordantly called SNPs were used to evaluate the phylogenetic relationship among the 14 accessions. The results showed that the divergence time between the Erianthus genus and the Saccharum genus was more than 10 million years ago (MYA). The Saccharum species separated from their common ancestors ranging from 0.19 to 1.65 MYA. The GBS pipelines including the reference sequences, alignment methods, sequence variant callers, and sequence depth were recommended and discussed for the Saccharum complex and other related species. A large number of sequence variations were discovered in the Saccharum complex, including SNPs, InDels, PAVs, and CNVs. Genome-wide SNPs were further used to illustrate sequence features of polyploid species and demonstrated the divergence of different species in the Saccharum complex. The results of this study showed that GBS was an effective NGS-based method to discover genomic sequence variations in highly polyploid and heterozygous species.« less

A mechanistic link between gene regulation and genome architecture in mammalian development.

PubMed

Bonora, Giancarlo; Plath, Kathrin; Denholtz, Matthew

2014-08-01

The organization of chromatin within the nucleus and the regulation of transcription are tightly linked. Recently, mechanisms underlying this relationship have been uncovered. By defining the organizational hierarchy of the genome, determining changes in chromatin organization associated with changes in cell identity, and describing chromatin organization within the context of linear genomic features (such as chromatin modifications and transcription factor binding) and architectural proteins (including Cohesin, CTCF, and Mediator), a new paradigm in genome biology was established wherein genomes are organized around gene regulatory factors that govern cell identity. As such, chromatin organization plays a central role in establishing and maintaining cell state during development, with gene regulation and genome organization being mutually dependent effectors of cell identity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nonequilibrium Chromosome Looping via Molecular Slip Links

NASA Astrophysics Data System (ADS)

Brackley, C. A.; Johnson, J.; Michieletto, D.; Morozov, A. N.; Nicodemi, M.; Cook, P. R.; Marenduzzo, D.

2017-09-01

We propose a model for the formation of chromatin loops based on the diffusive sliding of molecular slip links. These mimic the behavior of molecules like cohesin, which, along with the CTCF protein, stabilize loops which contribute to organizing the genome. By combining 3D Brownian dynamics simulations and 1D exactly solvable nonequilibrium models, we show that diffusive sliding is sufficient to account for the strong bias in favor of convergent CTCF-mediated chromosome loops observed experimentally. We also find that the diffusive motion of multiple slip links along chromatin is rectified by an intriguing ratchet effect that arises if slip links bind to the chromatin at a preferred "loading site." This emergent collective behavior favors the extrusion of loops which are much larger than the ones formed by single slip links.

Extreme Sensory Complexity Encoded in the 10-Megabase Draft Genome Sequence of the Chromatically Acclimating Cyanobacterium Tolypothrix sp. PCC 7601

PubMed Central

Yerrapragada, Shaila; Shukla, Animesh; Hallsworth-Pepin, Kymberlie; Choi, Kwangmin; Wollam, Aye; Clifton, Sandra; Qin, Xiang; Muzny, Donna; Raghuraman, Sriram; Ashki, Haleh; Uzman, Akif; Highlander, Sarah K.; Fryszczyn, Bartlomiej G.; Fox, George E.; Tirumalai, Madhan R.; Liu, Yamei; Kim, Sun

2015-01-01

Tolypothrix sp. PCC 7601 is a freshwater filamentous cyanobacterium with complex responses to environmental conditions. Here, we present its 9.96-Mbp draft genome sequence, containing 10,065 putative protein-coding sequences, including 305 predicted two-component system proteins and 27 putative phytochrome-class photoreceptors, the most such proteins in any sequenced genome. PMID:25953173

Assessment of antibody library diversity through next generation sequencing and technical error compensation

PubMed Central

Lisi, Simonetta; Chirichella, Michele; Arisi, Ivan; Goracci, Martina; Cremisi, Federico; Cattaneo, Antonino

2017-01-01

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its complexity (also known as diversity), i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library complexity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Complexity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring complexity from such a small sampling is, however, very rudimental and gives limited information about the real diversity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library complexity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the complexity, taking in consideration the sequencing error. PMID:28505201

Assessment of antibody library diversity through next generation sequencing and technical error compensation.

PubMed

Fantini, Marco; Pandolfini, Luca; Lisi, Simonetta; Chirichella, Michele; Arisi, Ivan; Terrigno, Marco; Goracci, Martina; Cremisi, Federico; Cattaneo, Antonino

2017-01-01

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its complexity (also known as diversity), i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library complexity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Complexity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring complexity from such a small sampling is, however, very rudimental and gives limited information about the real diversity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library complexity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the complexity, taking in consideration the sequencing error.

Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome

USDA-ARS?s Scientific Manuscript database

Modern biological analyses are often assisted by recent technologies making the sequencing of complex genomes both technically possible and feasible. We recently sequenced the tomato genome that, like many eukaryotic genomes, is large and complex. Current sequencing technologies allow the developmen...

Sequence co-evolution gives 3D contacts and structures of protein complexes

PubMed Central

Hopf, Thomas A; Schärfe, Charlotta P I; Rodrigues, João P G L M; Green, Anna G; Kohlbacher, Oliver; Sander, Chris; Bonvin, Alexandre M J J; Marks, Debora S

2014-01-01

Protein–protein interactions are fundamental to many biological processes. Experimental screens have identified tens of thousands of interactions, and structural biology has provided detailed functional insight for select 3D protein complexes. An alternative rich source of information about protein interactions is the evolutionary sequence record. Building on earlier work, we show that analysis of correlated evolutionary sequence changes across proteins identifies residues that are close in space with sufficient accuracy to determine the three-dimensional structure of the protein complexes. We evaluate prediction performance in blinded tests on 76 complexes of known 3D structure, predict protein–protein contacts in 32 complexes of unknown structure, and demonstrate how evolutionary couplings can be used to distinguish between interacting and non-interacting protein pairs in a large complex. With the current growth of sequences, we expect that the method can be generalized to genome-wide elucidation of protein–protein interaction networks and used for interaction predictions at residue resolution. DOI: http://dx.doi.org/10.7554/eLife.03430.001 PMID:25255213

Extreme Sensory Complexity Encoded in the 10-Megabase Draft Genome Sequence of the Chromatically Acclimating Cyanobacterium Tolypothrix sp. PCC 7601.

PubMed

Yerrapragada, Shaila; Shukla, Animesh; Hallsworth-Pepin, Kymberlie; Choi, Kwangmin; Wollam, Aye; Clifton, Sandra; Qin, Xiang; Muzny, Donna; Raghuraman, Sriram; Ashki, Haleh; Uzman, Akif; Highlander, Sarah K; Fryszczyn, Bartlomiej G; Fox, George E; Tirumalai, Madhan R; Liu, Yamei; Kim, Sun; Kehoe, David M; Weinstock, George M

2015-05-07

Tolypothrix sp. PCC 7601 is a freshwater filamentous cyanobacterium with complex responses to environmental conditions. Here, we present its 9.96-Mbp draft genome sequence, containing 10,065 putative protein-coding sequences, including 305 predicted two-component system proteins and 27 putative phytochrome-class photoreceptors, the most such proteins in any sequenced genome. Copyright © 2015 Yerrapragada et al.

Age effects on discrimination of timing in auditory sequences

NASA Astrophysics Data System (ADS)

Fitzgibbons, Peter J.; Gordon-Salant, Sandra

2004-08-01

The experiments examined age-related changes in temporal sensitivity to increments in the interonset intervals (IOI) of components in tonal sequences. Discrimination was examined using reference sequences consisting of five 50-ms tones separated by silent intervals; tone frequencies were either fixed at 4 kHz or varied within a 2-4-kHz range to produce spectrally complex patterns. The tonal IOIs within the reference sequences were either equal (200 or 600 ms) or varied individually with an average value of 200 or 600 ms to produce temporally complex patterns. The difference limen (DL) for increments of IOI was measured. Comparison sequences featured either equal increments in all tonal IOIs or increments in a single target IOI, with the sequential location of the target changing randomly across trials. Four groups of younger and older adults with and without sensorineural hearing loss participated. Results indicated that DLs for uniform changes of sequence rate were smaller than DLs for single target intervals, with the largest DLs observed for single targets embedded within temporally complex sequences. Older listeners performed more poorly than younger listeners in all conditions, but the largest age-related differences were observed for temporally complex stimulus conditions. No systematic effects of hearing loss were observed.

[Inverted meiosis and its place in the evolution of sexual reproduction pathways].

PubMed

Bogdanov, Yu F

2016-05-01

Inverted meiosis is observed in plants (Cyperaceae and Juncaceae) and insects (Coccoidea, Aphididae) with holocentric chromosomes, the centromeres of which occupy from 70 to 90% of the metaphase chromosome length. In the first meiotic division (meiosis I), chiasmata are formed and rodlike bivalents orient equationally, and in anaphase I, sister chromatids segregate to the poles; the diploid chromosome number is maintained. Non-sister chromatids of homologous chromosomes remain in contact during interkinesis and prophase II and segregate in anaphase II, forming haploid chromosome sets. The segregation of sister chromatids in meiosis I was demonstrated by example of three plant species that were heterozygous for chromosomal rearrangements. In these species, sister chromatids, marked with rearrangement, segregated in anaphase I. Using fluorescent antibodies, it was demonstrated that meiotic recombination enzymes Spo11 and Rad5l, typical of canonical meiosis, functioned at the meiotic prophase I of pollen mother cells of Luzula elegance and Rhynchospora pubera. Moreover, antibodies to synaptonemal complexes proteins ASY1 and ZYP1 were visualized as filamentous structures, pointing to probable formation of synaptonemal complexes. In L. elegance, chiasmata are formed by means of chromatin threads containing satellite DNA. According to the hypothesis of the author of this review, equational division of sister chromatids at meiosis I in the organisms with inverted meiosis can be explained by the absence of specific meiotic proteins (shugoshins). These proteins are able to protect cohesins of holocentric centromeres from hydrolysis by separases at meiosis I, as occurs in the organisms with monocentric chromosomes and canonical meiosis. The basic type of inverted meiosis was described in Coccoidea and Aphididae males. In their females, the variants of parthenogenesis were also observed. Until now, the methods of molecular cytogenetics were not applied for the analysis of inverted meiosis in Coccoidea and Aphididae. Evolutionary, inverted meiosis is thought to have appeared secondarily as an adaptation of the molecular mechanisms of canonical meiosis to chromosome holocentrism.

Computational design of enzyme-ligand binding using a combined energy function and deterministic sequence optimization algorithm.

PubMed

Tian, Ye; Huang, Xiaoqiang; Zhu, Yushan

2015-08-01

Enzyme amino-acid sequences at ligand-binding interfaces are evolutionarily optimized for reactions, and the natural conformation of an enzyme-ligand complex must have a low free energy relative to alternative conformations in native-like or non-native sequences. Based on this assumption, a combined energy function was developed for enzyme design and then evaluated by recapitulating native enzyme sequences at ligand-binding interfaces for 10 enzyme-ligand complexes. In this energy function, the electrostatic interaction between polar or charged atoms at buried interfaces is described by an explicitly orientation-dependent hydrogen-bonding potential and a pairwise-decomposable generalized Born model based on the general side chain in the protein design framework. The energy function is augmented with a pairwise surface-area based hydrophobic contribution for nonpolar atom burial. Using this function, on average, 78% of the amino acids at ligand-binding sites were predicted correctly in the minimum-energy sequences, whereas 84% were predicted correctly in the most-similar sequences, which were selected from the top 20 sequences for each enzyme-ligand complex. Hydrogen bonds at the enzyme-ligand binding interfaces in the 10 complexes were usually recovered with the correct geometries. The binding energies calculated using the combined energy function helped to discriminate the active sequences from a pool of alternative sequences that were generated by repeatedly solving a series of mixed-integer linear programming problems for sequence selection with increasing integer cuts.

Development of a Single Locus Sequence Typing (SLST) Scheme for Typing Bacterial Species Directly from Complex Communities.

PubMed

Scholz, Christian F P; Jensen, Anders

2017-01-01

The protocol describes a computational method to develop a Single Locus Sequence Typing (SLST) scheme for typing bacterial species. The resulting scheme can be used to type bacterial isolates as well as bacterial species directly from complex communities using next-generation sequencing technologies.

Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica.

PubMed

Shabbir Hussain, Murtaza; Gambill, Lauren; Smith, Spencer; Blenner, Mark A

2016-03-18

Eukaryotic promoters have a complex architecture to control both the strength and timing of gene transcription spanning up to thousands of bases from the initiation site. This complexity makes rational fine-tuning of promoters in fungi difficult to predict; however, this very same complexity enables multiple possible strategies for engineering promoter strength. Here, we studied promoter architecture in the oleaginous yeast, Yarrowia lipolytica. While recent studies have focused on upstream activating sequences, we systematically examined various components common in fungal promoters. Here, we examine several promoter components including upstream activating sequences, proximal promoter sequences, core promoters, and the TATA box in autonomously replicating expression plasmids and integrated into the genome. Our findings show that promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences. Additionally, we identified a previously unreported oleic acid responsive transcription enhancement in the XPR2 upstream activating sequences, which illustrates the complexity of fungal promoters. The promoters engineered here provide new genetic tools for metabolic engineering in Y. lipolytica and provide promoter engineering strategies that may be useful in engineering other non-model fungal systems.

Binding of resveratrol to the minor groove of DNA sequences with AATT and TTAA segments induces differential stability.

PubMed

Nair, Maya S; D'Mello, Samar; Pant, Rashmi; Poluri, Krishna Mohan

2017-05-01

Interactions of a natural stilbene compound, resveratrol with two DNA sequences containing AATT/TTAA segments have been studied. Resveratrol is found to interact with both the sequences. The mode of interaction has been studied using absorption, steady state fluorescence and circular dichroism spectroscopic techniques. UV-visible absorption and fluorescence studies provided the information regarding the binding constants and the stoichiometry of binding, whereas circular dichroism studies depicted the structural changes in DNA upon resveratrol binding. Our results evidenced that, though resveratrol showed similar affinity to both the sequences, the mode of interactions was different. The binding constants of resveratrol to AATT/TTAA sequences were found to be 7.55×10 5 M -1 and 5.42×10 5 M -1 respectively. Spectroscopic data evidenced for a groove binding interaction. Melting studies showed that the binding of resveratrol induces differential stability to the DNA sequences d(CGTTAACG) 2 and d(CGAATTCG) 2 . Fluorescence data showed a stoichiometry of 1:1 for d(CGAATTCG) 2 -resveratrol complex and 1:4 for d(CGTTAACG) 2 -resveratrol complex. Molecular docking studies demonstrated that resveratrol binds to the minor groove region of both the sequences to form stable complexes with varied atomic contacts to the DNA bases or backbone. Both the complexes are stabilized by hydrogen bond formation. Our results evidenced that modulation of DNA sequence within the same bases can greatly alter the binding geometry and stability of the complex upon binding to small molecule inhibitor compounds like resveratrol. Copyright © 2017 Elsevier B.V. All rights reserved.

QRS complex detection based on continuous density hidden Markov models using univariate observations

NASA Astrophysics Data System (ADS)

Sotelo, S.; Arenas, W.; Altuve, M.

2018-04-01

In the electrocardiogram (ECG), the detection of QRS complexes is a fundamental step in the ECG signal processing chain since it allows the determination of other characteristics waves of the ECG and provides information about heart rate variability. In this work, an automatic QRS complex detector based on continuous density hidden Markov models (HMM) is proposed. HMM were trained using univariate observation sequences taken either from QRS complexes or their derivatives. The detection approach is based on the log-likelihood comparison of the observation sequence with a fixed threshold. A sliding window was used to obtain the observation sequence to be evaluated by the model. The threshold was optimized by receiver operating characteristic curves. Sensitivity (Sen), specificity (Spc) and F1 score were used to evaluate the detection performance. The approach was validated using ECG recordings from the MIT-BIH Arrhythmia database. A 6-fold cross-validation shows that the best detection performance was achieved with 2 states HMM trained with QRS complexes sequences (Sen = 0.668, Spc = 0.360 and F1 = 0.309). We concluded that these univariate sequences provide enough information to characterize the QRS complex dynamics from HMM. Future works are directed to the use of multivariate observations to increase the detection performance.

SMRT sequencing of the Vitis vinifera cv. ‘Flame seedless’ genome using a SMRTbell-free library preparation from Swift Biosciences

USDA-ARS?s Scientific Manuscript database

Single Molecule Real-Time (SMRT) sequencing provides advantages to the sequencing of complex genomes. The long reads generated are superior for resolving complex genomic regions and provide highly contiguous de novo assemblies. Current SMRTbell libraries generate average read lengths of 10-15kb. How...

Detection and isolation of nucleic acid sequences using a bifunctional hybridization probe

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

2000-01-01

A method for detecting and isolating a target sequence in a sample of nucleic acids is provided using a bifunctional hybridization probe capable of hybridizing to the target sequence that includes a detectable marker and a first complexing agent capable of forming a binding pair with a second complexing agent. A kit is also provided for detecting a target sequence in a sample of nucleic acids using a bifunctional hybridization probe according to this method.

BAC sequencing using pooled methods.

PubMed

Saski, Christopher A; Feltus, F Alex; Parida, Laxmi; Haiminen, Niina

2015-01-01

Shotgun sequencing and assembly of a large, complex genome can be both expensive and challenging to accurately reconstruct the true genome sequence. Repetitive DNA arrays, paralogous sequences, polyploidy, and heterozygosity are main factors that plague de novo genome sequencing projects that typically result in highly fragmented assemblies and are difficult to extract biological meaning. Targeted, sub-genomic sequencing offers complexity reduction by removing distal segments of the genome and a systematic mechanism for exploring prioritized genomic content through BAC sequencing. If one isolates and sequences the genome fraction that encodes the relevant biological information, then it is possible to reduce overall sequencing costs and efforts that target a genomic segment. This chapter describes the sub-genome assembly protocol for an organism based upon a BAC tiling path derived from a genome-scale physical map or from fine mapping using BACs to target sub-genomic regions. Methods that are described include BAC isolation and mapping, DNA sequencing, and sequence assembly.

On the equivalence of some spectral sequences for Serre fibrations

NASA Astrophysics Data System (ADS)

Onishchenko, Aleksandr Yu; Popelenskii, Fedor Yu

2011-04-01

Several different constructions of a spectral sequence for a Serre fibration \\pi\\colon E \\to B over a compact simply connected manifold B are considered in this paper. Namely, we consider the spectral sequence for the minimal model (\\Lambda V\\otimes \\Lambda W,d) of the fibration, along with the spectral sequences arising from the Čech filtration in the complexes \\check{C}^*(\\mathscr{U}, A_{PL}^*(\\pi^{-1}(U))) and \\check{C}^*(\\mathscr{U}, S^*(\\pi^{-1}(U))), where \\mathscr{U}=\\{U\\} is a covering of the base B. It is known that all these spectral sequences have the same terms E_2^{*,*}=H^*(X)\\otimes H^*(F) and converge to the cohomology of the total space E. A new natural isomorphism of these spectral sequences is constructed in every term E_r with r\\ge2. It is also proved that in the case of a smooth locally trivial fibration these spectral sequences are isomorphic to the spectral sequences of the complex of smooth forms \\Omega^*(E) and of the Čech-de Rham complex. It is therefore established that all these constructions give the same spectral sequence, starting from the E_2 term. Bibliography: 9 titles.

Study designs for identification of rare disease variants in complex diseases: the utility of family-based designs.

PubMed

Ionita-Laza, Iuliana; Ottman, Ruth

2011-11-01

The recent progress in sequencing technologies makes possible large-scale medical sequencing efforts to assess the importance of rare variants in complex diseases. The results of such efforts depend heavily on the use of efficient study designs and analytical methods. We introduce here a unified framework for association testing of rare variants in family-based designs or designs based on unselected affected individuals. This framework allows us to quantify the enrichment in rare disease variants in families containing multiple affected individuals and to investigate the optimal design of studies aiming to identify rare disease variants in complex traits. We show that for many complex diseases with small values for the overall sibling recurrence risk ratio, such as Alzheimer's disease and most cancers, sequencing affected individuals with a positive family history of the disease can be extremely advantageous for identifying rare disease variants. In contrast, for complex diseases with large values of the sibling recurrence risk ratio, sequencing unselected affected individuals may be preferable.

Computational complexity of algorithms for sequence comparison, short-read assembly and genome alignment.

PubMed

Baichoo, Shakuntala; Ouzounis, Christos A

A multitude of algorithms for sequence comparison, short-read assembly and whole-genome alignment have been developed in the general context of molecular biology, to support technology development for high-throughput sequencing, numerous applications in genome biology and fundamental research on comparative genomics. The computational complexity of these algorithms has been previously reported in original research papers, yet this often neglected property has not been reviewed previously in a systematic manner and for a wider audience. We provide a review of space and time complexity of key sequence analysis algorithms and highlight their properties in a comprehensive manner, in order to identify potential opportunities for further research in algorithm or data structure optimization. The complexity aspect is poised to become pivotal as we will be facing challenges related to the continuous increase of genomic data on unprecedented scales and complexity in the foreseeable future, when robust biological simulation at the cell level and above becomes a reality. Copyright © 2017 Elsevier B.V. All rights reserved.

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE PAGES

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; ...

2016-03-09

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

A new complexity measure for time series analysis and classification

NASA Astrophysics Data System (ADS)

Nagaraj, Nithin; Balasubramanian, Karthi; Dey, Sutirth

2013-07-01

Complexity measures are used in a number of applications including extraction of information from data such as ecological time series, detection of non-random structure in biomedical signals, testing of random number generators, language recognition and authorship attribution etc. Different complexity measures proposed in the literature like Shannon entropy, Relative entropy, Lempel-Ziv, Kolmogrov and Algorithmic complexity are mostly ineffective in analyzing short sequences that are further corrupted with noise. To address this problem, we propose a new complexity measure ETC and define it as the "Effort To Compress" the input sequence by a lossless compression algorithm. Here, we employ the lossless compression algorithm known as Non-Sequential Recursive Pair Substitution (NSRPS) and define ETC as the number of iterations needed for NSRPS to transform the input sequence to a constant sequence. We demonstrate the utility of ETC in two applications. ETC is shown to have better correlation with Lyapunov exponent than Shannon entropy even with relatively short and noisy time series. The measure also has a greater rate of success in automatic identification and classification of short noisy sequences, compared to entropy and a popular measure based on Lempel-Ziv compression (implemented by Gzip).

Long-range barcode labeling-sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Feng; Zhang, Tao; Singh, Kanwar K.

Methods for sequencing single large DNA molecules by clonal multiple displacement amplification using barcoded primers. Sequences are binned based on barcode sequences and sequenced using a microdroplet-based method for sequencing large polynucleotide templates to enable assembly of haplotype-resolved complex genomes and metagenomes.

Learning of goal-relevant and -irrelevant complex visual sequences in human V1.

PubMed

Rosenthal, Clive R; Mallik, Indira; Caballero-Gaudes, Cesar; Sereno, Martin I; Soto, David

2018-06-12

Learning and memory are supported by a network involving the medial temporal lobe and linked neocortical regions. Emerging evidence indicates that primary visual cortex (i.e., V1) may contribute to recognition memory, but this has been tested only with a single visuospatial sequence as the target memorandum. The present study used functional magnetic resonance imaging to investigate whether human V1 can support the learning of multiple, concurrent complex visual sequences involving discontinous (second-order) associations. Two peripheral, goal-irrelevant but structured sequences of orientated gratings appeared simultaneously in fixed locations of the right and left visual fields alongside a central, goal-relevant sequence that was in the focus of spatial attention. Pseudorandom sequences were introduced at multiple intervals during the presentation of the three structured visual sequences to provide an online measure of sequence-specific knowledge at each retinotopic location. We found that a network involving the precuneus and V1 was involved in learning the structured sequence presented at central fixation, whereas right V1 was modulated by repeated exposure to the concurrent structured sequence presented in the left visual field. The same result was not found in left V1. These results indicate for the first time that human V1 can support the learning of multiple concurrent sequences involving complex discontinuous inter-item associations, even peripheral sequences that are goal-irrelevant. Copyright © 2018. Published by Elsevier Inc.

ATRX contributes to epigenetic asymmetry and silencing of major satellite transcripts in the maternal genome of the mouse embryo

PubMed Central

De La Fuente, Rabindranath; Baumann, Claudia; Viveiros, Maria M.

2015-01-01

A striking proportion of human cleavage-stage embryos exhibit chromosome instability (CIN). Notably, until now, no experimental model has been described to determine the origin and mechanisms of complex chromosomal rearrangements. Here, we examined mouse embryos deficient for the chromatin remodeling protein ATRX to determine the cellular mechanisms activated in response to CIN. We demonstrate that ATRX is required for silencing of major satellite transcripts in the maternal genome, where it confers epigenetic asymmetry to pericentric heterochromatin during the transition to the first mitosis. This stage is also characterized by a striking kinetochore size asymmetry established by differences in CENP-C protein between the parental genomes. Loss of ATRX results in increased centromeric mitotic recombination, a high frequency of sister chromatid exchanges and double strand DNA breaks, indicating the formation of mitotic recombination break points. ATRX-deficient embryos exhibit a twofold increase in transcripts for aurora kinase B, the centromeric cohesin ESCO2, DNMT1, the ubiquitin-ligase (DZIP3) and the histone methyl transferase (EHMT1). Thus, loss of ATRX activates a pathway that integrates epigenetic modifications and DNA repair in response to chromosome breaks. These results reveal the cellular response of the cleavage-stage embryo to CIN and uncover a mechanism by which centromeric fission induces the formation of large-scale chromosomal rearrangements. Our results have important implications to determine the epigenetic origins of CIN that lead to congenital birth defects and early pregnancy loss, as well as the mechanisms involved in the oocyte to embryo transition. PMID:25926359

History, rare, and multiple events of mechanical unfolding of repeat proteins

NASA Astrophysics Data System (ADS)

Sumbul, Fidan; Marchesi, Arin; Rico, Felix

2018-03-01

Mechanical unfolding of proteins consisting of repeat domains is an excellent tool to obtain large statistics. Force spectroscopy experiments using atomic force microscopy on proteins presenting multiple domains have revealed that unfolding forces depend on the number of folded domains (history) and have reported intermediate states and rare events. However, the common use of unspecific attachment approaches to pull the protein of interest holds important limitations to study unfolding history and may lead to discarding rare and multiple probing events due to the presence of unspecific adhesion and uncertainty on the pulling site. Site-specific methods that have recently emerged minimize this uncertainty and would be excellent tools to probe unfolding history and rare events. However, detailed characterization of these approaches is required to identify their advantages and limitations. Here, we characterize a site-specific binding approach based on the ultrastable complex dockerin/cohesin III revealing its advantages and limitations to assess the unfolding history and to investigate rare and multiple events during the unfolding of repeated domains. We show that this approach is more robust, reproducible, and provides larger statistics than conventional unspecific methods. We show that the method is optimal to reveal the history of unfolding from the very first domain and to detect rare events, while being more limited to assess intermediate states. Finally, we quantify the forces required to unfold two molecules pulled in parallel, difficult when using unspecific approaches. The proposed method represents a step forward toward more reproducible measurements to probe protein unfolding history and opens the door to systematic probing of rare and multiple molecule unfolding mechanisms.

Radiation hybrid maps of D-genome of Aegilops tauschii and their application in sequence assembly of large and complex plant genomes

USDA-ARS?s Scientific Manuscript database

The large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high-resolution genome maps saturated with ordered markers to assist in anchoring and orienting BAC contigs/ sequence scaffolds for whole genome sequence assembly. Radiation hybrid (RH) mapping has proven to be an e...

A combination of LongSAGE with Solexa sequencing is well suited to explore the depth and the complexity of transcriptome

PubMed Central

Hanriot, Lucie; Keime, Céline; Gay, Nadine; Faure, Claudine; Dossat, Carole; Wincker, Patrick; Scoté-Blachon, Céline; Peyron, Christelle; Gandrillon, Olivier

2008-01-01

Background "Open" transcriptome analysis methods allow to study gene expression without a priori knowledge of the transcript sequences. As of now, SAGE (Serial Analysis of Gene Expression), LongSAGE and MPSS (Massively Parallel Signature Sequencing) are the mostly used methods for "open" transcriptome analysis. Both LongSAGE and MPSS rely on the isolation of 21 pb tag sequences from each transcript. In contrast to LongSAGE, the high throughput sequencing method used in MPSS enables the rapid sequencing of very large libraries containing several millions of tags, allowing deep transcriptome analysis. However, a bias in the complexity of the transcriptome representation obtained by MPSS was recently uncovered. Results In order to make a deep analysis of mouse hypothalamus transcriptome avoiding the limitation introduced by MPSS, we combined LongSAGE with the Solexa sequencing technology and obtained a library of more than 11 millions of tags. We then compared it to a LongSAGE library of mouse hypothalamus sequenced with the Sanger method. Conclusion We found that Solexa sequencing technology combined with LongSAGE is perfectly suited for deep transcriptome analysis. In contrast to MPSS, it gives a complex representation of transcriptome as reliable as a LongSAGE library sequenced by the Sanger method. PMID:18796152

Taxonomy of the Rhizopogon vinicolor species complex based on analysis of ITS sequences and microsatellite loci.

Treesearch

Annette M. Kretzer; Daniel L. Luoma; Randy Molina; Joseph W. Spatafora

2003-01-01

We are re-addressing species concepts in the Rhizopogon vinicolor species complex (Boletales, Basidiomycota) using sequence data from the interna transcribed spacer (ITS) region of the nuclear ribosomal repeat, as well as genoLypic data from five microsatellite loci. The R. vinicolor species complex by our definition includes,...

How proteins bind to DNA: target discrimination and dynamic sequence search by the telomeric protein TRF1

PubMed Central

2017-01-01

Abstract Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein–DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation. PMID:28633355

Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

PubMed Central

Elmore, Joshua R.; Sheppard, Nolan F.; Ramia, Nancy; Deighan, Trace; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

2016-01-01

CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM. PMID:26848045

On the equivalence of some spectral sequences for Serre fibrations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Onishchenko, Aleksandr Yu; Popelenskii, Fedor Yu

2011-04-30

Several different constructions of a spectral sequence for a Serre fibration {pi}:E{yields}B over a compact simply connected manifold B are considered in this paper. Namely, we consider the spectral sequence for the minimal model ({Lambda}Vx{Lambda}W,d) of the fibration, along with the spectral sequences arising from the Cech filtration in the complexes C*(U,A*{sub PL}({pi}{sup -1}(U))) and C*(U,S*({pi}{sup -1}(U))), where U=(U) is a covering of the base B. It is known that all these spectral sequences have the same terms E{sub 2}*{sup ,}*=H*(X)xH{sup *}(F) and converge to the cohomology of the total space E. A new natural isomorphism of these spectral sequencesmore » is constructed in every term E{sub r} with r{>=}2. It is also proved that in the case of a smooth locally trivial fibration these spectral sequences are isomorphic to the spectral sequences of the complex of smooth forms {Omega}*(E) and of the Cech-de Rham complex. It is therefore established that all these constructions give the same spectral sequence, starting from the E{sub 2} term. Bibliography: 9 titles.« less

Mutation of Hip’s Carboxy-Terminal Region Inhibits a Transitional Stage of Progesterone Receptor Assembly

PubMed Central

Prapapanich, Viravan; Chen, Shiying; Smith, David F.

1998-01-01

Steroid receptor complexes are assembled through an ordered, multistep pathway involving multiple components of the cytoplasmic chaperone machinery. Two of these components are Hsp70-binding proteins, Hip and Hop, that have some limited homology in their C-terminal regions, outside the sequences mapped for Hsp70 binding. Within this region of Hip is a DPEV sequence that occurs twice; in Hop, one DPEV sequence plus a partial second sequence occurs. In an effort to better understand Hip function as it relates to assembly of progesterone receptor complexes, the DPEV region of Hip was targeted for mutations. Each DPEV sequence was mutated to an APAV sequence, singly or in combination. The combined mutation, APAV2, was further combined with a deletion of Hip’s tetratricopeptide repeat region that is required for Hsp70 binding or with a deletion of Hip’s GGMP repeat. An additional mutant was prepared by truncation of Hip’s DPEV-containing C terminus. By comparing interactions of various Hip forms with Hsp70, it was determined that mutation of the DPEV sequences created a dominant inhibitory form of Hip. The mutant Hip-Hsp70 complex was not prevented from interacting with progesterone receptor, but the mutant caused a dose-dependent inhibition of receptor assembly with Hsp90. The behavior of the Hip mutant is consistent with a model in which Hip and Hop are required to facilitate the transition from an early receptor complex with Hsp70 into later complexes containing Hsp90. PMID:9447991

Recognition of platinum-DNA adducts by HMGB1a.

PubMed

Ramachandran, Srinivas; Temple, Brenda; Alexandrova, Anastassia N; Chaney, Stephen G; Dokholyan, Nikolay V

2012-09-25

Cisplatin (CP) and oxaliplatin (OX), platinum-based drugs used widely in chemotherapy, form adducts on intrastrand guanines (5'GG) in genomic DNA. DNA damage recognition proteins, transcription factors, mismatch repair proteins, and DNA polymerases discriminate between CP- and OX-GG DNA adducts, which could partly account for differences in the efficacy, toxicity, and mutagenicity of CP and OX. In addition, differential recognition of CP- and OX-GG adducts is highly dependent on the sequence context of the Pt-GG adduct. In particular, DNA binding protein domain HMGB1a binds to CP-GG DNA adducts with up to 53-fold greater affinity than to OX-GG adducts in the TGGA sequence context but shows much smaller differences in binding in the AGGC or TGGT sequence contexts. Here, simulations of the HMGB1a-Pt-DNA complex in the three sequence contexts revealed a higher number of interface contacts for the CP-DNA complex in the TGGA sequence context than in the OX-DNA complex. However, the number of interface contacts was similar in the TGGT and AGGC sequence contexts. The higher number of interface contacts in the CP-TGGA sequence context corresponded to a larger roll of the Pt-GG base pair step. Furthermore, geometric analysis of stacking of phenylalanine 37 in HMGB1a (Phe37) with the platinated guanines revealed more favorable stacking modes correlated with a larger roll of the Pt-GG base pair step in the TGGA sequence context. These data are consistent with our previous molecular dynamics simulations showing that the CP-TGGA complex was able to sample larger roll angles than the OX-TGGA complex or either CP- or OX-DNA complexes in the AGGC or TGGT sequences. We infer that the high binding affinity of HMGB1a for CP-TGGA is due to the greater flexibility of CP-TGGA compared to OX-TGGA and other Pt-DNA adducts. This increased flexibility is reflected in the ability of CP-TGGA to sample larger roll angles, which allows for a higher number of interface contacts between the Pt-DNA adduct and HMGB1a.

Exploiting three kinds of interface propensities to identify protein binding sites.

PubMed

Liu, Bin; Wang, Xiaolong; Lin, Lei; Dong, Qiwen; Wang, Xuan

2009-08-01

Predicting the binding sites between two interacting proteins provides important clues to the function of a protein. In this study, we present a building block of proteins called order profiles to use the evolutionary information of the protein sequence frequency profiles and apply this building block to produce a class of propensities called order profile interface propensities. For comparisons, we revisit the usage of residue interface propensities and binary profile interface propensities for protein binding site prediction. Each kind of propensities combined with sequence profiles and accessible surface areas are inputted into SVM. When tested on four types of complexes (hetero-permanent complexes, hetero-transient complexes, homo-permanent complexes and homo-transient complexes), experimental results show that the order profile interface propensities are better than residue interface propensities and binary profile interface propensities. Therefore, order profile is a suitable profile-level building block of the protein sequences and can be widely used in many tasks of computational biology, such as the sequence alignment, the prediction of domain boundary, the designation of knowledge-based potentials and the protein remote homology detection.

Nature and provenance of the Beishan Complex, southernmost Central Asian Orogenic Belt

NASA Astrophysics Data System (ADS)

Zheng, Rongguo; Li, Jinyi; Xiao, Wenjiao; Zhang, Jin

2018-03-01

The ages and origins of metasedimentary rocks, which were previously mapped as Precambrian, are critical in rebuilding the orogenic process and better understanding the Phanerozoic continental growth in the Central Asian Orogenic Belt (CAOB). The Beishan Complex was widely distributed in the southern Beishan Orogenic Collage, southernmost CAOB, and their ages and tectonic affinities are still in controversy. The Beishan Complex was previously proposed as fragments drifted from the Tarim Craton, Neoproterozoic Block or Phanerozoic accretionary complex. In this study, we employ detrital zircon age spectra to constrain ages and provenances of metasedimentary sequences of the Beishan Complex in the Chuanshanxun area. The metasedimentary rocks here are dominated by zircons with Paleoproterozoic-Mesoproterozoic age ( 1160-2070 Ma), and yield two peak ages at 1454 and 1760 Ma. One sample yielded a middle Permian peak age (269 Ma), which suggests that the metasedimentary sequences were deposited in the late Paleozoic. The granitoid and dioritic dykes, intruding into the metasedimentary sequences, exhibit zircon U-Pb ages of 268 and 261 Ma, respectively, which constrain the minimum deposit age of the metasedimentary sequences. Zircon U-Pb ages of amphibolite (274 and 216 Ma) indicate that they might be affected by multi-stage metamorphic events. The Beishan Complex was not a fragment drifted from the Tarim Block or Dunhuang Block, and none of cratons or blocks surrounding Beishan Orogenic Collage was the sole material source of the Beishan Complex due to obviously different age spectra. Instead, 1.4 Ga marginal accretionary zones of the Columbia supercontinent might have existed in the southern CAOB, and may provide the main source materials for the sedimentary sequences in the Beishan Complex.

Complexity and Entropy Analysis of DNMT1 Gene

USDA-ARS?s Scientific Manuscript database

Background: The application of complexity information on DNA sequence and protein in biological processes are well established in this study. Available sequences for DNMT1 gene, which is a maintenance methyltransferase is responsible for copying DNA methylation patterns to the daughter strands durin...

Cryptic breakpoint identified by whole-genome mate-pair sequencing in a rare paternally inherited complex chromosomal rearrangement.

PubMed

Aristidou, Constantia; Theodosiou, Athina; Ketoni, Andria; Bak, Mads; Mehrjouy, Mana M; Tommerup, Niels; Sismani, Carolina

2018-01-01

Precise characterization of apparently balanced complex chromosomal rearrangements in non-affected individuals is crucial as they may result in reproductive failure, recurrent miscarriages or affected offspring. We present a family, where the non-affected father and daughter were found, using FISH and karyotyping, to be carriers of a three-way complex chromosomal rearrangement [t(6;7;10)(q16.2;q34;q26.1), de novo in the father]. The family suffered from two stillbirths, one miscarriage, and has a son with severe intellectual disability. In the present study, the family was revisited using whole-genome mate-pair sequencing. Interestingly, whole-genome mate-pair sequencing revealed a cryptic breakpoint on derivative (der) chromosome 6 rendering the rearrangement even more complex. FISH using a chromosome (chr) 6 custom-designed probe and a chr10 control probe confirmed that the interstitial chr6 segment, created by the two chr6 breakpoints, was translocated onto der(10). Breakpoints were successfully validated with Sanger sequencing, and small imbalances as well as microhomology were identified. Finally, the complex chromosomal rearrangement breakpoints disrupted the SIM1 , GRIK2 , CNTNAP2 , and PTPRE genes without causing any phenotype development. In contrast to the majority of maternally transmitted complex chromosomal rearrangement cases, our study investigated a rare case where a complex chromosomal rearrangement, which most probably resulted from a Type IV hexavalent during the pachytene stage of meiosis I, was stably transmitted from a fertile father to his non-affected daughter. Whole-genome mate-pair sequencing proved highly successful in identifying cryptic complexity, which consequently provided further insight into the meiotic segregation of chromosomes and the increased reproductive risk in individuals carrying the specific complex chromosomal rearrangement. We propose that such complex rearrangements should be characterized in detail using a combination of conventional cytogenetic and NGS-based approaches to aid in better prenatal preimplantation genetic diagnosis and counseling in couples with reproductive problems.

An additional function of the rough endoplasmic reticulum protein complex prolyl 3-hydroxylase 1·cartilage-associated protein·cyclophilin B: the CXXXC motif reveals disulfide isomerase activity in vitro.

PubMed

Ishikawa, Yoshihiro; Bächinger, Hans Peter

2013-11-01

Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.

RNACompress: Grammar-based compression and informational complexity measurement of RNA secondary structure.

PubMed

Liu, Qi; Yang, Yu; Chen, Chun; Bu, Jiajun; Zhang, Yin; Ye, Xiuzi

2008-03-31

With the rapid emergence of RNA databases and newly identified non-coding RNAs, an efficient compression algorithm for RNA sequence and structural information is needed for the storage and analysis of such data. Although several algorithms for compressing DNA sequences have been proposed, none of them are suitable for the compression of RNA sequences with their secondary structures simultaneously. This kind of compression not only facilitates the maintenance of RNA data, but also supplies a novel way to measure the informational complexity of RNA structural data, raising the possibility of studying the relationship between the functional activities of RNA structures and their complexities, as well as various structural properties of RNA based on compression. RNACompress employs an efficient grammar-based model to compress RNA sequences and their secondary structures. The main goals of this algorithm are two fold: (1) present a robust and effective way for RNA structural data compression; (2) design a suitable model to represent RNA secondary structure as well as derive the informational complexity of the structural data based on compression. Our extensive tests have shown that RNACompress achieves a universally better compression ratio compared with other sequence-specific or common text-specific compression algorithms, such as Gencompress, winrar and gzip. Moreover, a test of the activities of distinct GTP-binding RNAs (aptamers) compared with their structural complexity shows that our defined informational complexity can be used to describe how complexity varies with activity. These results lead to an objective means of comparing the functional properties of heteropolymers from the information perspective. A universal algorithm for the compression of RNA secondary structure as well as the evaluation of its informational complexity is discussed in this paper. We have developed RNACompress, as a useful tool for academic users. Extensive tests have shown that RNACompress is a universally efficient algorithm for the compression of RNA sequences with their secondary structures. RNACompress also serves as a good measurement of the informational complexity of RNA secondary structure, which can be used to study the functional activities of RNA molecules.

RNACompress: Grammar-based compression and informational complexity measurement of RNA secondary structure

PubMed Central

Liu, Qi; Yang, Yu; Chen, Chun; Bu, Jiajun; Zhang, Yin; Ye, Xiuzi

2008-01-01

Background With the rapid emergence of RNA databases and newly identified non-coding RNAs, an efficient compression algorithm for RNA sequence and structural information is needed for the storage and analysis of such data. Although several algorithms for compressing DNA sequences have been proposed, none of them are suitable for the compression of RNA sequences with their secondary structures simultaneously. This kind of compression not only facilitates the maintenance of RNA data, but also supplies a novel way to measure the informational complexity of RNA structural data, raising the possibility of studying the relationship between the functional activities of RNA structures and their complexities, as well as various structural properties of RNA based on compression. Results RNACompress employs an efficient grammar-based model to compress RNA sequences and their secondary structures. The main goals of this algorithm are two fold: (1) present a robust and effective way for RNA structural data compression; (2) design a suitable model to represent RNA secondary structure as well as derive the informational complexity of the structural data based on compression. Our extensive tests have shown that RNACompress achieves a universally better compression ratio compared with other sequence-specific or common text-specific compression algorithms, such as Gencompress, winrar and gzip. Moreover, a test of the activities of distinct GTP-binding RNAs (aptamers) compared with their structural complexity shows that our defined informational complexity can be used to describe how complexity varies with activity. These results lead to an objective means of comparing the functional properties of heteropolymers from the information perspective. Conclusion A universal algorithm for the compression of RNA secondary structure as well as the evaluation of its informational complexity is discussed in this paper. We have developed RNACompress, as a useful tool for academic users. Extensive tests have shown that RNACompress is a universally efficient algorithm for the compression of RNA sequences with their secondary structures. RNACompress also serves as a good measurement of the informational complexity of RNA secondary structure, which can be used to study the functional activities of RNA molecules. PMID:18373878

Engineering multi-functional bacterial outer membrane vesicles as modular nanodevices for biosensing and bioimaging.

PubMed

Chen, Qi; Rozovsky, Sharon; Chen, Wilfred

2017-07-04

Outer membrane vesicles (OMVs) are proteoliposomes derived from the outer membrane and periplasmic space of many Gram-negative bacteria including E. coli as part of their natural growth cycle. Inspired by the natural ability of E. coli to sort proteins to both the exterior and interior of OMVs, we reported here a one-pot synthesis approach to engineer multi-functionalized OMV-based sensors for both antigen binding and signal generation. SlyB, a native lipoprotein, was used a fusion partner to package nanoluciferase (Nluc) within OMVs, while a previously developed INP-Scaf3 surface scaffold was fused to the Z-domain for antibody recruiting. The multi-functionalized OMVs were used for thrombin detection with a detection limit of 0.5 nM, comparable to other detection methods. Using the cohesin domains inserted between the Z-domain and INP, these engineered OMVs were further functionalized with a dockerin-tagged GFP for cancer cell imaging.

Centromere pairing precedes meiotic chromosome pairing in plants.

PubMed

Zhang, Jing; Han, Fangpu

2017-11-01

Meiosis is a specialized eukaryotic cell division, in which diploid cells undergo a single round of DNA replication and two rounds of nuclear division to produce haploid gametes. In most eukaryotes, the core events of meiotic prophase I are chromosomal pairing, synapsis and recombination. To ensure accurate chromosomal segregation, homologs have to identify and align along each other at the onset of meiosis. Although much progress has been made in elucidating meiotic processes, information on the mechanisms underlying chromosome pairing is limited in contrast to the meiotic recombination and synapsis events. Recent research in many organisms indicated that centromere interactions during early meiotic prophase facilitate homologous chromosome pairing, and functional centromere is a prerequisite for centromere pairing such as in maize. Here, we summarize the recent achievements of chromosome pairing research on plants and other organisms, and outline centromere interactions, nuclear chromosome orientation, and meiotic cohesin, as main determinants of chromosome pairing in early meiotic prophase.

Centromeric Heterochromatin: The Primordial Segregation Machine

PubMed Central

Bloom, Kerry S.

2014-01-01

Centromeres are specialized domains of heterochromatin that provide the foundation for the kinetochore. Centromeric heterochromatin is characterized by specific histone modifications, a centromere-specific histone H3 variant (CENP-A), and the enrichment of cohesin, condensin, and topo-isomerase II. Centromere DNA varies orders of magnitude in size from 125 bp (budding yeast) to several megabases (human). In metaphase, sister kinetochores on the surface of replicated chromosomes face away from each other, where they establish microtubule attachment and bi-orientation. Despite the disparity in centromere size, the distance between separated sister kinetochores is remarkably conserved (approximately 1 μm) throughout phylogeny. The centromere functions as a molecular spring that resists microtubule-based extensional forces in mitosis. This review explores the physical properties of DNA in order to understand how the molecular spring is built and how it contributes to the fidelity of chromosome segregation. PMID:25251850

Deep sequencing with intronic capture enables identification of an APC exon 10 inversion in a patient with polyposis.

PubMed

Shirts, Brian H; Salipante, Stephen J; Casadei, Silvia; Ryan, Shawnia; Martin, Judith; Jacobson, Angela; Vlaskin, Tatyana; Koehler, Karen; Livingston, Robert J; King, Mary-Claire; Walsh, Tom; Pritchard, Colin C

2014-10-01

Single-exon inversions have rarely been described in clinical syndromes and are challenging to detect using Sanger sequencing. We report the case of a 40-year-old woman with adenomatous colon polyps too numerous to count and who had a complex inversion spanning the entire exon 10 in APC (the gene encoding for adenomatous polyposis coli), causing exon skipping and resulting in a frameshift and premature protein truncation. In this study, we employed complete APC gene sequencing using high-coverage next-generation sequencing by ColoSeq, analysis with BreakDancer and SLOPE software, and confirmatory transcript analysis. ColoSeq identified a complex small genomic rearrangement consisting of an inversion that results in translational skipping of exon 10 in the APC gene. This mutation would not have been detected by traditional sequencing or gene-dosage methods. We report a case of adenomatous polyposis resulting from a complex single-exon inversion. Our report highlights the benefits of large-scale sequencing methods that capture intronic sequences with high enough depth of coverage-as well as the use of informatics tools-to enable detection of small pathogenic structural rearrangements.

Short-Range Temporal Interactions in Sleep; Hippocampal Spike Avalanches Support a Large Milieu of Sequential Activity Including Replay

PubMed Central

Mahoney, J. Matthew; Titiz, Ali S.; Hernan, Amanda E.; Scott, Rod C.

2016-01-01

Hippocampal neural systems consolidate multiple complex behaviors into memory. However, the temporal structure of neural firing supporting complex memory consolidation is unknown. Replay of hippocampal place cells during sleep supports the view that a simple repetitive behavior modifies sleep firing dynamics, but does not explain how multiple episodes could be integrated into associative networks for recollection during future cognition. Here we decode sequential firing structure within spike avalanches of all pyramidal cells recorded in sleeping rats after running in a circular track. We find that short sequences that combine into multiple long sequences capture the majority of the sequential structure during sleep, including replay of hippocampal place cells. The ensemble, however, is not optimized for maximally producing the behavior-enriched episode. Thus behavioral programming of sequential correlations occurs at the level of short-range interactions, not whole behavioral sequences and these short sequences are assembled into a large and complex milieu that could support complex memory consolidation. PMID:26866597

3D RNA and functional interactions from evolutionary couplings

PubMed Central

Weinreb, Caleb; Riesselman, Adam; Ingraham, John B.; Gross, Torsten; Sander, Chris; Marks, Debora S.

2016-01-01

Summary Non-coding RNAs are ubiquitous, but the discovery of new RNA gene sequences far outpaces research on their structure and functional interactions. We mine the evolutionary sequence record to derive precise information about function and structure of RNAs and RNA-protein complexes. As in protein structure prediction, we use maximum entropy global probability models of sequence co-variation to infer evolutionarily constrained nucleotide-nucleotide interactions within RNA molecules, and nucleotide-amino acid interactions in RNA-protein complexes. The predicted contacts allow all-atom blinded 3D structure prediction at good accuracy for several known RNA structures and RNA-protein complexes. For unknown structures, we predict contacts in 160 non-coding RNA families. Beyond 3D structure prediction, evolutionary couplings help identify important functional interactions, e.g., at switch points in riboswitches and at a complex nucleation site in HIV. Aided by accelerating sequence accumulation, evolutionary coupling analysis can accelerate the discovery of functional interactions and 3D structures involving RNA. PMID:27087444

Complex dissemination of the diversified mcr-1-harbouring plasmids in Escherichia coli of different sequence types

PubMed Central

Lin, Jingxia; Wang, Xiuna; Deng, Xianbo; Feng, Youjun

2016-01-01

The emergence of the mobilized colistin resistance gene, representing a novel mechanism for bacterial drug resistance, challenges the last resort against the severe infections by Gram-negative bacteria with multi-drug resistances. Very recently, we showed the diversity in the mcr-1-carrying plasmid reservoirs from the gut microbiota. Here, we reported that a similar but more complex scenario is present in the healthy swine populations, Southern China, 2016. Amongst the 1026 pieces of Escherichia coli isolates from 3 different pig farms, 302 E. coli isolates were determined to be positive for the mcr-1 gene (30%, 302/1026). Multi-locus sequence typing assigned no less than 11 kinds of sequence types including one novel Sequence Type to these mcr-1-positive strains. PCR analyses combined with the direct DNA sequencing revealed unexpected complexity of the mcr-1-harbouring plasmids whose backbones are at least grouped into 6 types four of which are new. Transcriptional analyses showed that the mcr-1 promoter of different origins exhibits similar activity. It seems likely that complex dissemination of the diversified mcr-1-bearing plasmids occurs amongst the various ST E. coli inhabiting the healthy swine populations, in Southern China. PMID:27741523

Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

PubMed

Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan; Tanaka Hall, Traci M; Goldstrohm, Aaron C; Campbell, Zachary T

2017-04-15

RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA. Copyright © 2016 Elsevier Inc. All rights reserved.

Ordovician volcanic and plutonic complexes of the Sakmara allochthon in the southern Urals

NASA Astrophysics Data System (ADS)

Ryazantsev, A. V.; Tolmacheva, T. Yu.

2016-11-01

The Ordovician terrigenous, volcanic-sedimentary and volcanic sequences that formed in rifts of the active continental margin and igneous complexes of intraoceanic suprasubduction settings structurally related to ophiolites are closely spaced in allochthons of the Sakmara Zone in the southern Urals. The stratigraphic relationships of the Ordovician sequences have been established. Their age and facies features have been specified on the basis of biostratigraphic and geochronological data. The gabbro-tonalite-trondhjemite complex and the basalt-andesite-rhyolite sequence with massive sulfide mineralization make up a volcanic-plutonic association. These rock complexes vary in age from Late Ordovician to Early Silurian in certain structural units of the Sakmara Allochthon and to the east in the southern Urals. The proposed geodynamic model for the Ordovician in Paleozoides of the southern Urals reconstructs the active continental margin, whose complexes formed under extension settings, and the intraoceanic suprasubduction structures. The intraoceanic complexes display the evolution of a volcanic arc, back-, or interarc trough.

An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes

NASA Astrophysics Data System (ADS)

Li, Huilin; Nguyen, Hong Hanh; Ogorzalek Loo, Rachel R.; Campuzano, Iain D. G.; Loo, Joseph A.

2018-02-01

Mass spectrometry (MS) has become a crucial technique for the analysis of protein complexes. Native MS has traditionally examined protein subunit arrangements, while proteomics MS has focused on sequence identification. These two techniques are usually performed separately without taking advantage of the synergies between them. Here we describe the development of an integrated native MS and top-down proteomics method using Fourier-transform ion cyclotron resonance (FTICR) to analyse macromolecular protein complexes in a single experiment. We address previous concerns of employing FTICR MS to measure large macromolecular complexes by demonstrating the detection of complexes up to 1.8 MDa, and we demonstrate the efficacy of this technique for direct acquirement of sequence to higher-order structural information with several large complexes. We then summarize the unique functionalities of different activation/dissociation techniques. The platform expands the ability of MS to integrate proteomics and structural biology to provide insights into protein structure, function and regulation.

Structurally complex and highly active RNA ligases derived from random RNA sequences

NASA Technical Reports Server (NTRS)

Ekland, E. H.; Szostak, J. W.; Bartel, D. P.

1995-01-01

Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.

Multicatalytic asymmetric synthesis of complex tetrahydrocarbazoles via a Diels-Alder/benzoin reaction sequence.

PubMed

Liu, Yankai; Nappi, Manuel; Escudero-Adán, Eduardo C; Melchiorre, Paolo

2012-03-02

Expanding upon the recently developed aminocatalytic asymmetric indole-2,3-quinodimethane strategy, a straightforward synthesis of structurally and stereochemically complex tetrahydrocarbazoles has been devised. The chemistry's complexity-generating power was further harnessed by designing a multicatalytic, one-pot Diels-Alder/benzoin reaction sequence to stereoselectively access trans-fused tetracyclic indole-based compounds having four stereogenic centers with very high fidelity. © 2012 American Chemical Society

Structure of homeodomain-leucine zipper/DNA complexes studied using hydroxyl radical cleavage of DNA and methylation interference.

PubMed

Tron, Adriana E; Comelli, Raúl N; Gonzalez, Daniel H

2005-12-27

Homeodomain-leucine zipper (HD-Zip) proteins, unlike most homeodomain proteins, bind a pseudopalindromic DNA sequence as dimers. We have investigated the structure of the DNA complexes formed by two HD-Zip proteins with different nucleotide preferences at the central position of the binding site using footprinting and interference methods. The results indicate that the respective complexes are not symmetric, with the strand bearing a central purine (top strand) showing higher protection around the central region and the bottom strand protected toward the 3' end. Binding to a sequence with a nonpreferred central base pair produces a decrease in protection in either the top or the bottom strand, depending upon the protein. Modeling studies derived from the complex formed by the monomeric Antennapedia homeodomain with DNA indicate that in the HD-Zip/DNA complex the recognition helix of one of the monomers is displaced within the major groove respective to the other one. This monomer seems to lose contacts with a part of the recognition sequence upon binding to the nonpreferred site. The results show that the structure of the complex formed by HD-Zip proteins with DNA is dependent upon both protein intrinsic characteristics and the nucleotides present at the central position of the recognition sequence.

Application of population sequencing (POPSEQ) for ordering and inputting genotyping-by-sequencing markers in hexaploid wheat

USDA-ARS?s Scientific Manuscript database

The advancement of next-generation sequencing technologies in conjunction with new bioinformatics tools enabled fine-tuning of sequence-based high resolution mapping strategies for complex genomes. Although genotyping-by-sequencing (GBS) provides a large number of markers, its application for assoc...

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

PubMed Central

Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

1998-01-01

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

SPARSE: quadratic time simultaneous alignment and folding of RNAs without sequence-based heuristics.

PubMed

Will, Sebastian; Otto, Christina; Miladi, Milad; Möhl, Mathias; Backofen, Rolf

2015-08-01

RNA-Seq experiments have revealed a multitude of novel ncRNAs. The gold standard for their analysis based on simultaneous alignment and folding suffers from extreme time complexity of [Formula: see text]. Subsequently, numerous faster 'Sankoff-style' approaches have been suggested. Commonly, the performance of such methods relies on sequence-based heuristics that restrict the search space to optimal or near-optimal sequence alignments; however, the accuracy of sequence-based methods breaks down for RNAs with sequence identities below 60%. Alignment approaches like LocARNA that do not require sequence-based heuristics, have been limited to high complexity ([Formula: see text] quartic time). Breaking this barrier, we introduce the novel Sankoff-style algorithm 'sparsified prediction and alignment of RNAs based on their structure ensembles (SPARSE)', which runs in quadratic time without sequence-based heuristics. To achieve this low complexity, on par with sequence alignment algorithms, SPARSE features strong sparsification based on structural properties of the RNA ensembles. Following PMcomp, SPARSE gains further speed-up from lightweight energy computation. Although all existing lightweight Sankoff-style methods restrict Sankoff's original model by disallowing loop deletions and insertions, SPARSE transfers the Sankoff algorithm to the lightweight energy model completely for the first time. Compared with LocARNA, SPARSE achieves similar alignment and better folding quality in significantly less time (speedup: 3.7). At similar run-time, it aligns low sequence identity instances substantially more accurate than RAF, which uses sequence-based heuristics. © The Author 2015. Published by Oxford University Press.

Isolation and characterization of a virus infecting the freshwater algae Chrysochromulina parva

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mirza, S.F.; Staniewski, M.A.; Short, C.M.

Water samples from Lake Ontario, Canada were tested for lytic activity against the freshwater haptophyte algae Chrysochromulina parva. A filterable lytic agent was isolated and identified as a virus via transmission electron microscopy and molecular methods. The virus, CpV-BQ1, is icosahedral, ca. 145 nm in diameter, assembled within the cytoplasm, and has a genome size of ca. 485 kb. Sequences obtained through PCR-amplification of DNA polymerase (polB) genes clustered among sequences from the family Phycodnaviridae, whereas major capsid protein (MCP) sequences clustered among sequences from either the Phycodnaviridae or Mimiviridae. Based on quantitative molecular assays, C. parva's abundance in Lakemore » Ontario was relatively stable, yet CpV-BQ1's abundance was variable suggesting complex virus-host dynamics. This study demonstrates that CpV-BQ1 is a member of the proposed order Megavirales with characteristics of both phycodnaviruses and mimiviruses indicating that, in addition to its complex ecological dynamics, it also has a complex evolutionary history. - Highlights: • A virus infecting the algae C. parva was isolated from Lake Ontario. • Virus characteristics demonstrated that this novel virus is an NCLDV. • The virus's polB sequence suggests taxonomic affiliation with the Phycodnaviridae. • The virus's capsid protein sequences also suggest Mimiviridae ancestry. • Surveys of host and virus natural abundances revealed complex host–virus dynamics.« less

Novel species including Mycobacterium fukienense sp. is found from tuberculosis patients in Fujian Province, China, using phylogenetic analysis of Mycobacterium chelonae/abscessus complex.

PubMed

Zhang, Yuan Yuan; Li, Yan Bing; Huang, Ming Xiang; Zhao, Xiu Qin; Zhang, Li Shui; Liu, Wen En; Wan, Kang Lin

2013-11-01

To identify the novel species 'Mycobacterium fukienense' sp. nov of Mycobacterium chelonae/abscessus complex from tuberculosis patients in Fujian Province, China. Five of 27 clinical Mycobacterium isolates (Cls) were previously identified as M. chelonae/abscessus complex by sequencing the hsp65, rpoB, 16S-23S rRNA internal transcribed spacer region (its), recA and sodA house-keeping genes commonly used to describe the molecular characteristics of Mycobacterium. Clinical Mycobacterium isolates were classified according to the gene sequence using a clustering analysis program. Sequence similarity within clusters and diversity between clusters were analyzed. The 5 isolates were identified with distinct sequences exhibiting 99.8% homology in the hsp65 gene. However, a complete lack of homology was observed among the sequences of the rpoB, 16S-23S rRNA internal transcribed spacer region (its), sodA, and recA genes as compared with the M. abscessus. Furthermore, no match for rpoB, sodA, and recA genes was identified among the published sequences. The novel species, Mycobacterium fukienense, is identified from tuberculosis patients in Fujian Province, China, which does not belong to any existing subspecies of M. chelonea/abscessus complex. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Haplotype resolution of leukocyte receptor complex in cattle through targeted enrichment and SMRT sequencing

USDA-ARS?s Scientific Manuscript database

The highly repetitive nature of cattle leukocyte receptor complex (LRC) has made it difficult to assemble and fully characterize this region with short reads used by second-generation sequencing. Previously, we reported the first two cattle killer immunoglobulin-like receptors (KIR) haplotypes; one ...

Structure-Function, Stability, and Chemical Modification of the Cyanobacterial Cytochrome b6f Complex from Nostoc sp. PCC 7120*

PubMed Central

Baniulis, Danas; Yamashita, Eiki; Whitelegge, Julian P.; Zatsman, Anna I.; Hendrich, Michael P.; Hasan, S. Saif; Ryan, Christopher M.; Cramer, William A.

2009-01-01

The crystal structure of the cyanobacterial cytochrome b6f complex has previously been solved to 3.0-Å resolution using the thermophilic Mastigocladus laminosus whose genome has not been sequenced. Several unicellular cyanobacteria, whose genomes have been sequenced and are tractable for mutagenesis, do not yield b6f complex in an intact dimeric state with significant electron transport activity. The genome of Nostoc sp. PCC 7120 has been sequenced and is closer phylogenetically to M. laminosus than are unicellular cyanobacteria. The amino acid sequences of the large core subunits and four small peripheral subunits of Nostoc are 88 and 80% identical to those in the M. laminosus b6f complex. Purified b6f complex from Nostoc has a stable dimeric structure, eight subunits with masses similar to those of M. laminosus, and comparable electron transport activity. The crystal structure of the native b6f complex, determined to a resolution of 3.0Å (PDB id: 2ZT9), is almost identical to that of M. laminosus. Two unique aspects of the Nostoc complex are: (i) a dominant conformation of heme bp that is rotated 180° about the α- and γ-meso carbon axis relative to the orientation in the M. laminosus complex and (ii) acetylation of the Rieske iron-sulfur protein (PetC) at the N terminus, a post-translational modification unprecedented in cyanobacterial membrane and electron transport proteins, and in polypeptides of cytochrome bc complexes from any source. The high spin electronic character of the unique heme cn is similar to that previously found in the b6f complex from other sources. PMID:19189962

Structure-Function, Stability, and Chemical Modification of the Cyanobacterial Cytochrome b[subscript 6]f Complex from Nostoc sp. PCC 7120

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baniulis, Danas; Yamashita, Eiki; Whitelegge, Julian P.

2009-06-08

The crystal structure of the cyanobacterial cytochrome b{sub 6}f complex has previously been solved to 3.0-{angstrom} resolution using the thermophilic Mastigocladus laminosus whose genome has not been sequenced. Several unicellular cyanobacteria, whose genomes have been sequenced and are tractable for mutagenesis, do not yield b{sub 6}f complex in an intact dimeric state with significant electron transport activity. The genome of Nostoc sp. PCC 7120 has been sequenced and is closer phylogenetically to M. laminosus than are unicellular cyanobacteria. The amino acid sequences of the large core subunits and four small peripheral subunits of Nostoc are 88 and 80% identical tomore » those in the M. laminosus b{sub 6}f complex. Purified b{sub 6}f complex from Nostoc has a stable dimeric structure, eight subunits with masses similar to those of M. laminosus, and comparable electron transport activity. The crystal structure of the native b{sub 6}f complex, determined to a resolution of 3.0{angstrom} (PDB id: 2ZT9), is almost identical to that of M. laminosus. Two unique aspects of the Nostoc complex are: (i) a dominant conformation of heme b{sub p} that is rotated 180 deg. about the {alpha}- and {gamma}-meso carbon axis relative to the orientation in the M. laminosus complex and (ii) acetylation of the Rieske iron-sulfur protein (PetC) at the N terminus, a post-translational modification unprecedented in cyanobacterial membrane and electron transport proteins, and in polypeptides of cytochrome bc complexes from any source. The high spin electronic character of the unique heme cn is similar to that previously found in the b{sub 6}f complex from other sources.« less

Improving protein complex classification accuracy using amino acid composition profile.

PubMed

Huang, Chien-Hung; Chou, Szu-Yu; Ng, Ka-Lok

2013-09-01

Protein complex prediction approaches are based on the assumptions that complexes have dense protein-protein interactions and high functional similarity between their subunits. We investigated those assumptions by studying the subunits' interaction topology, sequence similarity and molecular function for human and yeast protein complexes. Inclusion of amino acids' physicochemical properties can provide better understanding of protein complex properties. Principal component analysis is carried out to determine the major features. Adopting amino acid composition profile information with the SVM classifier serves as an effective post-processing step for complexes classification. Improvement is based on primary sequence information only, which is easy to obtain. Copyright © 2013 Elsevier Ltd. All rights reserved.

The spatial proximity effect of beta-glucosidase and cellulosomes on cellulose degradation.

PubMed

Li, Xiaoyi; Xiao, Yan; Feng, Yingang; Li, Bin; Li, Wenli; Cui, Qiu

2018-08-01

Low-cost saccharification is one of the key bottlenecks hampering the further application of lignocellulosic biomass. Clostridium thermocellum is a naturally ideal cellulose degrading bacterium armed with cellulosomes, which are multienzyme complexes that are capable of efficiently degrading cellulose. However, under controlled condition, the inhibition effect of hydrolysate cellobiose severely restricts the hydrolytic ability of cellulosomes. Although the addition of beta-glucosidase (Bgl) could effectively relieve this inhibition, the spatial proximity effect of Bgl and cellulosomes on cellulose degradation is still unclear. To address this issue, free Bgl from Caldicellulosiruptor sp. F32 (CaBglA), carbohydrate-binding module (CBM) fused CaBglA (CaBglA-CBM) and cellulosomal type II cohesin module (CohII) fused to CaBglA (CaBglA-CohII) were successfully constructed, and their enzymatic activities, binding abilities and saccharification efficiencies were systematically investigated in vitro and in vivo. In vivo, with the adjacency of CaBglA to cellulosomes, the saccharification efficiency of microcrystalline cellulose increased from 40% to 50%. For the pretreated wheat straw, the degradation rate of the combination of cells and the CaBglA-CohII or the CaBglA-CBM was as efficient as that of the free CaBglA (approximately 60%). This study demonstrated that the proximity of CaBglA to cellulosomes had a positive effect on microcrystalline cellulose but not on pretreated wheat straw, which may result from the nonproductive adsorption of lignin and the decreased thermostability of CaBglA-CBM and CaBglA-CohII compared to that of CaBglA. The above results will contribute to the design of cost-effective Bgls for industrial cellulose degradation. Copyright © 2018. Published by Elsevier Inc.

Nipbl and mediator cooperatively regulate gene expression to control limb development.

PubMed

Muto, Akihiko; Ikeda, Shingo; Lopez-Burks, Martha E; Kikuchi, Yutaka; Calof, Anne L; Lander, Arthur D; Schilling, Thomas F

2014-09-01

Haploinsufficiency for Nipbl, a cohesin loading protein, causes Cornelia de Lange Syndrome (CdLS), the most common "cohesinopathy". It has been proposed that the effects of Nipbl-haploinsufficiency result from disruption of long-range communication between DNA elements. Here we use zebrafish and mouse models of CdLS to examine how transcriptional changes caused by Nipbl deficiency give rise to limb defects, a common condition in individuals with CdLS. In the zebrafish pectoral fin (forelimb), knockdown of Nipbl expression led to size reductions and patterning defects that were preceded by dysregulated expression of key early limb development genes, including fgfs, shha, hand2 and multiple hox genes. In limb buds of Nipbl-haploinsufficient mice, transcriptome analysis revealed many similar gene expression changes, as well as altered expression of additional classes of genes that play roles in limb development. In both species, the pattern of dysregulation of hox-gene expression depended on genomic location within the Hox clusters. In view of studies suggesting that Nipbl colocalizes with the mediator complex, which facilitates enhancer-promoter communication, we also examined zebrafish deficient for the Med12 Mediator subunit, and found they resembled Nipbl-deficient fish in both morphology and gene expression. Moreover, combined partial reduction of both Nipbl and Med12 had a strongly synergistic effect, consistent with both molecules acting in a common pathway. In addition, three-dimensional fluorescent in situ hybridization revealed that Nipbl and Med12 are required to bring regions containing long-range enhancers into close proximity with the zebrafish hoxda cluster. These data demonstrate a crucial role for Nipbl in limb development, and support the view that its actions on multiple gene pathways result from its influence, together with Mediator, on regulation of long-range chromosomal interactions.

Unusual association of non-anaplastic Wilms tumor and Cornelia de Lange syndrome: case report.

PubMed

Santoro, Claudia; Apicella, Andrea; Casale, Fiorina; La Manna, Angela; Di Martino, Martina; Di Pinto, Daniela; Indolfi, Cristiana; Perrotta, Silverio

2016-06-13

Cornelia de Lange syndrome is the prototype for cohesinopathy disorders, which are characterized by defects in chromosome segregation. Kidney malformations, including nephrogenic rests, are common in Cornelia de Lange syndrome. Only one post-mortem case report has described an association between Wilms tumor and Cornelia de Lange syndrome. Here, we describe the first case of a living child with both diseases. Non-anaplastic triphasic nephroblastoma was diagnosed in a patient carrying a not yet reported mutation in NIPBL (c.4920 G > A). The patient had the typical facial appearance and intellectual disability associated with Cornelia de Lange syndrome in absence of limb involvement. The child's kidneys were examined by ultrasound at 2 years of age to exclude kidney abnormalities associated with the syndrome. She underwent pre-operative chemotherapy and nephrectomy. Seven months later she was healthy and without residual detectable disease. The previous report of such co-occurrence, together with our report and previous reports of nephrogenic rests, led us to wonder if there may be any causal relationship between these two rare entities. The wingless/integrated (Wnt) pathway, which is implicated in kidney development, is constitutively activated in approximately 15-20 % of all non-anaplastic Wilms tumors. Interestingly, the Wnt pathway was recently found to be perturbed in a zebrafish model of Cornelia de Lange syndrome. Mutations in cohesin complex genes and regulators have also been identified in several types of cancers. On the other hand, there is no clear evidence of an increased risk of cancer in Cornelia de Lange syndrome, and no other similar cases have been published since the fist one reported by Cohen, and this prompts to think Wilms tumor and Cornelia de Lange syndrome occurred together in our patient by chance.

Complete Genome Sequence of the Complex Carbohydrate-Degrading Marine Bacterium, Saccharophagus degradans Strain 2-40T

PubMed Central

Weiner, Ronald M.; Taylor, Larry E.; Henrissat, Bernard; Hauser, Loren; Land, Miriam; Coutinho, Pedro M.; Rancurel, Corinne; Saunders, Elizabeth H.; Longmire, Atkinson G.; Zhang, Haitao; Bayer, Edward A.; Gilbert, Harry J.; Larimer, Frank; Zhulin, Igor B.; Ekborg, Nathan A.; Lamed, Raphael; Richardson, Paul M.; Borovok, Ilya; Hutcheson, Steven

2008-01-01

The marine bacterium Saccharophagus degradans strain 2-40 (Sde 2-40) is emerging as a vanguard of a recently discovered group of marine and estuarine bacteria that recycles complex polysaccharides. We report its complete genome sequence, analysis of which identifies an unusually large number of enzymes that degrade >10 complex polysaccharides. Not only is this an extraordinary range of catabolic capability, many of the enzymes exhibit unusual architecture including novel combinations of catalytic and substrate-binding modules. We hypothesize that many of these features are adaptations that facilitate depolymerization of complex polysaccharides in the marine environment. This is the first sequenced genome of a marine bacterium that can degrade plant cell walls, an important component of the carbon cycle that is not well-characterized in the marine environment. PMID:18516288

Learning predictive statistics from temporal sequences: Dynamics and strategies

PubMed Central

Wang, Rui; Shen, Yuan; Tino, Peter; Welchman, Andrew E.; Kourtzi, Zoe

2017-01-01

Human behavior is guided by our expectations about the future. Often, we make predictions by monitoring how event sequences unfold, even though such sequences may appear incomprehensible. Event structures in the natural environment typically vary in complexity, from simple repetition to complex probabilistic combinations. How do we learn these structures? Here we investigate the dynamics of structure learning by tracking human responses to temporal sequences that change in structure unbeknownst to the participants. Participants were asked to predict the upcoming item following a probabilistic sequence of symbols. Using a Markov process, we created a family of sequences, from simple frequency statistics (e.g., some symbols are more probable than others) to context-based statistics (e.g., symbol probability is contingent on preceding symbols). We demonstrate the dynamics with which individuals adapt to changes in the environment's statistics—that is, they extract the behaviorally relevant structures to make predictions about upcoming events. Further, we show that this structure learning relates to individual decision strategy; faster learning of complex structures relates to selection of the most probable outcome in a given context (maximizing) rather than matching of the exact sequence statistics. Our findings provide evidence for alternate routes to learning of behaviorally relevant statistics that facilitate our ability to predict future events in variable environments. PMID:28973111

Learning predictive statistics from temporal sequences: Dynamics and strategies.

PubMed

Wang, Rui; Shen, Yuan; Tino, Peter; Welchman, Andrew E; Kourtzi, Zoe

2017-10-01

Human behavior is guided by our expectations about the future. Often, we make predictions by monitoring how event sequences unfold, even though such sequences may appear incomprehensible. Event structures in the natural environment typically vary in complexity, from simple repetition to complex probabilistic combinations. How do we learn these structures? Here we investigate the dynamics of structure learning by tracking human responses to temporal sequences that change in structure unbeknownst to the participants. Participants were asked to predict the upcoming item following a probabilistic sequence of symbols. Using a Markov process, we created a family of sequences, from simple frequency statistics (e.g., some symbols are more probable than others) to context-based statistics (e.g., symbol probability is contingent on preceding symbols). We demonstrate the dynamics with which individuals adapt to changes in the environment's statistics-that is, they extract the behaviorally relevant structures to make predictions about upcoming events. Further, we show that this structure learning relates to individual decision strategy; faster learning of complex structures relates to selection of the most probable outcome in a given context (maximizing) rather than matching of the exact sequence statistics. Our findings provide evidence for alternate routes to learning of behaviorally relevant statistics that facilitate our ability to predict future events in variable environments.

Structural and functional analysis of an enhancer GPEI having a phorbol 12-O-tetradecanoate 13-acetate responsive element-like sequence found in the rat glutathione transferase P gene.

PubMed

Okuda, A; Imagawa, M; Maeda, Y; Sakai, M; Muramatsu, M

1989-10-05

We have recently identified a typical enhancer, termed GPEI, located about 2.5 kilobases upstream from the transcription initiation site of the rat glutathione transferase P gene. Analyses of 5' and 3' deletion mutants revealed that the cis-acting sequence of GPEI contained the phorbol 12-O-tetradecanoate 13-acetate responsive element (TRE)-like sequence in it. For the maximal activity, however, GPEI required an adjacent upstream sequence of about 19 base pairs in addition to the TRE-like sequence. With the DNA binding gel-shift assay, we could detect protein(s) that specifically binds to the TRE-like sequence of GPEI fragment, which was possibly c-jun.c-fos complex or a similar protein complex. The sequence immediately upstream of the TRE-like sequence did not have any activity by itself, but augmented the latter activity by about 5-fold.

Determination of haplotypes at structurally complex regions using emulsion haplotype fusion PCR.

PubMed

Tyson, Jess; Armour, John A L

2012-12-11

Genotyping and massively-parallel sequencing projects result in a vast amount of diploid data that is only rarely resolved into its constituent haplotypes. It is nevertheless this phased information that is transmitted from one generation to the next and is most directly associated with biological function and the genetic causes of biological effects. Despite progress made in genome-wide sequencing and phasing algorithms and methods, problems assembling (and reconstructing linear haplotypes in) regions of repetitive DNA and structural variation remain. These dynamic and structurally complex regions are often poorly understood from a sequence point of view. Regions such as these that are highly similar in their sequence tend to be collapsed onto the genome assembly. This is turn means downstream determination of the true sequence haplotype in these regions poses a particular challenge. For structurally complex regions, a more focussed approach to assembling haplotypes may be required. In order to investigate reconstruction of spatial information at structurally complex regions, we have used an emulsion haplotype fusion PCR approach to reproducibly link sequences of up to 1kb in length to allow phasing of multiple variants from neighbouring loci, using allele-specific PCR and sequencing to detect the phase. By using emulsion systems linking flanking regions to amplicons within the CNV, this led to the reconstruction of a 59kb haplotype across the DEFA1A3 CNV in HapMap individuals. This study has demonstrated a novel use for emulsion haplotype fusion PCR in addressing the issue of reconstructing structural haplotypes at multiallelic copy variable regions, using the DEFA1A3 locus as an example.

Guiding principles for peptide nanotechnology through directed discovery.

PubMed

Lampel, A; Ulijn, R V; Tuttle, T

2018-05-21

Life's diverse molecular functions are largely based on only a small number of highly conserved building blocks - the twenty canonical amino acids. These building blocks are chemically simple, but when they are organized in three-dimensional structures of tremendous complexity, new properties emerge. This review explores recent efforts in the directed discovery of functional nanoscale systems and materials based on these same amino acids, but that are not guided by copying or editing biological systems. The review summarises insights obtained using three complementary approaches of searching the sequence space to explore sequence-structure relationships for assembly, reactivity and complexation, namely: (i) strategic editing of short peptide sequences; (ii) computational approaches to predicting and comparing assembly behaviours; (iii) dynamic peptide libraries that explore the free energy landscape. These approaches give rise to guiding principles on controlling order/disorder, complexation and reactivity by peptide sequence design.

Earthquake forecasting during the complex Amatrice-Norcia seismic sequence

PubMed Central

Marzocchi, Warner; Taroni, Matteo; Falcone, Giuseppe

2017-01-01

Earthquake forecasting is the ultimate challenge for seismologists, because it condenses the scientific knowledge about the earthquake occurrence process, and it is an essential component of any sound risk mitigation planning. It is commonly assumed that, in the short term, trustworthy earthquake forecasts are possible only for typical aftershock sequences, where the largest shock is followed by many smaller earthquakes that decay with time according to the Omori power law. We show that the current Italian operational earthquake forecasting system issued statistically reliable and skillful space-time-magnitude forecasts of the largest earthquakes during the complex 2016–2017 Amatrice-Norcia sequence, which is characterized by several bursts of seismicity and a significant deviation from the Omori law. This capability to deliver statistically reliable forecasts is an essential component of any program to assist public decision-makers and citizens in the challenging risk management of complex seismic sequences. PMID:28924610

Earthquake forecasting during the complex Amatrice-Norcia seismic sequence.

PubMed

Marzocchi, Warner; Taroni, Matteo; Falcone, Giuseppe

2017-09-01

Earthquake forecasting is the ultimate challenge for seismologists, because it condenses the scientific knowledge about the earthquake occurrence process, and it is an essential component of any sound risk mitigation planning. It is commonly assumed that, in the short term, trustworthy earthquake forecasts are possible only for typical aftershock sequences, where the largest shock is followed by many smaller earthquakes that decay with time according to the Omori power law. We show that the current Italian operational earthquake forecasting system issued statistically reliable and skillful space-time-magnitude forecasts of the largest earthquakes during the complex 2016-2017 Amatrice-Norcia sequence, which is characterized by several bursts of seismicity and a significant deviation from the Omori law. This capability to deliver statistically reliable forecasts is an essential component of any program to assist public decision-makers and citizens in the challenging risk management of complex seismic sequences.

A complexity measure based method for studying the dependance of 222Rn concentration time series on indoor air temperature and humidity.

PubMed

Mihailovic, D T; Udovičić, V; Krmar, M; Arsenić, I

2014-02-01

We have suggested a complexity measure based method for studying the dependence of measured (222)Rn concentration time series on indoor air temperature and humidity. This method is based on the Kolmogorov complexity (KL). We have introduced (i) the sequence of the KL, (ii) the Kolmogorov complexity highest value in the sequence (KLM) and (iii) the KL of the product of time series. The noticed loss of the KLM complexity of (222)Rn concentration time series can be attributed to the indoor air humidity that keeps the radon daughters in air. © 2013 Published by Elsevier Ltd.

Grammatical complexity for two-dimensional maps

NASA Astrophysics Data System (ADS)

Hagiwara, Ryouichi; Shudo, Akira

2004-11-01

We calculate the grammatical complexity of the symbol sequences generated from the Hénon map and the Lozi map using the recently developed methods to construct the pruning front. When the map is hyperbolic, the language of symbol sequences is regular in the sense of the Chomsky hierarchy and the corresponding grammatical complexity takes finite values. It is found that the complexity exhibits a self-similar structure as a function of the system parameter, and the similarity of the pruning fronts is discussed as an origin of such self-similarity. For non-hyperbolic cases, it is observed that the complexity monotonically increases as we increase the resolution of the pruning front.

Cloning and expression of recombinant adhesive protein Mefp-1 of the blue mussel, Mytilus edulis

DOEpatents

Silverman, Heather G.; Roberto, Francisco F.

2006-01-17

The present invention comprises a Mytilus edulis cDNA sequenc having a nucleotide sequence that encodes for the Mytilus edulis foot protein-1 (Mefp-1), an example of a mollusk foot protein. Mefp-1 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-1 gene will allow researchers to produce Mefp-1 protein using genetic engineering techniques. The discovery of Mefp-1 gene sequence will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

Observing complex action sequences: The role of the fronto-parietal mirror neuron system.

PubMed

Molnar-Szakacs, Istvan; Kaplan, Jonas; Greenfield, Patricia M; Iacoboni, Marco

2006-11-15

A fronto-parietal mirror neuron network in the human brain supports the ability to represent and understand observed actions allowing us to successfully interact with others and our environment. Using functional magnetic resonance imaging (fMRI), we wanted to investigate the response of this network in adults during observation of hierarchically organized action sequences of varying complexity that emerge at different developmental stages. We hypothesized that fronto-parietal systems may play a role in coding the hierarchical structure of object-directed actions. The observation of all action sequences recruited a common bilateral network including the fronto-parietal mirror neuron system and occipito-temporal visual motion areas. Activity in mirror neuron areas varied according to the motoric complexity of the observed actions, but not according to the developmental sequence of action structures, possibly due to the fact that our subjects were all adults. These results suggest that the mirror neuron system provides a fairly accurate simulation process of observed actions, mimicking internally the level of motoric complexity. We also discuss the results in terms of the links between mirror neurons, language development and evolution.

Uncovering Neuronal Networks Defined by Consistent Between-Neuron Spike Timing from Neuronal Spike Recordings

PubMed Central

2018-01-01

Abstract It is widely assumed that distributed neuronal networks are fundamental to the functioning of the brain. Consistent spike timing between neurons is thought to be one of the key principles for the formation of these networks. This can involve synchronous spiking or spiking with time delays, forming spike sequences when the order of spiking is consistent. Finding networks defined by their sequence of time-shifted spikes, denoted here as spike timing networks, is a tremendous challenge. As neurons can participate in multiple spike sequences at multiple between-spike time delays, the possible complexity of networks is prohibitively large. We present a novel approach that is capable of (1) extracting spike timing networks regardless of their sequence complexity, and (2) that describes their spiking sequences with high temporal precision. We achieve this by decomposing frequency-transformed neuronal spiking into separate networks, characterizing each network’s spike sequence by a time delay per neuron, forming a spike sequence timeline. These networks provide a detailed template for an investigation of the experimental relevance of their spike sequences. Using simulated spike timing networks, we show network extraction is robust to spiking noise, spike timing jitter, and partial occurrences of the involved spike sequences. Using rat multineuron recordings, we demonstrate the approach is capable of revealing real spike timing networks with sub-millisecond temporal precision. By uncovering spike timing networks, the prevalence, structure, and function of complex spike sequences can be investigated in greater detail, allowing us to gain a better understanding of their role in neuronal functioning. PMID:29789811

Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato.

PubMed Central

Millar, A H; Knorpp, C; Leaver, C J; Hill, S A

1998-01-01

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) tuber mitochondria was purified 40-fold to a specific activity of 5.60 micromol/min per mg of protein. The activity of the complex depended on pyruvate, divalent cations, NAD+ and CoA and was competitively inhibited by both NADH and acetyl-CoA. SDS/PAGE revealed the complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa. N-terminal sequencing revealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were alpha subunits of pyruvate dehydrogenase, and the 37 kDa protein was the beta subunit of pyruvate dehydrogenase. N-terminal sequencing of the 55 kDa protein band yielded two protein sequences: one was another E3; the other was similar to the sequence of E2 from plant and yeast sources but was distinctly different from the sequence of the 78 kDa protein. Incubation of the mPDC with [2-14C]pyruvate resulted in the acetylation of both the 78 and 55 kDa proteins. PMID:9729464

Complex Sequencing Rules of Birdsong Can be Explained by Simple Hidden Markov Processes

PubMed Central

Katahira, Kentaro; Suzuki, Kenta; Okanoya, Kazuo; Okada, Masato

2011-01-01

Complex sequencing rules observed in birdsongs provide an opportunity to investigate the neural mechanism for generating complex sequential behaviors. To relate the findings from studying birdsongs to other sequential behaviors such as human speech and musical performance, it is crucial to characterize the statistical properties of the sequencing rules in birdsongs. However, the properties of the sequencing rules in birdsongs have not yet been fully addressed. In this study, we investigate the statistical properties of the complex birdsong of the Bengalese finch (Lonchura striata var. domestica). Based on manual-annotated syllable labeles, we first show that there are significant higher-order context dependencies in Bengalese finch songs, that is, which syllable appears next depends on more than one previous syllable. We then analyze acoustic features of the song and show that higher-order context dependencies can be explained using first-order hidden state transition dynamics with redundant hidden states. This model corresponds to hidden Markov models (HMMs), well known statistical models with a large range of application for time series modeling. The song annotation with these models with first-order hidden state dynamics agreed well with manual annotation, the score was comparable to that of a second-order HMM, and surpassed the zeroth-order model (the Gaussian mixture model; GMM), which does not use context information. Our results imply that the hierarchical representation with hidden state dynamics may underlie the neural implementation for generating complex behavioral sequences with higher-order dependencies. PMID:21915345

Methylene blue binding to DNA with alternating AT base sequence: minor groove binding is favored over intercalation.

PubMed

Rohs, Remo; Sklenar, Heinz

2004-04-01

The results presented in this paper on methylene blue (MB) binding to DNA with AT alternating base sequence complement the data obtained in two former modeling studies of MB binding to GC alternating DNA. In the light of the large amount of experimental data for both systems, this theoretical study is focused on a detailed energetic analysis and comparison in order to understand their different behavior. Since experimental high-resolution structures of the complexes are not available, the analysis is based on energy minimized structural models of the complexes in different binding modes. For both sequences, four different intercalation structures and two models for MB binding in the minor and major groove have been proposed. Solvent electrostatic effects were included in the energetic analysis by using electrostatic continuum theory, and the dependence of MB binding on salt concentration was investigated by solving the non-linear Poisson-Boltzmann equation. We find that the relative stability of the different complexes is similar for the two sequences, in agreement with the interpretation of spectroscopic data. Subtle differences, however, are seen in energy decompositions and can be attributed to the change from symmetric 5'-YpR-3' intercalation to minor groove binding with increasing salt concentration, which is experimentally observed for the AT sequence at lower salt concentration than for the GC sequence. According to our results, this difference is due to the significantly lower non-electrostatic energy for the minor groove complex with AT alternating DNA, whereas the slightly lower binding energy to this sequence is caused by a higher deformation energy of DNA. The energetic data are in agreement with the conclusions derived from different spectroscopic studies and can also be structurally interpreted on the basis of the modeled complexes. The simple static modeling technique and the neglect of entropy terms and of non-electrostatic solute-solvent interactions, which are assumed to be nearly constant for the compared complexes of MB with DNA, seem to be justified by the results.

Using Full Genomic Information to Predict Disease: Breaking Down the Barriers Between Complex and Mendelian Diseases.

PubMed

Jordan, Daniel M; Do, Ron

2018-04-11

While sequence-based genetic tests have long been available for specific loci, especially for Mendelian disease, the rapidly falling costs of genome-wide genotyping arrays, whole-exome sequencing, and whole-genome sequencing are moving us toward a future where full genomic information might inform the prognosis and treatment of a variety of diseases, including complex disease. Similarly, the availability of large populations with full genomic information has enabled new insights about the etiology and genetic architecture of complex disease. Insights from the latest generation of genomic studies suggest that our categorization of diseases as complex may conceal a wide spectrum of genetic architectures and causal mechanisms that ranges from Mendelian forms of complex disease to complex regulatory structures underlying Mendelian disease. Here, we review these insights, along with advances in the prediction of disease risk and outcomes from full genomic information. Expected final online publication date for the Annual Review of Genomics and Human Genetics Volume 19 is August 31, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Gentle Masking of Low-Complexity Sequences Improves Homology Search

PubMed Central

Frith, Martin C.

2011-01-01

Detection of sequences that are homologous, i.e. descended from a common ancestor, is a fundamental task in computational biology. This task is confounded by low-complexity tracts (such as atatatatatat), which arise frequently and independently, causing strong similarities that are not homologies. There has been much research on identifying low-complexity tracts, but little research on how to treat them during homology search. We propose to find homologies by aligning sequences with “gentle” masking of low-complexity tracts. Gentle masking means that the match score involving a masked letter is , where is the unmasked score. Gentle masking slightly but noticeably improves the sensitivity of homology search (compared to “harsh” masking), without harming specificity. We show examples in three useful homology search problems: detection of NUMTs (nuclear copies of mitochondrial DNA), recruitment of metagenomic DNA reads to reference genomes, and pseudogene detection. Gentle masking is currently the best way to treat low-complexity tracts during homology search. PMID:22205972

Implicit Sequence Learning in Dyslexia: A Within-Sequence Comparison of First- and Higher-Order Information

ERIC Educational Resources Information Center

Du, Wenchong; Kelly, Steve W.

2013-01-01

The present study examines implicit sequence learning in adult dyslexics with a focus on comparing sequence transitions with different statistical complexities. Learning of a 12-item deterministic sequence was assessed in 12 dyslexic and 12 non-dyslexic university students. Both groups showed equivalent standard reaction time increments when the…

SSAW: A new sequence similarity analysis method based on the stationary discrete wavelet transform.

PubMed

Lin, Jie; Wei, Jing; Adjeroh, Donald; Jiang, Bing-Hua; Jiang, Yue

2018-05-02

Alignment-free sequence similarity analysis methods often lead to significant savings in computational time over alignment-based counterparts. A new alignment-free sequence similarity analysis method, called SSAW is proposed. SSAW stands for Sequence Similarity Analysis using the Stationary Discrete Wavelet Transform (SDWT). It extracts k-mers from a sequence, then maps each k-mer to a complex number field. Then, the series of complex numbers formed are transformed into feature vectors using the stationary discrete wavelet transform. After these steps, the original sequence is turned into a feature vector with numeric values, which can then be used for clustering and/or classification. Using two different types of applications, namely, clustering and classification, we compared SSAW against the the-state-of-the-art alignment free sequence analysis methods. SSAW demonstrates competitive or superior performance in terms of standard indicators, such as accuracy, F-score, precision, and recall. The running time was significantly better in most cases. These make SSAW a suitable method for sequence analysis, especially, given the rapidly increasing volumes of sequence data required by most modern applications.

The language of geometry: Fast comprehension of geometrical primitives and rules in human adults and preschoolers.

PubMed

Amalric, Marie; Wang, Liping; Pica, Pierre; Figueira, Santiago; Sigman, Mariano; Dehaene, Stanislas

2017-01-01

During language processing, humans form complex embedded representations from sequential inputs. Here, we ask whether a "geometrical language" with recursive embedding also underlies the human ability to encode sequences of spatial locations. We introduce a novel paradigm in which subjects are exposed to a sequence of spatial locations on an octagon, and are asked to predict future locations. The sequences vary in complexity according to a well-defined language comprising elementary primitives and recursive rules. A detailed analysis of error patterns indicates that primitives of symmetry and rotation are spontaneously detected and used by adults, preschoolers, and adult members of an indigene group in the Amazon, the Munduruku, who have a restricted numerical and geometrical lexicon and limited access to schooling. Furthermore, subjects readily combine these geometrical primitives into hierarchically organized expressions. By evaluating a large set of such combinations, we obtained a first view of the language needed to account for the representation of visuospatial sequences in humans, and conclude that they encode visuospatial sequences by minimizing the complexity of the structured expressions that capture them.

Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing

PubMed Central

Tsai, Yu-Chih; Deming, Clayton; Segre, Julia A.; Kong, Heidi H.; Korlach, Jonas

2016-01-01

ABSTRACT Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation. PMID:26861018

The language of geometry: Fast comprehension of geometrical primitives and rules in human adults and preschoolers

PubMed Central

Amalric, Marie; Wang, Liping; Figueira, Santiago; Sigman, Mariano; Dehaene, Stanislas

2017-01-01

During language processing, humans form complex embedded representations from sequential inputs. Here, we ask whether a “geometrical language” with recursive embedding also underlies the human ability to encode sequences of spatial locations. We introduce a novel paradigm in which subjects are exposed to a sequence of spatial locations on an octagon, and are asked to predict future locations. The sequences vary in complexity according to a well-defined language comprising elementary primitives and recursive rules. A detailed analysis of error patterns indicates that primitives of symmetry and rotation are spontaneously detected and used by adults, preschoolers, and adult members of an indigene group in the Amazon, the Munduruku, who have a restricted numerical and geometrical lexicon and limited access to schooling. Furthermore, subjects readily combine these geometrical primitives into hierarchically organized expressions. By evaluating a large set of such combinations, we obtained a first view of the language needed to account for the representation of visuospatial sequences in humans, and conclude that they encode visuospatial sequences by minimizing the complexity of the structured expressions that capture them. PMID:28125595

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes.

PubMed

Kachhap, Sangita; Singh, Balvinder

2015-01-01

In most of homeodomain-DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1-DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein-DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein-DNA complexes. The order of stability of protein-DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein-DNA complexes. Among specific protein-DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.

GBA manager: an online tool for querying low-complexity regions in proteins.

PubMed

Bandyopadhyay, Nirmalya; Kahveci, Tamer

2010-01-01

Abstract We developed GBA Manager, an online software that facilitates the Graph-Based Algorithm (GBA) we proposed in our earlier work. GBA identifies the low-complexity regions (LCR) of protein sequences. GBA exploits a similarity matrix, such as BLOSUM62, to compute the complexity of the subsequences of the input protein sequence. It uses a graph-based algorithm to accurately compute the regions that have low complexities. GBA Manager is a user friendly web-service that enables online querying of protein sequences using GBA. In addition to querying capabilities of the existing GBA algorithm, GBA Manager computes the p-values of the LCR identified. The p-value gives an estimate of the possibility that the region appears by chance. GBA Manager presents the output in three different understandable formats. GBA Manager is freely accessible at http://bioinformatics.cise.ufl.edu/GBA/GBA.htm .

Isolation and characterization of major histocompatibility complex class II B genes in cranes.

PubMed

Kohyama, Tetsuo I; Akiyama, Takuya; Nishida, Chizuko; Takami, Kazutoshi; Onuma, Manabu; Momose, Kunikazu; Masuda, Ryuichi

2015-11-01

In this study, we isolated and characterized the major histocompatibility complex (MHC) class II B genes in cranes. Genomic sequences spanning exons 1 to 4 were amplified and determined in 13 crane species and three other species closely related to cranes. In all, 55 unique sequences were identified, and at least two polymorphic MHC class II B loci were found in most species. An analysis of sequence polymorphisms showed the signature of positive selection and recombination. A phylogenetic reconstruction based on exon 2 sequences indicated that trans-species polymorphism has persisted for at least 10 million years, whereas phylogenetic analyses of the sequences flanking exon 2 revealed a pattern of concerted evolution. These results suggest that both balancing selection and recombination play important roles in the crane MHC evolution.

Modular probes for enriching and detecting complex nucleic acid sequences

NASA Astrophysics Data System (ADS)

Wang, Juexiao Sherry; Yan, Yan Helen; Zhang, David Yu

2017-12-01

Complex DNA sequences are difficult to detect and profile, but are important contributors to human health and disease. Existing hybridization probes lack the capability to selectively bind and enrich hypervariable, long or repetitive sequences. Here, we present a generalized strategy for constructing modular hybridization probes (M-Probes) that overcomes these challenges. We demonstrate that M-Probes can tolerate sequence variations of up to 7 nt at prescribed positions while maintaining single nucleotide sensitivity at other positions. M-Probes are also shown to be capable of sequence-selectively binding a continuous DNA sequence of more than 500 nt. Furthermore, we show that M-Probes can detect genes with triplet repeats exceeding a programmed threshold. As a demonstration of this technology, we have developed a hybrid capture method to determine the exact triplet repeat expansion number in the Huntington's gene of genomic DNA using quantitative PCR.

Closed Genome Sequences of Seven Histophilus somni Isolates from Beef Calves with Bovine Respiratory Disease Complex.

PubMed

Harhay, Gregory P; Harhay, Dayna M; Bono, James L; Smith, Timothy P L; Capik, Sarah F; DeDonder, Keith D; Apley, Michael D; Lubbers, Brian V; White, Bradley J; Larson, Robert L

2017-10-05

Histophilus somni is a fastidious Gram-negative opportunistic pathogenic Pasteurellaceae that affects multiple organ systems and is one of the principal bacterial species contributing to bovine respiratory disease complex (BRDC) in feed yard cattle. Here, we present seven closed genome sequences isolated from three beef calves showing sign of BRDC.

The Development of Mental Models for Auditory Events: Relational Complexity and Discrimination of Pitch and Duration

ERIC Educational Resources Information Center

Stevens, Catherine; Gallagher, Melinda

2004-01-01

This experiment investigated relational complexity and relational shift in judgments of auditory patterns. Pitch and duration values were used to construct two-note perceptually similar sequences (unary relations) and four-note relationally similar sequences (binary relations). It was hypothesized that 5-, 8- and 11-year-old children would perform…

Professionally Responsible Disclosure of Genomic Sequencing Results in Pediatric Practice

PubMed Central

Brothers, Kyle B.; Chung, Wendy K.; Joffe, Steven; Koenig, Barbara A.; Wilfond, Benjamin; Yu, Joon-Ho

2015-01-01

Genomic sequencing is being rapidly introduced into pediatric clinical practice. The results of sequencing are distinctive for their complexity and subsequent challenges of interpretation for generalist and specialist pediatricians, parents, and patients. Pediatricians therefore need to prepare for the professionally responsible disclosure of sequencing results to parents and patients and guidance of parents and patients in the interpretation and use of these results, including managing uncertain data. This article provides an ethical framework to guide and evaluate the professionally responsible disclosure of the results of genomic sequencing in pediatric practice. The ethical framework comprises 3 core concepts of pediatric ethics: the best interests of the child standard, parental surrogate decision-making, and pediatric assent. When recommending sequencing, pediatricians should explain the nature of the proposed test, its scope and complexity, the categories of results, and the concept of a secondary or incidental finding. Pediatricians should obtain the informed permission of parents and the assent of mature adolescents about the scope of sequencing to be performed and the return of results. PMID:26371191

Determination of haplotypes at structurally complex regions using emulsion haplotype fusion PCR

PubMed Central

2012-01-01

Background Genotyping and massively-parallel sequencing projects result in a vast amount of diploid data that is only rarely resolved into its constituent haplotypes. It is nevertheless this phased information that is transmitted from one generation to the next and is most directly associated with biological function and the genetic causes of biological effects. Despite progress made in genome-wide sequencing and phasing algorithms and methods, problems assembling (and reconstructing linear haplotypes in) regions of repetitive DNA and structural variation remain. These dynamic and structurally complex regions are often poorly understood from a sequence point of view. Regions such as these that are highly similar in their sequence tend to be collapsed onto the genome assembly. This is turn means downstream determination of the true sequence haplotype in these regions poses a particular challenge. For structurally complex regions, a more focussed approach to assembling haplotypes may be required. Results In order to investigate reconstruction of spatial information at structurally complex regions, we have used an emulsion haplotype fusion PCR approach to reproducibly link sequences of up to 1kb in length to allow phasing of multiple variants from neighbouring loci, using allele-specific PCR and sequencing to detect the phase. By using emulsion systems linking flanking regions to amplicons within the CNV, this led to the reconstruction of a 59kb haplotype across the DEFA1A3 CNV in HapMap individuals. Conclusion This study has demonstrated a novel use for emulsion haplotype fusion PCR in addressing the issue of reconstructing structural haplotypes at multiallelic copy variable regions, using the DEFA1A3 locus as an example. PMID:23231411

NADH:ubiquinone oxidoreductase from bovine heart mitochondria. cDNA sequences of the import precursors of the nuclear-encoded 39 kDa and 42 kDa subunits.

PubMed Central

Fearnley, I M; Finel, M; Skehel, J M; Walker, J E

1991-01-01

The 39 kDa and 42 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria are nuclear-coded components of the hydrophobic protein fraction of the enzyme. Their amino acid sequences have been deduced from the sequences of overlapping cDNA clones. These clones were amplified from total bovine heart cDNA by means of the polymerase chain reaction, with the use of complex mixtures of oligonucleotide primers based upon fragments of protein sequence determined at the N-terminals of the proteins and at internal sites. The protein sequences of the 39 kDa and 42 kDa subunits are 345 and 320 amino acid residues long respectively, and their calculated molecular masses are 39,115 Da and 36,693 Da. Both proteins are predominantly hydrophilic, but each contains one or two hydrophobic segments that could possibly be folded into transmembrane alpha-helices. The bovine 39 kDa protein sequence is related to that of a 40 kDa subunit from complex I from Neurospora crassa mitochondria; otherwise, it is not related significantly to any known sequence, including redox proteins and two polypeptides involved in import of proteins into mitochondria, known as the mitochondrial processing peptidase and the processing-enhancing protein. Therefore the functions of the 39 kDa and 42 kDa subunits of complex I are unknown. The mitochondrial gene product, ND4, a hydrophobic component of complex I with an apparent molecular mass of about 39 kDa, has been identified in preparations of the enzyme. This subunit stains faintly with Coomassie Blue dye, and in many gel systems it is not resolved from the nuclearcoded 36 kDa subunit. Images Fig. 1. PMID:1832859

Conservation of coevolving protein interfaces bridges prokaryote–eukaryote homologies in the twilight zone

PubMed Central

Rodriguez-Rivas, Juan; Marsili, Simone; Juan, David; Valencia, Alfonso

2016-01-01

Protein–protein interactions are fundamental for the proper functioning of the cell. As a result, protein interaction surfaces are subject to strong evolutionary constraints. Recent developments have shown that residue coevolution provides accurate predictions of heterodimeric protein interfaces from sequence information. So far these approaches have been limited to the analysis of families of prokaryotic complexes for which large multiple sequence alignments of homologous sequences can be compiled. We explore the hypothesis that coevolution points to structurally conserved contacts at protein–protein interfaces, which can be reliably projected to homologous complexes with distantly related sequences. We introduce a domain-centered protocol to study the interplay between residue coevolution and structural conservation of protein–protein interfaces. We show that sequence-based coevolutionary analysis systematically identifies residue contacts at prokaryotic interfaces that are structurally conserved at the interface of their eukaryotic counterparts. In turn, this allows the prediction of conserved contacts at eukaryotic protein–protein interfaces with high confidence using solely mutational patterns extracted from prokaryotic genomes. Even in the context of high divergence in sequence (the twilight zone), where standard homology modeling of protein complexes is unreliable, our approach provides sequence-based accurate information about specific details of protein interactions at the residue level. Selected examples of the application of prokaryotic coevolutionary analysis to the prediction of eukaryotic interfaces further illustrate the potential of this approach. PMID:27965389

Conservation of coevolving protein interfaces bridges prokaryote-eukaryote homologies in the twilight zone.

PubMed

Rodriguez-Rivas, Juan; Marsili, Simone; Juan, David; Valencia, Alfonso

2016-12-27

Protein-protein interactions are fundamental for the proper functioning of the cell. As a result, protein interaction surfaces are subject to strong evolutionary constraints. Recent developments have shown that residue coevolution provides accurate predictions of heterodimeric protein interfaces from sequence information. So far these approaches have been limited to the analysis of families of prokaryotic complexes for which large multiple sequence alignments of homologous sequences can be compiled. We explore the hypothesis that coevolution points to structurally conserved contacts at protein-protein interfaces, which can be reliably projected to homologous complexes with distantly related sequences. We introduce a domain-centered protocol to study the interplay between residue coevolution and structural conservation of protein-protein interfaces. We show that sequence-based coevolutionary analysis systematically identifies residue contacts at prokaryotic interfaces that are structurally conserved at the interface of their eukaryotic counterparts. In turn, this allows the prediction of conserved contacts at eukaryotic protein-protein interfaces with high confidence using solely mutational patterns extracted from prokaryotic genomes. Even in the context of high divergence in sequence (the twilight zone), where standard homology modeling of protein complexes is unreliable, our approach provides sequence-based accurate information about specific details of protein interactions at the residue level. Selected examples of the application of prokaryotic coevolutionary analysis to the prediction of eukaryotic interfaces further illustrate the potential of this approach.

SPARSE: quadratic time simultaneous alignment and folding of RNAs without sequence-based heuristics

PubMed Central

Will, Sebastian; Otto, Christina; Miladi, Milad; Möhl, Mathias; Backofen, Rolf

2015-01-01

Motivation: RNA-Seq experiments have revealed a multitude of novel ncRNAs. The gold standard for their analysis based on simultaneous alignment and folding suffers from extreme time complexity of O(n6). Subsequently, numerous faster ‘Sankoff-style’ approaches have been suggested. Commonly, the performance of such methods relies on sequence-based heuristics that restrict the search space to optimal or near-optimal sequence alignments; however, the accuracy of sequence-based methods breaks down for RNAs with sequence identities below 60%. Alignment approaches like LocARNA that do not require sequence-based heuristics, have been limited to high complexity (≥ quartic time). Results: Breaking this barrier, we introduce the novel Sankoff-style algorithm ‘sparsified prediction and alignment of RNAs based on their structure ensembles (SPARSE)’, which runs in quadratic time without sequence-based heuristics. To achieve this low complexity, on par with sequence alignment algorithms, SPARSE features strong sparsification based on structural properties of the RNA ensembles. Following PMcomp, SPARSE gains further speed-up from lightweight energy computation. Although all existing lightweight Sankoff-style methods restrict Sankoff’s original model by disallowing loop deletions and insertions, SPARSE transfers the Sankoff algorithm to the lightweight energy model completely for the first time. Compared with LocARNA, SPARSE achieves similar alignment and better folding quality in significantly less time (speedup: 3.7). At similar run-time, it aligns low sequence identity instances substantially more accurate than RAF, which uses sequence-based heuristics. Availability and implementation: SPARSE is freely available at http://www.bioinf.uni-freiburg.de/Software/SPARSE. Contact: backofen@informatik.uni-freiburg.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25838465

Exact distribution of a pattern in a set of random sequences generated by a Markov source: applications to biological data

PubMed Central

2010-01-01

Background In bioinformatics it is common to search for a pattern of interest in a potentially large set of rather short sequences (upstream gene regions, proteins, exons, etc.). Although many methodological approaches allow practitioners to compute the distribution of a pattern count in a random sequence generated by a Markov source, no specific developments have taken into account the counting of occurrences in a set of independent sequences. We aim to address this problem by deriving efficient approaches and algorithms to perform these computations both for low and high complexity patterns in the framework of homogeneous or heterogeneous Markov models. Results The latest advances in the field allowed us to use a technique of optimal Markov chain embedding based on deterministic finite automata to introduce three innovative algorithms. Algorithm 1 is the only one able to deal with heterogeneous models. It also permits to avoid any product of convolution of the pattern distribution in individual sequences. When working with homogeneous models, Algorithm 2 yields a dramatic reduction in the complexity by taking advantage of previous computations to obtain moment generating functions efficiently. In the particular case of low or moderate complexity patterns, Algorithm 3 exploits power computation and binary decomposition to further reduce the time complexity to a logarithmic scale. All these algorithms and their relative interest in comparison with existing ones were then tested and discussed on a toy-example and three biological data sets: structural patterns in protein loop structures, PROSITE signatures in a bacterial proteome, and transcription factors in upstream gene regions. On these data sets, we also compared our exact approaches to the tempting approximation that consists in concatenating the sequences in the data set into a single sequence. Conclusions Our algorithms prove to be effective and able to handle real data sets with multiple sequences, as well as biological patterns of interest, even when the latter display a high complexity (PROSITE signatures for example). In addition, these exact algorithms allow us to avoid the edge effect observed under the single sequence approximation, which leads to erroneous results, especially when the marginal distribution of the model displays a slow convergence toward the stationary distribution. We end up with a discussion on our method and on its potential improvements. PMID:20205909

Exact distribution of a pattern in a set of random sequences generated by a Markov source: applications to biological data.

PubMed

Nuel, Gregory; Regad, Leslie; Martin, Juliette; Camproux, Anne-Claude

2010-01-26

In bioinformatics it is common to search for a pattern of interest in a potentially large set of rather short sequences (upstream gene regions, proteins, exons, etc.). Although many methodological approaches allow practitioners to compute the distribution of a pattern count in a random sequence generated by a Markov source, no specific developments have taken into account the counting of occurrences in a set of independent sequences. We aim to address this problem by deriving efficient approaches and algorithms to perform these computations both for low and high complexity patterns in the framework of homogeneous or heterogeneous Markov models. The latest advances in the field allowed us to use a technique of optimal Markov chain embedding based on deterministic finite automata to introduce three innovative algorithms. Algorithm 1 is the only one able to deal with heterogeneous models. It also permits to avoid any product of convolution of the pattern distribution in individual sequences. When working with homogeneous models, Algorithm 2 yields a dramatic reduction in the complexity by taking advantage of previous computations to obtain moment generating functions efficiently. In the particular case of low or moderate complexity patterns, Algorithm 3 exploits power computation and binary decomposition to further reduce the time complexity to a logarithmic scale. All these algorithms and their relative interest in comparison with existing ones were then tested and discussed on a toy-example and three biological data sets: structural patterns in protein loop structures, PROSITE signatures in a bacterial proteome, and transcription factors in upstream gene regions. On these data sets, we also compared our exact approaches to the tempting approximation that consists in concatenating the sequences in the data set into a single sequence. Our algorithms prove to be effective and able to handle real data sets with multiple sequences, as well as biological patterns of interest, even when the latter display a high complexity (PROSITE signatures for example). In addition, these exact algorithms allow us to avoid the edge effect observed under the single sequence approximation, which leads to erroneous results, especially when the marginal distribution of the model displays a slow convergence toward the stationary distribution. We end up with a discussion on our method and on its potential improvements.

GWAS and fine-mapping of 35 production, reproduction and conformation traits with imputed sequences of 27K Holstein bulls

USDA-ARS?s Scientific Manuscript database

Fine-mapping of causal variants is becoming feasible for complex traits in livestock GWAS, as an increasing number of animals are sequenced. Imputation has been routinely applied to ascertain sequence variants in large genotyped populations based on small reference populations of sequenced animals. ...

Genomic Sequencing: Assessing The Health Care System, Policy, And Big-Data Implications

PubMed Central

Phillips, Kathryn A.; Trosman, Julia; Kelley, Robin K.; Pletcher, Mark J.; Douglas, Michael P.; Weldon, Christine B.

2014-01-01

New genomic sequencing technologies enable the high-speed analysis of multiple genes simultaneously, including all of those in a person's genome. Sequencing is a prominent example of a “big data” technology because of the massive amount of information it produces and its complexity, diversity, and timeliness. Our objective in this article is to provide a policy primer on sequencing and illustrate how it can affect health care system and policy issues. Toward this end, we developed an easily applied classification of sequencing based on inputs, methods, and outputs. We used it to examine the implications of sequencing for three health care system and policy issues: making care more patient-centered, developing coverage and reimbursement policies, and assessing economic value. We conclude that sequencing has great promise but that policy challenges include how to optimize patient engagement as well as privacy, develop coverage policies that distinguish research from clinical uses and account for bioinformatics costs, and determine the economic value of sequencing through complex economic models that take into account multiple findings and downstream costs. PMID:25006153

Genomic sequencing: assessing the health care system, policy, and big-data implications.

PubMed

Phillips, Kathryn A; Trosman, Julia R; Kelley, Robin K; Pletcher, Mark J; Douglas, Michael P; Weldon, Christine B

2014-07-01

New genomic sequencing technologies enable the high-speed analysis of multiple genes simultaneously, including all of those in a person's genome. Sequencing is a prominent example of a "big data" technology because of the massive amount of information it produces and its complexity, diversity, and timeliness. Our objective in this article is to provide a policy primer on sequencing and illustrate how it can affect health care system and policy issues. Toward this end, we developed an easily applied classification of sequencing based on inputs, methods, and outputs. We used it to examine the implications of sequencing for three health care system and policy issues: making care more patient-centered, developing coverage and reimbursement policies, and assessing economic value. We conclude that sequencing has great promise but that policy challenges include how to optimize patient engagement as well as privacy, develop coverage policies that distinguish research from clinical uses and account for bioinformatics costs, and determine the economic value of sequencing through complex economic models that take into account multiple findings and downstream costs. Project HOPE—The People-to-People Health Foundation, Inc.

A sequence-based survey of the complex structural organization of tumor genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collins, Colin; Raphael, Benjamin J.; Volik, Stanislav

2008-04-03

The genomes of many epithelial tumors exhibit extensive chromosomal rearrangements. All classes of genome rearrangements can be identified using End Sequencing Profiling (ESP), which relies on paired-end sequencing of cloned tumor genomes. In this study, brain, breast, ovary and prostate tumors along with three breast cancer cell lines were surveyed with ESP yielding the largest available collection of sequence-ready tumor genome breakpoints and providing evidence that some rearrangements may be recurrent. Sequencing and fluorescence in situ hybridization (FISH) confirmed translocations and complex tumor genome structures that include coamplification and packaging of disparate genomic loci with associated molecular heterogeneity. Comparison ofmore » the tumor genomes suggests recurrent rearrangements. Some are likely to be novel structural polymorphisms, whereas others may be bona fide somatic rearrangements. A recurrent fusion transcript in breast tumors and a constitutional fusion transcript resulting from a segmental duplication were identified. Analysis of end sequences for single nucleotide polymorphisms (SNPs) revealed candidate somatic mutations and an elevated rate of novel SNPs in an ovarian tumor. These results suggest that the genomes of many epithelial tumors may be far more dynamic and complex than previously appreciated and that genomic fusions including fusion transcripts and proteins may be common, possibly yielding tumor-specific biomarkers and therapeutic targets.« less

Direct mapping of symbolic DNA sequence into frequency domain in global repeat map algorithm

PubMed Central

Glunčić, Matko; Paar, Vladimir

2013-01-01

The main feature of global repeat map (GRM) algorithm (www.hazu.hr/grm/software/win/grm2012.exe) is its ability to identify a broad variety of repeats of unbounded length that can be arbitrarily distant in sequences as large as human chromosomes. The efficacy is due to the use of complete set of a K-string ensemble which enables a new method of direct mapping of symbolic DNA sequence into frequency domain, with straightforward identification of repeats as peaks in GRM diagram. In this way, we obtain very fast, efficient and highly automatized repeat finding tool. The method is robust to substitutions and insertions/deletions, as well as to various complexities of the sequence pattern. We present several case studies of GRM use, in order to illustrate its capabilities: identification of α-satellite tandem repeats and higher order repeats (HORs), identification of Alu dispersed repeats and of Alu tandems, identification of Period 3 pattern in exons, implementation of ‘magnifying glass’ effect, identification of complex HOR pattern, identification of inter-tandem transitional dispersed repeat sequences and identification of long segmental duplications. GRM algorithm is convenient for use, in particular, in cases of large repeat units, of highly mutated and/or complex repeats, and of global repeat maps for large genomic sequences (chromosomes and genomes). PMID:22977183

Recognition of the Xenopus ribosomal core promoter by the transcription factor xUBF involves multiple HMG box domains and leads to an xUBF interdomain interaction.

PubMed

Leblanc, B; Read, C; Moss, T

1993-02-01

The interaction of the ribosomal transcription factor xUBF with the RNA polymerase I core promoter of Xenopus laevis has been studied both at the DNA and protein levels. It is shown that a single xUBF-DNA complex forms over the 40S initiation site (+1) and involves at least the DNA sequences between -20 and +60 bp. DNA sequences upstream of +10 and downstream of +18 are each sufficient to direct complex formation independently. HMG box 1 of xUBF independently recognizes the sequences -20 to -1 and +1 to +22 and the addition of the N-terminal dimerization domain to HMG box 1 stabilizes its interaction with these sequences approximately 10-fold. HMG boxes 2/3 interact with the DNA downstream of +22 and can independently position xUBF across the initiation site. The C-terminal segment of xUBF, HMG boxes 4, 5 or the acidic domain, directly or indirectly interact with HMG box 1, making the core promoter sequences between -11 and -15 hypersensitive to DNase. This interaction also requires the DNA sequences between +17 and +32, i.e. the HMG box 2/3 binding site. The data suggest extensive folding of the core promoter within the xUBF complex.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes.

PubMed

Srinivasulu, Yerukala Sathipati; Wang, Jyun-Rong; Hsu, Kai-Ti; Tsai, Ming-Ju; Charoenkwan, Phasit; Huang, Wen-Lin; Huang, Hui-Ling; Ho, Shinn-Ying

2015-01-01

Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

PubMed Central

2015-01-01

Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes. PMID:26681483

A Code Division Multiple Access Communication System for the Low Frequency Band.

DTIC Science & Technology

1983-04-01

frequency channels spread-spectrum communication / complex sequences, orthogonal codes impulsive noise 20. ABSTRACT (Continue an reverse side It...their transmissions with signature sequences. Our LF/CDMA scheme is different in that each user’s signature sequence set consists of M orthogonal ...signature sequences. Our LF/CDMA scheme is different in that each user’s signature sequence set consists of M orthogonal sequences and thus log 2 M

Using Video Modeling to Teach Complex Social Sequences to Children with Autism

ERIC Educational Resources Information Center

Nikopoulos, Christos K.; Keenan, Mickey

2007-01-01

This study comprised of two experiments was designed to teach complex social sequences to children with autism. Experimental control was achieved by collecting data using means of within-system design methodology. Across a number of conditions children were taken to a room to view one of the four short videos of two people engaging in a simple…

Closed Genome Sequences of Seven Histophilus somni Isolates from Beef Calves with Bovine Respiratory Disease Complex

PubMed Central

Harhay, Dayna M.; Bono, James L.; Smith, Timothy P. L.; Capik, Sarah F.; DeDonder, Keith D.; Apley, Michael D.; Lubbers, Brian V.; White, Bradley J.; Larson, Robert L.

2017-01-01

ABSTRACT Histophilus somni is a fastidious Gram-negative opportunistic pathogenic Pasteurellaceae that affects multiple organ systems and is one of the principal bacterial species contributing to bovine respiratory disease complex (BRDC) in feed yard cattle. Here, we present seven closed genome sequences isolated from three beef calves showing sign of BRDC. PMID:28983006

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex

PubMed Central

Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Karch, Francois

2015-01-01

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C. PMID:26303531

Model-based quality assessment and base-calling for second-generation sequencing data.

PubMed

Bravo, Héctor Corrada; Irizarry, Rafael A

2010-09-01

Second-generation sequencing (sec-gen) technology can sequence millions of short fragments of DNA in parallel, making it capable of assembling complex genomes for a small fraction of the price and time of previous technologies. In fact, a recently formed international consortium, the 1000 Genomes Project, plans to fully sequence the genomes of approximately 1200 people. The prospect of comparative analysis at the sequence level of a large number of samples across multiple populations may be achieved within the next five years. These data present unprecedented challenges in statistical analysis. For instance, analysis operates on millions of short nucleotide sequences, or reads-strings of A,C,G, or T's, between 30 and 100 characters long-which are the result of complex processing of noisy continuous fluorescence intensity measurements known as base-calling. The complexity of the base-calling discretization process results in reads of widely varying quality within and across sequence samples. This variation in processing quality results in infrequent but systematic errors that we have found to mislead downstream analysis of the discretized sequence read data. For instance, a central goal of the 1000 Genomes Project is to quantify across-sample variation at the single nucleotide level. At this resolution, small error rates in sequencing prove significant, especially for rare variants. Sec-gen sequencing is a relatively new technology for which potential biases and sources of obscuring variation are not yet fully understood. Therefore, modeling and quantifying the uncertainty inherent in the generation of sequence reads is of utmost importance. In this article, we present a simple model to capture uncertainty arising in the base-calling procedure of the Illumina/Solexa GA platform. Model parameters have a straightforward interpretation in terms of the chemistry of base-calling allowing for informative and easily interpretable metrics that capture the variability in sequencing quality. Our model provides these informative estimates readily usable in quality assessment tools while significantly improving base-calling performance. © 2009, The International Biometric Society.

Morphological and molecular identification of cryptic species in the Sergentomyia bailyi (Sinton, 1931) complex in Sri Lanka.

PubMed

Tharmatha, T; Gajapathy, K; Ramasamy, R; Surendran, S N

2017-02-01

The correct identification of sand fly vectors of leishmaniasis is important for controlling the disease. Genetic, particularly DNA sequence data, has lately become an important adjunct to the use of morphological criteria for this purpose. A recent DNA sequencing study revealed the presence of two cryptic species in the Sergentomyia bailyi species complex in India. The present study was undertaken to ascertain the presence of cryptic species in the Se. bailyi complex in Sri Lanka using morphological characteristics and DNA sequences from cytochrome c oxidase subunits. Sand flies were collected from leishmaniasis endemic and non-endemic dry zone districts of Sri Lanka. A total of 175 Se. bailyi specimens were initially screened for morphological variations and the identified samples formed two groups, tentatively termed as Se. bailyi species A and B, based on the relative length of the sensilla chaeticum and antennal flagellomere. DNA sequences from the mitochondrial cytochrome c oxidase subunit I (COI) and subunit II (COII) genes of morphologically identified Se. bailyi species A and B were subsequently analyzed. The two species showed differences in the COI and COII gene sequences and were placed in two separate clades by phylogenetic analysis. An allele specific polymerase chain reaction assay based on sequence variation in the COI gene accurately differentiated species A and B. The study therefore describes the first morphological and genetic evidence for the presence of two cryptic species within the Se. bailyi complex in Sri Lanka and a DNA-based laboratory technique for differentiating them.

Unusual chromosomal organization of telomeric sequences and expeditious karyotypic differentiation in the recently evolved Mus terricolor complex.

PubMed

Sharma, G G; Sharma, T

1998-01-01

The Mus terricolor complex displays a stable homozygous arrangement of autosomal heterochromatin variations in the form of accretion of definitive autosomal short arms among three nonoverlapping populations, in concert with an expeditious evolutionary differentiation into three chromosomal species: M. terricolor I, II, and III. In contrast to the highly conservative M. musculus-like chromosomes in the coexisting sibling species, M. booduga, reshuffling and differentiation of centric heterochromatin has occurred in harmony with a revision of centric configurations, resulting in acrocentric and submetacentric autosomes. The chromosomal distribution of the prevalent vertebrate telomeric sequence (TTAGGG)n was examined by fluorescence in situ hybridization to metaphase cells of M. terricolor I, II, and III. An unusual centric organization of internal telomeric sequences was detected in all the submetacentric and acrocentric autosomes. An auxiliary role of these presumably fragile, recombinogenic telomeric sequences in the evolutionary revision of centric configurations in the terricolor complex is hypothesized.

Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.

PubMed Central

Steiner, G; Hartmuth, K; Skriner, K; Maurer-Fogy, I; Sinski, A; Thalmann, E; Hassfeld, W; Barta, A; Smolen, J S

1992-01-01

RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen. Images PMID:1522214

Nucleic acid sequence detection using multiplexed oligonucleotide PCR

DOEpatents

Nolan, John P [Santa Fe, NM; White, P Scott [Los Alamos, NM

2006-12-26

Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

Annotation-based genome-wide SNP discovery in the large and complex Aegilops tauschii genome using next-generation sequencing without a reference genome sequence

PubMed Central

2011-01-01

Background Many plants have large and complex genomes with an abundance of repeated sequences. Many plants are also polyploid. Both of these attributes typify the genome architecture in the tribe Triticeae, whose members include economically important wheat, rye and barley. Large genome sizes, an abundance of repeated sequences, and polyploidy present challenges to genome-wide SNP discovery using next-generation sequencing (NGS) of total genomic DNA by making alignment and clustering of short reads generated by the NGS platforms difficult, particularly in the absence of a reference genome sequence. Results An annotation-based, genome-wide SNP discovery pipeline is reported using NGS data for large and complex genomes without a reference genome sequence. Roche 454 shotgun reads with low genome coverage of one genotype are annotated in order to distinguish single-copy sequences and repeat junctions from repetitive sequences and sequences shared by paralogous genes. Multiple genome equivalents of shotgun reads of another genotype generated with SOLiD or Solexa are then mapped to the annotated Roche 454 reads to identify putative SNPs. A pipeline program package, AGSNP, was developed and used for genome-wide SNP discovery in Aegilops tauschii-the diploid source of the wheat D genome, and with a genome size of 4.02 Gb, of which 90% is repetitive sequences. Genomic DNA of Ae. tauschii accession AL8/78 was sequenced with the Roche 454 NGS platform. Genomic DNA and cDNA of Ae. tauschii accession AS75 was sequenced primarily with SOLiD, although some Solexa and Roche 454 genomic sequences were also generated. A total of 195,631 putative SNPs were discovered in gene sequences, 155,580 putative SNPs were discovered in uncharacterized single-copy regions, and another 145,907 putative SNPs were discovered in repeat junctions. These SNPs were dispersed across the entire Ae. tauschii genome. To assess the false positive SNP discovery rate, DNA containing putative SNPs was amplified by PCR from AL8/78 and AS75 and resequenced with the ABI 3730 xl. In a sample of 302 randomly selected putative SNPs, 84.0% in gene regions, 88.0% in repeat junctions, and 81.3% in uncharacterized regions were validated. Conclusion An annotation-based genome-wide SNP discovery pipeline for NGS platforms was developed. The pipeline is suitable for SNP discovery in genomic libraries of complex genomes and does not require a reference genome sequence. The pipeline is applicable to all current NGS platforms, provided that at least one such platform generates relatively long reads. The pipeline package, AGSNP, and the discovered 497,118 Ae. tauschii SNPs can be accessed at (http://avena.pw.usda.gov/wheatD/agsnp.shtml). PMID:21266061

Cell proteins bind to multiple sites within the 5' untranslated region of poliovirus RNA.

PubMed Central

del Angel, R M; Papavassiliou, A G; Fernández-Tomás, C; Silverstein, S J; Racaniello, V R

1989-01-01

The 5' noncoding region of poliovirus RNA contains sequences necessary for translation and replication. These functions are probably carried out by recognition of poliovirus RNA by cellular and/or viral proteins. Using a mobility-shift electrophoresis assay and 1,10-phenanthroline/Cu+ footprinting, we demonstrate specific binding of cytoplasmic factors with a sequence from nucleotides 510-629 within the 5' untranslated region (UTR). Complex formation was also observed with a second sequence (nucleotides 97-182) within the 5' UTR. These two regions of the 5' UTR appear to be recognized by distinct cell factors as determined by competition analysis and the effects of ionic strength on complex formation. However, both complexes contain eukaryotic initiation factor 2 alpha, as revealed by their reaction with specific antibody. Images PMID:2554308

Targeted Re-Sequencing Emulsion PCR Panel for Myopathies: Results in 94 Cases.

PubMed

Punetha, Jaya; Kesari, Akanchha; Uapinyoying, Prech; Giri, Mamta; Clarke, Nigel F; Waddell, Leigh B; North, Kathryn N; Ghaoui, Roula; O'Grady, Gina L; Oates, Emily C; Sandaradura, Sarah A; Bönnemann, Carsten G; Donkervoort, Sandra; Plotz, Paul H; Smith, Edward C; Tesi-Rocha, Carolina; Bertorini, Tulio E; Tarnopolsky, Mark A; Reitter, Bernd; Hausmanowa-Petrusewicz, Irena; Hoffman, Eric P

2016-05-27

Molecular diagnostics in the genetic myopathies often requires testing of the largest and most complex transcript units in the human genome (DMD, TTN, NEB). Iteratively targeting single genes for sequencing has traditionally entailed high costs and long turnaround times. Exome sequencing has begun to supplant single targeted genes, but there are concerns regarding coverage and needed depth of the very large and complex genes that frequently cause myopathies. To evaluate efficiency of next-generation sequencing technologies to provide molecular diagnostics for patients with previously undiagnosed myopathies. We tested a targeted re-sequencing approach, using a 45 gene emulsion PCR myopathy panel, with subsequent sequencing on the Illumina platform in 94 undiagnosed patients. We compared the targeted re-sequencing approach to exome sequencing for 10 of these patients studied. We detected likely pathogenic mutations in 33 out of 94 patients with a molecular diagnostic rate of approximately 35%. The remaining patients showed variants of unknown significance (35/94 patients) or no mutations detected in the 45 genes tested (26/94 patients). Mutation detection rates for targeted re-sequencing vs. whole exome were similar in both methods; however exome sequencing showed better distribution of reads and fewer exon dropouts. Given that costs of highly parallel re-sequencing and whole exome sequencing are similar, and that exome sequencing now takes considerably less laboratory processing time than targeted re-sequencing, we recommend exome sequencing as the standard approach for molecular diagnostics of myopathies.

Geologic map of the Bonners Ferry 30' x 60' quadrangle, Idaho and Montana

USGS Publications Warehouse

Miller, Fred K.; Burmester, Russell F.

2003-01-01

This data set maps and describes the geology of the Bonners Ferry 30' x 60' quadrangle, Idaho and Montana. The bedrock geology of the Bonners Ferry quadrangle consists of sedimentary, metamorphic, and granitic rocks ranging in age from Middle Proterozoic to Eocene. Bedrock units include rocks of (1) the Middle Proterozoic Belt Supergroup (2) the Middle Proterozoic Deer Trail Group, (3) the Late Proterozoic Windermere Group, (4) miogeoclinal or shelf facies lower Paleozoic rocks, and (5) Mesozoic and Tertiary granitic rocks. The Belt Supergroup, a thick sequence of argillite, siltite, quartzite, and impure carbonate rocks up to 9,000 m thick, occurs in two non-contiguous sequences in the quadrangle: (1) the Clark Fork-Eastport Sequence east of the Purcell trench and (2) the Newport Sequence in the hanging wall of the Newport Fault. Only the two lowest Belt formations of the Newport Sequence are found in the Bonners Ferry quadrangle, but these two units are part of a continuous section, which extends southwestward to the town of Newport. Belt Supergroup rocks of the Clark Fork-Eastport Sequence are separated from those of the Newport Sequence by the Newport Fault, Priest River Complex, and Purcell Trench Fault. Some formations of the Belt Supergroup show differences in thickness and (or) lithofacies from one sequence to the other that are greater than those predicted from an empirical depositional model for the distances currently separating the sequences. These anomalous thickness and facies differences suggest that there has been a net contraction along structures separating the sequences despite Eocene extension associated with emplacement of the Priest River Complex. In addition to these two Belt sequences, probable Belt rocks are present in the Priest River Complex as high metamorphic grade crystalline schist and gneiss. Northwest of the Newport Sequence of Belt Supergroup is the Deer Trail Group, a distinct Middle Proterozoic sequence of argillite, siltite, quartzite, and carbonate rocks lithostratigraphically similar to the Belt Supergroup, but separated from all Belt Supergroup rocks by the Jumpoff Joe Fault. Rocks of the Deer Trail Group are pervasively phyllitic and noticeably more deformed than rocks in the Belt Supergroup sequences. Lithostratigraphically the Deer Trail Group is equivalent to part of the upper part of the Belt Supergroup. Differences in lithostratigraphy and thickness between individual Deer Trail and Belt units and between the Deer Trail and Belt sequences as a whole indicate that they were probably much farther apart when they were deposited. The Windermere Group is a lithologically varied sequence of volcanic rocks and coarse-grained, mostly immature, clastic sedimentary rocks up to 8,000 m thick. It is characterized by extreme differences in thickness and lithofacies over short distances caused by syndepositional faulting associated with initial stages of continental rifting in the Late Proterozoic. Strata of the Windermere Group unconformably overlie only the Deer Trail Group, and are nowhere found in depositional contact with Belt Supergroup rocks. Paleozoic rocks in the Bonners Ferry quadrangle consist of a thin, fault-bounded remnant preserved within the Clark Fork-Eastport Belt Supergroup Sequence. Mesozoic granitic rocks underlie at least 50 percent of the Bonners Ferry quadrangle. They fall into two petrogenetic suites, hornblende-biotite plutons and muscovite-biotite (two-mica) plutons, most of which are Cretaceous in age. Both suites are represented in the mid-crustal Priest River Complex and in the higher level plutons that flank the complex; by far the majority of the Priest River Complex are Cretaceous, two-mica bodies. Tertiary rocks are restricted to a single small stock, numerous hypabyssal dikes that are too small to show at the scale of the map, and to cataclastic rocks related to the Newport Fault. Quaternary deposits include unconsolidated to poorl

The use of network analysis to study complex animal communication systems: a study on nightingale song.

PubMed

Weiss, Michael; Hultsch, Henrike; Adam, Iris; Scharff, Constance; Kipper, Silke

2014-06-22

The singing of song birds can form complex signal systems comprised of numerous subunits sung with distinct combinatorial properties that have been described as syntax-like. This complexity has inspired inquiries into similarities of bird song to human language; but the quantitative analysis and description of song sequences is a challenging task. In this study, we analysed song sequences of common nightingales (Luscinia megarhynchos) by means of a network analysis. We translated long nocturnal song sequences into networks of song types with song transitions as connectors. As network measures, we calculated shortest path length and transitivity and identified the 'small-world' character of nightingale song networks. Besides comparing network measures with conventional measures of song complexity, we also found a correlation between network measures and age of birds. Furthermore, we determined the numbers of in-coming and out-going edges of each song type, characterizing transition patterns. These transition patterns were shared across males for certain song types. Playbacks with different transition patterns provided first evidence that these patterns are responded to differently and thus play a role in singing interactions. We discuss potential functions of the network properties of song sequences in the framework of vocal leadership. Network approaches provide biologically meaningful parameters to describe the song structure of species with extremely large repertoires and complex rules of song retrieval.

The use of network analysis to study complex animal communication systems: a study on nightingale song

PubMed Central

Weiss, Michael; Hultsch, Henrike; Adam, Iris; Scharff, Constance; Kipper, Silke

2014-01-01

The singing of song birds can form complex signal systems comprised of numerous subunits sung with distinct combinatorial properties that have been described as syntax-like. This complexity has inspired inquiries into similarities of bird song to human language; but the quantitative analysis and description of song sequences is a challenging task. In this study, we analysed song sequences of common nightingales (Luscinia megarhynchos) by means of a network analysis. We translated long nocturnal song sequences into networks of song types with song transitions as connectors. As network measures, we calculated shortest path length and transitivity and identified the ‘small-world’ character of nightingale song networks. Besides comparing network measures with conventional measures of song complexity, we also found a correlation between network measures and age of birds. Furthermore, we determined the numbers of in-coming and out-going edges of each song type, characterizing transition patterns. These transition patterns were shared across males for certain song types. Playbacks with different transition patterns provided first evidence that these patterns are responded to differently and thus play a role in singing interactions. We discuss potential functions of the network properties of song sequences in the framework of vocal leadership. Network approaches provide biologically meaningful parameters to describe the song structure of species with extremely large repertoires and complex rules of song retrieval. PMID:24807258

The genome sequence of 'Mycobacterium massiliense' strain CIP 108297 suggests the independent taxonomic status of the Mycobacterium abscessus complex at the subspecies level.

PubMed

Cho, Yong-Joon; Yi, Hana; Chun, Jongsik; Cho, Sang-Nae; Daley, Charles L; Koh, Won-Jung; Shin, Sung Jae

2013-01-01

Members of the Mycobacterium abscessus complex are rapidly growing mycobacteria that are emerging as human pathogens. The M. abscessus complex was previously composed of three species, namely M. abscessus sensu stricto, 'M. massiliense', and 'M. bolletii'. In 2011, 'M. massiliense' and 'M. bolletii' were united and reclassified as a single subspecies within M. abscessus: M. abscessus subsp. bolletii. However, the placement of 'M. massiliense' within the boundary of M. abscessus subsp. bolletii remains highly controversial with regard to clinical aspects. In this study, we revisited the taxonomic status of members of the M. abscessus complex based on comparative analysis of the whole-genome sequences of 53 strains. The genome sequence of the previous type strain of 'Mycobacterium massiliense' (CIP 108297) was determined using next-generation sequencing. The genome tree based on average nucleotide identity (ANI) values supported the differentiation of 'M. bolletii' and 'M. massiliense' at the subspecies level. The genome tree also clearly illustrated that 'M. bolletii' and 'M. massiliense' form a distinct phylogenetic clade within the radiation of the M. abscessus complex. The genomic distances observed in this study suggest that the current M. abscessus subsp. bolletii taxon should be divided into two subspecies, M. abscessus subsp. massiliense subsp. nov. and M. abscessus subsp. bolletii, to correspondingly accommodate the previously known 'M. massiliense' and 'M. bolletii' strains.

Rhipicephalus microplus dataset of nonredundant raw sequence reads from 454 GS FLX sequencing of Cot-selected (Cot = 660) genomic DNA

USDA-ARS?s Scientific Manuscript database

A reassociation kinetics-based approach was used to reduce the complexity of genomic DNA from the Deutsch laboratory strain of the cattle tick, Rhipicephalus microplus, to facilitate genome sequencing. Selected genomic DNA (Cot value = 660) was sequenced using 454 GS FLX technology, resulting in 356...

DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes.

PubMed

Iftikhar, Romana; Ashfaq, Muhammad; Rasool, Akhtar; Hebert, Paul D N

2016-01-01

Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5' (DNA barcode) region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN) system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27%) at COI, while intraspecific distances averaged 0.6% (range = 0.0% - 7.6%). BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci), and one predatory thrips (Aeolothrips intermedius) showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips.

Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample.

PubMed

Luo, Chengwei; Tsementzi, Despina; Kyrpides, Nikos; Read, Timothy; Konstantinidis, Konstantinos T

2012-01-01

Next-generation sequencing (NGS) is commonly used in metagenomic studies of complex microbial communities but whether or not different NGS platforms recover the same diversity from a sample and their assembled sequences are of comparable quality remain unclear. We compared the two most frequently used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA) II, on the same DNA sample obtained from a complex freshwater planktonic community. Despite the substantial differences in read length and sequencing protocols, the platforms provided a comparable view of the community sampled. For instance, derived assemblies overlapped in ~90% of their total sequences and in situ abundances of genes and genotypes (estimated based on sequence coverage) correlated highly between the two platforms (R(2)>0.9). Evaluation of base-call error, frameshift frequency, and contig length suggested that Illumina offered equivalent, if not better, assemblies than Roche 454. The results from metagenomic samples were further validated against DNA samples of eighteen isolate genomes, which showed a range of genome sizes and G+C% content. We also provide quantitative estimates of the errors in gene and contig sequences assembled from datasets characterized by different levels of complexity and G+C% content. For instance, we noted that homopolymer-associated, single-base errors affected ~1% of the protein sequences recovered in Illumina contigs of 10× coverage and 50% G+C; this frequency increased to ~3% when non-homopolymer errors were also considered. Collectively, our results should serve as a useful practical guide for choosing proper sampling strategies and data possessing protocols for future metagenomic studies.

Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing

PubMed Central

Be, Nicholas A.; Thissen, James B.; Gardner, Shea N.; McLoughlin, Kevin S.; Fofanov, Viacheslav Y.; Koshinsky, Heather; Ellingson, Sally R.; Brettin, Thomas S.; Jackson, Paul J.; Jaing, Crystal J.

2013-01-01

Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy. PMID:24039948

Systematic exploration of essential yeast gene function with temperature-sensitive mutants

PubMed Central

Li, Zhijian; Vizeacoumar, Franco J; Bahr, Sondra; Li, Jingjing; Warringer, Jonas; Vizeacoumar, Frederick S; Min, Renqiang; VanderSluis, Benjamin; Bellay, Jeremy; DeVit, Michael; Fleming, James A; Stephens, Andrew; Haase, Julian; Lin, Zhen-Yuan; Baryshnikova, Anastasia; Lu, Hong; Yan, Zhun; Jin, Ke; Barker, Sarah; Datti, Alessandro; Giaever, Guri; Nislow, Corey; Bulawa, Chris; Myers, Chad L; Costanzo, Michael; Gingras, Anne-Claude; Zhang, Zhaolei; Blomberg, Anders; Bloom, Kerry; Andrews, Brenda; Boone, Charles

2012-01-01

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (~45%) of the 1,101 essential yeast genes, with ~30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)–based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes. PMID:21441928

Meiosis I chromosome segregation is established through regulation of microtubule–kinetochore interactions

PubMed Central

Miller, Matthew P; Ünal, Elçin; Brar, Gloria A; Amon, Angelika

2012-01-01

During meiosis, a single round of DNA replication is followed by two consecutive rounds of nuclear divisions called meiosis I and meiosis II. In meiosis I, homologous chromosomes segregate, while sister chromatids remain together. Determining how this unusual chromosome segregation behavior is established is central to understanding germ cell development. Here we show that preventing microtubule–kinetochore interactions during premeiotic S phase and prophase I is essential for establishing the meiosis I chromosome segregation pattern. Premature interactions of kinetochores with microtubules transform meiosis I into a mitosis-like division by disrupting two key meiosis I events: coorientation of sister kinetochores and protection of centromeric cohesin removal from chromosomes. Furthermore we find that restricting outer kinetochore assembly contributes to preventing premature engagement of microtubules with kinetochores. We propose that inhibition of microtubule–kinetochore interactions during premeiotic S phase and prophase I is central to establishing the unique meiosis I chromosome segregation pattern. DOI: http://dx.doi.org/10.7554/eLife.00117.001 PMID:23275833

Benchmarking database performance for genomic data.

PubMed

Khushi, Matloob

2015-06-01

Genomic regions represent features such as gene annotations, transcription factor binding sites and epigenetic modifications. Performing various genomic operations such as identifying overlapping/non-overlapping regions or nearest gene annotations are common research needs. The data can be saved in a database system for easy management, however, there is no comprehensive database built-in algorithm at present to identify overlapping regions. Therefore I have developed a novel region-mapping (RegMap) SQL-based algorithm to perform genomic operations and have benchmarked the performance of different databases. Benchmarking identified that PostgreSQL extracts overlapping regions much faster than MySQL. Insertion and data uploads in PostgreSQL were also better, although general searching capability of both databases was almost equivalent. In addition, using the algorithm pair-wise, overlaps of >1000 datasets of transcription factor binding sites and histone marks, collected from previous publications, were reported and it was found that HNF4G significantly co-locates with cohesin subunit STAG1 (SA1).Inc. © 2015 Wiley Periodicals, Inc.

Chromosomal Organization by an Interplay of Loop Extrusion and Compartment Interaction

NASA Astrophysics Data System (ADS)

Nuebler, Johannes; Fudenberg, Geoffrey; Imakaev, Maxim; Lu, Carolyn; Goloborodko, Anton; Abdennur, Nezar; Mirny, Leonid

The chromatin fiber in eukaryotic nuclei is far from being simply a confined but otherwise randomly arranged polymer. Rather, it shows a high degree of spatial organization on all length scales, from individual nucleosomes up to well-segregated chromosome territories. On intermediate scales, chromosome conformation capture techniques have revealed two ubiquitous modes of organization: an alternating structure of A/B compartments, where each type preferentially associates with other base pairs of its type, and, typically on a smaller scale, the formation of topologically associating domains (TADs) with increased association within each domain but not across boundaries. The mechanisms behind this organization are only beginning to emerge. We review how the model of active loop extrusion can explain in a unified way such diverse phenomena as TAD formation and mitotic compaction and segregation, and we address in particular to what extent the interplay of active loop extrusion and compartment structure is compatible with recent experiments that interfere with the loading of the proposed loop extrusion factor cohesin. 4D Nucleome.

Evaluation of targeted exome sequencing for 28 protein-based blood group systems, including the homologous gene systems, for blood group genotyping.

PubMed

Schoeman, Elizna M; Lopez, Genghis H; McGowan, Eunike C; Millard, Glenda M; O'Brien, Helen; Roulis, Eileen V; Liew, Yew-Wah; Martin, Jacqueline R; McGrath, Kelli A; Powley, Tanya; Flower, Robert L; Hyland, Catherine A

2017-04-01

Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next-generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS. Donor samples (n = 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems. The average sequencing depth per target region was 66.2 ± 39.8. Each sample harbored on average 43 ± 9 variants, of which 10 ± 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement. The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting. © 2017 AABB.

The 3of5 web application for complex and comprehensive pattern matching in protein sequences.

PubMed

Seiler, Markus; Mehrle, Alexander; Poustka, Annemarie; Wiemann, Stefan

2006-03-16

The identification of patterns in biological sequences is a key challenge in genome analysis and in proteomics. Frequently such patterns are complex and highly variable, especially in protein sequences. They are frequently described using terms of regular expressions (RegEx) because of the user-friendly terminology. Limitations arise for queries with the increasing complexity of patterns and are accompanied by requirements for enhanced capabilities. This is especially true for patterns containing ambiguous characters and positions and/or length ambiguities. We have implemented the 3of5 web application in order to enable complex pattern matching in protein sequences. 3of5 is named after a special use of its main feature, the novel n-of-m pattern type. This feature allows for an extensive specification of variable patterns where the individual elements may vary in their position, order, and content within a defined stretch of sequence. The number of distinct elements can be constrained by operators, and individual characters may be excluded. The n-of-m pattern type can be combined with common regular expression terms and thus also allows for a comprehensive description of complex patterns. 3of5 increases the fidelity of pattern matching and finds ALL possible solutions in protein sequences in cases of length-ambiguous patterns instead of simply reporting the longest or shortest hits. Grouping and combined search for patterns provides a hierarchical arrangement of larger patterns sets. The algorithm is implemented as internet application and freely accessible. The application is available at http://dkfz.de/mga2/3of5/3of5.html. The 3of5 application offers an extended vocabulary for the definition of search patterns and thus allows the user to comprehensively specify and identify peptide patterns with variable elements. The n-of-m pattern type offers an improved accuracy for pattern matching in combination with the ability to find all solutions, without compromising the user friendliness of regular expression terms.

Mapping and phasing of structural variation in patient genomes using nanopore sequencing.

PubMed

Cretu Stancu, Mircea; van Roosmalen, Markus J; Renkens, Ivo; Nieboer, Marleen M; Middelkamp, Sjors; de Ligt, Joep; Pregno, Giulia; Giachino, Daniela; Mandrile, Giorgia; Espejo Valle-Inclan, Jose; Korzelius, Jerome; de Bruijn, Ewart; Cuppen, Edwin; Talkowski, Michael E; Marschall, Tobias; de Ridder, Jeroen; Kloosterman, Wigard P

2017-11-06

Despite improvements in genomics technology, the detection of structural variants (SVs) from short-read sequencing still poses challenges, particularly for complex variation. Here we analyse the genomes of two patients with congenital abnormalities using the MinION nanopore sequencer and a novel computational pipeline-NanoSV. We demonstrate that nanopore long reads are superior to short reads with regard to detection of de novo chromothripsis rearrangements. The long reads also enable efficient phasing of genetic variations, which we leveraged to determine the parental origin of all de novo chromothripsis breakpoints and to resolve the structure of these complex rearrangements. Additionally, genome-wide surveillance of inherited SVs reveals novel variants, missed in short-read data sets, a large proportion of which are retrotransposon insertions. We provide a first exploration of patient genome sequencing with a nanopore sequencer and demonstrate the value of long-read sequencing in mapping and phasing of SVs for both clinical and research applications.

Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ)

PubMed Central

Mascher, Martin; Muehlbauer, Gary J; Rokhsar, Daniel S; Chapman, Jarrod; Schmutz, Jeremy; Barry, Kerrie; Muñoz-Amatriaín, María; Close, Timothy J; Wise, Roger P; Schulman, Alan H; Himmelbach, Axel; Mayer, Klaus FX; Scholz, Uwe; Poland, Jesse A; Stein, Nils; Waugh, Robbie

2013-01-01

Next-generation whole-genome shotgun assemblies of complex genomes are highly useful, but fail to link nearby sequence contigs with each other or provide a linear order of contigs along individual chromosomes. Here, we introduce a strategy based on sequencing progeny of a segregating population that allows de novo production of a genetically anchored linear assembly of the gene space of an organism. We demonstrate the power of the approach by reconstructing the chromosomal organization of the gene space of barley, a large, complex and highly repetitive 5.1 Gb genome. We evaluate the robustness of the new assembly by comparison to a recently released physical and genetic framework of the barley genome, and to various genetically ordered sequence-based genotypic datasets. The method is independent of the need for any prior sequence resources, and will enable rapid and cost-efficient establishment of powerful genomic information for many species. PMID:23998490

Human structural variation: mechanisms of chromosome rearrangements

PubMed Central

Weckselblatt, Brooke; Rudd, M. Katharine

2015-01-01

Chromosome structural variation (SV) is a normal part of variation in the human genome, but some classes of SV can cause neurodevelopmental disorders. Analysis of the DNA sequence at SV breakpoints can reveal mutational mechanisms and risk factors for chromosome rearrangement. Large-scale SV breakpoint studies have become possible recently owing to advances in next-generation sequencing (NGS) including whole-genome sequencing (WGS). These findings have shed light on complex forms of SV such as triplications, inverted duplications, insertional translocations, and chromothripsis. Sequence-level breakpoint data resolve SV structure and determine how genes are disrupted, fused, and/or misregulated by breakpoints. Recent improvements in breakpoint sequencing have also revealed non-allelic homologous recombination (NAHR) between paralogous long interspersed nuclear element (LINE) or human endogenous retrovirus (HERV) repeats as a cause of deletions, duplications, and translocations. This review covers the genomic organization of simple and complex constitutional SVs, as well as the molecular mechanisms of their formation. PMID:26209074

Aircraft stress sequence development: A complex engineering process made simple

NASA Technical Reports Server (NTRS)

Schrader, K. H.; Butts, D. G.; Sparks, W. A.

1994-01-01

Development of stress sequences for critical aircraft structure requires flight measured usage data, known aircraft loads, and established relationships between aircraft flight loads and structural stresses. Resulting cycle-by-cycle stress sequences can be directly usable for crack growth analysis and coupon spectra tests. Often, an expert in loads and spectra development manipulates the usage data into a typical sequence of representative flight conditions for which loads and stresses are calculated. For a fighter/trainer type aircraft, this effort is repeated many times for each of the fatigue critical locations (FCL) resulting in expenditure of numerous engineering hours. The Aircraft Stress Sequence Computer Program (ACSTRSEQ), developed by Southwest Research Institute under contract to San Antonio Air Logistics Center, presents a unique approach for making complex technical computations in a simple, easy to use method. The program is written in Microsoft Visual Basic for the Microsoft Windows environment.

Sequence-controlled methacrylic multiblock copolymers via sulfur-free RAFT emulsion polymerization

NASA Astrophysics Data System (ADS)

Engelis, Nikolaos G.; Anastasaki, Athina; Nurumbetov, Gabit; Truong, Nghia P.; Nikolaou, Vasiliki; Shegiwal, Ataulla; Whittaker, Michael R.; Davis, Thomas P.; Haddleton, David M.

2017-02-01

Translating the precise monomer sequence control achieved in nature over macromolecular structure (for example, DNA) to whole synthetic systems has been limited due to the lack of efficient synthetic methodologies. So far, chemists have only been able to synthesize monomer sequence-controlled macromolecules by means of complex, time-consuming and iterative chemical strategies such as solid-state Merrifield-type approaches or molecularly dissolved solution-phase systems. Here, we report a rapid and quantitative synthesis of sequence-controlled multiblock polymers in discrete stable nanoscale compartments via an emulsion polymerization approach in which a vinyl-terminated macromolecule is used as an efficient chain-transfer agent. This approach is environmentally friendly, fully translatable to industry and thus represents a significant advance in the development of complex macromolecule synthesis, where a high level of molecular precision or monomer sequence control confers potential for molecular targeting, recognition and biocatalysis, as well as molecular information storage.

TIA-1 RRM23 binding and recognition of target oligonucleotides

PubMed Central

Waris, Saboora; García-Mauriño, Sofía M.; Sivakumaran, Andrew; Beckham, Simone A.; Loughlin, Fionna E.; Gorospe, Myriam; Díaz-Moreno, Irene; Wilce, Matthew C.J.

2017-01-01

Abstract TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression. PMID:28184449

TIA-1 RRM23 binding and recognition of target oligonucleotides.

PubMed

Waris, Saboora; García-Mauriño, Sofía M; Sivakumaran, Andrew; Beckham, Simone A; Loughlin, Fionna E; Gorospe, Myriam; Díaz-Moreno, Irene; Wilce, Matthew C J; Wilce, Jacqueline A

2017-05-05

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Causes of complexity in a fallout dominated plinian eruption sequence: 312 ka Fasnia Member, Diego Hernández Formation, Tenerife, Spain

NASA Astrophysics Data System (ADS)

Edgar, C. J.; Cas, R. A. F.; Olin, P. H.; Wolff, J. A.; Martí, J.; Simmons, J. M.

2017-10-01

The 312 ka Fasnia eruption from the Las Cañadas Caldera on Tenerife, Canary Islands, Spain, produced a complex sequence of twenty-two intercalated units, including 7 pumice fall, 7 ignimbrite and 8 ash surge and fall deposits that define two distinct eruption sequences (Lower and Upper Fasnia sequences). The fallout units themselves are internally complex, reflecting waxing and waning of the eruption column, while many of the ignimbrites reflect multiple intra-plinian partial column collapse events associated with the injection of lithic clasts into the eruption column. The Lower and Upper Fasnia eruption phases were each terminated by caldera collapse and complete column collapse events. Probable blockage of the conduit and vent system during Lower Fasnia caldera collapse event briefly terminated the eruption, resulting in a short-lived period of erosion and sedimentation prior to the onset of the Upper Fasnia phase. The transition to the Upper Fasnia eruption phase coincided with the eruption of more geochemically homogeneous pyroclasts. In total, 62 km3 of tephra were erupted, including 49 km3 of juvenile clasts and > 12 km3 of lithic clasts. The DRE volume of magma erupted was 13 km3 (Lower Fasnia > 5 km3, Upper Fasnia > 8 km3), two thirds of which ( 9-10 km3) was deposited purely by fallout. The Fasnia Member is one of the most complex plinian sequences known.

Serotype IV Sequence Type 468 Group B Streptococcus Neonatal Invasive Disease, Minnesota, USA.

PubMed

Teatero, Sarah; Ferrieri, Patricia; Fittipaldi, Nahuel

2016-11-01

To further understand the emergence of serotype IV group B Streptococcus (GBS) invasive disease, we used whole-genome sequencing to characterize 3 sequence type 468 strains isolated from neonates in Minnesota, USA. We found that strains of tetracycline-resistant sequence type 468 GBS have acquired virulence genes from a putative clonal complex 17 GBS donor by recombination.

BASiNET-BiologicAl Sequences NETwork: a case study on coding and non-coding RNAs identification.

PubMed

Ito, Eric Augusto; Katahira, Isaque; Vicente, Fábio Fernandes da Rocha; Pereira, Luiz Filipe Protasio; Lopes, Fabrício Martins

2018-06-05

With the emergence of Next Generation Sequencing (NGS) technologies, a large volume of sequence data in particular de novo sequencing was rapidly produced at relatively low costs. In this context, computational tools are increasingly important to assist in the identification of relevant information to understand the functioning of organisms. This work introduces BASiNET, an alignment-free tool for classifying biological sequences based on the feature extraction from complex network measurements. The method initially transform the sequences and represents them as complex networks. Then it extracts topological measures and constructs a feature vector that is used to classify the sequences. The method was evaluated in the classification of coding and non-coding RNAs of 13 species and compared to the CNCI, PLEK and CPC2 methods. BASiNET outperformed all compared methods in all adopted organisms and datasets. BASiNET have classified sequences in all organisms with high accuracy and low standard deviation, showing that the method is robust and non-biased by the organism. The proposed methodology is implemented in open source in R language and freely available for download at https://cran.r-project.org/package=BASiNET.

Reducing assembly complexity of microbial genomes with single-molecule sequencing.

PubMed

Koren, Sergey; Harhay, Gregory P; Smith, Timothy P L; Bono, James L; Harhay, Dayna M; Mcvey, Scott D; Radune, Diana; Bergman, Nicholas H; Phillippy, Adam M

2013-01-01

The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem. To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads. Automated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.

Employment of Near Full-Length Ribosome Gene TA-Cloning and Primer-Blast to Detect Multiple Species in a Natural Complex Microbial Community Using Species-Specific Primers Designed with Their Genome Sequences.

PubMed

Zhang, Huimin; He, Hongkui; Yu, Xiujuan; Xu, Zhaohui; Zhang, Zhizhou

2016-11-01

It remains an unsolved problem to quantify a natural microbial community by rapidly and conveniently measuring multiple species with functional significance. Most widely used high throughput next-generation sequencing methods can only generate information mainly for genus-level taxonomic identification and quantification, and detection of multiple species in a complex microbial community is still heavily dependent on approaches based on near full-length ribosome RNA gene or genome sequence information. In this study, we used near full-length rRNA gene library sequencing plus Primer-Blast to design species-specific primers based on whole microbial genome sequences. The primers were intended to be specific at the species level within relevant microbial communities, i.e., a defined genomics background. The primers were tested with samples collected from the Daqu (also called fermentation starters) and pit mud of a traditional Chinese liquor production plant. Sixteen pairs of primers were found to be suitable for identification of individual species. Among them, seven pairs were chosen to measure the abundance of microbial species through quantitative PCR. The combination of near full-length ribosome RNA gene library sequencing and Primer-Blast may represent a broadly useful protocol to quantify multiple species in complex microbial population samples with species-specific primers.

DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis.

PubMed

MacConnell, Andrew B; McEnaney, Patrick J; Cavett, Valerie J; Paegel, Brian M

2015-09-14

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10(4) molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75,645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries.

Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis.

PubMed

Nemati, Sara; Fazaeli, Asghar; Hajjaran, Homa; Khamesipour, Ali; Anbaran, Mohsen Falahati; Bozorgomid, Arezoo; Zarei, Fatah

2017-08-01

Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.

Mixed-complexity artificial grammar learning in humans and macaque monkeys: evaluating learning strategies.

PubMed

Wilson, Benjamin; Smith, Kenny; Petkov, Christopher I

2015-03-01

Artificial grammars (AG) can be used to generate rule-based sequences of stimuli. Some of these can be used to investigate sequence-processing computations in non-human animals that might be related to, but not unique to, human language. Previous AG learning studies in non-human animals have used different AGs to separately test for specific sequence-processing abilities. However, given that natural language and certain animal communication systems (in particular, song) have multiple levels of complexity, mixed-complexity AGs are needed to simultaneously evaluate sensitivity to the different features of the AG. Here, we tested humans and Rhesus macaques using a mixed-complexity auditory AG, containing both adjacent (local) and non-adjacent (longer-distance) relationships. Following exposure to exemplary sequences generated by the AG, humans and macaques were individually tested with sequences that were either consistent with the AG or violated specific adjacent or non-adjacent relationships. We observed a considerable level of cross-species correspondence in the sensitivity of both humans and macaques to the adjacent AG relationships and to the statistical properties of the sequences. We found no significant sensitivity to the non-adjacent AG relationships in the macaques. A subset of humans was sensitive to this non-adjacent relationship, revealing interesting between- and within-species differences in AG learning strategies. The results suggest that humans and macaques are largely comparably sensitive to the adjacent AG relationships and their statistical properties. However, in the presence of multiple cues to grammaticality, the non-adjacent relationships are less salient to the macaques and many of the humans. © 2015 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

High-precision relocation for aftershocks of the 2016 ML 5.8 Gyeongju earthquake in South Korea: Stress partitioning controlled by complex fault systems

NASA Astrophysics Data System (ADS)

Woo, J. U.; Rhie, J.; Kang, T. S.; Kim, S.; Chai, G.; Cho, E.

2017-12-01

Complex inherent fault system is one of key factors controlling the main shock occurrence and the pattern of aftershock sequence. Many field studies have shown that the fault systems in the Korean Peninsula are complex because they formed by various tectonic events since Proterozoic. Apart from that the mainshock is the largest one (ML 5.8) ever recorded in South Korea, the Gyeongju earthquake sequence shows particularly interesting features: ML 5.1 event preceded ML 5.8 event by 50 min and they are located closely to each other ( 1 km). In addition, ML 4.5 event occurred 2 3 km away from the two events after a week of the mainshock. Considering reported focal mechanisms and hypocenters of the three major events, it is unlikely that the earthquake sequence occurs on a single fault plane. To depict the detailed fault geometry associated with the sequence, we precisely determine the relative locations of 1,400 aftershocks recorded by 27 broadband stations, which started to be deployed less than one hour after the mainshock. Double difference algorithm is applied using relative travel time measurements by a waveform cross-correlation method. Relocated hypocenters show that a major fault striking NE-SW and some minor faults get involved in the sequence. In particular, aftershocks immediately following ML 4.5 event seem to occur on a fault striking NW-SE, which is orthogonal to the strike of a major fault. We expect that the Gyeongju earthquake sequence resulted from the stress transfer controlled by the complex inherent fault system in this region.

Adaptive decoding of convolutional codes

NASA Astrophysics Data System (ADS)

Hueske, K.; Geldmacher, J.; Götze, J.

2007-06-01

Convolutional codes, which are frequently used as error correction codes in digital transmission systems, are generally decoded using the Viterbi Decoder. On the one hand the Viterbi Decoder is an optimum maximum likelihood decoder, i.e. the most probable transmitted code sequence is obtained. On the other hand the mathematical complexity of the algorithm only depends on the used code, not on the number of transmission errors. To reduce the complexity of the decoding process for good transmission conditions, an alternative syndrome based decoder is presented. The reduction of complexity is realized by two different approaches, the syndrome zero sequence deactivation and the path metric equalization. The two approaches enable an easy adaptation of the decoding complexity for different transmission conditions, which results in a trade-off between decoding complexity and error correction performance.

Complete and Draft Genome Sequences of Nine Lactobacillus sakei Strains Selected from the Three Known Phylogenetic Lineages and Their Main Clonal Complexes.

PubMed

Loux, Valentin; Coeuret, Gwendoline; Zagorec, Monique; Champomier Vergès, Marie-Christine; Chaillou, Stéphane

2018-04-19

We present here the complete and draft genome sequences of nine Lactobacillus sakei strains, selected from the entire range of clonal complexes from the three known lineages of the species. The strains were chosen to provide a wide view of pangenomic and plasmidic diversity for this important foodborne species. Copyright © 2018 Loux et al.

MicroRNA expression profile in bovine cumulus–oocyte complexes: Possible role of let-7 and miR-106a in the development of bovine oocytes

USDA-ARS?s Scientific Manuscript database

The expression of microRNAs (miRs) in bovine cumulus-oocyte complexes (COCs) during late oogenesis was profiled to determine the potential for regulation of maternal mRNAs by this class of small RNAs. A cDNA cloning and sequencing strategy resulted in 1812 putative miR sequences, representing 72 di...

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex.

PubMed

Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Schedl, Paul; Karch, Francois

2015-11-01

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Molecular Structure and Sequence in Complex Coacervates

NASA Astrophysics Data System (ADS)

Sing, Charles; Lytle, Tyler; Madinya, Jason; Radhakrishna, Mithun

Oppositely-charged polyelectrolytes in aqueous solution can undergo associative phase separation, in a process known as complex coacervation. This results in a polyelectrolyte-dense phase (coacervate) and polyelectrolyte-dilute phase (supernatant). There remain challenges in understanding this process, despite a long history in polymer physics. We use Monte Carlo simulation to demonstrate that molecular features (charge spacing, size) play a crucial role in governing the equilibrium in coacervates. We show how these molecular features give rise to strong monomer sequence effects, due to a combination of counterion condensation and correlation effects. We distinguish between structural and sequence-based correlations, which can be designed to tune the phase diagram of coacervation. Sequence effects further inform the physical understanding of coacervation, and provide the basis for new coacervation models that take monomer-level features into account.

Isotopic constraints on contamination processes in the Tonian Goiás Stratiform Complex

NASA Astrophysics Data System (ADS)

Giovanardi, Tommaso; Mazzucchelli, Maurizio; Lugli, Federico; Girardi, Vicente A. V.; Correia, Ciro T.; Tassinari, Colombo C. G.; Cipriani, Anna

2018-06-01

The Tonian Goiás Stratiform Complex (TGSC, Goiás, central Brazil), is one of the largest mafic-ultramafic layered complexes in the world, emplaced during the geotectonic events that led to the Gondwana accretion. In this study, we present trace elements and in-situ U/Pb-Lu-Hf analyses of zircons and 87Sr/86Sr ratios of plagioclases from anorthosites and gabbros of the TGSC. Although formed by three isolated bodies (Cana Brava, Niquelândia and Barro Alto), and characterized by a Lower and Upper Sequence (LS and US), our new U/Pb zircon data confirm recent geochemical, geochronological, and structural evidences that the TGSC has originated from a single intrusive body in the Neoproterozoic. New Hf and Sr isotope ratios construe a complex contamination history for the TGSC, with different geochemical signatures in the two sequences. The low Hf and high Sr isotope ratios of the Lower Sequence (εHf(t) from -4.2 down to -27.5; 87Sr/86Sr = 0.706605-0.729226), suggest the presence of a crustal component and are consistent with contamination from meta-pelitic and calc-silicate rocks found as xenoliths within the Sequence. The more radiogenic Hf isotope ratios and low Sr isotope composition of the Upper Sequence (εHf(t) from 11.3 down to -8.4; 87Sr/86Sr = 0.702368-0.702452), suggest a contamination from mantle-derived metabasalts in agreement with the occurrences of amphibolite xenoliths in the US stratigraphy. The differential contamination of the two sequences is explained by the intrusion of the TGSC in a stratified crust dominated by metasedimentary rocks in its deeper part and metavolcanics at shallower levels. Moreover, the differential thermal gradient in the two crystallizing sequences might have contributed to the preservation and recrystallization of inherited zircon grains in the US and total dissolution or magmatic overgrowth of the LS zircons via melt/rock reaction processes.

Genotyping-by-sequencing (GBS) revealed molecular genetic diversity of Iranian wheat landraces and cultivars

USDA-ARS?s Scientific Manuscript database

Genetic diversity is an essential resource for breeders to improve new cultivars with desirable characteristics. Recently genotyping-by-sequencing (GBS), a next generation sequencing (NGS) based technology that can simplify complex genomes, has been used as a high-throughput and cost-effective molec...

High-Throughput resequencing of maize landraces at genomic regions associated with flowering time

USDA-ARS?s Scientific Manuscript database

Despite the reduction in the price of sequencing, it remains expensive to sequence and assemble whole, complex genomes of multiple samples for population studies, particularly for large genomes like those of many crop species. Enrichment of target genome regions coupled with next generation sequenci...

Genotyping-by-sequencing in three octoploid cultivated strawberry families

USDA-ARS?s Scientific Manuscript database

With the goal of evaluating genotyping-by-sequencing (GBS) in a species with a complex octoploid genome, GBS was used to survey genome-wide single-nucleotide polymorphisms (SNPs) in three biparental strawberry (Fragaria ×ananassa) populations. GBS sequence data were aligned to the F. vesca ‘Fvb’ ref...

Transcriptome sequencing of diverse peanut (arachis) wild species and the cultivated species reveals a wealth of untapped genetic variability

USDA-ARS?s Scientific Manuscript database

Next generation sequencing technologies and improved bioinformatics methods have provided opportunities to study sequence variability in complex polyploid transcriptomes. In this study, we used a diverse panel of twenty-two Arachis accessions representing seven Arachis hypogaea market classes, A-, B...

The Contribution of Short Repeats of Low Sequence Complexity to Large Conifer Genomes

Treesearch

A. Schmidt; R.L. Doudrick; J.S. Heslop-Harrison; T. Schmidt

2000-01-01

Abstract: The abundance and genomic organization of six simple sequence repeats, consisting of di-, tri-, and tetranucleotide sequence motifs, and a minisatellite repeat have been analyzed in different gymnosperms by Southern hybridization. Within the gymnosperm genomes investigated, the abundance and genomic organization of micro- and...

Quantification of fetal heart rate regularity using symbolic dynamics

NASA Astrophysics Data System (ADS)

van Leeuwen, P.; Cysarz, D.; Lange, S.; Geue, D.; Groenemeyer, D.

2007-03-01

Fetal heart rate complexity was examined on the basis of RR interval time series obtained in the second and third trimester of pregnancy. In each fetal RR interval time series, short term beat-to-beat heart rate changes were coded in 8bit binary sequences. Redundancies of the 28 different binary patterns were reduced by two different procedures. The complexity of these sequences was quantified using the approximate entropy (ApEn), resulting in discrete ApEn values which were used for classifying the sequences into 17 pattern sets. Also, the sequences were grouped into 20 pattern classes with respect to identity after rotation or inversion of the binary value. There was a specific, nonuniform distribution of the sequences in the pattern sets and this differed from the distribution found in surrogate data. In the course of gestation, the number of sequences increased in seven pattern sets, decreased in four and remained unchanged in six. Sequences that occurred less often over time, both regular and irregular, were characterized by patterns reflecting frequent beat-to-beat reversals in heart rate. They were also predominant in the surrogate data, suggesting that these patterns are associated with stochastic heart beat trains. Sequences that occurred more frequently over time were relatively rare in the surrogate data. Some of these sequences had a high degree of regularity and corresponded to prolonged heart rate accelerations or decelerations which may be associated with directed fetal activity or movement or baroreflex activity. Application of the pattern classes revealed that those sequences with a high degree of irregularity correspond to heart rate patterns resulting from complex physiological activity such as fetal breathing movements. The results suggest that the development of the autonomic nervous system and the emergence of fetal behavioral states lead to increases in not only irregular but also regular heart rate patterns. Using symbolic dynamics to examine the cardiovascular system may thus lead to new insight with respect to fetal development.

DNA barcoding of human-biting black flies (Diptera: Simuliidae) in Thailand.

PubMed

Pramual, Pairot; Thaijarern, Jiraporn; Wongpakam, Komgrit

2016-12-01

Black flies (Diptera: Simuliidae) are important insect vectors and pests of humans and animals. Accurate identification, therefore, is important for control and management. In this study, we used mitochondrial cytochrome oxidase I (COI) barcoding sequences to test the efficiency of species identification for the human-biting black flies in Thailand. We used human-biting specimens because they enabled us to link information with previous studies involving the immature stages. Three black fly taxa, Simulium nodosum, S. nigrogilvum and S. doipuiense complex, were collected. The S. doipuiense complex was confirmed for the first time as having human-biting habits. The COI sequences revealed considerable genetic diversity in all three species. Comparisons to a COI sequence library of black flies in Thailand and in a public database indicated a high efficiency for specimen identification for S. nodosum and S. nigrogilvum, but this method was not successful for the S. doipuiense complex. Phylogenetic analyses revealed two divergent lineages in the S. doipuiense complex. Human-biting specimens formed a separate clade from other members of this complex. The results are consistent with the Barcoding Index Number System (BINs) analysis that found six BINs in the S. doipuiense complex. Further taxonomic work is needed to clarify the species status of these human-biting specimens. Copyright © 2016 Elsevier B.V. All rights reserved.

The intrinsic combinatorial organization and information theoretic content of a sequence are correlated to the DNA encoded nucleosome organization of eukaryotic genomes.

PubMed

Utro, Filippo; Di Benedetto, Valeria; Corona, Davide F V; Giancarlo, Raffaele

2016-03-15

Thanks to research spanning nearly 30 years, two major models have emerged that account for nucleosome organization in chromatin: statistical and sequence specific. The first is based on elegant, easy to compute, closed-form mathematical formulas that make no assumptions of the physical and chemical properties of the underlying DNA sequence. Moreover, they need no training on the data for their computation. The latter is based on some sequence regularities but, as opposed to the statistical model, it lacks the same type of closed-form formulas that, in this case, should be based on the DNA sequence only. We contribute to close this important methodological gap between the two models by providing three very simple formulas for the sequence specific one. They are all based on well-known formulas in Computer Science and Bioinformatics, and they give different quantifications of how complex a sequence is. In view of how remarkably well they perform, it is very surprising that measures of sequence complexity have not even been considered as candidates to close the mentioned gap. We provide experimental evidence that the intrinsic level of combinatorial organization and information-theoretic content of subsequences within a genome are strongly correlated to the level of DNA encoded nucleosome organization discovered by Kaplan et al Our results establish an important connection between the intrinsic complexity of subsequences in a genome and the intrinsic, i.e. DNA encoded, nucleosome organization of eukaryotic genomes. It is a first step towards a mathematical characterization of this latter 'encoding'. Supplementary data are available at Bioinformatics online. futro@us.ibm.com. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Pyrosequencing as a tool for the identification of common isolates of Mycobacterium sp.

PubMed

Tuohy, Marion J; Hall, Gerri S; Sholtis, Mary; Procop, Gary W

2005-04-01

Pyrosequencing technology, sequencing by addition, was evaluated for categorization of mycobacterial isolates. One hundred and eighty-nine isolates, including 18 ATCC and Trudeau Mycobacterial Culture Collection (TMC) strains, were studied. There were 38 Mycobacterium tuberculosis complex, 27 M. kansasii, 27 MAI complex, 21 M. marinum, 14 M. gordonae, 20 M. chelonae-abscessus group, 10 M. fortuitum, 5 M. xenopi, 3 M. celatum, 2 M. terrae complex, 20 M. mucogenicum, and 2 M. scrofulaceum. Nucleic acid extracts were prepared from solid media or MGIT broth. Traditional PCR was performed with one of the primers biotinylated; the assay targeted a portion of the 16S rRNA gene that contains a hypervariable region, which has been previously shown to be useful for the identification of mycobacteria. The PSQ Sample Preparation Kit was used, and the biotinylated PCR product was processed to a single-stranded DNA template. The sequencing primer was hybridized to the DNA template in a PSQ96 plate. Incorporation of the complementary nucleotides resulted in light generation peaks, forming a pyrogram, which was evaluated by the instrument software. Thirty basepairs were used for isolate categorization. Manual interpretation of the sequences was performed if the quality of the 30-bp sequence was in doubt or if more than 4 bp homopolymers were recognized. Sequences with more than 5 bp of bad quality were deemed unacceptable. When blasted against GenBank, 179 of 189 sequences (94.7%) assigned isolates to the correct molecular genus or group. Ten M. gordonae isolates had more than 5 bp of bad quality sequence and were not accepted. Pyrosequencing of this hypervariable region afforded rapid and acceptable characterization of common, routinely isolated clinical Mycobacterium sp. Algorithms are recommended for further differentiation with an additional sequencing primer or additional biochemicals.

DNA–DNA kissing complexes as a new tool for the assembly of DNA nanostructures

PubMed Central

Barth, Anna; Kobbe, Daniela; Focke, Manfred

2016-01-01

Kissing-loop annealing of nucleic acids occurs in nature in several viruses and in prokaryotic replication, among other circumstances. Nucleobases of two nucleic acid strands (loops) interact with each other, although the two strands cannot wrap around each other completely because of the adjacent double-stranded regions (stems). In this study, we exploited DNA kissing-loop interaction for nanotechnological application. We functionalized the vertices of DNA tetrahedrons with DNA stem-loop sequences. The complementary loop sequence design allowed the hybridization of different tetrahedrons via kissing-loop interaction, which might be further exploited for nanotechnology applications like cargo transport and logical elements. Importantly, we were able to manipulate the stability of those kissing-loop complexes based on the choice and concentration of cations, the temperature and the number of complementary loops per tetrahedron either at the same or at different vertices. Moreover, variations in loop sequences allowed the characterization of necessary sequences within the loop as well as additional stability control of the kissing complexes. Therefore, the properties of the presented nanostructures make them an important tool for DNA nanotechnology. PMID:26773051

Conserved structures formed by heterogeneous RNA sequences drive silencing of an inflammation responsive post-transcriptional operon

PubMed Central

Basu, Abhijit; Jain, Niyati; Tolbert, Blanton S.; Komar, Anton A.

2017-01-01

Abstract RNA–protein interactions with physiological outcomes usually rely on conserved sequences within the RNA element. By contrast, activity of the diverse gamma-interferon-activated inhibitor of translation (GAIT)-elements relies on the conserved RNA folding motifs rather than the conserved sequence motifs. These elements drive the translational silencing of a group of chemokine (CC/CXC) and chemokine receptor (CCR) mRNAs, thereby helping to resolve physiological inflammation. Despite sequence dissimilarity, these RNA elements adopt common secondary structures (as revealed by 2D-1H NMR spectroscopy), providing a basis for their interaction with the RNA-binding GAIT complex. However, many of these elements (e.g. those derived from CCL22, CXCL13, CCR4 and ceruloplasmin (Cp) mRNAs) have substantially different affinities for GAIT complex binding. Toeprinting analysis shows that different positions within the overall conserved GAIT element structure contribute to differential affinities of the GAIT protein complex towards the elements. Thus, heterogeneity of GAIT elements may provide hierarchical fine-tuning of the resolution of inflammation. PMID:29069516

A distinct alleles and genetic recombination of pmrCAB operon in species of Acinetobacter baumannii complex isolates.

PubMed

Kim, Dae Hun; Ko, Kwan Soo

2015-07-01

To investigate pmrCAB sequence divergence in 5 species of Acinetobacter baumannii complex, a total of 80 isolates from a Korean hospital were explored. We evaluated nucleotide and amino acid polymorphisms of pmrCAB operon, and phylogenetic trees were constructed for each gene of prmCAB operon. Colistin and polymyxin B susceptibility was determined for all isolates, and multilocus sequence typing was also performed for A. baumannii isolates. Our results showed that each species of A. baumannii complex has divergent pmrCAB operon sequences. We identified a distinct pmrCAB allele allied with Acinetobacter nosocomialis in gene trees. Different grouping in each gene tree suggests sporadic recombination or emergence of pmrCAB genes among Acinetobacter species. Sequence polymorphisms among Acinetobacter species might not be associated with colistin resistance. We revealed that a distinct pmrCAB allele may be widespread across the continents such as North America and Asia and that sporadic genetic recombination or emergence of pmrCAB genes might occur. Copyright © 2015 Elsevier Inc. All rights reserved.

DNA condensing effects and sequence selectivity of DNA binding of antitumor noncovalent polynuclear platinum complexes.

PubMed

Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor

2014-02-03

The noncovalent analogues of antitumor polynuclear platinum complexes represent a structurally discrete class of platinum drugs. Their chemical and biological properties differ significantly from those of most platinum chemotherapeutics, which bind to DNA in a covalent manner by formation of Pt-DNA adducts. In spite of the fact that these noncovalent polynuclear platinum complexes contain no leaving groups, they have been shown to bind to DNA with high affinity. We report here on the DNA condensation properties of a series of noncovalent analogues of antitumor polynuclear platinum complexes described by biophysical and biochemical methods. The results demonstrate that these polynuclear platinum compounds are capable of inducing DNA condensation at more than 1 order of magnitude lower concentrations than conventional spermine. Atomic force microscopy studies of DNA condensation confined to a mica substrate have revealed that the DNA morphologies become more compact with increasing concentration of the platinum complexes. Moreover, we also found that the noncovalent polynuclear platinum complex [{Pt(NH3)3}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}](6+) (TriplatinNC-A) binds to DNA in a sequence-dependent manner, namely, to A/T-rich sequences and A-tract regions, and that noncovalent polynuclear platinum complexes protect DNA from enzymatic cleavage by DNase I. The results suggest that mechanisms of antitumor and cytotoxic activities of these complexes may be associated with their unique ability to condense DNA along with their sequence-specific DNA binding. Owing to their high cellular accumulation, it is also reasonable to suggest that their mechanism of action is based on the competition with naturally occurring DNA condensing agents, such as polyamines spermine, spermidine, and putrescine, for intracellular binding sites, resulting in the disturbance of the correct binding of regulatory proteins initiating the onset of apoptosis.

Deciphering the glycosaminoglycan code with the help of microarrays.

PubMed

de Paz, Jose L; Seeberger, Peter H

2008-07-01

Carbohydrate microarrays have become a powerful tool to elucidate the biological role of complex sugars. Microarrays are particularly useful for the study of glycosaminoglycans (GAGs), a key class of carbohydrates. The high-throughput chip format enables rapid screening of large numbers of potential GAG sequences produced via a complex biosynthesis while consuming very little sample. Here, we briefly highlight the most recent advances involving GAG microarrays built with synthetic or naturally derived oligosaccharides. These chips are powerful tools for characterizing GAG-protein interactions and determining structure-activity relationships for specific sequences. Thereby, they contribute to decoding the information contained in specific GAG sequences.

Cloning and expression of recombinant adhesive protein MEFP-2 of the blue mussel, Mytilus edulis

DOEpatents

Silverman, Heather G.; Roberto, Francisco F.

2006-02-07

The present invention includes a Mytilus edulis cDNA having a nucleotide sequence that encodes for the Mytilus edulis foot protein-2 (Mefp-2), an example of a mollusk foot protein. Mefp-2 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-2 gene will allow researchers to produce Mefp-2 protein using genetic engineering techniques. The discovery of Mefp-2 gene sequences will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

Three perspectives on complexity: entropy, compression, subsymmetry

NASA Astrophysics Data System (ADS)

Nagaraj, Nithin; Balasubramanian, Karthi

2017-12-01

There is no single universally accepted definition of `Complexity'. There are several perspectives on complexity and what constitutes complex behaviour or complex systems, as opposed to regular, predictable behaviour and simple systems. In this paper, we explore the following perspectives on complexity: effort-to-describe (Shannon entropy H, Lempel-Ziv complexity LZ), effort-to-compress (ETC complexity) and degree-of-order (Subsymmetry or SubSym). While Shannon entropy and LZ are very popular and widely used, ETC is relatively a new complexity measure. In this paper, we also propose a novel normalized complexity measure SubSym based on the existing idea of counting the number of subsymmetries or palindromes within a sequence. We compare the performance of these complexity measures on the following tasks: (A) characterizing complexity of short binary sequences of lengths 4 to 16, (B) distinguishing periodic and chaotic time series from 1D logistic map and 2D Hénon map, (C) analyzing the complexity of stochastic time series generated from 2-state Markov chains, and (D) distinguishing between tonic and irregular spiking patterns generated from the `Adaptive exponential integrate-and-fire' neuron model. Our study reveals that each perspective has its own advantages and uniqueness while also having an overlap with each other.

Structure of Franciscan complex in the Stanley Mountain window, Southern Coast ranges, California

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korsch, R.J.

1982-11-01

Three sets of deformational events are recognized in the Franciscan Complex of the Stanley Mt. area, S. Coast ranges, California. First, in pre-melange time, shortening of the relatively cohesive sequence of interbedded graywacke and mudstone formed isoclinal folds and an axial-plane slaty cleavage. Second, fragmentation of the once cohesive sequence, probably over a considerable period of time, produced the configuration now considered a melange. Third, after the melange developed, the Franciscan Complex was deformed along with the surrounding upper Mesozoic Great Valley sequence into the Stanley Mt. antiform. In the cohesive Upper Cretaceous Carrie Creek Formation, macroscopic and mesoscopic foldsmore » have 2 predominant orientations. The less cohesive Franciscan Complex attempted to fold, as shown by the distribution of shear foliations on stereographic projections, but lack of lithologic continuity and slip along previously formed shear fractures prevents the recognition of macroscopic folds. Hence, in the Franciscan Complex of the Stanley Mt. window, several lines of evidence show that the melange structure is tectonic in origin, not just a tectonic imprint superimposed upon already chaotic rocks of sedimentary origin (olistostromes). 43 references.« less

Sequencing the Black Aspergilli species complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuo, Alan; Salamov, Asaf; Zhou, Kemin

2011-03-11

The ~15 members of the Aspergillus section Nigri species complex (the "Black Aspergilli") are significant as platforms for bioenergy and bioindustrial technology, as members of soil microbial communities and players in the global carbon cycle, and as food processing and spoilage agents and agricultural toxigens. Despite their utility and ubiquity, the morphological and metabolic distinctiveness of the complex's members, and thus their taxonomy, is poorly defined. We are using short read pyrosequencing technology (Roche/454 and Illumina/Solexa) to rapidly scale up genomic and transcriptomic analysis of this species complex. To date we predict 11197 genes in Aspergillus niger, 11624 genes inmore » A. carbonarius, and 10845 genes in A. aculeatus. A. aculeatus is our most recent genome, and was assembled primarily from 454-sequenced reads and annotated with the aid of >2 million 454 ESTs and >300 million Solexa ESTs. To most effectively deploy these very large numbers of ESTs we developed 2 novel methods for clustering the ESTs into assemblies. We have also developed a pipeline to propose orthologies and paralogies among genes in the species complex. In the near future we will apply these methods to additional species of Black Aspergilli that are currently in our sequencing pipeline.« less

SNP discovery by high-throughput sequencing in soybean

PubMed Central

2010-01-01

Background With the advance of new massively parallel genotyping technologies, quantitative trait loci (QTL) fine mapping and map-based cloning become more achievable in identifying genes for important and complex traits. Development of high-density genetic markers in the QTL regions of specific mapping populations is essential for fine-mapping and map-based cloning of economically important genes. Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation existing between any diverse genotypes that are usually used for QTL mapping studies. The massively parallel sequencing technologies (Roche GS/454, Illumina GA/Solexa, and ABI/SOLiD), have been widely applied to identify genome-wide sequence variations. However, it is still remains unclear whether sequence data at a low sequencing depth are enough to detect the variations existing in any QTL regions of interest in a crop genome, and how to prepare sequencing samples for a complex genome such as soybean. Therefore, with the aims of identifying SNP markers in a cost effective way for fine-mapping several QTL regions, and testing the validation rate of the putative SNPs predicted with Solexa short sequence reads at a low sequencing depth, we evaluated a pooled DNA fragment reduced representation library and SNP detection methods applied to short read sequences generated by Solexa high-throughput sequencing technology. Results A total of 39,022 putative SNPs were identified by the Illumina/Solexa sequencing system using a reduced representation DNA library of two parental lines of a mapping population. The validation rates of these putative SNPs predicted with low and high stringency were 72% and 85%, respectively. One hundred sixty four SNP markers resulted from the validation of putative SNPs and have been selectively chosen to target a known QTL, thereby increasing the marker density of the targeted region to one marker per 42 K bp. Conclusions We have demonstrated how to quickly identify large numbers of SNPs for fine mapping of QTL regions by applying massively parallel sequencing combined with genome complexity reduction techniques. This SNP discovery approach is more efficient for targeting multiple QTL regions in a same genetic population, which can be applied to other crops. PMID:20701770

[Analysis of Conformational Features of Watson-Crick Duplex Fragments by Molecular Mechanics and Quantum Mechanics Methods].

PubMed

Poltev, V I; Anisimov, V M; Sanchez, C; Deriabina, A; Gonzalez, E; Garcia, D; Rivas, F; Polteva, N A

2016-01-01

It is generally accepted that the important characteristic features of the Watson-Crick duplex originate from the molecular structure of its subunits. However, it still remains to elucidate what properties of each subunit are responsible for the significant characteristic features of the DNA structure. The computations of desoxydinucleoside monophosphates complexes with Na-ions using density functional theory revealed a pivotal role of DNA conformational properties of single-chain minimal fragments in the development of unique features of the Watson-Crick duplex. We found that directionality of the sugar-phosphate backbone and the preferable ranges of its torsion angles, combined with the difference between purines and pyrimidines. in ring bases, define the dependence of three-dimensional structure of the Watson-Crick duplex on nucleotide base sequence. In this work, we extended these density functional theory computations to the minimal' fragments of DNA duplex, complementary desoxydinucleoside monophosphates complexes with Na-ions. Using several computational methods and various functionals, we performed a search for energy minima of BI-conformation for complementary desoxydinucleoside monophosphates complexes with different nucleoside sequences. Two sequences are optimized using ab initio method at the MP2/6-31++G** level of theory. The analysis of torsion angles, sugar ring puckering and mutual base positions of optimized structures demonstrates that the conformational characteristic features of complementary desoxydinucleoside monophosphates complexes with Na-ions remain within BI ranges and become closer to the corresponding characteristic features of the Watson-Crick duplex crystals. Qualitatively, the main characteristic features of each studied complementary desoxydinucleoside monophosphates complex remain invariant when different computational methods are used, although the quantitative values of some conformational parameters could vary lying within the limits typical for the corresponding family. We observe that popular functionals in density functional theory calculations lead to the overestimated distances between base pairs, while MP2 computations and the newer complex functionals produce the structures that have too close atom-atom contacts. A detailed study of some complementary desoxydinucleoside monophosphate complexes with Na-ions highlights the existence of several energy minima corresponding to BI-conformations, in other words, the complexity of the relief pattern of the potential energy surface of complementary desoxydinucleoside monophosphate complexes. This accounts for variability of conformational parameters of duplex fragments with the same base sequence. Popular molecular mechanics force fields AMBER and CHARMM reproduce most of the conformational characteristics of desoxydinucleoside monophosphates and their complementary complexes with Na-ions but fail to reproduce some details of the dependence of the Watson-Crick duplex conformation on the nucleotide sequence.

A computational proposal for designing structured RNA pools for in vitro selection of RNAs.

PubMed

Kim, Namhee; Gan, Hin Hark; Schlick, Tamar

2007-04-01

Although in vitro selection technology is a versatile experimental tool for discovering novel synthetic RNA molecules, finding complex RNA molecules is difficult because most RNAs identified from random sequence pools are simple motifs, consistent with recent computational analysis of such sequence pools. Thus, enriching in vitro selection pools with complex structures could increase the probability of discovering novel RNAs. Here we develop an approach for engineering sequence pools that links RNA sequence space regions with corresponding structural distributions via a "mixing matrix" approach combined with a graph theory analysis. We define five classes of mixing matrices motivated by covariance mutations in RNA; these constructs define nucleotide transition rates and are applied to chosen starting sequences to yield specific nonrandom pools. We examine the coverage of sequence space as a function of the mixing matrix and starting sequence via clustering analysis. We show that, in contrast to random sequences, which are associated only with a local region of sequence space, our designed pools, including a structured pool for GTP aptamers, can target specific motifs. It follows that experimental synthesis of designed pools can benefit from using optimized starting sequences, mixing matrices, and pool fractions associated with each of our constructed pools as a guide. Automation of our approach could provide practical tools for pool design applications for in vitro selection of RNAs and related problems.

TUMOR HAPLOTYPE ASSEMBLY ALGORITHMS FOR CANCER GENOMICS

PubMed Central

AGUIAR, DEREK; WONG, WENDY S.W.; ISTRAIL, SORIN

2014-01-01

The growing availability of inexpensive high-throughput sequence data is enabling researchers to sequence tumor populations within a single individual at high coverage. But, cancer genome sequence evolution and mutational phenomena like driver mutations and gene fusions are difficult to investigate without first reconstructing tumor haplotype sequences. Haplotype assembly of single individual tumor populations is an exceedingly difficult task complicated by tumor haplotype heterogeneity, tumor or normal cell sequence contamination, polyploidy, and complex patterns of variation. While computational and experimental haplotype phasing of diploid genomes has seen much progress in recent years, haplotype assembly in cancer genomes remains uncharted territory. In this work, we describe HapCompass-Tumor a computational modeling and algorithmic framework for haplotype assembly of copy number variable cancer genomes containing haplotypes at different frequencies and complex variation. We extend our polyploid haplotype assembly model and present novel algorithms for (1) complex variations, including copy number changes, as varying numbers of disjoint paths in an associated graph, (2) variable haplotype frequencies and contamination, and (3) computation of tumor haplotypes using simple cycles of the compass graph which constrain the space of haplotype assembly solutions. The model and algorithm are implemented in the software package HapCompass-Tumor which is available for download from http://www.brown.edu/Research/Istrail_Lab/. PMID:24297529

Characterization of Aftershock Sequences from Large Strike-Slip Earthquakes Along Geometrically Complex Faults

NASA Astrophysics Data System (ADS)

Sexton, E.; Thomas, A.; Delbridge, B. G.

2017-12-01

Large earthquakes often exhibit complex slip distributions and occur along non-planar fault geometries, resulting in variable stress changes throughout the region of the fault hosting aftershocks. To better discern the role of geometric discontinuities on aftershock sequences, we compare areas of enhanced and reduced Coulomb failure stress and mean stress for systematic differences in the time dependence and productivity of these aftershock sequences. In strike-slip faults, releasing structures, including stepovers and bends, experience an increase in both Coulomb failure stress and mean stress during an earthquake, promoting fluid diffusion into the region and further failure. Conversely, Coulomb failure stress and mean stress decrease in restraining bends and stepovers in strike-slip faults, and fluids diffuse away from these areas, discouraging failure. We examine spatial differences in seismicity patterns along structurally complex strike-slip faults which have hosted large earthquakes, such as the 1992 Mw 7.3 Landers, the 2010 Mw 7.2 El-Mayor Cucapah, the 2014 Mw 6.0 South Napa, and the 2016 Mw 7.0 Kumamoto events. We characterize the behavior of these aftershock sequences with the Epidemic Type Aftershock-Sequence Model (ETAS). In this statistical model, the total occurrence rate of aftershocks induced by an earthquake is λ(t) = λ_0 + \\sum_{i:t_i

Extraordinary Structured Noncoding RNAs Revealed by Bacterial Metagenome Analysis

PubMed Central

Weinberg, Zasha; Perreault, Jonathan; Meyer, Michelle M.; Breaker, Ronald R.

2012-01-01

Estimates of the total number of bacterial species1-3 suggest that existing DNA sequence databases carry only a tiny fraction of the total amount of DNA sequence space represented by this division of life. Indeed, environmental DNA samples have been shown to encode many previously unknown classes of proteins4 and RNAs5. Bioinformatics searches6-10 of genomic DNA from bacteria commonly identify novel noncoding RNAs (ncRNAs)10-12 such as riboswitches13,14. In rare instances, RNAs that exhibit more extensive sequence and structural conservation across a wide range of bacteria are encountered15,16. Given that large structured RNAs are known to carry out complex biochemical functions such as protein synthesis and RNA processing reactions, identifying more RNAs of great size and intricate structure is likely to reveal additional biochemical functions that can be achieved by RNA. We applied an updated computational pipeline17 to discover ncRNAs that rival the known large ribozymes in size and structural complexity or that are among the most abundant RNAs in bacteria that encode them. These RNAs would have been difficult or impossible to detect without examining environmental DNA sequences, suggesting that numerous RNAs with extraordinary size, structural complexity, or other exceptional characteristics remain to be discovered in unexplored sequence space. PMID:19956260

Structural basis of UGUA recognition by the Nudix protein CFIm25 and implications for a regulatory role in mRNA 3′ processing

PubMed Central

Yang, Qin; Gilmartin, Gregory M.; Doublié, Sylvie

2010-01-01

Human Cleavage Factor Im (CFIm) is an essential component of the pre-mRNA 3′ processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFIm25) of the CFIm complex possesses a characteristic α/β/α Nudix fold, CFIm25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFIm25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFIm25 is the first Nudix protein to be reported to bind RNA in a sequence-specific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson–Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap4A (diadenosine tetraphosphate) by CFIm25 suggests a potential role for small molecules in the regulation of mRNA 3′ processing. PMID:20479262

Structural basis of UGUA recognition by the Nudix protein CFI(m)25 and implications for a regulatory role in mRNA 3' processing.

PubMed

Yang, Qin; Gilmartin, Gregory M; Doublié, Sylvie

2010-06-01

Human Cleavage Factor Im (CFI(m)) is an essential component of the pre-mRNA 3' processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFI(m)25) of the CFI(m) complex possesses a characteristic alpha/beta/alpha Nudix fold, CFI(m)25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFI(m)25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFI(m)25 is the first Nudix protein to be reported to bind RNA in a sequence-specific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson-Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap(4)A (diadenosine tetraphosphate) by CFI(m)25 suggests a potential role for small molecules in the regulation of mRNA 3' processing.

Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC

PubMed Central

Kong, Daochun; Coleman, Thomas R.; DePamphilis, Melvin L.

2003-01-01

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe. PMID:12840006

Next-generation sequencing strategies enable routine detection of balanced chromosome rearrangements for clinical diagnostics and genetic research.

PubMed

Talkowski, Michael E; Ernst, Carl; Heilbut, Adrian; Chiang, Colby; Hanscom, Carrie; Lindgren, Amelia; Kirby, Andrew; Liu, Shangtao; Muddukrishna, Bhavana; Ohsumi, Toshiro K; Shen, Yiping; Borowsky, Mark; Daly, Mark J; Morton, Cynthia C; Gusella, James F

2011-04-08

The contribution of balanced chromosomal rearrangements to complex disorders remains unclear because they are not detected routinely by genome-wide microarrays and clinical localization is imprecise. Failure to consider these events bypasses a potentially powerful complement to single nucleotide polymorphism and copy-number association approaches to complex disorders, where much of the heritability remains unexplained. To capitalize on this genetic resource, we have applied optimized sequencing and analysis strategies to test whether these potentially high-impact variants can be mapped at reasonable cost and throughput. By using a whole-genome multiplexing strategy, rearrangement breakpoints could be delineated at a fraction of the cost of standard sequencing. For rearrangements already mapped regionally by karyotyping and fluorescence in situ hybridization, a targeted approach enabled capture and sequencing of multiple breakpoints simultaneously. Importantly, this strategy permitted capture and unique alignment of up to 97% of repeat-masked sequences in the targeted regions. Genome-wide analyses estimate that only 3.7% of bases should be routinely omitted from genomic DNA capture experiments. Illustrating the power of these approaches, the rearrangement breakpoints were rapidly defined to base pair resolution and revealed unexpected sequence complexity, such as co-occurrence of inversion and translocation as an underlying feature of karyotypically balanced alterations. These findings have implications ranging from genome annotation to de novo assemblies and could enable sequencing screens for structural variations at a cost comparable to that of microarrays in standard clinical practice. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

PubMed Central

Fuller, Carl W.; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Bibillo, Arek; Stranges, P. Benjamin; Dorwart, Michael; Tao, Chuanjuan; Li, Zengmin; Guo, Wenjing; Shi, Shundi; Korenblum, Daniel; Trans, Andrew; Aguirre, Anne; Liu, Edward; Harada, Eric T.; Pollard, James; Bhat, Ashwini; Cech, Cynthia; Yang, Alexander; Arnold, Cleoma; Palla, Mirkó; Hovis, Jennifer; Chen, Roger; Morozova, Irina; Kalachikov, Sergey; Russo, James J.; Kasianowicz, John J.; Davis, Randy; Roever, Stefan; Church, George M.; Ju, Jingyue

2016-01-01

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods. PMID:27091962

Using the self-select paradigm to delineate the nature of speech motor programming.

PubMed

Wright, David L; Robin, Don A; Rhee, Jooyhun; Vaculin, Amber; Jacks, Adam; Guenther, Frank H; Fox, Peter T

2009-06-01

The authors examined the involvement of 2 speech motor programming processes identified by S. T. Klapp (1995, 2003) during the articulation of utterances differing in syllable and sequence complexity. According to S. T. Klapp, 1 process, INT, resolves the demands of the programmed unit, whereas a second process, SEQ, oversees the serial order demands of longer sequences. A modified reaction time paradigm was used to assess INT and SEQ demands. Specifically, syllable complexity was dependent on syllable structure, whereas sequence complexity involved either repeated or unique syllabi within an utterance. INT execution was slowed when articulating single syllables in the form CCCV compared to simpler CV syllables. Planning unique syllables within a multisyllabic utterance rather than repetitions of the same syllable slowed INT but not SEQ. The INT speech motor programming process, important for mental syllabary access, is sensitive to changes in both syllable structure and the number of unique syllables in an utterance.

Method for Constructing Composite Response Surfaces by Combining Neural Networks with Polynominal Interpolation or Estimation Techniques

NASA Technical Reports Server (NTRS)

Rai, Man Mohan (Inventor); Madavan, Nateri K. (Inventor)

2007-01-01

A method and system for data modeling that incorporates the advantages of both traditional response surface methodology (RSM) and neural networks is disclosed. The invention partitions the parameters into a first set of s simple parameters, where observable data are expressible as low order polynomials, and c complex parameters that reflect more complicated variation of the observed data. Variation of the data with the simple parameters is modeled using polynomials; and variation of the data with the complex parameters at each vertex is analyzed using a neural network. Variations with the simple parameters and with the complex parameters are expressed using a first sequence of shape functions and a second sequence of neural network functions. The first and second sequences are multiplicatively combined to form a composite response surface, dependent upon the parameter values, that can be used to identify an accurate mode

Data compression of discrete sequence: A tree based approach using dynamic programming

NASA Technical Reports Server (NTRS)

Shivaram, Gurusrasad; Seetharaman, Guna; Rao, T. R. N.

1994-01-01

A dynamic programming based approach for data compression of a ID sequence is presented. The compression of an input sequence of size N to that of a smaller size k is achieved by dividing the input sequence into k subsequences and replacing the subsequences by their respective average values. The partitioning of the input sequence is carried with the intention of reducing the mean squared error in the reconstructed sequence. The complexity involved in finding the partitions which would result in such an optimal compressed sequence is reduced by using the dynamic programming approach, which is presented.

Pierre Robin sequence

MedlinePlus

Pierre Robin syndrome; Pierre Robin complex; Pierre Robin anomaly ... The exact causes of Pierre Robin sequence are unknown. It may be part of many genetic syndromes. The lower jaw develops slowly before birth, but may grow ...

Sequencing Complex Genomic Regions

ScienceCinema

Eichler, Evan

2018-02-12

Evan Eichler, Howard Hughes Medical Investigator at the University of Washington, gives the May 28, 2009 keynote speech at the "Sequencing, Finishing, Analysis in the Future" meeting in Santa Fe, NM. Part 1 of 2

Protein Crystal Eco R1 Endonulease-DNA Complex

NASA Technical Reports Server (NTRS)

1998-01-01

Type II restriction enzymes, such as Eco R1 endonulease, present a unique advantage for the study of sequence-specific recognition because they leave a record of where they have been in the form of the cleaved ends of the DNA sites where they were bound. The differential behavior of a sequence -specific protein at sites of differing base sequence is the essence of the sequence-specificity; the core question is how do these proteins discriminate between different DNA sequences especially when the two sequences are very similar. Principal Investigator: Dan Carter/New Century Pharmaceuticals

Sequence modelling and an extensible data model for genomic database

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Peter Wei-Der

1992-01-01

The Human Genome Project (HGP) plans to sequence the human genome by the beginning of the next century. It will generate DNA sequences of more than 10 billion bases and complex marker sequences (maps) of more than 100 million markers. All of these information will be stored in database management systems (DBMSs). However, existing data models do not have the abstraction mechanism for modelling sequences and existing DBMS's do not have operations for complex sequences. This work addresses the problem of sequence modelling in the context of the HGP and the more general problem of an extensible object data modelmore » that can incorporate the sequence model as well as existing and future data constructs and operators. First, we proposed a general sequence model that is application and implementation independent. This model is used to capture the sequence information found in the HGP at the conceptual level. In addition, abstract and biological sequence operators are defined for manipulating the modelled sequences. Second, we combined many features of semantic and object oriented data models into an extensible framework, which we called the Extensible Object Model'', to address the need of a modelling framework for incorporating the sequence data model with other types of data constructs and operators. This framework is based on the conceptual separation between constructors and constraints. We then used this modelling framework to integrate the constructs for the conceptual sequence model. The Extensible Object Model is also defined with a graphical representation, which is useful as a tool for database designers. Finally, we defined a query language to support this model and implement the query processor to demonstrate the feasibility of the extensible framework and the usefulness of the conceptual sequence model.« less

Sequence modelling and an extensible data model for genomic database

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Peter Wei-Der

1992-01-01

The Human Genome Project (HGP) plans to sequence the human genome by the beginning of the next century. It will generate DNA sequences of more than 10 billion bases and complex marker sequences (maps) of more than 100 million markers. All of these information will be stored in database management systems (DBMSs). However, existing data models do not have the abstraction mechanism for modelling sequences and existing DBMS`s do not have operations for complex sequences. This work addresses the problem of sequence modelling in the context of the HGP and the more general problem of an extensible object data modelmore » that can incorporate the sequence model as well as existing and future data constructs and operators. First, we proposed a general sequence model that is application and implementation independent. This model is used to capture the sequence information found in the HGP at the conceptual level. In addition, abstract and biological sequence operators are defined for manipulating the modelled sequences. Second, we combined many features of semantic and object oriented data models into an extensible framework, which we called the ``Extensible Object Model``, to address the need of a modelling framework for incorporating the sequence data model with other types of data constructs and operators. This framework is based on the conceptual separation between constructors and constraints. We then used this modelling framework to integrate the constructs for the conceptual sequence model. The Extensible Object Model is also defined with a graphical representation, which is useful as a tool for database designers. Finally, we defined a query language to support this model and implement the query processor to demonstrate the feasibility of the extensible framework and the usefulness of the conceptual sequence model.« less

In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

PubMed

Paca-Uccaralertkun, S; Zhao, L J; Adya, N; Cross, J V; Cullen, B R; Boros, I M; Giam, C Z

1994-01-01

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.

NMR and computational methods applied to the 3- dimensional structure determination of DNA and ligand-DNA complexes in solution

NASA Astrophysics Data System (ADS)

Smith, Jarrod Anson

2D homonuclear 1H NMR methods and restrained molecular dynamics (rMD) calculations have been applied to determining the three-dimensional structures of DNA and minor groove-binding ligand-DNA complexes in solution. The structure of the DNA decamer sequence d(GCGTTAACGC)2 has been solved both with a distance-based rMD protocol and an NOE relaxation matrix backcalculation-based protocol in order to probe the relative merits of the different refinement methods. In addition, three minor groove binding ligand-DNA complexes have been examined. The solution structure of the oligosaccharide moiety of the antitumor DNA scission agent calicheamicin γ1I has been determined in complex with a decamer duplex containing its high affinity 5'-TCCT- 3' binding sequence. The structure of the complex reinforces the belief that the oligosaccharide moiety is responsible for the sequence selective minor-groove binding activity of the agent, and critical intermolecular contacts are revealed. The solution structures of both the (+) and (-) enantiomers of the minor groove binding DNA alkylating agent duocarmycin SA have been determined in covalent complex with the undecamer DNA duplex d(GACTAATTGTC).d(GAC AATTAGTC). The results support the proposal that the alkylation activity of the duocarmycin antitumor antibiotics is catalyzed by a binding-induced conformational change in the ligand which activates the cyclopropyl group for reaction with the DNA. Comparisons between the structures of the two enantiomers covalently bound to the same DNA sequence at the same 5'-AATTA-3 ' site have provided insight into the binding orientation and site selectivity, as well as the relative rates of reactivity of these two agents.

Targeted parallel sequencing of the Musa species: searching for an alternative model system for polyploidy studies

USDA-ARS?s Scientific Manuscript database

Modern day genomics holds the promise of solving the complexities of basic plant sciences, and of catalyzing practical advances in plant breeding. While contiguous, "base perfect" deep sequencing is a key module of any genome project, recent advances in parallel next generation sequencing technologi...

Enabling next-gen sequencing and analysis at the USDA-ARS U.S. Meat Animal Research Center with MiniLIMS

USDA-ARS?s Scientific Manuscript database

There is a growing need to combine DNA sequencing technologies to address complex problems in genome biology. These genomic studies routinely generate voluminous image, sequence, and mapping files that should be associated with quality control information (gels, spectra, etc.), and other important ...

Endophyte Microbiome Diversity in Micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

PubMed Central

Lucero, Mary E.; Unc, Adrian; Cooke, Peter; Dowd, Scot; Sun, Shulei

2011-01-01

Microbial diversity associated with micropropagated Atriplex species was assessed using microscopy, isolate culturing, and sequencing. Light, electron, and confocal microscopy revealed microbial cells in aseptically regenerated leaves and roots. Clone libraries and tag-encoded FLX amplicon pyrosequencing (TEFAP) analysis amplified sequences from callus homologous to diverse fungal and bacterial taxa. Culturing isolated some seed borne endophyte taxa which could be readily propagated apart from the host. Microbial cells were observed within biofilm-like residues associated with plant cell surfaces and intercellular spaces. Various universal primers amplified both plant and microbial sequences, with different primers revealing different patterns of fungal diversity. Bacterial and fungal TEFAP followed by alignment with sequences from curated databases revealed 7 bacterial and 17 ascomycete taxa in A. canescens, and 5 bacterial taxa in A. torreyi. Additional diversity was observed among isolates and clone libraries. Micropropagated Atriplex retains a complex, intimately associated microbiome which includes diverse strains well poised to interact in manners that influence host physiology. Microbiome analysis was facilitated by high throughput sequencing methods, but primer biases continue to limit recovery of diverse sequences from even moderately complex communities. PMID:21437280

On the derivatives of unimodular polynomials

NASA Astrophysics Data System (ADS)

Nevai, P.; Erdélyi, T.

2016-04-01

Let D be the open unit disk of the complex plane; its boundary, the unit circle of the complex plane, is denoted by \\partial D. Let \\mathscr P_n^c denote the set of all algebraic polynomials of degree at most n with complex coefficients. For λ ≥ 0, let {\\mathscr K}_n^λ \\stackrel{{def}}{=} \\biggl\\{P_n: P_n(z) = \\sumk=0^n{ak k^λ z^k}, ak \\in { C}, |a_k| = 1 \\biggr\\} \\subset {\\mathscr P}_n^c.The class \\mathscr K_n^0 is often called the collection of all (complex) unimodular polynomials of degree n. Given a sequence (\\varepsilon_n) of positive numbers tending to 0, we say that a sequence (P_n) of polynomials P_n\\in\\mathscr K_n^λ is \\{λ, (\\varepsilon_n)\\}-ultraflat if \\displaystyle (1-\\varepsilon_n)\\frac{nλ+1/2}{\\sqrt{2λ+1}}≤\\ve......a +1/2}}{\\sqrt{2λ +1}},\\qquad z \\in \\partial D,\\quad n\\in N_0.Although we do not know, in general, whether or not \\{λ, (\\varepsilon_n)\\}-ultraflat sequences of polynomials P_n\\in\\mathscr K_n^λ exist for each fixed λ>0, we make an effort to prove various interesting properties of them. These allow us to conclude that there are no sequences (P_n) of either conjugate, or plain, or skew reciprocal unimodular polynomials P_n\\in\\mathscr K_n^0 such that (Q_n) with Q_n(z)\\stackrel{{def}}{=} zP_n'(z)+1 is a \\{1,(\\varepsilon_n)\\}-ultraflat sequence of polynomials.Bibliography: 18 titles.

Comparison of a High-Resolution Melting Assay to Next-Generation Sequencing for Analysis of HIV Diversity

PubMed Central

Cousins, Matthew M.; Ou, San-San; Wawer, Maria J.; Munshaw, Supriya; Swan, David; Magaret, Craig A.; Mullis, Caroline E.; Serwadda, David; Porcella, Stephen F.; Gray, Ronald H.; Quinn, Thomas C.; Donnell, Deborah; Eshleman, Susan H.

2012-01-01

Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution. PMID:22785188

High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency

PubMed Central

Calvo, Sarah E; Tucker, Elena J; Compton, Alison G; Kirby, Denise M; Crawford, Gabriel; Burtt, Noel P; Rivas, Manuel A; Guiducci, Candace; Bruno, Damien L; Goldberger, Olga A; Redman, Michelle C; Wiltshire, Esko; Wilson, Callum J; Altshuler, David; Gabriel, Stacey B; Daly, Mark J; Thorburn, David R; Mootha, Vamsi K

2010-01-01

Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing, and experimental validation to uncover the molecular basis of mitochondrial complex I (CI) disorders. We created five pools of DNA from a cohort of 103 patients and then performed deep sequencing of 103 candidate genes to spotlight 151 rare variants predicted to impact protein function. We used confirmatory experiments to establish genetic diagnoses in 22% of previously unsolved cases, and discovered that defects in NUBPL and FOXRED1 can cause CI deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can reveal novel disease-causing mutations in individual patients. PMID:20818383

The instant sequencing task: Toward constraint-checking a complex spacecraft command sequence interactively

NASA Technical Reports Server (NTRS)

Horvath, Joan C.; Alkalaj, Leon J.; Schneider, Karl M.; Amador, Arthur V.; Spitale, Joseph N.

1993-01-01

Robotic spacecraft are controlled by sets of commands called 'sequences.' These sequences must be checked against mission constraints. Making our existing constraint checking program faster would enable new capabilities in our uplink process. Therefore, we are rewriting this program to run on a parallel computer. To do so, we had to determine how to run constraint-checking algorithms in parallel and create a new method of specifying spacecraft models and constraints. This new specification gives us a means of representing flight systems and their predicted response to commands which could be used in a variety of applications throughout the command process, particularly during anomaly or high-activity operations. This commonality could reduce operations cost and risk for future complex missions. Lessons learned in applying some parts of this system to the TOPEX/Poseidon mission will be described.

Basic quantitative polymerase chain reaction using real-time fluorescence measurements.

PubMed

Ares, Manuel

2014-10-01

This protocol uses quantitative polymerase chain reaction (qPCR) to measure the number of DNA molecules containing a specific contiguous sequence in a sample of interest (e.g., genomic DNA or cDNA generated by reverse transcription). The sample is subjected to fluorescence-based PCR amplification and, theoretically, during each cycle, two new duplex DNA molecules are produced for each duplex DNA molecule present in the sample. The progress of the reaction during PCR is evaluated by measuring the fluorescence of dsDNA-dye complexes in real time. In the early cycles, DNA duplication is not detected because inadequate amounts of DNA are made. At a certain threshold cycle, DNA-dye complexes double each cycle for 8-10 cycles, until the DNA concentration becomes so high and the primer concentration so low that the reassociation of the product strands blocks efficient synthesis of new DNA and the reaction plateaus. There are two types of measurements: (1) the relative change of the target sequence compared to a reference sequence and (2) the determination of molecule number in the starting sample. The first requires a reference sequence, and the second requires a sample of the target sequence with known numbers of the molecules of sequence to generate a standard curve. By identifying the threshold cycle at which a sample first begins to accumulate DNA-dye complexes exponentially, an estimation of the numbers of starting molecules in the sample can be extrapolated. © 2014 Cold Spring Harbor Laboratory Press.

Can you sequence ecology? Metagenomics of adaptive diversification.

PubMed

Marx, Christopher J

2013-01-01

Few areas of science have benefited more from the expansion in sequencing capability than the study of microbial communities. Can sequence data, besides providing hypotheses of the functions the members possess, detect the evolutionary and ecological processes that are occurring? For example, can we determine if a species is adapting to one niche, or if it is diversifying into multiple specialists that inhabit distinct niches? Fortunately, adaptation of populations in the laboratory can serve as a model to test our ability to make such inferences about evolution and ecology from sequencing. Even adaptation to a single niche can give rise to complex temporal dynamics due to the transient presence of multiple competing lineages. If there are multiple niches, this complexity is augmented by segmentation of the population into multiple specialists that can each continue to evolve within their own niche. For a known example of parallel diversification that occurred in the laboratory, sequencing data gave surprisingly few obvious, unambiguous signs of the ecological complexity present. Whereas experimental systems are open to direct experimentation to test hypotheses of selection or ecological interaction, the difficulty in "seeing ecology" from sequencing for even such a simple system suggests translation to communities like the human microbiome will be quite challenging. This will require both improved empirical methods to enhance the depth and time resolution for the relevant polymorphisms and novel statistical approaches to rigorously examine time-series data for signs of various evolutionary and ecological phenomena within and between species.

Structure-affinity relationships for the binding of actinomycin D to DNA

NASA Astrophysics Data System (ADS)

Gallego, José; Ortiz, Angel R.; de Pascual-Teresa, Beatriz; Gago, Federico

1997-03-01

Molecular models of the complexes between actinomycin D and 14 different DNA hexamers were built based on the X-ray crystal structure of the actinomycin-d(GAAGCTTC)2 complex. The DNA sequences included the canonical GpC binding step flanked by different base pairs, nonclassical binding sites such as GpG and GpT, and sites containing 2,6-diamino- purine. A good correlation was found between the intermolecular interaction energies calculated for the refined complexes and the relative preferences of actinomycin binding to standard and modified DNA. A detailed energy decomposition into van der Waals and electrostatic components for the interactions between the DNA base pairs and either the chromophore or the peptidic part of the antibiotic was performed for each complex. The resulting energy matrix was then subjected to principal component analysis, which showed that actinomycin D discriminates among different DNA sequences by an interplay of hydrogen bonding and stacking interactions. The structure-affinity relationships for this important antitumor drug are thus rationalized and may be used to advantage in the design of novel sequence-specific DNA-binding agents.

A Targeted Oligonucleotide Enhancer of SMN2 Exon 7 Splicing Forms Competing Quadruplex and Protein Complexes in Functional Conditions

PubMed Central

Smith, Lindsay D.; Dickinson, Rachel L.; Lucas, Christian M.; Cousins, Alex; Malygin, Alexey A.; Weldon, Carika; Perrett, Andrew J.; Bottrill, Andrew R.; Searle, Mark S.; Burley, Glenn A.; Eperon, Ian C.

2014-01-01

Summary The use of oligonucleotides to activate the splicing of selected exons is limited by a poor understanding of the mechanisms affected. A targeted bifunctional oligonucleotide enhancer of splicing (TOES) anneals to SMN2 exon 7 and carries an exonic splicing enhancer (ESE) sequence. We show that it stimulates splicing specifically of intron 6 in the presence of repressing sequences in intron 7. Complementarity to the 5′ end of exon 7 increases U2AF65 binding, but the ESE sequence is required for efficient recruitment of U2 snRNP. The ESE forms at least three coexisting discrete states: a quadruplex, a complex containing only hnRNP F/H, and a complex enriched in the activator SRSF1. Neither hnRNP H nor quadruplex formation contributes to ESE activity. The results suggest that splicing limited by weak signals can be rescued by rapid exchange of TOES oligonucleotides in various complexes and raise the possibility that SR proteins associate transiently with ESEs. PMID:25263560

Genomic Analysis of Complex Microbial Communities in Wounds

DTIC Science & Technology

2009-07-01

Actinobacteria — were the most commonly misclassified [25]. The 16S sequences used in the current study were all greater than or equal to 200 bases...with most (89.1%) of the sequences falling into Firmicutes, Proteobac- teria, and Actinobacteria phyla. High percentages of the Firmicutes and... Actinobacteria sequences were successfully assigned to the genus level, 88.0% and 82.3%, respectively; however, only 53.0% of the Proteobacteria sequences

Plastome Sequence Determination and Comparative Analysis for Members of the Lolium-Festuca Grass Species Complex

PubMed Central

Hand, Melanie L.; Spangenberg, German C.; Forster, John W.; Cogan, Noel O. I.

2013-01-01

Chloroplast genome sequences are of broad significance in plant biology, due to frequent use in molecular phylogenetics, comparative genomics, population genetics, and genetic modification studies. The present study used a second-generation sequencing approach to determine and assemble the plastid genomes (plastomes) of four representatives from the agriculturally important Lolium-Festuca species complex of pasture grasses (Lolium multiflorum, Festuca pratensis, Festuca altissima, and Festuca ovina). Total cellular DNA was extracted from either roots or leaves, was sequenced, and the output was filtered for plastome-related reads. A comparison between sources revealed fewer plastome-related reads from root-derived template but an increase in incidental bacterium-derived sequences. Plastome assembly and annotation indicated high levels of sequence identity and a conserved organization and gene content between species. However, frequent deletions within the F. ovina plastome appeared to contribute to a smaller plastid genome size. Comparative analysis with complete plastome sequences from other members of the Poaceae confirmed conservation of most grass-specific features. Detailed analysis of the rbcL–psaI intergenic region, however, revealed a “hot-spot” of variation characterized by independent deletion events. The evolutionary implications of this observation are discussed. The complete plastome sequences are anticipated to provide the basis for potential organelle-specific genetic modification of pasture grasses. PMID:23550121

Campbell's monkeys concatenate vocalizations into context-specific call sequences

PubMed Central

Ouattara, Karim; Lemasson, Alban; Zuberbühler, Klaus

2009-01-01

Primate vocal behavior is often considered irrelevant in modeling human language evolution, mainly because of the caller's limited vocal control and apparent lack of intentional signaling. Here, we present the results of a long-term study on Campbell's monkeys, which has revealed an unrivaled degree of vocal complexity. Adult males produced six different loud call types, which they combined into various sequences in highly context-specific ways. We found stereotyped sequences that were strongly associated with cohesion and travel, falling trees, neighboring groups, nonpredatory animals, unspecific predatory threat, and specific predator classes. Within the responses to predators, we found that crowned eagles triggered four and leopards three different sequences, depending on how the caller learned about their presence. Callers followed a number of principles when concatenating sequences, such as nonrandom transition probabilities of call types, addition of specific calls into an existing sequence to form a different one, or recombination of two sequences to form a third one. We conclude that these primates have overcome some of the constraints of limited vocal control by combinatorial organization. As the different sequences were so tightly linked to specific external events, the Campbell's monkey call system may be the most complex example of ‘proto-syntax’ in animal communication known to date. PMID:20007377

Molecular Species Delimitation in the Racomitrium canescens Complex (Grimmiaceae) and Implications for DNA Barcoding of Species Complexes in Mosses

PubMed Central

Stech, Michael; Veldman, Sarina; Larraín, Juan; Muñoz, Jesús; Quandt, Dietmar; Hassel, Kristian; Kruijer, Hans

2013-01-01

In bryophytes a morphological species concept is still most commonly employed, but delimitation of closely related species based on morphological characters is often difficult. Here we test morphological species circumscriptions in a species complex of the moss genus Racomitrium, the R. canescens complex, based on variable DNA sequence markers from the plastid (rps4-trnT-trnL region) and nuclear (nrITS) genomes. The extensive morphological variability within the complex has led to different opinions about the number of species and intraspecific taxa to be distinguished. Molecular phylogenetic reconstructions allowed to clearly distinguish all eight currently recognised species of the complex plus a ninth species that was inferred to belong to the complex in earlier molecular analyses. The taxonomic significance of intraspecific sequence variation is discussed. The present molecular data do not support the division of the R. canescens complex into two groups of species (subsections or sections). Most morphological characters, albeit being in part difficult to apply, are reliable for species identification in the R. canescens complex. However, misidentification of collections that were morphologically intermediate between species questioned the suitability of leaf shape as diagnostic character. Four partitions of the molecular markers (rps4-trnT, trnT-trnL, ITS1, ITS2) that could potentially be used for molecular species identification (DNA barcoding) performed almost equally well concerning amplification and sequencing success. Of these, ITS1 provided the highest species discrimination capacity and should be considered as a DNA barcoding marker for mosses, especially in complexes of closely related species. Molecular species identification should be complemented by redefining morphological characters, to develop a set of easy-to-use molecular and non-molecular identification tools for improving biodiversity assessments and ecological research including mosses. PMID:23341927

The major histocompatibility complex of tassel-eared squirrels. II. Genetic diversity associated with Abert squirrels.

PubMed

Wettstein, P J; States, J S

1986-01-01

The extent of polymorphism and the rate of divergence of class I and class II sequences mapping to the mammalian major histocompatibility complex (MHC) have been the subject of experimentation and speculation. To provide further insight into the evolution of the MHC we have initiated the analysis of two geographically isolated subspecies of tassel-eared squirrels. In the preceding communication we described the number and polymorphism of TSLA class I and class II sequences in Kaibab squirrels (S. aberti kaibabensis), which live north of the Grand Canyon. In this report we present a parallel analysis of Abert squirrels (S. aberti aberti), which live south of the Grand Canyon in northern Arizona. Genomic DNA from 12 Abert squirrels was digested with restriction enzymes, electrophoresed, blotted, and hybridized with DR alpha, DR beta, DQ alpha, DQ beta, and HLA-B7 probes. The results of these hybridizations were remarkably similar to those obtained in Kaibab squirrels. The majority of class I and class II bands were identical in size and number, suggesting that Abert and Kaibab squirrels have not significantly diverged in the TSLA complex despite their geographical separation. Relative polymorphism of class II sequences was similar to that observed with Kaibab squirrels: beta sequences exhibited higher polymorphism than alpha sequences. As in Kaibab squirrels, a number of alpha and beta sequences were apparently carried on the same fragments. In comparison to class II beta sequences, there was limited polymorphism in class I sequences, although a diverse number of class I genotypes were observed. Attempts to identify segregating TSLA haplotypes were futile in that the only families of sequences with concordant distributions were DQ alpha and DQ beta. These observations and those obtained with Kaibab squirrels suggest that the present-day TSLA haplotypes of both subspecies are derived from a limited number of common, progenitor haplotypes through repeated intra-TSLA recombination.

Novel Insights into Tree Biology and Genome Evolution as Revealed Through Genomics.

PubMed

Neale, David B; Martínez-García, Pedro J; De La Torre, Amanda R; Montanari, Sara; Wei, Xiao-Xin

2017-04-28

Reference genome sequences are the key to the discovery of genes and gene families that determine traits of interest. Recent progress in sequencing technologies has enabled a rapid increase in genome sequencing of tree species, allowing the dissection of complex characters of economic importance, such as fruit and wood quality and resistance to biotic and abiotic stresses. Although the number of reference genome sequences for trees lags behind those for other plant species, it is not too early to gain insight into the unique features that distinguish trees from nontree plants. Our review of the published data suggests that, although many gene families are conserved among herbaceous and tree species, some gene families, such as those involved in resistance to biotic and abiotic stresses and in the synthesis and transport of sugars, are often expanded in tree genomes. As the genomes of more tree species are sequenced, comparative genomics will further elucidate the complexity of tree genomes and how this relates to traits unique to trees.

Genetic diversity based on 28S rDNA sequences among populations of Culex quinquefasciatus collected at different locations in Tamil Nadu, India.

PubMed

Sakthivelkumar, S; Ramaraj, P; Veeramani, V; Janarthanan, S

2015-09-01

The basis of the present study was to distinguish the existence of any genetic variability among populations of Culex quinquefasciatus which would be a valuable tool in the management of mosquito control programmes. In the present study, population of Cx. quinquefasciatus collected at different locations in Tamil Nadu were analyzed for their genetic variation based on 28S rDNA D2 region nucleotide sequences. A high degree of genetic polymorphism was detected in the sequences of D2 region of 28S rDNA on the predicted secondary structures in spite of high nucleotide sequence similarity. The findings based on secondary structure using rDNA sequences suggested the existence of a complex genotypic diversity of Cx. quinquefasciatus population collected at different locations of Tamil Nadu, India. This complexity in genetic diversity in a single mosquito population collected at different locations is considered an important issue towards their influence and nature of vector potential of these mosquitoes.

Crosslinking transcription factors to their recognition sequences with PtII complexes

NASA Technical Reports Server (NTRS)

Chu, B. C.; Orgel, L. E.

1992-01-01

We have prepared phosphorothioate-containing cyclic oligodeoxynucleotides that fold into 'dumbbells' containing CRE and TRE sequences, the binding sequences for the CREB and JUN proteins, respectively. Six phosphorothioate residues were introduced into each of the recognition sequences. K2PtCl4 crosslinks CRE to CREB and TRE to JUN. The extent of crosslinking is about eight times greater than that observed with standard oligodeoxynucleotides and amounts to 30-50% of the efficiency of non-covalent association as estimated by gel-shift assays. Crosslinking is reversed by incubation with NaCN. The crosslinking reaction is specific--a dumbbell oligonucleotide with six phosphorothioate groups introduced into the Sp1 recognition sequence could not be crosslinked efficiently to CREB or JUN proteins with K2PtCl4. The binding of TRE to CREB is not strong enough for effective detection by gel-shift assays, but the TRE-CREB complex is crosslinked efficiently by K2PtCl4 and can then readily be detected.

A spin echo sequence with a single-sided bipolar diffusion gradient pulse to obtain snapshot diffusion weighted images in moving media

NASA Astrophysics Data System (ADS)

Freidlin, R. Z.; Kakareka, J. W.; Pohida, T. J.; Komlosh, M. E.; Basser, P. J.

2012-08-01

In vivo MRI data can be corrupted by motion. Motion artifacts are particularly troublesome in Diffusion Weighted MRI (DWI), since the MR signal attenuation due to Brownian motion can be much less than the signal loss due to dephasing from other types of complex tissue motion, which can significantly degrade the estimation of self-diffusion coefficients, diffusion tensors, etc. This paper describes a snapshot DWI sequence, which utilizes a novel single-sided bipolar diffusion sensitizing gradient pulse within a spin echo sequence. The proposed method shortens the diffusion time by applying a single refocused bipolar diffusion gradient on one side of a refocusing RF pulse, instead of a set of diffusion sensitizing gradients, separated by a refocusing RF pulse, while reducing the impact of magnetic field inhomogeneity by using a spin echo sequence. A novel MRI phantom that can exhibit a range of complex motions was designed to demonstrate the robustness of the proposed DWI sequence.

Haplotag: Software for Haplotype-Based Genotyping-by-Sequencing Analysis

PubMed Central

Tinker, Nicholas A.; Bekele, Wubishet A.; Hattori, Jiro

2016-01-01

Genotyping-by-sequencing (GBS), and related methods, are based on high-throughput short-read sequencing of genomic complexity reductions followed by discovery of single nucleotide polymorphisms (SNPs) within sequence tags. This provides a powerful and economical approach to whole-genome genotyping, facilitating applications in genomics, diversity analysis, and molecular breeding. However, due to the complexity of analyzing large data sets, applications of GBS may require substantial time, expertise, and computational resources. Haplotag, the novel GBS software described here, is freely available, and operates with minimal user-investment on widely available computer platforms. Haplotag is unique in fulfilling the following set of criteria: (1) operates without a reference genome; (2) can be used in a polyploid species; (3) provides a discovery mode, and a production mode; (4) discovers polymorphisms based on a model of tag-level haplotypes within sequenced tags; (5) reports SNPs as well as haplotype-based genotypes; and (6) provides an intuitive visual “passport” for each inferred locus. Haplotag is optimized for use in a self-pollinating plant species. PMID:26818073

Chromosome rearrangements via template switching between diverged repeated sequences

PubMed Central

Anand, Ranjith P.; Tsaponina, Olga; Greenwell, Patricia W.; Lee, Cheng-Sheng; Du, Wei; Petes, Thomas D.

2014-01-01

Recent high-resolution genome analyses of cancer and other diseases have revealed the occurrence of microhomology-mediated chromosome rearrangements and copy number changes. Although some of these rearrangements appear to involve nonhomologous end-joining, many must have involved mechanisms requiring new DNA synthesis. Models such as microhomology-mediated break-induced replication (MM-BIR) have been invoked to explain these rearrangements. We examined BIR and template switching between highly diverged sequences in Saccharomyces cerevisiae, induced during repair of a site-specific double-strand break (DSB). Our data show that such template switches are robust mechanisms that give rise to complex rearrangements. Template switches between highly divergent sequences appear to be mechanistically distinct from the initial strand invasions that establish BIR. In particular, such jumps are less constrained by sequence divergence and exhibit a different pattern of microhomology junctions. BIR traversing repeated DNA sequences frequently results in complex translocations analogous to those seen in mammalian cells. These results suggest that template switching among repeated genes is a potent driver of genome instability and evolution. PMID:25367035

The Representation of Prediction Error in Auditory Cortex

PubMed Central

Rubin, Jonathan; Ulanovsky, Nachum; Tishby, Naftali

2016-01-01

To survive, organisms must extract information from the past that is relevant for their future. How this process is expressed at the neural level remains unclear. We address this problem by developing a novel approach from first principles. We show here how to generate low-complexity representations of the past that produce optimal predictions of future events. We then illustrate this framework by studying the coding of ‘oddball’ sequences in auditory cortex. We find that for many neurons in primary auditory cortex, trial-by-trial fluctuations of neuronal responses correlate with the theoretical prediction error calculated from the short-term past of the stimulation sequence, under constraints on the complexity of the representation of this past sequence. In some neurons, the effect of prediction error accounted for more than 50% of response variability. Reliable predictions often depended on a representation of the sequence of the last ten or more stimuli, although the representation kept only few details of that sequence. PMID:27490251

BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes.

PubMed

Staňková, Helena; Hastie, Alex R; Chan, Saki; Vrána, Jan; Tulpová, Zuzana; Kubaláková, Marie; Visendi, Paul; Hayashi, Satomi; Luo, Mingcheng; Batley, Jacqueline; Edwards, David; Doležel, Jaroslav; Šimková, Hana

2016-07-01

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC-by-BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high-resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high-resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome-scale analysis of repetitive sequences and revealed a ~800-kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone-by-clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC-contig physical map and validate sequence assembly on a chromosome-arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome-by-chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Optimization of parameter values for complex pulse sequences by simulated annealing: application to 3D MP-RAGE imaging of the brain.

PubMed

Epstein, F H; Mugler, J P; Brookeman, J R

1994-02-01

A number of pulse sequence techniques, including magnetization-prepared gradient echo (MP-GRE), segmented GRE, and hybrid RARE, employ a relatively large number of variable pulse sequence parameters and acquire the image data during a transient signal evolution. These sequences have recently been proposed and/or used for clinical applications in the brain, spine, liver, and coronary arteries. Thus, the need for a method of deriving optimal pulse sequence parameter values for this class of sequences now exists. Due to the complexity of these sequences, conventional optimization approaches, such as applying differential calculus to signal difference equations, are inadequate. We have developed a general framework for adapting the simulated annealing algorithm to pulse sequence parameter value optimization, and applied this framework to the specific case of optimizing the white matter-gray matter signal difference for a T1-weighted variable flip angle 3D MP-RAGE sequence. Using our algorithm, the values of 35 sequence parameters, including the magnetization-preparation RF pulse flip angle and delay time, 32 flip angles in the variable flip angle gradient-echo acquisition sequence, and the magnetization recovery time, were derived. Optimized 3D MP-RAGE achieved up to a 130% increase in white matter-gray matter signal difference compared with optimized 3D RF-spoiled FLASH with the same total acquisition time. The simulated annealing approach was effective at deriving optimal parameter values for a specific 3D MP-RAGE imaging objective, and may be useful for other imaging objectives and sequences in this general class.

Differentials on graph complexes II: hairy graphs

NASA Astrophysics Data System (ADS)

Khoroshkin, Anton; Willwacher, Thomas; Živković, Marko

2017-10-01

We study the cohomology of the hairy graph complexes which compute the rational homotopy of embedding spaces, generalizing the Vassiliev invariants of knot theory. We provide spectral sequences converging to zero whose first pages contain the hairy graph cohomology. Our results yield a way to construct many nonzero hairy graph cohomology classes out of (known) non-hairy classes by studying the cancellations in those sequences. This provide a first glimpse at the tentative global structure of the hairy graph cohomology.

Structural brain aging and speech production: a surface-based brain morphometry study.

PubMed

Tremblay, Pascale; Deschamps, Isabelle

2016-07-01

While there has been a growing number of studies examining the neurofunctional correlates of speech production over the past decade, the neurostructural correlates of this immensely important human behaviour remain less well understood, despite the fact that previous studies have established links between brain structure and behaviour, including speech and language. In the present study, we thus examined, for the first time, the relationship between surface-based cortical thickness (CT) and three different behavioural indexes of sublexical speech production: response duration, reaction times and articulatory accuracy, in healthy young and older adults during the production of simple and complex meaningless sequences of syllables (e.g., /pa-pa-pa/ vs. /pa-ta-ka/). The results show that each behavioural speech measure was sensitive to the complexity of the sequences, as indicated by slower reaction times, longer response durations and decreased articulatory accuracy in both groups for the complex sequences. Older adults produced longer speech responses, particularly during the production of complex sequence. Unique age-independent and age-dependent relationships between brain structure and each of these behavioural measures were found in several cortical and subcortical regions known for their involvement in speech production, including the bilateral anterior insula, the left primary motor area, the rostral supramarginal gyrus, the right inferior frontal sulcus, the bilateral putamen and caudate, and in some region less typically associated with speech production, such as the posterior cingulate cortex.

Contribution of PCR Denaturing Gradient Gel Electrophoresis Combined with Mixed Chromatogram Software Separation for Complex Urinary Sample Analysis.

PubMed

Kotásková, Iva; Mališová, Barbora; Obručová, Hana; Holá, Veronika; Peroutková, Tereza; Růžička, Filip; Freiberger, Tomáš

2017-01-01

Complex samples are a challenge for sequencing-based broad-range diagnostics. We analysed 19 urinary catheter, ureteral Double-J catheter, and urine samples using 3 methodological approaches. Out of the total 84 operational taxonomic units, 37, 61, and 88% were identified by culture, PCR-DGGE-SS (PCR denaturing gradient gel electrophoresis followed by Sanger sequencing), and PCR-DGGE-RM (PCR- DGGE combined with software chromatogram separation by RipSeq Mixed tool), respectively. The latter approach was shown to be an efficient tool to complement culture in complex sample assessment. © 2017 S. Karger AG, Basel.

Triple helix purification and sequencing

DOEpatents

Wang, Renfeng; Smith, Lloyd M.; Tong, Xinchun E.

1995-01-01

Disclosed herein are methods, kits, and equipment for purifying single stranded circular DNA and then using the DNA for DNA sequencing purposes. Templates are provided with an insert having a hybridization region. An elongated oligonucleotide has two regions that are complementary to the insert and the oligo is bound to a magnetic anchor. The oligo hybridizes to the insert on two sides to form a stable triple helix complex. The anchor can then be used to drag the template out of solution using a magnet. The system can purify sequencing templates, and if desired the triple helix complex can be opened up to a double helix so that the oligonucleotide will act as a primer for further DNA synthesis.

Triple helix purification and sequencing

DOEpatents

Wang, R.; Smith, L.M.; Tong, X.E.

1995-03-28

Disclosed herein are methods, kits, and equipment for purifying single stranded circular DNA and then using the DNA for DNA sequencing purposes. Templates are provided with an insert having a hybridization region. An elongated oligonucleotide has two regions that are complementary to the insert and the oligo is bound to a magnetic anchor. The oligo hybridizes to the insert on two sides to form a stable triple helix complex. The anchor can then be used to drag the template out of solution using a magnet. The system can purify sequencing templates, and if desired the triple helix complex can be opened up to a double helix so that the oligonucleotide will act as a primer for further DNA synthesis. 4 figures.

DNA barcoding of Bemisia tabaci complex (Hemiptera: Aleyrodidae) reveals southerly expansion of the dominant whitefly species on cotton in Pakistan.

PubMed

Ashfaq, Muhammad; Hebert, Paul D N; Mirza, M Sajjad; Khan, Arif M; Mansoor, Shahid; Shah, Ghulam S; Zafar, Yusuf

2014-01-01

Although whiteflies (Bemisia tabaci complex) are an important pest of cotton in Pakistan, its taxonomic diversity is poorly understood. As DNA barcoding is an effective tool for resolving species complexes and analyzing species distributions, we used this approach to analyze genetic diversity in the B. tabaci complex and map the distribution of B. tabaci lineages in cotton growing areas of Pakistan. Sequence diversity in the DNA barcode region (mtCOI-5') was examined in 593 whiteflies from Pakistan to determine the number of whitefly species and their distributions in the cotton-growing areas of Punjab and Sindh provinces. These new records were integrated with another 173 barcode sequences for B. tabaci, most from India, to better understand regional whitefly diversity. The Barcode Index Number (BIN) System assigned the 766 sequences to 15 BINs, including nine from Pakistan. Representative specimens of each Pakistan BIN were analyzed for mtCOI-3' to allow their assignment to one of the putative species in the B. tabaci complex recognized on the basis of sequence variation in this gene region. This analysis revealed the presence of Asia II 1, Middle East-Asia Minor 1, Asia 1, Asia II 5, Asia II 7, and a new lineage "Pakistan". The first two taxa were found in both Punjab and Sindh, but Asia 1 was only detected in Sindh, while Asia II 5, Asia II 7 and "Pakistan" were only present in Punjab. The haplotype networks showed that most haplotypes of Asia II 1, a species implicated in transmission of the cotton leaf curl virus, occurred in both India and Pakistan. DNA barcodes successfully discriminated cryptic species in B. tabaci complex. The dominant haplotypes in the B. tabaci complex were shared by India and Pakistan. Asia II 1 was previously restricted to Punjab, but is now the dominant lineage in southern Sindh; its southward spread may have serious implications for cotton plantations in this region.

Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery.

PubMed

Upadhya, Archana; Sangave, Preeti C

2016-10-01

Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa-arginine (R 8 ) and CHK 6 HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep-3, flanked with CH 3 on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide-plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non-covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide-plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

BAC-pool sequencing and analysis of large segments of A12 and D12 homoeologous chromosomes in Upland cotton

USDA-ARS?s Scientific Manuscript database

New and emerging next generation sequencing technologies have reduced sequencing costs, but there is room for additional approaches that can be applied to complex polyploid plant genomes. Large (about 2.5GB) and highly repetitive tetraploid genome of G. hirsutum is still cost-intensive with traditi...

Acquisition of Initial /s/-Stop and Stop-/s/ Sequences in Greek

ERIC Educational Resources Information Center

Syrika, Asimina; Nicolaidis, Katerina; Edwards, Jan; Beckman, Mary E.

2011-01-01

Previous work on children's acquisition of complex sequences points to a tendency for affricates to be acquired before clusters, but there is no clear evidence of a difference in order of acquisition between clusters with /s/ that violate the Sonority Sequencing Principle (SSP), such as /s/ followed by stop in onset position, and other clusters…

Evaluation of a dkgB linked intergenic sequence ribotyping (ISR) method for assigning serotype to Salmonella enterica isolated from poultry environmental samples.

USDA-ARS?s Scientific Manuscript database

The Kauffman White (KW) serotyping method requires more than 250 antisera to characterize more than 2,500 Salmonella serovars. The complexity of serotyping could be overcome using molecular methods. In this study, a dkgB-linked intergenic sequence ribotyping (ISR) method that generates sequence occu...

Draft Genome Sequence of Fish Pathogen Aeromonas bestiarum GA97-22.

PubMed

Kumru, Salih; Tekedar, Hasan C; Griffin, Matt J; Waldbieser, Geoffrey C; Liles, Mark R; Sonstegard, Tad; Schroeder, Steven G; Lawrence, Mark L; Karsi, Attila

2018-06-14

Aeromonas bestiarum is a Gram-negative mesophilic motile bacterium causing acute hemorrhagic septicemia or chronic skin ulcers in fish. Here, we report the draft genome sequence of A. bestiarum strain GA97-22, which was isolated from rainbow trout in 1997. This genome sequence will improve our understanding of the complex taxonomy of motile aeromonads.

Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shevtsov, M. B.; Streeter, S. D.; Thresh, S.-J.

2015-02-01

The structure of the new class of controller proteins (exemplified by C.Csp231I) in complex with its 21 bp DNA-recognition sequence is presented, and the molecular basis of sequence recognition in this class of proteins is discussed. An unusual extended spacer between the dimer binding sites suggests a novel interaction between the two C-protein dimers. In a wide variety of bacterial restriction–modification systems, a regulatory ‘controller’ protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class ofmore » controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Å resolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.« less

High-resolution record of the environmental response to climatic variations during the Last Interglacial-Glacial cycle in Central Europe: the loess-palaeosol sequence of Dolní Věstonice (Czech Republic)

NASA Astrophysics Data System (ADS)

Antoine, Pierre; Rousseau, Denis-Didier; Degeai, Jean-Philippe; Moine, Olivier; Lagroix, France; kreutzer, Sebastian; Fuchs, Markus; Hatté, Christine; Gauthier, Caroline; Svoboda, Jiri; Lisá, Lenka

2013-05-01

High-resolution multidisciplinary investigation of key European loess-palaeosols profiles have demonstrated that loess sequences result from rapid and cyclic aeolian sedimentation which is reflected in variations of loess grain size indexes and correlated with Greenland ice-core dust records. This correlation suggests a global connection between North Atlantic and west-European air masses. Herein, we present a revised stratigraphy and a continuous high-resolution record of grain-size, magnetic susceptibility and organic carbon δ13C of the famous of Dolní Vestonice (DV) loess sequence in the Moravian region of the Czech Republic. A new set of quartz OSL ages provides a reliable and accurate chronology of the sequence's main pedosedimentary events. The grain size record shows strongly contrasting variations with numerous abrupt coarse-grained events, especially in the upper part of the sequence between ca 20-30 ka. This time period is also characterised by a progressive coarsening of the loess deposits as already observed in other western European sequences. The base of the DV sequence exhibits an exceptionally well-preserved soil complex composed of three chernozem soil horizons and 5 aeolian silt layers (marker silts). This complex is, at present, the most complete record of environmental variations and dust deposition in the European loess belt for the Weichselian Early-glacial period spanning about 110 to 70 ka, allowing correlations with various global palaeoclimatic records. OSL ages combined with sedimentological and palaeopedological observations lead to the conclusion that this soil complex recorded all of the main climatic events expressed in the North GRIP record from Greenland Interstadials (GIS) 25 to 19.

Inaugural Genomics Automation Congress and the coming deluge of sequencing data.

PubMed

Creighton, Chad J

2010-10-01

Presentations at Select Biosciences's first 'Genomics Automation Congress' (Boston, MA, USA) in 2010 focused on next-generation sequencing and the platforms and methodology around them. The meeting provided an overview of sequencing technologies, both new and emerging. Speakers shared their recent work on applying sequencing to profile cells for various levels of biomolecular complexity, including DNA sequences, DNA copy, DNA methylation, mRNA and microRNA. With sequencing time and costs continuing to drop dramatically, a virtual explosion of very large sequencing datasets is at hand, which will probably present challenges and opportunities for high-level data analysis and interpretation, as well as for information technology infrastructure.

Assessing information content and interactive relationships of subgenomic DNA sequences of the MHC using complexity theory approaches based on the non-extensive statistical mechanics

NASA Astrophysics Data System (ADS)

Karakatsanis, L. P.; Pavlos, G. P.; Iliopoulos, A. C.; Pavlos, E. G.; Clark, P. M.; Duke, J. L.; Monos, D. S.

2018-09-01

This study combines two independent domains of science, the high throughput DNA sequencing capabilities of Genomics and complexity theory from Physics, to assess the information encoded by the different genomic segments of exonic, intronic and intergenic regions of the Major Histocompatibility Complex (MHC) and identify possible interactive relationships. The dynamic and non-extensive statistical characteristics of two well characterized MHC sequences from the homozygous cell lines, PGF and COX, in addition to two other genomic regions of comparable size, used as controls, have been studied using the reconstructed phase space theorem and the non-extensive statistical theory of Tsallis. The results reveal similar non-linear dynamical behavior as far as complexity and self-organization features. In particular, the low-dimensional deterministic nonlinear chaotic and non-extensive statistical character of the DNA sequences was verified with strong multifractal characteristics and long-range correlations. The nonlinear indices repeatedly verified that MHC sequences, whether exonic, intronic or intergenic include varying levels of information and reveal an interaction of the genes with intergenic regions, whereby the lower the number of genes in a region, the less the complexity and information content of the intergenic region. Finally we showed the significance of the intergenic region in the production of the DNA dynamics. The findings reveal interesting content information in all three genomic elements and interactive relationships of the genes with the intergenic regions. The results most likely are relevant to the whole genome and not only to the MHC. These findings are consistent with the ENCODE project, which has now established that the non-coding regions of the genome remain to be of relevance, as they are functionally important and play a significant role in the regulation of expression of genes and coordination of the many biological processes of the cell.

Address the Major Societal Challenges

NASA Astrophysics Data System (ADS)

Laubichler, Manfred

In his famous historical account about the origins of molecular biology Gunther Stent introduced a three phase sequence that turns out to be characteristic for many newly emerging paradigms within science. New ideas, according to Stent, follow a sequence of romantic, dogmatic, and academic phases. One can easily see that complex systems science followed this path. The question now is whether we are in an extended academic phase of gradually expanding both theoretical and practical knowledge, or whether we are entering a new transformation of complex systems science that might well bring about a new romantic phase. I would argue that complexity science, indeed, is at the dawn of a new period - let's call it complexity 3.0. The last academic phase has seen the application of complex systems ideas and methods in a variety of different domains. It has been to a large extent business as usual...

Rise and fall of political complexity in island South-East Asia and the Pacific.

PubMed

Currie, Thomas E; Greenhill, Simon J; Gray, Russell D; Hasegawa, Toshikazu; Mace, Ruth

2010-10-14

There is disagreement about whether human political evolution has proceeded through a sequence of incremental increases in complexity, or whether larger, non-sequential increases have occurred. The extent to which societies have decreased in complexity is also unclear. These debates have continued largely in the absence of rigorous, quantitative tests. We evaluated six competing models of political evolution in Austronesian-speaking societies using phylogenetic methods. Here we show that in the best-fitting model political complexity rises and falls in a sequence of small steps. This is closely followed by another model in which increases are sequential but decreases can be either sequential or in bigger drops. The results indicate that large, non-sequential jumps in political complexity have not occurred during the evolutionary history of these societies. This suggests that, despite the numerous contingent pathways of human history, there are regularities in cultural evolution that can be detected using computational phylogenetic methods.

A complex of RAG-1 and RAG-2 proteins persists on DNA after single-strand cleavage at V(D)J recombination signal sequences.

PubMed Central

Grawunder, U; Lieber, M R

1997-01-01

The recombination activating gene (RAG) 1 and 2 proteins are required for initiation of V(D)J recombination in vivo and have been shown to be sufficient to introduce DNA double-strand breaks at recombination signal sequences (RSSs) in a cell-free assay in vitro. RSSs consist of a highly conserved palindromic heptamer that is separated from a slightly less conserved A/T-rich nonamer by either a 12 or 23 bp spacer of random sequence. Despite the high sequence specificity of RAG-mediated cleavage at RSSs, direct binding of the RAG proteins to these sequences has been difficult to demonstrate by standard methods. Even when this can be demonstrated, questions about the order of events for an individual RAG-RSS complex will require methods that monitor aspects of the complex during transitions from one step of the reaction to the next. Here we have used template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) in order to assess occupancy of the reaction intermediates by the RAG complex during the reaction. In addition, this approach allows analysis of the accessibility of end products of a RAG-catalyzed cleavage reaction for N nucleotide addition. The results indicate that RAG proteins form a long-lived complex with the RSS once the initial nick is generated, because the 3'-OH group at the nick remains obstructed for TdT-catalyzed N nucleotide addition. In contrast, the 3'-OH group generated at the signal end after completion of the cleavage reaction can be efficiently tailed by TdT, suggesting that the RAG proteins disassemble from the signal end after DNA double-strand cleavage has been completed. Therefore, a single RAG complex maintains occupancy from the first step (nick formation) to the second step (cleavage). In addition, the results suggest that N region diversity at V(D)J junctions within rearranged immunoglobulin and T cell receptor gene loci can only be introduced after the generation of RAG-catalyzed DNA double-strand breaks, i.e. during the DNA end joining phase of the V(D)J recombination reaction. PMID:9060432

Fast social-like learning of complex behaviors based on motor motifs.

PubMed

Calvo Tapia, Carlos; Tyukin, Ivan Y; Makarov, Valeri A

2018-05-01

Social learning is widely observed in many species. Less experienced agents copy successful behaviors exhibited by more experienced individuals. Nevertheless, the dynamical mechanisms behind this process remain largely unknown. Here we assume that a complex behavior can be decomposed into a sequence of n motor motifs. Then a neural network capable of activating motor motifs in a given sequence can drive an agent. To account for (n-1)! possible sequences of motifs in a neural network, we employ the winnerless competition approach. We then consider a teacher-learner situation: one agent exhibits a complex movement, while another one aims at mimicking the teacher's behavior. Despite the huge variety of possible motif sequences we show that the learner, equipped with the provided learning model, can rewire "on the fly" its synaptic couplings in no more than (n-1) learning cycles and converge exponentially to the durations of the teacher's motifs. We validate the learning model on mobile robots. Experimental results show that the learner is indeed capable of copying the teacher's behavior composed of six motor motifs in a few learning cycles. The reported mechanism of learning is general and can be used for replicating different functions, including, for example, sound patterns or speech.

Fast social-like learning of complex behaviors based on motor motifs

NASA Astrophysics Data System (ADS)

Calvo Tapia, Carlos; Tyukin, Ivan Y.; Makarov, Valeri A.

2018-05-01

Social learning is widely observed in many species. Less experienced agents copy successful behaviors exhibited by more experienced individuals. Nevertheless, the dynamical mechanisms behind this process remain largely unknown. Here we assume that a complex behavior can be decomposed into a sequence of n motor motifs. Then a neural network capable of activating motor motifs in a given sequence can drive an agent. To account for (n -1 )! possible sequences of motifs in a neural network, we employ the winnerless competition approach. We then consider a teacher-learner situation: one agent exhibits a complex movement, while another one aims at mimicking the teacher's behavior. Despite the huge variety of possible motif sequences we show that the learner, equipped with the provided learning model, can rewire "on the fly" its synaptic couplings in no more than (n -1 ) learning cycles and converge exponentially to the durations of the teacher's motifs. We validate the learning model on mobile robots. Experimental results show that the learner is indeed capable of copying the teacher's behavior composed of six motor motifs in a few learning cycles. The reported mechanism of learning is general and can be used for replicating different functions, including, for example, sound patterns or speech.

Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity

PubMed Central

Rand, Tim A.; Ginalski, Krzysztof; Grishin, Nick V.; Wang, Xiaodong

2004-01-01

RNA interference is carried out by the small double-stranded RNA-induced silencing complex (RISC). The RISC-bound small RNA guides the RISC complex to identify and cleave mRNAs with complementary sequences. The proteins that make up the RISC complex and cleave mRNA have not been unequivocally defined. Here, we report the biochemical purification of RISC activity to homogeneity from Drosophila Schnieder 2 cell extracts. Argonaute 2 (Ago-2) is the sole protein component present in the purified, functional RISC. By using a bioinformatics method that combines sequence-profile analysis with predicted protein secondary structure, we found homology between the PIWI domain of Ago-2 and endonuclease V and identified potential active-site amino acid residues within the PIWI domain of Ago-2. PMID:15452342

Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity.

PubMed

Rand, Tim A; Ginalski, Krzysztof; Grishin, Nick V; Wang, Xiaodong

2004-10-05

RNA interference is carried out by the small double-stranded RNA-induced silencing complex (RISC). The RISC-bound small RNA guides the RISC complex to identify and cleave mRNAs with complementary sequences. The proteins that make up the RISC complex and cleave mRNA have not been unequivocally defined. Here, we report the biochemical purification of RISC activity to homogeneity from Drosophila Schnieder 2 cell extracts. Argonaute 2 (Ago-2) is the sole protein component present in the purified, functional RISC. By using a bioinformatics method that combines sequence-profile analysis with predicted protein secondary structure, we found homology between the PIWI domain of Ago-2 and endonuclease V and identified potential active-site amino acid residues within the PIWI domain of Ago-2.

Complexity of genetic sequences modified by horizontal gene transfer and degraded-DNA uptake

NASA Astrophysics Data System (ADS)

Tremberger, George; Dehipawala, S.; Nguyen, A.; Cheung, E.; Sullivan, R.; Holden, T.; Lieberman, D.; Cheung, T.

2015-09-01

Horizontal gene transfer has been a major vehicle for efficient transfer of genetic materials among living species and could be one of the sources for noncoding DNA incorporation into a genome. Our previous study of lnc- RNA sequence complexity in terms of fractal dimension and information entropy shows a tight regulation among the studied genes in numerous diseases. The role of sequence complexity in horizontal transferred genes was investigated with Mealybug in symbiotic relation with a 139K genome microbe and Deinococcus radiodurans as examples. The fractal dimension and entropy showed correlation R-sq of 0.82 (N = 6) for the studied Deinococcus radiodurans sequences. For comparison the Deinococcus radiodurans oxidative stress tolerant catalase and superoxide dismutase genes under extracellular dGMP growth condition showed R-sq ~ 0.42 (N = 6); and the studied arsenate reductase horizontal transferred genes for toxicity survival in several microorganisms showed no correlation. Simulation results showed that R-sq < 0.4 would be improbable at less than one percent chance, suggestive of additional selection pressure when compared to the R-sq ~ 0.29 (N = 21) in the studied transferred genes in Mealybug. The mild correlation of R-sq ~ 0.5 for fractal dimension versus transcription level in the studied Deinococcus radiodurans sequences upon extracellular dGMP growth condition would suggest that lower fractal dimension with less electron density fluctuation favors higher transcription level.

Molecular dynamics studies on the DNA-binding process of ERG.

PubMed

Beuerle, Matthias G; Dufton, Neil P; Randi, Anna M; Gould, Ian R

2016-11-15

The ETS family of transcription factors regulate gene targets by binding to a core GGAA DNA-sequence. The ETS factor ERG is required for homeostasis and lineage-specific functions in endothelial cells, some subset of haemopoietic cells and chondrocytes; its ectopic expression is linked to oncogenesis in multiple tissues. To date details of the DNA-binding process of ERG including DNA-sequence recognition outside the core GGAA-sequence are largely unknown. We combined available structural and experimental data to perform molecular dynamics simulations to study the DNA-binding process of ERG. In particular we were able to reproduce the ERG DNA-complex with a DNA-binding simulation starting in an unbound configuration with a final root-mean-square-deviation (RMSD) of 2.1 Å to the core ETS domain DNA-complex crystal structure. This allowed us to elucidate the relevance of amino acids involved in the formation of the ERG DNA-complex and to identify Arg385 as a novel key residue in the DNA-binding process. Moreover we were able to show that water-mediated hydrogen bonds are present between ERG and DNA in our simulations and that those interactions have the potential to achieve sequence recognition outside the GGAA core DNA-sequence. The methodology employed in this study shows the promising capabilities of modern molecular dynamics simulations in the field of protein DNA-interactions.

Polymorphism and selection in the major histocompatibility complex DRA and DQA genes in the family Equidae.

PubMed

Janova, Eva; Matiasovic, Jan; Vahala, Jiri; Vodicka, Roman; Van Dyk, Enette; Horin, Petr

2009-07-01

The major histocompatibility complex genes coding for antigen binding and presenting molecules are the most polymorphic genes in the vertebrate genome. We studied the DRA and DQA gene polymorphism of the family Equidae. In addition to 11 previously reported DRA and 24 DQA alleles, six new DRA sequences and 13 new DQA alleles were identified in the genus Equus. Phylogenetic analysis of both DRA and DQA sequences provided evidence for trans-species polymorphism in the family Equidae. The phylogenetic trees differed from species relationships defined by standard taxonomy of Equidae and from trees based on mitochondrial or neutral gene sequence data. Analysis of selection showed differences between the less variable DRA and more variable DQA genes. DRA alleles were more often shared by more species. The DQA sequences analysed showed strong amongst-species positive selection; the selected amino acid positions mostly corresponded to selected positions in rodent and human DQA genes.

Issues with RNA-seq analysis in non-model organisms: A salmonid example.

PubMed

Sundaram, Arvind; Tengs, Torstein; Grimholt, Unni

2017-10-01

High throughput sequencing (HTS) is useful for many purposes as exemplified by the other topics included in this special issue. The purpose of this paper is to look into the unique challenges of using this technology in non-model organisms where resources such as genomes, functional genome annotations or genome complexity provide obstacles not met in model organisms. To describe these challenges, we narrow our scope to RNA sequencing used to study differential gene expression in response to pathogen challenge. As a demonstration species we chose Atlantic salmon, which has a sequenced genome with poor annotation and an added complexity due to many duplicated genes. We find that our RNA-seq analysis pipeline deciphers between duplicates despite high sequence identity. However, annotation issues provide problems in linking differentially expressed genes to pathways. Also, comparing results between approaches and species are complicated due to lack of standardized annotation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Next-generation sequencing yields the complete mitochondrial genome of the flathead mullet, Mugil cephalus cryptic species in East Australia (Teleostei: Mugilidae).

PubMed

Shen, Kang-Ning; Chen, Ching-Hung; Hsiao, Chung-Der; Durand, Jean-Dominique

2016-09-01

In this study, the complete mitogenome sequence of a cryptic species from East Australia (Mugil sp. H) belonging to the worldwide Mugil cephalus species complex (Teleostei: Mugilidae) has been sequenced by next-generation sequencing method. The assembled mitogenome, consisting of 16,845 bp, had the typical vertebrate mitochondrial gene arrangement, including 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs genes and a non-coding control region of D-loop. D-loop consists of 1067 bp length, and is located between tRNA-Pro and tRNA-Phe. The overall base composition of East Australia M. cephalus is 28.4% for A, 29.3% for C, 15.4% for G and 26.9% for T. The complete mitogenome may provide essential and important DNA molecular data for further phylogenetic and evolutionary analysis for flathead mullet species complex.

Hyperdiversity of Genes Encoding Integral Light-Harvesting Proteins in the Dinoflagellate Symbiodinium sp

PubMed Central

Boldt, Lynda; Yellowlees, David; Leggat, William

2012-01-01

The superfamily of light-harvesting complex (LHC) proteins is comprised of proteins with diverse functions in light-harvesting and photoprotection. LHC proteins bind chlorophyll (Chl) and carotenoids and include a family of LHCs that bind Chl a and c. Dinophytes (dinoflagellates) are predominantly Chl c binding algal taxa, bind peridinin or fucoxanthin as the primary carotenoid, and can possess a number of LHC subfamilies. Here we report 11 LHC sequences for the chlorophyll a-chlorophyll c 2-peridinin protein complex (acpPC) subfamily isolated from Symbiodinium sp. C3, an ecologically important peridinin binding dinoflagellate taxa. Phylogenetic analysis of these proteins suggests the acpPC subfamily forms at least three clades within the Chl a/c binding LHC family; Clade 1 clusters with rhodophyte, cryptophyte and peridinin binding dinoflagellate sequences, Clade 2 with peridinin binding dinoflagellate sequences only and Clades 3 with heterokontophytes, fucoxanthin and peridinin binding dinoflagellate sequences. PMID:23112815

KGCAK: a K-mer based database for genome-wide phylogeny and complexity evaluation.

PubMed

Wang, Dapeng; Xu, Jiayue; Yu, Jun

2015-09-16

The K-mer approach, treating genomic sequences as simple characters and counting the relative abundance of each string upon a fixed K, has been extensively applied to phylogeny inference for genome assembly, annotation, and comparison. To meet increasing demands for comparing large genome sequences and to promote the use of the K-mer approach, we develop a versatile database, KGCAK ( http://kgcak.big.ac.cn/KGCAK/ ), containing ~8,000 genomes that include genome sequences of diverse life forms (viruses, prokaryotes, protists, animals, and plants) and cellular organelles of eukaryotic lineages. It builds phylogeny based on genomic elements in an alignment-free fashion and provides in-depth data processing enabling users to compare the complexity of genome sequences based on K-mer distribution. We hope that KGCAK becomes a powerful tool for exploring relationship within and among groups of species in a tree of life based on genomic data.

A Fly-Inspired Mushroom Bodies Model for Sensory-Motor Control Through Sequence and Subsequence Learning.

PubMed

Arena, Paolo; Calí, Marco; Patané, Luca; Portera, Agnese; Strauss, Roland

2016-09-01

Classification and sequence learning are relevant capabilities used by living beings to extract complex information from the environment for behavioral control. The insect world is full of examples where the presentation time of specific stimuli shapes the behavioral response. On the basis of previously developed neural models, inspired by Drosophila melanogaster, a new architecture for classification and sequence learning is here presented under the perspective of the Neural Reuse theory. Classification of relevant input stimuli is performed through resonant neurons, activated by the complex dynamics generated in a lattice of recurrent spiking neurons modeling the insect Mushroom Bodies neuropile. The network devoted to context formation is able to reconstruct the learned sequence and also to trace the subsequences present in the provided input. A sensitivity analysis to parameter variation and noise is reported. Experiments on a roving robot are reported to show the capabilities of the architecture used as a neural controller.

Reconsideration of systematic relationships within the order Euplotida (Protista, Ciliophora) using new sequences of the gene coding for small-subunit rRNA and testing the use of combined data sets to construct phylogenies of the Diophrys-complex.

PubMed

Yi, Zhenzhen; Song, Weibo; Clamp, John C; Chen, Zigui; Gao, Shan; Zhang, Qianqian

2009-03-01

Comprehensive molecular analyses of phylogenetic relationships within euplotid ciliates are relatively rare, and the relationships among some families remain questionable. We performed phylogenetic analyses of the order Euplotida based on new sequences of the gene coding for small-subunit RNA (SSrRNA) from a variety of taxa across the entire order as well as sequences from some of these taxa of other genes (ITS1-5.8S-ITS2 region and histone H4) that have not been included in previous analyses. Phylogenetic trees based on SSrRNA gene sequences constructed with four different methods had a consistent branching pattern that included the following features: (1) the "typical" euplotids comprised a paraphyletic assemblage composed of two divergent clades (family Uronychiidae and families Euplotidae-Certesiidae-Aspidiscidae-Gastrocirrhidae), (2) in the family Uronychiidae, the genera Uronychia and Paradiophrys formed a clearly outlined, well-supported clade that seemed to be rather divergent from Diophrys and Diophryopsis, suggesting that the Diophrys-complex may have had a longer and more separate evolutionary history than previously supposed, (3) inclusion of 12 new SSrRNA sequences in analyses of Euplotidae revealed two new clades of species within the family and cast additional doubt on the present classification of genera within the family, and (4) the intraspecific divergence among five species of Aspidisca was far greater than those of closely related genera. The ITS1-5.8S-ITS2 coding regions and partial histone H4 genes of six morphospecies in the Diophrys-complex were sequenced along with their SSrRNA genes and used to compare phylogenies constructed from single data sets to those constructed from combined sets. Results indicated that combined analyses could be used to construct more reliable, less ambiguous phylogenies of complex groups like the order Euplotida, because they provide a greater amount and diversity of information.

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed Central

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-01-01

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions. Images PMID:2556266

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-12-20

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.

Spectroscopic studies of the binding of Cu(II) complexes of oxicam NSAIDs to alternating G-C and homopolymeric G-C sequences

NASA Astrophysics Data System (ADS)

Chakraborty, Sreeja; Bose, Madhuparna; Sarkar, Munna

2014-03-01

Drugs belonging to the Non-steroidal anti-inflammatory (NSAID) group are not only used as anti-inflammatory, analgesic and anti-pyretic agents, but also show anti-cancer effects. Complexing them with a bioactive metal like copper, show an enhancement in their anti-cancer effects compared to the bare drugs, whose exact mechanism of action is not yet fully understood. For the first time, it was shown by our group that Cu(II)-NSAIDs can directly bind to the DNA backbone. The ability of the copper complexes of NSAIDs namely meloxicam and piroxicam to bind to the DNA backbone could be a possible molecular mechanism behind their enhanced anticancer effects. Elucidating base sequence specific interaction of Cu(II)-NSAIDs to the DNA will provide information on their possible binding sites in the genome sequence. In this work, we present how these complexes respond to differences in structure and hydration pattern of GC rich sequences. For this, binding studies of Cu(II) complexes of piroxicam [Cu(II)-(Px)2 (L)2] and meloxicam [Cu(II)-(Mx)2 (L)] with alternating GC (polydG-dC) and homopolymeric GC (polydG-polydC) sequences were carried out using a combination of spectroscopic techniques that include UV-Vis absorption, fluorescence and circular dichroism (CD) spectroscopy. The Cu(II)-NSAIDs show strong binding affinity to both polydG-dC and polydG-polydC. The role reversal of Cu(II)-meloxicam from a strong binder of polydG-dC (Kb = 11.5 × 103 M-1) to a weak binder of polydG-polydC (Kb = 5.02 × 103 M-1), while Cu(II)-piroxicam changes from a strong binder of polydG-polydC (Kb = 8.18 × 103 M-1) to a weak one of polydG-dC (Kb = 2.18 × 103 M-1), point to the sensitivity of these complexes to changes in the backbone structures/hydration. Changes in the profiles of UV absorption band and CD difference spectra, upon complex binding to polynucleotides and the results of competitive binding assay using ethidium bromide (EtBr) fluorescence indicate different binding modes in each case.

Characteristics of aperiodic sequence of slip events caused by interaction between seismic patches and that caused be self-organized stress heterogeneity

NASA Astrophysics Data System (ADS)

Kato, N.

2017-12-01

Numerical simulations of earthquake cycles are conducted to investigate the origin of complexity of earthquake recurrence. There are two main causes of the complexity. One is self-organized stress heterogeneity due to dynamical effect. The other is the effect of interaction between some fault patches. In the model, friction on the fault is assumed to obey a rate- and state-dependent friction law. Circular patches of velocity-weakening frictional property are assumed on the fault. On the remaining areas of the fault, velocity-strengthening friction is assumed. We consider three models: Single patch model, two-patch model, and three-patch model. In the first model, the dynamical effect is mainly examined. The latter two models take into consideration the effect of interaction as well as the dynamical effect. Complex multiperiodic or aperiodic sequences of slip events occur when slip behavior changes from the seismic to aseismic, and when the degree of interaction between seismic patches is intermediate. The former is observed in all the models, and the latter is observed in the two-patch model and the three-patch model. Evolution of spatial distribution of shear stress on the fault suggests that aperiodicity at the transition from seismic to aseismic slip is caused by self-organized stress heterogeneity. The iteration maps of recurrence intervals of slip events in aperiodic sequences are examined, and they are approximately expressed by simple curves for aperiodicity at the transition from seismic to aseismic slip. In contrast, the iteration maps for aperiodic sequences caused by interaction between seismic patches are scattered and they are not expressed by simple curves. This result suggests that complex sequences caused by different mechanisms may be distinguished.

Massively parallel sequencing of forensic STRs: Considerations of the DNA commission of the International Society for Forensic Genetics (ISFG) on minimal nomenclature requirements.

PubMed

Parson, Walther; Ballard, David; Budowle, Bruce; Butler, John M; Gettings, Katherine B; Gill, Peter; Gusmão, Leonor; Hares, Douglas R; Irwin, Jodi A; King, Jonathan L; Knijff, Peter de; Morling, Niels; Prinz, Mechthild; Schneider, Peter M; Neste, Christophe Van; Willuweit, Sascha; Phillips, Christopher

2016-05-01

The DNA Commission of the International Society for Forensic Genetics (ISFG) is reviewing factors that need to be considered ahead of the adoption by the forensic community of short tandem repeat (STR) genotyping by massively parallel sequencing (MPS) technologies. MPS produces sequence data that provide a precise description of the repeat allele structure of a STR marker and variants that may reside in the flanking areas of the repeat region. When a STR contains a complex arrangement of repeat motifs, the level of genetic polymorphism revealed by the sequence data can increase substantially. As repeat structures can be complex and include substitutions, insertions, deletions, variable tandem repeat arrangements of multiple nucleotide motifs, and flanking region SNPs, established capillary electrophoresis (CE) allele descriptions must be supplemented by a new system of STR allele nomenclature, which retains backward compatibility with the CE data that currently populate national DNA databases and that will continue to be produced for the coming years. Thus, there is a pressing need to produce a standardized framework for describing complex sequences that enable comparison with currently used repeat allele nomenclature derived from conventional CE systems. It is important to discern three levels of information in hierarchical order (i) the sequence, (ii) the alignment, and (iii) the nomenclature of STR sequence data. We propose a sequence (text) string format the minimal requirement of data storage that laboratories should follow when adopting MPS of STRs. We further discuss the variant annotation and sequence comparison framework necessary to maintain compatibility among established and future data. This system must be easy to use and interpret by the DNA specialist, based on a universally accessible genome assembly, and in place before the uptake of MPS by the general forensic community starts to generate sequence data on a large scale. While the established nomenclature for CE-based STR analysis will remain unchanged in the future, the nomenclature of sequence-based STR genotypes will need to follow updated rules and be generated by expert systems that translate MPS sequences to match CE conventions in order to guarantee compatibility between the different generations of STR data. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Methods and compositions for efficient nucleic acid sequencing

DOEpatents

Drmanac, Radoje

2006-07-04

Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

Methods and compositions for efficient nucleic acid sequencing

DOEpatents

Drmanac, Radoje

2002-01-01

Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

Spitzer Space Telescope Sequencing Operations Software, Strategies, and Lessons Learned

NASA Technical Reports Server (NTRS)

Bliss, David A.

2006-01-01

The Space Infrared Telescope Facility (SIRTF) was launched in August, 2003, and renamed to the Spitzer Space Telescope in 2004. Two years of observing the universe in the wavelength range from 3 to 180 microns has yielded enormous scientific discoveries. Since this magnificent observatory has a limited lifetime, maximizing science viewing efficiency (ie, maximizing time spent executing activities directly related to science observations) was the key operational objective. The strategy employed for maximizing science viewing efficiency was to optimize spacecraft flexibility, adaptability, and use of observation time. The selected approach involved implementation of a multi-engine sequencing architecture coupled with nondeterministic spacecraft and science execution times. This approach, though effective, added much complexity to uplink operations and sequence development. The Jet Propulsion Laboratory (JPL) manages Spitzer s operations. As part of the uplink process, Spitzer s Mission Sequence Team (MST) was tasked with processing observatory inputs from the Spitzer Science Center (SSC) into efficiently integrated, constraint-checked, and modeled review and command products which accommodated the complexity of non-deterministic spacecraft and science event executions without increasing operations costs. The MST developed processes, scripts, and participated in the adaptation of multi-mission core software to enable rapid processing of complex sequences. The MST was also tasked with developing a Downlink Keyword File (DKF) which could instruct Deep Space Network (DSN) stations on how and when to configure themselves to receive Spitzer science data. As MST and uplink operations developed, important lessons were learned that should be applied to future missions, especially those missions which employ command-intensive operations via a multi-engine sequence architecture.

Identification of distal silencing elements in the murine interferon-A11 gene promoter.

PubMed

Roffet, P; Lopez, S; Navarro, S; Bandu, M T; Coulombel, C; Vignal, M; Doly, J; Vodjdani, G

1996-08-01

The murine interferon-A11 (Mu IFN-A11) gene is a member of the IFN-A multigenic family. In mouse L929 cells, the weak response of the gene's promoter to viral induction is due to a combination of both a point mutation in the virus responsive element (VRE) and the presence of negatively regulating sequences surrounding the VRE. In the distal part of the promoter, the negatively acting E1E2 sequence was delimited. This sequence displays an inhibitory effect in either orientation or position on the inducibility of a virus-responsive heterologous promoter. It selectively represses VRE-dependent transcription but is not able to reduce the transcriptional activity of a VRE-lacking promoter. In a transient transfection assay, an E1E2-containing DNA competitor was able to derepress the native Mu IFN-A11 promoter. Specific nuclear factors bind to this sequence; thus the binding of trans-regulators participates in the repression of the Mu IFN-A11 gene. The E1E2 sequence contains an IFN regulatory factor (IRF)-binding site. Recombinant IRF2 binds this sequence and anti-IRF2 antibodies supershift a major complex formed with nuclear extracts. The protein composing the complex is 50 kDa in size, indicating the presence of IRF2 or antigenically related proteins in the complex. The Mu IFN-A11 gene is the first example within the murine IFN-A family, in which a distal promoter element has been identified that can negatively modulate the transcriptional response to viral induction.