Nostoc PCC7524, a cyanobacterium which contains five sequence-specific deoxyribonucleases

NARCIS (Netherlands)

Reaston, J.; Duybesteyn, M.G.C.; Waard, Adrian de

1982-01-01

Five nucleotide sequence-specific deoxyribonucleases present in cell-free extracts of the filamentous cyanobacterium Nostoc PCC7524 have been purified and characterized. One of these enzymes, designated Nsp(7524)I cleaves at a new kind of nucleotide sequence i.e. 5'-PuCATG λ Py-3'. The other four

Specific Deficit in Implicit Motor Sequence Learning following Spinal Cord Injury.

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Ayala Bloch

Full Text Available Physical and psychosocial rehabilitation following spinal cord injury (SCI leans heavily on learning and practicing new skills. However, despite research relating motor sequence learning to spinal cord activity and clinical observations of impeded skill-learning after SCI, implicit procedural learning following spinal cord damage has not been examined.To test the hypothesis that spinal cord injury (SCI in the absence of concomitant brain injury is associated with a specific implicit motor sequence learning deficit that cannot be explained by depression or impairments in other cognitive measures.Ten participants with SCI in T1-T11, unharmed upper limb motor and sensory functioning, and no concomitant brain injury were compared to ten matched control participants on measures derived from the serial reaction time (SRT task, which was used to assess implicit motor sequence learning. Explicit generation of the SRT sequence, depression, and additional measures of learning, memory, and intelligence were included to explore the source and specificity of potential learning deficits.There was no between-group difference in baseline reaction time, indicating that potential differences between the learning curves of the two groups could not be attributed to an overall reduction in response speed in the SCI group. Unlike controls, the SCI group showed no decline in reaction time over the first six blocks of the SRT task and no advantage for the initially presented sequence over the novel interference sequence. Meanwhile, no group differences were found in explicit learning, depression, or any additional cognitive measures.The dissociation between impaired implicit learning and intact declarative memory represents novel empirical evidence of a specific implicit procedural learning deficit following SCI, with broad implications for rehabilitation and adjustment.

First-order and higher order sequence learning in specific language impairment.

Science.gov (United States)

Clark, Gillian M; Lum, Jarrad A G

2017-02-01

A core claim of the procedural deficit hypothesis of specific language impairment (SLI) is that the disorder is associated with poor implicit sequence learning. This study investigated whether implicit sequence learning problems in SLI are present for first-order conditional (FOC) and higher order conditional (HOC) sequences. Twenty-five children with SLI and 27 age-matched, nonlanguage-impaired children completed 2 serial reaction time tasks. On 1 version, the sequence to be implicitly learnt comprised a FOC sequence and on the other a HOC sequence. Results showed that the SLI group learned the HOC sequence (η p ² = .285, p = .005) but not the FOC sequence (η p ² = .099, p = .118). The control group learned both sequences (FOC η p ² = .497, HOC η p 2= .465, ps < .001). The SLI group's difficulty learning the FOC sequence is consistent with the procedural deficit hypothesis. However, the study provides new evidence that multiple mechanisms may underpin the learning of FOC and HOC sequences. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Automated sequence-specific protein NMR assignment using the memetic algorithm MATCH

International Nuclear Information System (INIS)

Volk, Jochen; Herrmann, Torsten; Wuethrich, Kurt

2008-01-01

MATCH (Memetic Algorithm and Combinatorial Optimization Heuristics) is a new memetic algorithm for automated sequence-specific polypeptide backbone NMR assignment of proteins. MATCH employs local optimization for tracing partial sequence-specific assignments within a global, population-based search environment, where the simultaneous application of local and global optimization heuristics guarantees high efficiency and robustness. MATCH thus makes combined use of the two predominant concepts in use for automated NMR assignment of proteins. Dynamic transition and inherent mutation are new techniques that enable automatic adaptation to variable quality of the experimental input data. The concept of dynamic transition is incorporated in all major building blocks of the algorithm, where it enables switching between local and global optimization heuristics at any time during the assignment process. Inherent mutation restricts the intrinsically required randomness of the evolutionary algorithm to those regions of the conformation space that are compatible with the experimental input data. Using intact and artificially deteriorated APSY-NMR input data of proteins, MATCH performed sequence-specific resonance assignment with high efficiency and robustness

CaMV-35S promoter sequence-specific DNA methylation in lettuce.

Science.gov (United States)

Okumura, Azusa; Shimada, Asahi; Yamasaki, Satoshi; Horino, Takuya; Iwata, Yuji; Koizumi, Nozomu; Nishihara, Masahiro; Mishiba, Kei-ichiro

2016-01-01

We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery. We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.

Sequence specificity and biological consequences of drugs that bind covalently in the minor groove of DNA

International Nuclear Information System (INIS)

Hurley, L.H.; Needham-VanDevanter, D.R.

1986-01-01

DNA ligands which bind within the minor groove of DNA exhibit varying degrees of sequence selectivity. Factors which contribute to nucleotide sequence recognition by minor groove ligands have been extensively investigated. Electrostatic interactions, ligand and DNA dehydration energies, hydrophobic interactions and steric factors all play significant roles in sequence selectivity in the minor groove. Interestingly, ligand recognition of nucleotide sequence in the minor groove does not involve significant hydrogen bonding. This is in sharp contrast to cellular enzyme and protein recognition of nucleotide sequence, which is achieved in the major groove via specific hydrogen bond formation between individual bases and the ligand. The ability to read nucleotide sequence via hydrogen bonding allows precise binding of proteins to specific DNA sequences. Minor groove ligands examined to date exhibit a much lower sequence specificity, generally binding to a subset of possible sequences, rather than a single sequence. 19 refs., 7 figs

Cellular specificity of HIV-1 replication can be controlled by LTR sequences

International Nuclear Information System (INIS)

Reed-Inderbitzin, Edward; Maury, Wendy

2003-01-01

Two well-established determinants of retroviral tropism are envelope sequences that regulate entry and LTR sequences that can regulate viral expression in a cell-specific manner. Studies with human immunodeficiency virus-1 (HIV-1) have demonstrated that tropism of this virus maps primarily to variable envelope sequences. Studies have demonstrated that T cell and macrophage-specific transcription factor binding motifs exist in the upstream region of the LTR U3; however, the ability of the core enhancer/promoter proximal elements (two NF-κB and three Sp1 sites) to function well in macrophages and T cells have led many to conclude that HIV LTR sequences are not primary determinants of HIV tropism. To determine if cellular specificity could be imparted to HIV by the core enhancer elements, the enhancer/promoter proximal region of the HIV LTR was substituted with motifs that control gene expression in a myeloid-specific manner. The enhancer region from equine infectious anemia virus (EIAV) when substituted for the HIV enhancer/promoter proximal region was found to drive expression in a macrophage-specific manner and was responsive to HIV Tat. The addition of a 5' methylation-dependent binding site (MDBP) and a promoter proximal Sp1 motif increased expression without altering cellular specificity. Spacing between the promoter proximal region and the TATA box was also found to influence LTR activity. Infectivity studies using chimeric LTRs within the context of a dual-tropic infectious molecular clone established that these LTRs directed HIV replication and production of infectious virions in macrophages but not primary T cells or T cell lines. This investigation demonstrates that cellular specificity can be imparted onto HIV-1 replication at the level of viral transcription and not entry

Flanking sequence determination and specific PCR identification of transgenic wheat B102-1-2.

Science.gov (United States)

Cao, Jijuan; Xu, Junyi; Zhao, Tongtong; Cao, Dongmei; Huang, Xin; Zhang, Piqiao; Luan, Fengxia

2014-01-01

The exogenous fragment sequence and flanking sequence between the exogenous fragment and recombinant chromosome of transgenic wheat B102-1-2 were successfully acquired using genome walking technology. The newly acquired exogenous fragment encoded the full-length sequence of transformed genes with transformed plasmid and corresponding functional genes including ubi, vector pBANF-bar, vector pUbiGUSPlus, vector HSP, reporter vector pUbiGUSPlus, promoter ubiquitin, and coli DH1. A specific polymerase chain reaction (PCR) identification method for transgenic wheat B102-1-2 was established on the basis of designed primers according to flanking sequence. This established specific PCR strategy was validated by using transgenic wheat, transgenic corn, transgenic soybean, transgenic rice, and non-transgenic wheat. A specifically amplified target band was observed only in transgenic wheat B102-1-2. Therefore, this method is characterized by high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of transgenic wheat B102-1-2.

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

Science.gov (United States)

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.

Templated Chemistry for Sequence-Specific Fluorogenic Detection of Duplex DNA

Science.gov (United States)

Li, Hao; Franzini, Raphael M.; Bruner, Christopher; Kool, Eric T.

2015-01-01

We describe the development of templated fluorogenic chemistry for detection of specific sequences of duplex DNA in solution. In this approach, two modified homopyrimidine oligodeoxynucleotide probes are designed to bind by triple helix formation at adjacent positions on a specific purine-rich target sequence of duplex DNA. One fluorescein-labeled probe contains an α-azidoether linker to a fluorescence quencher; the second (trigger) probe carries a triarylphosphine, designed to reduce the azide and cleave the linker. The data showed that at pH 5.6 these probes yielded a strong fluorescence signal within minutes on addition to a complementary homopurine duplex DNA target. The signal increased by a factor of ca. 60, and was completely dependent on the presence of the target DNA. Replacement of cytosine in the probes with pseudoisocytosine allowed the templated chemistry to proceed readily at pH 7. Single nucleotide mismatches in the target oligonucleotide slowed the templated reaction considerably, demonstrating high sequence selectivity. The use of templated fluorogenic chemistry for detection of duplex DNAs has not been previously reported and may allow detection of double stranded DNA, at least for homopurine-homopyrimidine target sites, under native, non-disturbing conditions. PMID:20859985

Targeting and tracing of specific DNA sequences with dTALEs in living cells

Science.gov (United States)

Thanisch, Katharina; Schneider, Katrin; Morbitzer, Robert; Solovei, Irina; Lahaye, Thomas; Bultmann, Sebastian; Leonhardt, Heinrich

2014-01-01

Epigenetic regulation of gene expression involves, besides DNA and histone modifications, the relative positioning of DNA sequences within the nucleus. To trace specific DNA sequences in living cells, we used programmable sequence-specific DNA binding of designer transcription activator-like effectors (dTALEs). We designed a recombinant dTALE (msTALE) with variable repeat domains to specifically bind a 19-bp target sequence of major satellite DNA. The msTALE was fused with green fluorescent protein (GFP) and stably expressed in mouse embryonic stem cells. Hybridization with a major satellite probe (3D-fluorescent in situ hybridization) and co-staining for known cellular structures confirmed in vivo binding of the GFP-msTALE to major satellite DNA present at nuclear chromocenters. Dual tracing of major satellite DNA and the replication machinery throughout S-phase showed co-localization during mid to late S-phase, directly demonstrating the late replication timing of major satellite DNA. Fluorescence bleaching experiments indicated a relatively stable but still dynamic binding, with mean residence times in the range of minutes. Fluorescently labeled dTALEs open new perspectives to target and trace DNA sequences and to monitor dynamic changes in subnuclear positioning as well as interactions with functional nuclear structures during cell cycle progression and cellular differentiation. PMID:24371265

Targeting and tracing of specific DNA sequences with dTALEs in living cells.

Science.gov (United States)

Thanisch, Katharina; Schneider, Katrin; Morbitzer, Robert; Solovei, Irina; Lahaye, Thomas; Bultmann, Sebastian; Leonhardt, Heinrich

2014-04-01

Epigenetic regulation of gene expression involves, besides DNA and histone modifications, the relative positioning of DNA sequences within the nucleus. To trace specific DNA sequences in living cells, we used programmable sequence-specific DNA binding of designer transcription activator-like effectors (dTALEs). We designed a recombinant dTALE (msTALE) with variable repeat domains to specifically bind a 19-bp target sequence of major satellite DNA. The msTALE was fused with green fluorescent protein (GFP) and stably expressed in mouse embryonic stem cells. Hybridization with a major satellite probe (3D-fluorescent in situ hybridization) and co-staining for known cellular structures confirmed in vivo binding of the GFP-msTALE to major satellite DNA present at nuclear chromocenters. Dual tracing of major satellite DNA and the replication machinery throughout S-phase showed co-localization during mid to late S-phase, directly demonstrating the late replication timing of major satellite DNA. Fluorescence bleaching experiments indicated a relatively stable but still dynamic binding, with mean residence times in the range of minutes. Fluorescently labeled dTALEs open new perspectives to target and trace DNA sequences and to monitor dynamic changes in subnuclear positioning as well as interactions with functional nuclear structures during cell cycle progression and cellular differentiation.

Organism-specific rRNA capture system for application in next-generation sequencing.

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Sai-Kam Li

Full Text Available RNA-sequencing is a powerful tool in studying RNomics. However, the highly abundance of ribosomal RNAs (rRNA and transfer RNA (tRNA have predominated in the sequencing reads, thereby hindering the study of lowly expressed genes. Therefore, rRNA depletion prior to sequencing is often performed in order to preserve the subtle alteration in gene expression especially those at relatively low expression levels. One of the commercially available methods is to use DNA or RNA probes to hybridize to the target RNAs. However, there is always a concern with the non-specific binding and unintended removal of messenger RNA (mRNA when the same set of probes is applied to different organisms. The degree of such unintended mRNA removal varies among organisms due to organism-specific genomic variation. We developed a computer-based method to design probes to deplete rRNA in an organism-specific manner. Based on the computation results, biotinylated-RNA-probes were produced by in vitro transcription and were used to perform rRNA depletion with subtractive hybridization. We demonstrated that the designed probes of 16S rRNAs and 23S rRNAs can efficiently remove rRNAs from Mycobacterium smegmatis. In comparison with a commercial subtractive hybridization-based rRNA removal kit, using organism-specific probes is better in preserving the RNA integrity and abundance. We believe the computer-based design approach can be used as a generic method in preparing RNA of any organisms for next-generation sequencing, particularly for the transcriptome analysis of microbes.

Increased sequence diversity coverage improves detection of HIV-Specific T cell responses

DEFF Research Database (Denmark)

Frahm, N.; Kaufmann, D.E.; Yusim, K.

2007-01-01

The accurate identification of HIV-specific T cell responses is important for determining the relationship between immune response, viral control, and disease progression. HIV-specific immune responses are usually measured using peptide sets based on consensus sequences, which frequently miss res...

Chromatin accessibility data sets show bias due to sequence specificity of the DNase I enzyme.

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Hashem Koohy

Full Text Available DNase I is an enzyme which cuts duplex DNA at a rate that depends strongly upon its chromatin environment. In combination with high-throughput sequencing (HTS technology, it can be used to infer genome-wide landscapes of open chromatin regions. Using this technology, systematic identification of hundreds of thousands of DNase I hypersensitive sites (DHS per cell type has been possible, and this in turn has helped to precisely delineate genomic regulatory compartments. However, to date there has been relatively little investigation into possible biases affecting this data.We report a significant degree of sequence preference spanning sites cut by DNase I in a number of published data sets. The two major protocols in current use each show a different pattern, but for a given protocol the pattern of sequence specificity seems to be quite consistent. The patterns are substantially different from biases seen in other types of HTS data sets, and in some cases the most constrained position lies outside the sequenced fragment, implying that this constraint must relate to the digestion process rather than events occurring during library preparation or sequencing.DNase I is a sequence-specific enzyme, with a specificity that may depend on experimental conditions. This sequence specificity is not taken into account by existing pipelines for identifying open chromatin regions. Care must be taken when interpreting DNase I results, especially when looking at the precise locations of the reads. Future studies may be able to improve the sensitivity and precision of chromatin state measurement by compensating for sequence bias.

The hydrophobic core of twin-arginine signal sequences orchestrates specific binding to Tat-pathway related chaperones.

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Anitha Shanmugham

Full Text Available Redox enzyme maturation proteins (REMPs bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA and TorD (specific for trimethylamine N-oxide reductase, TorA. Green fluorescent protein (GFP was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function.

Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization

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Redkar Rajendra

2004-02-01

Full Text Available Abstract Background Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH was used to identify specific chromosomal sequences unique to B. anthracis. Results Two SSH libraries were generated. Genomic DNA from plasmid-cured B. anthracis was used as the tester DNA in both libraries, while genomic DNA from either B. cereus or B. thuringiensis served as the driver DNA. Progressive screening of the libraries by colony filter and Southern blot analyses identified 29 different clones that were specific for the B. anthracis chromosome relative not only to the respective driver DNAs, but also to seven other different strains of B. cereus and B. thuringiensis included in the process. The nucleotide sequences of the clones were compared with those found in genomic databases, revealing that over half of the clones were located into 2 regions on the B. anthracis chromosome. Conclusions Genes encoding potential cell wall synthesis proteins dominated one region, while bacteriophage-related sequences dominated the other region. The latter supports the hypothesis that acquisition of these bacteriophage sequences occurred during or after speciation of B. anthracis relative to B. cereus and B. thuringiensis. This study provides insight into the chromosomal differences between B. anthracis and its closest phylogenetic relatives.

Flanking sequence determination and event-specific detection of genetically modified wheat B73-6-1.

Science.gov (United States)

Xu, Junyi; Cao, Jijuan; Cao, Dongmei; Zhao, Tongtong; Huang, Xin; Zhang, Piqiao; Luan, Fengxia

2013-05-01

In order to establish a specific identification method for genetically modified (GM) wheat, exogenous insert DNA and flanking sequence between exogenous fragment and recombinant chromosome of GM wheat B73-6-1 were successfully acquired by means of conventional polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR strategies. Newly acquired exogenous fragment covered the full-length sequence of transformed genes such as transformed plasmid and corresponding functional genes including marker uidA, herbicide-resistant bar, ubiquitin promoter, and high-molecular-weight gluten subunit. The flanking sequence between insert DNA revealed high similarity with Triticum turgidum A gene (GenBank: AY494981.1). A specific PCR detection method for GM wheat B73-6-1 was established on the basis of primers designed according to the flanking sequence. This specific PCR method was validated by GM wheat, GM corn, GM soybean, GM rice, and non-GM wheat. The specifically amplified target band was observed only in GM wheat B73-6-1. This method is of high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of GM wheat B73-6-1.

Structural basis for sequence-specific recognition of DNA by TAL effectors

KAUST Repository

Deng, Dong; Yan, Chuangye; Pan, Xiaojing; Mahfouz, Magdy M.; Wang, Jiawei; Zhu, Jiankang; Shi, Yi Gong; Yan, Nieng

2012-01-01

TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved amino acids and targets a specific base pair

Analysis of B Cell Repertoire Dynamics Following Hepatitis B Vaccination in Humans, and Enrichment of Vaccine-specific Antibody Sequences.

Science.gov (United States)

Galson, Jacob D; Trück, Johannes; Fowler, Anna; Clutterbuck, Elizabeth A; Münz, Márton; Cerundolo, Vincenzo; Reinhard, Claudia; van der Most, Robbert; Pollard, Andrew J; Lunter, Gerton; Kelly, Dominic F

2015-12-01

Generating a diverse B cell immunoglobulin repertoire is essential for protection against infection. The repertoire in humans can now be comprehensively measured by high-throughput sequencing. Using hepatitis B vaccination as a model, we determined how the total immunoglobulin sequence repertoire changes following antigen exposure in humans, and compared this to sequences from vaccine-specific sorted cells. Clonal sequence expansions were seen 7 days after vaccination, which correlated with vaccine-specific plasma cell numbers. These expansions caused an increase in mutation, and a decrease in diversity and complementarity-determining region 3 sequence length in the repertoire. We also saw an increase in sequence convergence between participants 14 and 21 days after vaccination, coinciding with an increase of vaccine-specific memory cells. These features allowed development of a model for in silico enrichment of vaccine-specific sequences from the total repertoire. Identifying antigen-specific sequences from total repertoire data could aid our understanding B cell driven immunity, and be used for disease diagnostics and vaccine evaluation.

Specific versus non-specific immune responses in an invertebrate species evidenced by a comparative de novo sequencing study.

Directory of Open Access Journals (Sweden)

Emeline Deleury

Full Text Available Our present understanding of the functioning and evolutionary history of invertebrate innate immunity derives mostly from studies on a few model species belonging to ecdysozoa. In particular, the characterization of signaling pathways dedicated to specific responses towards fungi and Gram-positive or Gram-negative bacteria in Drosophila melanogaster challenged our original view of a non-specific immunity in invertebrates. However, much remains to be elucidated from lophotrochozoan species. To investigate the global specificity of the immune response in the fresh-water snail Biomphalaria glabrata, we used massive Illumina sequencing of 5'-end cDNAs to compare expression profiles after challenge by Gram-positive or Gram-negative bacteria or after a yeast challenge. 5'-end cDNA sequencing of the libraries yielded over 12 millions high quality reads. To link these short reads to expressed genes, we prepared a reference transcriptomic database through automatic assembly and annotation of the 758,510 redundant sequences (ESTs, mRNAs of B. glabrata available in public databases. Computational analysis of Illumina reads followed by multivariate analyses allowed identification of 1685 candidate transcripts differentially expressed after an immune challenge, with a two fold ratio between transcripts showing a challenge-specific expression versus a lower or non-specific differential expression. Differential expression has been validated using quantitative PCR for a subset of randomly selected candidates. Predicted functions of annotated candidates (approx. 700 unisequences belonged to a large extend to similar functional categories or protein types. This work significantly expands upon previous gene discovery and expression studies on B. glabrata and suggests that responses to various pathogens may involve similar immune processes or signaling pathways but different genes belonging to multigenic families. These results raise the question of the importance

Sequence motifs in MADS transcription factors responsible for specificity and diversification of protein-protein interaction.

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Aalt D J van Dijk

Full Text Available Protein sequences encompass tertiary structures and contain information about specific molecular interactions, which in turn determine biological functions of proteins. Knowledge about how protein sequences define interaction specificity is largely missing, in particular for paralogous protein families with high sequence similarity, such as the plant MADS domain transcription factor family. In comparison to the situation in mammalian species, this important family of transcription regulators has expanded enormously in plant species and contains over 100 members in the model plant species Arabidopsis thaliana. Here, we provide insight into the mechanisms that determine protein-protein interaction specificity for the Arabidopsis MADS domain transcription factor family, using an integrated computational and experimental approach. Plant MADS proteins have highly similar amino acid sequences, but their dimerization patterns vary substantially. Our computational analysis uncovered small sequence regions that explain observed differences in dimerization patterns with reasonable accuracy. Furthermore, we show the usefulness of the method for prediction of MADS domain transcription factor interaction networks in other plant species. Introduction of mutations in the predicted interaction motifs demonstrated that single amino acid mutations can have a large effect and lead to loss or gain of specific interactions. In addition, various performed bioinformatics analyses shed light on the way evolution has shaped MADS domain transcription factor interaction specificity. Identified protein-protein interaction motifs appeared to be strongly conserved among orthologs, indicating their evolutionary importance. We also provide evidence that mutations in these motifs can be a source for sub- or neo-functionalization. The analyses presented here take us a step forward in understanding protein-protein interactions and the interplay between protein sequences and

A survey of the sequence-specific interaction of damaging agents with DNA: emphasis on antitumor agents.

Science.gov (United States)

Murray, V

1999-01-01

This article reviews the literature concerning the sequence specificity of DNA-damaging agents. DNA-damaging agents are widely used in cancer chemotherapy. It is important to understand fully the determinants of DNA sequence specificity so that more effective DNA-damaging agents can be developed as antitumor drugs. There are five main methods of DNA sequence specificity analysis: cleavage of end-labeled fragments, linear amplification with Taq DNA polymerase, ligation-mediated polymerase chain reaction (PCR), single-strand ligation PCR, and footprinting. The DNA sequence specificity in purified DNA and in intact mammalian cells is reviewed for several classes of DNA-damaging agent. These include agents that form covalent adducts with DNA, free radical generators, topoisomerase inhibitors, intercalators and minor groove binders, enzymes, and electromagnetic radiation. The main sites of adduct formation are at the N-7 of guanine in the major groove of DNA and the N-3 of adenine in the minor groove, whereas free radical generators abstract hydrogen from the deoxyribose sugar and topoisomerase inhibitors cause enzyme-DNA cross-links to form. Several issues involved in the determination of the DNA sequence specificity are discussed. The future directions of the field, with respect to cancer chemotherapy, are also examined.

Analysis of B Cell Repertoire Dynamics Following Hepatitis B Vaccination in Humans, and Enrichment of Vaccine-specific Antibody Sequences

Directory of Open Access Journals (Sweden)

Jacob D. Galson

2015-12-01

Full Text Available Generating a diverse B cell immunoglobulin repertoire is essential for protection against infection. The repertoire in humans can now be comprehensively measured by high-throughput sequencing. Using hepatitis B vaccination as a model, we determined how the total immunoglobulin sequence repertoire changes following antigen exposure in humans, and compared this to sequences from vaccine-specific sorted cells. Clonal sequence expansions were seen 7 days after vaccination, which correlated with vaccine-specific plasma cell numbers. These expansions caused an increase in mutation, and a decrease in diversity and complementarity-determining region 3 sequence length in the repertoire. We also saw an increase in sequence convergence between participants 14 and 21 days after vaccination, coinciding with an increase of vaccine-specific memory cells. These features allowed development of a model for in silico enrichment of vaccine-specific sequences from the total repertoire. Identifying antigen-specific sequences from total repertoire data could aid our understanding B cell driven immunity, and be used for disease diagnostics and vaccine evaluation.

Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

Directory of Open Access Journals (Sweden)

TIJANA SAVIC

2006-02-01

Full Text Available In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

Sequence specific electronic conduction through polyion-stabilized double-stranded DNA in nanoscale break junctions

International Nuclear Information System (INIS)

Mahapatro, Ajit K; Jeong, Kyung J; Lee, Gil U; Janes, David B

2007-01-01

This paper presents a study of sequence specific electronic conduction through short (15-base-pair) double-stranded (ds) DNA molecules, measured by immobilizing 3 ' -thiol-derivatized DNAs in nanometre scale gaps between gold electrodes. The polycation spermidine was used to stabilize the ds-DNA structure, allowing electrical measurements to be performed in a dry state. For specific sequences, the conductivity was observed to scale with the surface density of immobilized DNA, which can be controlled by the buffer concentration. A series of 15-base DNA oligonucleotide pairs, in which the centre sequence of five base pairs was changed from G:C to A:T pairs, has been studied. The conductivity per molecule is observed to decrease exponentially with the number of adjacent A:T pairs replacing G:C pairs, consistent with a barrier at the A:T sites. Conductance-based devices for short DNA sequences could provide sensing approaches with direct electrical readout, as well as label-free detection

Micropatterning stretched and aligned DNA for sequence-specific nanolithography

Science.gov (United States)

Petit, Cecilia Anna Paulette

Techniques for fabricating nanostructured materials can be categorized as either "top-down" or "bottom-up". Top-down techniques use lithography and contact printing to create patterned surfaces and microfluidic channels that can corral and organize nanoscale structures, such as molecules and nanorods in contrast; bottom-up techniques use self-assembly or molecular recognition to direct the organization of materials. A central goal in nanotechnology is the integration of bottom-up and top-down assembly strategies for materials development, device design; and process integration. With this goal in mind, we have developed strategies that will allow this integration by using DNA as a template for nanofabrication; two top-down approaches allow the placement of these templates, while the bottom-up technique uses the specific sequence of bases to pattern materials along each strand of DNA. Our first top-down approach, termed combing of molecules in microchannels (COMMIC), produces microscopic patterns of stretched and aligned molecules of DNA on surfaces. This process consists of passing an air-water interface over end adsorbed molecules inside microfabricated channels. The geometry of the microchannel directs the placement of the DNA molecules, while the geometry of the airwater interface directs the local orientation and curvature of the molecules. We developed another top-down strategy for creating micropatterns of stretched and aligned DNA using surface chemistry. Because DNA stretching occurs on hydrophobic surfaces, this technique uses photolithography to pattern vinyl-terminated silanes on glass When these surface-, are immersed in DNA solution, molecules adhere preferentially to the silanized areas. This approach has also proven useful in patterning protein for cell adhesion studies. Finally, we describe the use of these stretched and aligned molecules of DNA as templates for the subsequent bottom-up construction of hetero-structures through hybridization

High-specificity detection of rare alleles with Paired-End Low Error Sequencing (PELE-Seq).

Science.gov (United States)

Preston, Jessica L; Royall, Ariel E; Randel, Melissa A; Sikkink, Kristin L; Phillips, Patrick C; Johnson, Eric A

2016-06-14

Polymorphic loci exist throughout the genomes of a population and provide the raw genetic material needed for a species to adapt to changes in the environment. The minor allele frequencies of rare Single Nucleotide Polymorphisms (SNPs) within a population have been difficult to track with Next-Generation Sequencing (NGS), due to the high error rate of standard methods such as Illumina sequencing. We have developed a wet-lab protocol and variant-calling method that identifies both sequencing and PCR errors, called Paired-End Low Error Sequencing (PELE-Seq). To test the specificity and sensitivity of the PELE-Seq method, we sequenced control E. coli DNA libraries containing known rare alleles present at frequencies ranging from 0.2-0.4 % of the total reads. PELE-Seq had higher specificity and sensitivity than standard libraries. We then used PELE-Seq to characterize rare alleles in a Caenorhabditis remanei nematode worm population before and after laboratory adaptation, and found that minor and rare alleles can undergo large changes in frequency during lab-adaptation. We have developed a method of rare allele detection that mitigates both sequencing and PCR errors, called PELE-Seq. PELE-Seq was evaluated using control E. coli populations and was then used to compare a wild C. remanei population to a lab-adapted population. The PELE-Seq method is ideal for investigating the dynamics of rare alleles in a broad range of reduced-representation sequencing methods, including targeted amplicon sequencing, RAD-Seq, ddRAD, and GBS. PELE-Seq is also well-suited for whole genome sequencing of mitochondria and viruses, and for high-throughput rare mutation screens.

Sequence specific motor performance gains after memory consolidation in children and adolescents.

Directory of Open Access Journals (Sweden)

Shoshi Dorfberger

Full Text Available Memory consolidation for a trained sequence of finger opposition movements, in 9- and 12-year-old children, was recently found to be significantly less susceptible to interference by a subsequent training experience, compared to that of 17-year-olds. It was suggested that, in children, the experience of training on any sequence of finger movements may affect the performance of the sequence elements, component movements, rather than the sequence as a unit; the latter has been implicated in the learning of the task by adults. This hypothesis implied a possible childhood advantage in the ability to transfer the gains from a trained to the reversed, untrained, sequence of movements. Here we report the results of transfer tests undertaken to test this proposal in 9-, 12-, and 17-year-olds after training in the finger-to-thumb opposition sequence (FOS learning task. Our results show that the performance gains in the trained sequence partially transferred from the left, trained hand, to the untrained hand at 48-hours after a single training session in the three age-groups tested. However, there was very little transfer of the gains from the trained to the untrained, reversed, sequence performed by either hand. The results indicate sequence specific post-training gains in FOS performance, as opposed to a general improvement in performance of the individual, component, movements that comprised both the trained and untrained sequences. These results do not support the proposal that the reduced susceptibility to interference, in children before adolescence, reflects a difference in movement syntax representation after training.

Sequence specific motor performance gains after memory consolidation in children and adolescents.

Science.gov (United States)

Dorfberger, Shoshi; Adi-Japha, Esther; Karni, Avi

2012-01-01

Memory consolidation for a trained sequence of finger opposition movements, in 9- and 12-year-old children, was recently found to be significantly less susceptible to interference by a subsequent training experience, compared to that of 17-year-olds. It was suggested that, in children, the experience of training on any sequence of finger movements may affect the performance of the sequence elements, component movements, rather than the sequence as a unit; the latter has been implicated in the learning of the task by adults. This hypothesis implied a possible childhood advantage in the ability to transfer the gains from a trained to the reversed, untrained, sequence of movements. Here we report the results of transfer tests undertaken to test this proposal in 9-, 12-, and 17-year-olds after training in the finger-to-thumb opposition sequence (FOS) learning task. Our results show that the performance gains in the trained sequence partially transferred from the left, trained hand, to the untrained hand at 48-hours after a single training session in the three age-groups tested. However, there was very little transfer of the gains from the trained to the untrained, reversed, sequence performed by either hand. The results indicate sequence specific post-training gains in FOS performance, as opposed to a general improvement in performance of the individual, component, movements that comprised both the trained and untrained sequences. These results do not support the proposal that the reduced susceptibility to interference, in children before adolescence, reflects a difference in movement syntax representation after training.

29 CFR 1926.752 - Site layout, site-specific erection plan and construction sequence.

Science.gov (United States)

2010-07-01

... 29 Labor 8 2010-07-01 2010-07-01 false Site layout, site-specific erection plan and construction... Steel Erection § 1926.752 Site layout, site-specific erection plan and construction sequence. (a... strength or sufficient strength to support the loads imposed during steel erection. (c) Site layout. The...

Chirality- and sequence-selective successive self-sorting via specific homo- and complementary-duplex formations

Science.gov (United States)

Makiguchi, Wataru; Tanabe, Junki; Yamada, Hidekazu; Iida, Hiroki; Taura, Daisuke; Ousaka, Naoki; Yashima, Eiji

2015-01-01

Self-recognition and self-discrimination within complex mixtures are of fundamental importance in biological systems, which entirely rely on the preprogrammed monomer sequences and homochirality of biological macromolecules. Here we report artificial chirality- and sequence-selective successive self-sorting of chiral dimeric strands bearing carboxylic acid or amidine groups joined by chiral amide linkers with different sequences through homo- and complementary-duplex formations. A mixture of carboxylic acid dimers linked by racemic-1,2-cyclohexane bis-amides with different amide sequences (NHCO or CONH) self-associate to form homoduplexes in a completely sequence-selective way, the structures of which are different from each other depending on the linker amide sequences. The further addition of an enantiopure amide-linked amidine dimer to a mixture of the racemic carboxylic acid dimers resulted in the formation of a single optically pure complementary duplex with a 100% diastereoselectivity and complete sequence specificity stabilized by the amidinium–carboxylate salt bridges, leading to the perfect chirality- and sequence-selective duplex formation. PMID:26051291

Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

DEFF Research Database (Denmark)

Izvolsky, K I; Demidov, V V; Nielsen, P E

2000-01-01

I restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate...... DNA duplexes in a virtually sequence-unrestricted manner....

BrEPS: a flexible and automatic protocol to compute enzyme-specific sequence profiles for functional annotation

Directory of Open Access Journals (Sweden)

Schomburg D

2010-12-01

Full Text Available Abstract Background Models for the simulation of metabolic networks require the accurate prediction of enzyme function. Based on a genomic sequence, enzymatic functions of gene products are today mainly predicted by sequence database searching and operon analysis. Other methods can support these techniques: We have developed an automatic method "BrEPS" that creates highly specific sequence patterns for the functional annotation of enzymes. Results The enzymes in the UniprotKB are identified and their sequences compared against each other with BLAST. The enzymes are then clustered into a number of trees, where each tree node is associated with a set of EC-numbers. The enzyme sequences in the tree nodes are aligned with ClustalW. The conserved columns of the resulting multiple alignments are used to construct sequence patterns. In the last step, we verify the quality of the patterns by computing their specificity. Patterns with low specificity are omitted and recomputed further down in the tree. The final high-quality patterns can be used for functional annotation. We ran our protocol on a recent Swiss-Prot release and show statistics, as well as a comparison to PRIAM, a probabilistic method that is also specialized on the functional annotation of enzymes. We determine the amount of true positive annotations for five common microorganisms with data from BRENDA and AMENDA serving as standard of truth. BrEPS is almost on par with PRIAM, a fact which we discuss in the context of five manually investigated cases. Conclusions Our protocol computes highly specific sequence patterns that can be used to support the functional annotation of enzymes. The main advantages of our method are that it is automatic and unsupervised, and quite fast once the patterns are evaluated. The results show that BrEPS can be a valuable addition to the reconstruction of metabolic networks.

Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project

Directory of Open Access Journals (Sweden)

Heinz Ruth A

2003-09-01

Full Text Available Abstract Background Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. Results Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4. The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60% did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences

Screening for SNPs with Allele-Specific Methylation based on Next-Generation Sequencing Data

OpenAIRE

Hu, Bo; Ji, Yuan; Xu, Yaomin; Ting, Angela H

2013-01-01

Allele-specific methylation (ASM) has long been studied but mainly documented in the context of genomic imprinting and X chromosome inactivation. Taking advantage of the next-generation sequencing technology, we conduct a high-throughput sequencing experiment with four prostate cell lines to survey the whole genome and identify single nucleotide polymorphisms (SNPs) with ASM. A Bayesian approach is proposed to model the counts of short reads for each SNP conditional on its genotypes of multip...

Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR.

Science.gov (United States)

D'Souza, T M; Boominathan, K; Reddy, C A

1996-01-01

Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum, Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases. PMID:8837429

Generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method

Directory of Open Access Journals (Sweden)

Sette Alessandro

2005-05-01

Full Text Available Abstract Background Many processes in molecular biology involve the recognition of short sequences of nucleic-or amino acids, such as the binding of immunogenic peptides to major histocompatibility complex (MHC molecules. From experimental data, a model of the sequence specificity of these processes can be constructed, such as a sequence motif, a scoring matrix or an artificial neural network. The purpose of these models is two-fold. First, they can provide a summary of experimental results, allowing for a deeper understanding of the mechanisms involved in sequence recognition. Second, such models can be used to predict the experimental outcome for yet untested sequences. In the past we reported the development of a method to generate such models called the Stabilized Matrix Method (SMM. This method has been successfully applied to predicting peptide binding to MHC molecules, peptide transport by the transporter associated with antigen presentation (TAP and proteasomal cleavage of protein sequences. Results Herein we report the implementation of the SMM algorithm as a publicly available software package. Specific features determining the type of problems the method is most appropriate for are discussed. Advantageous features of the package are: (1 the output generated is easy to interpret, (2 input and output are both quantitative, (3 specific computational strategies to handle experimental noise are built in, (4 the algorithm is designed to effectively handle bounded experimental data, (5 experimental data from randomized peptide libraries and conventional peptides can easily be combined, and (6 it is possible to incorporate pair interactions between positions of a sequence. Conclusion Making the SMM method publicly available enables bioinformaticians and experimental biologists to easily access it, to compare its performance to other prediction methods, and to extend it to other applications.

Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence

International Nuclear Information System (INIS)

Millan, J.L.; Driscoll, C.E.; LeVan, K.M.; Goldberg, E.

1987-01-01

The sequence and structure of human testis-specific L-lactate dehydrogenase [LDHC 4 , LDHX; (L)-lactate:NAD + oxidoreductase, EC 1.1.1.27] has been derived from analysis of a complementary DNA (cDNA) clone comprising the complete protein coding region of the enzyme. From the deduced amino acid sequence, human LDHC 4 is as different from rodent LDHC 4 (73% homology) as it is from human LDHA 4 (76% homology) and porcine LDHB 4 (68% homology). Subunit homologies are consistent with the conclusion that the LDHC gene arose by at least two independent duplication events. Furthermore, the lower degree of homology between mouse and human LDHC 4 and the appearance of this isozyme late in evolution suggests a higher rate of mutation in the mammalian LDHC genes than in the LDHA and -B genes. Comparison of exposed amino acid residues of discrete anti-genic determinants of mouse and human LDHC 4 reveals significant differences. Knowledge of the human LDHC 4 sequence will help design human-specific peptides useful in the development of a contraceptive vaccine

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

Science.gov (United States)

Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

Antitumor activity of sequence-specific alkylating agents: pyrolle-imidazole CBI conjugates with indole linker.

Science.gov (United States)

Shinohara, Ken-ichi; Bando, Toshikazu; Sasaki, Shunta; Sakakibara, Yogo; Minoshima, Masafumi; Sugiyama, Hiroshi

2006-03-01

DNA-targeting agents, including cisplatin, bleomycin and mitomycin C, are used routinely in cancer treatments. However, these drugs are extremely toxic, attacking normal cells and causing severe side effects. One important question to consider in designing anticancer agents is whether the introduction of sequence selectivity to DNA-targeting agents can improve their efficacy as anticancer agents. In the present study, the growth inhibition activities of an indole-seco 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) (1) and five conjugates with hairpin pyrrole-imidazole polyamides (2-6), which have different sequence specificities for DNA alkylation, were compared using 10 different cell lines. The average values of -log GI50 (50% growth inhibition concentration) for compounds 1-6 against the 10 cell lines were 8.33, 8.56, 8.29, 8.04, 8.23 and 8.83, showing that all of these compounds strongly inhibit cell growth. Interestingly, each alkylating agent caused significantly different growth inhibition patterns with each cell line. In particular, the correlation coefficients between the -log GI50 of compound 1 and its conjugates 2-6 showed extremely low values (Ralkylation lead to marked differences in biological activity. Comparison of the correlation coefficients between compounds 6 and 7, with the same sequence specificity as 6, and MS-247, with sequence specificity different from 6, when used against a panel of 37 human cancer cell lines further confirmed the above hypothesis.

Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR

Energy Technology Data Exchange (ETDEWEB)

D`Souza, T.M.; Boominathan, K.; Reddy, C.A. [Michigan State Univ., East Lansing, MI (United States)

1996-10-01

Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequences of each of the PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum, Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases. 36 refs., 6 figs., 2 tabs.

Molecular Simulations of Sequence-Specific Association of Transmembrane Proteins in Lipid Bilayers

Science.gov (United States)

Doxastakis, Manolis; Prakash, Anupam; Janosi, Lorant

2011-03-01

Association of membrane proteins is central in material and information flow across the cellular membranes. Amino-acid sequence and the membrane environment are two critical factors controlling association, however, quantitative knowledge on such contributions is limited. In this work, we study the dimerization of helices in lipid bilayers using extensive parallel Monte Carlo simulations with recently developed algorithms. The dimerization of Glycophorin A is examined employing a coarse-grain model that retains a level of amino-acid specificity, in three different phospholipid bilayers. Association is driven by a balance of protein-protein and lipid-induced interactions with the latter playing a major role at short separations. Following a different approach, the effect of amino-acid sequence is studied using the four transmembrane domains of the epidermal growth factor receptor family in identical lipid environments. Detailed characterization of dimer formation and estimates of the free energy of association reveal that these helices present significant affinity to self-associate with certain dimers forming non-specific interfaces.

Molecular-Level Thermodynamic Switch Controls Chemical Equilibrium in Sequence-Specific Hydrophobic Interaction of 35 Dipeptide Pairs

OpenAIRE

Chun, Paul W.

2003-01-01

Applying the Planck-Benzinger methodology, the sequence-specific hydrophobic interactions of 35 dipeptide pairs were examined over a temperature range of 273–333 K, based on data reported by Nemethy and Scheraga in 1962. The hydrophobic interaction in these sequence-specific dipeptide pairs is highly similar in its thermodynamic behavior to that of other biological systems. The results imply that the negative Gibbs free energy change minimum at a well-defined stable temperature, 〈Ts〉, where t...

Screening for sequence-specific RNA-BPs by comprehensive UV crosslinking

Directory of Open Access Journals (Sweden)

Le Meuth-Metzinger Valerie

2002-06-01

Full Text Available Abstract Background Specific cis-elements and the associated trans-acting factors have been implicated in the post-transcriptional regulation of gene expression. In the era of genome wide analyses identifying novel trans-acting factors and cis-regulatory elements is a step towards understanding coordinated gene expression. UV-crosslink analysis is a standard method used to identify RNA-binding proteins. Uridine is traditionally used to radiolabel substrate RNAs, however, proteins binding to cis-elments particularly uridine poor will be weakly or not detected. We evaluate here the possibility of using UV-crosslinking with RNA substrates radiolabeled with each of the four ribonucleotides as an approach for screening for novel sequence specific RNA-binding proteins. Results The radiolabeled RNA substrates were derived from the 3'UTRs of the cloned Eg and c-mos Xenopus laevis maternal mRNAs. Specific, but not identical, uv-crosslinking signals were obtained, some of which corresponded to already identified proteins. A signal for a novel 90 kDa protein was observed with the c-mos 3'UTR radiolabeled with both CTP and GTP but not with UTP. The binding site of the 90 kDa RNA-binding protein was localised to a 59-nucleotide portion of the c-mos 3'UTR. Conclusion That the 90 kDa signal was detected with RNAs radiolabeled with CTP or GTP but not UTP illustrates the advantage of radiolabeling all four nucleotides in a UV-crosslink based screen. This method can be used for both long and short RNAs and does not require knowledge of the cis-acting sequence. It should be amenable to high throughput screening for RNA binding proteins.

Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

KAUST Repository

Lee, Seung-Joo

2010-03-28

DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5\\'-TGGTC-3\\') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain. The Author(s) 2010. Published by Oxford University Press.

The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes

NARCIS (Netherlands)

Skaletsky, Helen; Kuroda-Kawaguchi, Tomoko; Minx, Patrick J.; Cordum, Holland S.; Hillier, LaDeana; Brown, Laura G.; Repping, Sjoerd; Pyntikova, Tatyana; Ali, Johar; Bieri, Tamberlyn; Chinwalla, Asif; Delehaunty, Andrew; Delehaunty, Kim; Du, Hui; Fewell, Ginger; Fulton, Lucinda; Fulton, Robert; Graves, Tina; Hou, Shun-Fang; Latrielle, Philip; Leonard, Shawn; Mardis, Elaine; Maupin, Rachel; McPherson, John; Miner, Tracie; Nash, William; Nguyen, Christine; Ozersky, Philip; Pepin, Kymberlie; Rock, Susan; Rohlfing, Tracy; Scott, Kelsi; Schultz, Brian; Strong, Cindy; Tin-Wollam, Aye; Yang, Shiaw-Pyng; Waterston, Robert H.; Wilson, Richard K.; Rozen, Steve; Page, David C.

2003-01-01

The male-specific region of the Y chromosome, the MSY, differentiates the sexes and comprises 95% of the chromosome's length. Here, we report that the MSY is a mosaic of heterochromatic sequences and three classes of euchromatic sequences: X-transposed, X-degenerate and ampliconic. These classes

Chimeric TALE recombinases with programmable DNA sequence specificity.

Science.gov (United States)

Mercer, Andrew C; Gaj, Thomas; Fuller, Roberta P; Barbas, Carlos F

2012-11-01

Site-specific recombinases are powerful tools for genome engineering. Hyperactivated variants of the resolvase/invertase family of serine recombinases function without accessory factors, and thus can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered zinc-finger proteins (ZFPs). However, imperfect modularity with particular domains, lack of high-affinity binding to all DNA triplets, and difficulty in construction has hindered the widespread adoption of ZFPs in unspecialized laboratories. The discovery of a novel type of DBD in transcription activator-like effector (TALE) proteins from Xanthomonas provides an alternative to ZFPs. Here we describe chimeric TALE recombinases (TALERs): engineered fusions between a hyperactivated catalytic domain from the DNA invertase Gin and an optimized TALE architecture. We use a library of incrementally truncated TALE variants to identify TALER fusions that modify DNA with efficiency and specificity comparable to zinc-finger recombinases in bacterial cells. We also show that TALERs recombine DNA in mammalian cells. The TALER architecture described herein provides a platform for insertion of customized TALE domains, thus significantly expanding the targeting capacity of engineered recombinases and their potential applications in biotechnology and medicine.

Implicit sequence-specific motor learning after sub-cortical stroke is associated with increased prefrontal brain activations: An fMRI study

Science.gov (United States)

Meehan, Sean K.; Randhawa, Bubblepreet; Wessel, Brenda; Boyd, Lara A.

2010-01-01

Implicit motor learning is preserved after stroke, but how the brain compensates for damage to facilitate learning is unclear. We used a random effects analysis to determine how stroke alters patterns of brain activity during implicit sequence-specific motor learning as compared to general improvements in motor control. Nine healthy participants and 9 individuals with chronic, right focal sub-cortical stroke performed a continuous joystick-based tracking task during an initial fMRI session, over 5 days of practice, and a retention test during a separate fMRI session. Sequence-specific implicit motor learning was differentiated from general improvements in motor control by comparing tracking performance on a novel, repeated tracking sequences during early practice and again at the retention test. Both groups demonstrated implicit sequence-specific motor learning at the retention test, yet substantial differences were apparent. At retention, healthy control participants demonstrated increased BOLD response in left dorsal premotor cortex (BA 6) but decreased BOLD response left dorsolateral prefrontal cortex (DLPFC; BA 9) during repeated sequence tracking. In contrast, at retention individuals with stroke did not show this reduction in DLPFC during repeated tracking. Instead implicit sequence-specific motor learning and general improvements in motor control were associated with increased BOLD response in the left middle frontal gyrus BA 8, regardless of sequence type after stroke. These data emphasize the potential importance of a prefrontal-based attentional network for implicit motor learning after stroke. The present study is the first to highlight the importance of the prefrontal cortex for implicit sequence-specific motor learning after stroke. PMID:20725908

Isolation and sequencing analysis on the seed-specific promoter from soybean

Institute of Scientific and Technical Information of China (English)

CAIYIN Qinggele; LI Mingchun; WEI Dongsheng; CAI Yi; XING Laijun

2007-01-01

The low level of foreign genes' expression in transgenic plants is a key factor that limits plant genetic engineering.Because of the critical regulatory activity of the promoters on gene transcription,they are studied extensively to improve the efficiency of the plant transgenic system.The constitutive promoters,such as CaMV 35S promoter,are usually used in plant genetic engineering.But those constitutive promoters continuously express their downstream genes during the whole life span in all the tissues of the host plants.This is not only wasteful to host plant's energy,but also harmful to host plants and usually affects their agronomic characteristics.In contrast,the seed-specific promoter only expresses its downstream genes from mid to late stage of seed maturation,and there is no expression or much lower expression in other tissues.So the seed-specific promoters are distinguished for their improvement and what they have brought to plant quality engineering.The aim of this article is to characterize a new seed-specific promoter and improve grain quality.The promoter region of β-conglycinin α-subunit gene was isolated from the genomic DNA of soybean Jilin 43 by PCR method,and successfully extended this fragment by TAIL PCR method and obtained the promoter fragment BCSP666.Sequencing analysis showed that the cloned fragment BCSP666 contained all of the motifs,such as RY repeat element,AG/CCCCA motif,TACACAT motif,ACGTmotif,A/T rich motif and E-box etc.,which constituted the seed-specific promoter activity.Based on this sequencing analysis,the seed-specific promoter activity of the fragment BCSP666 was predicted.And then the seed-specific expression vector pBI121-666,which contained GUS reporter gene,was constructed with the fragment BCSP666.Transformation of Arabidopsis thaliana plants by Agrobacterium-mediated floral-dip method with the recombined vector pBI121-666was conducted.The transgenic plants were selected on the kanamycin-resistant MS medium

Automatic Sequences and Zip-Specifications

NARCIS (Netherlands)

Grabmayer, C.A.; Endrullis, J.; Hendriks, D.; Klop, J.W.; Moss, L.S.

2012-01-01

We consider infinite sequences of symbols, also known as streams, and the decidability question for equality of streams defined in a restricted format. This restricted format consists of prefixing a symbol at the head of a stream, of the stream function `zip', and recursion variables. Here `zip'

Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

KAUST Repository

Lee, Seung-Joo; Zhu, Bin; Hamdan, Samir; Richardson, Charles C.

2010-01-01

DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical

Sequence-specific inhibition of Dicer measured with a force-based microarray for RNA ligands.

Science.gov (United States)

Limmer, Katja; Aschenbrenner, Daniela; Gaub, Hermann E

2013-04-01

Malfunction of protein translation causes many severe diseases, and suitable correction strategies may become the basis of effective therapies. One major regulatory element of protein translation is the nuclease Dicer that cuts double-stranded RNA independently of the sequence into pieces of 19-22 base pairs starting the RNA interference pathway and activating miRNAs. Inhibiting Dicer is not desirable owing to its multifunctional influence on the cell's gene regulation. Blocking specific RNA sequences by small-molecule binding, however, is a promising approach to affect the cell's condition in a controlled manner. A label-free assay for the screening of site-specific interference of small molecules with Dicer activity is thus needed. We used the Molecular Force Assay (MFA), recently developed in our lab, to measure the activity of Dicer. As a model system, we used an RNA sequence that forms an aptamer-binding site for paromomycin, a 615-dalton aminoglycoside. We show that Dicer activity is modulated as a function of concentration and incubation time: the addition of paromomycin leads to a decrease of Dicer activity according to the amount of ligand. The measured dissociation constant of paromomycin to its aptamer was found to agree well with literature values. The parallel format of the MFA allows a large-scale search and analysis for ligands for any RNA sequence.

Simultaneous discrimination of species and strains in Lactobacillus rhamnosus using species-specific PCR combined with multiplex mini-sequencing technology.

Science.gov (United States)

Huang, Chien-Hsun; Chang, Mu-Tzu; Huang, Lina; Chu, Wen-Shen

2015-12-01

This study described the use of species-specific PCR in combination with SNaPshot mini-sequencing to achieve species identification and strain differentiation in Lactobacillus rhamnosus. To develop species-specific PCR and strain subtyping primers, the dnaJ gene was used as a target, and its corresponding sequences were analyzed both in Lb. rhamnosus and in a subset of its phylogenetically closest species. The results indicated that the species-specific primer pair was indeed specific for Lb. rhamnosus, and the mini-sequencing assay was able to unambiguously distinguish Lb. rhamnosus strains into different haplotypes. In conclusion, we have successfully developed a rapid, accurate and cost-effective assay for inter- and intraspecies discrimination of Lb. rhamnosus, which can be applied to achieve efficient quality control of probiotic products. Copyright © 2015 Elsevier Ltd. All rights reserved.

The minimum information about a genome sequence (MIGS) specification

DEFF Research Database (Denmark)

Field, D; Garrity, G; Gray, T

2008-01-01

With the quantity of genomic data increasing at an exponential rate, it is imperative that these data be captured electronically, in a standard format. Standardization activities must proceed within the auspices of open-access and international working bodies. To tackle the issues surrounding the...... that will be required to develop improved mechanisms of metadata capture and exchange. As part of its wider goals, the GSC also supports improving the 'transparency' of the information contained in existing genomic databases....... the development of better descriptions of genomic investigations, we have formed the Genomic Standards Consortium (GSC). Here, we introduce the minimum information about a genome sequence (MIGS) specification with the intent of promoting participation in its development and discussing the resources...

Development of taxon-specific sequence characterized amplified region (SCAR) markers based on actin sequences and DNA amplification fingerprinting (DAF): a case study in the Phoma exigua species complex.

Science.gov (United States)

Aveskamp, Maikel M; Woudenberg, Joyce H C; de Gruyter, Johannes; Turco, Elena; Groenewald, Johannes Z; Crous, Pedro W

2009-05-01

Phoma exigua is considered to be an assemblage of at least nine varieties that are mainly distinguished on the basis of host specificity and pathogenicity. However, these varieties are also reported to be weak pathogens and secondary invaders on non-host tissue. In practice, it is difficult to distinguish P. exigua from its close relatives and to correctly identify isolates up to the variety level, because of their low genetic variation and high morphological similarity. Because of quarantine issues and phytosanitary measures, a robust DNA-based tool is required for accurate and rapid identification of the separate taxa in this species complex. The present study therefore aims to develop such a tool based on unique nucleotide sequence identifiers. More than 60 strains of P. exigua and related species were compared in terms of partial actin gene sequences, or analysed using DNA amplification fingerprinting (DAF) with short, arbitrary, mini-hairpin primers. Fragments in the fingerprint unique to a single taxon were identified, purified and sequenced. Alignment of the sequence data and subsequent primer trials led to the identification of taxon-specific sequence characterized amplified regions (SCARs), and to a set of specific oligonucleotide combinations that can be used to identify these organisms in plant quarantine inspections.

Sequence specificity of mutagenesis in the cI gene of bacteriophage lambda

International Nuclear Information System (INIS)

Skopek, T.R.; Wood, R.D.; Hutchinson, F.

1985-01-01

Studies of DNA base sequence alterations have shown that for every agent the mutagenic process is specific with respect to the types of base changes induced and the location of the changes in the DNA. Analysis of the types of mutations produced by mutagenic agents can provide insight into the mechanism of mutation and can suggest which DNA lesions may be involved in the actual mutagenic event. We have developed a system for the analysis of chemically induced base sequence alterations in the cI repressor gene of bacteriophage lambda using DNA sequencing techniques. To illustrate the utility of this type of analysis, we present the results obtained with ultraviolet light (UV). Irradiation of target DNA with UV alone, or UV followed by photoreactivating light (which removes dimers), produces mostly transitions at pyrimidine-pyrimidine sites. Conversely, irradiation with 313 nm light plus acetophenone (which produces only thymine dimers) produces mostly transversions at low efficiency. This and other evidence suggests that the actual premutagenic UV lesion in E. coli may not be pyrimidine-pyrimidine dimers, but rather pyr(6-4)pyo photoproducts

Stem loop sequences specific to transposable element IS605 are found linked to lipoprotein genes in Borrelia plasmids.

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Nicholas Delihas

Full Text Available BACKGROUND: Plasmids of Borrelia species are dynamic structures that contain a large number of repetitive genes, gene fragments, and gene fusions. In addition, the transposable element IS605/200 family, as well as degenerate forms of this IS element, are prevalent. In Helicobacter pylori, flanking regions of the IS605 transposase gene contain sequences that fold into identical small stem loops. These function in transposition at the single-stranded DNA level. METHODOLOGY/PRINCIPAL FINDINGS: In work reported here, bioinformatics techniques were used to scan Borrelia plasmid genomes for IS605 transposable element specific stem loop sequences. Two variant stem loop motifs are found in the left and right flanking regions of the transposase gene. Both motifs appear to have dispersed in plasmid genomes and are found "free-standing" and phylogenetically conserved without the associated IS605 transposase gene or the adjacent flanking sequence. Importantly, IS605 specific stem loop sequences are also found at the 3' ends of lipoprotein genes (PFam12 and PFam60, however the left and right sequences appear to develop their own evolutionary patterns. The lipoprotein gene-linked left stem loop sequences maintain the IS605 stem loop motif in orthologs but only at the RNA level. These show mutations whereby variants fold into phylogenetically conserved RNA-type stem loops that contain the wobble non-Watson-Crick G-U base-pairing. The right flanking sequence is associated with the family lipoprotein-1 genes. A comparison of homologs shows that the IS605 stem loop motif rapidly dissipates, but a more elaborate secondary structure appears to develop in its place. CONCLUSIONS/SIGNIFICANCE: Stem loop sequences specific to the transposable element IS605 are present in plasmid regions devoid of a transposase gene and significantly, are found linked to lipoprotein genes in Borrelia plasmids. These sequences are evolutionarily conserved and/or structurally developed in

Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons.

Science.gov (United States)

Locati, Mauro D; Pagano, Johanna F B; Ensink, Wim A; van Olst, Marina; van Leeuwen, Selina; Nehrdich, Ulrike; Zhu, Kongju; Spaink, Herman P; Girard, Geneviève; Rauwerda, Han; Jonker, Martijs J; Dekker, Rob J; Breit, Timo M

2017-04-01

5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo, and adult tissue identified maternal-type 5S rRNA that is exclusively accumulated during oogenesis, replaced throughout the embryogenesis by a somatic-type, and thus virtually absent in adult somatic tissue. The maternal-type 5S rDNA contains several thousands of gene copies on chromosome 4 in tandem repeats with small intergenic regions, whereas the somatic-type is present in only 12 gene copies on chromosome 18 with large intergenic regions. The nine-nucleotide variation between the two 5S rRNA types likely affects TFIII binding and riboprotein L5 binding, probably leading to storage of maternal-type rRNA. Remarkably, these sequence differences are located exactly at the sequence-specific target site for genome integration by the 5S rRNA-specific Mutsu retrotransposon family. Thus, we could define maternal- and somatic-type MutsuDr subfamilies. Furthermore, we identified four additional maternal-type and two new somatic-type MutsuDr subfamilies, each with their own target sequence. This target-site specificity, frequently intact maternal-type retrotransposon elements, plus specific presence of Mutsu retrotransposon RNA and piRNA in egg and adult tissue, suggest an involvement of retrotransposons in achieving the differential copy number of the two types of 5S rDNA loci. © 2017 Locati et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

The minimum information about a genome sequence (MIGS) specification

Science.gov (United States)

Field, Dawn; Garrity, George; Gray, Tanya; Morrison, Norman; Selengut, Jeremy; Sterk, Peter; Tatusova, Tatiana; Thomson, Nicholas; Allen, Michael J; Angiuoli, Samuel V; Ashburner, Michael; Axelrod, Nelson; Baldauf, Sandra; Ballard, Stuart; Boore, Jeffrey; Cochrane, Guy; Cole, James; Dawyndt, Peter; De Vos, Paul; dePamphilis, Claude; Edwards, Robert; Faruque, Nadeem; Feldman, Robert; Gilbert, Jack; Gilna, Paul; Glöckner, Frank Oliver; Goldstein, Philip; Guralnick, Robert; Haft, Dan; Hancock, David; Hermjakob, Henning; Hertz-Fowler, Christiane; Hugenholtz, Phil; Joint, Ian; Kagan, Leonid; Kane, Matthew; Kennedy, Jessie; Kowalchuk, George; Kottmann, Renzo; Kolker, Eugene; Kravitz, Saul; Kyrpides, Nikos; Leebens-Mack, Jim; Lewis, Suzanna E; Li, Kelvin; Lister, Allyson L; Lord, Phillip; Maltsev, Natalia; Markowitz, Victor; Martiny, Jennifer; Methe, Barbara; Mizrachi, Ilene; Moxon, Richard; Nelson, Karen; Parkhill, Julian; Proctor, Lita; White, Owen; Sansone, Susanna-Assunta; Spiers, Andrew; Stevens, Robert; Swift, Paul; Taylor, Chris; Tateno, Yoshio; Tett, Adrian; Turner, Sarah; Ussery, David; Vaughan, Bob; Ward, Naomi; Whetzel, Trish; Gil, Ingio San; Wilson, Gareth; Wipat, Anil

2008-01-01

With the quantity of genomic data increasing at an exponential rate, it is imperative that these data be captured electronically, in a standard format. Standardization activities must proceed within the auspices of open-access and international working bodies. To tackle the issues surrounding the development of better descriptions of genomic investigations, we have formed the Genomic Standards Consortium (GSC). Here, we introduce the minimum information about a genome sequence (MIGS) specification with the intent of promoting participation in its development and discussing the resources that will be required to develop improved mechanisms of metadata capture and exchange. As part of its wider goals, the GSC also supports improving the ‘transparency’ of the information contained in existing genomic databases. PMID:18464787

Segment-specific terminal sequences of Bunyamwera bunyavirus regulate genome replication

International Nuclear Information System (INIS)

Barr, John N.; Elliott, Richard M.; Dunn, Ewan F.; Wertz, Gail W.

2003-01-01

Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the Bunyaviridae family of segmented negative sense RNA viruses. The tripartite BUNV genome consists of small (S), medium (M), and large (L) segments that are transcribed to give a single mRNA and replicated to generate an antigenome that is the template for synthesis of further genomic RNA strands. We modified an existing cDNA-derived RNA synthesis system to allow identification of BUNV RNA replication and transcription products by direct metabolic labeling. Direct RNA analysis allowed us to distinguish between template activities that affected either RNA replication or mRNA transcription, an ability that was not possible using previous reporter gene expression assays. We generated genome analogs containing the entire nontranslated terminal sequences of the S, M, and L BUNV segments surrounding a common sequence. Analysis of RNAs synthesized from these templates revealed that the relative abilities of BUNV segments to perform RNA replication was M > L > S. Exchange of segment-specific terminal nucleotides identified a 12-nt region located within both the 3' and 5' termini of the M segment that correlated with its high replication ability

Sequence-specific DNA binding by MYC/MAX to low-affinity non-E-box motifs.

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Michael Allevato

Full Text Available The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX bind Enhancer box (E-box DNA elements (CANNTG and have the greatest affinity for the canonical MYC E-box (CME CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a "non-specific" fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87% of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.

Characterization of shark complement factor I gene(s): genomic analysis of a novel shark-specific sequence.

Science.gov (United States)

Shin, Dong-Ho; Webb, Barbara M; Nakao, Miki; Smith, Sylvia L

2009-07-01

Complement factor I is a crucial regulator of mammalian complement activity. Very little is known of complement regulators in non-mammalian species. We isolated and sequenced four highly similar complement factor I cDNAs from the liver of the nurse shark (Ginglymostoma cirratum), designated as GcIf-1, GcIf-2, GcIf-3 and GcIf-4 (previously referred to as nsFI-a, -b, -c and -d) which encode 689, 673, 673 and 657 amino acid residues, respectively. They share 95% (shark-specific sequence between the leader peptide (LP) and the factor I membrane attack complex (FIMAC) domain. The cDNA sequences differ only in the size and composition of the shark-specific region (SSR). Sequence analysis of each SSR has identified within the region two novel short sequences (SS1 and SS2) and three repeat sequences (RS1-3). Genomic analysis has revealed the existence of three introns between the leader peptide and the FIMAC domain, tentatively designated intron 1, intron 2, and intron 3 which span 4067, 2293 and 2082bp, respectively. Southern blot analysis suggests the presence of a single gene copy for each cDNA type. Phylogenetic analysis suggests that complement factor I of cartilaginous fish diverged prior to the emergence of mammals. All four GcIf cDNA species are expressed in four different tissues and the liver is the main tissue in which expression level of all four is high. This suggests that the expression of GcIf isotypes is tissue-dependent.

High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

Science.gov (United States)

van Koningsbruggen, Silvana; Gierlinski, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J; Ariyurek, Yavuz; den Dunnen, Johan T; Lamond, Angus I

2010-11-01

The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

C-terminal sequences of hsp70 and hsp90 as non-specific anchors for tetratricopeptide repeat (TPR) proteins.

Science.gov (United States)

Ramsey, Andrew J; Russell, Lance C; Chinkers, Michael

2009-10-12

Steroid-hormone-receptor maturation is a multi-step process that involves several TPR (tetratricopeptide repeat) proteins that bind to the maturation complex via the C-termini of hsp70 (heat-shock protein 70) and hsp90 (heat-shock protein 90). We produced a random T7 peptide library to investigate the roles played by the C-termini of the two heat-shock proteins in the TPR-hsp interactions. Surprisingly, phages with the MEEVD sequence, found at the C-terminus of hsp90, were not recovered from our biopanning experiments. However, two groups of phages were isolated that bound relatively tightly to HsPP5 (Homo sapiens protein phosphatase 5) TPR. Multiple copies of phages with a C-terminal sequence of LFG were isolated. These phages bound specifically to the TPR domain of HsPP5, although mutation studies produced no evidence that they bound to the domain's hsp90-binding groove. However, the most abundant family obtained in the initial screen had an aspartate residue at the C-terminus. Two members of this family with a C-terminal sequence of VD appeared to bind with approximately the same affinity as the hsp90 C-12 control. A second generation pseudo-random phage library produced a large number of phages with an LD C-terminus. These sequences acted as hsp70 analogues and had relatively low affinities for hsp90-specific TPR domains. Unfortunately, we failed to identify residues near hsp90's C-terminus that impart binding specificity to individual hsp90-TPR interactions. The results suggest that the C-terminal sequences of hsp70 and hsp90 act primarily as non-specific anchors for TPR proteins.

Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein.

Directory of Open Access Journals (Sweden)

2006-05-01

Full Text Available Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP, a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function.

Putative and unique gene sequence utilization for the design of species specific probes as modeled by Lactobacillus plantarum

Science.gov (United States)

The concept of utilizing putative and unique gene sequences for the design of species specific probes was tested. The abundance profile of assigned functions within the Lactobacillus plantarum genome was used for the identification of the putative and unique gene sequence, csh. The targeted gene (cs...

Prediction of membrane transport proteins and their substrate specificities using primary sequence information.

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Nitish K Mishra

Full Text Available Membrane transport proteins (transporters move hydrophilic substrates across hydrophobic membranes and play vital roles in most cellular functions. Transporters represent a diverse group of proteins that differ in topology, energy coupling mechanism, and substrate specificity as well as sequence similarity. Among the functional annotations of transporters, information about their transporting substrates is especially important. The experimental identification and characterization of transporters is currently costly and time-consuming. The development of robust bioinformatics-based methods for the prediction of membrane transport proteins and their substrate specificities is therefore an important and urgent task.Support vector machine (SVM-based computational models, which comprehensively utilize integrative protein sequence features such as amino acid composition, dipeptide composition, physico-chemical composition, biochemical composition, and position-specific scoring matrices (PSSM, were developed to predict the substrate specificity of seven transporter classes: amino acid, anion, cation, electron, protein/mRNA, sugar, and other transporters. An additional model to differentiate transporters from non-transporters was also developed. Among the developed models, the biochemical composition and PSSM hybrid model outperformed other models and achieved an overall average prediction accuracy of 76.69% with a Mathews correlation coefficient (MCC of 0.49 and a receiver operating characteristic area under the curve (AUC of 0.833 on our main dataset. This model also achieved an overall average prediction accuracy of 78.88% and MCC of 0.41 on an independent dataset.Our analyses suggest that evolutionary information (i.e., the PSSM and the AAIndex are key features for the substrate specificity prediction of transport proteins. In comparison, similarity-based methods such as BLAST, PSI-BLAST, and hidden Markov models do not provide accurate predictions

Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.

Science.gov (United States)

Miyazaki, Nobuo; Kiyose, Norihiko; Akazawa, Yoko; Takashima, Mizuki; Hagihara, Yosihisa; Inoue, Naokazu; Matsuda, Tomonari; Ogawa, Ryu; Inoue, Seiya; Ito, Yuji

2015-09-01

The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Virus-specific DNA sequences present in cells which carry the herpes simplex virus thymidine kinase gene.

Science.gov (United States)

Minson, A C; Darby, G K; Wildy, P

1979-11-01

Two independently derived cell lines which carry the herpes simplex type 2 thymidine kinase gene have been examined for the presence of HSV-2-specific DNA sequences. Both cell lines contained 1 to 3 copies per cell of a sequence lying within map co-ordinates 0.2 to 0.4 of the HSV-2 genome. Revertant cells, which contained no detectable thymidine kinase, did not contain this DNA sequence. The failure of EcoR1-restricted HSV-2 DNA to act as a donor of the thymidine kinase gene in transformation experiments suggests that the gene lies close to the EcoR1 restriction site within this sequence at a map position of approx. 0.3. The HSV-2 kinase gene is therefore approximately co-linear with the HSV-1 gene.

Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

Science.gov (United States)

Efstathiou, S; Minson, A C; Field, H J; Anderson, J R; Wildy, P

1986-02-01

Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations.

Using sequence-specific chemical and structural properties of DNA to predict transcription factor binding sites.

Directory of Open Access Journals (Sweden)

Amy L Bauer

2010-11-01

Full Text Available An important step in understanding gene regulation is to identify the DNA binding sites recognized by each transcription factor (TF. Conventional approaches to prediction of TF binding sites involve the definition of consensus sequences or position-specific weight matrices and rely on statistical analysis of DNA sequences of known binding sites. Here, we present a method called SiteSleuth in which DNA structure prediction, computational chemistry, and machine learning are applied to develop models for TF binding sites. In this approach, binary classifiers are trained to discriminate between true and false binding sites based on the sequence-specific chemical and structural features of DNA. These features are determined via molecular dynamics calculations in which we consider each base in different local neighborhoods. For each of 54 TFs in Escherichia coli, for which at least five DNA binding sites are documented in RegulonDB, the TF binding sites and portions of the non-coding genome sequence are mapped to feature vectors and used in training. According to cross-validation analysis and a comparison of computational predictions against ChIP-chip data available for the TF Fis, SiteSleuth outperforms three conventional approaches: Match, MATRIX SEARCH, and the method of Berg and von Hippel. SiteSleuth also outperforms QPMEME, a method similar to SiteSleuth in that it involves a learning algorithm. The main advantage of SiteSleuth is a lower false positive rate.

Characterization of upstream sequences of the LIM2 gene that bind developmentally regulated and lens-specific proteins

Institute of Scientific and Technical Information of China (English)

HSU Heng; Robert L. CHURCH

2004-01-01

During lens development, lens epithelial cells differentiate into fiber cells. To date, four major lens fiber cell intrinsic membrane proteins (MIP) ranging in size from 70 kD to 19 kD have been characterized. The second most abundant lens fiber cell intrinsic membrane protein is MP19. This protein probably is involved with lens cell communication and relates with cataractogenesis. The aim of this research is to characterize upstream sequences of the MP19 (also called LIM2) gene that bind developmentally regulated and lens-specific proteins. We have used the gel mobility assays and corresponding competition experiments to identify and characterize cis elements within approximately 500 bases of LIM2 upstream sequences. Our studies locate the positions of some cis elements, including a "CA" repeat, a methylation Hha I island, an FnuD II site, an Ap1 and an Ap2 consensus sequences, and identify some specific cis elements which relate to lens-specific transcription of LIM2. Our experiments also preliminarily identify trans factors which bind to specific cis elements of the LIM2 promoter and/or regulate transcription of LIM2. We conclude that developmental regulation and coordination of the MP 19 gene in ocular lens fiber cells is controlled by the presence of specific cis elements that bind regulatory trans factors that affect LIM2 gene expression. DNA methylation is one mechanism of controlling LIM2 gene expression during lens development.

Molecular Design of Antifouling Polymer Brushes Using Sequence-Specific Peptoids.

Science.gov (United States)

Lau, King Hang Aaron; Sileika, Tadas S; Park, Sung Hyun; Sousa, Ana Maria Leal; Burch, Patrick; Szleifer, Igal; Messersmith, Phillip B

2015-01-07

Material systems that can be used to flexibly and precisely define the chemical nature and molecular arrangement of a surface would be invaluable for the control of complex biointerfacial interactions. For example, progress in antifouling polymer biointerfaces that prevent non-specific protein adsorption and cell attachment, which can significantly improve the performance of an array of biomedical and industrial applications, is hampered by a lack of chemical models to identify the molecular features conferring their properties. Poly(N-substituted glycine) "peptoids" are peptidomimetic polymers that can be conveniently synthesized with specific monomer sequences and chain lengths, and are presented as a versatile platform for investigating the molecular design of antifouling polymer brushes. Zwitterionic antifouling polymer brushes have captured significant recent attention, and a targeted library of zwitterionic peptoid brushes with a different charge densities, hydration, separations between charged groups, chain lengths, and grafted chain densities, is quantitatively evaluated for their antifouling properties through a range of protein adsorption and cell attachment assays. Specific zwitterionic brush designs were found to give rise to distinct but subtle differences in properties. The results also point to the dominant roles of the grafted chain density and chain length in determining the performance of antifouling polymer brushes.

A framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates.

Science.gov (United States)

Helbling, Damian E; Johnson, David R; Lee, Tae Kwon; Scheidegger, Andreas; Fenner, Kathrin

2015-03-01

The rates at which wastewater treatment plant (WWTP) microbial communities biotransform specific substrates can differ by orders of magnitude among WWTP communities. Differences in taxonomic compositions among WWTP communities may predict differences in the rates of some types of biotransformations. In this work, we present a novel framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates. We selected ten WWTPs with substantial variation in their environmental and operational metrics and measured the in situ ammonia biotransformation rate constants in nine of them. We isolated total RNA from samples from each WWTP and analyzed 16S rRNA sequence reads. We then developed multivariate models between the measured abundances of specific bacterial 16S rRNA sequence reads and the ammonia biotransformation rate constants. We constructed model scenarios that systematically explored the effects of model regularization, model linearity and non-linearity, and aggregation of 16S rRNA sequences into operational taxonomic units (OTUs) as a function of sequence dissimilarity threshold (SDT). A large percentage (greater than 80%) of model scenarios resulted in well-performing and significant models at intermediate SDTs of 0.13-0.14 and 0.26. The 16S rRNA sequences consistently selected into the well-performing and significant models at those SDTs were classified as Nitrosomonas and Nitrospira groups. We then extend the framework by applying it to the biotransformation rate constants of ten micropollutants measured in batch reactors seeded with the ten WWTP communities. We identified phylogenetic groups that were robustly selected into all well-performing and significant models constructed with biotransformation rates of isoproturon, propachlor, ranitidine, and venlafaxine. These phylogenetic groups can be used as predictive biomarkers of WWTP microbial community activity towards these specific

ATRX mutation in two adult brothers with non-specific moderate intellectual disability identified by exome sequencing.

Science.gov (United States)

Moncini, S; Bedeschi, M F; Castronovo, P; Crippa, M; Calvello, M; Garghentino, R R; Scuvera, G; Finelli, P; Venturin, M

2013-12-01

In this report, we describe two adult brothers affected by moderate non-specific intellectual disability (ID). They showed minor facial anomalies, not clearly ascribable to any specific syndromic patterns, microcephaly, brachydactyly and broad toes. Both brothers presented seizures. Karyotype, subtelomeric and FMR1 analysis were normal in both cases. We performed array-CGH analysis that revealed no copy-number variations potentially associated with ID. Subsequent exome sequence analysis allowed the identification of the ATRX c.109C>T (p.R37X) mutation in both the affected brothers. Sanger sequencing confirmed the presence of the mutation in the brothers and showed that the mother is a healthy carrier. Mutations in the ATRX gene cause the X-linked alpha thalassemia/mental retardation (ATR-X) syndrome (MIM #301040), a severe clinical condition usually associated with profound ID, facial dysmorphism and alpha thalassemia. However, the syndrome is clinically heterogeneous and some mutations, including the c.109C>T, are associated with a broad phenotypic spectrum, with patients displaying a less severe phenotype with only mild-moderate ID. In the case presented here, exome sequencing provided an effective strategy to achieve the molecular diagnosis of ATR-X syndrome, which otherwise would have been difficult to consider due to the mild non-specific phenotype and the absence of a family history with typical severe cases.

Structural basis for sequence-specific recognition of DNA by TAL effectors

KAUST Repository

Deng, Dong

2012-01-05

TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved amino acids and targets a specific base pair by using two hypervariable residues [known as repeat variable diresidues (RVDs)] at positions 12 and 13. Here, we report the crystal structures of an 11.5-repeat TAL effector in both DNA-free and DNA-bound states. Each TAL repeat comprises two helices connected by a short RVD-containing loop. The 11.5 repeats form a right-handed, superhelical structure that tracks along the sense strand of DNA duplex, with RVDs contacting the major groove. The 12th residue stabilizes the RVD loop, whereas the 13th residue makes a base-specific contact. Understanding DNA recognition by TAL effectors may facilitate rational design of DNA-binding proteins with biotechnological applications.

An algorithm and program for finding sequence specific oligo-nucleotide probes for species identification

Directory of Open Access Journals (Sweden)

Tautz Diethard

2002-03-01

Full Text Available Abstract Background The identification of species or species groups with specific oligo-nucleotides as molecular signatures is becoming increasingly popular for bacterial samples. However, it shows also great promise for other small organisms that are taxonomically difficult to tract. Results We have devised here an algorithm that aims to find the optimal probes for any given set of sequences. The program requires only a crude alignment of these sequences as input and is optimized for performance to deal also with very large datasets. The algorithm is designed such that the position of mismatches in the probes influences the selection and makes provision of single nucleotide outloops. Program implementations are available for Linux and Windows.

The specificity of memory for a highly trained finger movement sequence: Change the ending, change all.

Science.gov (United States)

Rozanov, Simon; Keren, Ofer; Karni, Avi

2010-05-17

How are highly trained movement sequences represented in long-term memory? Here we show that the gains attained in the performance of a well-trained sequence of finger movements can be expressed only when the order of the movements is exactly as practiced. Ten young adults were trained to perform a given 5-element sequence of finger-to-thumb opposition movements with their left hand. Movements were analyzed using video based tracking. Three weeks of training resulted, along with improved accuracy, in robustly shortened movement times as well as shorter finger-to-thumb touch times. However, there was little transfer of these gains in speed to the execution of the same component movements arranged in a new order. Moreover, even when the only change was the omission of the one before final movement of the trained sequence (Omit sequence), the initial movements of the sequence were significantly slowed down, although these movements were identical to the initial movements of the trained sequence. Our results support the notion that a well-trained sequence of finger movements can be represented, in the adult motor system, as a singular, co-articulated, unit of movement, in which even the initial component movements are contingent on the subsequent, anticipated, ones. Because of co-articulation related anticipatory effects, gains in fluency and accuracy acquired in training on a specific movement sequence cannot be expressed in full in the execution of the trained component movements or of a full segment of the trained sequence, if followed by a different ending segment. Copyright 2010. Published by Elsevier B.V.

Versatile Gene-Specific Sequence Tags for Arabidopsis Functional Genomics: Transcript Profiling and Reverse Genetics Applications

Science.gov (United States)

Hilson, Pierre; Allemeersch, Joke; Altmann, Thomas; Aubourg, Sébastien; Avon, Alexandra; Beynon, Jim; Bhalerao, Rishikesh P.; Bitton, Frédérique; Caboche, Michel; Cannoot, Bernard; Chardakov, Vasil; Cognet-Holliger, Cécile; Colot, Vincent; Crowe, Mark; Darimont, Caroline; Durinck, Steffen; Eickhoff, Holger; de Longevialle, Andéol Falcon; Farmer, Edward E.; Grant, Murray; Kuiper, Martin T.R.; Lehrach, Hans; Léon, Céline; Leyva, Antonio; Lundeberg, Joakim; Lurin, Claire; Moreau, Yves; Nietfeld, Wilfried; Paz-Ares, Javier; Reymond, Philippe; Rouzé, Pierre; Sandberg, Goran; Segura, Maria Dolores; Serizet, Carine; Tabrett, Alexandra; Taconnat, Ludivine; Thareau, Vincent; Van Hummelen, Paul; Vercruysse, Steven; Vuylsteke, Marnik; Weingartner, Magdalena; Weisbeek, Peter J.; Wirta, Valtteri; Wittink, Floyd R.A.; Zabeau, Marc; Small, Ian

2004-01-01

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics. PMID:15489341

Interspecies hybridization on DNA resequencing microarrays: efficiency of sequence recovery and accuracy of SNP detection in human, ape, and codfish mitochondrial DNA genomes sequenced on a human-specific MitoChip

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Carr Steven M

2007-09-01

Full Text Available Abstract Background Iterative DNA "resequencing" on oligonucleotide microarrays offers a high-throughput method to measure intraspecific biodiversity, one that is especially suited to SNP-dense gene regions such as vertebrate mitochondrial (mtDNA genomes. However, costs of single-species design and microarray fabrication are prohibitive. A cost-effective, multi-species strategy is to hybridize experimental DNAs from diverse species to a common microarray that is tiled with oligonucleotide sets from multiple, homologous reference genomes. Such a strategy requires that cross-hybridization between the experimental DNAs and reference oligos from the different species not interfere with the accurate recovery of species-specific data. To determine the pattern and limits of such interspecific hybridization, we compared the efficiency of sequence recovery and accuracy of SNP identification by a 15,452-base human-specific microarray challenged with human, chimpanzee, gorilla, and codfish mtDNA genomes. Results In the human genome, 99.67% of the sequence was recovered with 100.0% accuracy. Accuracy of SNP identification declines log-linearly with sequence divergence from the reference, from 0.067 to 0.247 errors per SNP in the chimpanzee and gorilla genomes, respectively. Efficiency of sequence recovery declines with the increase of the number of interspecific SNPs in the 25b interval tiled by the reference oligonucleotides. In the gorilla genome, which differs from the human reference by 10%, and in which 46% of these 25b regions contain 3 or more SNP differences from the reference, only 88% of the sequence is recoverable. In the codfish genome, which differs from the reference by > 30%, less than 4% of the sequence is recoverable, in short islands ≥ 12b that are conserved between primates and fish. Conclusion Experimental DNAs bind inefficiently to homologous reference oligonucleotide sets on a re-sequencing microarray when their sequences differ by

Generalized min-max bound-based MRI pulse sequence design framework for wide-range T1 relaxometry: A case study on the tissue specific imaging sequence.

Directory of Open Access Journals (Sweden)

Yang Liu

Full Text Available This paper proposes a new design strategy for optimizing MRI pulse sequences for T1 relaxometry. The design strategy optimizes the pulse sequence parameters to minimize the maximum variance of unbiased T1 estimates over a range of T1 values using the Cramér-Rao bound. In contrast to prior sequences optimized for a single nominal T1 value, the optimized sequence using our bound-based strategy achieves improved precision and accuracy for a broad range of T1 estimates within a clinically feasible scan time. The optimization combines the downhill simplex method with a simulated annealing process. To show the effectiveness of the proposed strategy, we optimize the tissue specific imaging (TSI sequence. Preliminary Monte Carlo simulations demonstrate that the optimized TSI sequence yields improved precision and accuracy over the popular driven-equilibrium single-pulse observation of T1 (DESPOT1 approach for normal brain tissues (estimated T1 700-2000 ms at 3.0T. The relative mean estimation error (MSE for T1 estimation is less than 1.7% using the optimized TSI sequence, as opposed to less than 7.0% using DESPOT1 for normal brain tissues. The optimized TSI sequence achieves good stability by keeping the MSE under 7.0% over larger T1 values corresponding to different lesion tissues and the cerebrospinal fluid (up to 5000 ms. The T1 estimation accuracy using the new pulse sequence also shows improvement, which is more pronounced in low SNR scenarios.

Sequence-specific high mobility group box factors recognize 10-12-base pair minor groove motifs

DEFF Research Database (Denmark)

van Beest, M; Dooijes, D; van De Wetering, M

2000-01-01

Sequence-specific high mobility group (HMG) box factors bind and bend DNA via interactions in the minor groove. Three-dimensional NMR analyses have provided the structural basis for this interaction. The cognate HMG domain DNA motif is generally believed to span 6-8 bases. However, alignment...

Domain-specific and domain-general constraints on word and sequence learning.

Science.gov (United States)

Archibald, Lisa M D; Joanisse, Marc F

2013-02-01

The relative influences of language-related and memory-related constraints on the learning of novel words and sequences were examined by comparing individual differences in performance of children with and without specific deficits in either language or working memory. Children recalled lists of words in a Hebbian learning protocol in which occasional lists repeated, yielding improved recall over the course of the task on the repeated lists. The task involved presentation of pictures of common nouns followed immediately by equivalent presentations of the spoken names. The same participants also completed a paired-associate learning task involving word-picture and nonword-picture pairs. Hebbian learning was observed for all groups. Domain-general working memory constrained immediate recall, whereas language abilities impacted recall in the auditory modality only. In addition, working memory constrained paired-associate learning generally, whereas language abilities disproportionately impacted novel word learning. Overall, all of the learning tasks were highly correlated with domain-general working memory. The learning of nonwords was additionally related to general intelligence, phonological short-term memory, language abilities, and implicit learning. The results suggest that distinct associations between language- and memory-related mechanisms support learning of familiar and unfamiliar phonological forms and sequences.

Screening for SNPs with Allele-Specific Methylation based on Next-Generation Sequencing Data.

Science.gov (United States)

Hu, Bo; Ji, Yuan; Xu, Yaomin; Ting, Angela H

2013-05-01

Allele-specific methylation (ASM) has long been studied but mainly documented in the context of genomic imprinting and X chromosome inactivation. Taking advantage of the next-generation sequencing technology, we conduct a high-throughput sequencing experiment with four prostate cell lines to survey the whole genome and identify single nucleotide polymorphisms (SNPs) with ASM. A Bayesian approach is proposed to model the counts of short reads for each SNP conditional on its genotypes of multiple subjects, leading to a posterior probability of ASM. We flag SNPs with high posterior probabilities of ASM by accounting for multiple comparisons based on posterior false discovery rates. Applying the Bayesian approach to the in-house prostate cell line data, we identify 269 SNPs as candidates of ASM. A simulation study is carried out to demonstrate the quantitative performance of the proposed approach.

Flow Cytometry-Assisted Cloning of Specific Sequence Motifs from Complex 16S rRNA Gene Libraries

DEFF Research Database (Denmark)

Nielsen, Jeppe Lund; Schramm, Andreas; Bernhard, Anne E.

2004-01-01

for Systems Biology,3 Seattle, Washington, and Department of Ecological Microbiology, University of Bayreuth, Bayreuth, Germany2 A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting......  FLOW CYTOMETRY-ASSISTED CLONING OF SPECIFIC SEQUENCE MOTIFS FROM COMPLEX 16S RRNA GENE LIBRARIES Jeppe L. Nielsen,1 Andreas Schramm,1,2 Anne E. Bernhard,1 Gerrit J. van den Engh,3 and David A. Stahl1* Department of Civil and Environmental Engineering, University of Washington,1 and Institute......-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.  ...

Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families

International Nuclear Information System (INIS)

Carbonell, Alberto; Martinez de Alba, Angel-Emilio; Flores, Ricardo; Gago, Selma

2008-01-01

Infection by viroids, non-protein-coding circular RNAs, occurs with the accumulation of 21-24 nt viroid-derived small RNAs (vd-sRNAs) with characteristic properties of small interfering RNAs (siRNAs) associated to RNA silencing. The vd-sRNAs most likely derive from dicer-like (DCL) enzymes acting on viroid-specific dsRNA, the key elicitor of RNA silencing, or on the highly structured genomic RNA. Previously, viral dsRNAs delivered mechanically or agroinoculated have been shown to interfere with virus infection in a sequence-specific manner. Here, we report similar results with members of the two families of nuclear- and chloroplast-replicating viroids. Moreover, homologous vd-sRNAs co-delivered mechanically also interfered with one of the viroids examined. The interference was sequence-specific, temperature-dependent and, in some cases, also dependent on the dose of the co-inoculated dsRNA or vd-sRNAs. The sequence-specific nature of these effects suggests the involvement of the RNA induced silencing complex (RISC), which provides sequence specificity to RNA silencing machinery. Therefore, viroid titer in natural infections might be regulated by the concerted action of DCL and RISC. Viroids could have evolved their secondary structure as a compromise between resistance to DCL and RISC, which act preferentially against RNAs with compact and relaxed secondary structures, respectively. In addition, compartmentation, association with proteins or active replication might also help viroids to elude their host RNA silencing machinery

Dog Y chromosomal DNA sequence: identification, sequencing and SNP discovery

Directory of Open Access Journals (Sweden)

Kirkness Ewen

2006-10-01

Full Text Available Abstract Background Population genetic studies of dogs have so far mainly been based on analysis of mitochondrial DNA, describing only the history of female dogs. To get a picture of the male history, as well as a second independent marker, there is a need for studies of biallelic Y-chromosome polymorphisms. However, there are no biallelic polymorphisms reported, and only 3200 bp of non-repetitive dog Y-chromosome sequence deposited in GenBank, necessitating the identification of dog Y chromosome sequence and the search for polymorphisms therein. The genome has been only partially sequenced for one male dog, disallowing mapping of the sequence into specific chromosomes. However, by comparing the male genome sequence to the complete female dog genome sequence, candidate Y-chromosome sequence may be identified by exclusion. Results The male dog genome sequence was analysed by Blast search against the human genome to identify sequences with a best match to the human Y chromosome and to the female dog genome to identify those absent in the female genome. Candidate sequences were then tested for male specificity by PCR of five male and five female dogs. 32 sequences from the male genome, with a total length of 24 kbp, were identified as male specific, based on a match to the human Y chromosome, absence in the female dog genome and male specific PCR results. 14437 bp were then sequenced for 10 male dogs originating from Europe, Southwest Asia, Siberia, East Asia, Africa and America. Nine haplotypes were found, which were defined by 14 substitutions. The genetic distance between the haplotypes indicates that they originate from at least five wolf haplotypes. There was no obvious trend in the geographic distribution of the haplotypes. Conclusion We have identified 24159 bp of dog Y-chromosome sequence to be used for population genetic studies. We sequenced 14437 bp in a worldwide collection of dogs, identifying 14 SNPs for future SNP analyses, and

Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases.

Science.gov (United States)

Citorik, Robert J; Mimee, Mark; Lu, Timothy K

2014-11-01

Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes or polymorphisms, including antibiotic resistance and virulence determinants in carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia coli. Delivery of RGNs significantly improves survival in a Galleria mellonella infection model. We also show that RGNs enable modulation of complex bacterial populations by selective knockdown of targeted strains based on genetic signatures. RGNs constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects and enable programmable remodeling of microbiota.

ATRX mutation in two adult brothers with non-specific moderate intellectual disability identified by exome sequencing

OpenAIRE

Moncini, S.; Bedeschi, M.F.; Castronovo, P.; Crippa, M.; Calvello, M.; Garghentino, R.R.; Scuvera, G.; Finelli, P.; Venturin, M.

2013-01-01

In this report, we describe two adult brothers affected by moderate non-specific intellectual disability (ID). They showed minor facial anomalies, not clearly ascribable to any specific syndromic patterns, microcephaly, brachydactyly and broad toes. Both brothers presented seizures. Karyotype, subtelomeric and FMR1 analysis were normal in both cases. We performed array-CGH analysis that revealed no copy-number variations potentially associated with ID. Subsequent exome sequence analysis allow...

Single-cell mRNA cytometry via sequence-specific nanoparticle clustering and trapping

Science.gov (United States)

Labib, Mahmoud; Mohamadi, Reza M.; Poudineh, Mahla; Ahmed, Sharif U.; Ivanov, Ivaylo; Huang, Ching-Lung; Moosavi, Maral; Sargent, Edward H.; Kelley, Shana O.

2018-05-01

Cell-to-cell variation in gene expression creates a need for techniques that can characterize expression at the level of individual cells. This is particularly true for rare circulating tumour cells, in which subtyping and drug resistance are of intense interest. Here we describe a method for cell analysis—single-cell mRNA cytometry—that enables the isolation of rare cells from whole blood as a function of target mRNA sequences. This approach uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of the target mRNA. Hybridization leads to the formation of large magnetic clusters that remain localized within the cells of interest, thereby enabling the cells to be magnetically separated. Targeting specific intracellular mRNAs enablescirculating tumour cells to be distinguished from normal haematopoietic cells. No polymerase chain reaction amplification is required to determine RNA expression levels and genotype at the single-cell level, and minimal cell manipulation is required. To demonstrate this approach we use single-cell mRNA cytometry to detect clinically important sequences in prostate cancer specimens.

One-year retention of general and sequence-specific skills in a probabilistic, serial reaction time task.

Science.gov (United States)

Romano, Jennifer C; Howard, James H; Howard, Darlene V

2010-05-01

Procedural skills such as riding a bicycle and playing a musical instrument play a central role in daily life. Such skills are learned gradually and are retained throughout life. The present study investigated 1-year retention of procedural skill in a version of the widely used serial reaction time task (SRTT) in young and older motor-skill experts and older controls in two experiments. The young experts were college-age piano and action video-game players, and the older experts were piano players. Previous studies have reported sequence-specific skill retention in the SRTT as long as 2 weeks but not at 1 year. Results indicated that both young and older experts and older non-experts revealed sequence-specific skill retention after 1 year with some evidence that general motor skill was retained as well. These findings are consistent with theoretical accounts of procedural skill learning such as the procedural reinstatement theory as well as with previous studies of retention of other motor skills.

The"minimum information about an environmental sequence" (MIENS) specification

Energy Technology Data Exchange (ETDEWEB)

Yilmaz, P.; Kottmann, R.; Field, D.; Knight, R.; Cole, J.R.; Amaral-Zettler, L.; Gilbert, J.A.; Karsch-Mizrachi, I.; Johnston, A.; Cochrane, G.; Vaughan, R.; Hunter, C.; Park, J.; Morrison, N.; Rocca-Serra, P.; Sterk, P.; Arumugam, M.; Baumgartner, L.; Birren, B.W.; Blaser, M.J.; Bonazzi, V.; Bork, P.; Buttigieg, P. L.; Chain, P.; Costello, E.K.; Huot-Creasy, H.; Dawyndt, P.; DeSantis, T.; Fierer, N.; Fuhrman, J.; Gallery, R.E.; Gibbs, R.A.; Giglio, M.G.; Gil, I. San; Gonzalez, A.; Gordon, J.I.; Guralnick, R.; Hankeln, W.; Highlander, S.; Hugenholtz, P.; Jansson, J.; Kennedy, J.; Knights, D.; Koren, O.; Kuczynski, J.; Kyrpides, N.; Larsen, R.; Lauber, C.L.; Legg, T.; Ley, R.E.; Lozupone, C.A.; Ludwig, W.; Lyons, D.; Maguire, E.; Methe, B.A.; Meyer, F.; Nakieny, S.; Nelson, K.E.; Nemergut, D.; Neufeld, J.D.; Pace, N.R.; Palanisamy, G.; Peplies, J.; Peterson, J.; Petrosino, J.; Proctor, L.; Raes, J.; Ratnasingham, S.; Ravel, J.; Relman, D.A.; Assunta-Sansone, S.; Schriml, L.; Sodergren, E.; Spor, A.; Stombaugh, J.; Tiedje, J.M.; Ward, D.V.; Weinstock, G.M.; Wendel, D.; White, O.; Wikle, A.; Wortman, J.R.; Glockner, F.O.; Bushman, F.D.; Charlson, E.; Gevers, D.; Kelley, S.T.; Neubold, L.K.; Oliver, A.E.; Pruesse, E.; Quast, C.; Schloss, P.D.; Sinha, R.; Whitely, A.

2010-10-15

We present the Genomic Standards Consortium's (GSC) 'Minimum Information about an ENvironmental Sequence' (MIENS) standard for describing marker genes. Adoption of MIENS will enhance our ability to analyze natural genetic diversity across the Tree of Life as it is currently being documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.

Genomic library screening for viruses from the human dental plaque revealed pathogen-specific lytic phage sequences.

Science.gov (United States)

Al-Jarbou, Ahmed Nasser

2012-01-01

Bacterial pathogenesis presents an astounding arsenal of virulence factors that allow them to conquer many different niches throughout the course of infection. Principally fascinating is the fact that some bacterial species are able to induce different diseases by expression of different combinations of virulence factors. Nevertheless, studies aiming at screening for the presence of bacteriophages in humans have been limited. Such screening procedures would eventually lead to identification of phage-encoded properties that impart increased bacterial fitness and/or virulence in a particular niche, and hence, would potentially be used to reverse the course of bacterial infections. As the human oral cavity represents a rich and dynamic ecosystem for several upper respiratory tract pathogens. However, little is known about virus diversity in human dental plaque which is an important reservoir. We applied the culture-independent approach to characterize virus diversity in human dental plaque making a library from a virus DNA fraction amplified using a multiple displacement method and sequenced 80 clones. The resulting sequence showed 44% significant identities to GenBank databases by TBLASTX analysis. TBLAST homology comparisons showed that 66% was viral; 18% eukarya; 10% bacterial; 6% mobile elements. These sequences were sorted into 6 contigs and 45 single sequences in which 4 contigs and a single sequence showed significant identity to a small region of a putative prophage in the Corynebacterium diphtheria genome. These findings interestingly highlight the uniqueness of over half of the sequences, whilst the dominance of a pathogen-specific prophage sequences imply their role in virulence.

Solution structure of d-GAATTCGAATTC by 2D NMR

International Nuclear Information System (INIS)

Hosur, R.V.; Ravikumar, M.; Chary, K.V.R.; Sheth, A.; Govil, G.

1986-01-01

A new approach based on the correlated spectroscopy (COSY) in 2D NMR has been described for determination of sugar geometries in oligonucleotides. Under the usual low resolution conditions employed in COSY, the intensities of cross peaks depend on the magnitudes of coupling constants. There are five vicinal coupling constants in a deoxyribose ring which are sensitive to the sugar geometry. The presence, absence and rough comparison of relative intensities of COSY cross peaks arising from such coupling constants enable one to fix the sugar conformation to a fair degree of precision. The methodology has been applied to d-GAATTCGAATTC. It is observed that ten out of the twelve nucleotide units in this sequence exhibit a rare O1'-endo geometry. The EcoRI cleavage sites in the dodecanucleotide show an interesting variation in the conformation with the two sugars attached to the Gs acquiring a geometry between C2'-endo and C4'-endo. (Auth.)

IDENTIFICATION OF AVIAN-SPECIFIC FECAL METAGENOMIC SEQUENCES USING GENOME FRAGMENT ENRICHMENTS

Science.gov (United States)

Sequence analysis of microbial genomes has provided biologists the opportunity to compare genetic differences between closely related microorganisms. While random sequencing has also been used to study natural microbial communities, metagenomic comparisons via sequencing analysis...

BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction.

Directory of Open Access Journals (Sweden)

Brad Thomas Townsley

2015-05-01

Full Text Available Next Generation Sequencing (NGS is driving rapid advancement in biological understanding and RNA-sequencing (RNA-seq has become an indispensable tool for biology and medicine. There is a growing need for access to these technologies although preparation of NGS libraries remains a bottleneck to wider adoption. Here we report a novel method for the production of strand specific RNA-seq libraries utilizing inherent properties of double-stranded cDNA to capture and incorporate a sequencing adapter. Breath Adapter Directional sequencing (BrAD-seq reduces sample handling and requires far fewer enzymatic steps than most available methods to produce high quality strand-specific RNA-seq libraries. The method we present is optimized for 3-prime Digital Gene Expression (DGE libraries and can easily extend to full transcript coverage shotgun (SHO type strand-specific libraries and is modularized to accommodate a diversity of RNA and DNA input materials. BrAD-seq offers a highly streamlined and inexpensive option for RNA-seq libraries.

Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA

Science.gov (United States)

Oberstrass, Florian C.; Fernandes, Louis E.; Bryant, Zev

2012-01-01

B-DNA becomes unstable under superhelical stress and is able to adopt a wide range of alternative conformations including strand-separated DNA and Z-DNA. Localized sequence-dependent structural transitions are important for the regulation of biological processes such as DNA replication and transcription. To directly probe the effect of sequence on structural transitions driven by torque, we have measured the torsional response of a panel of DNA sequences using single molecule assays that employ nanosphere rotational probes to achieve high torque resolution. The responses of Z-forming d(pGpC)n sequences match our predictions based on a theoretical treatment of cooperative transitions in helical polymers. “Bubble” templates containing 50–100 bp mismatch regions show cooperative structural transitions similar to B-DNA, although less torque is required to disrupt strand–strand interactions. Our mechanical measurements, including direct characterization of the torsional rigidity of strand-separated DNA, establish a framework for quantitative predictions of the complex torsional response of arbitrary sequences in their biological context. PMID:22474350

Integration of hepatitis B virus DNA in chromosome-specific satellite sequences

International Nuclear Information System (INIS)

Shaul, Y.; Garcia, P.D.; Schonberg, S.; Rutter, W.J.

1986-01-01

The authors previously reported the cloning and detailed analysis of the integrated hepatitis B virus sequences in a human hepatoma cell line. They report here the integration of at least one of hepatitis B virus at human satellite DNA sequences. The majority of the cellular sequences identified by this satellite were organized as a multimeric composition of a 0.6-kilobase EcoRI fragment. This clone hybridized in situ almost exclusively to the centromeric heterochromatin of chromosomes 1 and 16 and to a lower extent to chromosome 2 and to the heterochromatic region of the Y chromosome. The immediate flanking host sequence appeared as a hierarchy of repeating units which were almost identical to a previously reported human satellite III DNA sequence

Thermodynamic Molecular Switch in Sequence-Specific Hydrophobic Interaction: Two Computational Models Compared

Directory of Open Access Journals (Sweden)

Paul Chun

2003-01-01

Full Text Available We have shown in our published work the existence of a thermodynamic switch in biological systems wherein a change of sign in ΔCp°(Treaction leads to a true negative minimum in the Gibbs free energy change of reaction, and hence, a maximum in the related Keq. We have examined 35 pair-wise, sequence-specific hydrophobic interactions over the temperature range of 273–333 K, based on data reported by Nemethy and Scheraga in 1962. A closer look at a single example, the pair-wise hydrophobic interaction of leucine-isoleucine, will demonstrate the significant differences when the data are analyzed using the Nemethy-Scheraga model or treated by the Planck-Benzinger methodology which we have developed. The change in inherent chemical bond energy at 0 K, ΔH°(T0 is 7.53 kcal mol-1 compared with 2.4 kcal mol-1, while ‹ts› is 365 K as compared with 355 K, for the Nemethy-Scheraga and Planck-Benzinger model, respectively. At ‹tm›, the thermal agitation energy is about five times greater than ΔH°(T0 in the Planck-Benzinger model, that is 465 K compared to 497 K in the Nemethy-Scheraga model. The results imply that the negative Gibbs free energy minimum at a well-defined ‹ts›, where TΔS° = 0 at about 355 K, has its origin in the sequence-specific hydrophobic interactions, which are highly dependent on details of molecular structure. The Nemethy-Scheraga model shows no evidence of the thermodynamic molecular switch that we have found to be a universal feature of biological interactions. The Planck-Benzinger method is the best known for evaluating the innate temperature-invariant enthalpy, ΔH°(T0, and provides for better understanding of the heat of reaction for biological molecules.

DNA minor groove electrostatic potential: influence of sequence-specific transitions of the torsion angle gamma and deoxyribose conformations.

Science.gov (United States)

Zhitnikova, M Y; Shestopalova, A V

2017-11-01

The structural adjustments of the sugar-phosphate DNA backbone (switching of the γ angle (O5'-C5'-C4'-C3') from canonical to alternative conformations and/or C2'-endo → C3'-endo transition of deoxyribose) lead to the sequence-specific changes in accessible surface area of both polar and non-polar atoms of the grooves and the polar/hydrophobic profile of the latter ones. The distribution of the minor groove electrostatic potential is likely to be changing as a result of such conformational rearrangements in sugar-phosphate DNA backbone. Our analysis of the crystal structures of the short free DNA fragments and calculation of their electrostatic potentials allowed us to determine: (1) the number of classical and alternative γ angle conformations in the free B-DNA; (2) changes in the minor groove electrostatic potential, depending on the conformation of the sugar-phosphate DNA backbone; (3) the effect of the DNA sequence on the minor groove electrostatic potential. We have demonstrated that the structural adjustments of the DNA double helix (the conformations of the sugar-phosphate backbone and the minor groove dimensions) induce changes in the distribution of the minor groove electrostatic potential and are sequence-specific. Therefore, these features of the minor groove sizes and distribution of minor groove electrostatic potential can be used as a signal for recognition of the target DNA sequence by protein in the implementation of the indirect readout mechanism.

Thermodynamics of sequence-specific binding of PNA to DNA

DEFF Research Database (Denmark)

Ratilainen, T; Holmén, A; Tuite, E

2000-01-01

For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and seq......For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes...

Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor

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Kareem Mohideen-Abdul

2017-06-01

Full Text Available Most nuclear receptors (NRs bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs involves specific protein–DNA and protein–protein interactions. The estrogen-related receptor (ERR belongs to the steroid hormone nuclear receptor (SHR family and shares strong similarity in its DNA-binding domain (DBD with that of the estrogen receptor (ER. In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3, but in vivo, it preferentially binds to single half-site REs extended at the 5′-end by 3 bp [estrogen-related response element (ERREs], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS, non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3, such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3, where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half

Preparation of genosensor for detection of specific DNA sequence of the hepatitis B virus

Science.gov (United States)

Honorato Castro, Ana C.; França, Erick G.; de Paula, Lucas F.; Soares, Marcia M. C. N.; Goulart, Luiz R.; Madurro, João M.; Brito-Madurro, Ana G.

2014-09-01

An electrochemical genosensor was constructed for detection of specific DNA sequence of the hepatitis B virus, based on graphite electrodes modified with poly(4-aminophenol) and incorporating a specific oligonucleotide probe. The modified electrode containing the probe was evaluated by differential pulse voltammetry, before and after incubation with the complementary oligonucleotide target. Detection was performed by monitoring oxidizable DNA bases (direct detection) or using ethidium bromide as indicator of the hybridization process (indirect detection). The device showed a detection limit for the oligonucleotide target of 2.61 nmol L-1. Indirect detection using ethidium bromide was promising in discriminating mismatches, which is a very desirable attribute for detection of disease-related point mutations. In addition, it was possible to observe differences between hybridized and non-hybridized surfaces by atomic force microscopy.

Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

DEFF Research Database (Denmark)

Ibaraki, K; Kozak, C A; Wewer, U M

1995-01-01

regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76......(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed...

Preparation of genosensor for detection of specific DNA sequence of the hepatitis B virus

International Nuclear Information System (INIS)

Honorato Castro, Ana C.; França, Erick G.; Paula, Lucas F. de; Soares, Marcia M.C.N.; Goulart, Luiz R.; Madurro, João M.; Brito-Madurro, Ana G.

2014-01-01

Graphical abstract: - Highlights: • Specific oligonucleotide detection for hepatitis B based on poly-4-aminophenol matrix. • Electrochemical detection of the gene specific using ethidium bromide as indicator. • The detection limit was 2.61 nmol L −1 , with a correlation coefficient of 0.998 (n = 3). • The system discriminates three-base mismatches and non-complementary target. - Abstract: An electrochemical genosensor was constructed for detection of specific DNA sequence of the hepatitis B virus, based on graphite electrodes modified with poly(4-aminophenol) and incorporating a specific oligonucleotide probe. The modified electrode containing the probe was evaluated by differential pulse voltammetry, before and after incubation with the complementary oligonucleotide target. Detection was performed by monitoring oxidizable DNA bases (direct detection) or using ethidium bromide as indicator of the hybridization process (indirect detection). The device showed a detection limit for the oligonucleotide target of 2.61 nmol L −1 . Indirect detection using ethidium bromide was promising in discriminating mismatches, which is a very desirable attribute for detection of disease-related point mutations. In addition, it was possible to observe differences between hybridized and non-hybridized surfaces by atomic force microscopy

Preparation of genosensor for detection of specific DNA sequence of the hepatitis B virus

Energy Technology Data Exchange (ETDEWEB)

Honorato Castro, Ana C.; França, Erick G. [Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia (Brazil); Paula, Lucas F. de [Institute of Chemistry, Federal University of Uberlândia, Uberlândia (Brazil); Soares, Marcia M.C.N. [Adolfo Lutz Institute, Regional Laboratory in São José do Rio Preto (Brazil); Goulart, Luiz R. [Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia (Brazil); Madurro, João M. [Institute of Chemistry, Federal University of Uberlândia, Uberlândia (Brazil); Brito-Madurro, Ana G., E-mail: agbrito@iqufu.ufu.br [Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia (Brazil)

2014-09-30

Graphical abstract: - Highlights: • Specific oligonucleotide detection for hepatitis B based on poly-4-aminophenol matrix. • Electrochemical detection of the gene specific using ethidium bromide as indicator. • The detection limit was 2.61 nmol L{sup −1}, with a correlation coefficient of 0.998 (n = 3). • The system discriminates three-base mismatches and non-complementary target. - Abstract: An electrochemical genosensor was constructed for detection of specific DNA sequence of the hepatitis B virus, based on graphite electrodes modified with poly(4-aminophenol) and incorporating a specific oligonucleotide probe. The modified electrode containing the probe was evaluated by differential pulse voltammetry, before and after incubation with the complementary oligonucleotide target. Detection was performed by monitoring oxidizable DNA bases (direct detection) or using ethidium bromide as indicator of the hybridization process (indirect detection). The device showed a detection limit for the oligonucleotide target of 2.61 nmol L{sup −1}. Indirect detection using ethidium bromide was promising in discriminating mismatches, which is a very desirable attribute for detection of disease-related point mutations. In addition, it was possible to observe differences between hybridized and non-hybridized surfaces by atomic force microscopy.

Sequence-specific capture of protein-DNA complexes for mass spectrometric protein identification.

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Cheng-Hsien Wu

Full Text Available The regulation of gene transcription is fundamental to the existence of complex multicellular organisms such as humans. Although it is widely recognized that much of gene regulation is controlled by gene-specific protein-DNA interactions, there presently exists little in the way of tools to identify proteins that interact with the genome at locations of interest. We have developed a novel strategy to address this problem, which we refer to as GENECAPP, for Global ExoNuclease-based Enrichment of Chromatin-Associated Proteins for Proteomics. In this approach, formaldehyde cross-linking is employed to covalently link DNA to its associated proteins; subsequent fragmentation of the DNA, followed by exonuclease digestion, produces a single-stranded region of the DNA that enables sequence-specific hybridization capture of the protein-DNA complex on a solid support. Mass spectrometric (MS analysis of the captured proteins is then used for their identification and/or quantification. We show here the development and optimization of GENECAPP for an in vitro model system, comprised of the murine insulin-like growth factor-binding protein 1 (IGFBP1 promoter region and FoxO1, a member of the forkhead rhabdomyosarcoma (FoxO subfamily of transcription factors, which binds specifically to the IGFBP1 promoter. This novel strategy provides a powerful tool for studies of protein-DNA and protein-protein interactions.

Mediators of exposure therapy for youth obsessive-compulsive disorder: specificity and temporal sequence of client and treatment factors.

Science.gov (United States)

Chu, Brian C; Colognori, Daniela B; Yang, Guang; Xie, Min-ge; Lindsey Bergman, R; Piacentini, John

2015-05-01

Behavioral engagement and cognitive coping have been hypothesized to mediate effectiveness of exposure-based therapies. Identifying which specific child factors mediate successful therapy and which therapist factors facilitate change can help make our evidence-based treatments more efficient and robust. The current study examines the specificity and temporal sequence of relations among hypothesized client and therapist mediators in exposure therapy for pediatric Obsessive Compulsive Disorder (OCD). Youth coping (cognitive, behavioral), youth safety behaviors (avoidance, escape, compulsive behaviors), therapist interventions (cognitive, exposure extensiveness), and youth anxiety were rated via observational ratings of therapy sessions of OCD youth (N=43; ages=8 - 17; 62.8% male) who had received Exposure and Response Prevention (ERP). Regression analysis using Generalized Estimation Equations and cross-lagged panel analysis (CLPA) were conducted to model anxiety change within and across sessions, to determine formal mediators of anxiety change, and to establish sequence of effects. Anxiety ratings decreased linearly across exposures within sessions. Youth coping and therapist interventions significantly mediated anxiety change across exposures, and youth-interfering behavior mediated anxiety change at the trend level. In CLPA, youth-interfering behaviors predicted, and were predicted by, changes in anxiety. Youth coping was predicted by prior anxiety change. The study provides a preliminary examination of specificity and temporal sequence among child and therapist behaviors in predicting youth anxiety. Results suggest that therapists should educate clients in the natural rebound effects of anxiety between sessions and should be aware of the negatively reinforcing properties of avoidance during exposure. Copyright © 2015. Published by Elsevier Ltd.

Multi-species sequence comparison reveals dynamic evolution of the elastin gene that has involved purifying selection and lineage-specific insertions/deletions

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Green Eric D

2004-05-01

Full Text Available Abstract Background The elastin gene (ELN is implicated as a factor in both supravalvular aortic stenosis (SVAS and Williams Beuren Syndrome (WBS, two diseases involving pronounced complications in mental or physical development. Although the complete spectrum of functional roles of the processed gene product remains to be established, these roles are inferred to be analogous in human and mouse. This view is supported by genomic sequence comparison, in which there are no large-scale differences in the ~1.8 Mb sequence block encompassing the common region deleted in WBS, with the exception of an overall reversed physical orientation between human and mouse. Results Conserved synteny around ELN does not translate to a high level of conservation in the gene itself. In fact, ELN orthologs in mammals show more sequence divergence than expected for a gene with a critical role in development. The pattern of divergence is non-conventional due to an unusually high ratio of gaps to substitutions. Specifically, multi-sequence alignments of eight mammalian sequences reveal numerous non-aligning regions caused by species-specific insertions and deletions, in spite of the fact that the vast majority of aligning sites appear to be conserved and undergoing purifying selection. Conclusions The pattern of lineage-specific, in-frame insertions/deletions in the coding exons of ELN orthologous genes is unusual and has led to unique features of the gene in each lineage. These differences may indicate that the gene has a slightly different functional mechanism in mammalian lineages, or that the corresponding regions are functionally inert. Identified regions that undergo purifying selection reflect a functional importance associated with evolutionary pressure to retain those features.

Representational difference analysis of Neisseria meningitidis identifies sequences that are specific for the hyper-virulent lineage III clone

NARCIS (Netherlands)

Bart, A.; Dankert, J.; van der Ende, A.

2000-01-01

Neisseria meningitidis may cause meningitis and septicemia. Since the early 1980s, an increased incidence of meningococcal disease has been caused by the lineage III clone in many countries in Europe and in New Zealand. We hypothesized that lineage III meningococci have specific DNA sequences,

Design of Tail-Clamp Peptide Nucleic Acid Tethered with Azobenzene Linker for Sequence-Specific Detection of Homopurine DNA

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Shinjiro Sawada

2017-10-01

Full Text Available DNA carries genetic information in its sequence of bases. Synthetic oligonucleotides that can sequence-specifically recognize a target gene sequence are a useful tool for regulating gene expression or detecting target genes. Among the many synthetic oligonucleotides, tail-clamp peptide nucleic acid (TC-PNA offers advantages since it has two homopyrimidine PNA strands connected via a flexible ethylene glycol-type linker that can recognize complementary homopurine sequences via Watson-Crick and Hoogsteen base pairings and form thermally-stable PNA/PNA/DNA triplex structures. Here, we synthesized a series of TC-PNAs that can possess different lengths of azobenzene-containing linkers and studied their binding behaviours to homopurine single-stranded DNA. Introduction of azobenzene at the N-terminus amine of PNA increased the thermal stability of PNA-DNA duplexes. Further extension of the homopyrimidine PNA strand at the N-terminus of PNA-AZO further increased the binding stability of the PNA/DNA/PNA triplex to the target homopurine sequence; however, it induced TC-PNA/DNA/TC-PNA complex formation. Among these TC-PNAs, 9W5H-C4-AZO consisting of nine Watson-Crick bases and five Hoogsteen bases tethered with a beta-alanine conjugated azobenzene linker gave a stable 1:1 TC-PNA/ssDNA complex and exhibited good mismatch recognition. Our design for TC-PNA-AZO can be utilized for detecting homopurine sequences in various genes.

Mutations of the kissing-loop dimerization sequence influence the site specificity of murine leukemia virus recombination in vivo

DEFF Research Database (Denmark)

Mikkelsen, J G; Lund, Anders Henrik; Duch, M

2000-01-01

synthesis in newly infected cells. We have previously shown that template shifts within the 5' leader of murine leukemia viruses occur preferentially within the kissing stem-loop motif, a cis element crucial for in vitro RNA dimer formation. By use of a forced recombination approach based on single......-cycle transfer of Akv murine leukemia virus-based vectors harboring defective primer binding site sequences, we now report that modifications of the kissing-loop structure, ranging from a deletion of the entire sequence to introduction of a single point mutation in the loop motif, significantly disturb site...... specificity of recombination within the highly structured 5' leader region. In addition, we find that an intact kissing-loop sequence favors optimal RNA encapsidation and vector transduction. Our data are consistent with the kissing-loop dimerization model and suggest that a direct intermolecular RNA...

Variations in CCL3L gene cluster sequence and non-specific gene copy numbers

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Edberg Jeffrey C

2010-03-01

Full Text Available Abstract Background Copy number variations (CNVs of the gene CC chemokine ligand 3-like1 (CCL3L1 have been implicated in HIV-1 susceptibility, but the association has been inconsistent. CCL3L1 shares homology with a cluster of genes localized to chromosome 17q12, namely CCL3, CCL3L2, and, CCL3L3. These genes are involved in host defense and inflammatory processes. Several CNV assays have been developed for the CCL3L1 gene. Findings Through pairwise and multiple alignments of these genes, we have shown that the homology between these genes ranges from 50% to 99% in complete gene sequences and from 70-100% in the exonic regions, with CCL3L1 and CCL3L3 being identical. By use of MEGA 4 and BioEdit, we aligned sense primers, anti-sense primers, and probes used in several previously described assays against pre-multiple alignments of all four chemokine genes. Each set of probes and primers aligned and matched with overlapping sequences in at least two of the four genes, indicating that previously utilized RT-PCR based CNV assays are not specific for only CCL3L1. The four available assays measured median copies of 2 and 3-4 in European and African American, respectively. The concordance between the assays ranged from 0.44-0.83 suggesting individual discordant calls and inconsistencies with the assays from the expected gene coverage from the known sequence. Conclusions This indicates that some of the inconsistencies in the association studies could be due to assays that provide heterogenous results. Sequence information to determine CNV of the three genes separately would allow to test whether their association with the pathogenesis of a human disease or phenotype is affected by an individual gene or by a combination of these genes.

Phase-Specific Vocalizations of Male Mice at the Initial Encounter during the Courtship Sequence.

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Yui K Matsumoto

Full Text Available Mice produce ultrasonic vocalizations featuring a variety of syllables. Vocalizations are observed during social interactions. In particular, males produce numerous syllables during courtship. Previous studies have shown that vocalizations change according to sexual behavior, suggesting that males vary their vocalizations depending on the phase of the courtship sequence. To examine this process, we recorded large sets of mouse vocalizations during male-female interactions and acoustically categorized these sounds into 12 vocal types. We found that males emitted predominantly short syllables during the first minute of interaction, more long syllables in the later phases, and mainly harmonic sounds during mounting. These context- and time-dependent changes in vocalization indicate that vocal communication during courtship in mice consists of at least three stages and imply that each vocalization type has a specific role in a phase of the courtship sequence. Our findings suggest that recording for a sufficiently long time and taking the phase of courtship into consideration could provide more insights into the role of vocalization in mouse courtship behavior in future study.

Somatic sex-specific transcriptome differences in Drosophila revealed by whole transcriptome sequencing

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Arbeitman Michelle N

2011-07-01

Full Text Available Abstract Background Understanding animal development and physiology at a molecular-biological level has been advanced by the ability to determine at high resolution the repertoire of mRNA molecules by whole transcriptome resequencing. This includes the ability to detect and quantify rare abundance transcripts and isoform-specific mRNA variants produced from a gene. The sex hierarchy consists of a pre-mRNA splicing cascade that directs the production of sex-specific transcription factors that specify nearly all sexual dimorphism. We have used deep RNA sequencing to gain insight into how the Drosophila sex hierarchy generates somatic sex differences, by examining gene and transcript isoform expression differences between the sexes in adult head tissues. Results Here we find 1,381 genes that differ in overall expression levels and 1,370 isoform-specific transcripts that differ between males and females. Additionally, we find 512 genes not regulated downstream of transformer that are significantly more highly expressed in males than females. These 512 genes are enriched on the × chromosome and reside adjacent to dosage compensation complex entry sites, which taken together suggests that their residence on the × chromosome might be sufficient to confer male-biased expression. There are no transcription unit structural features, from a set of features, that are robustly significantly different in the genes with significant sex differences in the ratio of isoform-specific transcripts, as compared to random isoform-specific transcripts, suggesting that there is no single molecular mechanism that generates isoform-specific transcript differences between the sexes, even though the sex hierarchy is known to include three pre-mRNA splicing factors. Conclusions We identify thousands of genes that show sex-specific differences in overall gene expression levels, and identify hundreds of additional genes that have differences in the abundance of isoform-specific

Sequence-Specific Model for Peptide Retention Time Prediction in Strong Cation Exchange Chromatography.

Science.gov (United States)

Gussakovsky, Daniel; Neustaeter, Haley; Spicer, Victor; Krokhin, Oleg V

2017-11-07

The development of a peptide retention prediction model for strong cation exchange (SCX) separation on a Polysulfoethyl A column is reported. Off-line 2D LC-MS/MS analysis (SCX-RPLC) of S. cerevisiae whole cell lysate was used to generate a retention dataset of ∼30 000 peptides, sufficient for identifying the major sequence-specific features of peptide retention mechanisms in SCX. In contrast to RPLC/hydrophilic interaction liquid chromatography (HILIC) separation modes, where retention is driven by hydrophobic/hydrophilic contributions of all individual residues, SCX interactions depend mainly on peptide charge (number of basic residues at acidic pH) and size. An additive model (incorporating the contributions of all 20 residues into the peptide retention) combined with a peptide length correction produces a 0.976 R 2 value prediction accuracy, significantly higher than the additive models for either HILIC or RPLC. Position-dependent effects on peptide retention for different residues were driven by the spatial orientation of tryptic peptides upon interaction with the negatively charged surface functional groups. The positively charged N-termini serve as a primary point of interaction. For example, basic residues (Arg, His, Lys) increase peptide retention when located closer to the N-terminus. We also found that hydrophobic interactions, which could lead to a mixed-mode separation mechanism, are largely suppressed at 20-30% of acetonitrile in the eluent. The accuracy of the final Sequence-Specific Retention Calculator (SSRCalc) SCX model (∼0.99 R 2 value) exceeds all previously reported predictors for peptide LC separations. This also provides a solid platform for method development in 2D LC-MS protocols in proteomics and peptide retention prediction filtering of false positive identifications.

Enactment versus observation: item-specific and relational processing in goal-directed action sequences (and lists of single actions.

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Janette Schult

Full Text Available What are the memory-related consequences of learning actions (such as "apply the patch" by enactment during study, as compared to action observation? Theories converge in postulating that enactment encoding increases item-specific processing, but not the processing of relational information. Typically, in the laboratory enactment encoding is studied for lists of unrelated single actions in which one action execution has no overarching purpose or relation with other actions. In contrast, real-life actions are usually carried out with the intention to achieve such a purpose. When actions are embedded in action sequences, relational information provides efficient retrieval cues. We contrasted memory for single actions with memory for action sequences in three experiments. We found more reliance on relational processing for action-sequences than single actions. To what degree can this relational information be used after enactment versus after the observation of an actor? We found indicators of superior relational processing after observation than enactment in ordered pair recall (Experiment 1A and in emerging subjective organization of repeated recall protocols (recall runs 2-3, Experiment 2. An indicator of superior item-specific processing after enactment compared to observation was recognition (Experiment 1B, Experiment 2. Similar net recall suggests that observation can be as good a learning strategy as enactment. We discuss possible reasons why these findings only partly converge with previous research and theorizing.

RNA-Mediated Gene Duplication and Retroposons: Retrogenes, LINEs, SINEs, and Sequence Specificity

Science.gov (United States)

2013-01-01

A substantial number of “retrogenes” that are derived from the mRNA of various intron-containing genes have been reported. A class of mammalian retroposons, long interspersed element-1 (LINE1, L1), has been shown to be involved in the reverse transcription of retrogenes (or processed pseudogenes) and non-autonomous short interspersed elements (SINEs). The 3′-end sequences of various SINEs originated from a corresponding LINE. As the 3′-untranslated regions of several LINEs are essential for retroposition, these LINEs presumably require “stringent” recognition of the 3′-end sequence of the RNA template. However, the 3′-ends of mammalian L1s do not exhibit any similarity to SINEs, except for the presence of 3′-poly(A) repeats. Since the 3′-poly(A) repeats of L1 and Alu SINE are critical for their retroposition, L1 probably recognizes the poly(A) repeats, thereby mobilizing not only Alu SINE but also cytosolic mRNA. Many flowering plants only harbor L1-clade LINEs and a significant number of SINEs with poly(A) repeats, but no homology to the LINEs. Moreover, processed pseudogenes have also been found in flowering plants. I propose that the ancestral L1-clade LINE in the common ancestor of green plants may have recognized a specific RNA template, with stringent recognition then becoming relaxed during the course of plant evolution. PMID:23984183

Polyvinyl-alcohol-based magnetic beads for rapid and efficient separation of specific or unspecific nucleic acid sequences

International Nuclear Information System (INIS)

Oster, J.; Parker, Jeffrey; Brassard, Lothar

2001-01-01

The versatile application of polyvinyl-alcohol-based magnetic M-PVA beads is demonstrated in the separation of genomic DNA, sequence specific nucleic acid purification, and binding of bacteria for subsequent DNA extraction and detection. It is shown that nucleic acids can be obtained in high yield and purity using M-PVA beads, making sample preparation efficient, fast and highly adaptable for automation processes

Minimum information about a marker gene sequence (MIMARKS) and minimum information about any (x) sequence (MIxS) specifications

DEFF Research Database (Denmark)

Yilmaz, Pelin; Kottmann, Renzo; Field, Dawn

2011-01-01

Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences--the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The 'environment...

Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy

Science.gov (United States)

Wong, Michael; Daly, Seth M.; Greenberg, David E.; Toprak, Erdal

2016-01-01

The lack of effective and well-tolerated therapies against antibiotic-resistant bacteria is a global public health problem leading to prolonged treatment and increased mortality. To improve the efficacy of existing antibiotic compounds, we introduce a new method for strategically inducing antibiotic hypersensitivity in pathogenic bacteria. Following the systematic verification that the AcrAB-TolC efflux system is one of the major determinants of the intrinsic antibiotic resistance levels in Escherichia coli, we have developed a short antisense oligomer designed to inhibit the expression of acrA and increase antibiotic susceptibility in E. coli. By employing this strategy, we can inhibit E. coli growth using 2- to 40-fold lower antibiotic doses, depending on the antibiotic compound utilized. The sensitizing effect of the antisense oligomer is highly specific to the targeted gene’s sequence, which is conserved in several bacterial genera, and the oligomer does not have any detectable toxicity against human cells. Finally, we demonstrate that antisense oligomers improve the efficacy of antibiotic combinations, allowing the combined use of even antagonistic antibiotic pairs that are typically not favored due to their reduced activities. PMID:27631336

Mouse Nkrp1-Clr gene cluster sequence and expression analyses reveal conservation of tissue-specific MHC-independent immunosurveillance.

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Qiang Zhang

Full Text Available The Nkrp1 (Klrb1-Clr (Clec2 genes encode a receptor-ligand system utilized by NK cells as an MHC-independent immunosurveillance strategy for innate immune responses. The related Ly49 family of MHC-I receptors displays extreme allelic polymorphism and haplotype plasticity. In contrast, previous BAC-mapping and aCGH studies in the mouse suggest the neighboring and related Nkrp1-Clr cluster is evolutionarily stable. To definitively compare the relative evolutionary rate of Nkrp1-Clr vs. Ly49 gene clusters, the Nkrp1-Clr gene clusters from two Ly49 haplotype-disparate inbred mouse strains, BALB/c and 129S6, were sequenced. Both Nkrp1-Clr gene cluster sequences are highly similar to the C57BL/6 reference sequence, displaying the same gene numbers and order, complete pseudogenes, and gene fragments. The Nkrp1-Clr clusters contain a strikingly dissimilar proportion of repetitive elements compared to the Ly49 clusters, suggesting that certain elements may be partly responsible for the highly disparate Ly49 vs. Nkrp1 evolutionary rate. Focused allelic polymorphisms were found within the Nkrp1b/d (Klrb1b, Nkrp1c (Klrb1c, and Clr-c (Clec2f genes, suggestive of possible immune selection. Cell-type specific transcription of Nkrp1-Clr genes in a large panel of tissues/organs was determined. Clr-b (Clec2d and Clr-g (Clec2i showed wide expression, while other Clr genes showed more tissue-specific expression patterns. In situ hybridization revealed specific expression of various members of the Clr family in leukocytes/hematopoietic cells of immune organs, various tissue-restricted epithelial cells (including intestinal, kidney tubular, lung, and corneal progenitor epithelial cells, as well as myocytes. In summary, the Nkrp1-Clr gene cluster appears to evolve more slowly relative to the related Ly49 cluster, and likely regulates innate immunosurveillance in a tissue-specific manner.

1H NMR studies of plastocyanin from Scenedesmus obliquus: Complete sequence-specific assignment, secondary structure analysis, and global fold

International Nuclear Information System (INIS)

Moore, J.M.; Chazin, W.J.; Wright, P.E.; Powls, R.

1988-01-01

Two-dimensional 1 H NMR methods have been used to make sequence-specific resonance assignments for the 97 amino acid residues of the plastocyanin from the green alga Scenedesmus obliquus. Assignments were obtained for all backbone protons and the majority of the side-chain protons. Spin system identification relied heavily on the observation of relayed connectivities to the backbone amide proton. Sequence-specific assignments were made by using the sequential assignment procedure. During this process, an extra valine residue was identified that had not been detected in the original amino acid sequence. Elements of regular secondary structure were identified from characteristic NOE connectivities between backbone protons, coupling constant values, and the observation of slowly exchanging amide protons. The protein in solution contains eight β-strands, one short segment of helix, five reverse turns, and five loops. The β-strands may be arranged into two βsheets on the basis of extensive cross-strand NOE connectivities. The chain-folding topology determined from the NMR experiments is that of a Greek key β-barrel and is similar to that observed for French bean plastocyanin in solution and poplar plastocyanin in the crystalline state. While the overall structures are similar, several differences in local structure between the S. obliquus and higher plant plastocyanins have been identified

Event-specific qualitative and quantitative PCR detection of the GMO carnation (Dianthus caryophyllus) variety Moonlite based upon the 5'-transgene integration sequence.

Science.gov (United States)

Li, P; Jia, J W; Jiang, L X; Zhu, H; Bai, L; Wang, J B; Tang, X M; Pan, A H

2012-04-27

To ensure the implementation of genetically modified organism (GMO)-labeling regulations, an event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic carnation variety Moonlite. The 5'-transgene integration sequence was isolated by thermal asymmetric interlaced PCR. Based upon the 5'-transgene integration sequence, the event-specific primers and TaqMan probe were designed to amplify the fragments, which spanned the exogenous DNA and carnation genomic DNA. Qualitative and quantitative PCR assays were developed employing the designed primers and probe. The detection limit of the qualitative PCR assay was 0.05% for Moonlite in 100 ng total carnation genomic DNA, corresponding to about 79 copies of the carnation haploid genome; the limit of detection and quantification of the quantitative PCR assay were estimated to be 38 and 190 copies of haploid carnation genomic DNA, respectively. Carnation samples with different contents of genetically modified components were quantified and the bias between the observed and true values of three samples were lower than the acceptance criterion (GMO detection method. These results indicated that these event-specific methods would be useful for the identification and quantification of the GMO carnation Moonlite.

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies

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Katherine E. Harris

2018-04-01

Full Text Available We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1. This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Diagnostic accuracy of unenhanced, contrast-enhanced perfusion and angiographic MRI sequences for pulmonary embolism diagnosis: results of independent sequence readings

Energy Technology Data Exchange (ETDEWEB)

Revel, Marie Pierre [Hopital Europeen Georges Pompidou, APHP, Departments of Radiology, Paris (France); Universite Paris Descartes Sorbonne Paris Cite, Paris (France); Hotel-Dieu, Service de Radiologie, Paris (France); Sanchez, Olivier; Meyer, Guy [Hopital Europeen Georges Pompidou, APHP, Respiratory and intensive care and, Paris (France); Universite Paris Descartes Sorbonne Paris Cite, Paris (France); INSERM Unite 765, Paris (France); Lefort, Catherine; Couchon, Sophie; Hernigou, Anne; Frija, Guy [Hopital Europeen Georges Pompidou, APHP, Departments of Radiology, Paris (France); Niarra, Ralph [Hopital Europeen Georges Pompidou, APHP, Clinical Epidemiology, Paris (France); Universite Paris Descartes Sorbonne Paris Cite, Paris (France); Chatellier, Gilles [Hopital Europeen Georges Pompidou, APHP, Clinical Epidemiology, Paris (France); Universite Paris Descartes Sorbonne Paris Cite, Paris (France); INSERM CIC-EC E4, Paris (France)

2013-09-15

To independently evaluate unenhanced, contrast-enhanced perfusion and angiographic MR sequences for pulmonary embolism (PE) diagnosis. Prospective investigation, including 274 patients who underwent perfusion, unenhanced 2D steady-state-free-precession (SSFP) and contrast-enhanced 3D angiographic MR sequences on a 1.5-T unit, in addition to CTA (CT angiography). Two independent readers evaluated each sequence independently in random order. Sensitivity, specificity, predictive values and inter-reader agreement were calculated for each sequence, excluding sequences judged inconclusive. Sensitivity was also calculated according to PE location. Contrast-enhanced angiographic sequences showed the highest sensitivity (82.9 and 89.7 %, reader 1 and reader 2, respectively), specificity (98.5 and 100 %) and agreement (kappa value 0.77). Unenhanced angiographic sequences, although less sensitive overall (68.7 and 76.4 %), were sensitive for the detection of proximal PE (92.7 and 100 %) and showed high specificity (96.1 and 99.1 %) and good agreement (kappa value 0.62). Perfusion sequences showed lower sensitivity (75.0 and 79.3 %), specificity (84.8 and 89.7 %) and agreement (kappa value 0.51), and a negative predictive value of 84.8 % at best. Compared with contrast-enhanced angiographic sequences, unenhanced sequences demonstrate lower sensitivity, except for proximal PE, but high specificity and agreement. The negative predictive value of perfusion sequences was insufficient to safely rule out PE. (orig.)

Development of a Species-specific PCR Assay for Three Xanthomonas Species, Causing Bulb and Flower Diseases, Based on Their Genome Sequences

Directory of Open Access Journals (Sweden)

Chang-Gi Back

2015-09-01

Full Text Available In this study, we developed a species-specific PCR assay for rapid and accurate detection of three Xanthomonas species, X. axonopodis pv. poinsettiicola (XAP, X. hyacinthi (XH and X. campestris pv. zantedeschiae (XCZ, based on their draft genome sequences. XAP, XH and XCZ genomes consist of single chromosomes that contain 5,221, 4,395 and 7,986 protein coding genes, respectively. Species-specific primers were designed from variable regions of the draft genome sequence data and assessed by a PCR-based detection method. These primers were also tested for specificity against 17 allied Xanthomonas species as well as against the host DNA and the microbial community of the host surface. Three primer sets were found to be very specific and no amplification product was obtained with the host DNA and the microbial community of the host surface. In addition, a detection limit of 1 pg/μl per PCR reaction was detected when these primer sets were used to amplify corresponding bacterial DNAs. Therefore, these primer sets and the developed species-specific PCR assay represent a valuable, sensitive, and rapid diagnostic tool that can be used to detect three specific pathogens at early stages of infection and may help control diseases.

Bellerophon: a program to detect chimeric sequences in multiple sequence alignments.

Science.gov (United States)

Huber, Thomas; Faulkner, Geoffrey; Hugenholtz, Philip

2004-09-22

Bellerophon is a program for detecting chimeric sequences in multiple sequence datasets by an adaption of partial treeing analysis. Bellerophon was specifically developed to detect 16S rRNA gene chimeras in PCR-clone libraries of environmental samples but can be applied to other nucleotide sequence alignments. Bellerophon is available as an interactive web server at http://foo.maths.uq.edu.au/~huber/bellerophon.pl

Structure and Sequence Analyses of Clustered Protocadherins Reveal Antiparallel Interactions that Mediate Homophilic Specificity.

Science.gov (United States)

Nicoludis, John M; Lau, Sze-Yi; Schärfe, Charlotta P I; Marks, Debora S; Weihofen, Wilhelm A; Gaudet, Rachelle

2015-11-03

Clustered protocadherin (Pcdh) proteins mediate dendritic self-avoidance in neurons via specific homophilic interactions in their extracellular cadherin (EC) domains. We determined crystal structures of EC1-EC3, containing the homophilic specificity-determining region, of two mouse clustered Pcdh isoforms (PcdhγA1 and PcdhγC3) to investigate the nature of the homophilic interaction. Within the crystal lattices, we observe antiparallel interfaces consistent with a role in trans cell-cell contact. Antiparallel dimerization is supported by evolutionary correlations. Two interfaces, located primarily on EC2-EC3, involve distinctive clustered Pcdh structure and sequence motifs, lack predicted glycosylation sites, and contain residues highly conserved in orthologs but not paralogs, pointing toward their biological significance as homophilic interaction interfaces. These two interfaces are similar yet distinct, reflecting a possible difference in interaction architecture between clustered Pcdh subfamilies. These structures initiate a molecular understanding of clustered Pcdh assemblies that are required to produce functional neuronal networks. Copyright © 2015 Elsevier Ltd. All rights reserved.

In silico site-directed mutagenesis informs species-specific predictions of chemical susceptibility derived from the Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool

Science.gov (United States)

The Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool was developed to address needs for rapid, cost effective methods of species extrapolation of chemical susceptibility. Specifically, the SeqAPASS tool compares the primary sequence (Level 1), functiona...

Endoplasmic reticulum targeting sequence enhances HBV-specific cytotoxic T lymphocytes induced by a CTL epitope-based DNA vaccine

International Nuclear Information System (INIS)

Xu Wei; Chu Yiwei; Zhang Ruihua; Xu Huanbin; Wang Ying; Xiong Sidong

2005-01-01

CD8 + T cells play a critical role in protective immunity against Hepatitis B Virus (HBV). Epitope-based DNA vaccines expressing HBV-dominant CTL epitopes can be used as candidate vaccines capable of inducing cytotoxic T Lymphocytes (CTL) responses. A plasmid DNA encoding a CTL epitope of HBV core antigen, HBc 18-27 , was constructed. Intramuscular immunization of C57BL/6 mice with this DNA vaccine resulted in successful induction of HBV-specific CTL responses. In order to promote transportation of the peptide into endoplasmic reticulum (ER) to bind to MHC class I molecules for optimal class I antigen presentation, an ER targeting sequence (ERTS) was fused with the C 18-27 encoding gene. ERTS fusion significantly enhanced specific CD8 + T cell responses in terms of CTL cytolysis as well as IFN-γ secretion. This enhancement was correlated with promoted epitope presentation on target cell surface. We report here an enhanced immunogenicity of an epitope-based DNA vaccine using an ER targeting signal sequence, which has significant implications for future design of therapeutic HBV vaccine

Leishmania-specific surface antigens show sub-genus sequence variation and immune recognition.

Directory of Open Access Journals (Sweden)

Daniel P Depledge

2010-09-01

Full Text Available A family of hydrophilic acylated surface (HASP proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic and intracellular (amastigote stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia have lost HASP genes from their genomes.We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia species, L. (V. braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L. mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o HASPs are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family.These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are

Differentiation of Actinobacillus pleuropneumoniae strains by sequence analysis of 16S rDNA and ribosomal intergenic regions, and development of a species specific oligonucleotide for in situ detection

DEFF Research Database (Denmark)

Fussing, Vivian; Paster, Bruce J.; Dewhirst, Floyd E.

1998-01-01

. The larger RIS's were different between the 3 species tested. The sequence of the 16S ribosomal gene was determined for 8 serotypes of A. pleuropneumoniae. These sequences showed only minor base differences, indicating a close genetic relatedness of these serotypes within the species. An oligonucleotide DNA...... probe designed from the 16S rRNA gene sequence of A. pleuropneumoniae was specific for all strains of the target species and did not cross react with A. lignieresii, the closest known relative of A. pleuropneumoniae. This species-specific DNA probe labeled with fluorescein was used for in situ......The aims of this study were to characterize and determine intraspecies and interspecies relatedness of Actinobacillus pleuropneumoniae to Actinobacillus lignieresii and Actinobacillus suis by sequence analysis of the ribosomal operon and to find a species-specific area for in situ detection of A...

Improved annotation of 3' untranslated regions and complex loci by combination of strand-specific direct RNA sequencing, RNA-Seq and ESTs.

Directory of Open Access Journals (Sweden)

Nicholas J Schurch

Full Text Available The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct and complete annotation in addition to the underlying genomic sequence is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3' untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3' polyadenylation sites to within +/- 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1 gene and 3' UTR re-annotation (including extension of one 3' UTR by 5.9 kb; (2 disentangling of gene expression in complex regions; (3 clearer interpretation of small RNA expression and (4 identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental data.

Impact of cadmium, cobalt and nickel on sequence-specific DNA binding of p63 and p73 in vitro and in cells

International Nuclear Information System (INIS)

Adámik, Matej; Bažantová, Pavla; Navrátilová, Lucie; Polášková, Alena; Pečinka, Petr; Holaňová, Lucie; Tichý, Vlastimil; Brázdová, Marie

2015-01-01

Highlights: • DNA binding of p53 family core domains is inhibited by cadmium, cobalt and nickel. • Binding to DNA protects p53 family core domains from metal induced inhibition. • Cadmium, cobalt and nickel induced inhibition was reverted by EDTA in vitro. - Abstract: Site-specific DNA recognition and binding activity belong to common attributes of all three members of tumor suppressor p53 family proteins: p53, p63 and p73. It was previously shown that heavy metals can affect p53 conformation, sequence-specific binding and suppress p53 response to DNA damage. Here we report for the first time that cadmium, nickel and cobalt, which have already been shown to disturb various DNA repair mechanisms, can also influence p63 and p73 sequence-specific DNA binding activity and transactivation of p53 family target genes. Based on results of electrophoretic mobility shift assay and luciferase reporter assay, we conclude that cadmium inhibits sequence-specific binding of all three core domains to p53 consensus sequences and abolishes transactivation of several promoters (e.g. BAX and MDM2) by 50 μM concentrations. In the presence of specific DNA, all p53 family core domains were partially protected against loss of DNA binding activity due to cadmium treatment. Effective cadmium concentration to abolish DNA–protein interactions was about two times higher for p63 and p73 proteins than for p53. Furthermore, we detected partial reversibility of cadmium inhibition for all p53 family members by EDTA. DTT was able to reverse cadmium inhibition only for p53 and p73. Nickel and cobalt abolished DNA–p53 interaction at sub-millimolar concentrations while inhibition of p63 and p73 DNA binding was observed at millimolar concentrations. In summary, cadmium strongly inhibits p53, p63 and p73 DNA binding in vitro and in cells in comparison to nickel and cobalt. The role of cadmium inhibition of p53 tumor suppressor family in carcinogenesis is discussed

Impact of cadmium, cobalt and nickel on sequence-specific DNA binding of p63 and p73 in vitro and in cells

Energy Technology Data Exchange (ETDEWEB)

Adámik, Matej [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Bažantová, Pavla [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 701 03 Ostrava (Czech Republic); Navrátilová, Lucie; Polášková, Alena [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Pečinka, Petr [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 701 03 Ostrava (Czech Republic); Holaňová, Lucie [Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 61242 Brno (Czech Republic); Tichý, Vlastimil [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Brázdová, Marie, E-mail: maruska@ibp.cz [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 61242 Brno (Czech Republic)

2015-01-02

Highlights: • DNA binding of p53 family core domains is inhibited by cadmium, cobalt and nickel. • Binding to DNA protects p53 family core domains from metal induced inhibition. • Cadmium, cobalt and nickel induced inhibition was reverted by EDTA in vitro. - Abstract: Site-specific DNA recognition and binding activity belong to common attributes of all three members of tumor suppressor p53 family proteins: p53, p63 and p73. It was previously shown that heavy metals can affect p53 conformation, sequence-specific binding and suppress p53 response to DNA damage. Here we report for the first time that cadmium, nickel and cobalt, which have already been shown to disturb various DNA repair mechanisms, can also influence p63 and p73 sequence-specific DNA binding activity and transactivation of p53 family target genes. Based on results of electrophoretic mobility shift assay and luciferase reporter assay, we conclude that cadmium inhibits sequence-specific binding of all three core domains to p53 consensus sequences and abolishes transactivation of several promoters (e.g. BAX and MDM2) by 50 μM concentrations. In the presence of specific DNA, all p53 family core domains were partially protected against loss of DNA binding activity due to cadmium treatment. Effective cadmium concentration to abolish DNA–protein interactions was about two times higher for p63 and p73 proteins than for p53. Furthermore, we detected partial reversibility of cadmium inhibition for all p53 family members by EDTA. DTT was able to reverse cadmium inhibition only for p53 and p73. Nickel and cobalt abolished DNA–p53 interaction at sub-millimolar concentrations while inhibition of p63 and p73 DNA binding was observed at millimolar concentrations. In summary, cadmium strongly inhibits p53, p63 and p73 DNA binding in vitro and in cells in comparison to nickel and cobalt. The role of cadmium inhibition of p53 tumor suppressor family in carcinogenesis is discussed.

Formal Specification and Description Language and Message Sequence Chart to Model and Validate Session Initiation Protocol Services

OpenAIRE

Sa'ed Abed; Mohammad H. Al Shayeji; Ovais Ahmed; Sahel Alouneh

2016-01-01

Session Initiation Protocol (SIP) is a signaling layer protocol for building, adjusting and ending sessions among participants including Internet conferences, telephone calls and multimedia distribution. SIP facilitates user movement by proxying and forwarding requests to the present location of the user. In this paper, we provide a formal Specification and Description Language (SDL) and Message Sequence Chart (MSC) to model and define the Internet Engineering Task Force (IETF) SIP protocol a...

Use of next generation sequencing data to develop a qPCR method for specific detection of EU-unauthorized genetically modified Bacillus subtilis overproducing riboflavin.

Science.gov (United States)

Barbau-Piednoir, Elodie; De Keersmaecker, Sigrid C J; Delvoye, Maud; Gau, Céline; Philipp, Patrick; Roosens, Nancy H

2015-11-11

Recently, the presence of an unauthorized genetically modified (GM) Bacillus subtilis bacterium overproducing vitamin B2 in a feed additive was notified by the Rapid Alert System for Food and Feed (RASFF). This has demonstrated that a contamination by a GM micro-organism (GMM) may occur in feed additives and has confronted for the first time,the enforcement laboratories with this type of RASFF. As no sequence information of this GMM nor any specific detection or identification method was available, Next GenerationSequencing (NGS) was used to generate sequence information. However, NGS data analysis often requires appropriate tools, involving bioinformatics expertise which is not alwayspresent in the average enforcement laboratory. This hampers the use of this technology to rapidly obtain critical sequence information in order to be able to develop a specific qPCRdetection method. Data generated by NGS were exploited using a simple BLAST approach. A TaqMan® qPCR method was developed and tested on isolated bacterial strains and on the feed additive directly. In this study, a very simple strategy based on the common BLAST tools that can be used by any enforcement lab without profound bioinformatics expertise, was successfully used toanalyse the B. subtilis data generated by NGS. The results were used to design and assess a new TaqMan® qPCR method, specifically detecting this GM vitamin B2 overproducing bacterium. The method complies with EU critical performance parameters for specificity, sensitivity, PCR efficiency and repeatability. The VitB2-UGM method also could detect the B. subtilis strain in genomic DNA extracted from the feed additive, without prior culturing step. The proposed method, provides a crucial tool for specifically and rapidly identifying this unauthorized GM bacterium in food and feed additives by enforcement laboratories. Moreover, this work can be seen as a case study to substantiate how the use of NGS data can offer an added value to easily

Targeted sequencing of clade-specific markers from skin microbiomes for forensic human identification.

Science.gov (United States)

Schmedes, Sarah E; Woerner, August E; Novroski, Nicole M M; Wendt, Frank R; King, Jonathan L; Stephens, Kathryn M; Budowle, Bruce

2018-01-01

The human skin microbiome is comprised of diverse communities of bacterial, eukaryotic, and viral taxa and contributes millions of additional genes to the repertoire of human genes, affecting human metabolism and immune response. Numerous genetic and environmental factors influence the microbiome composition and as such contribute to individual-specific microbial signatures which may be exploited for forensic applications. Previous studies have demonstrated the potential to associate skin microbial profiles collected from touched items to their individual owner, mainly using unsupervised methods from samples collected over short time intervals. Those studies utilize either targeted 16S rRNA or shotgun metagenomic sequencing to characterize skin microbiomes; however, these approaches have limited species and strain resolution and susceptibility to stochastic effects, respectively. Clade-specific markers from the skin microbiome, using supervised learning, can predict individual identity using skin microbiomes from their respective donors with high accuracy. In this study the hidSkinPlex is presented, a novel targeted sequencing method using skin microbiome markers developed for human identification. The hidSkinPlex (comprised of 286 bacterial (and phage) family-, genus-, species-, and subspecies-level markers), initially was evaluated on three bacterial control samples represented in the panel (i.e., Propionibacterium acnes, Propionibacterium granulosum, and Rothia dentocariosa) to assess the performance of the multiplex. The hidSkinPlex was further evaluated for prediction purposes. The hidSkinPlex markers were used to attribute skin microbiomes collected from eight individuals from three body sites (i.e., foot (Fb), hand (Hp) and manubrium (Mb)) to their host donor. Supervised learning, specifically regularized multinomial logistic regression and 1-nearest-neighbor classification were used to classify skin microbiomes to their hosts with up to 92% (Fb), 96% (Mb

Locomotor sequence learning in visually guided walking

DEFF Research Database (Denmark)

Choi, Julia T; Jensen, Peter; Nielsen, Jens Bo

2016-01-01

walking. In addition, we determined how age (i.e., healthy young adults vs. children) and biomechanical factors (i.e., walking speed) affected the rate and magnitude of locomotor sequence learning. The results showed that healthy young adults (age 24 ± 5 years, N = 20) could learn a specific sequence...... of step lengths over 300 training steps. Younger children (age 6-10 years, N = 8) have lower baseline performance, but their magnitude and rate of sequence learning was the same compared to older children (11-16 years, N = 10) and healthy adults. In addition, learning capacity may be more limited...... to modify step length from one trial to the next. Our sequence learning paradigm is derived from the serial reaction-time (SRT) task that has been used in upper limb studies. Both random and ordered sequences of step lengths were used to measure sequence-specific and sequence non-specific learning during...

Sequence specificity of alkali-labile DNA damage photosensitized by suprofen.

Science.gov (United States)

Starrs, S M; Davies, R J

2000-09-01

On irradiation at UVB wavelengths, in aerated neutral aqueous solution, the anti-inflammatory drug suprofen (SP) photosensitizes the production of alkali-labile cleavage sites in DNA much more efficiently than direct strand breaks. It is active at submillimolar concentrations despite having no significant binding affinity for DNA. Gel sequencing studies utilizing 32P-end-labeled oligonucleotides have revealed that piperidine-sensitive lesions are formed predominantly at the positions of guanine (G) bases, with the extent of modification being UV dose- and SP concentration-dependent. Quite distinct patterns of G-specific damage are observed in single-stranded and duplex DNA molecules. The uniform attack at all G residues in single-stranded DNA, which is enhanced in D2O, is compatible with a Type-II mechanism. SP is a known generator of singlet oxygen whose participation in the reaction is supported by the effects of quenchers and scavengers. In duplex DNA, piperidine-induced cleavage occurs with high selectivity at the 5'-G of GG and (less prominently) GA doublets. This behavior is characteristic of a Type-I process involving electron transfer from DNA to photoexcited SP molecules. The ability of SP to sensitize the formation of Type-I and Type-II photo-oxidation products from 2'-deoxyguanosine attests to the feasibility of competing mechanisms in DNA.

The CRISPR Spacer Space Is Dominated by Sequences from Species-Specific Mobilomes.

Science.gov (United States)

Shmakov, Sergey A; Sitnik, Vassilii; Makarova, Kira S; Wolf, Yuri I; Severinov, Konstantin V; Koonin, Eugene V

2017-09-19

Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas) systems store the memory of past encounters with foreign DNA in unique spacers that are inserted between direct repeats in CRISPR arrays. For only a small fraction of the spacers, homologous sequences, called protospacers, are detectable in viral, plasmid, and microbial genomes. The rest of the spacers remain the CRISPR "dark matter." We performed a comprehensive analysis of the spacers from all CRISPR- cas loci identified in bacterial and archaeal genomes, and we found that, depending on the CRISPR-Cas subtype and the prokaryotic phylum, protospacers were detectable for 1% to about 19% of the spacers (~7% global average). Among the detected protospacers, the majority, typically 80 to 90%, originated from viral genomes, including proviruses, and among the rest, the most common source was genes that are integrated into microbial chromosomes but are involved in plasmid conjugation or replication. Thus, almost all spacers with identifiable protospacers target mobile genetic elements (MGE). The GC content, as well as dinucleotide and tetranucleotide compositions, of microbial genomes, their spacer complements, and the cognate viral genomes showed a nearly perfect correlation and were almost identical. Given the near absence of self-targeting spacers, these findings are most compatible with the possibility that the spacers, including the dark matter, are derived almost completely from the species-specific microbial mobilomes. IMPORTANCE The principal function of CRISPR-Cas systems is thought to be protection of bacteria and archaea against viruses and other parasitic genetic elements. The CRISPR defense function is mediated by sequences from parasitic elements, known as spacers, that are inserted into CRISPR arrays and then transcribed and employed as guides to identify and inactivate the cognate parasitic genomes. However, only a small fraction of the CRISPR spacers

SATB1 regulates SPARC expression in K562 cell line through binding to a specific sequence in the third intron

International Nuclear Information System (INIS)

Li, K.; Cai, R.; Dai, B.B.; Zhang, X.Q.; Wang, H.J.; Ge, S.F.; Xu, W.R.; Lu, J.

2007-01-01

Special AT-rich binding protein 1 (SATB1), a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, tethers to a specific DNA sequence and regulates gene expression through chromatin remodeling and HDAC (histone deacetylase complex) recruitment. In this study, a SATB1 eukaryotic expression plasmid was transfected into the human erythroleukemia K562 cell line and individual clones that stably over-expressed the SATB1 protein were isolated. Microarray analysis revealed that hundreds of genes were either up- or down-regulated in the SATB1 over-expressing K562 cell lines. One of these was the extra-cellular matrix glycoprotein, SPARC (human secreted protein acidic and rich in cysteine). siRNA knock-down of SATB1 also reduced SPARC expression, which was consistent with elevated SPARC levels in the SATB1 over-expressing cell line. Bioinformatics software Mat-inspector showed that a 17 bp DNA sequence in the third intron of SPARC possessed a high potential for SATB1 binding; a finding confirmed by Chromatin immunoprecipitation (ChIP) with anti-SATB1 antibody. Our results show for the first time that forced-expression of SATB1 in K562 cells triggers SPARC up-regulation by binding to a 17 bp DNA sequence in the third intron

Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

Directory of Open Access Journals (Sweden)

Knut Rudi

2003-01-01

Full Text Available Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

Concordance of the ForenSeq™ system and characterisation of sequence-specific autosomal STR alleles across two major population groups.

Science.gov (United States)

Devesse, Laurence; Ballard, David; Davenport, Lucinda; Riethorst, Immy; Mason-Buck, Gabriella; Syndercombe Court, Denise

2018-05-01

By using sequencing technology to genotype loci of forensic interest it is possible to simultaneously target autosomal, X and Y STRs as well as identity, ancestry and phenotypic informative SNPs, resulting in a breadth of data obtained from a single run that is considerable when compared to that generated with standard technologies. It is important however that this information aligns with the genotype data currently obtained using commercially available kits for CE-based investigations such that results are compatible with existing databases and hence can be of use to the forensic community. In this work, 400 samples were typed using commercially available STR kits and CE, as well as using the Ilumina ForenSeq™ DNA Signature Prep Kit and MiSeq ® FGx to assess concordance of autosomal STRs and population variability. Results show a concordance rate between the two technologies exceeding 99.98% while numerous novel sequence based alleles are described. In order to make use of the sequence variation observed, sequence specific allele frequencies were generated for White British and British Chinese populations. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27.

Science.gov (United States)

Kim, Min Jung; Hwang, Kyung Hwan; Lee, Young-Seok; Park, Jae-Yoon; Kook, Joong-Ki

2011-03-01

The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Sequence specific DNA binding by P53 is enhanced by ionizing radiation and is mediated via DNA-PK activity

International Nuclear Information System (INIS)

Kachnic, L.A.; Wunsch, H.; Mekeel, K.L.; De Frank, J.S.; Powell, S.N.

1996-01-01

Purpose: P53 is known to be involved in the cellular response to DNA damage. It mediates many of its effects by acting as a transcription factor via sequence-specific DNA binding. The half-life of p53 is prolonged following DNA damage, and this results in elevated levels of p53 for a period of 2-8 hours. The increase in p53 is often relatively small, but this produces significant stimulation of a downstream gene such as p21(WAF1/cip1). We investigated post-translational modification of p53 following ionizing radiation damage. Materials and Methods: The response of normal Balb-C mouse fibroblasts (FC) to ionizing radiation (IR, 8 Gy) was measured at 0,3,6,9 and 24 hours, by the levels of p53, p21, flow cytometry and the electrophoretic mobility shift assay (EMSA). EMSA utilized a 26 bp consensus sequence end-labeled oligonucleotide to measure sequence-specific p53 binding. P53 specificity was confirmed by an enhanced mobility shift (retardation) when using p53 antibody. Comparison was made with scid fibroblasts (FS) and FC cells transfected with a plasmid (CX3) containing mutant p53 (alanine-143) or infected with a retrovirus containing the E6 protein of human papilloma virus type 16. Results: The response of p53 to DNA damage shows a 3-fold increase at 3-6 hours, and was not significantly different between FC and FS. FC-CX3 showed detectable basal levels of p53, and a 2-fold further induction of p53 after IR. FC-E6 showed no detectable levels of p53 before or after IR. No induction of p21 or G1/S arrest was seen in FC-CX3 or FC-E6, as has been observed previously. The induction of p21 in FS cells was attenuated and delayed: a 2-3-fold increase seen maximally at 9 hours, compared with a 5-fold increase seen maximally at 3-6 hours in FC cells. The accumulation of cells at the G1/S junction after IR showed the same kinetics as p21 induction: the peak of cells in G1 occurs at 3-6 hours in FC, but not until 9-24 hours in FS. The response is reminiscent of that seen in

Bamgineer: Introduction of simulated allele-specific copy number variants into exome and targeted sequence data sets.

Science.gov (United States)

Samadian, Soroush; Bruce, Jeff P; Pugh, Trevor J

2018-03-01

Somatic copy number variations (CNVs) play a crucial role in development of many human cancers. The broad availability of next-generation sequencing data has enabled the development of algorithms to computationally infer CNV profiles from a variety of data types including exome and targeted sequence data; currently the most prevalent types of cancer genomics data. However, systemic evaluation and comparison of these tools remains challenging due to a lack of ground truth reference sets. To address this need, we have developed Bamgineer, a tool written in Python to introduce user-defined haplotype-phased allele-specific copy number events into an existing Binary Alignment Mapping (BAM) file, with a focus on targeted and exome sequencing experiments. As input, this tool requires a read alignment file (BAM format), lists of non-overlapping genome coordinates for introduction of gains and losses (bed file), and an optional file defining known haplotypes (vcf format). To improve runtime performance, Bamgineer introduces the desired CNVs in parallel using queuing and parallel processing on a local machine or on a high-performance computing cluster. As proof-of-principle, we applied Bamgineer to a single high-coverage (mean: 220X) exome sequence file from a blood sample to simulate copy number profiles of 3 exemplar tumors from each of 10 tumor types at 5 tumor cellularity levels (20-100%, 150 BAM files in total). To demonstrate feasibility beyond exome data, we introduced read alignments to a targeted 5-gene cell-free DNA sequencing library to simulate EGFR amplifications at frequencies consistent with circulating tumor DNA (10, 1, 0.1 and 0.01%) while retaining the multimodal insert size distribution of the original data. We expect Bamgineer to be of use for development and systematic benchmarking of CNV calling algorithms by users using locally-generated data for a variety of applications. The source code is freely available at http://github.com/pughlab/bamgineer.

Bamgineer: Introduction of simulated allele-specific copy number variants into exome and targeted sequence data sets.

Directory of Open Access Journals (Sweden)

Soroush Samadian

2018-03-01

Full Text Available Somatic copy number variations (CNVs play a crucial role in development of many human cancers. The broad availability of next-generation sequencing data has enabled the development of algorithms to computationally infer CNV profiles from a variety of data types including exome and targeted sequence data; currently the most prevalent types of cancer genomics data. However, systemic evaluation and comparison of these tools remains challenging due to a lack of ground truth reference sets. To address this need, we have developed Bamgineer, a tool written in Python to introduce user-defined haplotype-phased allele-specific copy number events into an existing Binary Alignment Mapping (BAM file, with a focus on targeted and exome sequencing experiments. As input, this tool requires a read alignment file (BAM format, lists of non-overlapping genome coordinates for introduction of gains and losses (bed file, and an optional file defining known haplotypes (vcf format. To improve runtime performance, Bamgineer introduces the desired CNVs in parallel using queuing and parallel processing on a local machine or on a high-performance computing cluster. As proof-of-principle, we applied Bamgineer to a single high-coverage (mean: 220X exome sequence file from a blood sample to simulate copy number profiles of 3 exemplar tumors from each of 10 tumor types at 5 tumor cellularity levels (20-100%, 150 BAM files in total. To demonstrate feasibility beyond exome data, we introduced read alignments to a targeted 5-gene cell-free DNA sequencing library to simulate EGFR amplifications at frequencies consistent with circulating tumor DNA (10, 1, 0.1 and 0.01% while retaining the multimodal insert size distribution of the original data. We expect Bamgineer to be of use for development and systematic benchmarking of CNV calling algorithms by users using locally-generated data for a variety of applications. The source code is freely available at http://github.com/pughlab/bamgineer.

Specific nuclear localizing sequence directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner.

Science.gov (United States)

Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

2012-01-01

Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

The value of qualitative and quantitative assessment of lesion to cerebral cortex signal ratio on double inversion recovery sequence in the differentiation of demyelinating plaques from non-specific T2 hyperintensities

Energy Technology Data Exchange (ETDEWEB)

Hamcan, Salih; Battal, Bilal; Akgun, Veysel; Sari, Sebahattin; Tasar, Mustafa [Gulhane Military Medical School, Department of Radiology, Etlik, Ankara (Turkey); Oz, Oguzhan; Tasdemir, Serdar [Gulhane Military Medical School, Department of Neurology, Ankara (Turkey); Bozkurt, Yalcin [Golcuk Military Hospital, Department of Radiology, Kocaeli (Turkey)

2017-02-15

To assess the usefulness of the visual assessment and to determine diagnostic value of the lesion-to-cerebral cortex signal ratio (LCSR) measurement in the differentiation of demyelinating plaques and non-specific T2 hyperintensities on double inversion recovery (DIR) sequence. DIR and fluid-attenuated inversion recovery (FLAIR) sequences of 25 clinically diagnosed multiple sclerosis (MS) patients and 25 non-MS patients with non-specific T2-hyperintense lesions were evaluated visually and LCSRs were measured by two observers independently. On DIR sequence, the calculated mean LCSR ± SD for demyelinating plaques and non-specific T2-hyperintense lesions were 1.60 ± 0.26 and 0.75 ± 0.19 for observer1, and 1.61 ± 0.27 and 0.74 ± 0.19 for observer2. LCSRs of demyelinating plaques were significantly higher than other non-specific T2-hyperintense lesions on DIR sequence. By using the visual assessment demyelinating plaques were differentiated from non-specific T2-hyperintensities with 92.8 % sensitivity, 97.5 % specificity and 95.1 % accuracy for observer1 and 92.8 % sensitivity, 95 % specificity and 93.9 % accuracy for observer2. Visual assessment and LCSR measurement on DIR sequence seems to be useful for differentiating demyelinating MS plaques from supratentorial non-specific T2 hyperintensities. This feature can be used for diagnosis of MS particularly in patients with only supratentorial T2-hyperintense lesions who are categorized as radiologically possible MS. (orig.)

SOLiD sequencing of four Vibrio vulnificus genomes enables comparative genomic analysis and identification of candidate clade-specific virulence genes

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Telonis-Scott Marina

2010-09-01

Full Text Available Abstract Background Vibrio vulnificus is the leading cause of reported death from consumption of seafood in the United States. Despite several decades of research on molecular pathogenesis, much remains to be learned about the mechanisms of virulence of this opportunistic bacterial pathogen. The two complete and annotated genomic DNA sequences of V. vulnificus belong to strains of clade 2, which is the predominant clade among clinical strains. Clade 2 strains generally possess higher virulence potential in animal models of disease compared with clade 1, which predominates among environmental strains. SOLiD sequencing of four V. vulnificus strains representing different clades (1 and 2 and biotypes (1 and 2 was used for comparative genomic analysis. Results Greater than 4,100,000 bases were sequenced of each strain, yielding approximately 100-fold coverage for each of the four genomes. Although the read lengths of SOLiD genomic sequencing were only 35 nt, we were able to make significant conclusions about the unique and shared sequences among the genomes, including identification of single nucleotide polymorphisms. Comparative analysis of the newly sequenced genomes to the existing reference genomes enabled the identification of 3,459 core V. vulnificus genes shared among all six strains and 80 clade 2-specific genes. We identified 523,161 SNPs among the six genomes. Conclusions We were able to glean much information about the genomic content of each strain using next generation sequencing. Flp pili, GGDEF proteins, and genomic island XII were identified as possible virulence factors because of their presence in virulent sequenced strains. Genomic comparisons also point toward the involvement of sialic acid catabolism in pathogenesis.

A strand specific high resolution normalization method for chip-sequencing data employing multiple experimental control measurements

DEFF Research Database (Denmark)

Enroth, Stefan; Andersson, Claes; Andersson, Robin

2012-01-01

High-throughput sequencing is becoming the standard tool for investigating protein-DNA interactions or epigenetic modifications. However, the data generated will always contain noise due to e.g. repetitive regions or non-specific antibody interactions. The noise will appear in the form of a backg......, the background is only used to adjust peak calling and not as a pre-processing step that aims at discerning the signal from the background noise. A normalization procedure that extracts the signal of interest would be of universal use when investigating genomic patterns....

Development of a functional cell-based assay that probes the specific interaction between influenza A virus NP and its packaging signal sequence RNA.

Science.gov (United States)

Woo, Jiwon; Yu, Kyung Lee; Lee, Sun Hee; You, Ji Chang

2015-02-06

Although cis-acting packaging signal RNA sequences for the influenza virus NP encoding vRNA have been identified recently though genetic studies, little is known about the interaction between NP and the vRNA packaging signals either in vivo or in vitro. Here, we provide evidence that NP is able to interact specifically with the vRNA packaging sequence RNA within living cells and that the specific RNA binding activity of NP in vivo requires both the N-terminal and central region of the protein. This assay established would be a valuable tool for further detailed studies of the NP-packaging signal RNA interaction in living cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Alu polymerase chain reaction: A method for rapid isolation of human-specific sequences from complex DNA sources

International Nuclear Information System (INIS)

Nelson, D.L.; Ledbetter, S.A.; Corbo, L.; Victoria, M.F.; Ramirez-Solis, R.; Webster, T.D.; Ledbetter, D.H.; Caskey, C.T.

1989-01-01

Current efforts to map the human genome are focused on individual chromosomes or smaller regions and frequently rely on the use of somatic cell hybrids. The authors report the application of the polymerase chain reaction to direct amplification of human DNA from hybrid cells containing regions of the human genome in rodent cell backgrounds using primers directed to the human Alu repeat element. They demonstrate Alu-directed amplification of a fragment of the human HPRT gene from both hybrid cell and cloned DNA and identify through sequence analysis the Alu repeats involved in this amplification. They also demonstrate the application of this technique to identify the chromosomal locations of large fragments of the human X chromosome cloned in a yeast artificial chromosome and the general applicability of the method to the preparation of DNA probes from cloned human sequences. The technique allows rapid gene mapping and provides a simple method for the isolation and analysis of specific chromosomal regions

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

KAUST Repository

Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; D'Aliberti, Deborah; Venza, Mario; Borgogni, Erica; Castellino, Flora; Biondo, Carmelo; D'Andrea, Daniel; Grassi, Luigi; Tramontano, Anna; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

2014-01-01

There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

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Maria Domina

Full Text Available There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

KAUST Repository

Domina, Maria

2014-12-04

There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Herpes simplex virus latency-associated transcript sequence downstream of the promoter influences type-specific reactivation and viral neurotropism.

Science.gov (United States)

Bertke, Andrea S; Patel, Amita; Krause, Philip R

2007-06-01

Herpes simplex virus (HSV) establishes latency in sensory nerve ganglia during acute infection and may later periodically reactivate to cause recurrent disease. HSV type 1 (HSV-1) reactivates more efficiently than HSV-2 from trigeminal ganglia while HSV-2 reactivates more efficiently than HSV-1 from lumbosacral dorsal root ganglia (DRG) to cause recurrent orofacial and genital herpes, respectively. In a previous study, a chimeric HSV-2 that expressed the latency-associated transcript (LAT) from HSV-1 reactivated similarly to wild-type HSV-1, suggesting that the LAT influences the type-specific reactivation phenotype of HSV-2. To further define the LAT region essential for type-specific reactivation, we constructed additional chimeric HSV-2 viruses by replacing the HSV-2 LAT promoter (HSV2-LAT-P1) or 2.5 kb of the HSV-2 LAT sequence (HSV2-LAT-S1) with the corresponding regions from HSV-1. HSV2-LAT-S1 was impaired for reactivation in the guinea pig genital model, while its rescuant and HSV2-LAT-P1 reactivated with a wild-type HSV-2 phenotype. Moreover, recurrences of HSV-2-LAT-S1 were frequently fatal, in contrast to the relatively mild recurrences of the other viruses. During recurrences, HSV2-LAT-S1 DNA increased more in the sacral cord compared to its rescuant or HSV-2. Thus, the LAT sequence region, not the LAT promoter region, provides essential elements for type-specific reactivation of HSV-2 and also plays a role in viral neurotropism. HSV-1 DNA, as quantified by real-time PCR, was more abundant in the lumbar spinal cord, while HSV-2 DNA was more abundant in the sacral spinal cord, which may provide insights into the mechanism for type-specific reactivation and different patterns of central nervous system infection of HSV-1 and HSV-2.

Ion-channel genosensor for the detection of specific DNA sequences derived from Plum Pox Virus in plant extracts.

Science.gov (United States)

Malecka, Kamila; Michalczuk, Lech; Radecka, Hanna; Radecki, Jerzy

2014-10-09

A DNA biosensor for detection of specific oligonucleotides sequences of Plum Pox Virus (PPV) in plant extracts and buffer is proposed. The working principles of a genosensor are based on the ion-channel mechanism. The NH2-ssDNA probe was deposited onto a glassy carbon electrode surface to form an amide bond between the carboxyl group of oxidized electrode surface and amino group from ssDNA probe. The analytical signals generated as a result of hybridization were registered in Osteryoung square wave voltammetry in the presence of [Fe(CN)6]3-/4- as a redox marker. The 22-mer and 42-mer complementary ssDNA sequences derived from PPV and DNA samples from plants infected with PPV were used as targets. Similar detection limits of 2.4 pM (31.0 pg/mL) and 2.3 pM (29.5 pg/mL) in the concentration range 1-8 pM were observed in the presence of the 22-mer ssDNA and 42-mer complementary ssDNA sequences of PPV, respectively. The genosensor was capable of discriminating between samples consisting of extracts from healthy plants and leaf extracts from infected plants in the concentration range 10-50 pg/mL. The detection limit was 12.8 pg/mL. The genosensor displayed good selectivity and sensitivity. The 20-mer partially complementary DNA sequences with four complementary bases and DNA samples from healthy plants used as negative controls generated low signal.

A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.

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Marc W Schmid

Full Text Available The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM, linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq. As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms.

[Isolation and identification of specific sequences correlated to cytoplasmic male sterility and fertile maintenance in cauliflower (Brassica oleracea var. botrytis)].

Science.gov (United States)

Wang, Chun Guo; Chen, Xiao Qiang; Li, Hui; Zhao, Qian Cheng; Sun, De Ling; Song, Wen Qin

2008-02-01

Analysis of ISSR (Inter-Simple Sequence Repeat) and DDRT-PCR (Differential Display Reverse Transcriptase Polymerase Chain Reaction) was performed between cytoplasmic male sterility cauliflower ogura-A and its corresponding maintainer line ogura-B. Totally, 306 detectable bands were obtained by ISSR using thirty oligonucleotide primers. Commonly, six to twelve bands were produced per primer. Among all these primers only the amplification of primer ISSR3 was polymorphic, an 1100 bp specific band was only detected in maintainer line, named ISSR3(1100). Analysis of this sequence indicated that ISSR3(1100) was high homologous with the corresponding sequences of mitochondrial genome in Brassica napus and Arabidopsis thaliana,which suggested that ISSR3(1100) may derive from mitochondrial genome in cauliflower. To carry out DDRT-PCR analysis, three anchor primers and fifteen random primers were selected to combine. Totally, 1122 bands from 1 000 bp to 50 bp were detected. However, only four bands, named ogura-A 205, ogura-A383, ogura-B307 and ogura-B352, were confirmed to be different display in both lines. This result was further identified by reverse Northern dot blotting analysis. Among these four bands, ogura-A205 and ogura-A383 only express in cytoplasmic male sterility line, while ogura-B307 and ogura-B352 were only detected in maintainer line. Analysis of these sequences indicated that it was the first time that these four sequences were reported in cauliflower. Interestingly, ogura-A205 and ogura-B307 did not exhibit any similarities to other reported sequences in other species, more investigations were required to obtain further information. ogura-A383 and ogura-B352 were also two new sequences, they showed high similarities to corresponding chloroplast sequences of Arabidopsis thaliana and Brassica rapa subsp. pekinensis. So we speculated that these two sequences may derive from chloroplast genome. All these results obtained in this study offer new and

Structure and Sequence Search on Aptamer-Protein Docking

Science.gov (United States)

Xiao, Jiajie; Bonin, Keith; Guthold, Martin; Salsbury, Freddie

2015-03-01

Interactions between proteins and deoxyribonucleic acid (DNA) play a significant role in the living systems, especially through gene regulation. However, short nucleic acids sequences (aptamers) with specific binding affinity to specific proteins exhibit clinical potential as therapeutics. Our capillary and gel electrophoresis selection experiments show that specific sequences of aptamers can be selected that bind specific proteins. Computationally, given the experimentally-determined structure and sequence of a thrombin-binding aptamer, we can successfully dock the aptamer onto thrombin in agreement with experimental structures of the complex. In order to further study the conformational flexibility of this thrombin-binding aptamer and to potentially develop a predictive computational model of aptamer-binding, we use GPU-enabled molecular dynamics simulations to both examine the conformational flexibility of the aptamer in the absence of binding to thrombin, and to determine our ability to fold an aptamer. This study should help further de-novo predictions of aptamer sequences by enabling the study of structural and sequence-dependent effects on aptamer-protein docking specificity.

Viewing multiple sequence alignments with the JavaScript Sequence Alignment Viewer (JSAV).

Science.gov (United States)

Martin, Andrew C R

2014-01-01

The JavaScript Sequence Alignment Viewer (JSAV) is designed as a simple-to-use JavaScript component for displaying sequence alignments on web pages. The display of sequences is highly configurable with options to allow alternative coloring schemes, sorting of sequences and 'dotifying' repeated amino acids. An option is also available to submit selected sequences to another web site, or to other JavaScript code. JSAV is implemented purely in JavaScript making use of the JQuery and JQuery-UI libraries. It does not use any HTML5-specific options to help with browser compatibility. The code is documented using JSDOC and is available from http://www.bioinf.org.uk/software/jsav/.

Violation of an evolutionarily conserved immunoglobulin diversity gene sequence preference promotes production of dsDNA-specific IgG antibodies.

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Aaron Silva-Sanchez

Full Text Available Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3, which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH gene segment sequence content by reading frame (RF is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1, which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.

Generic and sequence-variant specific molecular assays for the detection of the highly variable Grapevine leafroll-associated virus 3.

Science.gov (United States)

Chooi, Kar Mun; Cohen, Daniel; Pearson, Michael N

2013-04-01

Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically important virus, which is found in all grapevine growing regions worldwide. Its accurate detection in nursery and field samples is of high importance for certification schemes and disease management programmes. To reduce false negatives that can be caused by sequence variability, a new universal primer pair was designed against a divergent sequence data set, targeting the open reading frame 4 (heat shock protein 70 homologue gene), and optimised for conventional one-step RT-PCR and one-step SYBR Green real-time RT-PCR assays. In addition, primer pairs for the simultaneous detection of specific GLRaV-3 variants from groups 1, 2, 6 (specifically NZ-1) and the outlier NZ2 variant, and the generic detection of variants from groups 1 to 5 were designed and optimised as a conventional one-step multiplex RT-PCR assay using the plant nad5 gene as an internal control (i.e. one-step hexaplex RT-PCR). Results showed that the generic and variant specific assays detected in vitro RNA transcripts from a range of 1×10(1)-1×10(8) copies of amplicon per μl diluted in healthy total RNA from Vitis vinifera cv. Cabernet Sauvignon. Furthermore, the assays were employed effectively to screen 157 germplasm and 159 commercial field samples. Thus results demonstrate that the GLRaV-3 generic and variant-specific assays are prospective tools that will be beneficial for certification schemes and disease management programmes, as well as biological and epidemiological studies of the divergent GLRaV-3 populations. Copyright © 2013 Elsevier B.V. All rights reserved.

Direct, rapid RNA sequence analysis

International Nuclear Information System (INIS)

Peattie, D.A.

1987-01-01

The original methods of RNA sequence analysis were based on enzymatic production and chromatographic separation of overlapping oligonucleotide fragments from within an RNA molecule followed by identification of the mononucleotides comprising the oligomer. Over the past decade the field of nucleic acid sequencing has changed dramatically, however, and RNA molecules now can be sequenced in a variety of more streamlined fashions. Most of the more recent advances in RNA sequencing have involved one-dimensional electrophoretic separation of 32 P-end-labeled oligoribonucleotides on polyacrylamide gels. In this chapter the author discusses two of these methods for determining the nucleotide sequences of RNA molecules rapidly: the chemical method and the enzymatic method. Both methods are direct and degradative, i.e., they rely on fragmatic and chemical approaches should be utilized. The single-strand-specific ribonucleases (A, T 1 , T 2 , and S 1 ) provide an efficient means to locate double-helical regions rapidly, and the chemical reactions provide a means to determine the RNA sequence within these regions. In addition, the chemical reactions allow one to assign interactions to specific atoms and to distinguish secondary interactions from tertiary ones. If the RNA molecule is small enough to be sequenced directly by the enzymatic or chemical method, the probing reactions can be done easily at the same time as sequencing reactions

Developing market class specific InDel markers from next generation sequence data in Phaseolus vulgaris L.

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Samira eMafi Moghaddam

2014-05-01

Full Text Available Next generation sequence data provides valuable information and tools for genetic and genomic research and offers new insights useful for marker development. This data is useful for the design of accurate and user-friendly molecular tools. Common bean (Phaseolus vulgaris L. is a diverse crop in which separate domestication events happened in each gene pool followed by race and market class diversification that has resulted in different morphological characteristics in each commercial market class. This has led to essentially independent breeding programs within each market class which in turn has resulted in limited within market class sequence variation. Sequence data from selected genotypes of five bean market classes (pinto, black, navy, and light and dark red kidney were used to develop InDel-based markers specific to each market class. Design of the InDel markers was conducted through a combination of assembly, alignment and primer design software using 1.6x to 5.1x coverage of Illumina GAII sequence data for each of the selected genotypes. The procedure we developed for primer design is fast, accurate, less error prone, and higher throughput than when they are designed manually. All InDel markers are easy to run and score with no need for PCR optimization. A total of 2,687 InDel markers distributed across the genome were developed. To highlight their usefulness, they were employed to construct a phylogenetic tree and a genetic map, showing that InDel markers are reliable, simple, and accurate.

Short sequence motifs, overrepresented in mammalian conservednon-coding sequences

Energy Technology Data Exchange (ETDEWEB)

Minovitsky, Simon; Stegmaier, Philip; Kel, Alexander; Kondrashov,Alexey S.; Dubchak, Inna

2007-02-21

Background: A substantial fraction of non-coding DNAsequences of multicellular eukaryotes is under selective constraint. Inparticular, ~;5 percent of the human genome consists of conservednon-coding sequences (CNSs). CNSs differ from other genomic sequences intheir nucleotide composition and must play important functional roles,which mostly remain obscure.Results: We investigated relative abundancesof short sequence motifs in all human CNSs present in the human/mousewhole-genome alignments vs. three background sets of sequences: (i)weakly conserved or unconserved non-coding sequences (non-CNSs); (ii)near-promoter sequences (located between nucleotides -500 and -1500,relative to a start of transcription); and (iii) random sequences withthe same nucleotide composition as that of CNSs. When compared tonon-CNSs and near-promoter sequences, CNSs possess an excess of AT-richmotifs, often containing runs of identical nucleotides. In contrast, whencompared to random sequences, CNSs contain an excess of GC-rich motifswhich, however, lack CpG dinucleotides. Thus, abundance of short sequencemotifs in human CNSs, taken as a whole, is mostly determined by theiroverall compositional properties and not by overrepresentation of anyspecific short motifs. These properties are: (i) high AT-content of CNSs,(ii) a tendency, probably due to context-dependent mutation, of A's andT's to clump, (iii) presence of short GC-rich regions, and (iv) avoidanceof CpG contexts, due to their hypermutability. Only a small number ofshort motifs, overrepresented in all human CNSs are similar to bindingsites of transcription factors from the FOX family.Conclusion: Human CNSsas a whole appear to be too broad a class of sequences to possess strongfootprints of any short sequence-specific functions. Such footprintsshould be studied at the level of functional subclasses of CNSs, such asthose which flank genes with a particular pattern of expression. Overallproperties of CNSs are affected by

Detection of nucleic acid sequences by invader-directed cleavage

Science.gov (United States)

Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

1999-01-01

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

Strain-specific and pooled genome sequences for populations of Drosophila melanogaster from three continents.

Science.gov (United States)

Bergman, Casey M; Haddrill, Penelope R

2015-01-01

To contribute to our general understanding of the evolutionary forces that shape variation in genome sequences in nature, we have sequenced genomes from 50 isofemale lines and six pooled samples from populations of Drosophila melanogaster on three continents. Analysis of raw and reference-mapped reads indicates the quality of these genomic sequence data is very high. Comparison of the predicted and experimentally-determined Wolbachia infection status of these samples suggests that strain or sample swaps are unlikely to have occurred in the generation of these data. Genome sequences are freely available in the European Nucleotide Archive under accession ERP009059. Isofemale lines can be obtained from the Drosophila Species Stock Center.

Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

Directory of Open Access Journals (Sweden)

Blackmon Barbara P

2011-07-01

Full Text Available Abstract Background BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library. Results This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Conclusions Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed.

Global Transcriptome Sequencing Identifies Chlamydospore Specific Markers in Candida albicans and Candida dubliniensis

LENUS (Irish Health Repository)

Palige, Katja

2013-04-15

Candida albicans and Candida dubliniensis are pathogenic fungi that are highly related but differ in virulence and in some phenotypic traits. During in vitro growth on certain nutrient-poor media, C. albicans and C. dubliniensis are the only yeast species which are able to produce chlamydospores, large thick-walled cells of unknown function. Interestingly, only C. dubliniensis forms pseudohyphae with abundant chlamydospores when grown on Staib medium, while C. albicans grows exclusively as a budding yeast. In order to further our understanding of chlamydospore development and assembly, we compared the global transcriptional profile of both species during growth in liquid Staib medium by RNA sequencing. We also included a C. albicans mutant in our study which lacks the morphogenetic transcriptional repressor Nrg1. This strain, which is characterized by its constitutive pseudohyphal growth, specifically produces masses of chlamydospores in Staib medium, similar to C. dubliniensis. This comparative approach identified a set of putatively chlamydospore-related genes. Two of the homologous C. albicans and C. dubliniensis genes (CSP1 and CSP2) which were most strongly upregulated during chlamydospore development were analysed in more detail. By use of the green fluorescent protein as a reporter, the encoded putative cell wall related proteins were found to exclusively localize to C. albicans and C. dubliniensis chlamydospores. Our findings uncover the first chlamydospore specific markers in Candida species and provide novel insights in the complex morphogenetic development of these important fungal pathogens.

Molecular dynamics simulations shed light on the enthalpic and entropic driving forces that govern the sequence specific recognition between netropsin and DNA.

Science.gov (United States)

Dolenc, Jozica; Gerster, Sarah; van Gunsteren, Wilfred F

2010-09-02

With the aim to gain a better understanding of the various driving forces that govern sequence specific DNA minor groove binding, we performed a thermodynamic analysis of netropsin binding to an AT-containing and to a set of six mixed AT/GC-containing binding sequences in the DNA minor groove. The relative binding free energies obtained using molecular dynamics simulations and free energy calculations show significant variations with the binding sequence. While the introduction of a GC base pair in the middle or close to the middle of the binding site is unfavorable for netropsin binding, a GC base pair at the end of the binding site appears to have no negative influence on the binding. The results of the structural and energetic analyses of the netropsin-DNA complexes reveal that the differences in the calculated binding affinities cannot be explained solely in terms of netropsin-DNA hydrogen-bonding or interaction energies. In addition, solvation effects and entropic contributions to the relative binding free energy provide a more complete picture of the various factors determining binding. Analysis of the relative binding entropy indicates that its magnitude is highly sequence-dependent, with the ratio |TDeltaDeltaS|/|DeltaDeltaH| ranging from 0.07 for the AAAGA to 1.7 for the AAGAG binding sequence, respectively.

Sequence-specific assignments in the 1H NMR spectrum of the human inflammatory protein C5a

International Nuclear Information System (INIS)

Zuiderweg, E.R.P.; Mollison, K.W.; Henkin, J.; Carter, G.W.

1988-01-01

Full sequence-specific assignments for the 1 H NMR lines of the backbone protons of the human complement factor C5a are described and documented. The results were obtained by largely following the methodology developed by Wuethrich et al. Assignments for the majority of the amino acid side chain protons were obtained by using a comparison of double- and triple-quantum-filtered two-dimensional correlated experiments together with the analysis of relayed coherence transfer spectra. The assignments provide the basis for the determination of the thus far unknown three-dimensional structure of C5a from nuclear Overhauser enhancement distance constraints

Sequencing Information Management System (SIMS). Final report

Energy Technology Data Exchange (ETDEWEB)

Fields, C.

1996-02-15

A feasibility study to develop a requirements analysis and functional specification for a data management system for large-scale DNA sequencing laboratories resulted in a functional specification for a Sequencing Information Management System (SIMS). This document reports the results of this feasibility study, and includes a functional specification for a SIMS relational schema. The SIMS is an integrated information management system that supports data acquisition, management, analysis, and distribution for DNA sequencing laboratories. The SIMS provides ad hoc query access to information on the sequencing process and its results, and partially automates the transfer of data between laboratory instruments, analysis programs, technical personnel, and managers. The SIMS user interfaces are designed for use by laboratory technicians, laboratory managers, and scientists. The SIMS is designed to run in a heterogeneous, multiplatform environment in a client/server mode. The SIMS communicates with external computational and data resources via the internet.

Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers.

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Stephan Pabinger

Full Text Available Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM. Here, we present a novel tool, called TABSAT, for analyzing targeted bisulfite sequencing data generated on Ion Torrent sequencers. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of Bismark to map the reads to a reference genome. The pipeline visualizes results as lollipop plots and is able to deduce specific methylation-patterns present in a sample. The obtained profiles are then summarized and compared between samples. In order to assess the performance of the targeted bisulfite sequencing workflow, 48 samples were used to generate 53 different Bisulfite-Sequencing PCR amplicons from each sample, resulting in 2,544 amplicon targets. We obtained a mean coverage of 282X using 1,196,822 aligned reads. Next, we compared the sequencing results of these targets to the methylation level of the corresponding sites on an Illumina 450k methylation chip. The calculated average Pearson correlation coefficient of 0.91 confirms the sequencing results with one of the industry-leading CpG methylation platforms and shows that targeted amplicon bisulfite sequencing provides an accurate and cost-efficient method for DNA methylation studies, e.g., to provide platform-independent confirmation of Illumina Infinium 450k methylation data. TABSAT offers a novel way to analyze data generated by Ion Torrent instruments and can also be used with data from the Illumina MiSeq platform. It can be easily accessed via the Platomics platform, which offers a web-based graphical user interface along with sample and parameter storage

Impact of cadmium, cobalt and nickel on sequence-specific DNA binding of p63 and p73 in vitro and in cells

Czech Academy of Sciences Publication Activity Database

Adámik, Matěj; Bažantová, Pavla; Navrátilová, Lucie; Polášková, Alena; Pečinka, Petr; Holanová, L.; Tichý, Vlastimil; Brázdová, Marie

2015-01-01

Roč. 456, č. 1 (2015), s. 29-34 ISSN 0006-291X R&D Projects: GA ČR(CZ) GA13-36108S Institutional support: RVO:68081707 Keywords : p53 protein family * Sequence-specific DNA binding * Heavy metals Subject RIV: BO - Biophysics Impact factor: 2.371, year: 2015

Integrated sequence analysis. Final report

International Nuclear Information System (INIS)

Andersson, K.; Pyy, P.

1998-02-01

The NKS/RAK subprojet 3 'integrated sequence analysis' (ISA) was formulated with the overall objective to develop and to test integrated methodologies in order to evaluate event sequences with significant human action contribution. The term 'methodology' denotes not only technical tools but also methods for integration of different scientific disciplines. In this report, we first discuss the background of ISA and the surveys made to map methods in different application fields, such as man machine system simulation software, human reliability analysis (HRA) and expert judgement. Specific event sequences were, after the surveys, selected for application and testing of a number of ISA methods. The event sequences discussed in the report were cold overpressure of BWR, shutdown LOCA of BWR, steam generator tube rupture of a PWR and BWR disturbed signal view in the control room after an external event. Different teams analysed these sequences by using different ISA and HRA methods. Two kinds of results were obtained from the ISA project: sequence specific and more general findings. The sequence specific results are discussed together with each sequence description. The general lessons are discussed under a separate chapter by using comparisons of different case studies. These lessons include areas ranging from plant safety management (design, procedures, instrumentation, operations, maintenance and safety practices) to methodological findings (ISA methodology, PSA,HRA, physical analyses, behavioural analyses and uncertainty assessment). Finally follows a discussion about the project and conclusions are presented. An interdisciplinary study of complex phenomena is a natural way to produce valuable and innovative results. This project came up with structured ways to perform ISA and managed to apply the in practice. The project also highlighted some areas where more work is needed. In the HRA work, development is required for the use of simulators and expert judgement as

Integrated sequence analysis. Final report

Energy Technology Data Exchange (ETDEWEB)

Andersson, K.; Pyy, P

1998-02-01

The NKS/RAK subprojet 3 `integrated sequence analysis` (ISA) was formulated with the overall objective to develop and to test integrated methodologies in order to evaluate event sequences with significant human action contribution. The term `methodology` denotes not only technical tools but also methods for integration of different scientific disciplines. In this report, we first discuss the background of ISA and the surveys made to map methods in different application fields, such as man machine system simulation software, human reliability analysis (HRA) and expert judgement. Specific event sequences were, after the surveys, selected for application and testing of a number of ISA methods. The event sequences discussed in the report were cold overpressure of BWR, shutdown LOCA of BWR, steam generator tube rupture of a PWR and BWR disturbed signal view in the control room after an external event. Different teams analysed these sequences by using different ISA and HRA methods. Two kinds of results were obtained from the ISA project: sequence specific and more general findings. The sequence specific results are discussed together with each sequence description. The general lessons are discussed under a separate chapter by using comparisons of different case studies. These lessons include areas ranging from plant safety management (design, procedures, instrumentation, operations, maintenance and safety practices) to methodological findings (ISA methodology, PSA,HRA, physical analyses, behavioural analyses and uncertainty assessment). Finally follows a discussion about the project and conclusions are presented. An interdisciplinary study of complex phenomena is a natural way to produce valuable and innovative results. This project came up with structured ways to perform ISA and managed to apply the in practice. The project also highlighted some areas where more work is needed. In the HRA work, development is required for the use of simulators and expert judgement as

Targeted assembly of short sequence reads.

Directory of Open Access Journals (Sweden)

René L Warren

Full Text Available As next-generation sequence (NGS production continues to increase, analysis is becoming a significant bottleneck. However, in situations where information is required only for specific sequence variants, it is not necessary to assemble or align whole genome data sets in their entirety. Rather, NGS data sets can be mined for the presence of sequence variants of interest by localized assembly, which is a faster, easier, and more accurate approach. We present TASR, a streamlined assembler that interrogates very large NGS data sets for the presence of specific variants by only considering reads within the sequence space of input target sequences provided by the user. The NGS data set is searched for reads with an exact match to all possible short words within the target sequence, and these reads are then assembled stringently to generate a consensus of the target and flanking sequence. Typically, variants of a particular locus are provided as different target sequences, and the presence of the variant in the data set being interrogated is revealed by a successful assembly outcome. However, TASR can also be used to find unknown sequences that flank a given target. We demonstrate that TASR has utility in finding or confirming genomic mutations, polymorphisms, fusions and integration events. Targeted assembly is a powerful method for interrogating large data sets for the presence of sequence variants of interest. TASR is a fast, flexible and easy to use tool for targeted assembly.

Use of PCR with Sequence-specific Primers for High-Resolution Human Leukocyte Antigen Typing of Patients with Narcolepsy

Science.gov (United States)

Woo, Hye In; Joo, Eun Yeon; Lee, Kyung Wha

2012-01-01

Background Narcolepsy is a neurologic disorder characterized by excessive daytime sleepiness, symptoms of abnormal rapid eye movement (REM) sleep, and a strong association with HLA-DRB1*1501, -DQA1*0102, and -DQB1*0602. Here, we investigated the clinico-physical characteristics of Korean patients with narcolepsy, their HLA types, and the clinical utility of high-resolution PCR with sequence-specific primers (PCR-SSP) as a simple typing method for identifying DRB1*15/16, DQA1, and DQB1 alleles. Methods The study population consisted of 67 consecutively enrolled patients having unexplained daytime sleepiness and diagnosed narcolepsy based on clinical and neurological findings. Clinical data and the results of the multiple sleep latency test and polysomnography were reviewed, and HLA typing was performed using both high-resolution PCR-SSP and sequence-based typing (SBT). Results The 44 narcolepsy patients with cataplexy displayed significantly higher frequencies of DRB1*1501 (Pc= 0.003), DQA1*0102 (Pc=0.001), and DQB1*0602 (Pc=0.014) than the patients without cataplexy. Among patients carrying DRB1*1501-DQB1*0602 or DQA1*0102, the frequencies of a mean REM sleep latency of less than 20 min in nocturnal polysomnography and clinical findings, including sleep paralysis and hypnagogic hallucination were significantly higher. SBT and PCR-SSP showed 100% concordance for high-resolution typing of DRB1*15/16 alleles and DQA1 and DQB1 loci. Conclusions The clinical characteristics and somnographic findings of narcolepsy patients were associated with specific HLA alleles, including DRB1*1501, DQA1*0102, and DQB1*0602. Application of high-resolution PCR-SSP, a reliable and simple method, for both allele- and locus-specific HLA typing of DRB1*15/16, DQA1, and DQB1 would be useful for characterizing clinical status among subjects with narcolepsy. PMID:22259780

SAAS-CNV: A Joint Segmentation Approach on Aggregated and Allele Specific Signals for the Identification of Somatic Copy Number Alterations with Next-Generation Sequencing Data.

Science.gov (United States)

Zhang, Zhongyang; Hao, Ke

2015-11-01

Cancer genomes exhibit profound somatic copy number alterations (SCNAs). Studying tumor SCNAs using massively parallel sequencing provides unprecedented resolution and meanwhile gives rise to new challenges in data analysis, complicated by tumor aneuploidy and heterogeneity as well as normal cell contamination. While the majority of read depth based methods utilize total sequencing depth alone for SCNA inference, the allele specific signals are undervalued. We proposed a joint segmentation and inference approach using both signals to meet some of the challenges. Our method consists of four major steps: 1) extracting read depth supporting reference and alternative alleles at each SNP/Indel locus and comparing the total read depth and alternative allele proportion between tumor and matched normal sample; 2) performing joint segmentation on the two signal dimensions; 3) correcting the copy number baseline from which the SCNA state is determined; 4) calling SCNA state for each segment based on both signal dimensions. The method is applicable to whole exome/genome sequencing (WES/WGS) as well as SNP array data in a tumor-control study. We applied the method to a dataset containing no SCNAs to test the specificity, created by pairing sequencing replicates of a single HapMap sample as normal/tumor pairs, as well as a large-scale WGS dataset consisting of 88 liver tumors along with adjacent normal tissues. Compared with representative methods, our method demonstrated improved accuracy, scalability to large cancer studies, capability in handling both sequencing and SNP array data, and the potential to improve the estimation of tumor ploidy and purity.

RISC RNA sequencing for context-specific identification of in vivo microRNA targets.

Science.gov (United States)

Matkovich, Scot J; Van Booven, Derek J; Eschenbacher, William H; Dorn, Gerald W

2011-01-07

MicroRNAs (miRs) are expanding our understanding of cardiac disease and have the potential to transform cardiovascular therapeutics. One miR can target hundreds of individual mRNAs, but existing methodologies are not sufficient to accurately and comprehensively identify these mRNA targets in vivo. To develop methods permitting identification of in vivo miR targets in an unbiased manner, using massively parallel sequencing of mouse cardiac transcriptomes in combination with sequencing of mRNA associated with mouse cardiac RNA-induced silencing complexes (RISCs). We optimized techniques for expression profiling small amounts of RNA without introducing amplification bias and applied this to anti-Argonaute 2 immunoprecipitated RISCs (RISC-Seq) from mouse hearts. By comparing RNA-sequencing results of cardiac RISC and transcriptome from the same individual hearts, we defined 1645 mRNAs consistently targeted to mouse cardiac RISCs. We used this approach in hearts overexpressing miRs from Myh6 promoter-driven precursors (programmed RISC-Seq) to identify 209 in vivo targets of miR-133a and 81 in vivo targets of miR-499. Consistent with the fact that miR-133a and miR-499 have widely differing "seed" sequences and belong to different miR families, only 6 targets were common to miR-133a- and miR-499-programmed hearts. RISC-sequencing is a highly sensitive method for general RISC profiling and individual miR target identification in biological context and is applicable to any tissue and any disease state.

Synthesis and evaluation of sequence-specific DNA alkylating agents: effect of alkylation subunits.

Science.gov (United States)

Shimizu, Tatsuhiko; Sasaki, Shunta; Minoshima, Masafumi; Shinohara, Ken-ichi; Bando, Toshikazu; Sugiyama, Hiroshi

2006-01-01

We have demonstrated that hairpin pyrrole (Py)- imidazole (Im) polyamide-CBI conjugates selectively alkylate predetermined sequences. In this study, we investigated the effect of alkylation subunits, for example conjugates 1-4 with three types of DNA alkylating units, and Py-Im polyamides with indole linker. Conjugate 3 and 4 selectively alkylated the predetermined sequences as described previously, while conjugates 1 and 2 alkylate at mismatched sites.

A modified strategy for sequence specific assignment of protein NMR spectra based on amino acid type selective experiments

International Nuclear Information System (INIS)

Schubert, Mario; Labudde, Dirk; Leitner, Dietmar; Oschkinat, Hartmut; Schmieder, Peter

2005-01-01

The determination of the three-dimensional structure of a protein or the study of protein-ligand interactions requires the assignment of all relevant nuclei as an initial step. This is nowadays almost exclusively performed using triple-resonance experiments. The conventional strategy utilizes one or more pairs of three dimensional spectra to obtain redundant information and thus reliable assignments. Here, a modified strategy for obtaining sequence specific assignments based on two dimensional amino acid type selective triple-resonance experiments is proposed. These experiments can be recorded with good resolution in a relatively short time. They provide very specific and redundant information, in particular on sequential connectivities, that drastically increases the ease and reliability of the assignment procedure, done either manually or in an automated fashion. The new strategy is demonstrated with the protein domain PB1 from yeast CDC24p

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.

Science.gov (United States)

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.

Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA

DEFF Research Database (Denmark)

Minero, Gabriel Khose Antonio; Nogueira, Catarina; Rizzi, Giovanni

2017-01-01

We report on an optomagnetic technique optimised for real-time molecular detection of Dengue fever virus under ideal as well as non-ideal laboratory conditions using two different detection approaches. The first approach is based on the detection of the hydrodynamic volume of streptavidin coated...... magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic...... claim detection of down to 100 fM of Dengue target after 20 min of LAMP with a contamination background....

Lanthanum-Based Metal-Organic Frameworks for Specific Detection of Sudan Virus RNA Conservative Sequences down to Single-Base Mismatch.

Science.gov (United States)

Yang, Shui-Ping; Zhao, Wei; Hu, Pei-Pei; Wu, Ke-Yang; Jiang, Zhi-Hong; Bai, Li-Ping; Li, Min-Min; Chen, Jin-Xiang

2017-12-18

Reactions of La(NO 3 ) 3 ·6H 2 O with the polar, tritopic quaternized carboxylate ligands N-carboxymethyl-3,5-dicarboxylpyridinium bromide (H 3 CmdcpBr) and N-(4-carboxybenzyl)-3,5-dicarboxylpyridinium bromide (H 3 CbdcpBr) afford two water-stable metal-organic frameworks (MOFs) of {[La 4 (Cmdcp) 6 (H 2 O) 9 ]} n (1, 3D) and {[La 2 (Cbdcp) 3 (H 2 O) 10 ]} n (2, 2D). MOFs 1 and 2 absorb the carboxyfluorescein (FAM)-tagged probe DNA (P-DNA) and quench the fluorescence of FAM via a photoinduced electron transfer (PET) process. The nonemissive P-DNA@MOF hybrids thus formed in turn function as sensing platforms to distinguish conservative linear, single-stranded RNA sequences of Sudan virus with high selectivity and low detection limits of 112 and 67 pM, respectively (at a signal-to-noise ratio of 3). These hybrids also exhibit high specificity and discriminate down to single-base mismatch RNA sequences.

CRISPR-Cas9 technology: applications in genome engineering, development of sequence-specific antimicrobials, and future prospects.

Science.gov (United States)

de la Fuente-Núñez, César; Lu, Timothy K

2017-02-20

The development of CRISPR-Cas9 technology has revolutionized our ability to edit DNA and to modulate expression levels of genes of interest, thus providing powerful tools to accelerate the precise engineering of a wide range of organisms. In addition, the CRISPR-Cas system can be harnessed to design "precision" antimicrobials that target bacterial pathogens in a DNA sequence-specific manner. This capability will enable killing of drug-resistant microbes by selectively targeting genes involved in antibiotic resistance, biofilm formation and virulence. Here, we review the origins and mechanistic basis of CRISPR-Cas systems, discuss how this technology can be leveraged to provide a range of applications in both eukaryotic and prokaryotic systems, and finish by outlining limitations and future prospects.

Next-generation sequencing of multiple individuals per barcoded library by deconvolution of sequenced amplicons using endonuclease fragment analysis

DEFF Research Database (Denmark)

Andersen, Jeppe D; Pereira, Vania; Pietroni, Carlotta

2014-01-01

The simultaneous sequencing of samples from multiple individuals increases the efficiency of next-generation sequencing (NGS) while also reducing costs. Here we describe a novel and simple approach for sequencing DNA from multiple individuals per barcode. Our strategy relies on the endonuclease...... digestion of PCR amplicons prior to library preparation, creating a specific fragment pattern for each individual that can be resolved after sequencing. By using both barcodes and restriction fragment patterns, we demonstrate the ability to sequence the human melanocortin 1 receptor (MC1R) genes from 72...... individuals using only 24 barcoded libraries....

Label-Free Detection of Sequence-Specific DNA Based on Fluorescent Silver Nanoclusters-Assisted Surface Plasmon-Enhanced Energy Transfer.

Science.gov (United States)

Ma, Jin-Liang; Yin, Bin-Cheng; Le, Huynh-Nhu; Ye, Bang-Ce

2015-06-17

We have developed a label-free method for sequence-specific DNA detection based on surface plasmon enhanced energy transfer (SPEET) process between fluorescent DNA/AgNC string and gold nanoparticles (AuNPs). DNA/AgNC string, prepared by a single-stranded DNA template encoded two emitter-nucleation sequences at its termini and an oligo spacer in the middle, was rationally designed to produce bright fluorescence emission. The proposed method takes advantage of two strategies. The first one is the difference in binding properties of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) toward AuNPs. The second one is SPEET process between fluorescent DNA/AgNC string and AuNPs, in which fluorescent DNA/AgNC string can be spontaneously adsorbed onto the surface of AuNPs and correspondingly AuNPs serve as "nanoquencher" to quench the fluorescence of DNA/AgNC string. In the presence of target DNA, the sensing probe hybridized with target DNA to form duplex DNA, leading to a salt-induced AuNP aggregation and subsequently weakened SPEET process between fluorescent DNA/AgNC string and AuNPs. A red-to-blue color change of AuNPs and a concomitant fluorescence increase were clearly observed in the sensing system, which had a concentration dependent manner with specific DNA. The proposed method achieved a detection limit of ∼2.5 nM, offering the following merits of simple design, convenient operation, and low experimental cost because of no chemical modification, organic dye, enzymatic reaction, or separation procedure involved.

Sex-specific markers developed by next-generation sequencing confirmed an XX/XY sex determination system in bighead carp (Hypophthalmichehys nobilis) and silver carp (Hypophthalmichthys molitrix).

Science.gov (United States)

Liu, Haiyang; Pang, Meixia; Yu, Xiaomu; Zhou, Ying; Tong, Jingou; Fu, Beide

2018-01-05

Sex-specific markers are powerful tools for identifying sex-determination system in various animals. Bighead carp (Hypophthalmichehys nobilis) and silver carp (Hypophthalmichthys molitrix) are two of the most important edible fish in Asia, which have a long juvenility period that can lasts for 4-5 years. In this study, we found one sex-specific marker by next-generation sequencing together with bioinformatics analysis in bighead carp. The male-specific markers were used to perform molecular sexing in the progenies of artificial gynogenetic diploids and found all progenies (n = 160) were females. Meanwhile, around 1 : 1 sex ratio was observed in a total of 579 juvenile offspring from three other families. To further extend the male-specific region, we performed genome walking and got a male-specific sequence of 8,661 bp. Five pairs of primers were designed and could be used to efficiently distinguish males from females in bighead carp and silver carp. The development of these male-specific markers and results of their molecular sexing in different populations provide strong evidence for a sex determination system of female homogametry or male heterogametry (XX/XY) in bighead carp and silver carp. To the best of our knowledge, this is the first report of effective sex-specific markers in these two large carp species. © The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene

International Nuclear Information System (INIS)

Knight, G.B.; Gudas, J.M.; Pardee, A.B.

1987-01-01

Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, the authors have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene

Sequence- and structure-dependent DNA base dynamics: Synthesis, structure, and dynamics of site and sequence specifically spin-labeled DNA

International Nuclear Information System (INIS)

Spaltenstein, A.; Robinson, B.H.; Hopkins, P.B.

1989-01-01

A nitroxide spin-labeled analogue of thymidine (1a), in which the methyl group is replaced by an acetylene-tethered nitroxide, was evaluated as a probe for structural and dynamics studies of sequence specifically spin-labeled DNA. Residue 1a was incorporated into synthetic deoxyoligonucleotides by using automated phosphite triester methods. 1 H NMR, CD, and thermal denaturation studies indicate that 1a (T) does not significantly alter the structure of 5'-d(CGCGAATT*CGCG) from that of the native dodecamer. EPR studies on monomer, single-stranded, and duplexed DNA show that 1a readily distinguishes environments of different rigidity. Comparison of the general line-shape features of the observed EPR spectra of several small duplexes (12-mer, 24-mer) with simulated EPR spectra assuming isotropic motion suggests that probe 1a monitors global tumbling of small duplexes. Increasing the length of the DNA oligomers results in significant deviation from isotropic motion, with line-shape features similar to those of calculated spectra of objects with isotropic rotational correlation times of 20-100 ns. EPR spectra of a spin-labeled GT mismatch and a T bulge in long DNAs are distinct from those of spin-labeled Watson-Crick paired DNAs, further demonstrating the value of EPR as a tool in the evaluation of local dynamic and structural features in macromolecules

Automated constraint checking of spacecraft command sequences

Science.gov (United States)

Horvath, Joan C.; Alkalaj, Leon J.; Schneider, Karl M.; Spitale, Joseph M.; Le, Dang

1995-01-01

Robotic spacecraft are controlled by onboard sets of commands called "sequences." Determining that sequences will have the desired effect on the spacecraft can be expensive in terms of both labor and computer coding time, with different particular costs for different types of spacecraft. Specification languages and appropriate user interface to the languages can be used to make the most effective use of engineering validation time. This paper describes one specification and verification environment ("SAVE") designed for validating that command sequences have not violated any flight rules. This SAVE system was subsequently adapted for flight use on the TOPEX/Poseidon spacecraft. The relationship of this work to rule-based artificial intelligence and to other specification techniques is discussed, as well as the issues that arise in the transfer of technology from a research prototype to a full flight system.

Triple helix-forming oligonucleotide corresponding to the polypyrimidine sequence in the rat alpha 1(I) collagen promoter specifically inhibits factor binding and transcription.

Science.gov (United States)

Kovacs, A; Kandala, J C; Weber, K T; Guntaka, R V

1996-01-19

Type I and III fibrillar collagens are the major structural proteins of the extracellular matrix found in various organs including the myocardium. Abnormal and progressive accumulation of fibrillar type I collagen in the interstitial spaces compromises organ function and therefore, the study of transcriptional regulation of this gene and specific targeting of its expression is of major interest. Transient transfection of adult cardiac fibroblasts indicate that the polypurine-polypyrimidine sequence of alpha 1(I) collagen promoter between nucleotides - 200 and -140 represents an overall positive regulatory element. DNase I footprinting and electrophoretic mobility shift assays suggest that multiple factors bind to different elements of this promoter region. We further demonstrate that the unique polypyrimidine sequence between -172 and -138 of the promoter represents a suitable target for a single-stranded polypurine oligonucleotide (TFO) to form a triple helix DNA structure. Modified electrophoretic mobility shift assays show that this TFO specifically inhibits the protein-DNA interaction within the target region. In vitro transcription assays and transient transfection experiments demonstrate that the transcriptional activity of the promoter is inhibited by this oligonucleotide. We propose that TFOs represent a therapeutic potential to specifically influence the expression of alpha 1(I) collagen gene in various disease states where abnormal type I collagen accumulation is known to occur.

Correlation between MCAT biology content specifications and topic scope and sequence of general education college biology textbooks.

Science.gov (United States)

Rissing, Steven W

2013-01-01

Most American colleges and universities offer gateway biology courses to meet the needs of three undergraduate audiences: biology and related science majors, many of whom will become biomedical researchers; premedical students meeting medical school requirements and preparing for the Medical College Admissions Test (MCAT); and students completing general education (GE) graduation requirements. Biology textbooks for these three audiences present a topic scope and sequence that correlates with the topic scope and importance ratings of the biology content specifications for the MCAT regardless of the intended audience. Texts for "nonmajors," GE courses appear derived directly from their publisher's majors text. Topic scope and sequence of GE texts reflect those of "their" majors text and, indirectly, the MCAT. MCAT term density of GE texts equals or exceeds that of their corresponding majors text. Most American universities require a GE curriculum to promote a core level of academic understanding among their graduates. This includes civic scientific literacy, recognized as an essential competence for the development of public policies in an increasingly scientific and technological world. Deriving GE biology and related science texts from majors texts designed to meet very different learning objectives may defeat the scientific literacy goals of most schools' GE curricula.

Correlation between MCAT Biology Content Specifications and Topic Scope and Sequence of General Education College Biology Textbooks

Science.gov (United States)

Rissing, Steven W.

2013-01-01

Most American colleges and universities offer gateway biology courses to meet the needs of three undergraduate audiences: biology and related science majors, many of whom will become biomedical researchers; premedical students meeting medical school requirements and preparing for the Medical College Admissions Test (MCAT); and students completing general education (GE) graduation requirements. Biology textbooks for these three audiences present a topic scope and sequence that correlates with the topic scope and importance ratings of the biology content specifications for the MCAT regardless of the intended audience. Texts for “nonmajors,” GE courses appear derived directly from their publisher's majors text. Topic scope and sequence of GE texts reflect those of “their” majors text and, indirectly, the MCAT. MCAT term density of GE texts equals or exceeds that of their corresponding majors text. Most American universities require a GE curriculum to promote a core level of academic understanding among their graduates. This includes civic scientific literacy, recognized as an essential competence for the development of public policies in an increasingly scientific and technological world. Deriving GE biology and related science texts from majors texts designed to meet very different learning objectives may defeat the scientific literacy goals of most schools’ GE curricula. PMID:24006392

Identification of a Flavivirus Sequence in a Marine Arthropod.

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Michael J Conway

Full Text Available Phylogenetic analysis has yet to uncover the early origins of flaviviruses. In this study, I mined a database of expressed sequence tags in order to discover novel flavivirus sequences. Flavivirus sequences were identified in a pool of mRNA extracted from the sea spider Endeis spinosa (Pycnogonida, Pantopoda. Reconstruction of the translated sequences and BLAST analysis matched the sequence to the flavivirus NS5 gene. Additional sequences corresponding to envelope and the NS5 MTase domain were also identified. Phylogenetic analysis of homologous NS5 sequences revealed that Endeis spinosa NS5 (ESNS5 is likely related to classical insect-specific flaviviruses. It is unclear if ESNS5 represents genetic material from an active viral infection or an integrated viral genome. These data raise the possibility that classical insect-specific flaviviruses and perhaps medically relevant flaviviruses, evolved from progenitors that infected marine arthropods.

Transcriptome sequencing of the Microarray Quality Control (MAQC RNA reference samples using next generation sequencing

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Thierry-Mieg Danielle

2009-06-01

Full Text Available Abstract Background Transcriptome sequencing using next-generation sequencing platforms will soon be competing with DNA microarray technologies for global gene expression analysis. As a preliminary evaluation of these promising technologies, we performed deep sequencing of cDNA synthesized from the Microarray Quality Control (MAQC reference RNA samples using Roche's 454 Genome Sequencer FLX. Results We generated more that 3.6 million sequence reads of average length 250 bp for the MAQC A and B samples and introduced a data analysis pipeline for translating cDNA read counts into gene expression levels. Using BLAST, 90% of the reads mapped to the human genome and 64% of the reads mapped to the RefSeq database of well annotated genes with e-values ≤ 10-20. We measured gene expression levels in the A and B samples by counting the numbers of reads that mapped to individual RefSeq genes in multiple sequencing runs to evaluate the MAQC quality metrics for reproducibility, sensitivity, specificity, and accuracy and compared the results with DNA microarrays and Quantitative RT-PCR (QRTPCR from the MAQC studies. In addition, 88% of the reads were successfully aligned directly to the human genome using the AceView alignment programs with an average 90% sequence similarity to identify 137,899 unique exon junctions, including 22,193 new exon junctions not yet contained in the RefSeq database. Conclusion Using the MAQC metrics for evaluating the performance of gene expression platforms, the ExpressSeq results for gene expression levels showed excellent reproducibility, sensitivity, and specificity that improved systematically with increasing shotgun sequencing depth, and quantitative accuracy that was comparable to DNA microarrays and QRTPCR. In addition, a careful mapping of the reads to the genome using the AceView alignment programs shed new light on the complexity of the human transcriptome including the discovery of thousands of new splice variants.

Sequence-specific 1H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin.

Science.gov (United States)

Roy, M; Lee, R W; Kaarsholm, N C; Thøgersen, H; Brange, J; Dunn, M F

1990-06-12

The aromatic region of the 1H-FT-NMR spectrum of the biologically fully-potent, monomeric human insulin mutant, B9 Ser----Asp, B27 Thr----Glu has been investigated in D2O. At 1 to 5 mM concentrations, this mutant insulin is monomeric above pH 7.5. Coupling and amino acid classification of all aromatic signals is established via a combination of homonuclear one- and two-dimensional methods, including COSY, multiple quantum filters, selective spin decoupling and pH titrations. By comparisons with other insulin mutants and with chemically modified native insulins, all resonances in the aromatic region are given sequence-specific assignments without any reliance on the various crystal structures reported for insulin. These comparisons also give the sequence-specific assignments of most of the aromatic resonances of the mutant insulins B16 Tyr----Glu, B27 Thr----Glu and B25 Phe----Asp and the chemically modified species des-(B23-B30) insulin and monoiodo-Tyr A14 insulin. Chemical dispersion of the assigned resonances, ring current perturbations and comparisons at high pH have made possible the assignment of the aromatic resonances of human insulin, and these studies indicate that the major structural features of the human insulin monomer (including those critical to biological function) are also present in the monomeric mutant.

Haematobia irritans dataset of raw sequence reads from Illumina-based transcriptome sequencing of specific tissues and life stages

Science.gov (United States)

Illumina HiSeq technology was used to sequence the transcriptome from various dissected tissues and life stages from the horn fly, Haematobia irritans. These samples include eggs (0, 2, 4, and 9 hours post-oviposition), adult fly gut, adult fly legs, adult fly malpighian tubule, adult fly ovary, adu...

Mapping of a Novel Race Specific Resistance Gene to Phytophthora Root Rot of Pepper (Capsicum annuum) Using Bulked Segregant Analysis Combined with Specific Length Amplified Fragment Sequencing Strategy.

Science.gov (United States)

Xu, Xiaomei; Chao, Juan; Cheng, Xueli; Wang, Rui; Sun, Baojuan; Wang, Hengming; Luo, Shaobo; Xu, Xiaowan; Wu, Tingquan; Li, Ying

2016-01-01

Phytophthora root rot caused by Phytophthora capsici (P. capsici) is a serious limitation to pepper production in Southern China, with high temperature and humidity. Mapping PRR resistance genes can provide linked DNA markers for breeding PRR resistant varieties by molecular marker-assisted selection (MAS). Two BC1 populations and an F2 population derived from a cross between P. capsici-resistant accession, Criollo de Morelos 334 (CM334) and P. capsici-susceptible accession, New Mexico Capsicum Accession 10399 (NMCA10399) were used to investigate the genetic characteristics of PRR resistance. PRR resistance to isolate Byl4 (race 3) was controlled by a single dominant gene, PhR10, that was mapped to an interval of 16.39Mb at the end of the long arm of chromosome 10. Integration of bulked segregant analysis (BSA) and Specific Length Amplified Fragment sequencing (SLAF-seq) provided an efficient genetic mapping strategy. Ten polymorphic Simple Sequence Repeat (SSR) markers were found within this region and used to screen the genotypes of 636 BC1 plants, delimiting PhR10 to a 2.57 Mb interval between markers P52-11-21 (1.5 cM away) and P52-11-41 (1.1 cM). A total of 163 genes were annotated within this region and 31 were predicted to be associated with disease resistance. PhR10 is a novel race specific gene for PRR, and this paper describes linked SSR markers suitable for marker-assisted selection of PRR resistant varieties, also laying a foundation for cloning the resistance gene.

Mapping of a Novel Race Specific Resistance Gene to Phytophthora Root Rot of Pepper (Capsicum annuum Using Bulked Segregant Analysis Combined with Specific Length Amplified Fragment Sequencing Strategy.

Directory of Open Access Journals (Sweden)

Xiaomei Xu

Full Text Available Phytophthora root rot caused by Phytophthora capsici (P. capsici is a serious limitation to pepper production in Southern China, with high temperature and humidity. Mapping PRR resistance genes can provide linked DNA markers for breeding PRR resistant varieties by molecular marker-assisted selection (MAS. Two BC1 populations and an F2 population derived from a cross between P. capsici-resistant accession, Criollo de Morelos 334 (CM334 and P. capsici-susceptible accession, New Mexico Capsicum Accession 10399 (NMCA10399 were used to investigate the genetic characteristics of PRR resistance. PRR resistance to isolate Byl4 (race 3 was controlled by a single dominant gene, PhR10, that was mapped to an interval of 16.39Mb at the end of the long arm of chromosome 10. Integration of bulked segregant analysis (BSA and Specific Length Amplified Fragment sequencing (SLAF-seq provided an efficient genetic mapping strategy. Ten polymorphic Simple Sequence Repeat (SSR markers were found within this region and used to screen the genotypes of 636 BC1 plants, delimiting PhR10 to a 2.57 Mb interval between markers P52-11-21 (1.5 cM away and P52-11-41 (1.1 cM. A total of 163 genes were annotated within this region and 31 were predicted to be associated with disease resistance. PhR10 is a novel race specific gene for PRR, and this paper describes linked SSR markers suitable for marker-assisted selection of PRR resistant varieties, also laying a foundation for cloning the resistance gene.

Exome sequencing in an admixed isolated population indicates NFXL1 variants confer a risk for specific language impairment.

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Pía Villanueva

2015-03-01

Full Text Available Children affected by Specific Language Impairment (SLI fail to acquire age appropriate language skills despite adequate intelligence and opportunity. SLI is highly heritable, but the understanding of underlying genetic mechanisms has proved challenging. In this study, we use molecular genetic techniques to investigate an admixed isolated founder population from the Robinson Crusoe Island (Chile, who are affected by a high incidence of SLI, increasing the power to discover contributory genetic factors. We utilize exome sequencing in selected individuals from this population to identify eight coding variants that are of putative significance. We then apply association analyses across the wider population to highlight a single rare coding variant (rs144169475, Minor Allele Frequency of 4.1% in admixed South American populations in the NFXL1 gene that confers a nonsynonymous change (N150K and is significantly associated with language impairment in the Robinson Crusoe population (p = 2.04 × 10-4, 8 variants tested. Subsequent sequencing of NFXL1 in 117 UK SLI cases identified four individuals with heterozygous variants predicted to be of functional consequence. We conclude that coding variants within NFXL1 confer an increased risk of SLI within a complex genetic model.

Optimization of sequence alignment for simple sequence repeat regions

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Ogbonnaya Francis C

2011-07-01

Full Text Available Abstract Background Microsatellites, or simple sequence repeats (SSRs, are tandemly repeated DNA sequences, including tandem copies of specific sequences no longer than six bases, that are distributed in the genome. SSR has been used as a molecular marker because it is easy to detect and is used in a range of applications, including genetic diversity, genome mapping, and marker assisted selection. It is also very mutable because of slipping in the DNA polymerase during DNA replication. This unique mutation increases the insertion/deletion (INDELs mutation frequency to a high ratio - more than other types of molecular markers such as single nucleotide polymorphism (SNPs. SNPs are more frequent than INDELs. Therefore, all designed algorithms for sequence alignment fit the vast majority of the genomic sequence without considering microsatellite regions, as unique sequences that require special consideration. The old algorithm is limited in its application because there are many overlaps between different repeat units which result in false evolutionary relationships. Findings To overcome the limitation of the aligning algorithm when dealing with SSR loci, a new algorithm was developed using PERL script with a Tk graphical interface. This program is based on aligning sequences after determining the repeated units first, and the last SSR nucleotides positions. This results in a shifting process according to the inserted repeated unit type. When studying the phylogenic relations before and after applying the new algorithm, many differences in the trees were obtained by increasing the SSR length and complexity. However, less distance between different linage had been observed after applying the new algorithm. Conclusions The new algorithm produces better estimates for aligning SSR loci because it reflects more reliable evolutionary relations between different linages. It reduces overlapping during SSR alignment, which results in a more realistic

Generation of species-specific DNA probes for Leishmania aethiopica

NARCIS (Netherlands)

Laskay, T.; Kiessling, R.; Rinke deWit, T. F.; Wirth, D. F.

1991-01-01

We report here the cloning of kinetoplast DNA (kDNA) sequences from Leishmania aethiopica in order to develop a specific and sensitive method for the identification of the parasite. Analysis of the cloned kDNA sequences showed different taxonomic specificities demonstrating sequence diversity within

Tracking TCRβ sequence clonotype expansions during antiviral therapy using high-throughput sequencing of the hypervariable region

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Mark W Robinson

2016-04-01

Full Text Available To maintain a persistent infection viruses such as hepatitis C virus (HCV employ a range of mechanisms that subvert protective T cell responses. The suppression of antigen-specific T cell responses by HCV hinders efforts to profile T cell responses during chronic infection and antiviral therapy. Conventional methods of detecting antigen-specific T cells utilise either antigen stimulation (e.g. ELISpot, proliferation assays, cytokine production or antigen-loaded tetramer staining. This limits the ability to profile T cell responses during chronic infection due to suppressed effector function and the requirement for prior knowledge of antigenic viral peptide sequences. Recently high-throughput sequencing (HTS technologies have been developed for the analysis of T cell repertoires. In the present study we have assessed the feasibility of HTS of the TCRβ complementarity determining region (CDR3 to track T cell expansions in an antigen-independent manner. Using sequential blood samples from HCV-infected individuals undergoing anti-viral therapy we were able to measure the population frequencies of >35,000 TCRβ sequence clonotypes in each individual over the course of 12 weeks. TRBV/TRBJ gene segment usage varied markedly between individuals but remained relatively constant within individuals across the course of therapy. Despite this stable TRBV/TRBJ gene segment usage, a number of TCRβ sequence clonotypes showed dramatic changes in read frequency. These changes could not be linked to therapy outcomes in the present study however the TCRβ CDR3 sequences with the largest fold changes did include sequences with identical TRBV/TRBJ gene segment usage and high joining region homology to previously published CDR3 sequences from HCV-specific T cells targeting the HLA-B*0801-restricted 1395HSKKKCDEL1403 and HLA-A*0101–restricted 1435ATDALMTGY1443 epitopes. The pipeline developed in this proof of concept study provides a platform for the design of

DNA minor groove targeted alkylating agents based on bisbenzimidazole carriers: synthesis, cytotoxicity and sequence-specificity of DNA alkylation.

Science.gov (United States)

Smaill, J B; Fan, J Y; Denny, W A

1998-12-01

A series of bisbenzimidazoles bearing a variety of alkylating agents [ortho- and meta-mustards, imidazolebis(hydroxymethyl), imidazolebis(methylcarbamate) and pyrrolebis(hydroxymethyl)], appended by a propyl linker chain, were prepared and investigated for sequence-specificity of DNA alkylation and their cytotoxicity. Previous work has shown that, for para-aniline mustards, a propyl linker is optimal for cytotoxicity. Alkaline cleavage assays using a variety of different labelled oligonucleotides showed that the preferred sequences for adenine alkylation were 5'-TTTANANAANN and 5'-ATTANANAANN (underlined bases show the drug alkylation sites), with AT-rich sequences required on both the 5' and 3' sides of the alkylated adenine. The different aniline mustards showed little variation in alkylation pattern and similar efficiencies of DNA cross-link formation despite the changes in orientation and positioning of the mustard, suggesting that the propyl linker has some flexibility. The imidazole- and pyrrolebis(hydroxymethyl) alkylators showed no DNA strand cleavage following base treatment, indicating that no guanine or adenine N3 or N7 adducts were formed. Using the PCR-based polymerase stop assay, these alkylators showed PCR blocks at 5'-C*G sites (the * nucleotide indicates the blocked site), particularly at 5'-TAC*GA 5'-AGC*GGA, and 5'-AGCC*GGT sequences, caused by guanine 2-NH2 lesions on the opposite strand. Only the (more reactive) imidazolebis(methylcarbamoyl) and pyrrolebis(hydroxymethyl) alkylators demonstrated interstrand cross-linking ability. All of the bifunctional mustards showed large (approximately 100-fold) increases in cytotoxicity over chlorambucil, with the corresponding monofunctional mustards being 20- to 60-fold less cytotoxic. These results suggest that in the mustards the propyl linker provides sufficient flexibility to achieve delivery of the alkylator to favoured (adenine N3) sites in the minor groove, regardless of its exact geometry with

Error Analysis of Deep Sequencing of Phage Libraries: Peptides Censored in Sequencing

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Wadim L. Matochko

2013-01-01

Full Text Available Next-generation sequencing techniques empower selection of ligands from phage-display libraries because they can detect low abundant clones and quantify changes in the copy numbers of clones without excessive selection rounds. Identification of errors in deep sequencing data is the most critical step in this process because these techniques have error rates >1%. Mechanisms that yield errors in Illumina and other techniques have been proposed, but no reports to date describe error analysis in phage libraries. Our paper focuses on error analysis of 7-mer peptide libraries sequenced by Illumina method. Low theoretical complexity of this phage library, as compared to complexity of long genetic reads and genomes, allowed us to describe this library using convenient linear vector and operator framework. We describe a phage library as N×1 frequency vector n=ni, where ni is the copy number of the ith sequence and N is the theoretical diversity, that is, the total number of all possible sequences. Any manipulation to the library is an operator acting on n. Selection, amplification, or sequencing could be described as a product of a N×N matrix and a stochastic sampling operator (Sa. The latter is a random diagonal matrix that describes sampling of a library. In this paper, we focus on the properties of Sa and use them to define the sequencing operator (Seq. Sequencing without any bias and errors is Seq=Sa IN, where IN is a N×N unity matrix. Any bias in sequencing changes IN to a nonunity matrix. We identified a diagonal censorship matrix (CEN, which describes elimination or statistically significant downsampling, of specific reads during the sequencing process.

Rapid microsatellite marker development for African mahogany (Khaya senegalensis, Meliaceae) using next-generation sequencing and assessment of its intra-specific genetic diversity.

Science.gov (United States)

Karan, M; Evans, D S; Reilly, D; Schulte, K; Wright, C; Innes, D; Holton, T A; Nikles, D G; Dickinson, G R

2012-03-01

Khaya senegalensis (African mahogany or dry-zone mahogany) is a high-value hardwood timber species with great potential for forest plantations in northern Australia. The species is distributed across the sub-Saharan belt from Senegal to Sudan and Uganda. Because of heavy exploitation and constraints on natural regeneration and sustainable planting, it is now classified as a vulnerable species. Here, we describe the development of microsatellite markers for K. senegalensis using next-generation sequencing to assess its intra-specific diversity across its natural range, which is a key for successful breeding programs and effective conservation management of the species. Next-generation sequencing yielded 93,943 sequences with an average read length of 234 bp. The assembled sequences contained 1030 simple sequence repeats, with primers designed for 522 microsatellite loci. Twenty-one microsatellite loci were tested with 11 showing reliable amplification and polymorphism in K. senegalensis. The 11 novel microsatellites, together with one previously published, were used to assess 73 accessions belonging to the Australian K. senegalensis domestication program, sampled from across the natural range of the species. STRUCTURE analysis shows two major clusters, one comprising mainly accessions from west Africa (Senegal to Benin) and the second based in the far eastern limits of the range in Sudan and Uganda. Higher levels of genetic diversity were found in material from western Africa. This suggests that new seed collections from this region may yield more diverse genotypes than those originating from Sudan and Uganda in eastern Africa. © 2011 Blackwell Publishing Ltd.

Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies

Energy Technology Data Exchange (ETDEWEB)

Catfish Genome Consortium; Wang, Shaolin; Peatman, Eric; Abernathy, Jason; Waldbieser, Geoff; Lindquist, Erika; Richardson, Paul; Lucas, Susan; Wang, Mei; Li, Ping; Thimmapuram, Jyothi; Liu, Lei; Vullaganti, Deepika; Kucuktas, Huseyin; Murdock, Christopher; Small, Brian C; Wilson, Melanie; Liu, Hong; Jiang, Yanliang; Lee, Yoona; Chen, Fei; Lu, Jianguo; Wang, Wenqi; Xu, Peng; Somridhivej, Benjaporn; Baoprasertkul, Puttharat; Quilang, Jonas; Sha, Zhenxia; Bao, Baolong; Wang, Yaping; Wang, Qun; Takano, Tomokazu; Nandi, Samiran; Liu, Shikai; Wong, Lilian; Kaltenboeck, Ludmilla; Quiniou, Sylvie; Bengten, Eva; Miller, Norman; Trant, John; Rokhsar, Daniel; Liu, Zhanjiang

2010-03-23

Background-Through the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energy's Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification. Results-A total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35percent of the unique sequences had significant similarities to known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis. Conclusions-This project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.

Comparative analyses of six solanaceous transcriptomes reveal a high degree of sequence conservation and species-specific transcripts

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Ouyang Shu

2005-09-01

Full Text Available Abstract Background The Solanaceae is a family of closely related species with diverse phenotypes that have been exploited for agronomic purposes. Previous studies involving a small number of genes suggested sequence conservation across the Solanaceae. The availability of large collections of Expressed Sequence Tags (ESTs for the Solanaceae now provides the opportunity to assess sequence conservation and divergence on a genomic scale. Results All available ESTs and Expressed Transcripts (ETs, 449,224 sequences for six Solanaceae species (potato, tomato, pepper, petunia, tobacco and Nicotiana benthamiana, were clustered and assembled into gene indices. Examination of gene ontologies revealed that the transcripts within the gene indices encode a similar suite of biological processes. Although the ESTs and ETs were derived from a variety of tissues, 55–81% of the sequences had significant similarity at the nucleotide level with sequences among the six species. Putative orthologs could be identified for 28–58% of the sequences. This high degree of sequence conservation was supported by expression profiling using heterologous hybridizations to potato cDNA arrays that showed similar expression patterns in mature leaves for all six solanaceous species. 16–19% of the transcripts within the six Solanaceae gene indices did not have matches among Solanaceae, Arabidopsis, rice or 21 other plant gene indices. Conclusion Results from this genome scale analysis confirmed a high level of sequence conservation at the nucleotide level of the coding sequence among Solanaceae. Additionally, the results indicated that part of the Solanaceae transcriptome is likely to be unique for each species.

AlignMe—a membrane protein sequence alignment web server

Science.gov (United States)

Stamm, Marcus; Staritzbichler, René; Khafizov, Kamil; Forrest, Lucy R.

2014-01-01

We present a web server for pair-wise alignment of membrane protein sequences, using the program AlignMe. The server makes available two operational modes of AlignMe: (i) sequence to sequence alignment, taking two sequences in fasta format as input, combining information about each sequence from multiple sources and producing a pair-wise alignment (PW mode); and (ii) alignment of two multiple sequence alignments to create family-averaged hydropathy profile alignments (HP mode). For the PW sequence alignment mode, four different optimized parameter sets are provided, each suited to pairs of sequences with a specific similarity level. These settings utilize different types of inputs: (position-specific) substitution matrices, secondary structure predictions and transmembrane propensities from transmembrane predictions or hydrophobicity scales. In the second (HP) mode, each input multiple sequence alignment is converted into a hydrophobicity profile averaged over the provided set of sequence homologs; the two profiles are then aligned. The HP mode enables qualitative comparison of transmembrane topologies (and therefore potentially of 3D folds) of two membrane proteins, which can be useful if the proteins have low sequence similarity. In summary, the AlignMe web server provides user-friendly access to a set of tools for analysis and comparison of membrane protein sequences. Access is available at http://www.bioinfo.mpg.de/AlignMe PMID:24753425

Protecting genomic sequence anonymity with generalization lattices.

Science.gov (United States)

Malin, B A

2005-01-01

Current genomic privacy technologies assume the identity of genomic sequence data is protected if personal information, such as demographics, are obscured, removed, or encrypted. While demographic features can directly compromise an individual's identity, recent research demonstrates such protections are insufficient because sequence data itself is susceptible to re-identification. To counteract this problem, we introduce an algorithm for anonymizing a collection of person-specific DNA sequences. The technique is termed DNA lattice anonymization (DNALA), and is based upon the formal privacy protection schema of k -anonymity. Under this model, it is impossible to observe or learn features that distinguish one genetic sequence from k-1 other entries in a collection. To maximize information retained in protected sequences, we incorporate a concept generalization lattice to learn the distance between two residues in a single nucleotide region. The lattice provides the most similar generalized concept for two residues (e.g. adenine and guanine are both purines). The method is tested and evaluated with several publicly available human population datasets ranging in size from 30 to 400 sequences. Our findings imply the anonymization schema is feasible for the protection of sequences privacy. The DNALA method is the first computational disclosure control technique for general DNA sequences. Given the computational nature of the method, guarantees of anonymity can be formally proven. There is room for improvement and validation, though this research provides the groundwork from which future researchers can construct genomics anonymization schemas tailored to specific datasharing scenarios.

Comparison and evaluation of two exome capture kits and sequencing platforms for variant calling.

Science.gov (United States)

Zhang, Guoqiang; Wang, Jianfeng; Yang, Jin; Li, Wenjie; Deng, Yutian; Li, Jing; Huang, Jun; Hu, Songnian; Zhang, Bing

2015-08-05

To promote the clinical application of next-generation sequencing, it is important to obtain accurate and consistent variants of target genomic regions at low cost. Ion Proton, the latest updated semiconductor-based sequencing instrument from Life Technologies, is designed to provide investigators with an inexpensive platform for human whole exome sequencing that achieves a rapid turnaround time. However, few studies have comprehensively compared and evaluated the accuracy of variant calling between Ion Proton and Illumina sequencing platforms such as HiSeq 2000, which is the most popular sequencing platform for the human genome. The Ion Proton sequencer combined with the Ion TargetSeq Exome Enrichment Kit together make up TargetSeq-Proton, whereas SureSelect-Hiseq is based on the Agilent SureSelect Human All Exon v4 Kit and the HiSeq 2000 sequencer. Here, we sequenced exonic DNA from four human blood samples using both TargetSeq-Proton and SureSelect-HiSeq. We then called variants in the exonic regions that overlapped between the two exome capture kits (33.6 Mb). The rates of shared variant loci called by two sequencing platforms were from 68.0 to 75.3% in four samples, whereas the concordance of co-detected variant loci reached 99%. Sanger sequencing validation revealed that the validated rate of concordant single nucleotide polymorphisms (SNPs) (91.5%) was higher than the SNPs specific to TargetSeq-Proton (60.0%) or specific to SureSelect-HiSeq (88.3%). With regard to 1-bp small insertions and deletions (InDels), the Sanger sequencing validated rates of concordant variants (100.0%) and SureSelect-HiSeq-specific (89.6%) were higher than those of TargetSeq-Proton-specific (15.8%). In the sequencing of exonic regions, a combination of using of two sequencing strategies (SureSelect-HiSeq and TargetSeq-Proton) increased the variant calling specificity for concordant variant loci and the sensitivity for variant loci called by any one platform. However, for the

The glycan-specific sulfotransferase (R77W)GalNAc-4-ST1 putatively responsible for peeling skin syndrome has normal properties consistent with a simple sequence polymorphisim.

Science.gov (United States)

Fiete, Dorothy; Mi, Yiling; Beranek, Mary; Baenziger, Nancy L; Baenziger, Jacques U

2017-05-01

Expanded access to DNA sequencing now fosters ready detection of site-specific human genome alterations whose actual significance requires in-depth functional study to rule in or out disease-causing mutations. This is a particular concern for genomic sequence differences in glycosyltransferases, whose implications are often difficult to assess. A recent whole-exome sequencing study identifies (c.229 C > T) in the GalNAc-4-ST1 glycosyltransferase (CHST8) as a disease-causing missense R77W mutation yielding the genodermatosis peeling skin syndrome (PSS) when homozygous. Cabral et al. (Genomics. 2012;99:202-208) cite this sequence change as reducing keratinocyte GalNAc-4-ST1 activity, thus decreasing glycosaminoglycan sulfation, as the mechanism for this blistering disorder. Such an identification could point toward potential clinical and/or prenatal diagnosis of a harmful medical condition. However, GalNAc-4-ST1 has minimal activity toward glycosaminoglycans, instead modifying terminal β1,4-linked GalNAc on N- and O-linked oligosaccharides on specific glycoproteins. We find expression, processing and catalytic activity of GalNAc-4-ST1 completely equivalent between wild type and (R77W) sulfotransferases. Moreover, keratinocytes have little or no GalNAc-4-ST1 mRNA, indicating that they do not express GalNAc-4-ST1. In addition, loss-of-function of GalNAc-4-ST1 primarily presents as reproductive system aberrations rather than skin effects. These findings, an allele frequency of 0.004357, and a 10-fold difference in prevalence of CHST8 (c.299 C > T, R77W) across different ethnic groups, suggest that this sequence represents a "passenger" distributed polymorphism, a simple sequence variant form of the enzyme having normal activity, rather than a "driver" disease-causing mutation that accounts for PSS. This study presents an example for guiding biomedical research initiatives, as well as medical and personal/family perspectives, regarding newly-identified genomic sequence

antaRNA: ant colony-based RNA sequence design.

Science.gov (United States)

Kleinkauf, Robert; Mann, Martin; Backofen, Rolf

2015-10-01

RNA sequence design is studied at least as long as the classical folding problem. Although for the latter the functional fold of an RNA molecule is to be found ,: inverse folding tries to identify RNA sequences that fold into a function-specific target structure. In combination with RNA-based biotechnology and synthetic biology ,: reliable RNA sequence design becomes a crucial step to generate novel biochemical components. In this article ,: the computational tool antaRNA is presented. It is capable of compiling RNA sequences for a given structure that comply in addition with an adjustable full range objective GC-content distribution ,: specific sequence constraints and additional fuzzy structure constraints. antaRNA applies ant colony optimization meta-heuristics and its superior performance is shown on a biological datasets. http://www.bioinf.uni-freiburg.de/Software/antaRNA CONTACT: backofen@informatik.uni-freiburg.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

Sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6, and V9 domains reveal highly species-specific variations within the genus Agrocybe.

Science.gov (United States)

Gonzalez, P; Labarère, J

1998-11-01

A comparative study of variable domains V4, V6, and V9 of the mitochondrial small-subunit (SSU) rRNA was carried out with the genus Agrocybe by PCR amplification of 42 wild isolates belonging to 10 species, Agrocybe aegerita, Agrocybe dura, Agrocybe chaxingu, Agrocybe erebia, Agrocybe firma, Agrocybe praecox, Agrocybe paludosa, Agrocybe pediades, Agrocybe alnetorum, and Agrocybe vervacti. Sequencing of the PCR products showed that the three domains in the isolates belonging to the same species were the same length and had the same sequence, while variations were found among the 10 species. Alignment of the sequences showed that nucleotide motifs encountered in the smallest sequence of each variable domain were also found in the largest sequence, indicating that the sequences evolved by insertion-deletion events. Determination of the secondary structure of each domain revealed that the insertion-deletion events commonly occurred in regions not directly involved in the secondary structure (i.e., the loops). Moreover, conserved sequences ranging from 4 to 25 nucleotides long were found at the beginning and end of each domain and could constitute genus-specific sequences. Comparisons of the V4, V6, and V9 secondary structures resulted in identification of the following four groups: (i) group I, which was characterized by the presence of additional P23-1 and P23-3 helices in the V4 domain and the lack of the P49-1 helix in V9 and included A. aegerita, A. chaxingu, and A. erebia; (ii) group II, which had the P23-3 helix in V4 and the P49-1 helix in V9 and included A. pediades; (iii) group III, which did not have additional helices in V4, had the P49-1 helix in V9 and included A. paludosa, A. firma, A. alnetorum, and A. praecox; and (iv) group IV, which lacked both the V4 additional helices and the P49-1 helix in V9 and included A. vervacti and A. dura. This grouping of species was supported by the structure of a consensus tree based on the variable domain sequences. The

On the optimal trimming of high-throughput mRNA sequence data

Directory of Open Access Journals (Sweden)

Matthew D MacManes

2014-01-01

Full Text Available The widespread and rapid adoption of high-throughput sequencing technologies has afforded researchers the opportunity to gain a deep understanding of genome level processes that underlie evolutionary change, and perhaps more importantly, the links between genotype and phenotype. In particular, researchers interested in functional biology and adaptation have used these technologies to sequence mRNA transcriptomes of specific tissues, which in turn are often compared to other tissues, or other individuals with different phenotypes. While these techniques are extremely powerful, careful attention to data quality is required. In particular, because high-throughput sequencing is more error-prone than traditional Sanger sequencing, quality trimming of sequence reads should be an important step in all data processing pipelines. While several software packages for quality trimming exist, no general guidelines for the specifics of trimming have been developed. Here, using empirically derived sequence data, I provide general recommendations regarding the optimal strength of trimming, specifically in mRNA-Seq studies. Although very aggressive quality trimming is common, this study suggests that a more gentle trimming, specifically of those nucleotides whose Phred score < 2 or < 5, is optimal for most studies across a wide variety of metrics.

SWORDS: A statistical tool for analysing large DNA sequences

Indian Academy of Sciences (India)

Unknown

These techniques are based on frequency distributions of DNA words in a large sequence, and have been packaged into a software called SWORDS. Using sequences available in ... tions with the cellular processes like recombination, replication .... in DNA sequences using certain specific probability laws. (Pevzner et al ...

Characterization and sequence analysis of cysteine and glycine-rich ...

African Journals Online (AJOL)

Primers specific for CSRP3 were designed using known cDNA sequences of Bos taurus published in database with different accession numbers. Polymerase chain reaction (PCR) was performed and products were purified and sequenced. Sequence analysis and alignment were carried out using CLUSTAL W (1.83).

The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection.

Science.gov (United States)

Winata, Patrick; Williams, Marissa; McGowan, Eileen; Nassif, Najah; van Zandwijk, Nico; Reid, Glen

2017-11-17

MicroRNAs are frequently downregulated in cancer, and restoring expression has tumour suppressive activity in tumour cells. Our recent phase I clinical trial investigated microRNA-based therapy in patients with malignant pleural mesothelioma. Treatment with TargomiRs, microRNA mimics with novel sequence packaged in EGFR antibody-targeted bacterial minicells, revealed clear signs of clinical activity. In order to detect delivery of microRNA mimics to tumour cells in future clinical trials, we tested hydrolysis probe-based assays specific for the sequence of the novel mimics in transfected mesothelioma cell lines using RT-qPCR. The custom assays efficiently and specifically amplified the consensus mimics. However, we found that these assays gave a signal when total RNA from untransfected and control mimic-transfected cells were used as templates. Further investigation revealed that the reverse transcription step using stem-loop primers appeared to introduce substantial non-specific amplification with either total RNA or synthetic RNA templates. This suggests that reverse transcription using stem-loop primers suffers from an intrinsic lack of specificity for the detection of highly similar microRNAs in the same family, especially when analysing total RNA. These results suggest that RT-qPCR is unlikely to be an effective means to detect delivery of microRNA mimic-based drugs to tumour cells in patients.

Stimulation of topoisomerase II mediated DNA cleavage at specific sequence elements by the 2-nitroimidazole Ro 15-0216

International Nuclear Information System (INIS)

Sorensen, B.S.; Jensen, P.S.; Andersen, A.H.; Christiansen, K.; Alsner, J.; Thomsen, B.; Westergaard, O.

1990-01-01

The effect of the 2-nitroimidazole Ro 15-0216 upon the interaction between purified topoisomerase II and its DNA substrate was investigated. The cleavage reaction in the presence of this DNA-nonintercalative drug took place with the hallmarks of a regular topoisomerase II mediated cleavage reaction, including covalent linkage of the enzyme to the cleaved DNA. In the presence of Ro 15-0216, topoisomerase II mediated cleavage was extensively stimulated at major cleavage sites of which only one existed in the 4363 base pair pBR322 molecule. The sites stimulated by Ro 15-0216 shared a pronounced sequence homology, indicating that a specific nucleotide sequence is crucial for the action of this drug. The effect of Ro 15-0216 thus differs from that of the clinically important topoisomerase II targeted agents such as mAMSA, VM26, and VP16, which enhance enzyme-mediated cleavage at a multiple number of sites. In contrast to the previous described drugs, Ro 15-0216 did not exert any inhibitory effect on the enzyme's catalytic activity. This observation might be ascribed to the low stability of the cleavage complexes formed in the presence of Ro 15-0216 as compared to the stability of the ones formed in the presence of traditional topoisomerase II targeted drugs

Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase

Science.gov (United States)

Morellet, Nelly; Li, Xianghong; Wieninger, Silke A; Taylor, Jennifer L; Bischerour, Julien; Moriau, Séverine; Lescop, Ewen; Bardiaux, Benjamin; Mathy, Nathalie; Assrir, Nadine; Bétermier, Mireille; Nilges, Michael; Hickman, Alison B; Dyda, Fred; Craig, Nancy L; Guittet, Eric

2018-01-01

Abstract The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure–function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5′-TGCGT-3′/3′-ACGCA-5′ motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity. PMID:29385532

Novel primer specific false terminations during DNA sequencing reactions: danger of inaccuracy of mutation analysis in molecular diagnostics

Science.gov (United States)

Anwar, R; Booth, A; Churchill, A J; Markham, A F

1996-01-01

The determination of nucleotide sequence is fundamental to the identification and molecular analysis of genes. Direct sequencing of PCR products is now becoming a commonplace procedure for haplotype analysis, and for defining mutations and polymorphism within genes, particularly for diagnostic purposes. A previously unrecognised phenomenon, primer related variability, observed in sequence data generated using Taq cycle sequencing and T7 Sequenase sequencing, is reported. This suggests that caution is necessary when interpreting DNA sequence data. This is particularly important in situations where treatment may be dependent on the accuracy of the molecular diagnosis. Images PMID:16696096

Nature and distribution of feline sarcoma virus nucleotide sequences.

Science.gov (United States)

Frankel, A E; Gilbert, J H; Porzig, K J; Scolnick, E M; Aaronson, S A

1979-01-01

The genomes of three independent isolates of feline sarcoma virus (FeSV) were compared by molecular hybridization techniques. Using complementary DNAs prepared from two strains, SM- and ST-FeSV, common complementary DNA'S were selected by sequential hybridization to FeSV and feline leukemia virus RNAs. These DNAs were shown to be highly related among the three independent sarcoma virus isolates. FeSV-specific complementary DNAs were prepared by selection for hybridization by the homologous FeSV RNA and against hybridization by fline leukemia virus RNA. Sarcoma virus-specific sequences of SM-FeSV were shown to differ from those of either ST- or GA-FeSV strains, whereas ST-FeSV-specific DNA shared extensive sequence homology with GA-FeSV. By molecular hybridization, each set of FeSV-specific sequences was demonstrated to be present in normal cat cellular DNA in approximately one copy per haploid genome and was conserved throughout Felidae. In contrast, FeSV-common sequences were present in multiple DNA copies and were found only in Mediterranean cats. The present results are consistent with the concept that each FeSV strain has arisen by a mechanism involving recombination between feline leukemia virus and cat cellular DNA sequences, the latter represented within the cat genome in a manner analogous to that of a cellular gene. PMID:225544

Facilitating genome navigation : survey sequencing and dense radiation-hybrid gene mapping

NARCIS (Netherlands)

Hitte, C; Madeoy, J; Kirkness, EF; Priat, C; Lorentzen, TD; Senger, F; Thomas, D; Derrien, T; Ramirez, C; Scott, C; Evanno, G; Pullar, B; Cadieu, E; Oza, [No Value; Lourgant, K; Jaffe, DB; Tacher, S; Dreano, S; Berkova, N; Andre, C; Deloukas, P; Fraser, C; Lindblad-Toh, K; Ostrander, EA; Galibert, F

Accurate and comprehensive sequence coverage for large genomes has been restricted to only a few species of specific interest. Lower sequence coverage (survey sequencing) of related species can yield a wealth of information about gene content and putative regulatory elements. But survey sequences

Differentiation of mycoplasmalike organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO.

Science.gov (United States)

Jarausch, W; Saillard, C; Dosba, F; Bové, J M

1994-01-01

A 1.8-kb chromosomal DNA fragment of the mycoplasmalike organism (MLO) associated with apple proliferation was sequenced. Three putative open reading frames were observed on this fragment. The protein encoded by open reading frame 2 shows significant homologies with bacterial nitroreductases. From the nucleotide sequence four primer pairs for PCR were chosen to specifically amplify DNA from MLOs associated with European diseases of fruit trees. Primer pairs specific for (i) Malus-affecting MLOs, (ii) Malus- and Prunus-affecting MLOs, and (iii) Malus-, Prunus-, and Pyrus-affecting MLOs were obtained. Restriction enzyme analysis of the amplification products revealed restriction fragment length polymorphisms between Malus-, Prunus, and Pyrus-affecting MLOs as well as between different isolates of the apple proliferation MLO. No amplification with either primer pair could be obtained with DNA from 12 different MLOs experimentally maintained in periwinkle. Images PMID:7916180

Machine-Checked Sequencer for Critical Embedded Code Generator

Science.gov (United States)

Izerrouken, Nassima; Pantel, Marc; Thirioux, Xavier

This paper presents the development of a correct-by-construction block sequencer for GeneAuto a qualifiable (according to DO178B/ED12B recommendation) automatic code generator. It transforms Simulink models to MISRA C code for safety critical systems. Our approach which combines classical development process and formal specification and verification using proof-assistants, led to preliminary fruitful exchanges with certification authorities. We present parts of the classical user and tools requirements and derived formal specifications, implementation and verification for the correctness and termination of the block sequencer. This sequencer has been successfully applied to real-size industrial use cases from various transportation domain partners and led to requirement errors detection and a correct-by-construction implementation.

Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro

International Nuclear Information System (INIS)

Kawasaki, E.S.; Clark, S.S.; Coyne, M.Y.; Smith, S.D.; Champlin, R.; Witte, O.N.; McCormick, F.P.

1988-01-01

The Philadelphia chromosome is present in more than 95% of chronic myeloid leukemia patients and 13% of acute lymphocytic leukemia patients. The Philadelphia translocation, t(9;22), fuses the BCR and ABL genes resulting in the expression of leukemia-specific, chimeric BCR-ABL messenger RNAs. To facilitate diagnosis of these leukemias, the authors have developed a method of amplifying and detecting only the unique mRNA sequences, using an extension of the polymerase chain reaction technique. Diagnosis of chronic myeloid and acute lymphocytic leukemias by this procedure is rapid, much more sensitive than existing protocols, and independent of the presence or absence of an identifiable Philadelphia chromosome

Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM™ Platform.

Science.gov (United States)

Vanni, Irene; Coco, Simona; Truini, Anna; Rusmini, Marta; Dal Bello, Maria Giovanna; Alama, Angela; Banelli, Barbara; Mora, Marco; Rijavec, Erika; Barletta, Giulia; Genova, Carlo; Biello, Federica; Maggioni, Claudia; Grossi, Francesco

2015-12-03

Next-generation sequencing (NGS) is a cost-effective technology capable of screening several genes simultaneously; however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE) blocks and circulating free DNA (cfDNA). Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC) patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs) and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.

Filovirus Glycoprotein Sequence, Structure and Virulence

OpenAIRE

Phillips, J. C.

2014-01-01

Leading Ebola subtypes exhibit a wide mortality range, here explained at the molecular level by using fractal hydropathic scaling of amino acid sequences based on protein self-organized criticality. Specific hydrophobic features in the hydrophilic mucin-like domain suffice to account for the wide mortality range. Significance statement: Ebola virus is spreading rapidly in Africa. The connection between protein amino acid sequence and mortality is identified here.

GuiTope: an application for mapping random-sequence peptides to protein sequences.

Science.gov (United States)

Halperin, Rebecca F; Stafford, Phillip; Emery, Jack S; Navalkar, Krupa Arun; Johnston, Stephen Albert

2012-01-03

Random-sequence peptide libraries are a commonly used tool to identify novel ligands for binding antibodies, other proteins, and small molecules. It is often of interest to compare the selected peptide sequences to the natural protein binding partners to infer the exact binding site or the importance of particular residues. The ability to search a set of sequences for similarity to a set of peptides may sometimes enable the prediction of an antibody epitope or a novel binding partner. We have developed a software application designed specifically for this task. GuiTope provides a graphical user interface for aligning peptide sequences to protein sequences. All alignment parameters are accessible to the user including the ability to specify the amino acid frequency in the peptide library; these frequencies often differ significantly from those assumed by popular alignment programs. It also includes a novel feature to align di-peptide inversions, which we have found improves the accuracy of antibody epitope prediction from peptide microarray data and shows utility in analyzing phage display datasets. Finally, GuiTope can randomly select peptides from a given library to estimate a null distribution of scores and calculate statistical significance. GuiTope provides a convenient method for comparing selected peptide sequences to protein sequences, including flexible alignment parameters, novel alignment features, ability to search a database, and statistical significance of results. The software is available as an executable (for PC) at http://www.immunosignature.com/software and ongoing updates and source code will be available at sourceforge.net.

GuiTope: an application for mapping random-sequence peptides to protein sequences

Directory of Open Access Journals (Sweden)

Halperin Rebecca F

2012-01-01

Full Text Available Abstract Background Random-sequence peptide libraries are a commonly used tool to identify novel ligands for binding antibodies, other proteins, and small molecules. It is often of interest to compare the selected peptide sequences to the natural protein binding partners to infer the exact binding site or the importance of particular residues. The ability to search a set of sequences for similarity to a set of peptides may sometimes enable the prediction of an antibody epitope or a novel binding partner. We have developed a software application designed specifically for this task. Results GuiTope provides a graphical user interface for aligning peptide sequences to protein sequences. All alignment parameters are accessible to the user including the ability to specify the amino acid frequency in the peptide library; these frequencies often differ significantly from those assumed by popular alignment programs. It also includes a novel feature to align di-peptide inversions, which we have found improves the accuracy of antibody epitope prediction from peptide microarray data and shows utility in analyzing phage display datasets. Finally, GuiTope can randomly select peptides from a given library to estimate a null distribution of scores and calculate statistical significance. Conclusions GuiTope provides a convenient method for comparing selected peptide sequences to protein sequences, including flexible alignment parameters, novel alignment features, ability to search a database, and statistical significance of results. The software is available as an executable (for PC at http://www.immunosignature.com/software and ongoing updates and source code will be available at sourceforge.net.

HDP2: a ribosomal DNA (NTS-ETS) sequence as a target for species-specific molecular diagnosis of intestinal taeniasis in humans.

Science.gov (United States)

Flores, María D; Gonzalez, Luis M; Hurtado, Carolina; Motta, Yamileth Monje; Domínguez-Hidalgo, Cristina; Merino, Francisco Jesús; Perteguer, María J; Gárate, Teresa

2018-02-27

Taenia solium, T. asiatica and T. saginata tapeworms cause human taeniasis and are the origin of porcine and bovine cysticercosis. Furthermore, T. solium eggs can cause human cysticercosis, with neurocysticercosis being the most serious form of the disease. These helminth infections are neglected tropical diseases and are endemic in several countries in the Americas, Asia and Africa. As a result of globalization, migration in particular, the infections have been extending to non-endemic territories. Species-specific diagnosis of taeniasis is subject to drawbacks that could be resolved using molecular approaches. In the present study, conventional and real-time amplification protocols (cPCR and qPCR) based on the T. saginata HDP2 sequence were applied in the differential diagnosis of taeniasis (T. saginata, T. solium) in both fecal samples and proglottids expelled by patients. The HDP2 homolog in T. solium was cloned and characterized. Semi-nested cPCR and qPCR (Sn-HDP2 cPCR and Sn-HDP2 qPCR) amplified T. saginata and T. solium DNA, with an analytical sensitivity of 40 and 400 fg, respectively, and identically in both protocols. Eighteen taeniasis patients were diagnosed directly with T. saginata or T. solium, either from proglottids or fecal samples with/without eggs (detected using microscopy), based on the optimized Sn-HDP2 qPCR. After cloning, the T. solium HDP2 homolog sequence was confirmed to be a ribosomal sequence. The HDP2 fragment corresponded to a non-transcribed sequence/external transcribed repeat (NTS/ETS) of ribosomal DNA. Compared with the T. saginata HDP2 homolog, the T solium HDP2 sequence lacked the first 900 nt at the 5' end and showed nucleotide substitutions and small deletions. Sn-HDP2 cPCR and Sn-HDP2 qPCR were set up for the diagnosis of human taeniasis, using proglottids and fecal samples from affected patients. The new Sn-HDP2 qPCR protocol was the best option, as it directly differentiated T. saginata from T. solium. The diagnosis of

Multineuronal Spike Sequences Repeat with Millisecond Precision

Directory of Open Access Journals (Sweden)

Koki eMatsumoto

2013-06-01

Full Text Available Cortical microcircuits are nonrandomly wired by neurons. As a natural consequence, spikes emitted by microcircuits are also nonrandomly patterned in time and space. One of the prominent spike organizations is a repetition of fixed patterns of spike series across multiple neurons. However, several questions remain unsolved, including how precisely spike sequences repeat, how the sequences are spatially organized, how many neurons participate in sequences, and how different sequences are functionally linked. To address these questions, we monitored spontaneous spikes of hippocampal CA3 neurons ex vivo using a high-speed functional multineuron calcium imaging technique that allowed us to monitor spikes with millisecond resolution and to record the location of spiking and nonspiking neurons. Multineuronal spike sequences were overrepresented in spontaneous activity compared to the statistical chance level. Approximately 75% of neurons participated in at least one sequence during our observation period. The participants were sparsely dispersed and did not show specific spatial organization. The number of sequences relative to the chance level decreased when larger time frames were used to detect sequences. Thus, sequences were precise at the millisecond level. Sequences often shared common spikes with other sequences; parts of sequences were subsequently relayed by following sequences, generating complex chains of multiple sequences.

Design of multi-specificity in protein interfaces.

Directory of Open Access Journals (Sweden)

Elisabeth L Humphris

2007-08-01

Full Text Available Interactions in protein networks may place constraints on protein interface sequences to maintain correct and avoid unwanted interactions. Here we describe a "multi-constraint" protein design protocol to predict sequences optimized for multiple criteria, such as maintaining sets of interactions, and apply it to characterize the mechanism and extent to which 20 multi-specific proteins are constrained by binding to multiple partners. We find that multi-specific binding is accommodated by at least two distinct patterns. In the simplest case, all partners share key interactions, and sequences optimized for binding to either single or multiple partners recover only a subset of native amino acid residues as optimal. More interestingly, for signaling interfaces functioning as network "hubs," we identify a different, "multi-faceted" mode, where each binding partner prefers its own subset of wild-type residues within the promiscuous binding site. Here, integration of preferences across all partners results in sequences much more "native-like" than seen in optimization for any single binding partner alone, suggesting these interfaces are substantially optimized for multi-specificity. The two strategies make distinct predictions for interface evolution and design. Shared interfaces may be better small molecule targets, whereas multi-faceted interactions may be more "designable" for altered specificity patterns. The computational methodology presented here is generalizable for examining how naturally occurring protein sequences have been selected to satisfy a variety of positive and negative constraints, as well as for rationally designing proteins to have desired patterns of altered specificity.

Advantage of whole exome sequencing over allele-specific and targeted segment sequencing in detection of novel TULP1 mutation in leber congenital amaurosis

DEFF Research Database (Denmark)

Guo, Yiran; Prokudin, Ivan; Yu, Cong

2015-01-01

by targeted segment sequencing of 61 regions in 14 causative genes was performed. Subsequently, exome sequencing was undertaken in the proband, unaffected consanguineous parents and two unaffected siblings. Bioinformatic analysis used two independent pipelines, BWA-GATK and SOAP, followed by Annovar and Snp...

Friend and Moloney murine leukemia viruses specifically recombine with different endogenous retroviral sequences to generate mink cell focus-forming viruses.

Science.gov (United States)

Evans, L H; Cloyd, M W

1985-01-01

A group of mink cell focus-forming (MCF) viruses was derived by inoculation of NFS/N mice with Moloney murine leukemia virus (Mo-MuLV 1387) and was compared to a similarly derived group of MCF viruses from mice inoculated with Friend MuLV (Fr-MuLV 57). Antigenic analyses using monoclonal antibodies specific for MCF virus and xenotropic MuLV envelope proteins and genomic structural analyses by RNase T1-resistant oligonucleotide finger-printing indicated that the Moloney and Friend MCF viruses arose by recombination of the respective ecotropic MuLVs with different endogenous retrovirus sequences of NFS mice.

Regulatory sequence of cupin family gene

Science.gov (United States)

Hood, Elizabeth; Teoh, Thomas

2017-07-25

This invention is in the field of plant biology and agriculture and relates to novel seed specific promoter regions. The present invention further provide methods of producing proteins and other products of interest and methods of controlling expression of nucleic acid sequences of interest using the seed specific promoter regions.

Risk assessment for long-term post-accident sequences

International Nuclear Information System (INIS)

Ellia-Hervy, A.; Ducamp, F.

1987-11-01

Probabilistic risk analysis, currently conducted by the CEA (French Atomic Energy Commission) for the French replicate series of 900 MWe power plants, has identified accident sequences requiring long-term operation of some systems after the initiating event. They have been named long-term sequences. Quantification of probabilities of such sequences cannot rely exclusively on equipment failure-on-demand data: it must also take into account operating failures, the probability of which increase with time. Specific studies have therefore been conducted for a number of plant systems actuated during these long-term sequences. This has required: - Definition of the most realistic equipment utilization strategies based on existing emergency procedures for 900 MWe French plants. - Evaluation of the potential to repair failed equipment, given accessibility, repair time, and specific radiation conditions for the given sequence. - Definition of the event bringing the long-term sequence to an end. - Establishment of an appropriate quantification method, capable of taking into account the evolution of assumptions concerning equipment utilization strategies or repair conditions over time. The accident sequence quantification method based on realistic scenarios has been used in the risk assessment of the initiating event loss of reactor coolant accident occurring at power and at shutdown. Compared with the results obtained from conventional methods, this method redistributes the relative weight of accident sequences and also demonstrates that the long term can be a significant contribution to the probability of core melt

Accurate Local-Ancestry Inference in Exome-Sequenced Admixed Individuals via Off-Target Sequence Reads

Science.gov (United States)

Hu, Youna; Willer, Cristen; Zhan, Xiaowei; Kang, Hyun Min; Abecasis, Gonçalo R.

2013-01-01

Estimates of the ancestry of specific chromosomal regions in admixed individuals are useful for studies of human evolutionary history and for genetic association studies. Previously, this ancestry inference relied on high-quality genotypes from genome-wide association study (GWAS) arrays. These high-quality genotypes are not always available when samples are exome sequenced, and exome sequencing is the strategy of choice for many ongoing genetic studies. Here we show that off-target reads generated during exome-sequencing experiments can be combined with on-target reads to accurately estimate the ancestry of each chromosomal segment in an admixed individual. To reconstruct local ancestry, our method SEQMIX models aligned bases directly instead of relying on hard genotype calls. We evaluate the accuracy of our method through simulations and analysis of samples sequenced by the 1000 Genomes Project and the NHLBI Grand Opportunity Exome Sequencing Project. In African Americans, we show that local-ancestry estimates derived by our method are very similar to those derived with Illumina’s Omni 2.5M genotyping array and much improved in relation to estimates that use only exome genotypes and ignore off-target sequencing reads. Software implementing this method, SEQMIX, can be applied to analysis of human population history or used for genetic association studies in admixed individuals. PMID:24210252

Preliminary hazard analysis using sequence tree method

International Nuclear Information System (INIS)

Huang Huiwen; Shih Chunkuan; Hung Hungchih; Chen Minghuei; Yih Swu; Lin Jiinming

2007-01-01

A system level PHA using sequence tree method was developed to perform Safety Related digital I and C system SSA. The conventional PHA is a brainstorming session among experts on various portions of the system to identify hazards through discussions. However, this conventional PHA is not a systematic technique, the analysis results strongly depend on the experts' subjective opinions. The analysis quality cannot be appropriately controlled. Thereby, this research developed a system level sequence tree based PHA, which can clarify the relationship among the major digital I and C systems. Two major phases are included in this sequence tree based technique. The first phase uses a table to analyze each event in SAR Chapter 15 for a specific safety related I and C system, such as RPS. The second phase uses sequence tree to recognize what I and C systems are involved in the event, how the safety related systems work, and how the backup systems can be activated to mitigate the consequence if the primary safety systems fail. In the sequence tree, the defense-in-depth echelons, including Control echelon, Reactor trip echelon, ESFAS echelon, and Indication and display echelon, are arranged to construct the sequence tree structure. All the related I and C systems, include digital system and the analog back-up systems are allocated in their specific echelon. By this system centric sequence tree based analysis, not only preliminary hazard can be identified systematically, the vulnerability of the nuclear power plant can also be recognized. Therefore, an effective simplified D3 evaluation can be performed as well. (author)

Technical Considerations for Reduced Representation Bisulfite Sequencing with Multiplexed Libraries

Science.gov (United States)

Chatterjee, Aniruddha; Rodger, Euan J.; Stockwell, Peter A.; Weeks, Robert J.; Morison, Ian M.

2012-01-01

Reduced representation bisulfite sequencing (RRBS), which couples bisulfite conversion and next generation sequencing, is an innovative method that specifically enriches genomic regions with a high density of potential methylation sites and enables investigation of DNA methylation at single-nucleotide resolution. Recent advances in the Illumina DNA sample preparation protocol and sequencing technology have vastly improved sequencing throughput capacity. Although the new Illumina technology is now widely used, the unique challenges associated with multiplexed RRBS libraries on this platform have not been previously described. We have made modifications to the RRBS library preparation protocol to sequence multiplexed libraries on a single flow cell lane of the Illumina HiSeq 2000. Furthermore, our analysis incorporates a bioinformatics pipeline specifically designed to process bisulfite-converted sequencing reads and evaluate the output and quality of the sequencing data generated from the multiplexed libraries. We obtained an average of 42 million paired-end reads per sample for each flow-cell lane, with a high unique mapping efficiency to the reference human genome. Here we provide a roadmap of modifications, strategies, and trouble shooting approaches we implemented to optimize sequencing of multiplexed libraries on an a RRBS background. PMID:23193365

Open questions in origin of life : Experimental studies on the origin of nucleic acids and proteins with specific and functional sequences by a chemical synthetic biology approach

NARCIS (Netherlands)

Adamala, K.; Anella, F.M.; Wieczorek, R.; Stano, P.; Chiarabelli, C.; Luisi, P.L.

2014-01-01

In this mini-review we present some experimental approaches to the important issue in the origin of life, namely the origin of nucleic acids and proteins with specific and functional sequences. The formation of macromolecules on prebiotic Earth faces practical and conceptual difficulties. From the

Sequence specificity of DNA cleavage by Micrococcus luteus γ endonuclease

International Nuclear Information System (INIS)

Hentosh, P.; Henner, W.D.; Reynolds, R.J.

1985-01-01

DNA fragments of defined sequence have been used to determine the sites of cleavage by γ-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus γ endonuclease and analyzed on irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to γ radiation

Supraspinatus tendon tears at 3.0 T shoulder MR arthrography: diagnosis with 3D isotropic turbo spin-echo SPACE sequence versus 2D conventional sequences

Energy Technology Data Exchange (ETDEWEB)

Jung, Joon-Yong; Jee, Won-Hee; Park, Michael Y.; Lee, So-Yeon [Seoul St. Mary' s Hospital, The Catholic University of Korea, Department of Radiology, Seoul (Korea, Republic of); Kim, Yang-Soo [Seoul St. Mary' s Hospital, The Catholic University of Korea, Department of Orthopedic Surgery, Seoul (Korea, Republic of)

2012-11-15

To assess the diagnostic performance of shoulder MR arthrography with 3D isotropic fat-suppressed (FS) turbo spin-echo sequence (TSE-SPACE) for supraspinatus tendon tears in comparison with 2D conventional sequences at 3.0 T. The study was HIPAA-compliant and approved by the institutional review board with a waiver of informed consent. Eighty-seven arthroscopically confirmed patients who underwent 3.0 T shoulder MR arthrography with 2D sequences and 3D TSE-SPACE were included in a consecutive fashion from March 2009 to February 2010. Two reviewers independently analyzed 2D sequences and 3D TSE-SPACE. Sensitivity, specificity, accuracy, and interobserver agreement ({kappa}) were compared between 2D sequences and 3D TSE-SPACE for full-thickness and partial-thickness supraspinatus tendon tears together and for partial-thickness supraspinatus tendon tears alone. There were 33 full-thickness tears and 28 partial-thickness tears of supraspinatus tendons. For full-thickness and partial-thickness supraspinatus tendon tears together, the mean sensitivity, specificity, and accuracy of both readers were 96, 92, and 94% on 2D sequences and 91, 84, and 89% on 3D TSE-SPACE. For partial-thickness supraspinatus tendon tears alone, the mean sensitivity, specificity, and accuracy were 95, 92, and 94% on 2D sequences and 84, 85, and 84% on 3D TSE-SPACE. There was no statistical difference between 2D sequences and 3D TSE-SPACE. Interobserver agreements were almost perfect on 2D conventional sequences and substantial on 3D TSE-SPACE. Compared with 2D conventional sequences, MR arthrography using 3D TSE-SPACE was comparable for diagnosing supraspinatus tendon tears despite limitations in detecting small partial-thickness tears and in discriminating between full-thickness and deep partial-thickness tears. (orig.)

Supraspinatus tendon tears at 3.0 T shoulder MR arthrography: diagnosis with 3D isotropic turbo spin-echo SPACE sequence versus 2D conventional sequences

International Nuclear Information System (INIS)

Jung, Joon-Yong; Jee, Won-Hee; Park, Michael Y.; Lee, So-Yeon; Kim, Yang-Soo

2012-01-01

To assess the diagnostic performance of shoulder MR arthrography with 3D isotropic fat-suppressed (FS) turbo spin-echo sequence (TSE-SPACE) for supraspinatus tendon tears in comparison with 2D conventional sequences at 3.0 T. The study was HIPAA-compliant and approved by the institutional review board with a waiver of informed consent. Eighty-seven arthroscopically confirmed patients who underwent 3.0 T shoulder MR arthrography with 2D sequences and 3D TSE-SPACE were included in a consecutive fashion from March 2009 to February 2010. Two reviewers independently analyzed 2D sequences and 3D TSE-SPACE. Sensitivity, specificity, accuracy, and interobserver agreement (κ) were compared between 2D sequences and 3D TSE-SPACE for full-thickness and partial-thickness supraspinatus tendon tears together and for partial-thickness supraspinatus tendon tears alone. There were 33 full-thickness tears and 28 partial-thickness tears of supraspinatus tendons. For full-thickness and partial-thickness supraspinatus tendon tears together, the mean sensitivity, specificity, and accuracy of both readers were 96, 92, and 94% on 2D sequences and 91, 84, and 89% on 3D TSE-SPACE. For partial-thickness supraspinatus tendon tears alone, the mean sensitivity, specificity, and accuracy were 95, 92, and 94% on 2D sequences and 84, 85, and 84% on 3D TSE-SPACE. There was no statistical difference between 2D sequences and 3D TSE-SPACE. Interobserver agreements were almost perfect on 2D conventional sequences and substantial on 3D TSE-SPACE. Compared with 2D conventional sequences, MR arthrography using 3D TSE-SPACE was comparable for diagnosing supraspinatus tendon tears despite limitations in detecting small partial-thickness tears and in discriminating between full-thickness and deep partial-thickness tears. (orig.)

An AU-rich element in the 3{prime} untranslated region of the spinach chloroplast petD gene participates in sequence-specific RNA-protein complex formation

Energy Technology Data Exchange (ETDEWEB)

Chen, Qiuyun; Adams, C.C.; Usack, L. [Cornell Univ., Ithaca, NY (United States)] [and others

1995-04-01

In chloroplasts, the 3{prime} untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3{prime}-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3{prime} IR-containing RNA (3{prime} IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b{sub 6}/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T{sub 1} digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3{prime} IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3{prime} untranslated region, only a single copy, which we have termed box II, appears to be essential for in vivo protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3{prime}-end processing and/or influence the stability of petD mRNA in chloroplasts. 48 refs., 9 figs., 2 tabs.

Hidden Markov model-derived structural alphabet for proteins: the learning of protein local shapes captures sequence specificity.

Science.gov (United States)

Camproux, A C; Tufféry, P

2005-08-05

Understanding and predicting protein structures depend on the complexity and the accuracy of the models used to represent them. We have recently set up a Hidden Markov Model to optimally compress protein three-dimensional conformations into a one-dimensional series of letters of a structural alphabet. Such a model learns simultaneously the shape of representative structural letters describing the local conformation and the logic of their connections, i.e. the transition matrix between the letters. Here, we move one step further and report some evidence that such a model of protein local architecture also captures some accurate amino acid features. All the letters have specific and distinct amino acid distributions. Moreover, we show that words of amino acids can have significant propensities for some letters. Perspectives point towards the prediction of the series of letters describing the structure of a protein from its amino acid sequence.

mirVAFC: A Web Server for Prioritizations of Pathogenic Sequence Variants from Exome Sequencing Data via Classifications.

Science.gov (United States)

Li, Zhongshan; Liu, Zhenwei; Jiang, Yi; Chen, Denghui; Ran, Xia; Sun, Zhong Sheng; Wu, Jinyu

2017-01-01

Exome sequencing has been widely used to identify the genetic variants underlying human genetic disorders for clinical diagnoses, but the identification of pathogenic sequence variants among the huge amounts of benign ones is complicated and challenging. Here, we describe a new Web server named mirVAFC for pathogenic sequence variants prioritizations from clinical exome sequencing (CES) variant data of single individual or family. The mirVAFC is able to comprehensively annotate sequence variants, filter out most irrelevant variants using custom criteria, classify variants into different categories as for estimated pathogenicity, and lastly provide pathogenic variants prioritizations based on classifications and mutation effects. Case studies using different types of datasets for different diseases from publication and our in-house data have revealed that mirVAFC can efficiently identify the right pathogenic candidates as in original work in each case. Overall, the Web server mirVAFC is specifically developed for pathogenic sequence variant identifications from family-based CES variants using classification-based prioritizations. The mirVAFC Web server is freely accessible at https://www.wzgenomics.cn/mirVAFC/. © 2016 WILEY PERIODICALS, INC.

DNA-directed alkylating ligands as potential antitumor agents: sequence specificity of alkylation by intercalating aniline mustards.

Science.gov (United States)

Prakash, A S; Denny, W A; Gourdie, T A; Valu, K K; Woodgate, P D; Wakelin, L P

1990-10-23

The sequence preferences for alkylation of a series of novel parasubstituted aniline mustards linked to the DNA-intercalating chromophore 9-aminoacridine by an alkyl chain of variable length were studied by using procedures analogous to Maxam-Gilbert reactions. The compounds alkylate DNA at both guanine and adenine sites. For mustards linked to the acridine by a short alkyl chain through a para O- or S-link group, 5'-GT sequences are the most preferred sites at which N7-guanine alkylation occurs. For analogues with longer chain lengths, the preference of 5'-GT sequences diminishes in favor of N7-adenine alkylation at the complementary 5'-AC sequence. Magnesium ions are shown to selectively inhibit alkylation at the N7 of adenine (in the major groove) by these compounds but not the alkylation at the N3 of adenine (in the minor groove) by the antitumor antibiotic CC-1065. Effects of chromophore variation were also studied by using aniline mustards linked to quinazoline and sterically hindered tert-butyl-9-aminoacridine chromophores. The results demonstrate that in this series of DNA-directed mustards the noncovalent interactions of the carrier chromophores with DNA significantly modify the sequence selectivity of alkylation by the mustard. Relationships between the DNA alkylation patterns of these compounds and their biological activities are discussed.

Mining candidate genes associated with powdery mildew resistance in cucumber via super-BSA by specific length amplified fragment (SLAF) sequencing.

Science.gov (United States)

Zhang, Peng; Zhu, Yuqiang; Wang, Lili; Chen, Liping; Zhou, Shengjun

2015-12-14

Powdery mildew (PM) is the most common fungal disease of cucumber and other cucurbit crops, while breeding the PM-resistant materials is the effective way to defense this disease, and the recent development of modern genetics and genomics make us aware of that studying the resistance genes is the essential way to breed the PM high-resistance plant. With the ever increasing throughput of next-generation sequencing (NGS), the development of specific length amplified fragment sequencing (SLAF-seq) as a high-resolution strategy for large-scale de novo SNP discovery is gradually applied for functional gene mining. Here we combined the bulked segregant analysis (BSA) with SLAF-seq to identify candidate genes associated with PM resistance in cucumber. A segregating population comprising 251 F2 individuals was developed using H136 (female parent) as susceptible parent and BK2 (male parent) as resistance donor. After PMR test, total genomic DNA was prepared from each plant. Systemic genomic analysis of the GC content, repeat sequence, etc. was carried out by prediction software SLAF_Predict to establish condition to ensure the uniformity and density of the molecular markers. After samples were gel purified, SLAFs were generated at Biomarker Technologies Corporation in Beijing. Based on SLAF tags and the PMR test result, the hot region were annotated. A total of 73,100 high-quality SLAF tags with an average depth of 99.11× were sequenced. Among these, 5,355 polymorphic tags were identified with a polymorphism rate of 7.34 %, including 7.09 % SNPs and other polymorphism types. Finally, 140 associated SLAFs were identified, and two main Hot Regions were detected on chromosome 1 and 6, which contained five genes invovled in defense response, toxin metabolism, cell stress response, and injury response in cucumber. Associated markers identified by super-BSA in this study, could not only speed up the study of the PMR genes, but also provide a feasible solution for breeding the

Genomic DNA Enrichment Using Sequence Capture Microarrays: a Novel Approach to Discover Sequence Nucleotide Polymorphisms (SNP) in Brassica napus L

Science.gov (United States)

Clarke, Wayne E.; Parkin, Isobel A.; Gajardo, Humberto A.; Gerhardt, Daniel J.; Higgins, Erin; Sidebottom, Christine; Sharpe, Andrew G.; Snowdon, Rod J.; Federico, Maria L.; Iniguez-Luy, Federico L.

2013-01-01

Targeted genomic selection methodologies, or sequence capture, allow for DNA enrichment and large-scale resequencing and characterization of natural genetic variation in species with complex genomes, such as rapeseed canola (Brassica napus L., AACC, 2n=38). The main goal of this project was to combine sequence capture with next generation sequencing (NGS) to discover single nucleotide polymorphisms (SNPs) in specific areas of the B. napus genome historically associated (via quantitative trait loci –QTL– analysis) to traits of agronomical and nutritional importance. A 2.1 million feature sequence capture platform was designed to interrogate DNA sequence variation across 47 specific genomic regions, representing 51.2 Mb of the Brassica A and C genomes, in ten diverse rapeseed genotypes. All ten genotypes were sequenced using the 454 Life Sciences chemistry and to assess the effect of increased sequence depth, two genotypes were also sequenced using Illumina HiSeq chemistry. As a result, 589,367 potentially useful SNPs were identified. Analysis of sequence coverage indicated a four-fold increased representation of target regions, with 57% of the filtered SNPs falling within these regions. Sixty percent of discovered SNPs corresponded to transitions while 40% were transversions. Interestingly, fifty eight percent of the SNPs were found in genic regions while 42% were found in intergenic regions. Further, a high percentage of genic SNPs was found in exons (65% and 64% for the A and C genomes, respectively). Two different genotyping assays were used to validate the discovered SNPs. Validation rates ranged from 61.5% to 84% of tested SNPs, underpinning the effectiveness of this SNP discovery approach. Most importantly, the discovered SNPs were associated with agronomically important regions of the B. napus genome generating a novel data resource for research and breeding this crop species. PMID:24312619

Methylation patterns of repetitive DNA sequences in germ cells of Mus musculus.

Science.gov (United States)

Sanford, J; Forrester, L; Chapman, V; Chandley, A; Hastie, N

1984-03-26

The major and the minor satellite sequences of Mus musculus were undermethylated in both sperm and oocyte DNAs relative to the amount of undermethylation observed in adult somatic tissue DNA. This hypomethylation was specific for satellite sequences in sperm DNA. Dispersed repetitive and low copy sequences show a high degree of methylation in sperm DNA; however, a dispersed repetitive sequence was undermethylated in oocyte DNA. This finding suggests a difference in the amount of total genomic DNA methylation between sperm and oocyte DNA. The methylation levels of the minor satellite sequences did not change during spermiogenesis, and were not associated with the onset of meiosis or a specific stage in sperm development.

Next-generation sequencing library preparation method for identification of RNA viruses on the Ion Torrent Sequencing Platform.

Science.gov (United States)

Chen, Guiqian; Qiu, Yuan; Zhuang, Qingye; Wang, Suchun; Wang, Tong; Chen, Jiming; Wang, Kaicheng

2018-05-09

Next generation sequencing (NGS) is a powerful tool for the characterization, discovery, and molecular identification of RNA viruses. There were multiple NGS library preparation methods published for strand-specific RNA-seq, but some methods are not suitable for identifying and characterizing RNA viruses. In this study, we report a NGS library preparation method to identify RNA viruses using the Ion Torrent PGM platform. The NGS sequencing adapters were directly inserted into the sequencing library through reverse transcription and polymerase chain reaction, without fragmentation and ligation of nucleic acids. The results show that this method is simple to perform, able to identify multiple species of RNA viruses in clinical samples.

Sequence-specific inhibition of microRNA-130a gene by CRISPR/Cas9 system in breast cancer cell line

Science.gov (United States)

Ainina Abdollah, Nur; Das Kumitaa, Theva; Yusof Narazah, Mohd; Razak, Siti Razila Abdul

2017-05-01

MicroRNAs (miRNAs) are short stranded noncoding RNA that play important roles in apoptosis, cell survival, development and cell proliferation. However, gene expression control via small regulatory RNA, particularly miRNA in breast cancer is still less explored. Therefore, this project aims to develop an approach to target microRNA-130a using the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system in MCF7, breast cancer cell line. The 20 bp sequences target at stem loop, 3ʹ and 5ʹ end of miR130a were cloned into pSpCas9(BB)-2A-GFP (PX458) plasmid, and the positive clones were confirmed by sequencing. A total of 5 μg of PX458-miR130a was transfected to MCF7 using Lipofectamine® 3000 according to manufacturer’s protocol. The transfected cells were maintained in the incubator at 37 °C under humidified 5% CO2. After 48 hours, cells were harvested and total RNA was extracted using miRNeasy Mini Kit (Qiagen). cDNAs were synthesised specific to miR-130a using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Then, qRT-PCR was carried out using TaqMan Universal Master Mix (Applied Biosystems) to quantify the knockdown level of mature miRNAs in the cells. Result showed that miR-130a-5p was significantly downregulated in MCF7 cell line. However, no significant changes were observed for sequences targeting miR-130a-3p and stem loop. Thus, this study showed that the expression of miR-130a-5p was successfully down-regulated using CRISPR silencing system. This technique may be useful to manipulate the level of miRNA in various cell types to answer clinical questions at the molecular level.

Next Generation Sequencing Methods for Diagnosis of Epilepsy Syndromes

Directory of Open Access Journals (Sweden)

Paul Dunn

2018-02-01

Full Text Available Epilepsy is a neurological disorder characterized by an increased predisposition for seizures. Although this definition suggests that it is a single disorder, epilepsy encompasses a group of disorders with diverse aetiologies and outcomes. A genetic basis for epilepsy syndromes has been postulated for several decades, with several mutations in specific genes identified that have increased our understanding of the genetic influence on epilepsies. With 70-80% of epilepsy cases identified to have a genetic cause, there are now hundreds of genes identified to be associated with epilepsy syndromes which can be analyzed using next generation sequencing (NGS techniques such as targeted gene panels, whole exome sequencing (WES and whole genome sequencing (WGS. For effective use of these methodologies, diagnostic laboratories and clinicians require information on the relevant workflows including analysis and sequencing depth to understand the specific clinical application and diagnostic capabilities of these gene sequencing techniques. As epilepsy is a complex disorder, the differences associated with each technique influence the ability to form a diagnosis along with an accurate detection of the genetic etiology of the disorder. In addition, for diagnostic testing, an important parameter is the cost-effectiveness and the specific diagnostic outcome of each technique. Here, we review these commonly used NGS techniques to determine their suitability for application to epilepsy genetic diagnostic testing.

A DNA sequence obtained by replacement of the dopamine RNA aptamer bases is not an aptamer.

Science.gov (United States)

Álvarez-Martos, Isabel; Ferapontova, Elena E

2017-08-05

A unique specificity of the aptamer-ligand biorecognition and binding facilitates bioanalysis and biosensor development, contributing to discrimination of structurally related molecules, such as dopamine and other catecholamine neurotransmitters. The aptamer sequence capable of specific binding of dopamine is a 57 nucleotides long RNA sequence reported in 1997 (Biochemistry, 1997, 36, 9726). Later, it was suggested that the DNA homologue of the RNA aptamer retains the specificity of dopamine binding (Biochem. Biophys. Res. Commun., 2009, 388, 732). Here, we show that the DNA sequence obtained by the replacement of the RNA aptamer bases for their DNA analogues is not able of specific biorecognition of dopamine, in contrast to the original RNA aptamer sequence. This DNA sequence binds dopamine and structurally related catecholamine neurotransmitters non-specifically, as any DNA sequence, and, thus, is not an aptamer and cannot be used neither for in vivo nor in situ analysis of dopamine in the presence of structurally related neurotransmitters. Copyright © 2017 Elsevier Inc. All rights reserved.

Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation

International Nuclear Information System (INIS)

Hanafusa, Tadashi; Shinji, Toshiyuki; Shiraha, Hidenori; Nouso, Kazuhiro; Iwasaki, Yoshiaki; Yumoto, Eichiro; Ono, Toshiro; Koide, Norio

2005-01-01

Insulin-like growth factor binding protein (IGFBP)-3 functions as a carrier of insulin-like growth factors (IGFs) in circulation and a mediator of the growth suppression signal in cells. There are two reported p53 regulatory regions in the IGFBP3 gene; one upstream of the promoter and one intronic. We previously reported a hot spot of promoter hypermethylation of IGFBP-3 in human hepatocellular carcinomas and derivative cell lines. As the hot spot locates at the putative upstream p53 consensus sequences, these p53 consensus sequences are really functional is a question to be answered. In this study, we examined the p53 consensus sequences upstream of the IGFBP-3 promoter for the p53 induced expression of IGFBP-3. Deletion, mutagenesis, and methylation constructs of IGFBP-3 promoter were assessed in the human hepatoblastoma cell line HepG2 for promoter activity. Deletions and mutations of these sequences completely abolished the expression of IGFBP-3 in the presence of p53 overexpression. In vitro methylation of these p53 consensus sequences also suppressed IGFBP-3 expression. In contrast, the expression of IGFBP-3 was not affected in the absence of p53 overexpression. Further, we observed by electrophoresis mobility shift assay that p53 binding to the promoter region was diminished when methylated. From these observations, we conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells

Memory for sequences of events impaired in typical aging

Science.gov (United States)

Allen, Timothy A.; Morris, Andrea M.; Stark, Shauna M.; Fortin, Norbert J.

2015-01-01

Typical aging is associated with diminished episodic memory performance. To improve our understanding of the fundamental mechanisms underlying this age-related memory deficit, we previously developed an integrated, cross-species approach to link converging evidence from human and animal research. This novel approach focuses on the ability to remember sequences of events, an important feature of episodic memory. Unlike existing paradigms, this task is nonspatial, nonverbal, and can be used to isolate different cognitive processes that may be differentially affected in aging. Here, we used this task to make a comprehensive comparison of sequence memory performance between younger (18–22 yr) and older adults (62–86 yr). Specifically, participants viewed repeated sequences of six colored, fractal images and indicated whether each item was presented “in sequence” or “out of sequence.” Several out of sequence probe trials were used to provide a detailed assessment of sequence memory, including: (i) repeating an item from earlier in the sequence (“Repeats”; e.g., ABADEF), (ii) skipping ahead in the sequence (“Skips”; e.g., ABDDEF), and (iii) inserting an item from a different sequence into the same ordinal position (“Ordinal Transfers”; e.g., AB3DEF). We found that older adults performed as well as younger controls when tested on well-known and predictable sequences, but were severely impaired when tested using novel sequences. Importantly, overall sequence memory performance in older adults steadily declined with age, a decline not detected with other measures (RAVLT or BPS-O). We further characterized this deficit by showing that performance of older adults was severely impaired on specific probe trials that required detailed knowledge of the sequence (Skips and Ordinal Transfers), and was associated with a shift in their underlying mnemonic representation of the sequences. Collectively, these findings provide unambiguous evidence that the

Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion

DEFF Research Database (Denmark)

Thomsen, Martin Christen Frølund; Nielsen, Morten

2012-01-01

Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino acid composition of binding motifs, active...... related to amino acid enrichment and depletion. Besides allowing input in the format of peptides and MSA, Seq2Logo accepts input as Blast sequence profiles, providing easy access for non-expert end-users to characterize and identify functionally conserved/variable amino acids in any given protein...... sites, etc. in biological sequences. Accurate generation of sequence logos is often compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation focus on displaying the position-specific enrichment of amino acids, discarding the equally...

Meniscal tear evaluation. Comparison of a conventional spin-echo proton density sequence with a fast spin-echo sequence utilizing a 512x358 matrix size

International Nuclear Information System (INIS)

Hopper, M.A.; Robinson, P.; Grainger, A.J.

2011-01-01

Aim: To determine the sensitivities, specificities, and receiver-operating characteristics (ROCs) for sagittal conventional spin-echo proton density (SE-PD) and fast spin-echo proton density (FSE-PD) sequences in the diagnosis of meniscal tears when compared to arthroscopic findings utilizing increased FSE matrix acquisition size. Method and materials: Magnetic resonance imaging (MRI) studies of 97 knees (194 menisci) were independently and prospectively interpreted by two experienced musculoskeletal radiologists over four separate readings at least 3 weeks apart. Readings 1 and 2 included images in all three planes in accordance with the standard protocol with either a SE or FSE sagittal PD, at readings 3 and 4 just the SE or FSE sagittal PD sequences were reported. The FSE sequence was acquired with an increased matrix size, compared to the SE sequence, to provide increased resolution. Menisci were graded for the presence of a tear and statistical analysis to calculate sensitivity and specificity was performed comparing to arthroscopy as the reference standard. ROC analysis for the diagnosis of meniscal tears on the SE and FSE sagittal sequences was also evaluated. Reader concordance for the SE and FSE sequences was calculated. Results: Sixty-seven tears were noted at arthroscopy; 60 were detected on SE and 56 on FSE. The sensitivity and specificity for SE was 90 and 90%, and for FSE was 84 and 94%, respectively, with no significant difference. ROC analysis showed no significant difference between the two sequences and kappa values demonstrated a higher level of reader agreement for the FSE than for the SE reading. Conclusion: Use of a FSE sagittal PD sequence with an increased matrix size provides comparable performance to conventional SE sagittal PD when evaluating meniscal disease with a modern system. The present study indicates an increased level of concordance between readers for the FSE sagittal sequence compared to the conventional SE.

Meniscal tear evaluation. Comparison of a conventional spin-echo proton density sequence with a fast spin-echo sequence utilizing a 512x358 matrix size

Energy Technology Data Exchange (ETDEWEB)

Hopper, M.A.; Robinson, P. [Leeds Teaching Hospitals NHS Trust, Leeds (United Kingdom); Grainger, A.J., E-mail: andrew.grainger@leedsth.nhs.u [Leeds Teaching Hospitals NHS Trust, Leeds (United Kingdom)

2011-04-15

Aim: To determine the sensitivities, specificities, and receiver-operating characteristics (ROCs) for sagittal conventional spin-echo proton density (SE-PD) and fast spin-echo proton density (FSE-PD) sequences in the diagnosis of meniscal tears when compared to arthroscopic findings utilizing increased FSE matrix acquisition size. Method and materials: Magnetic resonance imaging (MRI) studies of 97 knees (194 menisci) were independently and prospectively interpreted by two experienced musculoskeletal radiologists over four separate readings at least 3 weeks apart. Readings 1 and 2 included images in all three planes in accordance with the standard protocol with either a SE or FSE sagittal PD, at readings 3 and 4 just the SE or FSE sagittal PD sequences were reported. The FSE sequence was acquired with an increased matrix size, compared to the SE sequence, to provide increased resolution. Menisci were graded for the presence of a tear and statistical analysis to calculate sensitivity and specificity was performed comparing to arthroscopy as the reference standard. ROC analysis for the diagnosis of meniscal tears on the SE and FSE sagittal sequences was also evaluated. Reader concordance for the SE and FSE sequences was calculated. Results: Sixty-seven tears were noted at arthroscopy; 60 were detected on SE and 56 on FSE. The sensitivity and specificity for SE was 90 and 90%, and for FSE was 84 and 94%, respectively, with no significant difference. ROC analysis showed no significant difference between the two sequences and kappa values demonstrated a higher level of reader agreement for the FSE than for the SE reading. Conclusion: Use of a FSE sagittal PD sequence with an increased matrix size provides comparable performance to conventional SE sagittal PD when evaluating meniscal disease with a modern system. The present study indicates an increased level of concordance between readers for the FSE sagittal sequence compared to the conventional SE.

Noncoding sequence classification based on wavelet transform analysis: part I

Science.gov (United States)

Paredes, O.; Strojnik, M.; Romo-Vázquez, R.; Vélez Pérez, H.; Ranta, R.; Garcia-Torales, G.; Scholl, M. K.; Morales, J. A.

2017-09-01

DNA sequences in human genome can be divided into the coding and noncoding ones. Coding sequences are those that are read during the transcription. The identification of coding sequences has been widely reported in literature due to its much-studied periodicity. Noncoding sequences represent the majority of the human genome. They play an important role in gene regulation and differentiation among the cells. However, noncoding sequences do not exhibit periodicities that correlate to their functions. The ENCODE (Encyclopedia of DNA elements) and Epigenomic Roadmap Project projects have cataloged the human noncoding sequences into specific functions. We study characteristics of noncoding sequences with wavelet analysis of genomic signals.

Genome sequences of lower Great Lakes Microcystis sp. reveal strain-specific genes that are present and expressed in western Lake Erie blooms.

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Kevin Anthony Meyer

Full Text Available Blooms of the potentially toxic cyanobacterium Microcystis are increasing worldwide. In the Laurentian Great Lakes they pose major socioeconomic, ecological, and human health threats, particularly in western Lake Erie. However, the interpretation of "omics" data is constrained by the highly variable genome of Microcystis and the small number of reference genome sequences from strains isolated from the Great Lakes. To address this, we sequenced two Microcystis isolates from Lake Erie (Microcystis aeruginosa LE3 and M. wesenbergii LE013-01 and one from upstream Lake St. Clair (M. cf aeruginosa LSC13-02, and compared these data to the genomes of seventeen Microcystis spp. from across the globe as well as one metagenome and seven metatranscriptomes from a 2014 Lake Erie Microcystis bloom. For the publically available strains analyzed, the core genome is ~1900 genes, representing ~11% of total genes in the pan-genome and ~45% of each strain's genome. The flexible genome content was related to Microcystis subclades defined by phylogenetic analysis of both housekeeping genes and total core genes. To our knowledge this is the first evidence that the flexible genome is linked to the core genome of the Microcystis species complex. The majority of strain-specific genes were present and expressed in bloom communities in Lake Erie. Roughly 8% of these genes from the lower Great Lakes are involved in genome plasticity (rapid gain, loss, or rearrangement of genes and resistance to foreign genetic elements (such as CRISPR-Cas systems. Intriguingly, strain-specific genes from Microcystis cultured from around the world were also present and expressed in the Lake Erie blooms, suggesting that the Microcystis pangenome is truly global. The presence and expression of flexible genes, including strain-specific genes, suggests that strain-level genomic diversity may be important in maintaining Microcystis abundance during bloom events.

Characterizing leader sequences of CRISPR loci

DEFF Research Database (Denmark)

Alkhnbashi, Omer; Shah, Shiraz Ali; Garrett, Roger Antony

2016-01-01

The CRISPR-Cas system is an adaptive immune system in many archaea and bacteria, which provides resistance against invading genetic elements. The first phase of CRISPR-Cas immunity is called adaptation, in which small DNA fragments are excised from genetic elements and are inserted into a CRISPR...... array generally adjacent to its so called leader sequence at one end of the array. It has been shown that transcription initiation and adaptation signals of the CRISPR array are located within the leader. However, apart from promoters, there is very little knowledge of sequence or structural motifs...... sequences by focusing on the consensus repeat of the adjacent CRISPR array and weak upstream conservation signals. We applied our tool to the analysis of a comprehensive genomic database and identified several characteristic properties of leader sequences specific to archaea and bacteria, ranging from...

Mutation of miRNA target sequences during human evolution

DEFF Research Database (Denmark)

Gardner, Paul P; Vinther, Jeppe

2008-01-01

It has long-been hypothesized that changes in non-protein-coding genes and the regulatory sequences controlling expression could undergo positive selection. Here we identify 402 putative microRNA (miRNA) target sequences that have been mutated specifically in the human lineage and show that genes...... containing such deletions are more highly expressed than their mouse orthologs. Our findings indicate that some miRNA target mutations are fixed by positive selection and might have been involved in the evolution of human-specific traits....

Comprehensive search for intra- and inter-specific sequence polymorphisms among coding envelope genes of retroviral origin found in the human genome: genes and pseudogenes

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Vasilescu Alexandre

2005-09-01

Full Text Available Abstract Background The human genome carries a high load of proviral-like sequences, called Human Endogenous Retroviruses (HERVs, which are the genomic traces of ancient infections by active retroviruses. These elements are in most cases defective, but open reading frames can still be found for the retroviral envelope gene, with sixteen such genes identified so far. Several of them are conserved during primate evolution, having possibly been co-opted by their host for a physiological role. Results To characterize further their status, we presently sequenced 12 of these genes from a panel of 91 Caucasian individuals. Genomic analyses reveal strong sequence conservation (only two non synonymous Single Nucleotide Polymorphisms [SNPs] for the two HERV-W and HERV-FRD envelope genes, i.e. for the two genes specifically expressed in the placenta and possibly involved in syncytiotrophoblast formation. We further show – using an ex vivo fusion assay for each allelic form – that none of these SNPs impairs the fusogenic function. The other envelope proteins disclose variable polymorphisms, with the occurrence of a stop codon and/or frameshift for most – but not all – of them. Moreover, the sequence conservation analysis of the orthologous genes that can be found in primates shows that three env genes have been maintained in a fully coding state throughout evolution including envW and envFRD. Conclusion Altogether, the present study strongly suggests that some but not all envelope encoding sequences are bona fide genes. It also provides new tools to elucidate the possible role of endogenous envelope proteins as susceptibility factors in a number of pathologies where HERVs have been suspected to be involved.

Cloning, sequencing, and sequence analysis of two novel plasmids from the thermophilic anaerobic bacterium Anaerocellum thermophilum

DEFF Research Database (Denmark)

Clausen, Anders; Mikkelsen, Marie Just; Schrøder, I.

2004-01-01

The nucleotide sequence of two novel plasmids isolated from the extreme thermophilic anaerobic bacterium Anaerocellum thermophilum DSM6725 (A. thermophilum), growing optimally at 70degreesC, has been determined. pBAS2 was found to be a 3653 bp plasmid with a GC content of 43%, and the sequence re...... with highest similarity to DNA repair protein from Campylobacter jejuni (25% aa). Orf34 showed similarity to sigma factors with highest similarity (28% aa) to the sporulation specific Sigma factor, Sigma 28(K) from Bacillus thuringiensis....

New concepts of fluorescent probes for specific detection of DNA sequences: bis-modified oligonucleotides in excimer and exciplex detection.

Science.gov (United States)

Gbaj, A; Bichenkova, Ev; Walsh, L; Savage, He; Sardarian, Ar; Etchells, Ll; Gulati, A; Hawisa, S; Douglas, Kt

2009-12-01

The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5'-bispyrene and 3'-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5'-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5'-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.

Exploration of noncoding sequences in metagenomes.

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Fabián Tobar-Tosse

Full Text Available Environment-dependent genomic features have been defined for different metagenomes, whose genes and their associated processes are related to specific environments. Identification of ORFs and their functional categories are the most common methods for association between functional and environmental features. However, this analysis based on finding ORFs misses noncoding sequences and, therefore, some metagenome regulatory or structural information could be discarded. In this work we analyzed 23 whole metagenomes, including coding and noncoding sequences using the following sequence patterns: (G+C content, Codon Usage (Cd, Trinucleotide Usage (Tn, and functional assignments for ORF prediction. Herein, we present evidence of a high proportion of noncoding sequences discarded in common similarity-based methods in metagenomics, and the kind of relevant information present in those. We found a high density of trinucleotide repeat sequences (TRS in noncoding sequences, with a regulatory and adaptive function for metagenome communities. We present associations between trinucleotide values and gene function, where metagenome clustering correlate with microorganism adaptations and kinds of metagenomes. We propose here that noncoding sequences have relevant information to describe metagenomes that could be considered in a whole metagenome analysis in order to improve their organization, classification protocols, and their relation with the environment.

A novel analysis strategy for HLA typing using a sequence-specific oligonucleotide probe method.

Science.gov (United States)

Won, D I

2017-11-01

The technique of reverse sequence-specific oligonucleotide probes (SSOPs) is commonly used in human leukocyte antigen (HLA) typing. In the conventional method for data analysis (exact pattern matching, EPM), the larger is the number of mismatched probes, the longer the time for final typing assignment. A novel strategy, filtering and scoring (FnS), has been developed to easily assign the best-fit allele pair. In the FnS method, candidate alleles and allele pairs were filtered based on (1) subject's ethnicity, and (2) the measured partial reaction pattern with only definitely negative or positive probes. Then, the complete reaction pattern for all probes (CRPoAPs) were compared between the raw sample and expected residual allele pairs to obtain mismatch scores. To compare the FnS and EPM methods, each analysis time (minutes:seconds) for reverse SSOP HLA typing with intermediate resolution (n = 507) was measured. The analysis time with FnS method was shorter than that of the EPM method [00:21 (00:08-01:47) and 01:04 (00:15-23:45), respectively, P typing in a comprehensive and quantitative comparison between measured and expected CRPoAPs of candidate allele pairs. Therefore, this analysis strategy might be useful in a clinical setting. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

MIPS: a database for genomes and protein sequences.

Science.gov (United States)

Mewes, H W; Frishman, D; Güldener, U; Mannhaupt, G; Mayer, K; Mokrejs, M; Morgenstern, B; Münsterkötter, M; Rudd, S; Weil, B

2002-01-01

The Munich Information Center for Protein Sequences (MIPS-GSF, Neuherberg, Germany) continues to provide genome-related information in a systematic way. MIPS supports both national and European sequencing and functional analysis projects, develops and maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences, and provides tools for the comprehensive analysis of protein sequences. This report updates the information on the yeast genome (CYGD), the Neurospora crassa genome (MNCDB), the databases for the comprehensive set of genomes (PEDANT genomes), the database of annotated human EST clusters (HIB), the database of complete cDNAs from the DHGP (German Human Genome Project), as well as the project specific databases for the GABI (Genome Analysis in Plants) and HNB (Helmholtz-Netzwerk Bioinformatik) networks. The Arabidospsis thaliana database (MATDB), the database of mitochondrial proteins (MITOP) and our contribution to the PIR International Protein Sequence Database have been described elsewhere [Schoof et al. (2002) Nucleic Acids Res., 30, 91-93; Scharfe et al. (2000) Nucleic Acids Res., 28, 155-158; Barker et al. (2001) Nucleic Acids Res., 29, 29-32]. All databases described, the protein analysis tools provided and the detailed descriptions of our projects can be accessed through the MIPS World Wide Web server (http://mips.gsf.de).

Sequence-dependent DNA deformability studied using molecular dynamics simulations.

Science.gov (United States)

Fujii, Satoshi; Kono, Hidetoshi; Takenaka, Shigeori; Go, Nobuhiro; Sarai, Akinori

2007-01-01

Proteins recognize specific DNA sequences not only through direct contact between amino acids and bases, but also indirectly based on the sequence-dependent conformation and deformability of the DNA (indirect readout). We used molecular dynamics simulations to analyze the sequence-dependent DNA conformations of all 136 possible tetrameric sequences sandwiched between CGCG sequences. The deformability of dimeric steps obtained by the simulations is consistent with that by the crystal structures. The simulation results further showed that the conformation and deformability of the tetramers can highly depend on the flanking base pairs. The conformations of xATx tetramers show the most rigidity and are not affected by the flanking base pairs and the xYRx show by contrast the greatest flexibility and change their conformations depending on the base pairs at both ends, suggesting tetramers with the same central dimer can show different deformabilities. These results suggest that analysis of dimeric steps alone may overlook some conformational features of DNA and provide insight into the mechanism of indirect readout during protein-DNA recognition. Moreover, the sequence dependence of DNA conformation and deformability may be used to estimate the contribution of indirect readout to the specificity of protein-DNA recognition as well as nucleosome positioning and large-scale behavior of nucleic acids.

Towards the identification of flower-specific genes in Citrus spp

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Marcelo Carnier Dornelas

2007-01-01

Full Text Available Citrus sinensis is a perennial woody species, for which genetic approaches to the study of reproductive development are not readily amenable. Here, the usefulness of the CitEST Expressed Sequence Tag (EST database is demonstrated as a reliable new resource for identifying novel genes exclusively related to Citrus reproductive biology. We performed the analysis of an EST dataset of the CitEST Project containing 4,330 flower-derived cDNA sequences. Relying on bioinformatics tools, sequences exclusively present in this flower-derived sequence collection were selected and used for the identification of Citrus putative flower-specific genes. Our analysis revealed several Citrus sequences showing significant similarity to conserved genes known to have flower-specific expression and possessing functions related to flower metabolism and/or reproductive development in diverse plant species. Comparison of the Citrus flower-specific sequences with all available plant peptide sequences unraveled 247 unique transcripts not identified elsewhere within the plant kingdom. Additionally, 49 transcripts, for which no biological function could be attributed by means of sequence comparisons, were found to be conserved among plant species. These results allow further gene expression analysis and possibly novel approaches to the understanding of reproductive development in Citrus.

Sequencing of 50 human exomes reveals adaptation to high altitude

DEFF Research Database (Denmark)

Yi, Xin; Liang, Yu; Huerta-Sanchez, Emilia

2010-01-01

Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which repres...... in genetic adaptation to high altitude.......Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which...... represent strong candidates for altitude adaptation, were identified. The strongest signal of natural selection came from endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1), a transcription factor involved in response to hypoxia. One single-nucleotide polymorphism (SNP) at EPAS1 shows a 78% frequency...

SeqCompress: an algorithm for biological sequence compression.

Science.gov (United States)

Sardaraz, Muhammad; Tahir, Muhammad; Ikram, Ataul Aziz; Bajwa, Hassan

2014-10-01

The growth of Next Generation Sequencing technologies presents significant research challenges, specifically to design bioinformatics tools that handle massive amount of data efficiently. Biological sequence data storage cost has become a noticeable proportion of total cost in the generation and analysis. Particularly increase in DNA sequencing rate is significantly outstripping the rate of increase in disk storage capacity, which may go beyond the limit of storage capacity. It is essential to develop algorithms that handle large data sets via better memory management. This article presents a DNA sequence compression algorithm SeqCompress that copes with the space complexity of biological sequences. The algorithm is based on lossless data compression and uses statistical model as well as arithmetic coding to compress DNA sequences. The proposed algorithm is compared with recent specialized compression tools for biological sequences. Experimental results show that proposed algorithm has better compression gain as compared to other existing algorithms. Copyright © 2014 Elsevier Inc. All rights reserved.

Local sequence information in cellular retinoic acid-binding protein I: specific residue roles in beta-turns.

Science.gov (United States)

Rotondi, Kenneth S; Gierasch, Lila M

2003-01-01

We have recently shown that two of the beta-turns (III and IV) in the ten-stranded, beta-clam protein, cellular retinoic acid-binding protein I (CRABP I), are favored in short peptide fragments, arguing that they are encoded by local interactions (K. S. Rotondi and L. M. Gierasch, Biochemistry, 2003, Vol. 42, pp. 7976-7985). In this paper we examine these turns in greater detail to dissect the specific local interactions responsible for their observed native conformational biases. Conformations of peptides corresponding to the turn III and IV fragments were examined under conditions designed to selectively disrupt stabilizing interactions, using pH variation, chaotrope addition, or mutagenesis to probe specific side-chain influences. We find that steric constraints imposed by excluded volume effects between near neighbor residues (i,i+2), favorable polar (i,i+2) interactions, and steric permissiveness of glycines are the principal factors accounting for the observed native bias in these turns. Longer-range stabilizing interactions across the beta-turns do not appear to play a significant role in turn stability in these short peptides, in contrast to their importance in hairpins. Additionally, our data add to a growing number of examples of the 3:5 type I turn with a beta-bulge as a class of turns with high propensity to form locally defined structure. Current work is directed at the interplay between the local sequence information in the turns and more long-range influences in the mechanism of folding of this predominantly beta-sheet protein. Copyright 2004 Wiley Periodicals, Inc.

A specific implicit sequence learning deficit as an underlying cause of dyslexia? Investigating the role of attention in implicit learning tasks.

Science.gov (United States)

Staels, Eva; Van den Broeck, Wim

2017-05-01

Recently, a general implicit sequence learning deficit was proposed as an underlying cause of dyslexia. This new hypothesis was investigated in the present study by including a number of methodological improvements, for example, the inclusion of appropriate control conditions. The second goal of the study was to explore the role of attentional functioning in implicit and explicit learning tasks. In a 2 × 2 within-subjects design 4 tasks were administered in 30 dyslexic and 38 control children: an implicit and explicit serial reaction time (RT) task and an implicit and explicit contextual cueing task. Attentional functioning was also administered. The entire learning curves of all tasks were analyzed using latent growth curve modeling in order to compare performances between groups and to examine the role of attentional functioning on the learning curves. The amount of implicit learning was similar for both groups. However, the dyslexic group showed slower RTs throughout the entire task. This group difference reduced and became nonsignificant after controlling for attentional functioning. Both implicit learning tasks, but none of the explicit learning tasks, were significantly affected by attentional functioning. Dyslexic children do not suffer from a specific implicit sequence learning deficit. The slower RTs of the dyslexic children throughout the entire implicit sequence learning process are caused by their comorbid attention problems and overall slowness. A key finding of the present study is that, in contrast to what was assumed for a long time, implicit learning relies on attentional resources, perhaps even more than explicit learning does. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Testing of Badminton-Specific Endurance.

Science.gov (United States)

Madsen, Christian M; Højlyng, Mads; Nybo, Lars

2016-09-01

Madsen, CM, Højlyng, M, and Nybo, L. Testing of badminton-specific endurance. J Strength Cond Res 30(9): 2582-2590, 2016-In the present study, a novel intermittent badminton endurance (B-ENDURANCE) test was developed and tested in elite (n = 17) and skilled (n = 9) badminton players and in age-matched physically active men (nonbadminton players; n = 8). In addition, B-ENDURANCE test-retest reproducibility was evaluated in 9 badminton players. The B-ENDURANCE test is an incremental test where each level consists of repeated sequences of badminton-specific actions toward the 4 corners of the court. The subject starts in the center of the court in front of a computer screen and within each sequence, he must, in a randomized order, complete 8 actions as dictated by the computer, providing the audiovisual input and verifying that the appropriate sensor is activated within the allocated time. Recovery time between each sequence is 10 seconds throughout the test, but the time to complete each sequence is gradually decreased until the subjects cannot follow the dictated tempo. The B-ENDURANCE test performance for elite players was better (p ≤ 0.05) compared with the skilled players and nonbadminton players. In addition, the B-ENDURANCE test performance correlated (r = 0.8 and p badminton-specific endurance but at least 1 familiarization trial is recommended if the test is used for evaluation of longitudinal changes, e.g., tracking training effects.

Abundant Trimethylornithine Lipids and Specific Gene Sequences Are Indicative of Planctomycete Importance at the Oxic/Anoxic Interface in Sphagnum-Dominated Northern Wetlands.

Science.gov (United States)

Moore, Eli K; Villanueva, Laura; Hopmans, Ellen C; Rijpstra, W Irene C; Mets, Anchelique; Dedysh, Svetlana N; Sinninghe Damsté, Jaap S

2015-09-01

Northern wetlands make up a substantial terrestrial carbon sink and are often dominated by decay-resistant Sphagnum mosses. Recent studies have shown that planctomycetes appear to be involved in degradation of Sphagnum-derived debris. Novel trimethylornithine (TMO) lipids have recently been characterized as abundant lipids in various Sphagnum wetland planctomycete isolates, but their occurrence in the environment has not yet been confirmed. We applied a combined intact polar lipid (IPL) and molecular analysis of peat cores collected from two northern wetlands (Saxnäs Mosse [Sweden] and Obukhovskoye [Russia]) in order to investigate the preferred niche and abundance of TMO-producing planctomycetes. TMOs were present throughout the profiles of Sphagnum bogs, but their concentration peaked at the oxic/anoxic interface, which coincided with a maximum abundance of planctomycete-specific 16S rRNA gene sequences. The sequences detected at the oxic/anoxic interface were affiliated with the Isosphaera group, while sequences present in the anoxic peat layers were related to an uncultured planctomycete group. Pyrosequencing-based analysis identified Planctomycetes as the major bacterial group at the oxic/anoxic interface at the Obukhovskoye peat (54% of total 16S rRNA gene sequence reads), followed by Acidobacteria (19% reads), while in the Saxnäs Mosse peat, Acidobacteria were dominant (46%), and Planctomycetes contributed to 6% of the total reads. The detection of abundant TMO lipids in planctomycetes isolated from peat bogs and the lack of TMO production by cultures of acidobacteria suggest that planctomycetes are the producers of TMOs in peat bogs. The higher accumulation of TMOs at the oxic/anoxic interface and the change in the planctomycete community with depth suggest that these IPLs could be synthesized as a response to changing redox conditions at the oxic/anoxic interface. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Folding and activity of hybrid sequence, disulfide-stabilized peptides

Energy Technology Data Exchange (ETDEWEB)

Pease, J.H.B.; Storrs, R.W.; Wemmer, D.E. (Univ. of California, Berkeley (USA))

1990-08-01

Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a {beta}-turn and an {alpha}-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. The authors show that in S peptide the {alpha}-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure - function relationships for many biologically active sequences.

Folding and activity of hybrid sequence, disulfide-stabilized peptides

International Nuclear Information System (INIS)

Pease, J.H.B.; Storrs, R.W.; Wemmer, D.E.

1990-01-01

Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a β-turn and an α-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. The authors show that in S peptide the α-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure - function relationships for many biologically active sequences

Sequencing and analysis of an Irish human genome.

LENUS (Irish Health Repository)

Tong, Pin

2010-01-01

Recent studies generating complete human sequences from Asian, African and European subgroups have revealed population-specific variation and disease susceptibility loci. Here, choosing a DNA sample from a population of interest due to its relative geographical isolation and genetic impact on further populations, we extend the above studies through the generation of 11-fold coverage of the first Irish human genome sequence.

Individual differences in implicit motor learning: task specificity in sensorimotor adaptation and sequence learning.

Science.gov (United States)

Stark-Inbar, Alit; Raza, Meher; Taylor, Jordan A; Ivry, Richard B

2017-01-01

In standard taxonomies, motor skills are typically treated as representative of implicit or procedural memory. We examined two emblematic tasks of implicit motor learning, sensorimotor adaptation and sequence learning, asking whether individual differences in learning are correlated between these tasks, as well as how individual differences within each task are related to different performance variables. As a prerequisite, it was essential to establish the reliability of learning measures for each task. Participants were tested twice on a visuomotor adaptation task and on a sequence learning task, either the serial reaction time task or the alternating reaction time task. Learning was evident in all tasks at the group level and reliable at the individual level in visuomotor adaptation and the alternating reaction time task but not in the serial reaction time task. Performance variability was predictive of learning in both domains, yet the relationship was in the opposite direction for adaptation and sequence learning. For the former, faster learning was associated with lower variability, consistent with models of sensorimotor adaptation in which learning rates are sensitive to noise. For the latter, greater learning was associated with higher variability and slower reaction times, factors that may facilitate the spread of activation required to form predictive, sequential associations. Interestingly, learning measures of the different tasks were not correlated. Together, these results oppose a shared process for implicit learning in sensorimotor adaptation and sequence learning and provide insight into the factors that account for individual differences in learning within each task domain. We investigated individual differences in the ability to implicitly learn motor skills. As a prerequisite, we assessed whether individual differences were reliable across test sessions. We found that two commonly used tasks of implicit learning, visuomotor adaptation and the

Characterization of Satellite DNA Sequences from the Commercially Important Marine Rotifers Brachionus rotundiformis and Brachionus plicatilis.

Science.gov (United States)

Boehm; Gibson; Lubzens

2000-01-01

This study was initiated to search for species-specific and strain-specific satellite DNA sequences for which oligonucleotide primers could be designed to differentiate between various commercially important strains of the marine monogonont rotifers Brachionus rotundiformis and Brachionus plicatilis. Two unrelated, highly reiterated satellite sequences were cloned and characterized. The eight sequenced monomers from B. rotundiformis and six from B. plicatilis had low intrarepeat variability and were similar in their overall lengths, A + T compositions, and high degrees of repeated motif substructure. However, hybridizations to 19 representative strains, sequence characterizations, and GenBank searches indicated that these two satellites are morphotype-specific and population-specific, respectively, and share little homology to each other or to other characterized sequences in the database. Primer pairs designed for the B. rotundiformis satellite confirmed hybridization specificities on polymerase chain reaction and could serve as a useful molecular diagnostic tool to identify strains belonging to the SS morphotype, which are gaining widespread usage as first feeds for marine fish in commercial production.

Nucleotide sequence composition and method for detection of neisseria gonorrhoeae

International Nuclear Information System (INIS)

Lo, A.; Yang, H.L.

1990-01-01

This patent describes a composition of matter that is specific for Neisseria gonorrhoeae. It comprises: at least one nucleotide sequence for which the ratio of the amount of the sequence which hybridizes to chromosomal DNA of Neisseria gonorrhoeae to the amount of the sequence which hybridizes to chromosomal DNA of Neisseria meningitidis is greater than about five. The ratio being obtained by a method described

Sequence diversity in haloalkane dehalogenases, as revealed by PCR using family-specific primers

Czech Academy of Sciences Publication Activity Database

Kotík, Michael; Faměrová, Veronika

2012-01-01

Roč. 88, č. 2 (2012), s. 212-217 ISSN 0167-7012 R&D Projects: GA ČR GAP504/10/0137; GA ČR GAP207/10/0135 Institutional research plan: CEZ:AV0Z50200510 Keywords : Dehalogenation * Consensus sequence * Degenerate PCR primer Subject RIV: EE - Microbiology, Virology Impact factor: 2.161, year: 2012

Specific primary sequence requirements for Aurora B kinase-mediated phosphorylation and subcellular localization of TMAP during mitosis.

Science.gov (United States)

Kim, Hyun-Jun; Kwon, Hye-Rim; Bae, Chang-Dae; Park, Joobae; Hong, Kyung U

2010-05-15

During mitosis, regulation of protein structures and functions by phosphorylation plays critical roles in orchestrating a series of complex events essential for the cell division process. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a novel player in spindle assembly and chromosome segregation. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis. However, the mechanisms and functional importance of phosphorylation at most of the sites identified are currently unknown. Here, we report that TMAP is a novel substrate of the Aurora B kinase. Ser627 of TMAP was specifically phosphorylated by Aurora B both in vitro and in vivo. Ser627 and neighboring conserved residues were strictly required for efficient phosphorylation of TMAP by Aurora B, as even minor amino acid substitutions of the phosphorylation motif significantly diminished the efficiency of the substrate phosphorylation. Nearly all mutations at the phosphorylation motif had dramatic effects on the subcellular localization of TMAP. Instead of being localized to the chromosome region during late mitosis, the mutants remained associated with microtubules and centrosomes throughout mitosis. However, the changes in the subcellular localization of these mutants could not be completely explained by the phosphorylation status on Ser627. Our findings suggest that the motif surrounding Ser627 ((625) RRSRRL (630)) is a critical part of a functionally important sequence motif which not only governs the kinase-substrate recognition, but also regulates the subcellular localization of TMAP during mitosis.

Bamgineer: Introduction of simulated allele-specific copy number variants into exome and targeted sequence data sets

OpenAIRE

Bruce, Jeff; Pugh, Trevor; Samadian, Soroush

2017-01-01

Somatic copy number variations (CNVs) play a crucial role in development of many human cancers. The broad availability of next-generation sequencing data has enabled the development of algorithms to computationally infer CNV profiles from a variety of data types including exome and targeted sequence data; currently the most prevalent types of cancer genomics data. However, systemic evaluation and comparison of these tools remains challenging due to a lack of ground truth reference sets. To ad...

Nucleotide sequence composition and method for detection of neisseria gonorrhoeae

Energy Technology Data Exchange (ETDEWEB)

Lo, A.; Yang, H.L.

1990-02-13

This patent describes a composition of matter that is specific for {ital Neisseria gonorrhoeae}. It comprises: at least one nucleotide sequence for which the ratio of the amount of the sequence which hybridizes to chromosomal DNA of {ital Neisseria gonorrhoeae} to the amount of the sequence which hybridizes to chromosomal DNA of {ital Neisseria meningitidis} is greater than about five. The ratio being obtained by a method described.

A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats.

Science.gov (United States)

Beloglazova, Natalia; Brown, Greg; Zimmerman, Matthew D; Proudfoot, Michael; Makarova, Kira S; Kudritska, Marina; Kochinyan, Samvel; Wang, Shuren; Chruszcz, Maksymilian; Minor, Wladek; Koonin, Eugene V; Edwards, Aled M; Savchenko, Alexei; Yakunin, Alexander F

2008-07-18

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.

Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

Science.gov (United States)

Hosokawa, Masahito; Nishikawa, Yohei; Kogawa, Masato; Takeyama, Haruko

2017-07-12

Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

A novel bioinformatics method for efficient knowledge discovery by BLSOM from big genomic sequence data.

Science.gov (United States)

Bai, Yu; Iwasaki, Yuki; Kanaya, Shigehiko; Zhao, Yue; Ikemura, Toshimichi

2014-01-01

With remarkable increase of genomic sequence data of a wide range of species, novel tools are needed for comprehensive analyses of the big sequence data. Self-Organizing Map (SOM) is an effective tool for clustering and visualizing high-dimensional data such as oligonucleotide composition on one map. By modifying the conventional SOM, we have previously developed Batch-Learning SOM (BLSOM), which allows classification of sequence fragments according to species, solely depending on the oligonucleotide composition. In the present study, we introduce the oligonucleotide BLSOM used for characterization of vertebrate genome sequences. We first analyzed pentanucleotide compositions in 100 kb sequences derived from a wide range of vertebrate genomes and then the compositions in the human and mouse genomes in order to investigate an efficient method for detecting differences between the closely related genomes. BLSOM can recognize the species-specific key combination of oligonucleotide frequencies in each genome, which is called a "genome signature," and the specific regions specifically enriched in transcription-factor-binding sequences. Because the classification and visualization power is very high, BLSOM is an efficient powerful tool for extracting a wide range of information from massive amounts of genomic sequences (i.e., big sequence data).

Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions.

Science.gov (United States)

Silvas, Tania V; Hou, Shurong; Myint, Wazo; Nalivaika, Ellen; Somasundaran, Mohan; Kelch, Brian A; Matsuo, Hiroshi; Kurt Yilmaz, Nese; Schiffer, Celia A

2018-05-14

The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A-ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A's involvement in mutation of endogenous or exogenous DNA.

EGNAS: an exhaustive DNA sequence design algorithm

Directory of Open Access Journals (Sweden)

Kick Alfred

2012-06-01

Full Text Available Abstract Background The molecular recognition based on the complementary base pairing of deoxyribonucleic acid (DNA is the fundamental principle in the fields of genetics, DNA nanotechnology and DNA computing. We present an exhaustive DNA sequence design algorithm that allows to generate sets containing a maximum number of sequences with defined properties. EGNAS (Exhaustive Generation of Nucleic Acid Sequences offers the possibility of controlling both interstrand and intrastrand properties. The guanine-cytosine content can be adjusted. Sequences can be forced to start and end with guanine or cytosine. This option reduces the risk of “fraying” of DNA strands. It is possible to limit cross hybridizations of a defined length, and to adjust the uniqueness of sequences. Self-complementarity and hairpin structures of certain length can be avoided. Sequences and subsequences can optionally be forbidden. Furthermore, sequences can be designed to have minimum interactions with predefined strands and neighboring sequences. Results The algorithm is realized in a C++ program. TAG sequences can be generated and combined with primers for single-base extension reactions, which were described for multiplexed genotyping of single nucleotide polymorphisms. Thereby, possible foldback through intrastrand interaction of TAG-primer pairs can be limited. The design of sequences for specific attachment of molecular constructs to DNA origami is presented. Conclusions We developed a new software tool called EGNAS for the design of unique nucleic acid sequences. The presented exhaustive algorithm allows to generate greater sets of sequences than with previous software and equal constraints. EGNAS is freely available for noncommercial use at http://www.chm.tu-dresden.de/pc6/EGNAS.

Complete cDNA sequence coding for human docking protein

Energy Technology Data Exchange (ETDEWEB)

Hortsch, M; Labeit, S; Meyer, D I

1988-01-11

Docking protein (DP, or SRP receptor) is a rough endoplasmic reticulum (ER)-associated protein essential for the targeting and translocation of nascent polypeptides across this membrane. It specifically interacts with a cytoplasmic ribonucleoprotein complex, the signal recognition particle (SRP). The nucleotide sequence of cDNA encoding the entire human DP and its deduced amino acid sequence are given.

Radiographer led supplementary anterior cruciate ligament MRI sequences: Technical report

International Nuclear Information System (INIS)

Richards, Paula J.; McCall, Iain; Kraus, Alexandra; Jones, Mary; Walley, Gayle; Gibson, Kathryn; Maffulli, Nicola

2012-01-01

sequences by the radiologist, and one ACL was normal and one had a full thickness tear at arthroscopy. The ACL evaluation for complete tears and a normal ACL on the volume sequences had a sensitivity of 100%, specificity of 97% and accuracy of 97%, excluding partial tears. Volume sequences including partial tears, had specificities and accuracies over 94%, with substantial interobserver agreement (Kappa 0.86, 95% CI 0.71–1.0). The limited oblique T1 sequence had sensitivities and negative predictive value of over 90%, but low specificity and positive predictive values and slight interobserver reliability (Kappa 0.42, 95% CI 0.2–0.6). Conclusions: Firstly, we have shown pragmatically that experienced radiographers identified almost all cases requiring supplementary MRI ACL sequences, without over scanning or needing supervision. Secondly, either volume sequence (DESS or FLASH) could be used as the supplementary sequence, to evaluate the ACL, but a limited oblique T1 sequence of the intercondylar notch cannot be recommended.

Sequence-specific electrochemical recognition of multiple species using nanoparticle labels

International Nuclear Information System (INIS)

Cai Hong; Shang, Chii; Hsing, I.-Ming

2004-01-01

In this work, we report an electrochemical methodology that enables the rapid identification of different DNA sequences on the microfabricated electrodes. Our approach starts with an electropolymerization process on a patterned indium tin oxide (ITO)-coated glass electrode, followed by a selective immobilization of biotin-tagged probes on individually addressable spots via the biotin-streptavidin linkage. An exemplary target mixture containing E. coli and Stachybotrys Chartarum, an airborne pathogen, is then introduced. Recognition of the DNA hybridization event of the immobilized probes with the target pathogen PCR products or synthetic oligonucleotides is achieved by a novel electrochemistry-based technique utilizing the preferential catalytic silver electrodeposition process on the DNA-linked nanogold shells. The ability to selectively immobilize different oligonucleotide probes together with a sensitive electrochemistry-based detection for multiple species, as demonstrated in this study, is an important step forward for the realization of a portable bioanalytical microdevice for the rapid detection of pathogens

Subfamily logos: visualization of sequence deviations at alignment positions with high information content

Directory of Open Access Journals (Sweden)

Beitz Eric

2006-06-01

Full Text Available Abstract Background Recognition of relevant sequence deviations can be valuable for elucidating functional differences between protein subfamilies. Interesting residues at highly conserved positions can then be mutated and experimentally analyzed. However, identification of such sites is tedious because automated approaches are scarce. Results Subfamily logos visualize subfamily-specific sequence deviations. The display is similar to classical sequence logos but extends into the negative range. Positive, upright characters correspond to residues which are characteristic for the subfamily, negative, upside-down characters to residues typical for the remaining sequences. The symbol height is adjusted to the information content of the alignment position. Residues which are conserved throughout do not appear. Conclusion Subfamily logos provide an intuitive display of relevant sequence deviations. The method has proven to be valid using a set of 135 aligned aquaporin sequences in which established subfamily-specific positions were readily identified by the algorithm.

Intra-species sequence comparisons for annotating genomes

Energy Technology Data Exchange (ETDEWEB)

Boffelli, Dario; Weer, Claire V.; Weng, Li; Lewis, Keith D.; Shoukry, Malak I.; Pachter, Lior; Keys, David N.; Rubin, Edward M.

2004-07-15

Analysis of sequence variation among members of a single species offers a potential approach to identify functional DNA elements responsible for biological features unique to that species. Due to its high rate of allelic polymorphism and ease of genetic manipulability, we chose the sea squirt, Ciona intestinalis, to explore intra-species sequence comparisons for genome annotation. A large number of C. intestinalis specimens were collected from four continents and a set of genomic intervals amplified, resequenced and analyzed to determine the mutation rates at each nucleotide in the sequence. We found that regions with low mutation rates efficiently demarcated functionally constrained sequences: these include a set of noncoding elements, which we showed in C intestinalis transgenic assays to act as tissue-specific enhancers, as well as the location of coding sequences. This illustrates that comparisons of multiple members of a species can be used for genome annotation, suggesting a path for the annotation of the sequenced genomes of organisms occupying uncharacterized phylogenetic branches of the animal kingdom and raises the possibility that the resequencing of a large number of Homo sapiens individuals might be used to annotate the human genome and identify sequences defining traits unique to our species. The sequence data from this study has been submitted to GenBank under accession nos. AY667278-AY667407.

Waiting Time Distributions for Pattern Occurrence in a Constrained Sequence

Directory of Open Access Journals (Sweden)

Valeri Stefanov

2007-01-01

Full Text Available A binary sequence of zeros and ones is called a (d,k-sequence if it does not contain runs of zeros of length either less than d or greater than k, where d and k are arbitrary, but fixed, non-negative integers and d < k. Such sequences find an abundance of applications in communications, in particular for magnetic and optical recording. Occasionally, one requires that (d,k-sequences do not contain a specific pattern w. Therefore, distribution results concerning pattern occurrence in (d,k-sequences are of interest. In this paper we study the distribution of the waiting time until the r th occurrence of a pattern w in a random (d,k-sequence generated by a Markov source. Numerical examples are also provided.

Entropic fluctuations in DNA sequences

Science.gov (United States)

Thanos, Dimitrios; Li, Wentian; Provata, Astero

2018-03-01

The Local Shannon Entropy (LSE) in blocks is used as a complexity measure to study the information fluctuations along DNA sequences. The LSE of a DNA block maps the local base arrangement information to a single numerical value. It is shown that despite this reduction of information, LSE allows to extract meaningful information related to the detection of repetitive sequences in whole chromosomes and is useful in finding evolutionary differences between organisms. More specifically, large regions of tandem repeats, such as centromeres, can be detected based on their low LSE fluctuations along the chromosome. Furthermore, an empirical investigation of the appropriate block sizes is provided and the relationship of LSE properties with the structure of the underlying repetitive units is revealed by using both computational and mathematical methods. Sequence similarity between the genomic DNA of closely related species also leads to similar LSE values at the orthologous regions. As an application, the LSE covariance function is used to measure the evolutionary distance between several primate genomes.

Prediction of Protein Structural Classes for Low-Similarity Sequences Based on Consensus Sequence and Segmented PSSM

Directory of Open Access Journals (Sweden)

Yunyun Liang

2015-01-01

Full Text Available Prediction of protein structural classes for low-similarity sequences is useful for understanding fold patterns, regulation, functions, and interactions of proteins. It is well known that feature extraction is significant to prediction of protein structural class and it mainly uses protein primary sequence, predicted secondary structure sequence, and position-specific scoring matrix (PSSM. Currently, prediction solely based on the PSSM has played a key role in improving the prediction accuracy. In this paper, we propose a novel method called CSP-SegPseP-SegACP by fusing consensus sequence (CS, segmented PsePSSM, and segmented autocovariance transformation (ACT based on PSSM. Three widely used low-similarity datasets (1189, 25PDB, and 640 are adopted in this paper. Then a 700-dimensional (700D feature vector is constructed and the dimension is decreased to 224D by using principal component analysis (PCA. To verify the performance of our method, rigorous jackknife cross-validation tests are performed on 1189, 25PDB, and 640 datasets. Comparison of our results with the existing PSSM-based methods demonstrates that our method achieves the favorable and competitive performance. This will offer an important complementary to other PSSM-based methods for prediction of protein structural classes for low-similarity sequences.

Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context.

Directory of Open Access Journals (Sweden)

Artyom A Alekseyenko

Full Text Available The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE. However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex and female Kc cells (which lack the complex, we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.

The consolidation of implicit sequence memory in obstructive sleep apnea.

Directory of Open Access Journals (Sweden)

Eszter Csabi

Full Text Available Obstructive Sleep Apnea (OSA Syndrome is a relatively frequent sleep disorder characterized by disrupted sleep patterns. It is a well-established fact that sleep has beneficial effect on memory consolidation by enhancing neural plasticity. Implicit sequence learning is a prominent component of skill learning. However, the formation and consolidation of this fundamental learning mechanism remains poorly understood in OSA. In the present study we examined the consolidation of different aspects of implicit sequence learning in patients with OSA. We used the Alternating Serial Reaction Time task to measure general skill learning and sequence-specific learning. There were two sessions: a learning phase and a testing phase, separated by a 10-hour offline period with sleep. Our data showed differences in offline changes of general skill learning between the OSA and control group. The control group demonstrated offline improvement from evening to morning, while the OSA group did not. In contrast, we did not observe differences between the groups in offline changes in sequence-specific learning. Our findings suggest that disrupted sleep in OSA differently affects neural circuits involved in the consolidation of sequence learning.

Low-Energy Electron-Induced Strand Breaks in Telomere-Derived DNA Sequences-Influence of DNA Sequence and Topology.

Science.gov (United States)

Rackwitz, Jenny; Bald, Ilko

2018-03-26

During cancer radiation therapy high-energy radiation is used to reduce tumour tissue. The irradiation produces a shower of secondary low-energy (DNA very efficiently by dissociative electron attachment. Recently, it was suggested that low-energy electron-induced DNA strand breaks strongly depend on the specific DNA sequence with a high sensitivity of G-rich sequences. Here, we use DNA origami platforms to expose G-rich telomere sequences to low-energy (8.8 eV) electrons to determine absolute cross sections for strand breakage and to study the influence of sequence modifications and topology of telomeric DNA on the strand breakage. We find that the telomeric DNA 5'-(TTA GGG) 2 is more sensitive to low-energy electrons than an intermixed sequence 5'-(TGT GTG A) 2 confirming the unique electronic properties resulting from G-stacking. With increasing length of the oligonucleotide (i.e., going from 5'-(GGG ATT) 2 to 5'-(GGG ATT) 4 ), both the variety of topology and the electron-induced strand break cross sections increase. Addition of K + ions decreases the strand break cross section for all sequences that are able to fold G-quadruplexes or G-intermediates, whereas the strand break cross section for the intermixed sequence remains unchanged. These results indicate that telomeric DNA is rather sensitive towards low-energy electron-induced strand breakage suggesting significant telomere shortening that can also occur during cancer radiation therapy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Salt bridges: geometrically specific, designable interactions.

Science.gov (United States)

Donald, Jason E; Kulp, Daniel W; DeGrado, William F

2011-03-01

Salt bridges occur frequently in proteins, providing conformational specificity and contributing to molecular recognition and catalysis. We present a comprehensive analysis of these interactions in protein structures by surveying a large database of protein structures. Salt bridges between Asp or Glu and His, Arg, or Lys display extremely well-defined geometric preferences. Several previously observed preferences are confirmed, and others that were previously unrecognized are discovered. Salt bridges are explored for their preferences for different separations in sequence and in space, geometric preferences within proteins and at protein-protein interfaces, co-operativity in networked salt bridges, inclusion within metal-binding sites, preference for acidic electrons, apparent conformational side chain entropy reduction on formation, and degree of burial. Salt bridges occur far more frequently between residues at close than distant sequence separations, but, at close distances, there remain strong preferences for salt bridges at specific separations. Specific types of complex salt bridges, involving three or more members, are also discovered. As we observe a strong relationship between the propensity to form a salt bridge and the placement of salt-bridging residues in protein sequences, we discuss the role that salt bridges might play in kinetically influencing protein folding and thermodynamically stabilizing the native conformation. We also develop a quantitative method to select appropriate crystal structure resolution and B-factor cutoffs. Detailed knowledge of these geometric and sequence dependences should aid de novo design and prediction algorithms. Copyright © 2010 Wiley-Liss, Inc.

In Silico Derivation of HLA-Specific Alloreactivity Potential from Whole Exome Sequencing of Stem Cell Transplant Donors and Recipients: Understanding the Quantitative Immunobiology of Allogeneic Transplantation

Directory of Open Access Journals (Sweden)

Max eJameson-Lee

2014-11-01

Full Text Available Donor T cell mediated graft versus host effects (GVH may result from the aggregate alloreactivity to minor histocompatibility antigens (mHA presented by the HLA molecules in each donor-recipient pair undergoing stem cell transplantation (SCT. Whole exome sequencing has previously demonstrated a large number of nonsynonymous single nucleotide polymorphisms (SNP present in HLA-matched recipients of SCT donors (GVH direction. The nucleotide sequence flanking each of these SNPs was obtained and the amino acid sequence determined. All the possible nonameric-peptides incorporating the variant amino acid resulting from these SNPs were interrogated in-silico for their likelihood to be presented by the HLA class I molecules using the Immune Epitope Database stabilized matrix method (SMM and NetMHCpan algorithms. The SMM algorithm predicted that a median of 18,396 peptides weakly bound HLA class I molecules in individual SCT recipients, and 2,254 peptides displayed strong binding. A similar library of presented peptides was identified when the data was interrogated using the NetMHCpan algorithm. The bioinformatic algorithm presented here demonstrates that there may be a high level of mHA variation in HLA-matched individuals, constituting an HLA-specific alloreactivity potential.

Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

Science.gov (United States)

Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

2017-01-01

Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

A method for selecting cis-acting regulatory sequences that respond to small molecule effectors

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Allas Ülar

2010-08-01

Full Text Available Abstract Background Several cis-acting regulatory sequences functioning at the level of mRNA or nascent peptide and specifically influencing transcription or translation have been described. These regulatory elements often respond to specific chemicals. Results We have developed a method that allows us to select cis-acting regulatory sequences that respond to diverse chemicals. The method is based on the β-lactamase gene containing a random sequence inserted into the beginning of the ORF. Several rounds of selection are used to isolate sequences that suppress β-lactamase expression in response to the compound under study. We have isolated sequences that respond to erythromycin, troleandomycin, chloramphenicol, meta-toluate and homoserine lactone. By introducing synonymous and non-synonymous mutations we have shown that at least in the case of erythromycin the sequences act at the peptide level. We have also tested the cross-activities of the constructs and found that in most cases the sequences respond most strongly to the compound on which they were isolated. Conclusions Several selected peptides showed ligand-specific changes in amino acid frequencies, but no consensus motif could be identified. This is consistent with previous observations on natural cis-acting peptides, showing that it is often impossible to demonstrate a consensus. Applying the currently developed method on a larger scale, by selecting and comparing an extended set of sequences, might allow the sequence rules underlying the activity of cis-acting regulatory peptides to be identified.

Compositions and methods for xylem-specific expression in plant cells

Energy Technology Data Exchange (ETDEWEB)

Han, Kyung-Hwan; Ko, Jae-Heung

2017-12-19

The invention provides promoter sequences that regulate specific expression of operably linked sequences in developing xylem cells and/or in developing xylem tissue. The developing xylem-specific sequences are exemplified by the DX5, DX8, DX11, and DX15 promoters, portions thereof, and homologs thereof. The invention further provides expression vectors, cells, tissues and plants that contain the invention's sequences. The compositions of the invention and methods of using them are useful in, for example, improving the quantity (biomass) and/or the quality (wood density, lignin content, sugar content etc.) of expressed biomass feedstock products that may be used for bioenergy, biorefinary, and generating wood products such as pulp, paper, and solid wood.

Application of Next-generation Sequencing in Clinical Molecular Diagnostics

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Morteza Seifi

2017-05-01

Full Text Available ABSTRACT Next-generation sequencing (NGS is the catch all terms that used to explain several different modern sequencing technologies which let us to sequence nucleic acids much more rapidly and cheaply than the formerly used Sanger sequencing, and as such have revolutionized the study of molecular biology and genomics with excellent resolution and accuracy. Over the past years, many academic companies and institutions have continued technological advances to expand NGS applications from research to the clinic. In this review, the performance and technical features of current NGS platforms were described. Furthermore, advances in the applying of NGS technologies towards the progress of clinical molecular diagnostics were emphasized. General advantages and disadvantages of each sequencing system are summarized and compared to guide the selection of NGS platforms for specific research aims.

Statistical method to compare massive parallel sequencing pipelines.

Science.gov (United States)

Elsensohn, M H; Leblay, N; Dimassi, S; Campan-Fournier, A; Labalme, A; Roucher-Boulez, F; Sanlaville, D; Lesca, G; Bardel, C; Roy, P

2017-03-01

Today, sequencing is frequently carried out by Massive Parallel Sequencing (MPS) that cuts drastically sequencing time and expenses. Nevertheless, Sanger sequencing remains the main validation method to confirm the presence of variants. The analysis of MPS data involves the development of several bioinformatic tools, academic or commercial. We present here a statistical method to compare MPS pipelines and test it in a comparison between an academic (BWA-GATK) and a commercial pipeline (TMAP-NextGENe®), with and without reference to a gold standard (here, Sanger sequencing), on a panel of 41 genes in 43 epileptic patients. This method used the number of variants to fit log-linear models for pairwise agreements between pipelines. To assess the heterogeneity of the margins and the odds ratios of agreement, four log-linear models were used: a full model, a homogeneous-margin model, a model with single odds ratio for all patients, and a model with single intercept. Then a log-linear mixed model was fitted considering the biological variability as a random effect. Among the 390,339 base-pairs sequenced, TMAP-NextGENe® and BWA-GATK found, on average, 2253.49 and 1857.14 variants (single nucleotide variants and indels), respectively. Against the gold standard, the pipelines had similar sensitivities (63.47% vs. 63.42%) and close but significantly different specificities (99.57% vs. 99.65%; p < 0.001). Same-trend results were obtained when only single nucleotide variants were considered (99.98% specificity and 76.81% sensitivity for both pipelines). The method allows thus pipeline comparison and selection. It is generalizable to all types of MPS data and all pipelines.

HEURISTIC METHODS FOR TEST SEQUENCING IN TELECOMMUNICATION SATELLITES

OpenAIRE

Boche-Sauvan , Ludivine; Cabon , Bertrand; Huguet , Marie-José; Hébrard , Emmanuel

2014-01-01

Colloque avec actes et comité de lecture. internationale.; International audience; The increasing complexity in telecommunication satellite payload design demands new test sequencing approaches to face the induced increase of validation complexity. In such a sequencing problem, each test requires specific payload equipment states at a dedicated temperature range. The industrial objectives are both to reduce the total time of tests and to keep the thermal stability of the payload. For solving ...

Controversy and debate on clinical genomics sequencing-paper 2: clinical genome-wide sequencing: don't throw out the baby with the bathwater!

Science.gov (United States)

Adam, Shelin; Friedman, Jan M

2017-12-01

Genome-wide (exome or whole genome) sequencing with appropriate genetic counseling should be considered for any patient with a suspected Mendelian disease that has not been identified by conventional testing. Clinical genome-wide sequencing provides a powerful and effective means of identifying specific genetic causes of serious disease and improving clinical care. Copyright © 2017 Elsevier Inc. All rights reserved.

AlignMiner: a Web-based tool for detection of divergent regions in multiple sequence alignments of conserved sequences

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Claros M Gonzalo

2010-06-01

Full Text Available Abstract Background Multiple sequence alignments are used to study gene or protein function, phylogenetic relations, genome evolution hypotheses and even gene polymorphisms. Virtually without exception, all available tools focus on conserved segments or residues. Small divergent regions, however, are biologically important for specific quantitative polymerase chain reaction, genotyping, molecular markers and preparation of specific antibodies, and yet have received little attention. As a consequence, they must be selected empirically by the researcher. AlignMiner has been developed to fill this gap in bioinformatic analyses. Results AlignMiner is a Web-based application for detection of conserved and divergent regions in alignments of conserved sequences, focusing particularly on divergence. It accepts alignments (protein or nucleic acid obtained using any of a variety of algorithms, which does not appear to have a significant impact on the final results. AlignMiner uses different scoring methods for assessing conserved/divergent regions, Entropy being the method that provides the highest number of regions with the greatest length, and Weighted being the most restrictive. Conserved/divergent regions can be generated either with respect to the consensus sequence or to one master sequence. The resulting data are presented in a graphical interface developed in AJAX, which provides remarkable user interaction capabilities. Users do not need to wait until execution is complete and can.even inspect their results on a different computer. Data can be downloaded onto a user disk, in standard formats. In silico and experimental proof-of-concept cases have shown that AlignMiner can be successfully used to designing specific polymerase chain reaction primers as well as potential epitopes for antibodies. Primer design is assisted by a module that deploys several oligonucleotide parameters for designing primers "on the fly". Conclusions AlignMiner can be used

Sequence analysis and over-expression of ribosomal protein S28 ...

African Journals Online (AJOL)

RPS28 is a component of the 40S small ribosomal subunit encoded by RPS28 gene, which is specific to eukaryotes. The cDNA and the genomic sequence of RPS28 were cloned successfully from the Giant Panda using RT-PCR technology and Touchdown-PCR, respectively. Both sequences were analyzed preliminarily ...

A programmable method for massively parallel targeted sequencing

Science.gov (United States)

Hopmans, Erik S.; Natsoulis, Georges; Bell, John M.; Grimes, Susan M.; Sieh, Weiva; Ji, Hanlee P.

2014-01-01

We have developed a targeted resequencing approach referred to as Oligonucleotide-Selective Sequencing. In this study, we report a series of significant improvements and novel applications of this method whereby the surface of a sequencing flow cell is modified in situ to capture specific genomic regions of interest from a sample and then sequenced. These improvements include a fully automated targeted sequencing platform through the use of a standard Illumina cBot fluidics station. Targeting optimization increased the yield of total on-target sequencing data 2-fold compared to the previous iteration, while simultaneously increasing the percentage of reads that could be mapped to the human genome. The described assays cover up to 1421 genes with a total coverage of 5.5 Megabases (Mb). We demonstrate a 10-fold abundance uniformity of greater than 90% in 1 log distance from the median and a targeting rate of up to 95%. We also sequenced continuous genomic loci up to 1.5 Mb while simultaneously genotyping SNPs and genes. Variants with low minor allele fraction were sensitively detected at levels of 5%. Finally, we determined the exact breakpoint sequence of cancer rearrangements. Overall, this approach has high performance for selective sequencing of genome targets, configuration flexibility and variant calling accuracy. PMID:24782526

Metal resistance sequences and transgenic plants

Science.gov (United States)

Meagher, Richard Brian; Summers, Anne O.; Rugh, Clayton L.

1999-10-12

The present invention provides nucleic acid sequences encoding a metal ion resistance protein, which are expressible in plant cells. The metal resistance protein provides for the enzymatic reduction of metal ions including but not limited to divalent Cu, divalent mercury, trivalent gold, divalent cadmium, lead ions and monovalent silver ions. Transgenic plants which express these coding sequences exhibit increased resistance to metal ions in the environment as compared with plants which have not been so genetically modified. Transgenic plants with improved resistance to organometals including alkylmercury compounds, among others, are provided by the further inclusion of plant-expressible organometal lyase coding sequences, as specifically exemplified by the plant-expressible merB coding sequence. Furthermore, these transgenic plants which have been genetically modified to express the metal resistance coding sequences of the present invention can participate in the bioremediation of metal contamination via the enzymatic reduction of metal ions. Transgenic plants resistant to organometals can further mediate remediation of organic metal compounds, for example, alkylmetal compounds including but not limited to methyl mercury, methyl lead compounds, methyl cadmium and methyl arsenic compounds, in the environment by causing the freeing of mercuric or other metal ions and the reduction of the ionic mercury or other metal ions to the less toxic elemental mercury or other metals.

Computational sequence analysis of predicted long dsRNA transcriptomes of major crops reveals sequence complementarity with human genes.

Science.gov (United States)

Jensen, Peter D; Zhang, Yuanji; Wiggins, B Elizabeth; Petrick, Jay S; Zhu, Jin; Kerstetter, Randall A; Heck, Gregory R; Ivashuta, Sergey I

2013-01-01

Long double-stranded RNAs (long dsRNAs) are precursors for the effector molecules of sequence-specific RNA-based gene silencing in eukaryotes. Plant cells can contain numerous endogenous long dsRNAs. This study demonstrates that such endogenous long dsRNAs in plants have sequence complementarity to human genes. Many of these complementary long dsRNAs have perfect sequence complementarity of at least 21 nucleotides to human genes; enough complementarity to potentially trigger gene silencing in targeted human cells if delivered in functional form. However, the number and diversity of long dsRNA molecules in plant tissue from crops such as lettuce, tomato, corn, soy and rice with complementarity to human genes that have a long history of safe consumption supports a conclusion that long dsRNAs do not present a significant dietary risk.

Outline of a genome navigation system based on the properties of GA-sequences and their flanks.

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Guenter Albrecht-Buehler

Full Text Available Introducing a new method to visualize large stretches of genomic DNA (see Appendix S1 the article reports that most GA-sequences [1] shared chains of tetra-GA-motifs and contained upstream poly(A-segments. Although not integral parts of them, Alu-elements were found immediately upstream of all human and chimpanzee GA-sequences with an upstream poly(A-segment. The article hypothesizes that genome navigation uses these properties of GA-sequences in the following way. (1 Poly(A binding proteins interact with the upstream poly(A-segments and arrange adjacent GA-sequences side-by-side ('GA-ribbon', while folding the intervening DNA sequences between them into loops ('associated DNA-loops'. (2 Genome navigation uses the GA-ribbon as a search path for specific target genes that is up to 730-fold shorter than the full-length chromosome. (3 As to the specificity of the search, each molecule of a target protein is assumed to catalyze the formation of specific oligomers from a set of transcription factors that recognize tetra-GA-motifs. Their specific combinations of tetra-GA motifs are assumed to be present in the particular GA-sequence whose associated loop contains the gene for the target protein. As long as the target protein is abundant in the cell it produces sufficient numbers of such oligomers which bind to their specific GA-sequences and, thereby, inhibit locally the transcription of the target protein in the associated loop. However, if the amount of target protein drops below a certain threshold, the resultant reduction of specific oligomers leaves the corresponding GA-sequence 'denuded'. In response, the associated DNA-loop releases its nucleosomes and allows transcription of the target protein to proceed. (4 The Alu-transcripts may help control the general background of protein synthesis proportional to the number of transcriptionally active associated loops, especially in stressed cells. (5 The model offers a new mechanism of co-regulation of

Methylation patterns of repetitive DNA sequences in germ cells of Mus musculus.

OpenAIRE

Sanford, J; Forrester, L; Chapman, V; Chandley, A; Hastie, N

1984-01-01

The major and the minor satellite sequences of Mus musculus were undermethylated in both sperm and oocyte DNAs relative to the amount of undermethylation observed in adult somatic tissue DNA. This hypomethylation was specific for satellite sequences in sperm DNA. Dispersed repetitive and low copy sequences show a high degree of methylation in sperm DNA; however, a dispersed repetitive sequence was undermethylated in oocyte DNA. This finding suggests a difference in the amount of total genomic...

Genome-Wide Single-Nucleotide Polymorphisms Discovery and High-Density Genetic Map Construction in Cauliflower Using Specific-Locus Amplified Fragment Sequencing

Science.gov (United States)

Zhao, Zhenqing; Gu, Honghui; Sheng, Xiaoguang; Yu, Huifang; Wang, Jiansheng; Huang, Long; Wang, Dan

2016-01-01

Molecular markers and genetic maps play an important role in plant genomics and breeding studies. Cauliflower is an important and distinctive vegetable; however, very few molecular resources have been reported for this species. In this study, a novel, specific-locus amplified fragment (SLAF) sequencing strategy was employed for large-scale single nucleotide polymorphism (SNP) discovery and high-density genetic map construction in a double-haploid, segregating population of cauliflower. A total of 12.47 Gb raw data containing 77.92 M pair-end reads were obtained after processing and 6815 polymorphic SLAFs between the two parents were detected. The average sequencing depths reached 52.66-fold for the female parent and 49.35-fold for the male parent. Subsequently, these polymorphic SLAFs were used to genotype the population and further filtered based on several criteria to construct a genetic linkage map of cauliflower. Finally, 1776 high-quality SLAF markers, including 2741 SNPs, constituted the linkage map with average data integrity of 95.68%. The final map spanned a total genetic length of 890.01 cM with an average marker interval of 0.50 cM, and covered 364.9 Mb of the reference genome. The markers and genetic map developed in this study could provide an important foundation not only for comparative genomics studies within Brassica oleracea species but also for quantitative trait loci identification and molecular breeding of cauliflower. PMID:27047515

Primate-Specific Evolution of an LDLR Enhancer

Energy Technology Data Exchange (ETDEWEB)

Wang, Qian-fei; Prabhakar, Shyam; Wang, Qianben; Moses, Alan M.; Chanan, Sumita; Brown, Myles; Eisen, Michael B.; Cheng, Jan-Fang; Rubin,Edward M.; Boffelli, Dario

2006-06-28

Sequence changes in regulatory regions have often beeninvoked to explain phenotypic divergence among species, but molecularexamples of this have been difficult to obtain. In this study, weidentified an anthropoid primate specific sequence element thatcontributed to the regulatory evolution of the LDL receptor. Using acombination of close and distant species genomic sequence comparisonscoupled with in vivo and in vitro studies, we show that a functionalcholesterol-sensing sequence motif arose and was fixed within apre-existing enhancer in the common ancestor of anthropoid primates. Ourstudy demonstrates one molecular mechanism by which ancestral mammalianregulatory elements can evolve to perform new functions in the primatelineage leading to human.

Efficiency to Discovery Transgenic Loci in GM Rice Using Next Generation Sequencing Whole Genome Re-sequencing

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Doori Park

2015-09-01

Full Text Available Molecular characterization technology in genetically modified organisms, in addition to how transgenic biotechnologies are developed now require full transparency to assess the risk to living modified and non-modified organisms. Next generation sequencing (NGS methodology is suggested as an effective means in genome characterization and detection of transgenic insertion locations. In the present study, we applied NGS to insert transgenic loci, specifically the epidermal growth factor (EGF in genetically modified rice cells. A total of 29.3 Gb (~72× coverage was sequenced with a 2 × 150 bp paired end method by Illumina HiSeq2500, which was consecutively mapped to the rice genome and T-vector sequence. The compatible pairs of reads were successfully mapped to 10 loci on the rice chromosome and vector sequences were validated to the insertion location by polymerase chain reaction (PCR amplification. The EGF transgenic site was confirmed only on chromosome 4 by PCR. Results of this study demonstrated the success of NGS data to characterize the rice genome. Bioinformatics analyses must be developed in association with NGS data to identify highly accurate transgenic sites.

cDNA sequences of two inducible T-cell genes

Energy Technology Data Exchange (ETDEWEB)

Kwon, B.S. (Indiana Univ. School of Medicine, Indianapolis (USA) Guthrie Research Institute, Sayre, PA (USA)); Weissman, S.M. (Yale Univ., New Haven, CT (USA))

1989-03-01

The authors have previously described a set of human T-lymphocyte-specific cDNA clones isolated by a modified differential screening procedure. Apparent full-length cDNAs containing the sequences of 14 of the 16 initial isolates were sequenced and were found to represent five different species of mRNA; three of the five species were identical to previously reported cDNA sequences of preproenkephalin, T-cell-replacing factor, and a serine esterase, respectively. The other two species, 4-1BB and L2G25B, were inducible sequences found in mRNA from both a cytolytic T-lymphocyte and a helper T-lymphocyte clone and were not previously described in T-cell mRNA; these mRNA sequences encode peptides of 256 and 92 amino acids, respectively. Both peptides contain putative leader sequences. The protein encoded by 4-1BB also has a potential membrane anchor segment and other features also seen in known receptor proteins.

High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic.

Science.gov (United States)

Sealfon, Rachel; Gire, Stephen; Ellis, Crystal; Calderwood, Stephen; Qadri, Firdausi; Hensley, Lisa; Kellis, Manolis; Ryan, Edward T; LaRocque, Regina C; Harris, Jason B; Sabeti, Pardis C

2012-09-11

Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x); four of the seven isolates were previously sequenced. Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961), 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.

High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic

Directory of Open Access Journals (Sweden)

Sealfon Rachel

2012-09-01

Full Text Available Abstract Background Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x; four of the seven isolates were previously sequenced. Results Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961, 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Conclusions Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.

System-level hazard analysis using the sequence-tree method

International Nuclear Information System (INIS)

Huang, H.-W.; Shih Chunkuan; Yih Swu; Chen, M.-H.

2008-01-01

A system-level PHA using the sequence-tree method is presented to perform safety-related digital I and C system SSA. The conventional PHA involves brainstorming among experts on various portions of the system to identify hazards through discussions. However, since the conventional PHA is not a systematic technique, the analysis results depend strongly on the experts' subjective opinions. The quality of analysis cannot be appropriately controlled. Therefore, this study presents a system-level sequence tree based PHA, which can clarify the relationship among the major digital I and C systems. This sequence-tree-based technique has two major phases. The first phase adopts a table to analyze each event in SAR Chapter 15 for a specific safety-related I and C system, such as RPS. The second phase adopts a sequence tree to recognize the I and C systems involved in the event, the working of the safety-related systems and how the backup systems can be activated to mitigate the consequence if the primary safety systems fail. The defense-in-depth echelons, namely the Control echelon, Reactor trip echelon, ESFAS echelon and Monitoring and indicator echelon, are arranged to build the sequence-tree structure. All the related I and C systems, including the digital systems and the analog back-up systems, are allocated in their specific echelons. This system-centric sequence-tree analysis not only systematically identifies preliminary hazards, but also vulnerabilities in a nuclear power plant. Hence, an effective simplified D3 evaluation can also be conducted

Low-pass shotgun sequencing of the barley genome facilitates rapid identification of genes, conserved non-coding sequences and novel repeats

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Graner Andreas

2008-10-01

Full Text Available Abstract Background Barley has one of the largest and most complex genomes of all economically important food crops. The rise of new short read sequencing technologies such as Illumina/Solexa permits such large genomes to be effectively sampled at relatively low cost. Based on the corresponding sequence reads a Mathematically Defined Repeat (MDR index can be generated to map repetitive regions in genomic sequences. Results We have generated 574 Mbp of Illumina/Solexa sequences from barley total genomic DNA, representing about 10% of a genome equivalent. From these sequences we generated an MDR index which was then used to identify and mark repetitive regions in the barley genome. Comparison of the MDR plots with expert repeat annotation drawing on the information already available for known repetitive elements revealed a significant correspondence between the two methods. MDR-based annotation allowed for the identification of dozens of novel repeat sequences, though, which were not recognised by hand-annotation. The MDR data was also used to identify gene-containing regions by masking of repetitive sequences in eight de-novo sequenced bacterial artificial chromosome (BAC clones. For half of the identified candidate gene islands indeed gene sequences could be identified. MDR data were only of limited use, when mapped on genomic sequences from the closely related species Triticum monococcum as only a fraction of the repetitive sequences was recognised. Conclusion An MDR index for barley, which was obtained by whole-genome Illumina/Solexa sequencing, proved as efficient in repeat identification as manual expert annotation. Circumventing the labour-intensive step of producing a specific repeat library for expert annotation, an MDR index provides an elegant and efficient resource for the identification of repetitive and low-copy (i.e. potentially gene-containing sequences regions in uncharacterised genomic sequences. The restriction that a particular

Third-Generation Sequencing and Analysis of Four Complete Pig Liver Esterase Gene Sequences in Clones Identified by Screening BAC Library.

Science.gov (United States)

Zhou, Qiongqiong; Sun, Wenjuan; Liu, Xiyan; Wang, Xiliang; Xiao, Yuncai; Bi, Dingren; Yin, Jingdong; Shi, Deshi

2016-01-01

Pig liver carboxylesterase (PLE) gene sequences in GenBank are incomplete, which has led to difficulties in studying the genetic structure and regulation mechanisms of gene expression of PLE family genes. The aim of this study was to obtain and analysis of complete gene sequences of PLE family by screening from a Rongchang pig BAC library and third-generation PacBio gene sequencing. After a number of existing incomplete PLE isoform gene sequences were analysed, primers were designed based on conserved regions in PLE exons, and the whole pig genome used as a template for Polymerase chain reaction (PCR) amplification. Specific primers were then selected based on the PCR amplification results. A three-step PCR screening method was used to identify PLE-positive clones by screening a Rongchang pig BAC library and PacBio third-generation sequencing was performed. BLAST comparisons and other bioinformatics methods were applied for sequence analysis. Five PLE-positive BAC clones, designated BAC-10, BAC-70, BAC-75, BAC-119 and BAC-206, were identified. Sequence analysis yielded the complete sequences of four PLE genes, PLE1, PLE-B9, PLE-C4, and PLE-G2. Complete PLE gene sequences were defined as those containing regulatory sequences, exons, and introns. It was found that, not only did the PLE exon sequences of the four genes show a high degree of homology, but also that the intron sequences were highly similar. Additionally, the regulatory region of the genes contained two 720bps reverse complement sequences that may have an important function in the regulation of PLE gene expression. This is the first report to confirm the complete sequences of four PLE genes. In addition, the study demonstrates that each PLE isoform is encoded by a single gene and that the various genes exhibit a high degree of sequence homology, suggesting that the PLE family evolved from a single ancestral gene. Obtaining the complete sequences of these PLE genes provides the necessary foundation for

Development of a real-time PCR for detection of Staphylococcus pseudintermedius using a novel automated comparison of whole-genome sequences.

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Koen M Verstappen

Full Text Available Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and cats and occasionally causes infections in humans. S. pseudintermedius is often resistant to multiple classes of antimicrobials. It requires a reliable detection so that it is not misidentified as S. aureus. Phenotypic and currently-used molecular-based diagnostic assays lack specificity or are labour-intensive using multiplex PCR or nucleic acid sequencing. The aim of this study was to identify a specific target for real-time PCR by comparing whole genome sequences of S. pseudintermedius and non-pseudintermedius.Genome sequences were downloaded from public repositories and supplemented by isolates that were sequenced in this study. A Perl-script was written that analysed 300-nt fragments from a reference genome sequence of S. pseudintermedius and checked if this sequence was present in other S. pseudintermedius genomes (n = 74 and non-pseudintermedius genomes (n = 138. Six sequences specific for S. pseudintermedius were identified (sequence length between 300-500 nt. One sequence, which was located in the spsJ gene, was used to develop primers and a probe. The real-time PCR showed 100% specificity when testing for S. pseudintermedius isolates (n = 54, and eight other staphylococcal species (n = 43. In conclusion, a novel approach by comparing whole genome sequences identified a sequence that is specific for S. pseudintermedius and provided a real-time PCR target for rapid and reliable detection of S. pseudintermedius.

Universal sequence map (USM of arbitrary discrete sequences

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Almeida Jonas S

2002-02-01

Full Text Available Abstract Background For over a decade the idea of representing biological sequences in a continuous coordinate space has maintained its appeal but not been fully realized. The basic idea is that any sequence of symbols may define trajectories in the continuous space conserving all its statistical properties. Ideally, such a representation would allow scale independent sequence analysis – without the context of fixed memory length. A simple example would consist on being able to infer the homology between two sequences solely by comparing the coordinates of any two homologous units. Results We have successfully identified such an iterative function for bijective mappingψ of discrete sequences into objects of continuous state space that enable scale-independent sequence analysis. The technique, named Universal Sequence Mapping (USM, is applicable to sequences with an arbitrary length and arbitrary number of unique units and generates a representation where map distance estimates sequence similarity. The novel USM procedure is based on earlier work by these and other authors on the properties of Chaos Game Representation (CGR. The latter enables the representation of 4 unit type sequences (like DNA as an order free Markov Chain transition table. The properties of USM are illustrated with test data and can be verified for other data by using the accompanying web-based tool:http://bioinformatics.musc.edu/~jonas/usm/. Conclusions USM is shown to enable a statistical mechanics approach to sequence analysis. The scale independent representation frees sequence analysis from the need to assume a memory length in the investigation of syntactic rules.

Properties of Sequence Conservation in Upstream Regulatory and Protein Coding Sequences among Paralogs in Arabidopsis thaliana

Science.gov (United States)

Richardson, Dale N.; Wiehe, Thomas

Whole genome duplication (WGD) has catalyzed the formation of new species, genes with novel functions, altered expression patterns, complexified signaling pathways and has provided organisms a level of genetic robustness. We studied the long-term evolution and interrelationships of 5’ upstream regulatory sequences (URSs), protein coding sequences (CDSs) and expression correlations (EC) of duplicated gene pairs in Arabidopsis. Three distinct methods revealed significant evolutionary conservation between paralogous URSs and were highly correlated with microarray-based expression correlation of the respective gene pairs. Positional information on exact matches between sequences unveiled the contribution of micro-chromosomal rearrangements on expression divergence. A three-way rank analysis of URS similarity, CDS divergence and EC uncovered specific gene functional biases. Transcription factor activity was associated with gene pairs exhibiting conserved URSs and divergent CDSs, whereas a broad array of metabolic enzymes was found to be associated with gene pairs showing diverged URSs but conserved CDSs.

Cascade detection for the extraction of localized sequence features; specificity results for HIV-1 protease and structure-function results for the Schellman loop.

Science.gov (United States)

Newell, Nicholas E

2011-12-15

The extraction of the set of features most relevant to function from classified biological sequence sets is still a challenging problem. A central issue is the determination of expected counts for higher order features so that artifact features may be screened. Cascade detection (CD), a new algorithm for the extraction of localized features from sequence sets, is introduced. CD is a natural extension of the proportional modeling techniques used in contingency table analysis into the domain of feature detection. The algorithm is successfully tested on synthetic data and then applied to feature detection problems from two different domains to demonstrate its broad utility. An analysis of HIV-1 protease specificity reveals patterns of strong first-order features that group hydrophobic residues by side chain geometry and exhibit substantial symmetry about the cleavage site. Higher order results suggest that favorable cooperativity is weak by comparison and broadly distributed, but indicate possible synergies between negative charge and hydrophobicity in the substrate. Structure-function results for the Schellman loop, a helix-capping motif in proteins, contain strong first-order features and also show statistically significant cooperativities that provide new insights into the design of the motif. These include a new 'hydrophobic staple' and multiple amphipathic and electrostatic pair features. CD should prove useful not only for sequence analysis, but also for the detection of multifactor synergies in cross-classified data from clinical studies or other sources. Windows XP/7 application and data files available at: https://sites.google.com/site/cascadedetect/home. nacnewell@comcast.net Supplementary information is available at Bioinformatics online.

Results of the event sequence reliability benchmark exercise

International Nuclear Information System (INIS)

Silvestri, E.

1990-01-01

The Event Sequence Reliability Benchmark Exercise is the fourth of a series of benchmark exercises on reliability and risk assessment, with specific reference to nuclear power plant applications, and is the logical continuation of the previous benchmark exercises on System Analysis Common Cause Failure and Human Factors. The reference plant is the Nuclear Power Plant at Grohnde Federal Republic of Germany a 1300 MW PWR plant of KWU design. The specific objective of the Exercise is to model, to quantify and to analyze such event sequences initiated by the occurrence of a loss of offsite power that involve the steam generator feed. The general aim is to develop a segment of a risk assessment, which ought to include all the specific aspects and models of quantification, such as common canal failure, Human Factors and System Analysis, developed in the previous reliability benchmark exercises, with the addition of the specific topics of dependences between homologous components belonging to different systems featuring in a given event sequence and of uncertainty quantification, to end up with an overall assessment of: - the state of the art in risk assessment and the relative influences of quantification problems in a general risk assessment framework. The Exercise has been carried out in two phases, both requiring modelling and quantification, with the second phase adopting more restrictive rules and fixing certain common data, as emerged necessary from the first phase. Fourteen teams have participated in the Exercise mostly from EEC countries, with one from Sweden and one from the USA. (author)

HomPPI: a class of sequence homology based protein-protein interface prediction methods

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Dobbs Drena

2011-06-01

Full Text Available Abstract Background Although homology-based methods are among the most widely used methods for predicting the structure and function of proteins, the question as to whether interface sequence conservation can be effectively exploited in predicting protein-protein interfaces has been a subject of debate. Results We studied more than 300,000 pair-wise alignments of protein sequences from structurally characterized protein complexes, including both obligate and transient complexes. We identified sequence similarity criteria required for accurate homology-based inference of interface residues in a query protein sequence. Based on these analyses, we developed HomPPI, a class of sequence homology-based methods for predicting protein-protein interface residues. We present two variants of HomPPI: (i NPS-HomPPI (Non partner-specific HomPPI, which can be used to predict interface residues of a query protein in the absence of knowledge of the interaction partner; and (ii PS-HomPPI (Partner-specific HomPPI, which can be used to predict the interface residues of a query protein with a specific target protein. Our experiments on a benchmark dataset of obligate homodimeric complexes show that NPS-HomPPI can reliably predict protein-protein interface residues in a given protein, with an average correlation coefficient (CC of 0.76, sensitivity of 0.83, and specificity of 0.78, when sequence homologs of the query protein can be reliably identified. NPS-HomPPI also reliably predicts the interface residues of intrinsically disordered proteins. Our experiments suggest that NPS-HomPPI is competitive with several state-of-the-art interface prediction servers including those that exploit the structure of the query proteins. The partner-specific classifier, PS-HomPPI can, on a large dataset of transient complexes, predict the interface residues of a query protein with a specific target, with a CC of 0.65, sensitivity of 0.69, and specificity of 0.70, when homologs of

A duplex DNA-gold nanoparticle probe composed as a colorimetric biosensor for sequence-specific DNA-binding proteins.

Science.gov (United States)

Ahn, Junho; Choi, Yeonweon; Lee, Ae-Ree; Lee, Joon-Hwa; Jung, Jong Hwa

2016-03-21

Using duplex DNA-AuNP aggregates, a sequence-specific DNA-binding protein, SQUAMOSA Promoter-binding-Like protein 12 (SPL-12), was directly determined by SPL-12-duplex DNA interaction-based colorimetric actions of DNA-Au assemblies. In order to prepare duplex DNA-Au aggregates, thiol-modified DNA 1 and DNA 2 were attached onto the surface of AuNPs, respectively, by the salt-aging method and then the DNA-attached AuNPs were mixed. Duplex-DNA-Au aggregates having the average size of 160 nm diameter and the maximum absorption at 529 nm were able to recognize SPL-12 and reached the equivalent state by the addition of ∼30 equivalents of SPL-12 accompanying a color change from red to blue with a red shift of the maximum absorption at 570 nm. As a result, the aggregation size grew to about 247 nm. Also, at higher temperatures of the mixture of duplex-DNA-Au aggregate solution and SPL-12, the equivalent state was reached rapidly. On the contrary, in the control experiment using Bovine Serum Albumin (BSA), no absorption band shift of duplex-DNA-Au aggregates was observed.

Second generation sequencing of the mesothelioma tumor genome.

Directory of Open Access Journals (Sweden)

Raphael Bueno

2010-05-01

Full Text Available The current paradigm for elucidating the molecular etiology of cancers relies on the interrogation of small numbers of genes, which limits the scope of investigation. Emerging second-generation massively parallel DNA sequencing technologies have enabled more precise definition of the cancer genome on a global scale. We examined the genome of a human primary malignant pleural mesothelioma (MPM tumor and matched normal tissue by using a combination of sequencing-by-synthesis and pyrosequencing methodologies to a 9.6X depth of coverage. Read density analysis uncovered significant aneuploidy and numerous rearrangements. Method-dependent informatics rules, which combined the results of different sequencing platforms, were developed to identify and validate candidate mutations of multiple types. Many more tumor-specific rearrangements than point mutations were uncovered at this depth of sequencing, resulting in novel, large-scale, inter- and intra-chromosomal deletions, inversions, and translocations. Nearly all candidate point mutations appeared to be previously unknown SNPs. Thirty tumor-specific fusions/translocations were independently validated with PCR and Sanger sequencing. Of these, 15 represented disrupted gene-encoding regions, including kinases, transcription factors, and growth factors. One large deletion in DPP10 resulted in altered transcription and expression of DPP10 transcripts in a set of 53 additional MPM tumors correlated with survival. Additionally, three point mutations were observed in the coding regions of NKX6-2, a transcription regulator, and NFRKB, a DNA-binding protein involved in modulating NFKB1. Several regions containing genes such as PCBD2 and DHFR, which are involved in growth factor signaling and nucleotide synthesis, respectively, were selectively amplified in the tumor. Second-generation sequencing uncovered all types of mutations in this MPM tumor, with DNA rearrangements representing the dominant type.

3D knee segmentation based on three MRI sequences from different planes.

Science.gov (United States)

Zhou, L; Chav, R; Cresson, T; Chartrand, G; de Guise, J

2016-08-01

In clinical practice, knee MRI sequences with 3.5~5 mm slice distance in sagittal, coronal, and axial planes are often requested for the knee examination since its acquisition is faster than high-resolution MRI sequence in a single plane, thereby reducing the probability of motion artifact. In order to take advantage of the three sequences from different planes, a 3D segmentation method based on the combination of three knee models obtained from the three sequences is proposed in this paper. In the method, the sub-segmentation is respectively performed with sagittal, coronal, and axial MRI sequence in the image coordinate system. With each sequence, an initial knee model is hierarchically deformed, and then the three deformed models are mapped to reference coordinate system defined by the DICOM standard and combined to obtain a patient-specific model. The experimental results verified that the three sub-segmentation results can complement each other, and their integration can compensate for the insufficiency of boundary information caused by 3.5~5 mm gap between consecutive slices. Therefore, the obtained patient-specific model is substantially more accurate than each sub-segmentation results.

Is sequence awareness mandatory for perceptual sequence learning: An assessment using a pure perceptual sequence learning design.

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Deroost, Natacha; Coomans, Daphné

2018-02-01

We examined the role of sequence awareness in a pure perceptual sequence learning design. Participants had to react to the target's colour that changed according to a perceptual sequence. By varying the mapping of the target's colour onto the response keys, motor responses changed randomly. The effect of sequence awareness on perceptual sequence learning was determined by manipulating the learning instructions (explicit versus implicit) and assessing the amount of sequence awareness after the experiment. In the explicit instruction condition (n = 15), participants were instructed to intentionally search for the colour sequence, whereas in the implicit instruction condition (n = 15), they were left uninformed about the sequenced nature of the task. Sequence awareness after the sequence learning task was tested by means of a questionnaire and the process-dissociation-procedure. The results showed that the instruction manipulation had no effect on the amount of perceptual sequence learning. Based on their report to have actively applied their sequence knowledge during the experiment, participants were subsequently regrouped in a sequence strategy group (n = 14, of which 4 participants from the implicit instruction condition and 10 participants from the explicit instruction condition) and a no-sequence strategy group (n = 16, of which 11 participants from the implicit instruction condition and 5 participants from the explicit instruction condition). Only participants of the sequence strategy group showed reliable perceptual sequence learning and sequence awareness. These results indicate that perceptual sequence learning depends upon the continuous employment of strategic cognitive control processes on sequence knowledge. Sequence awareness is suggested to be a necessary but not sufficient condition for perceptual learning to take place. Copyright © 2018 Elsevier B.V. All rights reserved.

Single-cell sequencing in stem cell biology.

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Wen, Lu; Tang, Fuchou

2016-04-15

Cell-to-cell variation and heterogeneity are fundamental and intrinsic characteristics of stem cell populations, but these differences are masked when bulk cells are used for omic analysis. Single-cell sequencing technologies serve as powerful tools to dissect cellular heterogeneity comprehensively and to identify distinct phenotypic cell types, even within a 'homogeneous' stem cell population. These technologies, including single-cell genome, epigenome, and transcriptome sequencing technologies, have been developing rapidly in recent years. The application of these methods to different types of stem cells, including pluripotent stem cells and tissue-specific stem cells, has led to exciting new findings in the stem cell field. In this review, we discuss the recent progress as well as future perspectives in the methodologies and applications of single-cell omic sequencing technologies.

Histoimmunogenetics Markup Language 1.0: Reporting next generation sequencing-based HLA and KIR genotyping.

Science.gov (United States)

Milius, Robert P; Heuer, Michael; Valiga, Daniel; Doroschak, Kathryn J; Kennedy, Caleb J; Bolon, Yung-Tsi; Schneider, Joel; Pollack, Jane; Kim, Hwa Ran; Cereb, Nezih; Hollenbach, Jill A; Mack, Steven J; Maiers, Martin

2015-12-01

We present an electronic format for exchanging data for HLA and KIR genotyping with extensions for next-generation sequencing (NGS). This format addresses NGS data exchange by refining the Histoimmunogenetics Markup Language (HML) to conform to the proposed Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines (miring.immunogenomics.org). Our refinements of HML include two major additions. First, NGS is supported by new XML structures to capture additional NGS data and metadata required to produce a genotyping result, including analysis-dependent (dynamic) and method-dependent (static) components. A full genotype, consensus sequence, and the surrounding metadata are included directly, while the raw sequence reads and platform documentation are externally referenced. Second, genotype ambiguity is fully represented by integrating Genotype List Strings, which use a hierarchical set of delimiters to represent allele and genotype ambiguity in a complete and accurate fashion. HML also continues to enable the transmission of legacy methods (e.g. site-specific oligonucleotide, sequence-specific priming, and Sequence Based Typing (SBT)), adding features such as allowing multiple group-specific sequencing primers, and fully leveraging techniques that combine multiple methods to obtain a single result, such as SBT integrated with NGS. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Deep sequencing identifies ethnicity-specific bacterial signatures in the oral microbiome.

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Matthew R Mason

Full Text Available Oral infections have a strong ethnic predilection; suggesting that ethnicity is a critical determinant of oral microbial colonization. Dental plaque and saliva samples from 192 subjects belonging to four major ethnicities in the United States were analyzed using terminal restriction fragment length polymorphism (t-RFLP and 16S pyrosequencing. Ethnicity-specific clustering of microbial communities was apparent in saliva and subgingival biofilms, and a machine-learning classifier was capable of identifying an individual's ethnicity from subgingival microbial signatures. The classifier identified African Americans with a 100% sensitivity and 74% specificity and Caucasians with a 50% sensitivity and 91% specificity. The data demonstrates a significant association between ethnic affiliation and the composition of the oral microbiome; to the extent that these microbial signatures appear to be capable of discriminating between ethnicities.

Strand specific RNA-sequencing and membrane lipid profiling reveals growth phase-dependent cold stress response mechanisms in Listeria monocytogenes

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Hingston, Patricia; Chen, Jessica; Allen, Kevin; Truelstrup Hansen, Lisbeth

2017-01-01

The human pathogen Listeria monocytogenes continues to pose a challenge in the food industry, where it is known to contaminate ready-to-eat foods and grow during refrigerated storage. Increased knowledge of the cold-stress response of this pathogen will enhance the ability to control it in the food-supply-chain. This study utilized strand-specific RNA sequencing and whole cell fatty acid (FA) profiling to characterize the bacterium’s cold stress response. RNA and FAs were extracted from a cold-tolerant strain at five time points between early lag phase and late stationary-phase, both at 4°C and 20°C. Overall, more genes (1.3×) were suppressed than induced at 4°C. Late stationary-phase cells exhibited the greatest number (n = 1,431) and magnitude (>1,000-fold) of differentially expressed genes (>2-fold, pmonocytogenes, the growth-phase dependency of its cold-stress regulon, and the active roles of antisense transcripts in regulating its cold stress response. PMID:28662112

Strand specific RNA-sequencing and membrane lipid profiling reveals growth phase-dependent cold stress response mechanisms in Listeria monocytogenes

DEFF Research Database (Denmark)

Hingston, Patricia; Chen, Jessica; Allen, Kevin

2017-01-01

The human pathogen Listeria monocytogenes continues to pose a challenge in the food industry, where it is known to contaminate ready-to-eat foods and grow during refrigerated storage. Increased knowledge of the cold-stress response of this pathogen will enhance the ability to control it in the food-supply-chain....... This study utilized strand-specific RNA sequencing and whole cell fatty acid (FA) profiling to characterize the bacterium’s cold stress response. RNA and FAs were extracted from a cold-tolerant strain at five time points between early lag phase and late stationary-phase, both at 4°C and 20°C. Overall, more...... genes (1.3×) were suppressed than induced at 4°C. Late stationary-phase cells exhibited the greatest number (n = 1,431) and magnitude (>1,000-fold) of differentially expressed genes (>2-fold, pcold. A core set of 22 genes was upregulated at all growth phases, including nine genes...

Googling DNA sequences on the World Wide Web.

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Hajibabaei, Mehrdad; Singer, Gregory A C

2009-11-10

New web-based technologies provide an excellent opportunity for sharing and accessing information and using web as a platform for interaction and collaboration. Although several specialized tools are available for analyzing DNA sequence information, conventional web-based tools have not been utilized for bioinformatics applications. We have developed a novel algorithm and implemented it for searching species-specific genomic sequences, DNA barcodes, by using popular web-based methods such as Google. We developed an alignment independent character based algorithm based on dividing a sequence library (DNA barcodes) and query sequence to words. The actual search is conducted by conventional search tools such as freely available Google Desktop Search. We implemented our algorithm in two exemplar packages. We developed pre and post-processing software to provide customized input and output services, respectively. Our analysis of all publicly available DNA barcode sequences shows a high accuracy as well as rapid results. Our method makes use of conventional web-based technologies for specialized genetic data. It provides a robust and efficient solution for sequence search on the web. The integration of our search method for large-scale sequence libraries such as DNA barcodes provides an excellent web-based tool for accessing this information and linking it to other available categories of information on the web.

Identification of S-glutathionylation sites in species-specific proteins by incorporating five sequence-derived features into the general pseudo-amino acid composition.

Science.gov (United States)

Zhao, Xiaowei; Ning, Qiao; Ai, Meiyue; Chai, Haiting; Yang, Guifu

2016-06-07

As a selective and reversible protein post-translational modification, S-glutathionylation generates mixed disulfides between glutathione (GSH) and cysteine residues, and plays an important role in regulating protein activity, stability, and redox regulation. To fully understand S-glutathionylation mechanisms, identification of substrates and specific S-Glutathionylated sites is crucial. Experimental identification of S-glutathionylated sites is labor-intensive and time consuming, so establishing an effective computational method is much desirable due to their convenient and fast speed. Therefore, in this study, a new bioinformatics tool named SSGlu (Species-Specific identification of Protein S-glutathionylation Sites) was developed to identify species-specific protein S-glutathionylated sites, utilizing support vector machines that combine multiple sequence-derived features with a two-step feature selection. By 5-fold cross validation, the performance of SSGlu was measured with an AUC of 0.8105 and 0.8041 for Homo sapiens and Mus musculus, respectively. Additionally, SSGlu was compared with the existing methods, and the higher MCC and AUC of SSGlu demonstrated that SSGlu was very promising to predict S-glutathionylated sites. Furthermore, a site-specific analysis showed that S-glutathionylation intimately correlated with the features derived from its surrounding sites. The conclusions derived from this study might help to understand more of the S-glutathionylation mechanism and guide the related experimental validation. For public access, SSGlu is freely accessible at http://59.73.198.144:8080/SSGlu/. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sequence Similarity Presenter: a tool for the graphic display of similarities of long sequences for use in presentations.

Science.gov (United States)

Fröhlich, K U

1994-04-01

A new method for the presentation of alignments of long sequences is described. The degree of identity for the aligned sequences is averaged for sections of a fixed number of residues. The resulting values are converted to shades of gray, with white corresponding to lack of identity and black corresponding to perfect identity. A sequence alignment is represented as a bar filled with varying shades of gray. The display is compact and allows for a fast and intuitive recognition of the distribution of regions with a high similarity. It is well suited for the presentation of alignments of long sequences, e.g. of protein superfamilies, in plenary lectures. The method is implemented as a HyperCard stack for Apple Macintosh computers. Several options for the modification of the output are available (e.g. background reduction, size of the summation window, consideration of amino acid similarity, inclusion of graphic markers to indicate specific domains). The output is a PostScript file which can be printed, imported as EPS or processed further with Adobe Illustrator.

Efficient DNA fingerprinting based on the targeted sequencing of active retrotransposon insertion sites using a bench-top high-throughput sequencing platform.

Science.gov (United States)

Monden, Yuki; Yamamoto, Ayaka; Shindo, Akiko; Tahara, Makoto

2014-10-01

In many crop species, DNA fingerprinting is required for the precise identification of cultivars to protect the rights of breeders. Many families of retrotransposons have multiple copies throughout the eukaryotic genome and their integrated copies are inherited genetically. Thus, their insertion polymorphisms among cultivars are useful for DNA fingerprinting. In this study, we conducted a DNA fingerprinting based on the insertion polymorphisms of active retrotransposon families (Rtsp-1 and LIb) in sweet potato. Using 38 cultivars, we identified 2,024 insertion sites in the two families with an Illumina MiSeq sequencing platform. Of these insertion sites, 91.4% appeared to be polymorphic among the cultivars and 376 cultivar-specific insertion sites were identified, which were converted directly into cultivar-specific sequence-characterized amplified region (SCAR) markers. A phylogenetic tree was constructed using these insertion sites, which corresponded well with known pedigree information, thereby indicating their suitability for genetic diversity studies. Thus, the genome-wide comparative analysis of active retrotransposon insertion sites using the bench-top MiSeq sequencing platform is highly effective for DNA fingerprinting without any requirement for whole genome sequence information. This approach may facilitate the development of practical polymerase chain reaction-based cultivar diagnostic system and could also be applied to the determination of genetic relationships. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

SDSS-IV MaNGA: Spatially Resolved Star Formation Main Sequence and LI(N)ER Sequence

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Hsieh, B. C.; Lin, Lihwai; Lin, J. H.; Pan, H. A.; Hsu, C. H.; Sánchez, S. F.; Cano-Díaz, M.; Zhang, K.; Yan, R.; Barrera-Ballesteros, J. K.; Boquien, M.; Riffel, R.; Brownstein, J.; Cruz-González, I.; Hagen, A.; Ibarra, H.; Pan, K.; Bizyaev, D.; Oravetz, D.; Simmons, A.

2017-12-01

We present our study on the spatially resolved Hα and M * relation for 536 star-forming and 424 quiescent galaxies taken from the MaNGA survey. We show that the star formation rate surface density ({{{Σ }}}{SFR}), derived based on the Hα emissions, is strongly correlated with the M * surface density ({{{Σ }}}* ) on kiloparsec scales for star-forming galaxies and can be directly connected to the global star-forming sequence. This suggests that the global main sequence may be a consequence of a more fundamental relation on small scales. On the other hand, our result suggests that ∼20% of quiescent galaxies in our sample still have star formation activities in the outer region with lower specific star formation rate (SSFR) than typical star-forming galaxies. Meanwhile, we also find a tight correlation between {{{Σ }}}{{H}α } and {{{Σ }}}* for LI(N)ER regions, named the resolved “LI(N)ER” sequence, in quiescent galaxies, which is consistent with the scenario that LI(N)ER emissions are primarily powered by the hot, evolved stars as suggested in the literature.

Antibody-mediated platelet phagocytosis by human macrophages is inhibited by siRNA specific for sequences in the SH2 tyrosine kinase, Syk.

Science.gov (United States)

Lu, Ying; Wang, Weiming; Mao, Huiming; Hu, Hai; Wu, Yanling; Chen, Bing-Guan; Liu, Zhongmin

2011-01-01

Immune thrombocytopenia depends upon Fc receptor-mediated phagocytosis that involves signaling through the SH2 tyrosine kinase, Syk. We designed small interfering (siRNA) sequences complementary to Syk coding regions to decrease the expression of Syk in the human macrophage cell line, THP-1. To evaluate the functional effect of siRNA on phagocytosis, we developed a new in vitro assay for antibody-mediated platelet ingestion by THP-1 cells. Incubation of THP-1 cells at 37°C with fluorescence-labeled platelets and anti-platelet antibody promoted ingestion of platelets that could be quantitated by flow cytometry. Transfection of THP-1 cells with Syk-specific siRNA resulted in a reduction in the amount of FcγRII-associated Syk protein. Coincident with decreased Syk expression, we observed inhibition of antibody-mediated platelet ingestion. These results confirm a key role for Syk in antibody-mediated phagocytosis and suggest Syk-specific siRNA as a possible therapeutic candidate for immune thrombocytopenia. Copyright © 2011 Elsevier Inc. All rights reserved.

Primate-specific evolution of an LDLR enhancer

Energy Technology Data Exchange (ETDEWEB)

Wang, Qian-Fei; Prabhakar, Shyam; Wang, Qianben; Moses, Alan M.; Chanan, Sumita; Brown, Myles; Eisen, Michael B.; Cheng, Jan-Fang; Rubin,Edward M.; Boffelli, Dario

2005-12-01

Sequence changes in regulatory regions have often been invoked to explain phenotypic divergence among species, but molecular examples of this have been difficult to obtain. In this study we identified an anthropoid primate-specific sequence element that contributed to the regulatory evolution of the low-density lipoprotein receptor. Using a combination of close and distant species genomic sequence comparisons coupled with in vivo and in vitro studies, we found that a functional cholesterol-sensing sequence motif arose and was fixed within a pre-existing enhancer in the common ancestor of anthropoid primates. Our study demonstrates one molecular mechanism by which ancestral mammalian regulatory elements can evolve to perform new functions in the primate lineage leading to human.

Small RNA sequence analysis of adenovirus VA RNA-derived miRNAs reveals an unexpected serotype-specific difference in structure and abundance.

Directory of Open Access Journals (Sweden)

Wael Kamel

Full Text Available Human adenoviruses (HAds encode for one or two highly abundant virus-associated RNAs, designated VA RNAI and VA RNAII, which fold into stable hairpin structures resembling miRNA precursors. Here we show that the terminal stem of the VA RNAs originating from Ad4, Ad5, Ad11 and Ad37, all undergo Dicer dependent processing into virus-specific miRNAs (so-called mivaRNAs. We further show that the mivaRNA duplex is subjected to a highly asymmetric RISC loading with the 3'-strand from all VA RNAs being the favored strand, except for the Ad37 VA RNAII, where the 5'-mivaRNAII strand was preferentially assembled into RISC. Although the mivaRNA seed sequences are not fully conserved between the HAds a bioinformatics prediction approach suggests that a large fraction of the VA RNAII-, but not the VA RNAI-derived mivaRNAs still are able to target the same cellular genes. Using small RNA deep sequencing we demonstrate that the Dicer processing event in the terminal stem of the VA RNAs is not unique and generates 3'-mivaRNAs with a slight variation of the position of the 5' terminal nucleotide in the RISC loaded guide strand. Also, we show that all analyzed VA RNAs, except Ad37 VA RNAI and Ad5 VA RNAII, utilize an alternative upstream A start site in addition to the classical +1 G start site. Further, the 5'-mivaRNAs with an A start appears to be preferentially incorporated into RISC. Although the majority of mivaRNA research has been done using Ad5 as the model system our analysis demonstrates that the mivaRNAs expressed in Ad11- and Ad37-infected cells are the most abundant mivaRNAs associated with Ago2-containing RISC. Collectively, our results show an unexpected variability in Dicer processing of the VA RNAs and a serotype-specific loading of mivaRNAs into Ago2-based RISC.

Sequence characterisation of deletion breakpoints in the dystrophin gene by PCR

Energy Technology Data Exchange (ETDEWEB)

Abbs, S.; Sandhu, S.; Bobrow, M. [Guy`s Hospital, London (United Kingdom)

1994-09-01

Partial deletions of the dystrophin gene account for 65% of cases of Duchenne muscular dystrophy. A high proportion of these structural changes are generated by new mutational events, and lie predominantly within two `hotspot` regions, yet the underlying reasons for this are not known. We are characterizing and sequencing the regions surrounding deletion breakpoints in order to: (i) investigate the mechanisms of deletion mutation, and (ii) enable the design of PCR assays to specifically amplify mutant and normal sequences, allowing us to search for the presence of somatic mosaicism in appropriate family members. Using this approach we have been able to demonstrate the presence of somatic mosaicism in a maternal grandfather of a DMD-affected male, deleted for exons 49-50. Three deletions, namely of exons 48-49, 49-50, and 50, have been characterized using a PCR approach that avoids any cloning procedures. Breakpoints were initially localized to within regions of a few kilobases using Southern blot restriction analyses with exon-specific probes and PCR amplification of exonic and intronic loci. Sequencing was performed directly on PCR products: (i) mutant sequences were obtained from long-range or inverse-PCR across the deletion junction fragments, and (ii) normal sequences were obtained from the products of standard PCR, vectorette PCR, or inverse-PCR performed on YACs. Further characterization of intronic sequences will allow us to amplify and sequence across other deletion breakpoints and increase our knowledge of the mechanisms of mutation in the dystophin gene.

Prostate cancer in magnetic resonance imaging: diagnostic utilites of spectroscopic sequences

International Nuclear Information System (INIS)

Caivano, Rocchina; Cirillo, Patrizia; Lotumolo, Antonella; Fortunato, Giovanna; Zandolino, Alexis; Cammarota, Aldo; Balestra, Antonio; Macarini, Luca; Vita, Giulia

2012-01-01

The aim of our work is to determine the efficacy of a combined study 3 Tesla Magnetic Resonance Imaging (3T MRI), with phased-array coil, for the detection of prostate cancer using magnetic resonance spectroscopy (MRS) and diffusion-weighted images (DWI) in identifying doubt nodules. In this study, we prospectively studied 46 patients who consecutively underwent digital-rectal exploration for high doses of prostate specific antigen (PSA), as well as a MRI examination and a subsequent rectal biopsy. The study of magnetic resonance imaging was performed with a Philips Achieva 3T scanner and phased-array coil. The images were obtained with turbo spin-echo sequences T2-weighted images, T1-weighted before and after the administration of contrast medium, DWI sequences and 3D spectroscopic sequences. The ultrasound-guided prostate biopsy was performed approximately 15 days after the MRI. The data obtained from MR images and spectroscopy were correlated with histological data. MRI revealed sensitivity and specificity of 88% and 61% respectively and positive predictive value (PPV) of 73%, negative predicted value (NPV) of 81% and accuracy of 76%. In identifying the location of prostate cancer, the sensitivity of 3T MRS was 92%, with a specificity of 89%, PPV of 87%, NPV of 88% and accuracy of 87%; DWI showed a sensitivity of 88%, specificity of 61%, PPV of 73%, NPV of 81% and accuracy of 76%. The 3T MR study with phased-array coil and the use of DWI and spectroscopic sequences, in addition to T2-weighted sequences, revealed to be accurate in the diagnosis of prostate cancer and in the identification of nodules to be biopsied. It may be indicated as a resolute way before biopsy in patients with elevated PSA value and can be proposed in the staging and follow-up.

Size, Shape, and Sequence-Dependent Immunogenicity of RNA Nanoparticles

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Sijin Guo

2017-12-01

Full Text Available RNA molecules have emerged as promising therapeutics. Like all other drugs, the safety profile and immune response are important criteria for drug evaluation. However, the literature on RNA immunogenicity has been controversial. Here, we used the approach of RNA nanotechnology to demonstrate that the immune response of RNA nanoparticles is size, shape, and sequence dependent. RNA triangle, square, pentagon, and tetrahedron with same shape but different sizes, or same size but different shapes were used as models to investigate the immune response. The levels of pro-inflammatory cytokines induced by these RNA nanoarchitectures were assessed in macrophage-like cells and animals. It was found that RNA polygons without extension at the vertexes were immune inert. However, when single-stranded RNA with a specific sequence was extended from the vertexes of RNA polygons, strong immune responses were detected. These immunostimulations are sequence specific, because some other extended sequences induced little or no immune response. Additionally, larger-size RNA square induced stronger cytokine secretion. 3D RNA tetrahedron showed stronger immunostimulation than planar RNA triangle. These results suggest that the immunogenicity of RNA nanoparticles is tunable to produce either a minimal immune response that can serve as safe therapeutic vectors, or a strong immune response for cancer immunotherapy or vaccine adjuvants.

From Conventional to Next Generation Sequencing of Epstein-Barr Virus Genomes.

Science.gov (United States)

Kwok, Hin; Chiang, Alan Kwok Shing

2016-02-24

Genomic sequences of Epstein-Barr virus (EBV) have been of interest because the virus is associated with cancers, such as nasopharyngeal carcinoma, and conditions such as infectious mononucleosis. The progress of whole-genome EBV sequencing has been limited by the inefficiency and cost of the first-generation sequencing technology. With the advancement of next-generation sequencing (NGS) and target enrichment strategies, increasing number of EBV genomes has been published. These genomes were sequenced using different approaches, either with or without EBV DNA enrichment. This review provides an overview of the EBV genomes published to date, and a description of the sequencing technology and bioinformatic analyses employed in generating these sequences. We further explored ways through which the quality of sequencing data can be improved, such as using DNA oligos for capture hybridization, and longer insert size and read length in the sequencing runs. These advances will enable large-scale genomic sequencing of EBV which will facilitate a better understanding of the genetic variations of EBV in different geographic regions and discovery of potentially pathogenic variants in specific diseases.

From Conventional to Next Generation Sequencing of Epstein-Barr Virus Genomes

Directory of Open Access Journals (Sweden)

Hin Kwok

2016-02-01

Full Text Available Genomic sequences of Epstein–Barr virus (EBV have been of interest because the virus is associated with cancers, such as nasopharyngeal carcinoma, and conditions such as infectious mononucleosis. The progress of whole-genome EBV sequencing has been limited by the inefficiency and cost of the first-generation sequencing technology. With the advancement of next-generation sequencing (NGS and target enrichment strategies, increasing number of EBV genomes has been published. These genomes were sequenced using different approaches, either with or without EBV DNA enrichment. This review provides an overview of the EBV genomes published to date, and a description of the sequencing technology and bioinformatic analyses employed in generating these sequences. We further explored ways through which the quality of sequencing data can be improved, such as using DNA oligos for capture hybridization, and longer insert size and read length in the sequencing runs. These advances will enable large-scale genomic sequencing of EBV which will facilitate a better understanding of the genetic variations of EBV in different geographic regions and discovery of potentially pathogenic variants in specific diseases.

Plantagora: modeling whole genome sequencing and assembly of plant genomes.

Directory of Open Access Journals (Sweden)

Roger Barthelson

Full Text Available BACKGROUND: Genomics studies are being revolutionized by the next generation sequencing technologies, which have made whole genome sequencing much more accessible to the average researcher. Whole genome sequencing with the new technologies is a developing art that, despite the large volumes of data that can be produced, may still fail to provide a clear and thorough map of a genome. The Plantagora project was conceived to address specifically the gap between having the technical tools for genome sequencing and knowing precisely the best way to use them. METHODOLOGY/PRINCIPAL FINDINGS: For Plantagora, a platform was created for generating simulated reads from several different plant genomes of different sizes. The resulting read files mimicked either 454 or Illumina reads, with varying paired end spacing. Thousands of datasets of reads were created, most derived from our primary model genome, rice chromosome one. All reads were assembled with different software assemblers, including Newbler, Abyss, and SOAPdenovo, and the resulting assemblies were evaluated by an extensive battery of metrics chosen for these studies. The metrics included both statistics of the assembly sequences and fidelity-related measures derived by alignment of the assemblies to the original genome source for the reads. The results were presented in a website, which includes a data graphing tool, all created to help the user compare rapidly the feasibility and effectiveness of different sequencing and assembly strategies prior to testing an approach in the lab. Some of our own conclusions regarding the different strategies were also recorded on the website. CONCLUSIONS/SIGNIFICANCE: Plantagora provides a substantial body of information for comparing different approaches to sequencing a plant genome, and some conclusions regarding some of the specific approaches. Plantagora also provides a platform of metrics and tools for studying the process of sequencing and assembly

Determination of specificity influencing residues for key transcription factor families

DEFF Research Database (Denmark)

Patel, Ronak Y.; Garde, Christian; Stormo, Gary D.

2015-01-01

Transcription factors (TFs) are major modulators of transcription and subsequent cellular processes. The binding of TFs to specific regulatory elements is governed by their specificity. Considering the gap between known TFs sequence and specificity, specificity prediction frameworks are highly de...

Identification of Y-Chromosome Sequences in Turner Syndrome.

Science.gov (United States)

Silva-Grecco, Roseane Lopes da; Trovó-Marqui, Alessandra Bernadete; Sousa, Tiago Alves de; Croce, Lilian Da; Balarin, Marly Aparecida Spadotto

2016-05-01

To investigate the presence of Y-chromosome sequences and determine their frequency in patients with Turner syndrome. The study included 23 patients with Turner syndrome from Brazil, who gave written informed consent for participating in the study. Cytogenetic analyses were performed in peripheral blood lymphocytes, with 100 metaphases per patient. Genomic DNA was also extracted from peripheral blood lymphocytes, and gene sequences DYZ1, DYZ3, ZFY and SRY were amplified by Polymerase Chain Reaction. The cytogenetic analysis showed a 45,X karyotype in 9 patients (39.2 %) and a mosaic pattern in 14 (60.8 %). In 8.7 % (2 out of 23) of the patients, Y-chromosome sequences were found. This prevalence is very similar to those reported previously. The initial karyotype analysis of these patients did not reveal Y-chromosome material, but they were found positive for Y-specific sequences in the lymphocyte DNA analysis. The PCR technique showed that 2 (8.7 %) of the patients with Turner syndrome had Y-chromosome sequences, both presenting marker chromosomes on cytogenetic analysis.

ALEA: a toolbox for allele-specific epigenomics analysis.

Science.gov (United States)

Younesy, Hamid; Möller, Torsten; Heravi-Moussavi, Alireza; Cheng, Jeffrey B; Costello, Joseph F; Lorincz, Matthew C; Karimi, Mohammad M; Jones, Steven J M

2014-04-15

The assessment of expression and epigenomic status using sequencing based methods provides an unprecedented opportunity to identify and correlate allelic differences with epigenomic status. We present ALEA, a computational toolbox for allele-specific epigenomics analysis, which incorporates allelic variation data within existing resources, allowing for the identification of significant associations between epigenetic modifications and specific allelic variants in human and mouse cells. ALEA provides a customizable pipeline of command line tools for allele-specific analysis of next-generation sequencing data (ChIP-seq, RNA-seq, etc.) that takes the raw sequencing data and produces separate allelic tracks ready to be viewed on genome browsers. The pipeline has been validated using human and hybrid mouse ChIP-seq and RNA-seq data. The package, test data and usage instructions are available online at http://www.bcgsc.ca/platform/bioinfo/software/alea CONTACT: : mkarimi1@interchange.ubc.ca or sjones@bcgsc.ca Supplementary information: Supplementary data are available at Bioinformatics online. © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

RDNAnalyzer: A tool for DNA secondary structure prediction and sequence analysis.

Science.gov (United States)

Afzal, Muhammad; Shahid, Ahmad Ali; Shehzadi, Abida; Nadeem, Shahid; Husnain, Tayyab

2012-01-01

RDNAnalyzer is an innovative computer based tool designed for DNA secondary structure prediction and sequence analysis. It can randomly generate the DNA sequence or user can upload the sequences of their own interest in RAW format. It uses and extends the Nussinov dynamic programming algorithm and has various application for the sequence analysis. It predicts the DNA secondary structure and base pairings. It also provides the tools for routinely performed sequence analysis by the biological scientists such as DNA replication, reverse compliment generation, transcription, translation, sequence specific information as total number of nucleotide bases, ATGC base contents along with their respective percentages and sequence cleaner. RDNAnalyzer is a unique tool developed in Microsoft Visual Studio 2008 using Microsoft Visual C# and Windows Presentation Foundation and provides user friendly environment for sequence analysis. It is freely available. http://www.cemb.edu.pk/sw.html RDNAnalyzer - Random DNA Analyser, GUI - Graphical user interface, XAML - Extensible Application Markup Language.

Cassini Mission Sequence Subsystem (MSS)

Science.gov (United States)

Alland, Robert

2011-01-01

This paper describes my work with the Cassini Mission Sequence Subsystem (MSS) team during the summer of 2011. It gives some background on the motivation for this project and describes the expected benefit to the Cassini program. It then introduces the two tasks that I worked on - an automatic system auditing tool and a series of corrections to the Cassini Sequence Generator (SEQ_GEN) - and the specific objectives these tasks were to accomplish. Next, it details the approach I took to meet these objectives and the results of this approach, followed by a discussion of how the outcome of the project compares with my initial expectations. The paper concludes with a summary of my experience working on this project, lists what the next steps are, and acknowledges the help of my Cassini colleagues.

Peptide de novo sequencing of mixture tandem mass spectra

DEFF Research Database (Denmark)

Gorshkov, Vladimir; Hotta, Stéphanie Yuki Kolbeck; Braga, Thiago Verano

2016-01-01

they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co-isolation and thus prone to false identifications. The deconvolution approach matched...... complementary b-, y-ions to each precursor peptide mass, which allowed the creation of virtual spectra containing sequence specific fragment ions of each co-isolated peptide. Deconvolution processing resulted in equally efficient identification rates but increased the absolute number of correctly sequenced...... peptides. The improvement was in the range of 20–35% additional peptide identifications for a HeLa lysate sample. Some correct sequences were identified only using unprocessed spectra; however, the number of these was lower than those where improvement was obtained by mass spectral deconvolution. Tight...

On the Specificity of Sequential Congruency Effects in Implicit Learning of Motor and Perceptual Sequences

Science.gov (United States)

D'Angelo, Maria C.; Jimenez, Luis; Milliken, Bruce; Lupianez, Juan

2013-01-01

Individuals experience less interference from conflicting information following events that contain conflicting information. Recently, Jimenez, Lupianez, and Vaquero (2009) demonstrated that such adaptations to conflict occur even when the source of conflict arises from implicit knowledge of sequences. There is accumulating evidence that momentary…

IDENTIFICATION OF SEQUENCE SPECIFIC PCR PRIMERS FOR DETECTION OF THE TOXIGENIC FUNGAL SPECIES STACHYBOTRYS CHARTARUM

Science.gov (United States)

The nucleotide sequence of a 936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal species Stachybotrys chartarum and for other species of Stachybotrys and the related genus Memnoniella. This information was used to infer the phylogenitic relati...

High-Throughput Sequencing Reveals Diverse Sets of Conserved, Nonconserved, and Species-Specific miRNAs in Jute

Directory of Open Access Journals (Sweden)

Md. Tariqul Islam

2015-01-01

Full Text Available MicroRNAs play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. A group of plant microRNAs are evolutionarily conserved; however, others are expressed in a species-specific manner. Jute is an agroeconomically important fibre crop; nonetheless, no practical information is available for microRNAs in jute to date. In this study, Illumina sequencing revealed a total of 227 known microRNAs and 17 potential novel microRNA candidates in jute, of which 164 belong to 23 conserved families and the remaining 63 belong to 58 nonconserved families. Among a total of 81 identified microRNA families, 116 potential target genes were predicted for 39 families and 11 targets were predicted for 4 among the 17 identified novel microRNAs. For understanding better the functions of microRNAs, target genes were analyzed by Gene Ontology and their pathways illustrated by KEGG pathway analyses. The presence of microRNAs identified in jute was validated by stem-loop RT-PCR followed by end point PCR and qPCR for randomly selected 20 known and novel microRNAs. This study exhaustively identifies microRNAs and their target genes in jute which will ultimately pave the way for understanding their role in this crop and other crops.

Generic Amplicon Deep Sequencing to Determine Ilarvirus Species Diversity in Australian Prunus.

Science.gov (United States)

Kinoti, Wycliff M; Constable, Fiona E; Nancarrow, Narelle; Plummer, Kim M; Rodoni, Brendan

2017-01-01

The distribution of Ilarvirus species populations amongst 61 Australian Prunus trees was determined by next generation sequencing (NGS) of amplicons generated using a genus-based generic RT-PCR targeting a conserved region of the Ilarvirus RNA2 component that encodes the RNA dependent RNA polymerase (RdRp) gene. Presence of Ilarvirus sequences in each positive sample was further validated by Sanger sequencing of cloned amplicons of regions of each of RNA1, RNA2 and/or RNA3 that were generated by species specific PCRs and by metagenomic NGS. Prunus necrotic ringspot virus (PNRSV) was the most frequently detected Ilarvirus , occurring in 48 of the 61 Ilarvirus -positive trees and Prune dwarf virus (PDV) and Apple mosaic virus (ApMV) were detected in three trees and one tree, respectively. American plum line pattern virus (APLPV) was detected in three trees and represents the first report of APLPV detection in Australia. Two novel and distinct groups of Ilarvirus -like RNA2 amplicon sequences were also identified in several trees by the generic amplicon NGS approach. The high read depth from the amplicon NGS of the generic PCR products allowed the detection of distinct RNA2 RdRp sequence variant populations of PNRSV, PDV, ApMV, APLPV and the two novel Ilarvirus -like sequences. Mixed infections of ilarviruses were also detected in seven Prunus trees. Sanger sequencing of specific RNA1, RNA2, and/or RNA3 genome segments of each virus and total nucleic acid metagenomics NGS confirmed the presence of PNRSV, PDV, ApMV and APLPV detected by RNA2 generic amplicon NGS. However, the two novel groups of Ilarvirus -like RNA2 amplicon sequences detected by the generic amplicon NGS could not be associated to the presence of sequence from RNA1 or RNA3 genome segments or full Ilarvirus genomes, and their origin is unclear. This work highlights the sensitivity of genus-specific amplicon NGS in detection of virus sequences and their distinct populations in multiple samples, and the

Generic Amplicon Deep Sequencing to Determine Ilarvirus Species Diversity in Australian Prunus

Directory of Open Access Journals (Sweden)

Wycliff M. Kinoti

2017-06-01

Full Text Available The distribution of Ilarvirus species populations amongst 61 Australian Prunus trees was determined by next generation sequencing (NGS of amplicons generated using a genus-based generic RT-PCR targeting a conserved region of the Ilarvirus RNA2 component that encodes the RNA dependent RNA polymerase (RdRp gene. Presence of Ilarvirus sequences in each positive sample was further validated by Sanger sequencing of cloned amplicons of regions of each of RNA1, RNA2 and/or RNA3 that were generated by species specific PCRs and by metagenomic NGS. Prunus necrotic ringspot virus (PNRSV was the most frequently detected Ilarvirus, occurring in 48 of the 61 Ilarvirus-positive trees and Prune dwarf virus (PDV and Apple mosaic virus (ApMV were detected in three trees and one tree, respectively. American plum line pattern virus (APLPV was detected in three trees and represents the first report of APLPV detection in Australia. Two novel and distinct groups of Ilarvirus-like RNA2 amplicon sequences were also identified in several trees by the generic amplicon NGS approach. The high read depth from the amplicon NGS of the generic PCR products allowed the detection of distinct RNA2 RdRp sequence variant populations of PNRSV, PDV, ApMV, APLPV and the two novel Ilarvirus-like sequences. Mixed infections of ilarviruses were also detected in seven Prunus trees. Sanger sequencing of specific RNA1, RNA2, and/or RNA3 genome segments of each virus and total nucleic acid metagenomics NGS confirmed the presence of PNRSV, PDV, ApMV and APLPV detected by RNA2 generic amplicon NGS. However, the two novel groups of Ilarvirus-like RNA2 amplicon sequences detected by the generic amplicon NGS could not be associated to the presence of sequence from RNA1 or RNA3 genome segments or full Ilarvirus genomes, and their origin is unclear. This work highlights the sensitivity of genus-specific amplicon NGS in detection of virus sequences and their distinct populations in multiple samples

Discriminating Microbial Species Using Protein Sequence Properties and Machine Learning

NARCIS (Netherlands)

Shahib, Ali Al-; Gilbert, David; Breitling, Rainer

2007-01-01

Much work has been done to identify species-specific proteins in sequenced genomes and hence to determine their function. We assumed that such proteins have specific physico-chemical properties that will discriminate them from proteins in other species. In this paper, we examine the validity of this

SMM-system: A mining tool to identify specific markers in Salmonella enterica.

Science.gov (United States)

Yu, Shuijing; Liu, Weibing; Shi, Chunlei; Wang, Dapeng; Dan, Xianlong; Li, Xiao; Shi, Xianming

2011-03-01

This report presents SMM-system, a software package that implements various personalized pre- and post-BLASTN tasks for mining specific markers of microbial pathogens. The main functionalities of SMM-system are summarized as follows: (i) converting multi-FASTA file, (ii) cutting interesting genomic sequence, (iii) automatic high-throughput BLASTN searches, and (iv) screening target sequences. The utility of SMM-system was demonstrated by using it to identify 214 Salmonella enterica-specific protein-coding sequences (CDSs). Eighteen primer pairs were designed based on eighteen S. enterica-specific CDSs, respectively. Seven of these primer pairs were validated with PCR assay, which showed 100% inclusivity for the 101 S. enterica genomes and 100% exclusivity of 30 non-S. enterica genomes. Three specific primer pairs were chosen to develop a multiplex PCR assay, which generated specific amplicons with a size of 180bp (SC1286), 238bp (SC1598) and 405bp (SC4361), respectively. This study demonstrates that SMM-system is a high-throughput specific marker generation tool that can be used to identify genus-, species-, serogroup- and even serovar-specific DNA sequences of microbial pathogens, which has a potential to be applied in food industries, diagnostics and taxonomic studies. SMM-system is freely available and can be downloaded from http://foodsafety.sjtu.edu.cn/SMM-system.html. Copyright © 2011 Elsevier B.V. All rights reserved.

Nucleation phenomena in protein folding: the modulating role of protein sequence

International Nuclear Information System (INIS)

Travasso, Rui D M; FaIsca, Patricia F N; Gama, Margarida M Telo da

2007-01-01

For the vast majority of naturally occurring, small, single-domain proteins, folding is often described as a two-state process that lacks detectable intermediates. This observation has often been rationalized on the basis of a nucleation mechanism for protein folding whose basic premise is the idea that, after completion of a specific set of contacts forming the so-called folding nucleus, the native state is achieved promptly. Here we propose a methodology to identify folding nuclei in small lattice polymers and apply it to the study of protein molecules with a chain length of N = 48. To investigate the extent to which protein topology is a robust determinant of the nucleation mechanism, we compare the nucleation scenario of a native-centric model with that of a sequence-specific model sharing the same native fold. To evaluate the impact of the sequence's finer details in the nucleation mechanism, we consider the folding of two non-homologous sequences. We conclude that, in a sequence-specific model, the folding nucleus is, to some extent, formed by the most stable contacts in the protein and that the less stable linkages in the folding nucleus are solely determined by the fold's topology. We have also found that, independently of the protein sequence, the folding nucleus performs the same 'topological' function. This unifying feature of the nucleation mechanism results from the residues forming the folding nucleus being distributed along the protein chain in a similar and well-defined manner that is determined by the fold's topological features

The complementarity-determining region sequences in IgY antivenom hypervariable regions

Directory of Open Access Journals (Sweden)

David Gitirana da Rocha

2017-08-01

Full Text Available The data presented in this article are related to the research article entitled "Development of IgY antibodies against anti-snake toxins endowed with highly lethal neutralizing activity" (da Rocha et al., 2017 [1]. Complementarity-determining region (CDR sequences are variable antibody (Ab sequences that respond with specificity, duration and strength to identify and bind to antigen (Ag epitopes. B lymphocytes isolated from hens immunized with Bitis arietans (Ba and anti-Crotalus durissus terrificus (Cdt venoms and expressing high specificity, affinity and toxicity neutralizing antibody titers were used as DNA sources. The VLF1, CDR1, CDR2, VLR1 and CDR3 sequences were validated by BLASTp, and values corresponding to IgY VL and VH anti-Ba or anti-Cdt venoms were identified, registered [Gallus gallus IgY Fv Light chain (GU815099/Gallus gallus IgY Fv Heavy chain (GU815098] and used for molecular modeling of IgY scFv anti-Ba. The resulting CDR1, CDR2 and CDR3 sequences were combined to construct the three - dimensional structure of the Ab paratope.

Should abdominal sequences be included in prostate cancer MR staging studies?

International Nuclear Information System (INIS)

McEvoy, S.H.; Lavelle, L.P.; Purcell, Y.M.; Quinlan, D.M.; Skehan, S.J.; Collins, C.D.; McMahon, C.J.

2015-01-01

Highlights: • ESUR guideline that abdominal MR sequences are reserved for high-risk prostate cancer is tested. • Routine abdominal sequences are of low yield in prostate cancer MR staging. • Routine abdominal staging sequences frequently result in incidental findings. • Abdominal staging sequences should be reserved for high-risk prostate cancer cases. - Abstract: Objectives: Prostate cancer staging MR examinations commonly include abdominal sequences to assess for non-regional (common iliac or para-aortic) nodal metastasis. In our experience the diagnostic yield of this is limited, but incidental findings are frequent, often necessitating further investigations. The aim of this study is to assess the diagnostic utility of abdominal sequences in routine prostate cancer MR staging studies. Methods: Findings on abdominal sequences of consecutive MRI prostate studies performed for staging newly diagnosed prostate cancer between September 2011 and September 2013 were reviewed with respect to adenopathy and additional incidental findings. Results were correlated with Gleason grade and serum prostate-specific antigen (PSA) level in each case. Results: 355 MRI prostate examinations were reviewed. 4 (1.1%) showed enlarged non-regional lymph nodes. Incidental findings were found in 82(23.1%) cases, neccessitating further investigation in 45 (12.7%) cases. Enlarged non-regional nodes were associated with higher PSA level and Gleason grade (p = 0.007, p = 0.005 respectively). With a combined threshold of PSA > 20 ng/mL and/or Gleason grade ≥8 the sensitivity, specificity, PPV and NPV were 100, 60, 3 and 100% respectively for predicting the presence of non-regional adenopathy. Conclusions: Routine abdominal sequences are of very low yield in routine prostate cancer MR staging, frequently resulting in incidental findings requiring further work-up and should be reserved for high-risk cases. Our experience supports the use of an abdominal staging sequence in high

Isolation of a sex-linked DNA sequence in cranes.

Science.gov (United States)

Duan, W; Fuerst, P A

2001-01-01

A female-specific DNA fragment (CSL-W; crane sex-linked DNA on W chromosome) was cloned from female whooping cranes (Grus americana). From the nucleotide sequence of CSL-W, a set of polymerase chain reaction (PCR) primers was identified which amplify a 227-230 bp female-specific fragment from all existing crane species and some other noncrane species. A duplicated versions of the DNA segment, which is found to have a larger size (231-235 bp) than CSL-W in both sexes, was also identified, and was designated CSL-NW (crane sex-linked DNA on non-W chromosome). The nucleotide similarity between the sequences of CSL-W and CSL-NW from whooping cranes was 86.3%. The CSL primers do not amplify any sequence from mammalian DNA, limiting the potential for contamination from human sources. Using the CSL primers in combination with a quick DNA extraction method allows the noninvasive identification of crane gender in less than 10 h. A test of the methodology was carried out on fully developed body feathers from 18 captive cranes and resulted in 100% successful identification.

77 FR 189 - Federal Acquisition Regulation; Brand-Name Specifications

Science.gov (United States)

2012-01-03

... 2006-0020, Sequence 26] RIN 9000-AK55 Federal Acquisition Regulation; Brand-Name Specifications... Management and Budget memoranda on brand-name specifications. DATES: Effective Date: February 2, 2012. FOR... brand- name specifications. Eight respondents submitted 32 comments in response to the interim rule. The...

Robustness of ancestral sequence reconstruction to phylogenetic uncertainty.

Science.gov (United States)

Hanson-Smith, Victor; Kolaczkowski, Bryan; Thornton, Joseph W

2010-09-01

Ancestral sequence reconstruction (ASR) is widely used to formulate and test hypotheses about the sequences, functions, and structures of ancient genes. Ancestral sequences are usually inferred from an alignment of extant sequences using a maximum likelihood (ML) phylogenetic algorithm, which calculates the most likely ancestral sequence assuming a probabilistic model of sequence evolution and a specific phylogeny--typically the tree with the ML. The true phylogeny is seldom known with certainty, however. ML methods ignore this uncertainty, whereas Bayesian methods incorporate it by integrating the likelihood of each ancestral state over a distribution of possible trees. It is not known whether Bayesian approaches to phylogenetic uncertainty improve the accuracy of inferred ancestral sequences. Here, we use simulation-based experiments under both simplified and empirically derived conditions to compare the accuracy of ASR carried out using ML and Bayesian approaches. We show that incorporating phylogenetic uncertainty by integrating over topologies very rarely changes the inferred ancestral state and does not improve the accuracy of the reconstructed ancestral sequence. Ancestral state reconstructions are robust to uncertainty about the underlying tree because the conditions that produce phylogenetic uncertainty also make the ancestral state identical across plausible trees; conversely, the conditions under which different phylogenies yield different inferred ancestral states produce little or no ambiguity about the true phylogeny. Our results suggest that ML can produce accurate ASRs, even in the face of phylogenetic uncertainty. Using Bayesian integration to incorporate this uncertainty is neither necessary nor beneficial.

Complete cDNA sequence and amino acid analysis of a bovine ribonuclease K6 gene.

Science.gov (United States)

Pietrowski, D; Förster, M

2000-01-01

The complete cDNA sequence of a ribonuclease k6 gene of Bos Taurus has been determined. It codes for a protein with 154 amino acids and contains the invariant cysteine, histidine and lysine residues as well as the characteristic motifs specific to ribonuclease active sites. The deduced protein sequence is 27 residues longer than other known ribonucleases k6 and shows amino acids exchanges which could reflect a strain specificity or polymorphism within the bovine genome. Based on sequence similarity we have termed the identified gene bovine ribonuclease k6 b (brk6b).

Using RAD-seq to recognize sex-specific markers and sex chromosome systems.

Science.gov (United States)

Gamble, Tony

2016-05-01

Next-generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. ). Among the most impressive of these sequencing innovations is restriction site-associated DNA sequencing or RAD-seq (Baird et al. ; Andrews et al. ). RAD-seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD-seq data has been to identify sex-specific genetic markers, markers found in one sex but not the other (Baxter et al. ; Gamble & Zarkower ). Sex-specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon ; Mossman & Waser ), the management and breeding of endangered species (Taberlet et al. ; Griffiths & Tiwari ; Robertson et al. ) and sexing embryonic material (Hacker et al. ; Smith et al. ). Furthermore, sex-specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank ; Gamble & Zarkower ). Thus, species with male-specific markers have male heterogamety (XY) while species with female-specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi () illustrate the ease by which RAD-seq data can generate sex-specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD-seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig. ), Fowler & Buonaccorsi () uncover shared sex-specific markers and a conserved sex chromosome system. © 2016 John Wiley & Sons Ltd.

DNA interactions with a Methylene Blue redox indicator depend on the DNA length and are sequence specific.

Science.gov (United States)

Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt V; Ferapontova, Elena E

2010-06-01

A DNA molecular beacon approach was used for the analysis of interactions between DNA and Methylene Blue (MB) as a redox indicator of a hybridization event. DNA hairpin structures of different length and guanine (G) content were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 5'-end. Binding of MB to the folded hairpin DNA was electrochemically studied and compared with binding to the duplex structure formed by hybridization of the hairpin DNA to a complementary DNA strand. Variation of the electrochemical signal from the DNA-MB complex was shown to depend primarily on the DNA length and sequence used: the G-C base pairs were the preferential sites of MB binding in the duplex. For short 20 nts long DNA sequences, the increased electrochemical response from MB bound to the duplex structure was consistent with the increased amount of bound and electrochemically readable MB molecules (i.e. MB molecules that are available for the electron transfer (ET) reaction with the electrode). With longer DNA sequences, the balance between the amounts of the electrochemically readable MB molecules bound to the hairpin DNA and to the hybrid was opposite: a part of the MB molecules bound to the long-sequence DNA duplex seem to be electrochemically mute due to long ET distance. The increasing electrochemical response from MB bound to the short-length DNA hybrid contrasts with the decreasing signal from MB bound to the long-length DNA hybrid and allows an "off"-"on" genosensor development.

New Concepts of Fluorescent Probes for Specific Detection of DNA Sequences: Bis-Modified Oligonucleotides in Excimer and Exciplex Detection

Directory of Open Access Journals (Sweden)

Gbaj A

2009-01-01

Full Text Available The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5’-bispyrene and 3’-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5’-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5'-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.

Sequencing and characterization of the guppy (Poecilia reticulata transcriptome

Directory of Open Access Journals (Sweden)

Rodd F Helen

2011-04-01

Full Text Available Abstract Background Next-generation sequencing is providing researchers with a relatively fast and affordable option for developing genomic resources for organisms that are not among the traditional genetic models. Here we present a de novo assembly of the guppy (Poecilia reticulata transcriptome using 454 sequence reads, and we evaluate potential uses of this transcriptome, including detection of sex-specific transcripts and deployment as a reference for gene expression analysis in guppies and a related species. Guppies have been model organisms in ecology, evolutionary biology, and animal behaviour for over 100 years. An annotated transcriptome and other genomic tools will facilitate understanding the genetic and molecular bases of adaptation and variation in a vertebrate species with a uniquely well known natural history. Results We generated approximately 336 Mbp of mRNA sequence data from male brain, male body, female brain, and female body. The resulting 1,162,670 reads assembled into 54,921 contigs, creating a reference transcriptome for the guppy with an average read depth of 28×. We annotated nearly 40% of this reference transcriptome by searching protein and gene ontology databases. Using this annotated transcriptome database, we identified candidate genes of interest to the guppy research community, putative single nucleotide polymorphisms (SNPs, and male-specific expressed genes. We also showed that our reference transcriptome can be used for RNA-sequencing-based analysis of differential gene expression. We identified transcripts that, in juveniles, are regulated differently in the presence and absence of an important predator, Rivulus hartii, including two genes implicated in stress response. For each sample in the RNA-seq study, >50% of high-quality reads mapped to unique sequences in the reference database with high confidence. In addition, we evaluated the use of the guppy reference transcriptome for gene expression analyses in

Host-Specific and Segment-Specific Evolutionary Dynamics of Avian and Human Influenza A Viruses: A Systematic Review

KAUST Repository

Kim, Kiyeon

2016-01-13

Understanding the evolutionary dynamics of influenza viruses is essential to control both avian and human influenza. Here, we analyze host-specific and segment-specific Tajima’s D trends of influenza A virus through a systematic review using viral sequences registered in the National Center for Biotechnology Information. To avoid bias from viral population subdivision, viral sequences were stratified according to their sampling locations and sampling years. As a result, we obtained a total of 580 datasets each of which consists of nucleotide sequences of influenza A viruses isolated from a single population of hosts at a single sampling site within a single year. By analyzing nucleotide sequences in the datasets, we found that Tajima’s D values of viral sequences were different depending on hosts and gene segments. Tajima’s D values of viruses isolated from chicken and human samples showed negative, suggesting purifying selection or a rapid population growth of the viruses. The negative Tajima’s D values in rapidly growing viral population were also observed in computer simulations. Tajima’s D values of PB2, PB1, PA, NP, and M genes of the viruses circulating in wild mallards were close to zero, suggesting that these genes have undergone neutral selection in constant-sized population. On the other hand, Tajima’s D values of HA and NA genes of these viruses were positive, indicating HA and NA have undergone balancing selection in wild mallards. Taken together, these results indicated the existence of unknown factors that maintain viral subtypes in wild mallards.

Host-Specific and Segment-Specific Evolutionary Dynamics of Avian and Human Influenza A Viruses: A Systematic Review

KAUST Repository

Kim, Kiyeon; Omori, Ryosuke; Ueno, Keisuke; Iida, Sayaka; Ito, Kimihito

2016-01-01

Understanding the evolutionary dynamics of influenza viruses is essential to control both avian and human influenza. Here, we analyze host-specific and segment-specific Tajima’s D trends of influenza A virus through a systematic review using viral sequences registered in the National Center for Biotechnology Information. To avoid bias from viral population subdivision, viral sequences were stratified according to their sampling locations and sampling years. As a result, we obtained a total of 580 datasets each of which consists of nucleotide sequences of influenza A viruses isolated from a single population of hosts at a single sampling site within a single year. By analyzing nucleotide sequences in the datasets, we found that Tajima’s D values of viral sequences were different depending on hosts and gene segments. Tajima’s D values of viruses isolated from chicken and human samples showed negative, suggesting purifying selection or a rapid population growth of the viruses. The negative Tajima’s D values in rapidly growing viral population were also observed in computer simulations. Tajima’s D values of PB2, PB1, PA, NP, and M genes of the viruses circulating in wild mallards were close to zero, suggesting that these genes have undergone neutral selection in constant-sized population. On the other hand, Tajima’s D values of HA and NA genes of these viruses were positive, indicating HA and NA have undergone balancing selection in wild mallards. Taken together, these results indicated the existence of unknown factors that maintain viral subtypes in wild mallards.

Cooperativity, Specificity, and Evolutionary Stability of Polycomb Targeting in Drosophila

Directory of Open Access Journals (Sweden)

Bernd Schuettengruber

2014-10-01

Full Text Available Summary: Metazoan genomes are partitioned into modular chromosomal domains containing active or repressive chromatin. In flies, Polycomb group (PcG response elements (PREs recruit PHO and other DNA-binding factors and act as nucleation sites for the formation of Polycomb repressive domains. The sequence specificity of PREs is not well understood. Here, we use comparative epigenomics and transgenic assays to show that Drosophila domain organization and PRE specification are evolutionarily conserved despite significant cis-element divergence within Polycomb domains, whereas cis-element evolution is strongly correlated with transcription factor binding divergence outside of Polycomb domains. Cooperative interactions of PcG complexes and their recruiting factor PHO stabilize PHO recruitment to low-specificity sequences. Consistently, PHO recruitment to sites within Polycomb domains is stabilized by PRC1. These data suggest that cooperative rather than hierarchical interactions among low-affinity sequences, DNA-binding factors, and the Polycomb machinery are giving rise to specific and strongly conserved 3D structures in Drosophila. : Schuettengruber et al. present an extensive comparative epigenomics data set, providing new insights into cis-driven versus buffered evolution of Polycomb recruitment and Polycomb domain specificity. Using chromatin immunoprecipitation sequencing and transgenic assays, they demonstrate an extremely high conservation of Polycomb repressive domains in five Drosophila species. Using Hi-C and knockout experiments, they challenge the standard hierarchical Polycomb recruitment model and demonstrate that cooperative rather than hierarchical interactions among DNA motifs, transcription factors, and Polycomb group complexes define Polycomb domains.

A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry.

Directory of Open Access Journals (Sweden)

Bárbara Angulo

Full Text Available The objective of this study is to compare two EGFR testing methodologies (a commercial real-time PCR kit and a specific EGFR mutant immunohistochemistry, with direct sequencing and to investigate the limit of detection (LOD of both PCR-based methods. We identified EGFR mutations in 21 (16% of the 136 tumours analyzed by direct sequencing. Interestingly, the Therascreen EGFR Mutation Test kit was able to characterize as wild-type one tumour that could not be analyzed by direct sequencing of the PCR product. We then compared the LOD of the kit and that of direct sequencing using the available mutant tumours. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in nine of the 18 tumours (50%, which tested positive with the real-time quantitative PCR method. In all cases, EGFR mutation was identified at a dilution of 5%. Where the mutant DNA represented 30% of the total DNA, sequencing was able to detect mutations in 12 out of 19 cases (63%. Additional experiments with genetically defined standards (EGFR ΔE746-A750/+ and EGFR L858R/+ yielded similar results. Immunohistochemistry (IHC staining with exon 19-specific antibody was seen in eight out of nine cases with E746-A750del detected by direct sequencing. Neither of the two tumours with complex deletions were positive. Of the five L858R-mutated tumours detected by the PCR methods, only two were positive for the exon 21-specific antibody. The specificity was 100% for both antibodies. The LOD of the real-time PCR method was lower than that of direct sequencing. The mutation specific IHC produced excellent specificity.

High-throughput sequencing of nematode communities from total soil DNA extractions

DEFF Research Database (Denmark)

Sapkota, Rumakanta; Nicolaisen, Mogens

2015-01-01

nematodes without the need for enrichment was developed. Using this strategy on DNA templates from a set of 22 agricultural soils, we obtained 64.4% sequences of nematode origin in total, whereas the remaining sequences were almost entirely from other metazoans. The nematode sequences were derived from...... in previous sequence-based studies are not nematode specific but also amplify other groups of organisms such as fungi and plantae, and thus require a nematode enrichment step that may introduce biases. Results: In this study an amplification strategy which selectively amplifies a fragment of the SSU from...... a broad taxonomic range and most sequences were from nematode taxa that have previously been found to be abundant in soil such as Tylenchida, Rhabditida, Dorylaimida, Triplonchida and Araeolaimida. Conclusions: Our amplification and sequencing strategy for assessing nematode diversity was able to collect...

SPHINX--an algorithm for taxonomic binning of metagenomic sequences.

Science.gov (United States)

Mohammed, Monzoorul Haque; Ghosh, Tarini Shankar; Singh, Nitin Kumar; Mande, Sharmila S

2011-01-01

Compared with composition-based binning algorithms, the binning accuracy and specificity of alignment-based binning algorithms is significantly higher. However, being alignment-based, the latter class of algorithms require enormous amount of time and computing resources for binning huge metagenomic datasets. The motivation was to develop a binning approach that can analyze metagenomic datasets as rapidly as composition-based approaches, but nevertheless has the accuracy and specificity of alignment-based algorithms. This article describes a hybrid binning approach (SPHINX) that achieves high binning efficiency by utilizing the principles of both 'composition'- and 'alignment'-based binning algorithms. Validation results with simulated sequence datasets indicate that SPHINX is able to analyze metagenomic sequences as rapidly as composition-based algorithms. Furthermore, the binning efficiency (in terms of accuracy and specificity of assignments) of SPHINX is observed to be comparable with results obtained using alignment-based algorithms. A web server for the SPHINX algorithm is available at http://metagenomics.atc.tcs.com/SPHINX/.

Quantum-Sequencing: Fast electronic single DNA molecule sequencing

Science.gov (United States)

Casamada Ribot, Josep; Chatterjee, Anushree; Nagpal, Prashant

2014-03-01

A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free, high-throughput and cost-effective, single-molecule sequencing method. Here, we present the first demonstration of unique ``electronic fingerprint'' of all nucleotides (A, G, T, C), with single-molecule DNA sequencing, using Quantum-tunneling Sequencing (Q-Seq) at room temperature. We show that the electronic state of the nucleobases shift depending on the pH, with most distinct states identified at acidic pH. We also demonstrate identification of single nucleotide modifications (methylation here). Using these unique electronic fingerprints (or tunneling data), we report a partial sequence of beta lactamase (bla) gene, which encodes resistance to beta-lactam antibiotics, with over 95% success rate. These results highlight the potential of Q-Seq as a robust technique for next-generation sequencing.

Continuous Online Sequence Learning with an Unsupervised Neural Network Model.

Science.gov (United States)

Cui, Yuwei; Ahmad, Subutar; Hawkins, Jeff

2016-09-14

The ability to recognize and predict temporal sequences of sensory inputs is vital for survival in natural environments. Based on many known properties of cortical neurons, hierarchical temporal memory (HTM) sequence memory recently has been proposed as a theoretical framework for sequence learning in the cortex. In this letter, we analyze properties of HTM sequence memory and apply it to sequence learning and prediction problems with streaming data. We show the model is able to continuously learn a large number of variableorder temporal sequences using an unsupervised Hebbian-like learning rule. The sparse temporal codes formed by the model can robustly handle branching temporal sequences by maintaining multiple predictions until there is sufficient disambiguating evidence. We compare the HTM sequence memory with other sequence learning algorithms, including statistical methods: autoregressive integrated moving average; feedforward neural networks-time delay neural network and online sequential extreme learning machine; and recurrent neural networks-long short-term memory and echo-state networks on sequence prediction problems with both artificial and real-world data. The HTM model achieves comparable accuracy to other state-of-the-art algorithms. The model also exhibits properties that are critical for sequence learning, including continuous online learning, the ability to handle multiple predictions and branching sequences with high-order statistics, robustness to sensor noise and fault tolerance, and good performance without task-specific hyperparameter tuning. Therefore, the HTM sequence memory not only advances our understanding of how the brain may solve the sequence learning problem but is also applicable to real-world sequence learning problems from continuous data streams.

SEQUENCING AND SEQUENCE ANALYSIS OF MYOSTATIN GENE IN THE EXON 1 OF THE CAMEL (CAMELUS DROMEDARIUS

Directory of Open Access Journals (Sweden)

M. G. SHAH, A. S. QURESHI1, M. REISSMANN2 AND H. J. SCHWARTZ3

2006-10-01

Full Text Available Myostatin, also called growth differentiation factor-8 (GDF-8, is a member of the mammalian growth transforming family (TGF-beta superfamily, which is expressed specifically in developing an adult skeletal muscle. Muscular hypertrophy allele (mh allele in the double muscle breeds involved mutation within the myostatin gene. Genomic DNA was isolated from the camel hair using NucleoSpin Tissue kit. Two animals of each of the six breeds namely, Marecha, Dhatti, Larri, Kohi, Sakrai and Cambelpuri were used for sequencing. For PCR amplification of the gene, a primer pair was designed from homolog regions of already published sequences of farm animals from GenBank. Results showed that camel myostatin possessed more than 90% homology with that of cattle, sheep and pig. Camel formed separate cluster from the pig in spite of having high homology (98% and showed 94% homology with cattle and sheep as reported in literature. Sequence analysis of the PCR amplified part of exon 1 (256 bp of the camel myostatin was identical among six camel breeds.

pyPaSWAS : Python-based multi-core CPU and GPU sequence alignment

NARCIS (Netherlands)

Warris, Sven; Timal, N Roshan N; Kempenaar, Marcel; Poortinga, Arne M; van de Geest, Henri; Varbanescu, Ana L; Nap, Jan-Peter

2018-01-01

BACKGROUND: Our previously published CUDA-only application PaSWAS for Smith-Waterman (SW) sequence alignment of any type of sequence on NVIDIA-based GPUs is platform-specific and therefore adopted less than could be. The OpenCL language is supported more widely and allows use on a variety of

Functional role of a highly repetitive DNA sequence in anchorage of the mouse genome.

Science.gov (United States)

Neuer-Nitsche, B; Lu, X N; Werner, D

1988-09-12

The major portion of the eukaryotic genome consists of various categories of repetitive DNA sequences which have been studied with respect to their base compositions, organizations, copy numbers, transcription and species specificities; their biological roles, however, are still unclear. A novel quality of a highly repetitive mouse DNA sequence is described which points to a functional role: All copies (approximately 50,000 per haploid genome) of this DNA sequence reside on genomic Alu I DNA fragments each associated with nuclear polypeptides that are not released from DNA by proteinase K, SDS and phenol extraction. By this quality the repetitive DNA sequence is classified as a member of the sub-set of DNA sequences involved in tight DNA-polypeptide complexes which have been previously shown to be components of the subnuclear structure termed 'nuclear matrix'. From these results it has to be concluded that the repetitive DNA sequence characterized in this report represents or comprises a signal for a large number of site specific attachment points of the mouse genome in the nuclear matrix.

Analysis of genetic diversity and population structure of oil palm (Elaeis guineensis) from China and Malaysia based on species-specific simple sequence repeat markers.