Quantitative statistical analysis of cis-regulatory sequences in ABA/VP1- and CBF/DREB1-regulated genes of Arabidopsis.

PubMed

Suzuki, Masaharu; Ketterling, Matthew G; McCarty, Donald R

2005-09-01

We have developed a simple quantitative computational approach for objective analysis of cis-regulatory sequences in promoters of coregulated genes. The program, designated MotifFinder, identifies oligo sequences that are overrepresented in promoters of coregulated genes. We used this approach to analyze promoter sequences of Viviparous1 (VP1)/abscisic acid (ABA)-regulated genes and cold-regulated genes, respectively, of Arabidopsis (Arabidopsis thaliana). We detected significantly enriched sequences in up-regulated genes but not in down-regulated genes. This result suggests that gene activation but not repression is mediated by specific and common sequence elements in promoters. The enriched motifs include several known cis-regulatory sequences as well as previously unidentified motifs. With respect to known cis-elements, we dissected the flanking nucleotides of the core sequences of Sph element, ABA response elements (ABREs), and the C repeat/dehydration-responsive element. This analysis identified the motif variants that may correlate with qualitative and quantitative differences in gene expression. While both VP1 and cold responses are mediated in part by ABA signaling via ABREs, these responses correlate with unique ABRE variants distinguished by nucleotides flanking the ACGT core. ABRE and Sph motifs are tightly associated uniquely in the coregulated set of genes showing a strict dependence on VP1 and ABA signaling. Finally, analysis of distribution of the enriched sequences revealed a striking concentration of enriched motifs in a proximal 200-base region of VP1/ABA and cold-regulated promoters. Overall, each class of coregulated genes possesses a discrete set of the enriched motifs with unique distributions in their promoters that may account for the specificity of gene regulation.

Sequence information gain based motif analysis.

PubMed

Maynou, Joan; Pairó, Erola; Marco, Santiago; Perera, Alexandre

2015-11-09

The detection of regulatory regions in candidate sequences is essential for the understanding of the regulation of a particular gene and the mechanisms involved. This paper proposes a novel methodology based on information theoretic metrics for finding regulatory sequences in promoter regions. This methodology (SIGMA) has been tested on genomic sequence data for Homo sapiens and Mus musculus. SIGMA has been compared with different publicly available alternatives for motif detection, such as MEME/MAST, Biostrings (Bioconductor package), MotifRegressor, and previous work such Qresiduals projections or information theoretic based detectors. Comparative results, in the form of Receiver Operating Characteristic curves, show how, in 70% of the studied Transcription Factor Binding Sites, the SIGMA detector has a better performance and behaves more robustly than the methods compared, while having a similar computational time. The performance of SIGMA can be explained by its parametric simplicity in the modelling of the non-linear co-variability in the binding motif positions. Sequence Information Gain based Motif Analysis is a generalisation of a non-linear model of the cis-regulatory sequences detection based on Information Theory. This generalisation allows us to detect transcription factor binding sites with maximum performance disregarding the covariability observed in the positions of the training set of sequences. SIGMA is freely available to the public at http://b2slab.upc.edu.

Using RSAT oligo-analysis and dyad-analysis tools to discover regulatory signals in nucleic sequences.

PubMed

Defrance, Matthieu; Janky, Rekin's; Sand, Olivier; van Helden, Jacques

2008-01-01

This protocol explains how to discover functional signals in genomic sequences by detecting over- or under-represented oligonucleotides (words) or spaced pairs thereof (dyads) with the Regulatory Sequence Analysis Tools (http://rsat.ulb.ac.be/rsat/). Two typical applications are presented: (i) predicting transcription factor-binding motifs in promoters of coregulated genes and (ii) discovering phylogenetic footprints in promoters of orthologous genes. The steps of this protocol include purging genomic sequences to discard redundant fragments, discovering over-represented patterns and assembling them to obtain degenerate motifs, scanning sequences and drawing feature maps. The main strength of the method is its statistical ground: the binomial significance provides an efficient control on the rate of false positives. In contrast with optimization-based pattern discovery algorithms, the method supports the detection of under- as well as over-represented motifs. Computation times vary from seconds (gene clusters) to minutes (whole genomes). The execution of the whole protocol should take approximately 1 h.

Markov model plus k-word distributions: a synergy that produces novel statistical measures for sequence comparison.

PubMed

Dai, Qi; Yang, Yanchun; Wang, Tianming

2008-10-15

Many proposed statistical measures can efficiently compare biological sequences to further infer their structures, functions and evolutionary information. They are related in spirit because all the ideas for sequence comparison try to use the information on the k-word distributions, Markov model or both. Motivated by adding k-word distributions to Markov model directly, we investigated two novel statistical measures for sequence comparison, called wre.k.r and S2.k.r. The proposed measures were tested by similarity search, evaluation on functionally related regulatory sequences and phylogenetic analysis. This offers the systematic and quantitative experimental assessment of our measures. Moreover, we compared our achievements with these based on alignment or alignment-free. We grouped our experiments into two sets. The first one, performed via ROC (receiver operating curve) analysis, aims at assessing the intrinsic ability of our statistical measures to search for similar sequences from a database and discriminate functionally related regulatory sequences from unrelated sequences. The second one aims at assessing how well our statistical measure is used for phylogenetic analysis. The experimental assessment demonstrates that our similarity measures intending to incorporate k-word distributions into Markov model are more efficient.

Genomic identification of regulatory elements by evolutionary sequence comparison and functional analysis.

PubMed

Loots, Gabriela G

2008-01-01

Despite remarkable recent advances in genomics that have enabled us to identify most of the genes in the human genome, comparable efforts to define transcriptional cis-regulatory elements that control gene expression are lagging behind. The difficulty of this task stems from two equally important problems: our knowledge of how regulatory elements are encoded in genomes remains elementary, and there is a vast genomic search space for regulatory elements, since most of mammalian genomes are noncoding. Comparative genomic approaches are having a remarkable impact on the study of transcriptional regulation in eukaryotes and currently represent the most efficient and reliable methods of predicting noncoding sequences likely to control the patterns of gene expression. By subjecting eukaryotic genomic sequences to computational comparisons and subsequent experimentation, we are inching our way toward a more comprehensive catalog of common regulatory motifs that lie behind fundamental biological processes. We are still far from comprehending how the transcriptional regulatory code is encrypted in the human genome and providing an initial global view of regulatory gene networks, but collectively, the continued development of comparative and experimental approaches will rapidly expand our knowledge of the transcriptional regulome.

RNA-Seq Atlas of Glycine max: a guide to the soybean transcriptome

USDA-ARS?s Scientific Manuscript database

A first analysis of the Glycine max (L.) Merr. (soybean) transcriptome using next generation sequencing technology and RNA-Sequencing (RNA-Seq) is presented. This analysis will provide an important resource for understanding transcription and gene co-regulatory networks in soybean, the most economic...

Using RSAT to scan genome sequences for transcription factor binding sites and cis-regulatory modules.

PubMed

Turatsinze, Jean-Valery; Thomas-Chollier, Morgane; Defrance, Matthieu; van Helden, Jacques

2008-01-01

This protocol shows how to detect putative cis-regulatory elements and regions enriched in such elements with the regulatory sequence analysis tools (RSAT) web server (http://rsat.ulb.ac.be/rsat/). The approach applies to known transcription factors, whose binding specificity is represented by position-specific scoring matrices, using the program matrix-scan. The detection of individual binding sites is known to return many false predictions. However, results can be strongly improved by estimating P value, and by searching for combinations of sites (homotypic and heterotypic models). We illustrate the detection of sites and enriched regions with a study case, the upstream sequence of the Drosophila melanogaster gene even-skipped. This protocol is also tested on random control sequences to evaluate the reliability of the predictions. Each task requires a few minutes of computation time on the server. The complete protocol can be executed in about one hour.

A genomic regulatory network for development

NASA Technical Reports Server (NTRS)

Davidson, Eric H.; Rast, Jonathan P.; Oliveri, Paola; Ransick, Andrew; Calestani, Cristina; Yuh, Chiou-Hwa; Minokawa, Takuya; Amore, Gabriele; Hinman, Veronica; Arenas-Mena, Cesar;

Transterm: a database to aid the analysis of regulatory sequences in mRNAs

PubMed Central

Jacobs, Grant H.; Chen, Augustine; Stevens, Stewart G.; Stockwell, Peter A.; Black, Michael A.; Tate, Warren P.; Brown, Chris M.

2009-01-01

Messenger RNAs, in addition to coding for proteins, may contain regulatory elements that affect how the protein is translated. These include protein and microRNA-binding sites. Transterm (http://mRNA.otago.ac.nz/Transterm.html) is a database of regions and elements that affect translation with two major unique components. The first is integrated results of analysis of general features that affect translation (initiation, elongation, termination) for species or strains in Genbank, processed through a standard pipeline. The second is curated descriptions of experimentally determined regulatory elements that function as translational control elements in mRNAs. Transterm focuses on protein binding sites, particularly those in 3′-untranslated regions (3′-UTR). For this release the interface has been extensively updated based on user feedback. The data is now accessible by strain rather than species, for example there are 10 Escherichia coli strains (genomes) analysed separately. In addition to providing a repository of data, the database also provides tools for users to query their own mRNA sequences. Users can search sequences for Transterm or user defined regulatory elements, including protein or miRNA targets. Transterm also provides a central core of links to related resources for complementary analyses. PMID:18984623

rVISTA 2.0: Evolutionary Analysis of Transcription Factor Binding Sites

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

Loots, G G; Ovcharenko, I

2004-01-28

Identifying and characterizing the patterns of DNA cis-regulatory modules represents a challenge that has the potential to reveal the regulatory language the genome uses to dictate transcriptional dynamics. Several studies have demonstrated that regulatory modules are under positive selection and therefore are often conserved between related species. Using this evolutionary principle we have created a comparative tool, rVISTA, for analyzing the regulatory potential of noncoding sequences. The rVISTA tool combines transcription factor binding site (TFBS) predictions, sequence comparisons and cluster analysis to identify noncoding DNA regions that are highly conserved and present in a specific configuration within an alignment. Heremore » we present the newly developed version 2.0 of the rVISTA tool that can process alignments generated by both zPicture and PipMaker alignment programs or use pre-computed pairwise alignments of seven vertebrate genomes available from the ECR Browser. The rVISTA web server is closely interconnected with the TRANSFAC database, allowing users to either search for matrices present in the TRANSFAC library collection or search for user-defined consensus sequences. rVISTA tool is publicly available at http://rvista.dcode.org/.« less

The identification of cis-regulatory elements: A review from a machine learning perspective.

PubMed

Li, Yifeng; Chen, Chih-Yu; Kaye, Alice M; Wasserman, Wyeth W

2015-12-01

The majority of the human genome consists of non-coding regions that have been called junk DNA. However, recent studies have unveiled that these regions contain cis-regulatory elements, such as promoters, enhancers, silencers, insulators, etc. These regulatory elements can play crucial roles in controlling gene expressions in specific cell types, conditions, and developmental stages. Disruption to these regions could contribute to phenotype changes. Precisely identifying regulatory elements is key to deciphering the mechanisms underlying transcriptional regulation. Cis-regulatory events are complex processes that involve chromatin accessibility, transcription factor binding, DNA methylation, histone modifications, and the interactions between them. The development of next-generation sequencing techniques has allowed us to capture these genomic features in depth. Applied analysis of genome sequences for clinical genetics has increased the urgency for detecting these regions. However, the complexity of cis-regulatory events and the deluge of sequencing data require accurate and efficient computational approaches, in particular, machine learning techniques. In this review, we describe machine learning approaches for predicting transcription factor binding sites, enhancers, and promoters, primarily driven by next-generation sequencing data. Data sources are provided in order to facilitate testing of novel methods. The purpose of this review is to attract computational experts and data scientists to advance this field. Crown Copyright © 2015. Published by Elsevier Ireland Ltd. All rights reserved.

Mechanistically Distinct Pathways of Divergent Regulatory DNA Creation Contribute to Evolution of Human-Specific Genomic Regulatory Networks Driving Phenotypic Divergence of Homo sapiens

PubMed Central

Glinsky, Gennadi V.

2016-01-01

Abstract Thousands of candidate human-specific regulatory sequences (HSRS) have been identified, supporting the hypothesis that unique to human phenotypes result from human-specific alterations of genomic regulatory networks. Collectively, a compendium of multiple diverse families of HSRS that are functionally and structurally divergent from Great Apes could be defined as the backbone of human-specific genomic regulatory networks. Here, the conservation patterns analysis of 18,364 candidate HSRS was carried out requiring that 100% of bases must remap during the alignments of human, chimpanzee, and bonobo sequences. A total of 5,535 candidate HSRS were identified that are: (i) highly conserved in Great Apes; (ii) evolved by the exaptation of highly conserved ancestral DNA; (iii) defined by either the acceleration of mutation rates on the human lineage or the functional divergence from non-human primates. The exaptation of highly conserved ancestral DNA pathway seems mechanistically distinct from the evolution of regulatory DNA segments driven by the species-specific expansion of transposable elements. Genome-wide proximity placement analysis of HSRS revealed that a small fraction of topologically associating domains (TADs) contain more than half of HSRS from four distinct families. TADs that are enriched for HSRS and termed rapidly evolving in humans TADs (revTADs) comprise 0.8–10.3% of 3,127 TADs in the hESC genome. RevTADs manifest distinct correlation patterns between placements of human accelerated regions, human-specific transcription factor-binding sites, and recombination rates. There is a significant enrichment within revTAD boundaries of hESC-enhancers, primate-specific CTCF-binding sites, human-specific RNAPII-binding sites, hCONDELs, and H3K4me3 peaks with human-specific enrichment at TSS in prefrontal cortex neurons (P < 0.0001 in all instances). Present analysis supports the idea that phenotypic divergence of Homo sapiens is driven by the evolution of human-specific genomic regulatory networks via at least two mechanistically distinct pathways of creation of divergent sequences of regulatory DNA: (i) recombination-associated exaptation of the highly conserved ancestral regulatory DNA segments; (ii) human-specific insertions of transposable elements. PMID:27503290

Prevalence of transcription promoters within archaeal operons and coding sequences

PubMed Central

Koide, Tie; Reiss, David J; Bare, J Christopher; Pang, Wyming Lee; Facciotti, Marc T; Schmid, Amy K; Pan, Min; Marzolf, Bruz; Van, Phu T; Lo, Fang-Yin; Pratap, Abhishek; Deutsch, Eric W; Peterson, Amelia; Martin, Dan; Baliga, Nitin S

2009-01-01

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ∼64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements. PMID:19536208

Prevalence of transcription promoters within archaeal operons and coding sequences.

PubMed

Koide, Tie; Reiss, David J; Bare, J Christopher; Pang, Wyming Lee; Facciotti, Marc T; Schmid, Amy K; Pan, Min; Marzolf, Bruz; Van, Phu T; Lo, Fang-Yin; Pratap, Abhishek; Deutsch, Eric W; Peterson, Amelia; Martin, Dan; Baliga, Nitin S

2009-01-01

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of approximately 64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein-DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3' ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes-events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.

Repression of enhancer II activity by a negative regulatory element in the hepatitis B virus genome.

PubMed Central

Lo, W Y; Ting, L P

1994-01-01

Enhancer II of human hepatitis B virus has dual functions in vivo. Located at nucleotides (nt) 1646 to 1741, it can stimulate the surface and X promoters from a downstream position. Moreover, the same sequence can also function as upstream regulatory element that activates the core promoter in a position- and orientation-dependent manner. In this study, we report the identification and characterization of a negative regulatory element (NRE) upstream of enhancer II (nt 1613 to 1636) which can repress both the enhancer and upstream stimulatory function of the enhancer II sequence in differentiated liver cells. This NRE has marginal inhibitory effect by itself but a strong repressive function in the presence of a functional enhancer II. Mutational analysis reveals that sequence from nt 1616 to 1621 is required for repression of enhancer activity by the NRE. Gel shift analysis reveals that this negative regulatory region can be recognized by a specific protein factor(s) present at the 0.4 M NaCl fraction of HepG2 nuclear extracts. The discovery of the NRE indicates that HBV gene transcription is controlled by combined effects of both positive and negative regulation. It also provides a unique system with which to study the mechanism of negative regulation of gene expression. Images PMID:8107237

PCR detection and DNA sequence analysis of the regulatory region of lymphotropic papovavirus in peripheral blood mononuclear cells of an immunocompromised rhesus macaque

NASA Technical Reports Server (NTRS)

Lednicky, John A.; Halvorson, Steven J.; Butel, Janet S.

2002-01-01

A lymphotropic papovavirus (LPV) archetypal regulatory region was amplified from DNA from the blood of an immunocompromised rhesus monkey. We believe this is the first nonserological evidence of LPV infection in rhesus monkeys.

Analysis of conserved noncoding DNA in Drosophila reveals similar constraints in intergenic and intronic sequences.

PubMed

Bergman, C M; Kreitman, M

2001-08-01

Comparative genomic approaches to gene and cis-regulatory prediction are based on the principle that differential DNA sequence conservation reflects variation in functional constraint. Using this principle, we analyze noncoding sequence conservation in Drosophila for 40 loci with known or suspected cis-regulatory function encompassing >100 kb of DNA. We estimate the fraction of noncoding DNA conserved in both intergenic and intronic regions and describe the length distribution of ungapped conserved noncoding blocks. On average, 22%-26% of noncoding sequences surveyed are conserved in Drosophila, with median block length approximately 19 bp. We show that point substitution in conserved noncoding blocks exhibits transition bias as well as lineage effects in base composition, and occurs more than an order of magnitude more frequently than insertion/deletion (indel) substitution. Overall, patterns of noncoding DNA structure and evolution differ remarkably little between intergenic and intronic conserved blocks, suggesting that the effects of transcription per se contribute minimally to the constraints operating on these sequences. The results of this study have implications for the development of alignment and prediction algorithms specific to noncoding DNA, as well as for models of cis-regulatory DNA sequence evolution.

Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism.

PubMed

van der Meulen, Sjoerd B; de Jong, Anne; Kok, Jan

2016-01-01

RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species.

Comparative genome sequencing of Drosophila pseudoobscura: Chromosomal, gene, and cis-element evolution

PubMed Central

Richards, Stephen; Liu, Yue; Bettencourt, Brian R.; Hradecky, Pavel; Letovsky, Stan; Nielsen, Rasmus; Thornton, Kevin; Hubisz, Melissa J.; Chen, Rui; Meisel, Richard P.; Couronne, Olivier; Hua, Sujun; Smith, Mark A.; Zhang, Peili; Liu, Jing; Bussemaker, Harmen J.; van Batenburg, Marinus F.; Howells, Sally L.; Scherer, Steven E.; Sodergren, Erica; Matthews, Beverly B.; Crosby, Madeline A.; Schroeder, Andrew J.; Ortiz-Barrientos, Daniel; Rives, Catharine M.; Metzker, Michael L.; Muzny, Donna M.; Scott, Graham; Steffen, David; Wheeler, David A.; Worley, Kim C.; Havlak, Paul; Durbin, K. James; Egan, Amy; Gill, Rachel; Hume, Jennifer; Morgan, Margaret B.; Miner, George; Hamilton, Cerissa; Huang, Yanmei; Waldron, Lenée; Verduzco, Daniel; Clerc-Blankenburg, Kerstin P.; Dubchak, Inna; Noor, Mohamed A.F.; Anderson, Wyatt; White, Kevin P.; Clark, Andrew G.; Schaeffer, Stephen W.; Gelbart, William; Weinstock, George M.; Gibbs, Richard A.

2005-01-01

We have sequenced the genome of a second Drosophila species, Drosophila pseudoobscura, and compared this to the genome sequence of Drosophila melanogaster, a primary model organism. Throughout evolution the vast majority of Drosophila genes have remained on the same chromosome arm, but within each arm gene order has been extensively reshuffled, leading to a minimum of 921 syntenic blocks shared between the species. A repetitive sequence is found in the D. pseudoobscura genome at many junctions between adjacent syntenic blocks. Analysis of this novel repetitive element family suggests that recombination between offset elements may have given rise to many paracentric inversions, thereby contributing to the shuffling of gene order in the D. pseudoobscura lineage. Based on sequence similarity and synteny, 10,516 putative orthologs have been identified as a core gene set conserved over 25–55 million years (Myr) since the pseudoobscura/melanogaster divergence. Genes expressed in the testes had higher amino acid sequence divergence than the genome-wide average, consistent with the rapid evolution of sex-specific proteins. Cis-regulatory sequences are more conserved than random and nearby sequences between the species—but the difference is slight, suggesting that the evolution of cis-regulatory elements is flexible. Overall, a pattern of repeat-mediated chromosomal rearrangement, and high coadaptation of both male genes and cis-regulatory sequences emerges as important themes of genome divergence between these species of Drosophila. PMID:15632085

Characterization of Cer-1 cis-regulatory region during early Xenopus development.

PubMed

Silva, Ana Cristina; Filipe, Mário; Steinbeisser, Herbert; Belo, José António

2011-05-01

Cerberus-related molecules are well-known Wnt, Nodal, and BMP inhibitors that have been implicated in different processes including anterior–posterior patterning and left–right asymmetry. In both mouse and frog, two Cerberus-related genes have been isolated, mCer-1 and mCer-2, and Xcer and Xcoco, respectively. Until now, little is known about the mechanisms involved in their transcriptional regulation. Here, we report a heterologous analysis of the mouse Cerberus-1 gene upstream regulatory regions, responsible for its expression in the visceral endodermal cells. Our analysis showed that the consensus sequences for a TATA, CAAT, or GC boxes were absent but a TGTGG sequence was present at position -172 to -168 bp, relative to the ATG. Using a series of deletion constructs and transient expression in Xenopus embryos, we found that a fragment of 1.4 kb of Cer-1 promoter sequence could reproduce the endogenous expression pattern of Xenopus cerberus. A 0.7-kb mcer-1 upstream region was able to drive reporter expression to the involuting mesendodermal cells, while further deletions abolished reporter gene expression. Our results suggest that although no sequence similarity was found between mouse and Xenopus cerberus cis-regulatory regions, the signaling cascades regulating cerberus expression, during gastrulation, is conserved.

Characterization of microRNAs Expressed during Secondary Wall Biosynthesis in Acacia mangium

PubMed Central

Ong, Seong Siang; Wickneswari, Ratnam

2012-01-01

MicroRNAs (miRNAs) play critical regulatory roles by acting as sequence specific guide during secondary wall formation in woody and non-woody species. Although thousands of plant miRNAs have been sequenced, there is no comprehensive view of miRNA mediated gene regulatory network to provide profound biological insights into the regulation of xylem development. Herein, we report the involvement of six highly conserved amg-miRNA families (amg-miR166, amg-miR172, amg-miR168, amg-miR159, amg-miR394, and amg-miR156) as the potential regulatory sequences of secondary cell wall biosynthesis. Within this highly conserved amg-miRNA family, only amg-miR166 exhibited strong differences in expression between phloem and xylem tissue. The functional characterization of amg-miR166 targets in various tissues revealed three groups of HD-ZIP III: ATHB8, ATHB15, and REVOLUTA which play pivotal roles in xylem development. Although these three groups vary in their functions, -psRNA target analysis indicated that miRNA target sequences of the nine different members of HD-ZIP III are always conserved. We found that precursor structures of amg-miR166 undergo exhaustive sequence variation even within members of the same family. Gene expression analysis showed three key lignin pathway genes: C4H, CAD, and CCoAOMT were upregulated in compression wood where a cascade of miRNAs was downregulated. This study offers a comprehensive analysis on the involvement of highly conserved miRNAs implicated in the secondary wall formation of woody plants. PMID:23251324

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-08-15

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2.

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed Central

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-01-01

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2. PMID:9693111

Molecular Characterization of Transgenic Events Using Next Generation Sequencing Approach.

PubMed

Guttikonda, Satish K; Marri, Pradeep; Mammadov, Jafar; Ye, Liang; Soe, Khaing; Richey, Kimberly; Cruse, James; Zhuang, Meibao; Gao, Zhifang; Evans, Clive; Rounsley, Steve; Kumpatla, Siva P

2016-01-01

Demand for the commercial use of genetically modified (GM) crops has been increasing in light of the projected growth of world population to nine billion by 2050. A prerequisite of paramount importance for regulatory submissions is the rigorous safety assessment of GM crops. One of the components of safety assessment is molecular characterization at DNA level which helps to determine the copy number, integrity and stability of a transgene; characterize the integration site within a host genome; and confirm the absence of vector DNA. Historically, molecular characterization has been carried out using Southern blot analysis coupled with Sanger sequencing. While this is a robust approach to characterize the transgenic crops, it is both time- and resource-consuming. The emergence of next-generation sequencing (NGS) technologies has provided highly sensitive and cost- and labor-effective alternative for molecular characterization compared to traditional Southern blot analysis. Herein, we have demonstrated the successful application of both whole genome sequencing and target capture sequencing approaches for the characterization of single and stacked transgenic events and compared the results and inferences with traditional method with respect to key criteria required for regulatory submissions.

Identification of a cis-Regulatory Element Involved in Phytochrome Down-Regulated Expression of the Pea Small GTPase Gene pra21

PubMed Central

Inaba, Takehito; Nagano, Yukio; Sakakibara, Toshihiro; Sasaki, Yukiko

1999-01-01

The pra2 gene encodes a pea (Pisum sativum) small GTPase belonging to the YPT/rab family, and its expression is down-regulated by light, mediated by phytochrome. We have isolated and characterized a genomic clone of this gene and constructed a fusion DNA of its 5′-upstream region in front of the gene for firefly luciferase. Using this construct in a transient assay, we determined a pra2 cis-regulatory region sufficient to direct the light down-regulation of the luciferase reporter gene. Both 5′- and internal deletion analyses revealed that the 93-bp sequence between −734 and −642 from the transcriptional start site was important for phytochrome down-regulation. Gain-of-function analysis showed that this 93-bp region could confer light down-regulation when fused to the cauliflower mosaic virus 35S promoter. Furthermore, linker-scanning analysis showed that a 12-bp sequence within the 93-bp region mediated phytochrome down-regulation. Gel-retardation analysis showed the presence of a nuclear factor that was specifically bound to the 12-bp sequence in vitro. These results indicate that this element is a cis-regulatory element involved in phytochrome down-regulated expression. PMID:10364400

Use of a Drosophila Genome-Wide Conserved Sequence Database to Identify Functionally Related cis-Regulatory Enhancers

PubMed Central

Brody, Thomas; Yavatkar, Amarendra S; Kuzin, Alexander; Kundu, Mukta; Tyson, Leonard J; Ross, Jermaine; Lin, Tzu-Yang; Lee, Chi-Hon; Awasaki, Takeshi; Lee, Tzumin; Odenwald, Ward F

2012-01-01

Background: Phylogenetic footprinting has revealed that cis-regulatory enhancers consist of conserved DNA sequence clusters (CSCs). Currently, there is no systematic approach for enhancer discovery and analysis that takes full-advantage of the sequence information within enhancer CSCs. Results: We have generated a Drosophila genome-wide database of conserved DNA consisting of >100,000 CSCs derived from EvoPrints spanning over 90% of the genome. cis-Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. The program first identifies conserved repeat elements within an input enhancer and then searches the database for CSCs that score highly against the input CSC. Scoring is based on shared repeats as well as uniquely shared matches, and includes measures of the balance of shared elements, a diagnostic that has proven to be useful in predicting cis-regulatory function. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization. Conclusions: cis-Decoder reveals that co-regulating enhancers consist of combinations of overlapping shared sequence elements, providing insights into the mode of integration of multiple regulating transcription factors. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process. Developmental Dynamics 241:169–189, 2012. © 2011 Wiley Periodicals, Inc. Key findings A genome-wide catalog of Drosophila conserved DNA sequence clusters. cis-Decoder discovers functionally related enhancers. Functionally related enhancers share balanced sequence element copy numbers. Many enhancers function during multiple phases of development. PMID:22174086

Mechanistically Distinct Pathways of Divergent Regulatory DNA Creation Contribute to Evolution of Human-Specific Genomic Regulatory Networks Driving Phenotypic Divergence of Homo sapiens.

PubMed

Glinsky, Gennadi V

2016-09-19

Thousands of candidate human-specific regulatory sequences (HSRS) have been identified, supporting the hypothesis that unique to human phenotypes result from human-specific alterations of genomic regulatory networks. Collectively, a compendium of multiple diverse families of HSRS that are functionally and structurally divergent from Great Apes could be defined as the backbone of human-specific genomic regulatory networks. Here, the conservation patterns analysis of 18,364 candidate HSRS was carried out requiring that 100% of bases must remap during the alignments of human, chimpanzee, and bonobo sequences. A total of 5,535 candidate HSRS were identified that are: (i) highly conserved in Great Apes; (ii) evolved by the exaptation of highly conserved ancestral DNA; (iii) defined by either the acceleration of mutation rates on the human lineage or the functional divergence from non-human primates. The exaptation of highly conserved ancestral DNA pathway seems mechanistically distinct from the evolution of regulatory DNA segments driven by the species-specific expansion of transposable elements. Genome-wide proximity placement analysis of HSRS revealed that a small fraction of topologically associating domains (TADs) contain more than half of HSRS from four distinct families. TADs that are enriched for HSRS and termed rapidly evolving in humans TADs (revTADs) comprise 0.8-10.3% of 3,127 TADs in the hESC genome. RevTADs manifest distinct correlation patterns between placements of human accelerated regions, human-specific transcription factor-binding sites, and recombination rates. There is a significant enrichment within revTAD boundaries of hESC-enhancers, primate-specific CTCF-binding sites, human-specific RNAPII-binding sites, hCONDELs, and H3K4me3 peaks with human-specific enrichment at TSS in prefrontal cortex neurons (P < 0.0001 in all instances). Present analysis supports the idea that phenotypic divergence of Homo sapiens is driven by the evolution of human-specific genomic regulatory networks via at least two mechanistically distinct pathways of creation of divergent sequences of regulatory DNA: (i) recombination-associated exaptation of the highly conserved ancestral regulatory DNA segments; (ii) human-specific insertions of transposable elements. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Transcriptomic sequencing reveals a set of unique genes activated by butyrate-induced histone modification

USDA-ARS?s Scientific Manuscript database

Butyrate is a nutritional element with strong epigenetic regulatory activity as an inhibitor of histone deacetylases (HDACs). Based on the analysis of differentially expressed genes induced by butyrate in the bovine epithelial cell using deep RNA-sequencing technology (RNA-seq), a set of unique gen...

In silico evolution of the Drosophila gap gene regulatory sequence under elevated mutational pressure.

PubMed

Chertkova, Aleksandra A; Schiffman, Joshua S; Nuzhdin, Sergey V; Kozlov, Konstantin N; Samsonova, Maria G; Gursky, Vitaly V

2017-02-07

Cis-regulatory sequences are often composed of many low-affinity transcription factor binding sites (TFBSs). Determining the evolutionary and functional importance of regulatory sequence composition is impeded without a detailed knowledge of the genotype-phenotype map. We simulate the evolution of regulatory sequences involved in Drosophila melanogaster embryo segmentation during early development. Natural selection evaluates gene expression dynamics produced by a computational model of the developmental network. We observe a dramatic decrease in the total number of transcription factor binding sites through the course of evolution. Despite a decrease in average sequence binding energies through time, the regulatory sequences tend towards organisations containing increased high affinity transcription factor binding sites. Additionally, the binding energies of separate sequence segments demonstrate ubiquitous mutual correlations through time. Fewer than 10% of initial TFBSs are maintained throughout the entire simulation, deemed 'core' sites. These sites have increased functional importance as assessed under wild-type conditions and their binding energy distributions are highly conserved. Furthermore, TFBSs within close proximity of core sites exhibit increased longevity, reflecting functional regulatory interactions with core sites. In response to elevated mutational pressure, evolution tends to sample regulatory sequence organisations with fewer, albeit on average, stronger functional transcription factor binding sites. These organisations are also shaped by the regulatory interactions among core binding sites with sites in their local vicinity.

Effect of regulatory peptides on gene transcription.

PubMed

Khavinson, V Kh; Shataeva, L K; Chernova, A A

2003-09-01

Experimental studies of geroprotective activity of synthetic oligopeptides and conformational analysis of the tetrapeptide Epithalon allowed us to hypothesize that regulatory oligopeptides directly initiate transcription of genes for vitally important proteins. Sequences of nucleotide pairs that can serve as binding sites for tetrapeptide Epithalon were identified in the promoter regions of retinal genes F379, telomerase, and RNA polymerase II.

Enhancer Linking by Methylation/Expression Relationships (ELMER) | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

R tool for analysis of DNA methylation and expression datasets. Integrative analysis allows reconstruction of in vivo transcription factor networks altered in cancer along with identification of the underlying gene regulatory sequences.

Functional identification and regulatory analysis of Δ6-fatty acid desaturase from the oleaginous fungus Mucor sp. EIM-10.

PubMed

Jiang, Xianzhang; Liu, Hongjiao; Niu, Yongchao; Qi, Feng; Zhang, Mingliang; Huang, Jianzhong

2017-03-01

To enlarge the diversity of the desaturases associated with PUFA biosynthesis and to better understand the transcriptional regulation of desaturases, a Δ 6 -desaturase gene (Md6) from Mucor sp. and its 5'-upstream sequence was functionally identified in Saccharomyces cerevisiae. Expression of the Δ 6 -fatty acid desaturase (Md6) in S. cerevisiae showed that Md6 could convert linolenic acid to γ-linolenic acid. Computational analysis of the promoter of Md6 suggested it contains several eukaryotic fundamental transcription regulatory elements. In vivo functional analysis of the promoter showed the 5'-upstream sequence of Md6 could initiate expression of GFP and Md6 itself in S. cerevisiae. A series deletion analysis of the promoter suggested that sequence between -919 to -784 bp (relative to start site) named as eMd6 is the key factor for high activity of Δ 6 -desaturase. The activity of Δ 6 -desaturase was increased by 2.8-fold and 2.5-fold when the eMd6 sequence was placed upstream of -434 with forward or reverse orientations respectively. To our best knowledge, the native promoter of Md6 from Mucor is the strongest promoter for Δ 6 -desaturase reported so far and the sequence between -919 to -784 bp is an enhancer for Δ 6 -desaturase activity.

The noncoding human genome and the future of personalised medicine.

PubMed

Cowie, Philip; Hay, Elizabeth A; MacKenzie, Alasdair

2015-01-30

Non-coding cis-regulatory sequences act as the 'eyes' of the genome and their role is to perceive, organise and relay cellular communication information to RNA polymerase II at gene promoters. The evolution of these sequences, that include enhancers, silencers, insulators and promoters, has progressed in multicellular organisms to the extent that cis-regulatory sequences make up as much as 10% of the human genome. Parallel evidence suggests that 75% of polymorphisms associated with heritable disease occur within predicted cis-regulatory sequences that effectively alter the 'perception' of cis-regulatory sequences or render them blind to cell communication cues. Cis-regulatory sequences also act as major functional targets of epigenetic modification thus representing an important conduit through which changes in DNA-methylation affects disease susceptibility. The objectives of the current review are (1) to describe what has been learned about identifying and characterising cis-regulatory sequences since the sequencing of the human genome; (2) to discuss their role in interpreting cell signalling pathways pathways; and (3) outline how this role may be altered by polymorphisms and epigenetic changes. We argue that the importance of the cis-regulatory genome for the interpretation of cellular communication pathways cannot be overstated and understanding its role in health and disease will be critical for the future development of personalised medicine.

Fractal landscape analysis of DNA walks

NASA Technical Reports Server (NTRS)

Peng, C. K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Sciortino, F.; Simons, M.; Stanley, H. E.

1992-01-01

By mapping nucleotide sequences onto a "DNA walk", we uncovered remarkably long-range power law correlations [Nature 356 (1992) 168] that imply a new scale invariant property of DNA. We found such long-range correlations in intron-containing genes and in non-transcribed regulatory DNA sequences, but not in cDNA sequences or intron-less genes. In this paper, we present more explicit evidences to support our findings.

High-resolution transcriptional analysis of the regulatory influence of cell-to-cell signalling reveals novel genes that contribute to Xanthomonas phytopathogenesis

PubMed Central

An, Shi-Qi; Febrer, Melanie; McCarthy, Yvonne; Tang, Dong-Jie; Clissold, Leah; Kaithakottil, Gemy; Swarbreck, David; Tang, Ji-Liang; Rogers, Jane; Dow, J Maxwell; Ryan, Robert P

2013-01-01

The bacterium Xanthomonas campestris is an economically important pathogen of many crop species and a model for the study of bacterial phytopathogenesis. In X. campestris, a regulatory system mediated by the signal molecule DSF controls virulence to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon a system comprising the sensor RpfC and regulator RpfG. Here we have addressed the action and role of Rpf/DSF signalling in phytopathogenesis by high-resolution transcriptional analysis coupled to functional genomics. We detected transcripts for many genes that were unidentified by previous computational analysis of the genome sequence. Novel transcribed regions included intergenic transcripts predicted as coding or non-coding as well as those that were antisense to coding sequences. In total, mutation of rpfF, rpfG and rpfC led to alteration in transcript levels (more than fourfold) of approximately 480 genes. The regulatory influence of RpfF and RpfC demonstrated considerable overlap. Contrary to expectation, the regulatory influence of RpfC and RpfG had limited overlap, indicating complexities of the Rpf signalling system. Importantly, functional analysis revealed over 160 new virulence factors within the group of Rpf-regulated genes. PMID:23617851

Elements in the transcriptional regulatory region flanking herpes simplex virus type 1 oriS stimulate origin function.

PubMed

Wong, S W; Schaffer, P A

1991-05-01

Like other DNA-containing viruses, the three origins of herpes simplex virus type 1 (HSV-1) DNA replication are flanked by sequences containing transcriptional regulatory elements. In a transient plasmid replication assay, deletion of sequences comprising the transcriptional regulatory elements of ICP4 and ICP22/47, which flank oriS, resulted in a greater than 80-fold decrease in origin function compared with a plasmid, pOS-822, which retains these sequences. In an effort to identify specific cis-acting elements responsible for this effect, we conducted systematic deletion analysis of the flanking region with plasmid pOS-822 and tested the resulting mutant plasmids for origin function. Stimulation by cis-acting elements was shown to be both distance and orientation dependent, as changes in either parameter resulted in a decrease in oriS function. Additional evidence for the stimulatory effect of flanking sequences on origin function was demonstrated by replacement of these sequences with the cytomegalovirus immediate-early promoter, resulting in nearly wild-type levels of oriS function. In competition experiments, cotransfection of cells with the test plasmid, pOS-822, and increasing molar concentrations of a competitor plasmid which contained the ICP4 and ICP22/47 transcriptional regulatory regions but lacked core origin sequences resulted in a significant reduction in the replication efficiency of pOS-822, demonstrating that factors which bind specifically to the oriS-flanking sequences are likely involved as auxiliary proteins in oriS function. Together, these studies demonstrate that trans-acting factors and the sites to which they bind play a critical role in the efficiency of HSV-1 DNA replication from oriS in transient-replication assays.

Mutations in the newly identified RAX regulatory sequence are not a frequent cause of micro/anophthalmia.

PubMed

Chassaing, Nicolas; Vigouroux, Adeline; Calvas, Patrick

2009-06-01

Microphthalmia and anophthalmia are at the severe end of the spectrum of abnormalities in ocular development. A few genes (SOX2, OTX2, RAX, and CHX10) have been implicated in isolated micro/anophthalmia, but causative mutations of these genes explain less than a quarter of these developmental defects. A specifically conserved SOX2/OTX2-mediated RAX expression regulatory sequence has recently been identified. We postulated that mutations in this sequence could lead to micro/anophthalmia, and thus we performed molecular screening of this regulatory element in patients suffering from micro/anophthalmia. Fifty-one patients suffering from nonsyndromic microphthalmia (n = 40) or anophthalmia (n = 11) were included in this study after negative molecular screening for SOX2, OTX2, RAX, and CHX10 mutations. Mutation screening of the RAX regulatory sequence was performed by direct sequencing for these patients. No mutations were identified in the highly conserved RAX regulatory sequence in any of the 51 patients. Mutations in the newly identified RAX regulatory sequence do not represent a frequent cause of nonsyndromic micro/anophthalmia.

PROSPECT improves cis-acting regulatory element prediction by integrating expression profile data with consensus pattern searches

PubMed Central

Fujibuchi, Wataru; Anderson, John S. J.; Landsman, David

2001-01-01

Consensus pattern and matrix-based searches designed to predict cis-acting transcriptional regulatory sequences have historically been subject to large numbers of false positives. We sought to decrease false positives by incorporating expression profile data into a consensus pattern-based search method. We have systematically analyzed the expression phenotypes of over 6000 yeast genes, across 121 expression profile experiments, and correlated them with the distribution of 14 known regulatory elements over sequences upstream of the genes. Our method is based on a metric we term probabilistic element assessment (PEA), which is a ranking of potential sites based on sequence similarity in the upstream regions of genes with similar expression phenotypes. For eight of the 14 known elements that we examined, our method had a much higher selectivity than a naïve consensus pattern search. Based on our analysis, we have developed a web-based tool called PROSPECT, which allows consensus pattern-based searching of gene clusters obtained from microarray data. PMID:11574681

Fungal Genes in Context: Genome Architecture Reflects Regulatory Complexity and Function

PubMed Central

Noble, Luke M.; Andrianopoulos, Alex

2013-01-01

Gene context determines gene expression, with local chromosomal environment most influential. Comparative genomic analysis is often limited in scope to conserved or divergent gene and protein families, and fungi are well suited to this approach with low functional redundancy and relatively streamlined genomes. We show here that one aspect of gene context, the amount of potential upstream regulatory sequence maintained through evolution, is highly predictive of both molecular function and biological process in diverse fungi. Orthologs with large upstream intergenic regions (UIRs) are strongly enriched in information processing functions, such as signal transduction and sequence-specific DNA binding, and, in the genus Aspergillus, include the majority of experimentally studied, high-level developmental and metabolic transcriptional regulators. Many uncharacterized genes are also present in this class and, by implication, may be of similar importance. Large intergenic regions also share two novel sequence characteristics, currently of unknown significance: they are enriched for plus-strand polypyrimidine tracts and an information-rich, putative regulatory motif that was present in the last common ancestor of the Pezizomycotina. Systematic consideration of gene UIR in comparative genomics, particularly for poorly characterized species, could help reveal organisms’ regulatory priorities. PMID:23699226

PlantPAN 2.0: an update of plant promoter analysis navigator for reconstructing transcriptional regulatory networks in plants.

PubMed

Chow, Chi-Nga; Zheng, Han-Qin; Wu, Nai-Yun; Chien, Chia-Hung; Huang, Hsien-Da; Lee, Tzong-Yi; Chiang-Hsieh, Yi-Fan; Hou, Ping-Fu; Yang, Tien-Yi; Chang, Wen-Chi

2016-01-04

Transcription factors (TFs) are sequence-specific DNA-binding proteins acting as critical regulators of gene expression. The Plant Promoter Analysis Navigator (PlantPAN; http://PlantPAN2.itps.ncku.edu.tw) provides an informative resource for detecting transcription factor binding sites (TFBSs), corresponding TFs, and other important regulatory elements (CpG islands and tandem repeats) in a promoter or a set of plant promoters. Additionally, TFBSs, CpG islands, and tandem repeats in the conserve regions between similar gene promoters are also identified. The current PlantPAN release (version 2.0) contains 16 960 TFs and 1143 TF binding site matrices among 76 plant species. In addition to updating of the annotation information, adding experimentally verified TF matrices, and making improvements in the visualization of transcriptional regulatory networks, several new features and functions are incorporated. These features include: (i) comprehensive curation of TF information (response conditions, target genes, and sequence logos of binding motifs, etc.), (ii) co-expression profiles of TFs and their target genes under various conditions, (iii) protein-protein interactions among TFs and their co-factors, (iv) TF-target networks, and (v) downstream promoter elements. Furthermore, a dynamic transcriptional regulatory network under various conditions is provided in PlantPAN 2.0. The PlantPAN 2.0 is a systematic platform for plant promoter analysis and reconstructing transcriptional regulatory networks. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Global versus Local Regulatory Roles for Lrp-Related Proteins: Haemophilus influenzae as a Case Study

PubMed Central

Friedberg, Devorah; Midkiff, Michael; Calvo, Joseph M.

2001-01-01

Lrp (leucine-responsive regulatory protein) plays a global regulatory role in Escherichia coli, affecting expression of dozens of operons. Numerous lrp-related genes have been identified in different bacteria and archaea, including asnC, an E. coli gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated with E. coli Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein from Haemophilus influenzae that shares 75% sequence identity with E. coli Lrp (highest sequence identity among 42 sequences compared). A strain of H. influenzae having an lrfB null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies with lrp+ and lrp E. coli strains by others showed 20 differences). The abundance of LrfB in H. influenzae, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per E. coli cell). LrfB expressed in E. coli replaced Lrp as a repressor of the lrp gene but acted only to a limited extent as an activator of the ilvIH operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function. PMID:11395465

Genome-wide identification of conserved intronic non-coding sequences using a Bayesian segmentation approach.

PubMed

Algama, Manjula; Tasker, Edward; Williams, Caitlin; Parslow, Adam C; Bryson-Richardson, Robert J; Keith, Jonathan M

2017-03-27

Computational identification of non-coding RNAs (ncRNAs) is a challenging problem. We describe a genome-wide analysis using Bayesian segmentation to identify intronic elements highly conserved between three evolutionarily distant vertebrate species: human, mouse and zebrafish. We investigate the extent to which these elements include ncRNAs (or conserved domains of ncRNAs) and regulatory sequences. We identified 655 deeply conserved intronic sequences in a genome-wide analysis. We also performed a pathway-focussed analysis on genes involved in muscle development, detecting 27 intronic elements, of which 22 were not detected in the genome-wide analysis. At least 87% of the genome-wide and 70% of the pathway-focussed elements have existing annotations indicative of conserved RNA secondary structure. The expression of 26 of the pathway-focused elements was examined using RT-PCR, providing confirmation that they include expressed ncRNAs. Consistent with previous studies, these elements are significantly over-represented in the introns of transcription factors. This study demonstrates a novel, highly effective, Bayesian approach to identifying conserved non-coding sequences. Our results complement previous findings that these sequences are enriched in transcription factors. However, in contrast to previous studies which suggest the majority of conserved sequences are regulatory factor binding sites, the majority of conserved sequences identified using our approach contain evidence of conserved RNA secondary structures, and our laboratory results suggest most are expressed. Functional roles at DNA and RNA levels are not mutually exclusive, and many of our elements possess evidence of both. Moreover, ncRNAs play roles in transcriptional and post-transcriptional regulation, and this may contribute to the over-representation of these elements in introns of transcription factors. We attribute the higher sensitivity of the pathway-focussed analysis compared to the genome-wide analysis to improved alignment quality, suggesting that enhanced genomic alignments may reveal many more conserved intronic sequences.

Nucleotide sequencing analysis of a LEU gene of Candida maltosa which complements leuB mutation of Escherichia coli and leu2 mutation of Saccharomyces cerevisiae.

PubMed

Takagi, M; Kobayashi, N; Sugimoto, M; Fujii, T; Watari, J; Yano, K

1987-01-01

The expression of a LEU gene from Candida maltosa (designated as C-LEU2) isolated previously (Kawamura et al. 1983) was shown to be regulated, when transferred into Saccharomyces cerevisiae, by leucine and threonine in the medium, as in the case of LEU2 gene of S. cerevisiae. The coding region together with the regulatory region was subcloned and the nucleotide sequence was determined. When the sequence of the coding region was compared with that of LEU2, the homology was 72% for base pairs and 76% for deduced amino acids. Comparison of the regulatory region of C-LEU2 with those of LEU1 and LEU2 suggested a few short consensus sequences which are involved in regulation of gene expression by leucine and threonine in the medium.

Scan for Motifs: a webserver for the analysis of post-transcriptional regulatory elements in the 3' untranslated regions (3' UTRs) of mRNAs.

PubMed

Biswas, Ambarish; Brown, Chris M

2014-06-08

Gene expression in vertebrate cells may be controlled post-transcriptionally through regulatory elements in mRNAs. These are usually located in the untranslated regions (UTRs) of mRNA sequences, particularly the 3'UTRs. Scan for Motifs (SFM) simplifies the process of identifying a wide range of regulatory elements on alignments of vertebrate 3'UTRs. SFM includes identification of both RNA Binding Protein (RBP) sites and targets of miRNAs. In addition to searching pre-computed alignments, the tool provides users the flexibility to search their own sequences or alignments. The regulatory elements may be filtered by expected value cutoffs and are cross-referenced back to their respective sources and literature. The output is an interactive graphical representation, highlighting potential regulatory elements and overlaps between them. The output also provides simple statistics and links to related resources for complementary analyses. The overall process is intuitive and fast. As SFM is a free web-application, the user does not need to install any software or databases. Visualisation of the binding sites of different classes of effectors that bind to 3'UTRs will facilitate the study of regulatory elements in 3' UTRs.

Inference of Expanded Lrp-Like Feast/Famine Transcription Factor Targets in a Non-Model Organism Using Protein Structure-Based Prediction

PubMed Central

Ashworth, Justin; Plaisier, Christopher L.; Lo, Fang Yin; Reiss, David J.; Baliga, Nitin S.

2014-01-01

Widespread microbial genome sequencing presents an opportunity to understand the gene regulatory networks of non-model organisms. This requires knowledge of the binding sites for transcription factors whose DNA-binding properties are unknown or difficult to infer. We adapted a protein structure-based method to predict the specificities and putative regulons of homologous transcription factors across diverse species. As a proof-of-concept we predicted the specificities and transcriptional target genes of divergent archaeal feast/famine regulatory proteins, several of which are encoded in the genome of Halobacterium salinarum. This was validated by comparison to experimentally determined specificities for transcription factors in distantly related extremophiles, chromatin immunoprecipitation experiments, and cis-regulatory sequence conservation across eighteen related species of halobacteria. Through this analysis we were able to infer that Halobacterium salinarum employs a divergent local trans-regulatory strategy to regulate genes (carA and carB) involved in arginine and pyrimidine metabolism, whereas Escherichia coli employs an operon. The prediction of gene regulatory binding sites using structure-based methods is useful for the inference of gene regulatory relationships in new species that are otherwise difficult to infer. PMID:25255272

Inference of expanded Lrp-like feast/famine transcription factor targets in a non-model organism using protein structure-based prediction.

PubMed

Ashworth, Justin; Plaisier, Christopher L; Lo, Fang Yin; Reiss, David J; Baliga, Nitin S

2014-01-01

Widespread microbial genome sequencing presents an opportunity to understand the gene regulatory networks of non-model organisms. This requires knowledge of the binding sites for transcription factors whose DNA-binding properties are unknown or difficult to infer. We adapted a protein structure-based method to predict the specificities and putative regulons of homologous transcription factors across diverse species. As a proof-of-concept we predicted the specificities and transcriptional target genes of divergent archaeal feast/famine regulatory proteins, several of which are encoded in the genome of Halobacterium salinarum. This was validated by comparison to experimentally determined specificities for transcription factors in distantly related extremophiles, chromatin immunoprecipitation experiments, and cis-regulatory sequence conservation across eighteen related species of halobacteria. Through this analysis we were able to infer that Halobacterium salinarum employs a divergent local trans-regulatory strategy to regulate genes (carA and carB) involved in arginine and pyrimidine metabolism, whereas Escherichia coli employs an operon. The prediction of gene regulatory binding sites using structure-based methods is useful for the inference of gene regulatory relationships in new species that are otherwise difficult to infer.

Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.

PubMed

Preissl, Sebastian; Fang, Rongxin; Huang, Hui; Zhao, Yuan; Raviram, Ramya; Gorkin, David U; Zhang, Yanxiao; Sos, Brandon C; Afzal, Veena; Dickel, Diane E; Kuan, Samantha; Visel, Axel; Pennacchio, Len A; Zhang, Kun; Ren, Bing

2018-03-01

Analysis of chromatin accessibility can reveal transcriptional regulatory sequences, but heterogeneity of primary tissues poses a significant challenge in mapping the precise chromatin landscape in specific cell types. Here we report single-nucleus ATAC-seq, a combinatorial barcoding-assisted single-cell assay for transposase-accessible chromatin that is optimized for use on flash-frozen primary tissue samples. We apply this technique to the mouse forebrain through eight developmental stages. Through analysis of more than 15,000 nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell types. We further define cell-type-specific transcriptional regulatory sequences, infer potential master transcriptional regulators and delineate developmental changes in forebrain cellular composition. Our results provide insight into the molecular and cellular dynamics that underlie forebrain development in the mouse and establish technical and analytical frameworks that are broadly applicable to other heterogeneous tissues.

Genomic Identification and Analysis of Shared Cis-regulator Elements in a Developmentally Critical homeobox Cluster

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

Chris Amemiya

2003-04-01

The goals of this project were to isolate, characterize, and sequence the Dlx3/Dlx7 bigene cluster from twelve different species of mammals. The Dlx3 and Dlx7 genes are known to encode homeobox transcription factors involved in patterning of structures in the vertebrate jaw as well as vertebrate limbs. Genomic sequences from the respective taxa will subsequently be compared in order to identify conserved non-coding sequences that are potential cis-regulatory elements. Based on the comparisons they will fashion transgenic mouse experiments to functionally test the strength of the potential cis-regulatory elements. A goal of the project is to attempt to identify thosemore » elements that may function in coordinately regulating both Dlx3 and Dlx7 functions.« less

Reconstitution of wild type viral DNA in simian cells transfected with early and late SV40 defective genomes.

PubMed

O'Neill, F J; Gao, Y; Xu, X

1993-11-01

The DNAs of polyomaviruses ordinarily exist as a single circular molecule of approximately 5000 base pairs. Variants of SV40, BKV and JCV have been described which contain two complementing defective DNA molecules. These defectives, which form a bipartite genome structure, contain either the viral early region or the late region. The defectives have the unique property of being able to tolerate variable sized reiterations of regulatory and terminus region sequences, and portions of the coding region. They can also exchange coding region sequences with other polyomaviruses. It has been suggested that the bipartite genome structure might be a stage in the evolution of polyomaviruses which can uniquely sustain genome and sequence diversity. However, it is not known if the regulatory and terminus region sequences are highly mutable. Also, it is not known if the bipartite genome structure is reversible and what the conditions might be which would favor restoration of the monomolecular genome structure. We addressed the first question by sequencing the reiterated regulatory and terminus regions of E- and L-SV40 DNAs. This revealed a large number of mutations in the regulatory regions of the defective genomes, including deletions, insertions, rearrangements and base substitutions. We also detected insertions and base substitutions in the T-antigen gene. We addressed the second question by introducing into permissive simian cells, E- and L-SV40 genomes which had been engineered to contain only a single regulatory region. Analysis of viral DNA from transfected cells demonstrated recombined genomes containing a wild type monomolecular DNA structure. However, the complete defectives, containing reiterated regulatory regions, could often compete away the wild type genomes. The recombinant monomolecular genomes were isolated, cloned and found to be infectious. All of the DNA alterations identified in one of the regulatory regions of E-SV40 DNA were present in the recombinant monomolecular genomes. These and other findings indicate that the bipartite genome state can sustain many mutations which wtSV40 cannot directly sustain. However, the mutations can later be introduced into the wild type genomes when the E- and L-SV40 DNAs recombine to generate a new monomolecular genome structure.

A Predictive Model of the Oxygen and Heme Regulatory Network in Yeast

PubMed Central

Kundaje, Anshul; Xin, Xiantong; Lan, Changgui; Lianoglou, Steve; Zhou, Mei; Zhang, Li; Leslie, Christina

2008-01-01

Deciphering gene regulatory mechanisms through the analysis of high-throughput expression data is a challenging computational problem. Previous computational studies have used large expression datasets in order to resolve fine patterns of coexpression, producing clusters or modules of potentially coregulated genes. These methods typically examine promoter sequence information, such as DNA motifs or transcription factor occupancy data, in a separate step after clustering. We needed an alternative and more integrative approach to study the oxygen regulatory network in Saccharomyces cerevisiae using a small dataset of perturbation experiments. Mechanisms of oxygen sensing and regulation underlie many physiological and pathological processes, and only a handful of oxygen regulators have been identified in previous studies. We used a new machine learning algorithm called MEDUSA to uncover detailed information about the oxygen regulatory network using genome-wide expression changes in response to perturbations in the levels of oxygen, heme, Hap1, and Co2+. MEDUSA integrates mRNA expression, promoter sequence, and ChIP-chip occupancy data to learn a model that accurately predicts the differential expression of target genes in held-out data. We used a novel margin-based score to extract significant condition-specific regulators and assemble a global map of the oxygen sensing and regulatory network. This network includes both known oxygen and heme regulators, such as Hap1, Mga2, Hap4, and Upc2, as well as many new candidate regulators. MEDUSA also identified many DNA motifs that are consistent with previous experimentally identified transcription factor binding sites. Because MEDUSA's regulatory program associates regulators to target genes through their promoter sequences, we directly tested the predicted regulators for OLE1, a gene specifically induced under hypoxia, by experimental analysis of the activity of its promoter. In each case, deletion of the candidate regulator resulted in the predicted effect on promoter activity, confirming that several novel regulators identified by MEDUSA are indeed involved in oxygen regulation. MEDUSA can reveal important information from a small dataset and generate testable hypotheses for further experimental analysis. Supplemental data are included. PMID:19008939

Functional analysis of regulatory single-nucleotide polymorphisms.

PubMed

Pampín, Sandra; Rodríguez-Rey, José C

2007-04-01

The identification of regulatory polymorphisms has become a key problem in human genetics. In the past few years there has been a conceptual change in the way in which regulatory single-nucleotide polymorphisms are studied. We revise the new approaches and discuss how gene expression studies can contribute to a better knowledge of the genetics of common diseases. New techniques for the association of single-nucleotide polymorphisms with changes in gene expression have been recently developed. This, together with a more comprehensive use of the old in-vitro methods, has produced a great amount of genetic information. When added to current databases, it will help to design better tools for the detection of regulatory single-nucleotide polymorphisms. The identification of functional regulatory single-nucleotide polymorphisms cannot be done by the simple inspection of DNA sequence. In-vivo techniques, based on primer-extension, and the more recently developed 'haploChIP' allow the association of gene variants to changes in gene expression. Gene expression analysis by conventional in-vitro techniques is the only way to identify the functional consequences of regulatory single-nucleotide polymorphisms. The amount of information produced in the last few years will help to refine the tools for the future analysis of regulatory gene variants.

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis.

PubMed

Kague, Erika; Bessling, Seneca L; Lee, Josephine; Hu, Gui; Passos-Bueno, Maria Rita; Fisher, Shannon

2010-01-15

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short. Copyright 2009 Elsevier Inc. All rights reserved.

Networking Omic Data to Envisage Systems Biological Regulation.

PubMed

Kalapanulak, Saowalak; Saithong, Treenut; Thammarongtham, Chinae

To understand how biological processes work, it is necessary to explore the systematic regulation governing the behaviour of the processes. Not only driving the normal behavior of organisms, the systematic regulation evidently underlies the temporal responses to surrounding environments (dynamics) and long-term phenotypic adaptation (evolution). The systematic regulation is, in effect, formulated from the regulatory components which collaboratively work together as a network. In the drive to decipher such a code of lives, a spectrum of technologies has continuously been developed in the post-genomic era. With current advances, high-throughput sequencing technologies are tremendously powerful for facilitating genomics and systems biology studies in the attempt to understand system regulation inside the cells. The ability to explore relevant regulatory components which infer transcriptional and signaling regulation, driving core cellular processes, is thus enhanced. This chapter reviews high-throughput sequencing technologies, including second and third generation sequencing technologies, which support the investigation of genomics and transcriptomics data. Utilization of this high-throughput data to form the virtual network of systems regulation is explained, particularly transcriptional regulatory networks. Analysis of the resulting regulatory networks could lead to an understanding of cellular systems regulation at the mechanistic and dynamics levels. The great contribution of the biological networking approach to envisage systems regulation is finally demonstrated by a broad range of examples.

SNPs in the 5'-regulatory region of the tyrosinase gene do not affect plumage color in ducks (Anas platyrhynchos).

PubMed

Zhang, N N; Hu, J W; Liu, H H; Xu, H Y; He, H; Li, L

2015-12-29

Tyrosinase, encoded by the TYR gene, is the rate-limiting enzyme in the production of melanin pigment. In this study, plumage color separation was observed in Cherry Valley duck line D and F1 and F2 hybrid generations of Liancheng white ducks. Gene sequencing and bioinformatic analysis were applied to the 5'-regulatory region of TYR, to explore the connection between TYR sequence variation and duck plumage color. Four SNPs were found in the 5'-regulatory region. The SNPs were in tight linkage and formed three haplotypes. However, the genotype distribution in groups with different plumage color was not significantly different, and there were no changes in the transcription factor binding sites between the different genotypes. In conclusion, these SNP variations may not cause the differences in feather color observed in this test group.

A provisional regulatory gene network for specification of endomesoderm in the sea urchin embryo

NASA Technical Reports Server (NTRS)

Davidson, Eric H.; Rast, Jonathan P.; Oliveri, Paola; Ransick, Andrew; Calestani, Cristina; Yuh, Chiou-Hwa; Minokawa, Takuya; Amore, Gabriele; Hinman, Veronica; Arenas-Mena, Cesar;

Molecular Cloning, Expression Profile and 5′ Regulatory Region Analysis of Two Chemosensory Protein Genes from the Diamondback Moth, Plutella xylostella

PubMed Central

Gong, Liang; Zhong, Guo-Hua; Hu, Mei-Ying; Luo, Qian; Ren, Zhen-Zhen

2010-01-01

Chemosensory proteins play an important role in transporting chemical compounds to their receptors on dendrite membranes. In this study, two full-length cDNA codings for chemosensory proteins of Plutella xylostella (Lepidoptera: Plutellidae) were obtained by RACE-PCR. PxylCSP3 and Pxyl-CSP4, with GenBank accession numbers ABM92663 and ABM92664, respectively, were cloned and sequenced. The gene sequences both consisted of three exons and two introns. RT-PCR analysis showed that Pxyl-CSP3 and Pxyl-CSP4 had different expression patterns in the examined developmental stages, but were expressed in all larval stages. Phylogenetic analysis indicated that lepidopteran insects consist of three branches, and Pxyl-CSP3 and Pxyl-CSP4 belong to different branches. The 5′regulatory regions of Pxyl-CSP3 and Pxyl-CSP4 were isolated and analyzed, and the results consist of not only the core promoter sequences (TATA-box), but also several transcriptional elements (BR-C Z4, Hb, Dfd, CF2-II, etc.). This study provides clues to better understanding the various physiological functions of CSPs in P. xylostella and other insects. PMID:21073345

Analysis of loss of decay-heat-removal sequences at Browns Ferry Unit One

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

Harrington, R.M.

1983-01-01

This paper summarizes the Oak Ridge National Laboratory (ORNL) report Loss of DHR Sequences at Browns Ferry Unit One - Accident Sequence Analysis (NUREG/CR-2973). The Loss of DHR investigation is the third in a series of accident studies concerning the BWR 4 - MK I containment plant design. These studies, sponsored by the Nuclear Regulatory Commission Severe Accident Sequence Analysis (SASA) program, have been conducted at ORNL with the full cooperation of the Tennessee Valley Authority (TVA). The purpose of the SASA studies is to predetermine the probable course of postulated severe accidents so as to establish the timing andmore » the sequence of events. The SASA studies also produce recommendations concerning the implementation of better system design and better emergency operating instructions and operator training. The ORNL studies also include a detailed, best-estimate calculation of the release and transport of radioactive fission products following postulated severe accidents.« less

Uncovering microRNA-mediated response to SO2 stress in Arabidopsis thaliana by deep sequencing.

PubMed

Li, Lihong; Xue, Meizhao; Yi, Huilan

2016-10-05

Sulfur dioxide (SO2) is a major air pollutant and has significant impacts on plants. MicroRNAs (miRNAs) are a class of gene expression regulators that play important roles in response to environmental stresses. In this study, deep sequencing was used for genome-wide identification of miRNAs and their expression profiles in response to SO2 stress in Arabidopsis thaliana shoots. A total of 27 conserved miRNAs and 5 novel miRNAs were found to be differentially expressed under SO2 stress. qRT-PCR analysis showed mostly negative correlation between miRNA accumulation and target gene mRNA abundance, suggesting regulatory roles of these miRNAs during SO2 exposure. The target genes of SO2-responsive miRNAs encode transcription factors and proteins that regulate auxin signaling and stress response, and the miRNAs-mediated suppression of these genes could improve plant resistance to SO2 stress. Promoter sequence analysis of genes encoding SO2-responsive miRNAs showed that stress-responsive and phytohormone-related cis-regulatory elements occurred frequently, providing additional evidence of the involvement of miRNAs in adaption to SO2 stress. This study represents a comprehensive expression profiling of SO2-responsive miRNAs in Arabidopsis and broads our perspective on the ubiquitous regulatory roles of miRNAs under stress conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Establishing glucose- and ABA-regulated transcription networks in Arabidopsis by microarray analysis and promoter classification using a Relevance Vector Machine.

PubMed

Li, Yunhai; Lee, Kee Khoon; Walsh, Sean; Smith, Caroline; Hadingham, Sophie; Sorefan, Karim; Cawley, Gavin; Bevan, Michael W

2006-03-01

Establishing transcriptional regulatory networks by analysis of gene expression data and promoter sequences shows great promise. We developed a novel promoter classification method using a Relevance Vector Machine (RVM) and Bayesian statistical principles to identify discriminatory features in the promoter sequences of genes that can correctly classify transcriptional responses. The method was applied to microarray data obtained from Arabidopsis seedlings treated with glucose or abscisic acid (ABA). Of those genes showing >2.5-fold changes in expression level, approximately 70% were correctly predicted as being up- or down-regulated (under 10-fold cross-validation), based on the presence or absence of a small set of discriminative promoter motifs. Many of these motifs have known regulatory functions in sugar- and ABA-mediated gene expression. One promoter motif that was not known to be involved in glucose-responsive gene expression was identified as the strongest classifier of glucose-up-regulated gene expression. We show it confers glucose-responsive gene expression in conjunction with another promoter motif, thus validating the classification method. We were able to establish a detailed model of glucose and ABA transcriptional regulatory networks and their interactions, which will help us to understand the mechanisms linking metabolism with growth in Arabidopsis. This study shows that machine learning strategies coupled to Bayesian statistical methods hold significant promise for identifying functionally significant promoter sequences.

SRD: a Staphylococcus regulatory RNA database.

PubMed

Sassi, Mohamed; Augagneur, Yoann; Mauro, Tony; Ivain, Lorraine; Chabelskaya, Svetlana; Hallier, Marc; Sallou, Olivier; Felden, Brice

2015-05-01

An overflow of regulatory RNAs (sRNAs) was identified in a wide range of bacteria. We designed and implemented a new resource for the hundreds of sRNAs identified in Staphylococci, with primary focus on the human pathogen Staphylococcus aureus. The "Staphylococcal Regulatory RNA Database" (SRD, http://srd.genouest.org/) compiled all published data in a single interface including genetic locations, sequences and other features. SRD proposes novel and simplified identifiers for Staphylococcal regulatory RNAs (srn) based on the sRNA's genetic location in S. aureus strain N315 which served as a reference. From a set of 894 sequences and after an in-depth cleaning, SRD provides a list of 575 srn exempt of redundant sequences. For each sRNA, their experimental support(s) is provided, allowing the user to individually assess their validity and significance. RNA-seq analysis performed on strains N315, NCTC8325, and Newman allowed us to provide further details, upgrade the initial annotation, and identified 159 RNA-seq independent transcribed sRNAs. The lists of 575 and 159 sRNAs sequences were used to predict the number and location of srns in 18 S. aureus strains and 10 other Staphylococci. A comparison of the srn contents within 32 Staphylococcal genomes revealed a poor conservation between species. In addition, sRNA structure predictions obtained with MFold are accessible. A BLAST server and the intaRNA program, which is dedicated to target prediction, were implemented. SRD is the first sRNA database centered on a genus; it is a user-friendly and scalable device with the possibility to submit new sequences that should spread in the literature. © 2015 Sassi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence

PubMed Central

Gordon, Kacy L.; Arthur, Robert K.; Ruvinsky, Ilya

2015-01-01

Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2) from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements. PMID:26020930

KinView: A visual comparative sequence analysis tool for integrated kinome research

PubMed Central

McSkimming, Daniel Ian; Dastgheib, Shima; Baffi, Timothy R.; Byrne, Dominic P.; Ferries, Samantha; Scott, Steven Thomas; Newton, Alexandra C.; Eyers, Claire E.; Kochut, Krzysztof J.; Eyers, Patrick A.

2017-01-01

Multiple sequence alignments (MSAs) are a fundamental analysis tool used throughout biology to investigate relationships between protein sequence, structure, function, evolutionary history, and patterns of disease-associated variants. However, their widespread application in systems biology research is currently hindered by the lack of user-friendly tools to simultaneously visualize, manipulate and query the information conceptualized in large sequence alignments, and the challenges in integrating MSAs with multiple orthogonal data such as cancer variants and post-translational modifications, which are often stored in heterogeneous data sources and formats. Here, we present the Multiple Sequence Alignment Ontology (MSAOnt), which represents a profile or consensus alignment in an ontological format. Subsets of the alignment are easily selected through the SPARQL Protocol and RDF Query Language for downstream statistical analysis or visualization. We have also created the Kinome Viewer (KinView), an interactive integrative visualization that places eukaryotic protein kinase cancer variants in the context of natural sequence variation and experimentally determined post-translational modifications, which play central roles in the regulation of cellular signaling pathways. Using KinView, we identified differential phosphorylation patterns between tyrosine and serine/threonine kinases in the activation segment, a major kinase regulatory region that is often mutated in proliferative diseases. We discuss cancer variants that disrupt phosphorylation sites in the activation segment, and show how KinView can be used as a comparative tool to identify differences and similarities in natural variation, cancer variants and post-translational modifications between kinase groups, families and subfamilies. Based on KinView comparisons, we identify and experimentally characterize a regulatory tyrosine (Y177PLK4) in the PLK4 C-terminal activation segment region termed the P+1 loop. To further demonstrate the application of KinView in hypothesis generation and testing, we formulate and validate a hypothesis explaining a novel predicted loss-of-function variant (D523NPKCβ) in the regulatory spine of PKCβ, a recently identified tumor suppressor kinase. KinView provides a novel, extensible interface for performing comparative analyses between subsets of kinases and for integrating multiple types of residue specific annotations in user friendly formats. PMID:27731453

Mammalian genomic regulatory regions predicted by utilizing human genomics, transcriptomics, and epigenetics data

PubMed Central

Nguyen, Quan H; Tellam, Ross L; Naval-Sanchez, Marina; Porto-Neto, Laercio R; Barendse, William; Reverter, Antonio; Hayes, Benjamin; Kijas, James; Dalrymple, Brian P

2018-01-01

Abstract Genome sequences for hundreds of mammalian species are available, but an understanding of their genomic regulatory regions, which control gene expression, is only beginning. A comprehensive prediction of potential active regulatory regions is necessary to functionally study the roles of the majority of genomic variants in evolution, domestication, and animal production. We developed a computational method to predict regulatory DNA sequences (promoters, enhancers, and transcription factor binding sites) in production animals (cows and pigs) and extended its broad applicability to other mammals. The method utilizes human regulatory features identified from thousands of tissues, cell lines, and experimental assays to find homologous regions that are conserved in sequences and genome organization and are enriched for regulatory elements in the genome sequences of other mammalian species. Importantly, we developed a filtering strategy, including a machine learning classification method, to utilize a very small number of species-specific experimental datasets available to select for the likely active regulatory regions. The method finds the optimal combination of sensitivity and accuracy to unbiasedly predict regulatory regions in mammalian species. Furthermore, we demonstrated the utility of the predicted regulatory datasets in cattle for prioritizing variants associated with multiple production and climate change adaptation traits and identifying potential genome editing targets. PMID:29618048

Mammalian genomic regulatory regions predicted by utilizing human genomics, transcriptomics, and epigenetics data.

PubMed

Nguyen, Quan H; Tellam, Ross L; Naval-Sanchez, Marina; Porto-Neto, Laercio R; Barendse, William; Reverter, Antonio; Hayes, Benjamin; Kijas, James; Dalrymple, Brian P

2018-03-01

Genome sequences for hundreds of mammalian species are available, but an understanding of their genomic regulatory regions, which control gene expression, is only beginning. A comprehensive prediction of potential active regulatory regions is necessary to functionally study the roles of the majority of genomic variants in evolution, domestication, and animal production. We developed a computational method to predict regulatory DNA sequences (promoters, enhancers, and transcription factor binding sites) in production animals (cows and pigs) and extended its broad applicability to other mammals. The method utilizes human regulatory features identified from thousands of tissues, cell lines, and experimental assays to find homologous regions that are conserved in sequences and genome organization and are enriched for regulatory elements in the genome sequences of other mammalian species. Importantly, we developed a filtering strategy, including a machine learning classification method, to utilize a very small number of species-specific experimental datasets available to select for the likely active regulatory regions. The method finds the optimal combination of sensitivity and accuracy to unbiasedly predict regulatory regions in mammalian species. Furthermore, we demonstrated the utility of the predicted regulatory datasets in cattle for prioritizing variants associated with multiple production and climate change adaptation traits and identifying potential genome editing targets.

Identification of cis-elements and evaluation of upstream regulatory region of a rice anther-specific gene, OSIPP3, conferring pollen-specific expression in Oryza sativa (L.) ssp. indica.

PubMed

Manimaran, P; Raghurami Reddy, M; Bhaskar Rao, T; Mangrauthia, Satendra K; Sundaram, R M; Balachandran, S M

2015-12-01

Pollen-specific expression. Promoters comprise of various cis-regulatory elements which control development and physiology of plants by regulating gene expression. To understand the promoter specificity and also identification of functional cis-acting elements, progressive 5' deletion analysis of the promoter fragments is widely used. We have evaluated the activity of regulatory elements of 5' promoter deletion sequences of anther-specific gene OSIPP3, viz. OSIPP3-∆1 (1504 bp), OSIPP3-∆2 (968 bp), OSIPP3-∆3 (388 bp) and OSIPP3-∆4 (286 bp) through the expression of transgene GUS in rice. In silico analysis of 1504-bp sequence harboring different copy number of cis-acting regulatory elements such as POLLENLELAT52, GTGANTG10, enhancer element of LAT52 and LAT56 indicated that they were essential for high level of expression in pollen. Histochemical GUS analysis of the transgenic plants revealed that 1504- and 968-bp fragments directed GUS expression in roots and anthers, while the 388- and 286-bp fragments restricted the GUS expression to only pollen, of which 388 bp conferred strong GUS expression. Further, GUS staining analysis of different panicle development stages (P1-P6) confirmed that the GUS gene was preferentially expressed only at P6 stage (late pollen stage). The qRT-PCR analysis of GUS transcript revealed 23-fold higher expression of GUS transcript in OSIPP3-Δ1 followed by OSIPP3-Δ2 (eightfold) and OSIPP3-Δ3 (threefold) when compared to OSIPP3-Δ4. Based on our results, we proposed that among the two smaller fragments, the 388-bp upstream regulatory region could be considered as a promising candidate for pollen-specific expression of agronomically important transgenes in rice.

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease.

PubMed

Carss, Keren J; Arno, Gavin; Erwood, Marie; Stephens, Jonathan; Sanchis-Juan, Alba; Hull, Sarah; Megy, Karyn; Grozeva, Detelina; Dewhurst, Eleanor; Malka, Samantha; Plagnol, Vincent; Penkett, Christopher; Stirrups, Kathleen; Rizzo, Roberta; Wright, Genevieve; Josifova, Dragana; Bitner-Glindzicz, Maria; Scott, Richard H; Clement, Emma; Allen, Louise; Armstrong, Ruth; Brady, Angela F; Carmichael, Jenny; Chitre, Manali; Henderson, Robert H H; Hurst, Jane; MacLaren, Robert E; Murphy, Elaine; Paterson, Joan; Rosser, Elisabeth; Thompson, Dorothy A; Wakeling, Emma; Ouwehand, Willem H; Michaelides, Michel; Moore, Anthony T; Webster, Andrew R; Raymond, F Lucy

2017-01-05

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in CHM in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease. Copyright © 2017. Published by Elsevier Inc.

Large Scale Comparative Visualisation of Regulatory Networks with TRNDiff

DOE PAGES

Chua, Xin-Yi; Buckingham, Lawrence; Hogan, James M.; ...

2015-06-01

The advent of Next Generation Sequencing (NGS) technologies has seen explosive growth in genomic datasets, and dense coverage of related organisms, supporting study of subtle, strain-specific variations as a determinant of function. Such data collections present fresh and complex challenges for bioinformatics, those of comparing models of complex relationships across hundreds and even thousands of sequences. Transcriptional Regulatory Network (TRN) structures document the influence of regulatory proteins called Transcription Factors (TFs) on associated Target Genes (TGs). TRNs are routinely inferred from model systems or iterative search, and analysis at these scales requires simultaneous displays of multiple networks well beyond thosemore » of existing network visualisation tools [1]. In this paper we describe TRNDiff, an open source system supporting the comparative analysis and visualization of TRNs (and similarly structured data) from many genomes, allowing rapid identification of functional variations within species. The approach is demonstrated through a small scale multiple TRN analysis of the Fur iron-uptake system of Yersinia, suggesting a number of candidate virulence factors; and through a larger study exploiting integration with the RegPrecise database (http://regprecise.lbl.gov; [2]) - a collection of hundreds of manually curated and predicted transcription factor regulons drawn from across the entire spectrum of prokaryotic organisms.« less

On the Concept of Cis-regulatory Information: From Sequence Motifs to Logic Functions

NASA Astrophysics Data System (ADS)

Tarpine, Ryan; Istrail, Sorin

The regulatory genome is about the “system level organization of the core genomic regulatory apparatus, and how this is the locus of causality underlying the twin phenomena of animal development and animal evolution” (E.H. Davidson. The Regulatory Genome: Gene Regulatory Networks in Development and Evolution, Academic Press, 2006). Information processing in the regulatory genome is done through regulatory states, defined as sets of transcription factors (sequence-specific DNA binding proteins which determine gene expression) that are expressed and active at the same time. The core information processing machinery consists of modular DNA sequence elements, called cis-modules, that interact with transcription factors. The cis-modules “read” the information contained in the regulatory state of the cell through transcription factor binding, “process” it, and directly or indirectly communicate with the basal transcription apparatus to determine gene expression. This endowment of each gene with the information-receiving capacity through their cis-regulatory modules is essential for the response to every possible regulatory state to which it might be exposed during all phases of the life cycle and in all cell types. We present here a set of challenges addressed by our CYRENE research project aimed at studying the cis-regulatory code of the regulatory genome. The CYRENE Project is devoted to (1) the construction of a database, the cis-Lexicon, containing comprehensive information across species about experimentally validated cis-regulatory modules; and (2) the software development of a next-generation genome browser, the cis-Browser, specialized for the regulatory genome. The presentation is anchored on three main computational challenges: the Gene Naming Problem, the Consensus Sequence Bottleneck Problem, and the Logic Function Inference Problem.

A 20 bp cis-acting element is both necessary and sufficient to mediate elicitor response of a maize PRms gene.

PubMed

Raventós, D; Jensen, A B; Rask, M B; Casacuberta, J M; Mundy, J; San Segundo, B

1995-01-01

Transient gene expression assays in barley aleurone protoplasts were used to identify a cis-regulatory element involved in the elicitor-responsive expression of the maize PRms gene. Analysis of transcriptional fusions between PRms 5' upstream sequences and a chloramphenicol acetyltransferase reporter gene, as well as chimeric promoters containing PRms promoter fragments or repeated oligonucleotides fused to a minimal promoter, delineated a 20 bp sequence which functioned as an elicitor-response element (ERE). This sequence contains a motif (-246 AATTGACC) similar to sequences found in promoters of other pathogen-responsive genes. The analysis also indicated that an enhancing sequence(s) between -397 and -296 is required for full PRms activation by elicitors. The protein kinase inhibitor staurosporine was found to completely block the transcriptional activation induced by elicitors. These data indicate that protein phosphorylation is involved in the signal transduction pathway leading to PRms expression.

Many human accelerated regions are developmental enhancers

PubMed Central

Capra, John A.; Erwin, Genevieve D.; McKinsey, Gabriel; Rubenstein, John L. R.; Pollard, Katherine S.

2013-01-01

The genetic changes underlying the dramatic differences in form and function between humans and other primates are largely unknown, although it is clear that gene regulatory changes play an important role. To identify regulatory sequences with potentially human-specific functions, we and others used comparative genomics to find non-coding regions conserved across mammals that have acquired many sequence changes in humans since divergence from chimpanzees. These regions are good candidates for performing human-specific regulatory functions. Here, we analysed the DNA sequence, evolutionary history, histone modifications, chromatin state and transcription factor (TF) binding sites of a combined set of 2649 non-coding human accelerated regions (ncHARs) and predicted that at least 30% of them function as developmental enhancers. We prioritized the predicted ncHAR enhancers using analysis of TF binding site gain and loss, along with the functional annotations and expression patterns of nearby genes. We then tested both the human and chimpanzee sequence for 29 ncHARs in transgenic mice, and found 24 novel developmental enhancers active in both species, 17 of which had very consistent patterns of activity in specific embryonic tissues. Of these ncHAR enhancers, five drove expression patterns suggestive of different activity for the human and chimpanzee sequence at embryonic day 11.5. The changes to human non-coding DNA in these ncHAR enhancers may modify the complex patterns of gene expression necessary for proper development in a human-specific manner and are thus promising candidates for understanding the genetic basis of human-specific biology. PMID:24218637

Sequences required for induction of neurotensin receptor gene expression during neuronal differentiation of N1E-115 neuroblastoma cells.

PubMed

Tavares, D; Tully, K; Dobner, P R

1999-10-15

The promoter region of the mouse high affinity neurotensin receptor (Ntr-1) gene was characterized, and sequences required for expression in neuroblastoma cell lines that express high affinity NT-binding sites were characterized. Me(2)SO-induced neuronal differentiation of N1E-115 neuroblastoma cells increased both the expression of the endogenous Ntr-1 gene and reporter genes driven by NTR-1 promoter sequences by 3-4-fold. Deletion analysis revealed that an 83-base pair promoter region containing the transcriptional start site is required for Me(2)SO activation. Detailed mutational analysis of this region revealed that a CACCC box and the central region of a large GC-rich palindrome are the crucial cis-regulatory elements required for Me(2)SO induction. The CACCC box is bound by at least one factor that is induced upon Me(2)SO treatment of N1E-115 cells. The Me(2)SO effect was found to be both selective and cell type-restricted. Basal expression in the neuroblastoma cell lines required a distinct set of sequences, including an Sp1-like sequence, and a sequence resembling an NGFI-A-binding site; however, a more distal 5' sequence was found to repress basal activity in N1E-115 cells. These results provide evidence that Ntr-1 gene regulation involves both positive and negative regulatory elements located in the 5'-flanking region and that Ntr-1 gene activation involves the coordinate activation or induction of several factors, including a CACCC box binding complex.

Computational identification of developmental enhancers:conservation and function of transcription factor binding-site clustersin drosophila melanogaster and drosophila psedoobscura

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

Berman, Benjamin P.; Pfeiffer, Barret D.; Laverty, Todd R.

2004-08-06

The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, we demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. Here, we report the results of in vivo functional analysis of 27 remaining clusters. We generated transgenic flies carrying each cluster attached to a basal promoter and reporter gene, and assayedmore » embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. We used the Drosophila pseudoobscura genome to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. We incorporated conservation of binding-site clustering into a new genome-wide enhancer screen, and predict several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns. Measuring conservation of sequence features closely linked to function--such as binding-site clustering--makes better use of comparative sequence data than commonly used methods that examine only sequence identity.« less

Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

PubMed Central

Fauteux, François; Strömvik, Martina V

2009-01-01

Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP) gene promoters from three plant families, namely Brassicaceae (mustards), Fabaceae (legumes) and Poaceae (grasses) using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L.) Heynh.), soybean (Glycine max (L.) Merr.) and rice (Oryza sativa L.) respectively. We have identified three conserved motifs (two RY-like and one ACGT-like) in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination of conserved motifs. The majority of discovered motifs match experimentally characterized cis-regulatory elements. These results provide a good starting point for further experimental analysis of plant seed-specific promoters and our methodology can be used to unravel more transcriptional regulatory mechanisms in plants and other eukaryotes. PMID:19843335

Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing.

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

Hong, R. L., Hamaguchi, L., Busch, M. A., and Weigel, D.

2003-06-01

OAK-B135 In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3 kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae, several other motifs, but not the LFY and WUS binding sites previously identified, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally importantmore » for activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection, but also highlight that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.« less

Initial deployment of the cardiogenic gene regulatory network in the basal chordate, Ciona intestinalis.

PubMed

Woznica, Arielle; Haeussler, Maximilian; Starobinska, Ella; Jemmett, Jessica; Li, Younan; Mount, David; Davidson, Brad

2012-08-01

The complex, partially redundant gene regulatory architecture underlying vertebrate heart formation has been difficult to characterize. Here, we dissect the primary cardiac gene regulatory network in the invertebrate chordate, Ciona intestinalis. The Ciona heart progenitor lineage is first specified by Fibroblast Growth Factor/Map Kinase (FGF/MapK) activation of the transcription factor Ets1/2 (Ets). Through microarray analysis of sorted heart progenitor cells, we identified the complete set of primary genes upregulated by FGF/Ets shortly after heart progenitor emergence. Combinatorial sequence analysis of these co-regulated genes generated a hypothetical regulatory code consisting of Ets binding sites associated with a specific co-motif, ATTA. Through extensive reporter analysis, we confirmed the functional importance of the ATTA co-motif in primary heart progenitor gene regulation. We then used the Ets/ATTA combination motif to successfully predict a number of additional heart progenitor gene regulatory elements, including an intronic element driving expression of the core conserved cardiac transcription factor, GATAa. This work significantly advances our understanding of the Ciona heart gene network. Furthermore, this work has begun to elucidate the precise regulatory architecture underlying the conserved, primary role of FGF/Ets in chordate heart lineage specification. Copyright © 2012 Elsevier Inc. All rights reserved.

PAZAR: a framework for collection and dissemination of cis-regulatory sequence annotation

PubMed Central

Portales-Casamar, Elodie; Kirov, Stefan; Lim, Jonathan; Lithwick, Stuart; Swanson, Magdalena I; Ticoll, Amy; Snoddy, Jay; Wasserman, Wyeth W

2007-01-01

PAZAR is an open-access and open-source database of transcription factor and regulatory sequence annotation with associated web interface and programming tools for data submission and extraction. Curated boutique data collections can be maintained and disseminated through the unified schema of the mall-like PAZAR repository. The Pleiades Promoter Project collection of brain-linked regulatory sequences is introduced to demonstrate the depth of annotation possible within PAZAR. PAZAR, located at , is open for business. PMID:17916232

PAZAR: a framework for collection and dissemination of cis-regulatory sequence annotation.

PubMed

Portales-Casamar, Elodie; Kirov, Stefan; Lim, Jonathan; Lithwick, Stuart; Swanson, Magdalena I; Ticoll, Amy; Snoddy, Jay; Wasserman, Wyeth W

2007-01-01

PAZAR is an open-access and open-source database of transcription factor and regulatory sequence annotation with associated web interface and programming tools for data submission and extraction. Curated boutique data collections can be maintained and disseminated through the unified schema of the mall-like PAZAR repository. The Pleiades Promoter Project collection of brain-linked regulatory sequences is introduced to demonstrate the depth of annotation possible within PAZAR. PAZAR, located at http://www.pazar.info, is open for business.

Scanning sequences after Gibbs sampling to find multiple occurrences of functional elements

PubMed Central

Tharakaraman, Kannan; Mariño-Ramírez, Leonardo; Sheetlin, Sergey L; Landsman, David; Spouge, John L

2006-01-01

Background Many DNA regulatory elements occur as multiple instances within a target promoter. Gibbs sampling programs for finding DNA regulatory elements de novo can be prohibitively slow in locating all instances of such an element in a sequence set. Results We describe an improvement to the A-GLAM computer program, which predicts regulatory elements within DNA sequences with Gibbs sampling. The improvement adds an optional "scanning step" after Gibbs sampling. Gibbs sampling produces a position specific scoring matrix (PSSM). The new scanning step resembles an iterative PSI-BLAST search based on the PSSM. First, it assigns an "individual score" to each subsequence of appropriate length within the input sequences using the initial PSSM. Second, it computes an E-value from each individual score, to assess the agreement between the corresponding subsequence and the PSSM. Third, it permits subsequences with E-values falling below a threshold to contribute to the underlying PSSM, which is then updated using the Bayesian calculus. A-GLAM iterates its scanning step to convergence, at which point no new subsequences contribute to the PSSM. After convergence, A-GLAM reports predicted regulatory elements within each sequence in order of increasing E-values, so users have a statistical evaluation of the predicted elements in a convenient presentation. Thus, although the Gibbs sampling step in A-GLAM finds at most one regulatory element per input sequence, the scanning step can now rapidly locate further instances of the element in each sequence. Conclusion Datasets from experiments determining the binding sites of transcription factors were used to evaluate the improvement to A-GLAM. Typically, the datasets included several sequences containing multiple instances of a regulatory motif. The improvements to A-GLAM permitted it to predict the multiple instances. PMID:16961919

Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades

PubMed Central

2009-01-01

Background Tardigrades represent an animal phylum with extraordinary resistance to environmental stress. Results To gain insights into their stress-specific adaptation potential, major clusters of related and similar proteins are identified, as well as specific functional clusters delineated comparing all tardigrades and individual species (Milnesium tardigradum, Hypsibius dujardini, Echiniscus testudo, Tulinus stephaniae, Richtersius coronifer) and functional elements in tardigrade mRNAs are analysed. We find that 39.3% of the total sequences clustered in 58 clusters of more than 20 proteins. Among these are ten tardigrade specific as well as a number of stress-specific protein clusters. Tardigrade-specific functional adaptations include strong protein, DNA- and redox protection, maintenance and protein recycling. Specific regulatory elements regulate tardigrade mRNA stability such as lox P DICE elements whereas 14 other RNA elements of higher eukaryotes are not found. Further features of tardigrade specific adaption are rapidly identified by sequence and/or pattern search on the web-tool tardigrade analyzer http://waterbear.bioapps.biozentrum.uni-wuerzburg.de. The work-bench offers nucleotide pattern analysis for promotor and regulatory element detection (tardigrade specific; nrdb) as well as rapid COG search for function assignments including species-specific repositories of all analysed data. Conclusion Different protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences. PMID:19821996

Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades.

PubMed

Förster, Frank; Liang, Chunguang; Shkumatov, Alexander; Beisser, Daniela; Engelmann, Julia C; Schnölzer, Martina; Frohme, Marcus; Müller, Tobias; Schill, Ralph O; Dandekar, Thomas

2009-10-12

Tardigrades represent an animal phylum with extraordinary resistance to environmental stress. To gain insights into their stress-specific adaptation potential, major clusters of related and similar proteins are identified, as well as specific functional clusters delineated comparing all tardigrades and individual species (Milnesium tardigradum, Hypsibius dujardini, Echiniscus testudo, Tulinus stephaniae, Richtersius coronifer) and functional elements in tardigrade mRNAs are analysed. We find that 39.3% of the total sequences clustered in 58 clusters of more than 20 proteins. Among these are ten tardigrade specific as well as a number of stress-specific protein clusters. Tardigrade-specific functional adaptations include strong protein, DNA- and redox protection, maintenance and protein recycling. Specific regulatory elements regulate tardigrade mRNA stability such as lox P DICE elements whereas 14 other RNA elements of higher eukaryotes are not found. Further features of tardigrade specific adaption are rapidly identified by sequence and/or pattern search on the web-tool tardigrade analyzer http://waterbear.bioapps.biozentrum.uni-wuerzburg.de. The work-bench offers nucleotide pattern analysis for promotor and regulatory element detection (tardigrade specific; nrdb) as well as rapid COG search for function assignments including species-specific repositories of all analysed data. Different protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences.

Enhanced Regulatory Sequence Prediction Using Gapped k-mer Features

PubMed Central

Mohammad-Noori, Morteza; Beer, Michael A.

2014-01-01

Abstract Oligomers of length k, or k-mers, are convenient and widely used features for modeling the properties and functions of DNA and protein sequences. However, k-mers suffer from the inherent limitation that if the parameter k is increased to resolve longer features, the probability of observing any specific k-mer becomes very small, and k-mer counts approach a binary variable, with most k-mers absent and a few present once. Thus, any statistical learning approach using k-mers as features becomes susceptible to noisy training set k-mer frequencies once k becomes large. To address this problem, we introduce alternative feature sets using gapped k-mers, a new classifier, gkm-SVM, and a general method for robust estimation of k-mer frequencies. To make the method applicable to large-scale genome wide applications, we develop an efficient tree data structure for computing the kernel matrix. We show that compared to our original kmer-SVM and alternative approaches, our gkm-SVM predicts functional genomic regulatory elements and tissue specific enhancers with significantly improved accuracy, increasing the precision by up to a factor of two. We then show that gkm-SVM consistently outperforms kmer-SVM on human ENCODE ChIP-seq datasets, and further demonstrate the general utility of our method using a Naïve-Bayes classifier. Although developed for regulatory sequence analysis, these methods can be applied to any sequence classification problem. PMID:25033408

Enhanced regulatory sequence prediction using gapped k-mer features.

PubMed

Ghandi, Mahmoud; Lee, Dongwon; Mohammad-Noori, Morteza; Beer, Michael A

2014-07-01

Oligomers of length k, or k-mers, are convenient and widely used features for modeling the properties and functions of DNA and protein sequences. However, k-mers suffer from the inherent limitation that if the parameter k is increased to resolve longer features, the probability of observing any specific k-mer becomes very small, and k-mer counts approach a binary variable, with most k-mers absent and a few present once. Thus, any statistical learning approach using k-mers as features becomes susceptible to noisy training set k-mer frequencies once k becomes large. To address this problem, we introduce alternative feature sets using gapped k-mers, a new classifier, gkm-SVM, and a general method for robust estimation of k-mer frequencies. To make the method applicable to large-scale genome wide applications, we develop an efficient tree data structure for computing the kernel matrix. We show that compared to our original kmer-SVM and alternative approaches, our gkm-SVM predicts functional genomic regulatory elements and tissue specific enhancers with significantly improved accuracy, increasing the precision by up to a factor of two. We then show that gkm-SVM consistently outperforms kmer-SVM on human ENCODE ChIP-seq datasets, and further demonstrate the general utility of our method using a Naïve-Bayes classifier. Although developed for regulatory sequence analysis, these methods can be applied to any sequence classification problem.

Building a dictionary for genomes: Identification of presumptive regulatory sites by statistical analysis

PubMed Central

Bussemaker, Harmen J.; Li, Hao; Siggia, Eric D.

2000-01-01

The availability of complete genome sequences and mRNA expression data for all genes creates new opportunities and challenges for identifying DNA sequence motifs that control gene expression. An algorithm, “MobyDick,” is presented that decomposes a set of DNA sequences into the most probable dictionary of motifs or words. This method is applicable to any set of DNA sequences: for example, all upstream regions in a genome or all genes expressed under certain conditions. Identification of words is based on a probabilistic segmentation model in which the significance of longer words is deduced from the frequency of shorter ones of various lengths, eliminating the need for a separate set of reference data to define probabilities. We have built a dictionary with 1,200 words for the 6,000 upstream regulatory regions in the yeast genome; the 500 most significant words (some with as few as 10 copies in all of the upstream regions) match 114 of 443 experimentally determined sites (a significance level of 18 standard deviations). When analyzing all of the genes up-regulated during sporulation as a group, we find many motifs in addition to the few previously identified by analyzing the subclusters individually to the expression subclusters. Applying MobyDick to the genes derepressed when the general repressor Tup1 is deleted, we find known as well as putative binding sites for its regulatory partners. PMID:10944202

Evolutionary conservation of regulatory elements in vertebrate HOX gene clusters

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

Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

2003-12-31

Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involvedmore » in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.« less

QuASAR-MPRA: accurate allele-specific analysis for massively parallel reporter assays.

PubMed

Kalita, Cynthia A; Moyerbrailean, Gregory A; Brown, Christopher; Wen, Xiaoquan; Luca, Francesca; Pique-Regi, Roger

2018-03-01

The majority of the human genome is composed of non-coding regions containing regulatory elements such as enhancers, which are crucial for controlling gene expression. Many variants associated with complex traits are in these regions, and may disrupt gene regulatory sequences. Consequently, it is important to not only identify true enhancers but also to test if a variant within an enhancer affects gene regulation. Recently, allele-specific analysis in high-throughput reporter assays, such as massively parallel reporter assays (MPRAs), have been used to functionally validate non-coding variants. However, we are still missing high-quality and robust data analysis tools for these datasets. We have further developed our method for allele-specific analysis QuASAR (quantitative allele-specific analysis of reads) to analyze allele-specific signals in barcoded read counts data from MPRA. Using this approach, we can take into account the uncertainty on the original plasmid proportions, over-dispersion, and sequencing errors. The provided allelic skew estimate and its standard error also simplifies meta-analysis of replicate experiments. Additionally, we show that a beta-binomial distribution better models the variability present in the allelic imbalance of these synthetic reporters and results in a test that is statistically well calibrated under the null. Applying this approach to the MPRA data, we found 602 SNPs with significant (false discovery rate 10%) allele-specific regulatory function in LCLs. We also show that we can combine MPRA with QuASAR estimates to validate existing experimental and computational annotations of regulatory variants. Our study shows that with appropriate data analysis tools, we can improve the power to detect allelic effects in high-throughput reporter assays. http://github.com/piquelab/QuASAR/tree/master/mpra. fluca@wayne.edu or rpique@wayne.edu. Supplementary data are available online at Bioinformatics. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

RNA2DMut: a web tool for the design and analysis of RNA structure mutations.

PubMed

Moss, Walter N

2018-03-01

With the widespread application of high-throughput sequencing, novel RNA sequences are being discovered at an astonishing rate. The analysis of function, however, lags behind. In both the cis - and trans -regulatory functions of RNA, secondary structure (2D base-pairing) plays essential regulatory roles. In order to test RNA function, it is essential to be able to design and analyze mutations that can affect structure. This was the motivation for the creation of the RNA2DMut web tool. With RNA2DMut, users can enter in RNA sequences to analyze, constrain mutations to specific residues, or limit changes to purines/pyrimidines. The sequence is analyzed at each base to determine the effect of every possible point mutation on 2D structure. The metrics used in RNA2DMut rely on the calculation of the Boltzmann structure ensemble and do not require a robust 2D model of RNA structure for designing mutations. This tool can facilitate a wide array of uses involving RNA: for example, in designing and evaluating mutants for biological assays, interrogating RNA-protein interactions, identifying key regions to alter in SELEX experiments, and improving RNA folding and crystallization properties for structural biology. Additional tools are available to help users introduce other mutations (e.g., indels and substitutions) and evaluate their effects on RNA structure. Example calculations are shown for five RNAs that require 2D structure for their function: the MALAT1 mascRNA, an influenza virus splicing regulatory motif, the EBER2 viral noncoding RNA, the Xist lncRNA repA region, and human Y RNA 5. RNA2DMut can be accessed at https://rna2dmut.bb.iastate.edu/. © 2018 Moss; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Transcriptomic analysis of rice aleurone cells identified a novel abscisic acid response element.

PubMed

Watanabe, Kenneth A; Homayouni, Arielle; Gu, Lingkun; Huang, Kuan-Ying; Ho, Tuan-Hua David; Shen, Qingxi J

2017-09-01

Seeds serve as a great model to study plant responses to drought stress, which is largely mediated by abscisic acid (ABA). The ABA responsive element (ABRE) is a key cis-regulatory element in ABA signalling. However, its consensus sequence (ACGTG(G/T)C) is present in the promoters of only about 40% of ABA-induced genes in rice aleurone cells, suggesting other ABREs may exist. To identify novel ABREs, RNA sequencing was performed on aleurone cells of rice seeds treated with 20 μM ABA. Gibbs sampling was used to identify enriched elements, and particle bombardment-mediated transient expression studies were performed to verify the function. Gene ontology analysis was performed to predict the roles of genes containing the novel ABREs. This study revealed 2443 ABA-inducible genes and a novel ABRE, designated as ABREN, which was experimentally verified to mediate ABA signalling in rice aleurone cells. Many of the ABREN-containing genes are predicted to be involved in stress responses and transcription. Analysis of other species suggests that the ABREN may be monocot specific. This study also revealed interesting expression patterns of genes involved in ABA metabolism and signalling. Collectively, this study advanced our understanding of diverse cis-regulatory sequences and the transcriptomes underlying ABA responses in rice aleurone cells. © 2017 John Wiley & Sons Ltd.

The PAZAR database of gene regulatory information coupled to the ORCA toolkit for the study of regulatory sequences

PubMed Central

Portales-Casamar, Elodie; Arenillas, David; Lim, Jonathan; Swanson, Magdalena I.; Jiang, Steven; McCallum, Anthony; Kirov, Stefan; Wasserman, Wyeth W.

2009-01-01

The PAZAR database unites independently created and maintained data collections of transcription factor and regulatory sequence annotation. The flexible PAZAR schema permits the representation of diverse information derived from experiments ranging from biochemical protein–DNA binding to cellular reporter gene assays. Data collections can be made available to the public, or restricted to specific system users. The data ‘boutiques’ within the shopping-mall-inspired system facilitate the analysis of genomics data and the creation of predictive models of gene regulation. Since its initial release, PAZAR has grown in terms of data, features and through the addition of an associated package of software tools called the ORCA toolkit (ORCAtk). ORCAtk allows users to rapidly develop analyses based on the information stored in the PAZAR system. PAZAR is available at http://www.pazar.info. ORCAtk can be accessed through convenient buttons located in the PAZAR pages or via our website at http://www.cisreg.ca/ORCAtk. PMID:18971253

A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design.

PubMed

Smith, Robin P; Riesenfeld, Samantha J; Holloway, Alisha K; Li, Qiang; Murphy, Karl K; Feliciano, Natalie M; Orecchia, Lorenzo; Oksenberg, Nir; Pollard, Katherine S; Ahituv, Nadav

2013-07-18

Large-scale annotation efforts have improved our ability to coarsely predict regulatory elements throughout vertebrate genomes. However, it is unclear how complex spatiotemporal patterns of gene expression driven by these elements emerge from the activity of short, transcription factor binding sequences. We describe a comprehensive promoter extension assay in which the regulatory potential of all 6 base-pair (bp) sequences was tested in the context of a minimal promoter. To enable this large-scale screen, we developed algorithms that use a reverse-complement aware decomposition of the de Bruijn graph to design a library of DNA oligomers incorporating every 6-bp sequence exactly once. Our library multiplexes all 4,096 unique 6-mers into 184 double-stranded 15-bp oligomers, which is sufficiently compact for in vivo testing. We injected each multiplexed construct into zebrafish embryos and scored GFP expression in 15 tissues at two developmental time points. Twenty-seven constructs produced consistent expression patterns, with the majority doing so in only one tissue. Functional sequences are enriched near biologically relevant genes, match motifs for developmental transcription factors, and are required for enhancer activity. By concatenating tissue-specific functional sequences, we generated completely synthetic enhancers for the notochord, epidermis, spinal cord, forebrain and otic lateral line, and show that short regulatory sequences do not always function modularly. This work introduces a unique in vivo catalog of short, functional regulatory sequences and demonstrates several important principles of regulatory element organization. Furthermore, we provide resources for designing compact, reverse-complement aware k-mer libraries.

Transcriptome Analysis of an Insecticide Resistant Housefly Strain: Insights about SNPs and Regulatory Elements in Cytochrome P450 Genes.

PubMed

Mahmood, Khalid; Højland, Dorte H; Asp, Torben; Kristensen, Michael

2016-01-01

Insecticide resistance in the housefly, Musca domestica, has been investigated for more than 60 years. It will enter a new era after the recent publication of the housefly genome and the development of multiple next generation sequencing technologies. The genetic background of the xenobiotic response can now be investigated in greater detail. Here, we investigate the 454-pyrosequencing transcriptome of the spinosad-resistant 791spin strain in relation to the housefly genome with focus on P450 genes. The de novo assembly of clean reads gave 35,834 contigs consisting of 21,780 sequences of the spinosad resistant strain. The 3,648 sequences were annotated with an enzyme code EC number and were mapped to 124 KEGG pathways with metabolic processes as most highly represented pathway. One hundred and twenty contigs were annotated as P450s covering 44 different P450 genes of housefly. Eight differentially expressed P450s genes were identified and investigated for SNPs, CpG islands and common regulatory motifs in promoter and coding regions. Functional annotation clustering of metabolic related genes and motif analysis of P450s revealed their association with epigenetic, transcription and gene expression related functions. The sequence variation analysis resulted in 12 SNPs and eight of them found in cyp6d1. There is variation in location, size and frequency of CpG islands and specific motifs were also identified in these P450s. Moreover, identified motifs were associated to GO terms and transcription factors using bioinformatic tools. Transcriptome data of a spinosad resistant strain provide together with genome data fundamental support for future research to understand evolution of resistance in houseflies. Here, we report for the first time the SNPs, CpG islands and common regulatory motifs in differentially expressed P450s. Taken together our findings will serve as a stepping stone to advance understanding of the mechanism and role of P450s in xenobiotic detoxification.

Non-random distribution and co-localization of purine/pyrimidine-encoded information and transcriptional regulatory domains.

PubMed

Povinelli, C M

1992-01-01

In order to detect sequence-based information predictive for the location of eukaryotic transcriptional regulatory domains, the frequencies and distributions of the 36 possible purine/pyrimidine reverse complement hexamer pairs was determined for test sets of real and random sequences. The distribution of one of the hexamer pairs (RRYYRR/YYRRYY, referred to as M1) was further examined in a larger set of sequences (> 32 genes, 230 kb). Predominant clusters of M1 and the locations of eukaryotic transcriptional regulatory domains were found to be associated and non-randomly distributed along the DNA consistent with a periodicity of approximately 1.2 kb. In the context of higher ordered chromatin this would align promoters, enhancers and the predominant clusters of M1 longitudinally along one face of a 30 nm fiber. Using only information about the distribution of the M1 motif, 50-70% of a sequence could be eliminated as being unlikely to contain transcriptional regulatory domains with an 87% recovery of the regulatory domains present.

Finding functional features in Saccharomyces genomes by phylogenetic footprinting.

PubMed

Cliften, Paul; Sudarsanam, Priya; Desikan, Ashwin; Fulton, Lucinda; Fulton, Bob; Majors, John; Waterston, Robert; Cohen, Barak A; Johnston, Mark

2003-07-04

The sifting and winnowing of DNA sequence that occur during evolution cause nonfunctional sequences to diverge, leaving phylogenetic footprints of functional sequence elements in comparisons of genome sequences. We searched for such footprints among the genome sequences of six Saccharomyces species and identified potentially functional sequences. Comparison of these sequences allowed us to revise the catalog of yeast genes and identify sequence motifs that may be targets of transcriptional regulatory proteins. Some of these conserved sequence motifs reside upstream of genes with similar functional annotations or similar expression patterns or those bound by the same transcription factor and are thus good candidates for functional regulatory sequences.

Genetic divergence between freshwater and marine morphs of alewife (Alosa pseudoharengus): a 'next-generation' sequencing analysis.

PubMed

Czesny, Sergiusz; Epifanio, John; Michalak, Pawel

2012-01-01

Alewife Alosa pseudoharengus, a small clupeid fish native to Atlantic Ocean, has recently (∼150 years ago) invaded the North American Great Lakes and despite challenges of freshwater environment its populations exploded and disrupted local food web structures. This range expansion has been accompanied by dramatic changes at all levels of organization. Growth rates, size at maturation, or fecundity are only a few of the most distinct morphological and life history traits that contrast the two alewife morphs. A question arises to what extent these rapidly evolving differences between marine and freshwater varieties result from regulatory (including phenotypic plasticity) or structural mutations. To gain insights into expression changes and sequence divergence between marine and freshwater alewives, we sequenced transcriptomes of individuals from Lake Michigan and Atlantic Ocean. Population specific single nucleotide polymorphisms were rare but interestingly occurred in sequences of genes that also tended to show large differences in expression. Our results show that the striking phenotypic divergence between anadromous and lake alewives can be attributed to massive regulatory modifications rather than coding changes.

Genetic Divergence between Freshwater and Marine Morphs of Alewife (Alosa pseudoharengus): A ‘Next-Generation’ Sequencing Analysis

PubMed Central

Czesny, Sergiusz; Epifanio, John; Michalak, Pawel

2012-01-01

Alewife Alosa pseudoharengus, a small clupeid fish native to Atlantic Ocean, has recently (∼150 years ago) invaded the North American Great Lakes and despite challenges of freshwater environment its populations exploded and disrupted local food web structures. This range expansion has been accompanied by dramatic changes at all levels of organization. Growth rates, size at maturation, or fecundity are only a few of the most distinct morphological and life history traits that contrast the two alewife morphs. A question arises to what extent these rapidly evolving differences between marine and freshwater varieties result from regulatory (including phenotypic plasticity) or structural mutations. To gain insights into expression changes and sequence divergence between marine and freshwater alewives, we sequenced transcriptomes of individuals from Lake Michigan and Atlantic Ocean. Population specific single nucleotide polymorphisms were rare but interestingly occurred in sequences of genes that also tended to show large differences in expression. Our results show that the striking phenotypic divergence between anadromous and lake alewives can be attributed to massive regulatory modifications rather than coding changes. PMID:22438868

DMINDA: an integrated web server for DNA motif identification and analyses

PubMed Central

Ma, Qin; Zhang, Hanyuan; Mao, Xizeng; Zhou, Chuan; Liu, Bingqiang; Chen, Xin; Xu, Ying

2014-01-01

DMINDA (DNA motif identification and analyses) is an integrated web server for DNA motif identification and analyses, which is accessible at http://csbl.bmb.uga.edu/DMINDA/. This web site is freely available to all users and there is no login requirement. This server provides a suite of cis-regulatory motif analysis functions on DNA sequences, which are important to elucidation of the mechanisms of transcriptional regulation: (i) de novo motif finding for a given set of promoter sequences along with statistical scores for the predicted motifs derived based on information extracted from a control set, (ii) scanning motif instances of a query motif in provided genomic sequences, (iii) motif comparison and clustering of identified motifs, and (iv) co-occurrence analyses of query motifs in given promoter sequences. The server is powered by a backend computer cluster with over 150 computing nodes, and is particularly useful for motif prediction and analyses in prokaryotic genomes. We believe that DMINDA, as a new and comprehensive web server for cis-regulatory motif finding and analyses, will benefit the genomic research community in general and prokaryotic genome researchers in particular. PMID:24753419

Using FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) to isolate active regulatory DNA

PubMed Central

Simon, Jeremy M.; Giresi, Paul G.; Davis, Ian J.; Lieb, Jason D.

2013-01-01

Eviction or destabilization of nucleosomes from chromatin is a hallmark of functional regulatory elements of the eukaryotic genome. Historically identified by nuclease hypersensitivity, these regulatory elements are typically bound by transcription factors or other regulatory proteins. FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) is an alternative approach to identify these genomic regions and has proven successful in a multitude of eukaryotic cell and tissue types. Cells or dissociated tissues are crosslinked briefly with formaldehyde, lysed, and sonicated. Sheared chromatin is subjected to phenol-chloroform extraction and the isolated DNA, typically encompassing 1–3% of the human genome, is purified. We provide guidelines for quantitative analysis by PCR, microarrays, or next-generation sequencing. Regulatory elements enriched by FAIRE display high concordance with those identified by nuclease hypersensitivity or ChIP, and the entire procedure can be completed in three days. FAIRE exhibits low technical variability, which allows its use in large-scale studies of chromatin from normal or diseased tissues. PMID:22262007

Evaluating the protein coding potential of exonized transposable element sequences

PubMed Central

Piriyapongsa, Jittima; Rutledge, Mark T; Patel, Sanil; Borodovsky, Mark; Jordan, I King

2007-01-01

Background Transposable element (TE) sequences, once thought to be merely selfish or parasitic members of the genomic community, have been shown to contribute a wide variety of functional sequences to their host genomes. Analysis of complete genome sequences have turned up numerous cases where TE sequences have been incorporated as exons into mRNAs, and it is widely assumed that such 'exonized' TEs encode protein sequences. However, the extent to which TE-derived sequences actually encode proteins is unknown and a matter of some controversy. We have tried to address this outstanding issue from two perspectives: i-by evaluating ascertainment biases related to the search methods used to uncover TE-derived protein coding sequences (CDS) and ii-through a probabilistic codon-frequency based analysis of the protein coding potential of TE-derived exons. Results We compared the ability of three classes of sequence similarity search methods to detect TE-derived sequences among data sets of experimentally characterized proteins: 1-a profile-based hidden Markov model (HMM) approach, 2-BLAST methods and 3-RepeatMasker. Profile based methods are more sensitive and more selective than the other methods evaluated. However, the application of profile-based search methods to the detection of TE-derived sequences among well-curated experimentally characterized protein data sets did not turn up many more cases than had been previously detected and nowhere near as many cases as recent genome-wide searches have. We observed that the different search methods used were complementary in the sense that they yielded largely non-overlapping sets of hits and differed in their ability to recover known cases of TE-derived CDS. The probabilistic analysis of TE-derived exon sequences indicates that these sequences have low protein coding potential on average. In particular, non-autonomous TEs that do not encode protein sequences, such as Alu elements, are frequently exonized but unlikely to encode protein sequences. Conclusion The exaptation of the numerous TE sequences found in exons as bona fide protein coding sequences may prove to be far less common than has been suggested by the analysis of complete genomes. We hypothesize that many exonized TE sequences actually function as post-transcriptional regulators of gene expression, rather than coding sequences, which may act through a variety of double stranded RNA related regulatory pathways. Indeed, their relatively high copy numbers and similarity to sequences dispersed throughout the genome suggests that exonized TE sequences could serve as master regulators with a wide scope of regulatory influence. Reviewers: This article was reviewed by Itai Yanai, Kateryna D. Makova, Melissa Wilson (nominated by Kateryna D. Makova) and Cedric Feschotte (nominated by John M. Logsdon Jr.). PMID:18036258

Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

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

Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong

2010-02-19

Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna wasmore » similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.« less

Analysis of xylem formation in pine by cDNA sequencing

NASA Technical Reports Server (NTRS)

Allona, I.; Quinn, M.; Shoop, E.; Swope, K.; St Cyr, S.; Carlis, J.; Riedl, J.; Retzel, E.; Campbell, M. M.; Sederoff, R.;

P2RP: a Web-based framework for the identification and analysis of regulatory proteins in prokaryotic genomes.

PubMed

Barakat, Mohamed; Ortet, Philippe; Whitworth, David E

2013-04-20

Regulatory proteins (RPs) such as transcription factors (TFs) and two-component system (TCS) proteins control how prokaryotic cells respond to changes in their external and/or internal state. Identification and annotation of TFs and TCSs is non-trivial, and between-genome comparisons are often confounded by different standards in annotation. There is a need for user-friendly, fast and convenient tools to allow researchers to overcome the inherent variability in annotation between genome sequences. We have developed the web-server P2RP (Predicted Prokaryotic Regulatory Proteins), which enables users to identify and annotate TFs and TCS proteins within their sequences of interest. Users can input amino acid or genomic DNA sequences, and predicted proteins therein are scanned for the possession of DNA-binding domains and/or TCS domains. RPs identified in this manner are categorised into families, unambiguously annotated, and a detailed description of their features generated, using an integrated software pipeline. P2RP results can then be outputted in user-specified formats. Biologists have an increasing need for fast and intuitively usable tools, which is why P2RP has been developed as an interactive system. As well as assisting experimental biologists to interrogate novel sequence data, it is hoped that P2RP will be built into genome annotation pipelines and re-annotation processes, to increase the consistency of RP annotation in public genomic sequences. P2RP is the first publicly available tool for predicting and analysing RP proteins in users' sequences. The server is freely available and can be accessed along with documentation at http://www.p2rp.org.

Network Biomarkers of Bladder Cancer Based on a Genome-Wide Genetic and Epigenetic Network Derived from Next-Generation Sequencing Data.

PubMed

Li, Cheng-Wei; Chen, Bor-Sen

2016-01-01

Epigenetic and microRNA (miRNA) regulation are associated with carcinogenesis and the development of cancer. By using the available omics data, including those from next-generation sequencing (NGS), genome-wide methylation profiling, candidate integrated genetic and epigenetic network (IGEN) analysis, and drug response genome-wide microarray analysis, we constructed an IGEN system based on three coupling regression models that characterize protein-protein interaction networks (PPINs), gene regulatory networks (GRNs), miRNA regulatory networks (MRNs), and epigenetic regulatory networks (ERNs). By applying system identification method and principal genome-wide network projection (PGNP) to IGEN analysis, we identified the core network biomarkers to investigate bladder carcinogenic mechanisms and design multiple drug combinations for treating bladder cancer with minimal side-effects. The progression of DNA repair and cell proliferation in stage 1 bladder cancer ultimately results not only in the derepression of miR-200a and miR-200b but also in the regulation of the TNF pathway to metastasis-related genes or proteins, cell proliferation, and DNA repair in stage 4 bladder cancer. We designed a multiple drug combination comprising gefitinib, estradiol, yohimbine, and fulvestrant for treating stage 1 bladder cancer with minimal side-effects, and another multiple drug combination comprising gefitinib, estradiol, chlorpromazine, and LY294002 for treating stage 4 bladder cancer with minimal side-effects.

A Framework for the Evaluation of Biosecurity, Commercial, Regulatory, and Scientific Impacts of Plant Viruses and Viroids Identified by NGS Technologies

PubMed Central

Massart, Sebastien; Candresse, Thierry; Gil, José; Lacomme, Christophe; Predajna, Lukas; Ravnikar, Maja; Reynard, Jean-Sébastien; Rumbou, Artemis; Saldarelli, Pasquale; Škorić, Dijana; Vainio, Eeva J.; Valkonen, Jari P. T.; Vanderschuren, Hervé; Varveri, Christina; Wetzel, Thierry

2017-01-01

Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the newly discovered viruses and the analysis of their impact at the biosecurity, commercial, regulatory, and scientific levels. This paper proposes a scaled and progressive scientific framework for efficient biological characterization and risk assessment when a previously known or a new plant virus is detected by next generation sequencing (NGS) technologies. Four case studies are also presented to illustrate the need for such a framework, and to discuss the scenarios. PMID:28174561

Modeling positional effects of regulatory sequences with spline transformations increases prediction accuracy of deep neural networks

PubMed Central

Avsec, Žiga; Cheng, Jun; Gagneur, Julien

2018-01-01

Abstract Motivation Regulatory sequences are not solely defined by their nucleic acid sequence but also by their relative distances to genomic landmarks such as transcription start site, exon boundaries or polyadenylation site. Deep learning has become the approach of choice for modeling regulatory sequences because of its strength to learn complex sequence features. However, modeling relative distances to genomic landmarks in deep neural networks has not been addressed. Results Here we developed spline transformation, a neural network module based on splines to flexibly and robustly model distances. Modeling distances to various genomic landmarks with spline transformations significantly increased state-of-the-art prediction accuracy of in vivo RNA-binding protein binding sites for 120 out of 123 proteins. We also developed a deep neural network for human splice branchpoint based on spline transformations that outperformed the current best, already distance-based, machine learning model. Compared to piecewise linear transformation, as obtained by composition of rectified linear units, spline transformation yields higher prediction accuracy as well as faster and more robust training. As spline transformation can be applied to further quantities beyond distances, such as methylation or conservation, we foresee it as a versatile component in the genomics deep learning toolbox. Availability and implementation Spline transformation is implemented as a Keras layer in the CONCISE python package: https://github.com/gagneurlab/concise. Analysis code is available at https://github.com/gagneurlab/Manuscript_Avsec_Bioinformatics_2017. Contact avsec@in.tum.de or gagneur@in.tum.de Supplementary information Supplementary data are available at Bioinformatics online. PMID:29155928

Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci.

PubMed

Canver, Matthew C; Lessard, Samuel; Pinello, Luca; Wu, Yuxuan; Ilboudo, Yann; Stern, Emily N; Needleman, Austen J; Galactéros, Frédéric; Brugnara, Carlo; Kutlar, Abdullah; McKenzie, Colin; Reid, Marvin; Chen, Diane D; Das, Partha Pratim; A Cole, Mitchel; Zeng, Jing; Kurita, Ryo; Nakamura, Yukio; Yuan, Guo-Cheng; Lettre, Guillaume; Bauer, Daniel E; Orkin, Stuart H

2017-04-01

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.

DMRT gene cluster analysis in the platypus: new insights into genomic organization and regulatory regions.

PubMed

El-Mogharbel, Nisrine; Wakefield, Matthew; Deakin, Janine E; Tsend-Ayush, Enkhjargal; Grützner, Frank; Alsop, Amber; Ezaz, Tariq; Marshall Graves, Jennifer A

2007-01-01

We isolated and characterized a cluster of platypus DMRT genes and compared their arrangement, location, and sequence across vertebrates. The DMRT gene cluster on human 9p24.3 harbors, in order, DMRT1, DMRT3, and DMRT2, which share a DM domain. DMRT1 is highly conserved and involved in sexual development in vertebrates, and deletions in this region cause sex reversal in humans. Sequence comparisons of DMRT genes between species have been valuable in identifying exons, control regions, and conserved nongenic regions (CNGs). The addition of platypus sequences is expected to be particularly valuable, since monotremes fill a gap in the vertebrate genome coverage. We therefore isolated and fully sequenced platypus BAC clones containing DMRT3 and DMRT2 as well as DMRT1 and then generated multispecies alignments and ran prediction programs followed by experimental verification to annotate this gene cluster. We found that the three genes have 58-66% identity to their human orthologues, lie in the same order as in other vertebrates, and colocate on 1 of the 10 platypus sex chromosomes, X5. We also predict that optimal annotation of the newly sequenced platypus genome will be challenging. The analysis of platypus sequence revealed differences in structure and sequence of the DMRT gene cluster. Multispecies comparison was particularly effective for detecting CNGs, revealing several novel potential regulatory regions within DMRT3 and DMRT2 as well as DMRT1. RT-PCR indicated that platypus DMRT1 and DMRT3 are expressed specifically in the adult testis (and not ovary), but DMRT2 has a wider expression profile, as it does for other mammals. The platypus DMRT1 expression pattern, and its location on an X chromosome, suggests an involvement in monotreme sexual development.

Computational identification of developmental enhancers:conservation and function of transcription factor binding-site clustersin drosophila melanogaster and drosophila psedoobscura

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

Berman, Benjamin P.; Pfeiffer, Barret D.; Laverty, Todd R.

2004-08-06

Background The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, we demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. Here, we report the results of in vivo functional analysis of 27 remaining clusters. Results We generated transgenic flies carrying each cluster attached to a basal promoter and reporter gene,more » and assayed embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. We used the Drosophila pseudoobscura genome to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. We incorporated conservation of binding-site clustering into a new genome-wide enhancer screen, and predict several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns. Conclusions Measuring conservation of sequence features closely linked to function - such as binding-site clustering - makes better use of comparative sequence data than commonly used methods that examine only sequence identity.« less

Isolation, characterization, and evaluation of three Citrus sinensis-derived constitutive gene promoters.

PubMed

Erpen, L; Tavano, E C R; Harakava, R; Dutt, M; Grosser, J W; Piedade, S M S; Mendes, B M J; Mourão Filho, F A A

2018-05-23

Regulatory sequences from the citrus constitutive genes cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1) were isolated, fused to the uidA gene, and qualitatively and quantitatively evaluated in transgenic sweet orange plants. The 5' upstream region of a gene (the promoter) is the most important component for the initiation and regulation of gene transcription of both native genes and transgenes in plants. The isolation and characterization of gene regulatory sequences are essential to the development of intragenic or cisgenic genetic manipulation strategies, which imply the use of genetic material from the same species or from closely related species. We describe herein the isolation and evaluation of the promoter sequence from three constitutively expressed citrus genes: cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1). The functionality of the promoters was confirmed by a histochemical GUS assay in leaves, stems, and roots of stably transformed citrus plants expressing the promoter-uidA construct. Lower uidA mRNA levels were detected when the transgene was under the control of citrus promoters as compared to the expression under the control of the CaMV35S promoter. The association of the uidA gene with the citrus-derived promoters resulted in mRNA levels of up to 60-41.8% of the value obtained with the construct containing CaMV35S driving the uidA gene. Moreover, a lower inter-individual variability in transgene expression was observed amongst the different transgenic lines, where gene constructs containing citrus-derived promoters were used. In silico analysis of the citrus-derived promoter sequences revealed that their activity may be controlled by several putative cis-regulatory elements. These citrus promoters will expand the availability of regulatory sequences for driving gene expression in citrus gene-modification programs.

Functional and mechanistic diversity of distal transcription enhancers

PubMed Central

Bulger, Michael; Groudine, Mark

2013-01-01

Biological differences among metazoans, and between cell types in a given organism, arise in large part due to differences in gene expression patterns. The sequencing of multiple metazoan genomes, coupled with recent advances in genome-wide analysis of histone modifications and transcription factor binding, has revealed that among regulatory DNA sequences, gene-distal enhancers appear to exhibit the greatest diversity and cell-type specificity. Moreover, such elements are emerging as important targets for mutations that can give rise to disease and to genetic variability that underlies evolutionary change. Studies of long-range interactions between distal genomic sequences in the nucleus indicate that enhancers are often important determinants of nuclear organization, contributing to a general model for enhancer function that involves direct enhancer-promoter contact. In a number of systems, however, mechanisms for enhancer function are emerging that do not fit solely within such a model, suggesting that enhancers as a class of DNA regulatory element may be functionally and mechanistically diverse. PMID:21295696

The Intolerance of Regulatory Sequence to Genetic Variation Predicts Gene Dosage Sensitivity

PubMed Central

Wang, Quanli; Halvorsen, Matt; Han, Yujun; Weir, William H.; Allen, Andrew S.; Goldstein, David B.

2015-01-01

Noncoding sequence contains pathogenic mutations. Yet, compared with mutations in protein-coding sequence, pathogenic regulatory mutations are notoriously difficult to recognize. Most fundamentally, we are not yet adept at recognizing the sequence stretches in the human genome that are most important in regulating the expression of genes. For this reason, it is difficult to apply to the regulatory regions the same kinds of analytical paradigms that are being successfully applied to identify mutations among protein-coding regions that influence risk. To determine whether dosage sensitive genes have distinct patterns among their noncoding sequence, we present two primary approaches that focus solely on a gene’s proximal noncoding regulatory sequence. The first approach is a regulatory sequence analogue of the recently introduced residual variation intolerance score (RVIS), termed noncoding RVIS, or ncRVIS. The ncRVIS compares observed and predicted levels of standing variation in the regulatory sequence of human genes. The second approach, termed ncGERP, reflects the phylogenetic conservation of a gene’s regulatory sequence using GERP++. We assess how well these two approaches correlate with four gene lists that use different ways to identify genes known or likely to cause disease through changes in expression: 1) genes that are known to cause disease through haploinsufficiency, 2) genes curated as dosage sensitive in ClinGen’s Genome Dosage Map, 3) genes judged likely to be under purifying selection for mutations that change expression levels because they are statistically depleted of loss-of-function variants in the general population, and 4) genes judged unlikely to cause disease based on the presence of copy number variants in the general population. We find that both noncoding scores are highly predictive of dosage sensitivity using any of these criteria. In a similar way to ncGERP, we assess two ensemble-based predictors of regional noncoding importance, ncCADD and ncGWAVA, and find both scores are significantly predictive of human dosage sensitive genes and appear to carry information beyond conservation, as assessed by ncGERP. These results highlight that the intolerance of noncoding sequence stretches in the human genome can provide a critical complementary tool to other genome annotation approaches to help identify the parts of the human genome increasingly likely to harbor mutations that influence risk of disease. PMID:26332131

Abundance and functional diversity of riboswitches in microbial communities

PubMed Central

Kazanov, Marat D; Vitreschak, Alexey G; Gelfand, Mikhail S

2007-01-01

Background Several recently completed large-scale enviromental sequencing projects produced a large amount of genetic information about microbial communities ('metagenomes') which is not biased towards cultured organisms. It is a good source for estimation of the abundance of genes and regulatory structures in both known and unknown members of microbial communities. In this study we consider the distribution of RNA regulatory structures, riboswitches, in the Sargasso Sea, Minnesota Soil and Whale Falls metagenomes. Results Over three hundred riboswitches were found in about 2 Gbp metagenome DNA sequences. The abundabce of riboswitches in metagenomes was highest for the TPP, B12 and GCVT riboswitches; the S-box, RFN, YKKC/YXKD, YYBP/YKOY regulatory elements showed lower but significant abundance, while the LYS, G-box, GLMS and YKOK riboswitches were rare. Regions downstream of identified riboswitches were scanned for open reading frames. Comparative analysis of identified ORFs revealed new riboswitch-regulated functions for several classes of riboswitches. In particular, we have observed phosphoserine aminotransferase serC (COG1932) and malate synthase glcB (COG2225) to be regulated by the glycine (GCVT) riboswitch; fatty acid desaturase ole1 (COG1398), by the cobalamin (B12) riboswitch; 5-methylthioribose-1-phosphate isomerase ykrS (COG0182), by the SAM-riboswitch. We also identified conserved riboswitches upstream of genes of unknown function: thiamine (TPP), cobalamine (B12), and glycine (GCVT, upstream of genes from COG4198). Conclusion This study demonstrates applicability of bioinformatics to the analysis of RNA regulatory structures in metagenomes. PMID:17908319

Next Generation Sequencing Technology and Genomewide Data Analysis: Perspectives for Retinal Research

PubMed Central

Chaitankar, Vijender; Karakülah, Gökhan; Ratnapriya, Rinki; Giuste, Felipe O.; Brooks, Matthew J.; Swaroop, Anand

2016-01-01

The advent of high throughput next generation sequencing (NGS) has accelerated the pace of discovery of disease-associated genetic variants and genomewide profiling of expressed sequences and epigenetic marks, thereby permitting systems-based analyses of ocular development and disease. Rapid evolution of NGS and associated methodologies presents significant challenges in acquisition, management, and analysis of large data sets and for extracting biologically or clinically relevant information. Here we illustrate the basic design of commonly used NGS-based methods, specifically whole exome sequencing, transcriptome, and epigenome profiling, and provide recommendations for data analyses. We briefly discuss systems biology approaches for integrating multiple data sets to elucidate gene regulatory or disease networks. While we provide examples from the retina, the NGS guidelines reviewed here are applicable to other tissues/cell types as well. PMID:27297499

[Analysis of cis-regulatory element distribution in gene promoters of Gossypium raimondii and Arabidopsis thaliana].

PubMed

Sun, Gao-Fei; He, Shou-Pu; Du, Xiong-Ming

2013-10-01

Cotton genomic studies have boomed since the release of Gossypium raimondii draft genome. In this study, cis-regulatory element (CRE) in 1 kb length sequence upstream 5' UTR of annotated genes were selected and scanned in the Arabidopsis thaliana (At) and Gossypium raimondii (Gr) genomes, based on the database of PLACE (Plant cis-acting Regulatory DNA Elements). According to the definition of this study, 44 (12.3%) and 57 (15.5%) CREs presented "peak-like" distribution in the 1 kb selected sequences of both genomes, respectively. Thirty-four of them were peak-like distributed in both genomes, which could be further categorized into 4 types based on their core sequences. The coincidence of TATABOX peak position and their actual position ((-) -30 bp) indicated that the position of a common CRE was conservative in different genes, which suggested that the peak position of these CREs was their possible actual position of transcription factors. The position of a common CRE was also different between the two genomes due to stronger length variation of 5' UTR in Gr than At. Furthermore, most of the peak-like CREs were located in the region of -110 bp-0 bp, which suggested that concentrated distribution might be conductive to the interaction of transcription factors, and then regulate the gene expression in downstream.

Sequence Evolution and Expression Regulation of Stress-Responsive Genes in Natural Populations of Wild Tomato

PubMed Central

Fischer, Iris; Steige, Kim A.; Stephan, Wolfgang; Mboup, Mamadou

2013-01-01

The wild tomato species Solanum chilense and S. peruvianum are a valuable non-model system for studying plant adaptation since they grow in diverse environments facing many abiotic constraints. Here we investigate the sequence evolution of regulatory regions of drought and cold responsive genes and their expression regulation. The coding regions of these genes were previously shown to exhibit signatures of positive selection. Expression profiles and sequence evolution of regulatory regions of members of the Asr (ABA/water stress/ripening induced) gene family and the dehydrin gene pLC30-15 were analyzed in wild tomato populations from contrasting environments. For S. chilense, we found that Asr4 and pLC30-15 appear to respond much faster to drought conditions in accessions from very dry environments than accessions from more mesic locations. Sequence analysis suggests that the promoter of Asr2 and the downstream region of pLC30-15 are under positive selection in some local populations of S. chilense. By investigating gene expression differences at the population level we provide further support of our previous conclusions that Asr2, Asr4, and pLC30-15 are promising candidates for functional studies of adaptation. Our analysis also demonstrates the power of the candidate gene approach in evolutionary biology research and highlights the importance of wild Solanum species as a genetic resource for their cultivated relatives. PMID:24205149

Consistency of biological networks inferred from microarray and sequencing data.

PubMed

Vinciotti, Veronica; Wit, Ernst C; Jansen, Rick; de Geus, Eco J C N; Penninx, Brenda W J H; Boomsma, Dorret I; 't Hoen, Peter A C

2016-06-24

Sparse Gaussian graphical models are popular for inferring biological networks, such as gene regulatory networks. In this paper, we investigate the consistency of these models across different data platforms, such as microarray and next generation sequencing, on the basis of a rich dataset containing samples that are profiled under both techniques as well as a large set of independent samples. Our analysis shows that individual node variances can have a remarkable effect on the connectivity of the resulting network. Their inconsistency across platforms and the fact that the variability level of a node may not be linked to its regulatory role mean that, failing to scale the data prior to the network analysis, leads to networks that are not reproducible across different platforms and that may be misleading. Moreover, we show how the reproducibility of networks across different platforms is significantly higher if networks are summarised in terms of enrichment amongst functional groups of interest, such as pathways, rather than at the level of individual edges. Careful pre-processing of transcriptional data and summaries of networks beyond individual edges can improve the consistency of network inference across platforms. However, caution is needed at this stage in the (over)interpretation of gene regulatory networks inferred from biological data.

Evolutionary Novelty in a Butterfly Wing Pattern through Enhancer Shuffling

PubMed Central

Pardo-Diaz, Carolina; Hanly, Joseph J.; Martin, Simon H.; Mallet, James; Dasmahapatra, Kanchon K.; Salazar, Camilo; Joron, Mathieu; Nadeau, Nicola; McMillan, W. Owen; Jiggins, Chris D.

2016-01-01

An important goal in evolutionary biology is to understand the genetic changes underlying novel morphological structures. We investigated the origins of a complex wing pattern found among Amazonian Heliconius butterflies. Genome sequence data from 142 individuals across 17 species identified narrow regions associated with two distinct red colour pattern elements, dennis and ray. We hypothesise that these modules in non-coding sequence represent distinct cis-regulatory loci that control expression of the transcription factor optix, which in turn controls red pattern variation across Heliconius. Phylogenetic analysis of the two elements demonstrated that they have distinct evolutionary histories and that novel adaptive morphological variation was created by shuffling these cis-regulatory modules through recombination between divergent lineages. In addition, recombination of modules into different combinations within species further contributes to diversity. Analysis of the timing of diversification in these two regions supports the hypothesis of introgression moving regulatory modules between species, rather than shared ancestral variation. The dennis phenotype introgressed into Heliconius melpomene at about the same time that ray originated in this group, while ray introgressed back into H. elevatus much more recently. We show that shuffling of existing enhancer elements both within and between species provides a mechanism for rapid diversification and generation of novel morphological combinations during adaptive radiation. PMID:26771987

Evolutionary Novelty in a Butterfly Wing Pattern through Enhancer Shuffling.

PubMed

Wallbank, Richard W R; Baxter, Simon W; Pardo-Diaz, Carolina; Hanly, Joseph J; Martin, Simon H; Mallet, James; Dasmahapatra, Kanchon K; Salazar, Camilo; Joron, Mathieu; Nadeau, Nicola; McMillan, W Owen; Jiggins, Chris D

2016-01-01

An important goal in evolutionary biology is to understand the genetic changes underlying novel morphological structures. We investigated the origins of a complex wing pattern found among Amazonian Heliconius butterflies. Genome sequence data from 142 individuals across 17 species identified narrow regions associated with two distinct red colour pattern elements, dennis and ray. We hypothesise that these modules in non-coding sequence represent distinct cis-regulatory loci that control expression of the transcription factor optix, which in turn controls red pattern variation across Heliconius. Phylogenetic analysis of the two elements demonstrated that they have distinct evolutionary histories and that novel adaptive morphological variation was created by shuffling these cis-regulatory modules through recombination between divergent lineages. In addition, recombination of modules into different combinations within species further contributes to diversity. Analysis of the timing of diversification in these two regions supports the hypothesis of introgression moving regulatory modules between species, rather than shared ancestral variation. The dennis phenotype introgressed into Heliconius melpomene at about the same time that ray originated in this group, while ray introgressed back into H. elevatus much more recently. We show that shuffling of existing enhancer elements both within and between species provides a mechanism for rapid diversification and generation of novel morphological combinations during adaptive radiation.

Rationalizing context-dependent performance of dynamic RNA regulatory devices.

PubMed

Kent, Ross; Halliwell, Samantha; Young, Kate; Swainston, Neil; Dixon, Neil

2018-06-21

The ability of RNA to sense, regulate and store information is an attractive attribute for a variety of functional applications including the development of regulatory control devices for synthetic biology. RNA folding and function is known to be highly context sensitive, which limits the modularity and reuse of RNA regulatory devices to control different heterologous sequences and genes. We explored the cause and effect of sequence context sensitivity for translational ON riboswitches located in the 5' UTR, by constructing and screening a library of N-terminal synonymous codon variants. By altering the N-terminal codon usage we were able to obtain RNA devices with a broad range of functional performance properties (ON, OFF, fold-change). Linear regression and calculated metrics were used to rationalize the major determining features leading to optimal riboswitch performance, and to identify multiple interactions between the explanatory metrics. Finally, partial least squared (PLS) analysis was employed in order to understand the metrics and their respective effect on performance. This PLS model was shown to provide good explanation of our library. This study provides a novel multi-variant analysis framework by which to rationalize the codon context performance of allosteric RNA-devices. The framework will also serve as a platform for future riboswitch context engineering endeavors.

oPOSSUM-3: Advanced Analysis of Regulatory Motif Over-Representation Across Genes or ChIP-Seq Datasets

PubMed Central

Kwon, Andrew T.; Arenillas, David J.; Hunt, Rebecca Worsley; Wasserman, Wyeth W.

2012-01-01

oPOSSUM-3 is a web-accessible software system for identification of over-represented transcription factor binding sites (TFBS) and TFBS families in either DNA sequences of co-expressed genes or sequences generated from high-throughput methods, such as ChIP-Seq. Validation of the system with known sets of co-regulated genes and published ChIP-Seq data demonstrates the capacity for oPOSSUM-3 to identify mediating transcription factors (TF) for co-regulated genes or co-recovered sequences. oPOSSUM-3 is available at http://opossum.cisreg.ca. PMID:22973536

oPOSSUM-3: advanced analysis of regulatory motif over-representation across genes or ChIP-Seq datasets.

PubMed

Kwon, Andrew T; Arenillas, David J; Worsley Hunt, Rebecca; Wasserman, Wyeth W

2012-09-01

oPOSSUM-3 is a web-accessible software system for identification of over-represented transcription factor binding sites (TFBS) and TFBS families in either DNA sequences of co-expressed genes or sequences generated from high-throughput methods, such as ChIP-Seq. Validation of the system with known sets of co-regulated genes and published ChIP-Seq data demonstrates the capacity for oPOSSUM-3 to identify mediating transcription factors (TF) for co-regulated genes or co-recovered sequences. oPOSSUM-3 is available at http://opossum.cisreg.ca.

Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12.

PubMed

Claverie-Martin, F; Diaz-Torres, M R; Kushner, S R

1987-01-01

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.

Sarcococca blight: Use of whole genome sequencing as a strategy for fungal disease diagnosis

USDA-ARS?s Scientific Manuscript database

Early and accurate diagnosis of new plant pathogens is vital for the rapid implementation of effective mitigation strategies and appropriate regulatory responses. Most commonly, pathogen identification relies on morphology and DNA marker analysis. However, for new diseases, these approaches may not...

cDNA cloning, genomic organization and expression analysis during somatic embryogenesis of the translationally controlled tumor protein (TCTP) gene from Japanese larch (Larix leptolepis).

PubMed

Zhang, Li-Feng; Li, Wan-Feng; Han, Su-Ying; Yang, Wen-Hua; Qi, Li-Wang

2013-10-15

A full-length cDNA and genomic sequences of a translationally controlled tumor protein (TCTP) gene were isolated from Japanese larch (Larix leptolepis) and designated LaTCTP. The length of the cDNA was 1, 043 bp and contained a 504 bp open reading frame that encodes a predicted protein of 167 amino acids, characterized by two signature sequences of the TCTP protein family. Analysis of the LaTCTP gene structure indicated four introns and five exons, and it is the largest of all currently known TCTP genes in plants. The 5'-flanking promoter region of LaTCTP was cloned using an improved TAIL-PCR technique. In this region we identified many important potential cis-acting elements, such as a Box-W1 (fungal elicitor responsive element), a CAT-box (cis-acting regulatory element related to meristem expression), a CGTCA-motif (cis-acting regulatory element involved in MeJA-responsiveness), a GT1-motif (light responsive element), a Skn-1-motif (cis-acting regulatory element required for endosperm expression) and a TGA-element (auxin-responsive element), suggesting that expression of LaTCTP is highly regulated. Expression analysis demonstrated ubiquitous localization of LaTCTP mRNA in the roots, stems and needles, high mRNA levels in the embryonal-suspensor mass (ESM), browning embryogenic cultures and mature somatic embryos, and low levels of mRNA at day five during somatic embryogenesis. We suggest that LaTCTP might participate in the regulation of somatic embryo development. These results provide a theoretical basis for understanding the molecular regulatory mechanism of LaTCTP and lay the foundation for artificial regulation of somatic embryogenesis. © 2013.

Detecting differential allelic expression using high-resolution melting curve analysis: application to the breast cancer susceptibility gene CHEK2

PubMed Central

2011-01-01

Background The gene CHEK2 encodes a checkpoint kinase playing a key role in the DNA damage pathway. Though CHEK2 has been identified as an intermediate breast cancer susceptibility gene, only a small proportion of high-risk families have been explained by genetic variants located in its coding region. Alteration in gene expression regulation provides a potential mechanism for generating disease susceptibility. The detection of differential allelic expression (DAE) represents a sensitive assay to direct the search for a functional sequence variant within the transcriptional regulatory elements of a candidate gene. We aimed to assess whether CHEK2 was subject to DAE in lymphoblastoid cell lines (LCLs) from high-risk breast cancer patients for whom no mutation in BRCA1 or BRCA2 had been identified. Methods We implemented an assay based on high-resolution melting (HRM) curve analysis and developed an analysis tool for DAE assessment. Results We observed allelic expression imbalance in 4 of the 41 LCLs examined. All four were carriers of the truncating mutation 1100delC. We confirmed previous findings that this mutation induces non-sense mediated mRNA decay. In our series, we ruled out the possibility of a functional sequence variant located in the promoter region or in a regulatory element of CHEK2 that would lead to DAE in the transcriptional regulatory milieu of freely proliferating LCLs. Conclusions Our results support that HRM is a sensitive and accurate method for DAE assessment. This approach would be of great interest for high-throughput mutation screening projects aiming to identify genes carrying functional regulatory polymorphisms. PMID:21569354

Sequence-dependent modelling of local DNA bending phenomena: curvature prediction and vibrational analysis.

PubMed

Vlahovicek, K; Munteanu, M G; Pongor, S

1999-01-01

Bending is a local conformational micropolymorphism of DNA in which the original B-DNA structure is only distorted but not extensively modified. Bending can be predicted by simple static geometry models as well as by a recently developed elastic model that incorporate sequence dependent anisotropic bendability (SDAB). The SDAB model qualitatively explains phenomena including affinity of protein binding, kinking, as well as sequence-dependent vibrational properties of DNA. The vibrational properties of DNA segments can be studied by finite element analysis of a model subjected to an initial bending moment. The frequency spectrum is obtained by applying Fourier analysis to the displacement values in the time domain. This analysis shows that the spectrum of the bending vibrations quite sensitively depends on the sequence, for example the spectrum of a curved sequence is characteristically different from the spectrum of straight sequence motifs of identical basepair composition. Curvature distributions are genome-specific, and pronounced differences are found between protein-coding and regulatory regions, respectively, that is, sites of extreme curvature and/or bendability are less frequent in protein-coding regions. A WWW server is set up for the prediction of curvature and generation of 3D models from DNA sequences (http:@www.icgeb.trieste.it/dna).

Sequence and expression variation in SUPPRESSOR of OVEREXPRESSION of CONSTANS 1 (SOC1): homeolog evolution in Indian Brassicas.

PubMed

Sri, Tanu; Mayee, Pratiksha; Singh, Anandita

2015-09-01

Whole genome sequence analyses allow unravelling such evolutionary consequences of meso-triplication event in Brassicaceae (∼14-20 million years ago (MYA)) as differential gene fractionation and diversification in homeologous sub-genomes. This study presents a simple gene-centric approach involving microsynteny and natural genetic variation analysis for understanding SUPPRESSOR of OVEREXPRESSION of CONSTANS 1 (SOC1) homeolog evolution in Brassica. Analysis of microsynteny in Brassica rapa homeologous regions containing SOC1 revealed differential gene fractionation correlating to reported fractionation status of sub-genomes of origin, viz. least fractionated (LF), moderately fractionated 1 (MF1) and most fractionated (MF2), respectively. Screening 18 cultivars of 6 Brassica species led to the identification of 8 genomic and 27 transcript variants of SOC1, including splice-forms. Co-occurrence of both interrupted and intronless SOC1 genes was detected in few Brassica species. In silico analysis characterised Brassica SOC1 as MADS intervening, K-box, C-terminal (MIKC(C)) transcription factor, with highly conserved MADS and I domains relative to K-box and C-terminal domain. Phylogenetic analyses and multiple sequence alignments depicting shared pattern of silent/non-silent mutations assigned Brassica SOC1 homologs into groups based on shared diploid base genome. In addition, a sub-genome structure in uncharacterised Brassica genomes was inferred. Expression analysis of putative MF2 and LF (Brassica diploid base genome A (AA)) sub-genome-specific SOC1 homeologs of Brassica juncea revealed near identical expression pattern. However, MF2-specific homeolog exhibited significantly higher expression implying regulatory diversification. In conclusion, evidence for polyploidy-induced sequence and regulatory evolution in Brassica SOC1 is being presented wherein differential homeolog expression is implied in functional diversification.

PAINT: a promoter analysis and interaction network generation tool for gene regulatory network identification.

PubMed

Vadigepalli, Rajanikanth; Chakravarthula, Praveen; Zak, Daniel E; Schwaber, James S; Gonye, Gregory E

2003-01-01

We have developed a bioinformatics tool named PAINT that automates the promoter analysis of a given set of genes for the presence of transcription factor binding sites. Based on coincidence of regulatory sites, this tool produces an interaction matrix that represents a candidate transcriptional regulatory network. This tool currently consists of (1) a database of promoter sequences of known or predicted genes in the Ensembl annotated mouse genome database, (2) various modules that can retrieve and process the promoter sequences for binding sites of known transcription factors, and (3) modules for visualization and analysis of the resulting set of candidate network connections. This information provides a substantially pruned list of genes and transcription factors that can be examined in detail in further experimental studies on gene regulation. Also, the candidate network can be incorporated into network identification methods in the form of constraints on feasible structures in order to render the algorithms tractable for large-scale systems. The tool can also produce output in various formats suitable for use in external visualization and analysis software. In this manuscript, PAINT is demonstrated in two case studies involving analysis of differentially regulated genes chosen from two microarray data sets. The first set is from a neuroblastoma N1E-115 cell differentiation experiment, and the second set is from neuroblastoma N1E-115 cells at different time intervals following exposure to neuropeptide angiotensin II. PAINT is available for use as an agent in BioSPICE simulation and analysis framework (www.biospice.org), and can also be accessed via a WWW interface at www.dbi.tju.edu/dbi/tools/paint/.

Primate-specific evolution of an LDLR enhancer

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

Wang, Qian-Fei; Prabhakar, Shyam; Wang, Qianben

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 elementsmore » can evolve to perform new functions in the primate lineage leading to human.« less

The Evolution of Bony Vertebrate Enhancers at Odds with Their Coding Sequence Landscape.

PubMed

Yousaf, Aisha; Sohail Raza, Muhammad; Ali Abbasi, Amir

2015-08-06

Enhancers lie at the heart of transcriptional and developmental gene regulation. Therefore, changes in enhancer sequences usually disrupt the target gene expression and result in disease phenotypes. Despite the well-established role of enhancers in development and disease, evolutionary sequence studies are lacking. The current study attempts to unravel the puzzle of bony vertebrates' conserved noncoding elements (CNE) enhancer evolution. Bayesian phylogenetics of enhancer sequences spotlights promising interordinal relationships among placental mammals, proposing a closer relationship between humans and laurasiatherians while placing rodents at the basal position. Clock-based estimates of enhancer evolution provided a dynamic picture of interspecific rate changes across the bony vertebrate lineage. Moreover, coelacanth in the study augmented our appreciation of the vertebrate cis-regulatory evolution during water-land transition. Intriguingly, we observed a pronounced upsurge in enhancer evolution in land-dwelling vertebrates. These novel findings triggered us to further investigate the evolutionary trend of coding as well as CNE nonenhancer repertoires, to highlight the relative evolutionary dynamics of diverse genomic landscapes. Surprisingly, the evolutionary rates of enhancer sequences were clearly at odds with those of the coding and the CNE nonenhancer sequences during vertebrate adaptation to land, with land vertebrates exhibiting significantly reduced rates of coding sequence evolution in comparison to their fast evolving regulatory landscape. The observed variation in tetrapod cis-regulatory elements caused the fine-tuning of associated gene regulatory networks. Therefore, the increased evolutionary rate of tetrapods' enhancer sequences might be responsible for the variation in developmental regulatory circuits during the process of vertebrate adaptation to land. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Both positive and negative regulatory elements mediate expression of a photoregulated CAB gene from Nicotiana plumbaginifolia.

PubMed Central

Castresana, C; Garcia-Luque, I; Alonso, E; Malik, V S; Cashmore, A R

1988-01-01

We have analyzed promoter regulatory elements from a photoregulated CAB gene (Cab-E) isolated from Nicotiana plumbaginifolia. These studies have been performed by introducing chimeric gene constructs into tobacco cells via Agrobacterium tumefaciens-mediated transformation. Expression studies on the regenerated transgenic plants have allowed us to characterize three positive and one negative cis-acting elements that influence photoregulated expression of the Cab-E gene. Within the upstream sequences we have identified two positive regulatory elements (PRE1 and PRE2) which confer maximum levels of photoregulated expression. These sequences contain multiple repeated elements related to the sequence-ACCGGCCCACTT-. We have also identified within the upstream region a negative regulatory element (NRE) extremely rich in AT sequences, which reduces the level of gene expression in the light. We have defined a light regulatory element (LRE) within the promoter region extending from -396 to -186 bp which confers photoregulated expression when fused to a constitutive nopaline synthase ('nos') promoter. Within this region there is a 132-bp element, extending from -368 to -234 bp, which on deletion from the Cab-E promoter reduces gene expression from high levels to undetectable levels. Finally, we have demonstrated for a full length Cab-E promoter conferring high levels of photoregulated expression, that sequences proximal to the Cab-E TATA box are not replaceable by corresponding sequences from a 'nos' promoter. This contrasts with the apparent equivalence of these Cab-E and 'nos' TATA box-proximal sequences in truncated promoters conferring low levels of photoregulated expression. Images PMID:2901343

RNA regulators responding to ribosomal protein S15 are frequent in sequence space

PubMed Central

Slinger, Betty L.; Meyer, Michelle M.

2016-01-01

There are several natural examples of distinct RNA structures that interact with the same ligand to regulate the expression of homologous genes in different organisms. One essential question regarding this phenomenon is whether such RNA regulators are the result of convergent or divergent evolution. Are the RNAs derived from some common ancestor and diverged to the point where we cannot identify the similarity, or have multiple solutions to the same biological problem arisen independently? A key variable in assessing these alternatives is how frequently such regulators arise within sequence space. Ribosomal protein S15 is autogenously regulated via an RNA regulator in many bacterial species; four apparently distinct regulators have been functionally validated in different bacterial phyla. Here, we explore how frequently such regulators arise within a partially randomized sequence population. We find many RNAs that interact specifically with ribosomal protein S15 from Geobacillus kaustophilus with biologically relevant dissociation constants. Furthermore, of the six sequences we characterize, four show regulatory activity in an Escherichia coli reporter assay. Subsequent footprinting and mutagenesis analysis indicates that protein binding proximal to regulatory features such as the Shine–Dalgarno sequence is sufficient to enable regulation, suggesting that regulation in response to S15 is relatively easily acquired. PMID:27580716

Conserved noncoding sequences conserve biological networks and influence genome evolution.

PubMed

Xie, Jianbo; Qian, Kecheng; Si, Jingna; Xiao, Liang; Ci, Dong; Zhang, Deqiang

2018-05-01

Comparative genomics approaches have identified numerous conserved cis-regulatory sequences near genes in plant genomes. Despite the identification of these conserved noncoding sequences (CNSs), our knowledge of their functional importance and selection remains limited. Here, we used a combination of DNA methylome analysis, microarray expression analyses, and functional annotation to study these sequences in the model tree Populus trichocarpa. Methylation in CG contexts and non-CG contexts was lower in CNSs, particularly CNSs in the 5'-upstream regions of genes, compared with other sites in the genome. We observed that CNSs are enriched in genes with transcription and binding functions, and this also associated with syntenic genes and those from whole-genome duplications, suggesting that cis-regulatory sequences play a key role in genome evolution. We detected a significant positive correlation between CNS number and protein interactions, suggesting that CNSs may have roles in the evolution and maintenance of biological networks. The divergence of CNSs indicates that duplication-degeneration-complementation drives the subfunctionalization of a proportion of duplicated genes from whole-genome duplication. Furthermore, population genomics confirmed that most CNSs are under strong purifying selection and only a small subset of CNSs shows evidence of adaptive evolution. These findings provide a foundation for future studies exploring these key genomic features in the maintenance of biological networks, local adaptation, and transcription.

Diversification of transcription factor-DNA interactions and the evolution of gene regulatory networks.

PubMed

Rogers, Julia M; Bulyk, Martha L

2018-04-25

Sequence-specific transcription factors (TFs) bind short DNA sequences in the genome to regulate the expression of target genes. In the last decade, numerous technical advances have enabled the determination of the DNA-binding specificities of many of these factors. Large-scale screens of many TFs enabled the creation of databases of TF DNA-binding specificities, typically represented as position weight matrices (PWMs). Although great progress has been made in determining and predicting binding specificities systematically, there are still many surprises to be found when studying a particular TF's interactions with DNA in detail. Paralogous TFs' binding specificities can differ in subtle ways, in a manner that is not immediately apparent from looking at their PWMs. These differences affect gene regulatory outputs and enable TFs to rewire transcriptional networks over evolutionary time. This review discusses recent observations made in the study of TF-DNA interactions that highlight the importance of continued in-depth analysis of TF-DNA interactions and their inherent complexity. This article is categorized under: Biological Mechanisms > Regulatory Biology. © 2018 Wiley Periodicals, Inc.

Comprehensive analysis of Arabidopsis expression level polymorphisms with simple inheritance

PubMed Central

Plantegenet, Stephanie; Weber, Johann; Goldstein, Darlene R; Zeller, Georg; Nussbaumer, Cindy; Thomas, Jérôme; Weigel, Detlef; Harshman, Keith; Hardtke, Christian S

2009-01-01

In Arabidopsis thaliana, gene expression level polymorphisms (ELPs) between natural accessions that exhibit simple, single locus inheritance are promising quantitative trait locus (QTL) candidates to explain phenotypic variability. It is assumed that such ELPs overwhelmingly represent regulatory element polymorphisms. However, comprehensive genome-wide analyses linking expression level, regulatory sequence and gene structure variation are missing, preventing definite verification of this assumption. Here, we analyzed ELPs observed between the Eil-0 and Lc-0 accessions. Compared with non-variable controls, 5′ regulatory sequence variation in the corresponding genes is indeed increased. However, ∼42% of all the ELP genes also carry major transcription unit deletions in one parent as revealed by genome tiling arrays, representing a >4-fold enrichment over controls. Within the subset of ELPs with simple inheritance, this proportion is even higher and deletions are generally more severe. Similar results were obtained from analyses of the Bay-0 and Sha accessions, using alternative technical approaches. Collectively, our results suggest that drastic structural changes are a major cause for ELPs with simple inheritance, corroborating experimentally observed indel preponderance in cloned Arabidopsis QTL. PMID:19225455

Comparative analysis on the structural features of the 5' flanking region of κ-casein genes from six different species

PubMed Central

Gerencsér, Ákos; Barta, Endre; Boa, Simon; Kastanis, Petros; Bösze, Zsuzsanna; Whitelaw, C Bruce A

2002-01-01

κ-casein plays an essential role in the formation, stabilisation and aggregation of milk micelles. Control of κ-casein expression reflects this essential role, although an understanding of the mechanisms involved lags behind that of the other milk protein genes. We determined the 5'-flanking sequences for the murine, rabbit and human κ-casein genes and compared them to the published ruminant sequences. The most conserved region was not the proximal promoter region but an approximately 400 bp long region centred 800 bp upstream of the TATA box. This region contained two highly conserved MGF/STAT5 sites with common spacing relative to each other. In this region, six conserved short stretches of similarity were also found which did not correspond to known transcription factor consensus sites. On the contrary to ruminant and human 5' regulatory sequences, the rabbit and murine 5'-flanking regions did not harbour any kind of repetitive elements. We generated a phylogenetic tree of the six species based on multiple alignment of the κ-casein sequences. This study identified conserved candidate transcriptional regulatory elements within the κ-casein gene promoter. PMID:11929628

DMINDA: an integrated web server for DNA motif identification and analyses.

PubMed

Ma, Qin; Zhang, Hanyuan; Mao, Xizeng; Zhou, Chuan; Liu, Bingqiang; Chen, Xin; Xu, Ying

2014-07-01

DMINDA (DNA motif identification and analyses) is an integrated web server for DNA motif identification and analyses, which is accessible at http://csbl.bmb.uga.edu/DMINDA/. This web site is freely available to all users and there is no login requirement. This server provides a suite of cis-regulatory motif analysis functions on DNA sequences, which are important to elucidation of the mechanisms of transcriptional regulation: (i) de novo motif finding for a given set of promoter sequences along with statistical scores for the predicted motifs derived based on information extracted from a control set, (ii) scanning motif instances of a query motif in provided genomic sequences, (iii) motif comparison and clustering of identified motifs, and (iv) co-occurrence analyses of query motifs in given promoter sequences. The server is powered by a backend computer cluster with over 150 computing nodes, and is particularly useful for motif prediction and analyses in prokaryotic genomes. We believe that DMINDA, as a new and comprehensive web server for cis-regulatory motif finding and analyses, will benefit the genomic research community in general and prokaryotic genome researchers in particular. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements

DOE PAGES

Henry, Kelli F.; Kawashima, Tomokazu; Goldberg, Robert B.

2015-03-22

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean ( Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we usemore » site-directed mutagenesis experiments in transgenic tobacco globularstage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. Lastly, a homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.« less

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements

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

Henry, Kelli F.; Kawashima, Tomokazu; Goldberg, Robert B.

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean ( Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we usemore » site-directed mutagenesis experiments in transgenic tobacco globularstage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. Lastly, a homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.« less

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements.

PubMed

Henry, Kelli F; Kawashima, Tomokazu; Goldberg, Robert B

2015-06-01

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean (Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we use site-directed mutagenesis experiments in transgenic tobacco globular-stage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. A homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.

Systematic elucidation and in vivo validation of sequences enriched in hindbrain transcriptional control

PubMed Central

Burzynski, Grzegorz M.; Reed, Xylena; Taher, Leila; Stine, Zachary E.; Matsui, Takeshi; Ovcharenko, Ivan; McCallion, Andrew S.

2012-01-01

Illuminating the primary sequence encryption of enhancers is central to understanding the regulatory architecture of genomes. We have developed a machine learning approach to decipher motif patterns of hindbrain enhancers and identify 40,000 sequences in the human genome that we predict display regulatory control that includes the hindbrain. Consistent with their roles in hindbrain patterning, MEIS1, NKX6-1, as well as HOX and POU family binding motifs contributed strongly to this enhancer model. Predicted hindbrain enhancers are overrepresented at genes expressed in hindbrain and associated with nervous system development, and primarily reside in the areas of open chromatin. In addition, 77 (0.2%) of these predictions are identified as hindbrain enhancers on the VISTA Enhancer Browser, and 26,000 (60%) overlap enhancer marks (H3K4me1 or H3K27ac). To validate these putative hindbrain enhancers, we selected 55 elements distributed throughout our predictions and six low scoring controls for evaluation in a zebrafish transgenic assay. When assayed in mosaic transgenic embryos, 51/55 elements directed expression in the central nervous system. Furthermore, 30/34 (88%) predicted enhancers analyzed in stable zebrafish transgenic lines directed expression in the larval zebrafish hindbrain. Subsequent analysis of sequence fragments selected based upon motif clustering further confirmed the critical role of the motifs contributing to the classifier. Our results demonstrate the existence of a primary sequence code characteristic to hindbrain enhancers. This code can be accurately extracted using machine-learning approaches and applied successfully for de novo identification of hindbrain enhancers. This study represents a critical step toward the dissection of regulatory control in specific neuronal subtypes. PMID:22759862

Discovery of precursor and mature microRNAs and their putative gene targets using high-throughput sequencing in pineapple (Ananas comosus var. comosus).

PubMed

Yusuf, Noor Hydayaty Md; Ong, Wen Dee; Redwan, Raimi Mohamed; Latip, Mariam Abd; Kumar, S Vijay

2015-10-15

MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that negatively regulate gene expression, resulting in the silencing of target mRNA transcripts through mRNA cleavage or translational inhibition. MiRNAs play significant roles in various biological and physiological processes in plants. However, the miRNA-mediated gene regulatory network in pineapple, the model tropical non-climacteric fruit, remains largely unexplored. Here, we report a complete list of pineapple mature miRNAs obtained from high-throughput small RNA sequencing and precursor miRNAs (pre-miRNAs) obtained from ESTs. Two small RNA libraries were constructed from pineapple fruits and leaves, respectively, using Illumina's Solexa technology. Sequence similarity analysis using miRBase revealed 579,179 reads homologous to 153 miRNAs from 41 miRNA families. In addition, a pineapple fruit transcriptome library consisting of approximately 30,000 EST contigs constructed using Solexa sequencing was used for the discovery of pre-miRNAs. In all, four pre-miRNAs were identified (MIR156, MIR399, MIR444 and MIR2673). Furthermore, the same pineapple transcriptome was used to dissect the function of the miRNAs in pineapple by predicting their putative targets in conjunction with their regulatory networks. In total, 23 metabolic pathways were found to be regulated by miRNAs in pineapple. The use of high-throughput sequencing in pineapples to unveil the presence of miRNAs and their regulatory pathways provides insight into the repertoire of miRNA regulation used exclusively in this non-climacteric model plant. Copyright © 2015 Elsevier B.V. All rights reserved.

Long-Range Control of Gene Expression: Emerging Mechanisms and Disruption in Disease

PubMed Central

Kleinjan, Dirk A.; van Heyningen, Veronica

2005-01-01

Transcriptional control is a major mechanism for regulating gene expression. The complex machinery required to effect this control is still emerging from functional and evolutionary analysis of genomic architecture. In addition to the promoter, many other regulatory elements are required for spatiotemporally and quantitatively correct gene expression. Enhancer and repressor elements may reside in introns or up- and downstream of the transcription unit. For some genes with highly complex expression patterns—often those that function as key developmental control genes—the cis-regulatory domain can extend long distances outside the transcription unit. Some of the earliest hints of this came from disease-associated chromosomal breaks positioned well outside the relevant gene. With the availability of wide-ranging genome sequence comparisons, strong conservation of many noncoding regions became obvious. Functional studies have shown many of these conserved sites to be transcriptional regulatory elements that sometimes reside inside unrelated neighboring genes. Such sequence-conserved elements generally harbor sites for tissue-specific DNA-binding proteins. Developmentally variable chromatin conformation can control protein access to these sites and can regulate transcription. Disruption of these finely tuned mechanisms can cause disease. Some regulatory element mutations will be associated with phenotypes distinct from any identified for coding-region mutations. PMID:15549674

The Regulatory Properties of Autonomous Subtelomeric P Elements Are Sensitive to a Suppressor of Variegation in Drosophila Melanogaster

PubMed Central

Ronsseray, S.; Lehmann, M.; Nouaud, D.; Anxolabehere, D.

1996-01-01

Genetic recombination was used in Drosophila melanogaster to isolate P elements, inserted at the telomeres of X chromosomes (cytological site 1A) from natural populations, in a genetic background devoid of other P elements. We show that complete maternally inherited P repression in the germline (P cytotype) can be elicited by only two autonomous P elements at 1A and that a single element at this site has partial regulatory properties. The analysis of the surrounding chromosomal regions of the P elements at 1A shows that in all cases these elements are flanked by Telomeric Associated Sequences, tandemly repetitive noncoding sequences that have properties of heterochromatin. In addition, we show that the regulatory properties of P elements at 1A can be inhibited by some of the mutant alleles of the Su(var)205 gene and by a deficiency of this gene. However, the regulatory properties of reference P strains (Harwich and Texas 007) are not impaired by Su(var)205 mutations. Su(var)205 encodes Heterochromatin Protein 1 (HP1). These results suggest that the HP1 dosage effect on the P element properties is site-dependent and could involve the structure of the chromatin. PMID:8844154

Sasquatch: predicting the impact of regulatory SNPs on transcription factor binding from cell- and tissue-specific DNase footprints

PubMed Central

Suciu, Maria C.; Telenius, Jelena

2017-01-01

In the era of genome-wide association studies (GWAS) and personalized medicine, predicting the impact of single nucleotide polymorphisms (SNPs) in regulatory elements is an important goal. Current approaches to determine the potential of regulatory SNPs depend on inadequate knowledge of cell-specific DNA binding motifs. Here, we present Sasquatch, a new computational approach that uses DNase footprint data to estimate and visualize the effects of noncoding variants on transcription factor binding. Sasquatch performs a comprehensive k-mer-based analysis of DNase footprints to determine any k-mer's potential for protein binding in a specific cell type and how this may be changed by sequence variants. Therefore, Sasquatch uses an unbiased approach, independent of known transcription factor binding sites and motifs. Sasquatch only requires a single DNase-seq data set per cell type, from any genotype, and produces consistent predictions from data generated by different experimental procedures and at different sequence depths. Here we demonstrate the effectiveness of Sasquatch using previously validated functional SNPs and benchmark its performance against existing approaches. Sasquatch is available as a versatile webtool incorporating publicly available data, including the human ENCODE collection. Thus, Sasquatch provides a powerful tool and repository for prioritizing likely regulatory SNPs in the noncoding genome. PMID:28904015

Method to transform algae, materials therefor, and products produced thereby

DOEpatents

Dunahay, T.G.; Roessler, P.G.; Jarvis, E.E.

1997-08-26

Disclosed is a method to transform chlorophyll C-containing algae. The method includes introducing a recombinant molecule comprising a nucleic acid molecule encoding a dominant selectable marker operatively linked to an algal regulatory control sequence into a chlorophyll C-containing alga in such a manner that the marker is produced by the alga. In a preferred embodiment the algal regulatory control sequence is derived from a diatom and preferably Cyclotella cryptica. Also disclosed is a chimeric molecule having one or more regulatory control sequences derived from one or more chlorophyll C-containing algae operatively linked to a nucleic acid molecule encoding a selectable marker, an RNA molecule and/or a protein, wherein the nucleic acid molecule does not normally occur with one or more of the regulatory control sequences. Further, specifically disclosed are molecules pACCNPT10, pACCNPT4.8 and pACCNPT5.1. The methods and materials of the present invention provide the ability to accomplish stable genetic transformation of chlorophyll C-containing algae. 2 figs.

Method to transform algae, materials therefor, and products produced thereby

DOEpatents

Dunahay, Terri Goodman; Roessler, Paul G.; Jarvis, Eric E.

1997-01-01

Disclosed is a method to transform chlorophyll C-containing algae which includes introducing a recombinant molecule comprising a nucleic acid molecule encoding a dominant selectable marker operatively linked to an algal regulatory control sequence into a chlorophyll C-containing alga in such a manner that the marker is produced by the alga. In a preferred embodiment the algal regulatory control sequence is derived from a diatom and preferably Cyclotella cryptica. Also disclosed is a chimeric molecule having one or more regulatory control sequences derived from one or more chlorophyll C-containing algae operatively linked to a nucleic acid molecule encoding a selectable marker, an RNA molecule and/or a protein, wherein the nucleic acid molecule does not normally occur with one or more of the regulatory control sequences. Further specifically disclosed are molecules pACCNPT10, pACCNPT4.8 and pACCNPT5.1. The methods and materials of the present invention provide the ability to accomplish stable genetic transformation of chlorophyll C-containing algae.

Plant nitrogen regulatory P-PII polypeptides

DOEpatents

Coruzzi, Gloria M.; Lam, Hon-Ming; Hsieh, Ming-Hsiun

2004-11-23

The present invention generally relates to plant nitrogen regulatory PII gene (hereinafter P-PII gene), a gene involved in regulating plant nitrogen metabolism. The invention provides P-PII nucleotide sequences, expression constructs comprising said nucleotide sequences, and host cells and plants having said constructs and, optionally expressing the P-PII gene from said constructs. The invention also provides substantially pure P-PII proteins. The P-PII nucleotide sequences and constructs of the invention may be used to engineer organisms to overexpress wild-type or mutant P-PII regulatory protein. Engineered plants that overexpress or underexpress P-PII regulatory protein may have increased nitrogen assimilation capacity. Engineered organisms may be used to produce P-PII proteins which, in turn, can be used for a variety of purposes including in vitro screening of herbicides. P-PII nucleotide sequences have additional uses as probes for isolating additional genomic clones having the promoters of P-PII gene. P-PII promoters are light- and/or sucrose-inducible and may be advantageously used in genetic engineering of plants.

Deciphering the transcriptional cis-regulatory code.

PubMed

Yáñez-Cuna, J Omar; Kvon, Evgeny Z; Stark, Alexander

2013-01-01

Information about developmental gene expression resides in defined regulatory elements, called enhancers, in the non-coding part of the genome. Although cells reliably utilize enhancers to orchestrate gene expression, a cis-regulatory code that would allow their interpretation has remained one of the greatest challenges of modern biology. In this review, we summarize studies from the past three decades that describe progress towards revealing the properties of enhancers and discuss how recent approaches are providing unprecedented insights into regulatory elements in animal genomes. Over the next years, we believe that the functional characterization of regulatory sequences in entire genomes, combined with recent computational methods, will provide a comprehensive view of genomic regulatory elements and their building blocks and will enable researchers to begin to understand the sequence basis of the cis-regulatory code. Copyright © 2012 Elsevier Ltd. All rights reserved.

The standard operating procedure of the DOE-JGI Metagenome Annotation Pipeline (MAP v.4)

DOE PAGES

Huntemann, Marcel; Ivanova, Natalia N.; Mavromatis, Konstantinos; ...

2016-02-24

The DOE-JGI Metagenome Annotation Pipeline (MAP v.4) performs structural and functional annotation for metagenomic sequences that are submitted to the Integrated Microbial Genomes with Microbiomes (IMG/M) system for comparative analysis. The pipeline runs on nucleotide sequences provide d via the IMG submission site. Users must first define their analysis projects in GOLD and then submit the associated sequence datasets consisting of scaffolds/contigs with optional coverage information and/or unassembled reads in fasta and fastq file formats. The MAP processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNAs, as well as CRISPR elements. Structural annotation ismore » followed by functional annotation including assignment of protein product names and connection to various protein family databases.« less

The standard operating procedure of the DOE-JGI Metagenome Annotation Pipeline (MAP v.4)

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

Huntemann, Marcel; Ivanova, Natalia N.; Mavromatis, Konstantinos

The DOE-JGI Metagenome Annotation Pipeline (MAP v.4) performs structural and functional annotation for metagenomic sequences that are submitted to the Integrated Microbial Genomes with Microbiomes (IMG/M) system for comparative analysis. The pipeline runs on nucleotide sequences provide d via the IMG submission site. Users must first define their analysis projects in GOLD and then submit the associated sequence datasets consisting of scaffolds/contigs with optional coverage information and/or unassembled reads in fasta and fastq file formats. The MAP processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNAs, as well as CRISPR elements. Structural annotation ismore » followed by functional annotation including assignment of protein product names and connection to various protein family databases.« less

Epigenome analysis of pluripotent stem cells

PubMed Central

Ricupero, Christopher L.; Swerdel, Mavis R.; Hart, Ronald P.

2015-01-01

Summary Mis-regulation of gene expression due to epigenetic abnormalities has been linked with complex genetic disorders, psychiatric illness and cancer. In addition, the dynamic epigenetic changes that occur in pluripotent stem cells are believed to impact regulatory networks essential for proper lineage development. Chromatin immunoprecipitation (ChIP) is a technique used to isolate and enrich chromatin fragments using antibodies against specific chromatin modifications, such as DNA binding proteins or covalent histone modifications. Until recently, many ChIP protocols required millions of cells for each immunoprecipitation. This severely limited analysis of rare cell populations or post-mitotic, differentiated cell lines. Here, we describe a low cell number ChIP protocol with next generation sequencing and analysis, that has the potential to uncover novel epigenetic regulatory pathways that were previously difficult or impossible to obtain. PMID:23546758

Apple miRNAs and tasiRNAs with novel regulatory networks

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) and their regulatory functions have been extensively characterized in model species but whether apple has evolved similar or unique regulatory features remains unknown. Results We performed deep small RNA-seq and identified 23 conserved, 10 less-conserved and 42 apple-specific miRNAs or families with distinct expression patterns. The identified miRNAs target 118 genes representing a wide range of enzymatic and regulatory activities. Apple also conserves two TAS gene families with similar but unique trans-acting small interfering RNA (tasiRNA) biogenesis profiles and target specificities. Importantly, we found that miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. These miRNA target sites are differentially conserved among MYBs, which is largely influenced by the location and conservation of the encoded amino acid residues in MYB factors. Finally, we found that 10 of the 19 miR828-targeted MYBs undergo small interfering RNA (siRNA) biogenesis at the 3' cleaved, highly divergent transcript regions, generating over 100 sequence-distinct siRNAs that potentially target over 70 diverse genes as confirmed by degradome analysis. Conclusions Our work identified and characterized apple miRNAs, their expression patterns, targets and regulatory functions. We also discovered that three miRNAs and the ensuing siRNAs exploit both conserved and divergent sequence features of MYB genes to initiate distinct regulatory networks targeting a multitude of genes inside and outside the MYB family. PMID:22704043

Thermodynamics-based models of transcriptional regulation with gene sequence.

PubMed

Wang, Shuqiang; Shen, Yanyan; Hu, Jinxing

2015-12-01

Quantitative models of gene regulatory activity have the potential to improve our mechanistic understanding of transcriptional regulation. However, the few models available today have been based on simplistic assumptions about the sequences being modeled or heuristic approximations of the underlying regulatory mechanisms. In this work, we have developed a thermodynamics-based model to predict gene expression driven by any DNA sequence. The proposed model relies on a continuous time, differential equation description of transcriptional dynamics. The sequence features of the promoter are exploited to derive the binding affinity which is derived based on statistical molecular thermodynamics. Experimental results show that the proposed model can effectively identify the activity levels of transcription factors and the regulatory parameters. Comparing with the previous models, the proposed model can reveal more biological sense.

Use of transcriptome sequencing to understand the pistillate flowering in hickory (Carya cathayensis Sarg.).

PubMed

Huang, You-Jun; Liu, Li-Li; Huang, Jian-Qin; Wang, Zheng-Jia; Chen, Fang-Fang; Zhang, Qi-Xiang; Zheng, Bing-Song; Chen, Ming

2013-10-10

Different from herbaceous plants, the woody plants undergo a long-period vegetative stage to achieve floral transition. They then turn into seasonal plants, flowering annually. In this study, a preliminary model of gene regulations for seasonal pistillate flowering in hickory (Carya cathayensis) was proposed. The genome-wide dynamic transcriptome was characterized via the joint-approach of RNA sequencing and microarray analysis. Differential transcript abundance analysis uncovered the dynamic transcript abundance patterns of flowering correlated genes and their major functions based on Gene Ontology (GO) analysis. To explore pistillate flowering mechanism in hickory, a comprehensive flowering gene regulatory network based on Arabidopsis thaliana was constructed by additional literature mining. A total of 114 putative flowering or floral genes including 31 with differential transcript abundance were identified in hickory. The locations, functions and dynamic transcript abundances were analyzed in the gene regulatory networks. A genome-wide co-expression network for the putative flowering or floral genes shows three flowering regulatory modules corresponding to response to light abiotic stimulus, cold stress, and reproductive development process, respectively. Totally 27 potential flowering or floral genes were recruited which are meaningful to understand the hickory specific seasonal flowering mechanism better. Flowering event of pistillate flower bud in hickory is triggered by several pathways synchronously including the photoperiod, autonomous, vernalization, gibberellin, and sucrose pathway. Totally 27 potential flowering or floral genes were recruited from the genome-wide co-expression network function module analysis. Moreover, the analysis provides a potential FLC-like gene based vernalization pathway and an 'AC' model for pistillate flower development in hickory. This work provides an available framework for pistillate flower development in hickory, which is significant for insight into regulation of flowering and floral development of woody plants.

Use of transcriptome sequencing to understand the pistillate flowering in hickory (Carya cathayensis Sarg.)

PubMed Central

2013-01-01

Background Different from herbaceous plants, the woody plants undergo a long-period vegetative stage to achieve floral transition. They then turn into seasonal plants, flowering annually. In this study, a preliminary model of gene regulations for seasonal pistillate flowering in hickory (Carya cathayensis) was proposed. The genome-wide dynamic transcriptome was characterized via the joint-approach of RNA sequencing and microarray analysis. Results Differential transcript abundance analysis uncovered the dynamic transcript abundance patterns of flowering correlated genes and their major functions based on Gene Ontology (GO) analysis. To explore pistillate flowering mechanism in hickory, a comprehensive flowering gene regulatory network based on Arabidopsis thaliana was constructed by additional literature mining. A total of 114 putative flowering or floral genes including 31 with differential transcript abundance were identified in hickory. The locations, functions and dynamic transcript abundances were analyzed in the gene regulatory networks. A genome-wide co-expression network for the putative flowering or floral genes shows three flowering regulatory modules corresponding to response to light abiotic stimulus, cold stress, and reproductive development process, respectively. Totally 27 potential flowering or floral genes were recruited which are meaningful to understand the hickory specific seasonal flowering mechanism better. Conclusions Flowering event of pistillate flower bud in hickory is triggered by several pathways synchronously including the photoperiod, autonomous, vernalization, gibberellin, and sucrose pathway. Totally 27 potential flowering or floral genes were recruited from the genome-wide co-expression network function module analysis. Moreover, the analysis provides a potential FLC-like gene based vernalization pathway and an 'AC’ model for pistillate flower development in hickory. This work provides an available framework for pistillate flower development in hickory, which is significant for insight into regulation of flowering and floral development of woody plants. PMID:24106755

Plant nitrogen regulatory P-PII genes

DOEpatents

Coruzzi, Gloria M.; Lam, Hon-Ming; Hsieh, Ming-Hsiun

2001-01-01

The present invention generally relates to plant nitrogen regulatory PII gene (hereinafter P-PII gene), a gene involved in regulating plant nitrogen metabolism. The invention provides P-PII nucleotide sequences, expression constructs comprising said nucleotide sequences, and host cells and plants having said constructs and, optionally expressing the P-PII gene from said constructs. The invention also provides substantially pure P-PII proteins. The P-PII nucleotide sequences and constructs of the

Transcriptome Analysis of an Insecticide Resistant Housefly Strain: Insights about SNPs and Regulatory Elements in Cytochrome P450 Genes

PubMed Central

Asp, Torben; Kristensen, Michael

2016-01-01

Background Insecticide resistance in the housefly, Musca domestica, has been investigated for more than 60 years. It will enter a new era after the recent publication of the housefly genome and the development of multiple next generation sequencing technologies. The genetic background of the xenobiotic response can now be investigated in greater detail. Here, we investigate the 454-pyrosequencing transcriptome of the spinosad-resistant 791spin strain in relation to the housefly genome with focus on P450 genes. Results The de novo assembly of clean reads gave 35,834 contigs consisting of 21,780 sequences of the spinosad resistant strain. The 3,648 sequences were annotated with an enzyme code EC number and were mapped to 124 KEGG pathways with metabolic processes as most highly represented pathway. One hundred and twenty contigs were annotated as P450s covering 44 different P450 genes of housefly. Eight differentially expressed P450s genes were identified and investigated for SNPs, CpG islands and common regulatory motifs in promoter and coding regions. Functional annotation clustering of metabolic related genes and motif analysis of P450s revealed their association with epigenetic, transcription and gene expression related functions. The sequence variation analysis resulted in 12 SNPs and eight of them found in cyp6d1. There is variation in location, size and frequency of CpG islands and specific motifs were also identified in these P450s. Moreover, identified motifs were associated to GO terms and transcription factors using bioinformatic tools. Conclusion Transcriptome data of a spinosad resistant strain provide together with genome data fundamental support for future research to understand evolution of resistance in houseflies. Here, we report for the first time the SNPs, CpG islands and common regulatory motifs in differentially expressed P450s. Taken together our findings will serve as a stepping stone to advance understanding of the mechanism and role of P450s in xenobiotic detoxification. PMID:27019205

Cis-regulatory landscapes of four cell types of the retina

PubMed Central

Hartl, Dominik; Jüttner, Josephine

2017-01-01

Abstract The retina is composed of ∼50 cell-types with specific functions for the process of vision. Identification of the cis-regulatory elements active in retinal cell-types is key to elucidate the networks controlling this diversity. Here, we combined transcriptome and epigenome profiling to map the regulatory landscape of four cell-types isolated from mouse retinas including rod and cone photoreceptors as well as rare inter-neuron populations such as horizontal and starburst amacrine cells. Integration of this information reveals sequence determinants and candidate transcription factors for controlling cellular specialization. Additionally, we refined parallel reporter assays to enable studying the transcriptional activity of large collection of sequences in individual cell-types isolated from a tissue. We provide proof of concept for this approach and its scalability by characterizing the transcriptional capacity of several hundred putative regulatory sequences within individual retinal cell-types. This generates a catalogue of cis-regulatory regions active in retinal cell types and we further demonstrate their utility as potential resource for cellular tagging and manipulation. PMID:29059322

Nucleotide sequence determination of guinea-pig casein B mRNA reveals homology with bovine and rat alpha s1 caseins and conservation of the non-coding regions of the mRNA.

PubMed Central

Hall, L; Laird, J E; Craig, R K

1984-01-01

Nucleotide sequence analysis of cloned guinea-pig casein B cDNA sequences has identified two casein B variants related to the bovine and rat alpha s1 caseins. Amino acid homology was largely confined to the known bovine or predicted rat phosphorylation sites and within the 'signal' precursor sequence. Comparison of the deduced nucleotide sequence of the guinea-pig and rat alpha s1 casein mRNA species showed greater sequence conservation in the non-coding than in the coding regions, suggesting a functional and possibly regulatory role for the non-coding regions of casein mRNA. The results provide insight into the evolution of the casein genes, and raise questions as to the role of conserved nucleotide sequences within the non-coding regions of mRNA species. Images Fig. 1. PMID:6548375

Discovery and Annotation of Plant Endogenous Target Mimicry Sequences from Public Transcriptome Libraries: A Case Study of Prunus persica.

PubMed

Karakülah, Gökhan

2017-06-28

Novel transcript discovery through RNA sequencing has substantially improved our understanding of the transcriptome dynamics of biological systems. Endogenous target mimicry (eTM) transcripts, a novel class of regulatory molecules, bind to their target microRNAs (miRNAs) by base pairing and block their biological activity. The objective of this study was to provide a computational analysis framework for the prediction of putative eTM sequences in plants, and as an example, to discover previously un-annotated eTMs in Prunus persica (peach) transcriptome. Therefore, two public peach transcriptome libraries downloaded from Sequence Read Archive (SRA) and a previously published set of long non-coding RNAs (lncRNAs) were investigated with multi-step analysis pipeline, and 44 putative eTMs were found. Additionally, an eTM-miRNA-mRNA regulatory network module associated with peach fruit organ development was built via integration of the miRNA target information and predicted eTM-miRNA interactions. My findings suggest that one of the most widely expressed miRNA families among diverse plant species, miR156, might be potentially sponged by seven putative eTMs. Besides, the study indicates eTMs potentially play roles in the regulation of development processes in peach fruit via targeting specific miRNAs. In conclusion, by following the step-by step instructions provided in this study, novel eTMs can be identified and annotated effectively in public plant transcriptome libraries.

Structural and functional analysis of mouse Msx1 gene promoter: sequence conservation with human MSX1 promoter points at potential regulatory elements.

PubMed

Gonzalez, S M; Ferland, L H; Robert, B; Abdelhay, E

1998-06-01

Vertebrate Msx genes are related to one of the most divergent homeobox genes of Drosophila, the muscle segment homeobox (msh) gene, and are expressed in a well-defined pattern at sites of tissue interactions. This pattern of expression is conserved in vertebrates as diverse as quail, zebrafish, and mouse in a range of sites including neural crest, appendages, and craniofacial structures. In the present work, we performed structural and functional analyses in order to identify potential cis-acting elements that may be regulating Msx1 gene expression. To this end, a 4.9-kb segment of the 5'-flanking region was sequenced and analyzed for transcription-factor binding sites. Four regions showing a high concentration of these sites were identified. Transfection assays with fragments of regulatory sequences driving the expression of the bacterial lacZ reporter gene showed that a region of 4 kb upstream of the transcription start site contains positive and negative elements responsible for controlling gene expression. Interestingly, a fragment of 130 bp seems to contain the minimal elements necessary for gene expression, as its removal completely abolishes gene expression in cultured cells. These results are reinforced by comparison of this region with the human Msx1 gene promoter, which shows extensive conservation, including many consensus binding sites, suggesting a regulatory role for them.

DNA sequence and characterization of GcvA, a LysR family regulatory protein for the Escherichia coli glycine cleavage enzyme system.

PubMed Central

Wilson, R L; Stauffer, G V

1994-01-01

The gene encoding GcvA, the trans-acting regulatory protein for the Escherichia coli glycine cleavage enzyme system, has been sequenced. The gcvA locus contains an open reading frame of 930 nucleotides that could encode a protein with a molecular mass of 34.4 kDa, consistent with the results of minicell analysis indicating that GcvA is a polypeptide of approximately 33 kDa. The deduced amino acid sequence of GcvA revealed that this protein shares similarity with the LysR family of activator proteins. The transcription start site was found to be 72 bp upstream of the presumed translation start site. A chromosomal deletion of gcvA resulted in the inability of cells to activate the expression of a gcvT-lacZ gene fusion when grown in the presence of glycine and an inability to repress gcvT-lacZ expression when grown in the presence of inosine. The regulation of gcvA was examined by constructing a gcvA-lacZ gene fusion in which beta-galactosidase synthesis is under the control of the gcvA regulatory region. Although gcvA expression appears to be autogenously regulated over a two- to threefold range, it is neither induced by glycine nor repressed by inosine. Images PMID:8188587

Strategies for Protein Overproduction in Escherichia coli.

ERIC Educational Resources Information Center

Mott, John E.

1984-01-01

Examines heterologous expression in Escherichia coli and the role of regulatory sequences which control gene expression at transcription resulting in abundant production of messenger RNA and regulatory sequences in mRNA which promote efficient translation. Also examines the role of E. coli cells in stabilizing mRNA and protein that is…

Search for 5'-leader regulatory RNA structures based on gene annotation aided by the RiboGap database.

PubMed

Naghdi, Mohammad Reza; Smail, Katia; Wang, Joy X; Wade, Fallou; Breaker, Ronald R; Perreault, Jonathan

2017-03-15

The discovery of noncoding RNAs (ncRNAs) and their importance for gene regulation led us to develop bioinformatics tools to pursue the discovery of novel ncRNAs. Finding ncRNAs de novo is challenging, first due to the difficulty of retrieving large numbers of sequences for given gene activities, and second due to exponential demands on calculation needed for comparative genomics on a large scale. Recently, several tools for the prediction of conserved RNA secondary structure were developed, but many of them are not designed to uncover new ncRNAs, or are too slow for conducting analyses on a large scale. Here we present various approaches using the database RiboGap as a primary tool for finding known ncRNAs and for uncovering simple sequence motifs with regulatory roles. This database also can be used to easily extract intergenic sequences of eubacteria and archaea to find conserved RNA structures upstream of given genes. We also show how to extend analysis further to choose the best candidate ncRNAs for experimental validation. Copyright © 2017 Elsevier Inc. All rights reserved.

Developmentally distinct MYB genes encode functionally equivalent proteins in Arabidopsis.

PubMed

Lee, M M; Schiefelbein, J

2001-05-01

The duplication and divergence of developmental control genes is thought to have driven morphological diversification during the evolution of multicellular organisms. To examine the molecular basis of this process, we analyzed the functional relationship between two paralogous MYB transcription factor genes, WEREWOLF (WER) and GLABROUS1 (GL1), in Arabidopsis. The WER and GL1 genes specify distinct cell types and exhibit non-overlapping expression patterns during Arabidopsis development. Nevertheless, reciprocal complementation experiments with a series of gene fusions showed that WER and GL1 encode functionally equivalent proteins, and their unique roles in plant development are entirely due to differences in their cis-regulatory sequences. Similar experiments with a distantly related MYB gene (MYB2) showed that its product cannot functionally substitute for WER or GL1. Furthermore, an analysis of the WER and GL1 proteins shows that conserved sequences correspond to specific functional domains. These results provide new insights into the evolution of the MYB gene family in Arabidopsis, and, more generally, they demonstrate that novel developmental gene function may arise solely by the modification of cis-regulatory sequences.

T-Reg Comparator: an analysis tool for the comparison of position weight matrices

PubMed Central

Roepcke, Stefan; Grossmann, Steffen; Rahmann, Sven; Vingron, Martin

2005-01-01

T-Reg Comparator is a novel software tool designed to support research into transcriptional regulation. Sequence motifs representing transcription factor binding sites are usually encoded as position weight matrices. The user inputs a set of such weight matrices or binding site sequences and our program matches them against the T-Reg database, which is presently built on data from the Transfac [E. Wingender (2004) In Silico Biol., 4, 55–61] and Jaspar [A. Sandelin, W. Alkema, P. Engstrom, W. W. Wasserman and B. Lenhard (2004) Nucleic Acids Res., 32, D91–D94]. Our tool delivers a detailed report on similarities between user-supplied motifs and motifs in the database. Apart from simple one-to-one relationships, T-Reg Comparator is also able to detect similarities between submatrices. In addition, we provide a user interface to a program for sequence scanning with weight matrices. Typical areas of application for T-Reg Comparator are motif and regulatory module finding and annotation of regulatory genomic regions. T-Reg Comparator is available at . PMID:15980506

T-Reg Comparator: an analysis tool for the comparison of position weight matrices.

PubMed

Roepcke, Stefan; Grossmann, Steffen; Rahmann, Sven; Vingron, Martin

2005-07-01

T-Reg Comparator is a novel software tool designed to support research into transcriptional regulation. Sequence motifs representing transcription factor binding sites are usually encoded as position weight matrices. The user inputs a set of such weight matrices or binding site sequences and our program matches them against the T-Reg database, which is presently built on data from the Transfac [E. Wingender (2004) In Silico Biol., 4, 55-61] and Jaspar [A. Sandelin, W. Alkema, P. Engstrom, W. W. Wasserman and B. Lenhard (2004) Nucleic Acids Res., 32, D91-D94]. Our tool delivers a detailed report on similarities between user-supplied motifs and motifs in the database. Apart from simple one-to-one relationships, T-Reg Comparator is also able to detect similarities between submatrices. In addition, we provide a user interface to a program for sequence scanning with weight matrices. Typical areas of application for T-Reg Comparator are motif and regulatory module finding and annotation of regulatory genomic regions. T-Reg Comparator is available at http://treg.molgen.mpg.de.

A High-Resolution Enhancer Atlas of the Developing Telencephalon

PubMed Central

Visel, Axel; Taher, Leila; Girgis, Hani; May, Dalit; Golonzhka, Olga; Hoch, Renee; McKinsey, Gabriel L.; Pattabiraman, Kartik; Silberberg, Shanni N.; Blow, Matthew J.; Hansen, David V.; Nord, Alex S.; Akiyama, Jennifer A.; Holt, Amy; Hosseini, Roya; Phouanenavong, Sengthavy; Plajzer-Frick, Ingrid; Shoukry, Malak; Afzal, Veena; Kaplan, Tommy; Kriegstein, Arnold R.; Rubin, Edward M.; Ovcharenko, Ivan; Pennacchio, Len A.; Rubenstein, John L. R.

2013-01-01

Summary The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. While many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified over 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising over 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders. PMID:23375746

Rhodobase, a meta-analytical tool for reconstructing gene regulatory networks in a model photosynthetic bacterium.

PubMed

Moskvin, Oleg V; Bolotin, Dmitry; Wang, Andrew; Ivanov, Pavel S; Gomelsky, Mark

2011-02-01

We present Rhodobase, a web-based meta-analytical tool for analysis of transcriptional regulation in a model anoxygenic photosynthetic bacterium, Rhodobacter sphaeroides. The gene association meta-analysis is based on the pooled data from 100 of R. sphaeroides whole-genome DNA microarrays. Gene-centric regulatory networks were visualized using the StarNet approach (Jupiter, D.C., VanBuren, V., 2008. A visual data mining tool that facilitates reconstruction of transcription regulatory networks. PLoS ONE 3, e1717) with several modifications. We developed a means to identify and visualize operons and superoperons. We designed a framework for the cross-genome search for transcription factor binding sites that takes into account high GC-content and oligonucleotide usage profile characteristic of the R. sphaeroides genome. To facilitate reconstruction of directional relationships between co-regulated genes, we screened upstream sequences (-400 to +20bp from start codons) of all genes for putative binding sites of bacterial transcription factors using a self-optimizing search method developed here. To test performance of the meta-analysis tools and transcription factor site predictions, we reconstructed selected nodes of the R. sphaeroides transcription factor-centric regulatory matrix. The test revealed regulatory relationships that correlate well with the experimentally derived data. The database of transcriptional profile correlations, the network visualization engine and the optimized search engine for transcription factor binding sites analysis are available at http://rhodobase.org. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Integrated analysis of 454 and Illumina transcriptomic sequencing characterizes carbon flux and energy source for fatty acid synthesis in developing Lindera glauca fruits for woody biodiesel.

PubMed

Lin, Zixin; An, Jiyong; Wang, Jia; Niu, Jun; Ma, Chao; Wang, Libing; Yuan, Guanshen; Shi, Lingling; Liu, Lili; Zhang, Jinsong; Zhang, Zhixiang; Qi, Ji; Lin, Shanzhi

2017-01-01

Lindera glauca fruit with high quality and quantity of oil has emerged as a novel potential source of biodiesel in China, but the molecular regulatory mechanism of carbon flux and energy source for oil biosynthesis in developing fruits is still unknown. To better develop fruit oils of L. glauca as woody biodiesel, a combination of two different sequencing platforms (454 and Illumina) and qRT-PCR analysis was used to define a minimal reference transcriptome of developing L. glauca fruits, and to construct carbon and energy metabolic model for regulation of carbon partitioning and energy supply for FA biosynthesis and oil accumulation. We first analyzed the dynamic patterns of growth tendency, oil content, FA compositions, biodiesel properties, and the contents of ATP and pyridine nucleotide of L. glauca fruits from seven different developing stages. Comprehensive characterization of transcriptome of the developing L. glauca fruit was performed using a combination of two different next-generation sequencing platforms, of which three representative fruit samples (50, 125, and 150 DAF) and one mixed sample from seven developing stages were selected for Illumina and 454 sequencing, respectively. The unigenes separately obtained from long and short reads (201, and 259, respectively, in total) were reconciled using TGICL software, resulting in a total of 60,031 unigenes (mean length = 1061.95 bp) to describe a transcriptome for developing L. glauca fruits. Notably, 198 genes were annotated for photosynthesis, sucrose cleavage, carbon allocation, metabolite transport, acetyl-CoA formation, oil synthesis, and energy metabolism, among which some specific transporters, transcription factors, and enzymes were identified to be implicated in carbon partitioning and energy source for oil synthesis by an integrated analysis of transcriptomic sequencing and qRT-PCR. Importantly, the carbon and energy metabolic model was well established for oil biosynthesis of developing L. glauca fruits, which could help to reveal the molecular regulatory mechanism of the increased oil production in developing fruits. This study presents for the first time the application of an integrated two different sequencing analyses (Illumina and 454) and qRT-PCR detection to define a minimal reference transcriptome for developing L. glauca fruits, and to elucidate the molecular regulatory mechanism of carbon flux control and energy provision for oil synthesis. Our results will provide a valuable resource for future fundamental and applied research on the woody biodiesel plants.

A generic Transcriptomics Reporting Framework (TRF) for 'omics data processing and analysis.

PubMed

Gant, Timothy W; Sauer, Ursula G; Zhang, Shu-Dong; Chorley, Brian N; Hackermüller, Jörg; Perdichizzi, Stefania; Tollefsen, Knut E; van Ravenzwaay, Ben; Yauk, Carole; Tong, Weida; Poole, Alan

2017-12-01

A generic Transcriptomics Reporting Framework (TRF) is presented that lists parameters that should be reported in 'omics studies used in a regulatory context. The TRF encompasses the processes from transcriptome profiling from data generation to a processed list of differentially expressed genes (DEGs) ready for interpretation. Included within the TRF is a reference baseline analysis (RBA) that encompasses raw data selection; data normalisation; recognition of outliers; and statistical analysis. The TRF itself does not dictate the methodology for data processing, but deals with what should be reported. Its principles are also applicable to sequencing data and other 'omics. In contrast, the RBA specifies a simple data processing and analysis methodology that is designed to provide a comparison point for other approaches and is exemplified here by a case study. By providing transparency on the steps applied during 'omics data processing and analysis, the TRF will increase confidence processing of 'omics data, and regulatory use. Applicability of the TRF is ensured by its simplicity and generality. The TRF can be applied to all types of regulatory 'omics studies, and it can be executed using different commonly available software tools. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

Analysis of C. elegans VIG-1 expression.

PubMed

Shin, Kyoung-Hwa; Choi, Boram; Park, Yang-Seo; Cho, Nam Jeong

2008-12-31

Double-stranded RNA (dsRNA) induces gene silencing in a sequence-specific manner by a process known as RNA interference (RNAi). The RNA-induced silencing complex (RISC) is a multi-subunit ribonucleoprotein complex that plays a key role in RNAi. VIG (Vasa intronic gene) has been identified as a component of Drosophila RISC; however, the role VIG plays in regulating RNAi is poorly understood. Here, we examined the spatial and temporal expression patterns of VIG-1, the C. elegans ortholog of Drosophila VIG, using a vig-1::gfp fusion construct. This construct contains the 908-bp region immediately upstream of vig-1 gene translation initiation site. Analysis by confocal microscopy demonstrated GFP-VIG-1 expression in a number of tissues including the pharynx, body wall muscle, hypodermis, intestine, reproductive system, and nervous system at the larval and adult stages. Furthermore, western blot analysis showed that VIG-1 is present in each developmental stage examined. To investigate regulatory sequences for vig-1 gene expression, we generated constructs containing deletions in the upstream region. It was determined that the GFP expression pattern of a deletion construct (delta-908 to -597) was generally similar to that of the non-deletion construct. In contrast, removal of a larger segment (delta-908 to -191) resulted in the loss of GFP expression in most cell types. Collectively, these results indicate that the 406-bp upstream region (-596 to -191) contains essential regulatory sequences required for VIG-1 expression.

RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay.

PubMed

Dean, Kimberly M; Grayhack, Elizabeth J

2012-12-01

We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.

Mutations in the PP2A regulatory subunit B family genes PPP2R5B, PPP2R5C and PPP2R5D cause human overgrowth.

PubMed

Loveday, Chey; Tatton-Brown, Katrina; Clarke, Matthew; Westwood, Isaac; Renwick, Anthony; Ramsay, Emma; Nemeth, Andrea; Campbell, Jennifer; Joss, Shelagh; Gardner, McKinlay; Zachariou, Anna; Elliott, Anna; Ruark, Elise; van Montfort, Rob; Rahman, Nazneen

2015-09-01

Overgrowth syndromes comprise a group of heterogeneous disorders characterised by excessive growth parameters, often in association with intellectual disability. To identify new causes of human overgrowth, we have been undertaking trio-based exome sequencing studies in overgrowth patients and their unaffected parents. Prioritisation of functionally relevant genes with multiple unique de novo mutations revealed four mutations in protein phosphatase 2A (PP2A) regulatory subunit B family genes protein phosphatase 2, regulatory Subunit B', beta (PPP2R5B); protein phosphatase 2, regulatory Subunit B', gamma (PPP2R5C); and protein phosphatase 2, regulatory Subunit B', delta (PPP2R5D). This observation in 3 related genes in 111 individuals with a similar phenotype is greatly in excess of the expected number, as determined from gene-specific de novo mutation rates (P = 1.43 × 10(-10)). Analysis of exome-sequencing data from a follow-up series of overgrowth probands identified a further pathogenic mutation, bringing the total number of affected individuals to 5. Heterozygotes shared similar phenotypic features including increased height, increased head circumference and intellectual disability. The mutations clustered within a region of nine amino acid residues in the aligned protein sequences (P = 1.6 × 10(-5)). We mapped the mutations onto the crystal structure of the PP2A holoenzyme complex to predict their molecular and functional consequences. These studies suggest that the mutations may affect substrate binding, thus perturbing the ability of PP2A to dephosphorylate particular protein substrates. PP2A is a major negative regulator of v-akt murine thymoma viral oncogene homolog 1 (AKT). Thus, our data further expand the list of genes encoding components of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signalling cascade that are disrupted in human overgrowth conditions. © The Author 2015. Published by Oxford University Press.

Computational Approaches to Identify Promoters and cis-Regulatory Elements in Plant Genomes1

PubMed Central

Rombauts, Stephane; Florquin, Kobe; Lescot, Magali; Marchal, Kathleen; Rouzé, Pierre; Van de Peer, Yves

2003-01-01

The identification of promoters and their regulatory elements is one of the major challenges in bioinformatics and integrates comparative, structural, and functional genomics. Many different approaches have been developed to detect conserved motifs in a set of genes that are either coregulated or orthologous. However, although recent approaches seem promising, in general, unambiguous identification of regulatory elements is not straightforward. The delineation of promoters is even harder, due to its complex nature, and in silico promoter prediction is still in its infancy. Here, we review the different approaches that have been developed for identifying promoters and their regulatory elements. We discuss the detection of cis-acting regulatory elements using word-counting or probabilistic methods (so-called “search by signal” methods) and the delineation of promoters by considering both sequence content and structural features (“search by content” methods). As an example of search by content, we explored in greater detail the association of promoters with CpG islands. However, due to differences in sequence content, the parameters used to detect CpG islands in humans and other vertebrates cannot be used for plants. Therefore, a preliminary attempt was made to define parameters that could possibly define CpG and CpNpG islands in Arabidopsis, by exploring the compositional landscape around the transcriptional start site. To this end, a data set of more than 5,000 gene sequences was built, including the promoter region, the 5′-untranslated region, and the first introns and coding exons. Preliminary analysis shows that promoter location based on the detection of potential CpG/CpNpG islands in the Arabidopsis genome is not straightforward. Nevertheless, because the landscape of CpG/CpNpG islands differs considerably between promoters and introns on the one side and exons (whether coding or not) on the other, more sophisticated approaches can probably be developed for the successful detection of “putative” CpG and CpNpG islands in plants. PMID:12857799

An integrative and applicable phylogenetic footprinting framework for cis-regulatory motifs identification in prokaryotic genomes.

PubMed

Liu, Bingqiang; Zhang, Hanyuan; Zhou, Chuan; Li, Guojun; Fennell, Anne; Wang, Guanghui; Kang, Yu; Liu, Qi; Ma, Qin

2016-08-09

Phylogenetic footprinting is an important computational technique for identifying cis-regulatory motifs in orthologous regulatory regions from multiple genomes, as motifs tend to evolve slower than their surrounding non-functional sequences. Its application, however, has several difficulties for optimizing the selection of orthologous data and reducing the false positives in motif prediction. Here we present an integrative phylogenetic footprinting framework for accurate motif predictions in prokaryotic genomes (MP(3)). The framework includes a new orthologous data preparation procedure, an additional promoter scoring and pruning method and an integration of six existing motif finding algorithms as basic motif search engines. Specifically, we collected orthologous genes from available prokaryotic genomes and built the orthologous regulatory regions based on sequence similarity of promoter regions. This procedure made full use of the large-scale genomic data and taxonomy information and filtered out the promoters with limited contribution to produce a high quality orthologous promoter set. The promoter scoring and pruning is implemented through motif voting by a set of complementary predicting tools that mine as many motif candidates as possible and simultaneously eliminate the effect of random noise. We have applied the framework to Escherichia coli k12 genome and evaluated the prediction performance through comparison with seven existing programs. This evaluation was systematically carried out at the nucleotide and binding site level, and the results showed that MP(3) consistently outperformed other popular motif finding tools. We have integrated MP(3) into our motif identification and analysis server DMINDA, allowing users to efficiently identify and analyze motifs in 2,072 completely sequenced prokaryotic genomes. The performance evaluation indicated that MP(3) is effective for predicting regulatory motifs in prokaryotic genomes. Its application may enhance progress in elucidating transcription regulation mechanism, thus provide benefit to the genomic research community and prokaryotic genome researchers in particular.

Direct activation of a notochord cis-regulatory module by Brachyury and FoxA in the ascidian Ciona intestinalis.

PubMed

Passamaneck, Yale J; Katikala, Lavanya; Perrone, Lorena; Dunn, Matthew P; Oda-Ishii, Izumi; Di Gregorio, Anna

2009-11-01

The notochord is a defining feature of the chordate body plan. Experiments in ascidian, frog and mouse embryos have shown that co-expression of Brachyury and FoxA class transcription factors is required for notochord development. However, studies on the cis-regulatory sequences mediating the synergistic effects of these transcription factors are complicated by the limited knowledge of notochord genes and cis-regulatory modules (CRMs) that are directly targeted by both. We have identified an easily testable model for such investigations in a 155-bp notochord-specific CRM from the ascidian Ciona intestinalis. This CRM contains functional binding sites for both Ciona Brachyury (Ci-Bra) and FoxA (Ci-FoxA-a). By combining point mutation analysis and misexpression experiments, we demonstrate that binding of both transcription factors to this CRM is necessary and sufficient to activate transcription. To gain insights into the cis-regulatory criteria controlling its activity, we investigated the organization of the transcription factor binding sites within the 155-bp CRM. The 155-bp sequence contains two Ci-Bra binding sites with identical core sequences but opposite orientations, only one of which is required for enhancer activity. Changes in both orientation and spacing of these sites substantially affect the activity of the CRM, as clusters of identical sites found in the Ciona genome with different arrangements are unable to activate transcription in notochord cells. This work presents the first evidence of a synergistic interaction between Brachyury and FoxA in the activation of an individual notochord CRM, and highlights the importance of transcription factor binding site arrangement for its function.

First Pass Annotation of Promoters on Human Chromosome 22

PubMed Central

Scherf, Matthias; Klingenhoff, Andreas; Frech, Kornelie; Quandt, Kerstin; Schneider, Ralf; Grote, Korbinian; Frisch, Matthias; Gailus-Durner, Valérie; Seidel, Alexander; Brack-Werner, Ruth; Werner, Thomas

2001-01-01

The publication of the first almost complete sequence of a human chromosome (chromosome 22) is a major milestone in human genomics. Together with the sequence, an excellent annotation of genes was published which certainly will serve as an information resource for numerous future projects. We noted that the annotation did not cover regulatory regions; in particular, no promoter annotation has been provided. Here we present an analysis of the complete published chromosome 22 sequence for promoters. A recent breakthrough in specific in silico prediction of promoter regions enabled us to attempt large-scale prediction of promoter regions on chromosome 22. Scanning of sequence databases revealed only 20 experimentally verified promoters, of which 10 were correctly predicted by our approach. Nearly 40% of our 465 predicted promoter regions are supported by the currently available gene annotation. Promoter finding also provides a biologically meaningful method for “chromosomal scaffolding”, by which long genomic sequences can be divided into segments starting with a gene. As one example, the combination of promoter region prediction with exon/intron structure predictions greatly enhances the specificity of de novo gene finding. The present study demonstrates that it is possible to identify promoters in silico on the chromosomal level with sufficient reliability for experimental planning and indicates that a wealth of information about regulatory regions can be extracted from current large-scale (megabase) sequencing projects. Results are available on-line at http://genomatix.gsf.de/chr22/. PMID:11230158

Phylogenetic shadowing of primate sequences to find functional regions of the human genome.

PubMed

Boffelli, Dario; McAuliffe, Jon; Ovcharenko, Dmitriy; Lewis, Keith D; Ovcharenko, Ivan; Pachter, Lior; Rubin, Edward M

2003-02-28

Nonhuman primates represent the most relevant model organisms to understand the biology of Homo sapiens. The recent divergence and associated overall sequence conservation between individual members of this taxon have nonetheless largely precluded the use of primates in comparative sequence studies. We used sequence comparisons of an extensive set of Old World and New World monkeys and hominoids to identify functional regions in the human genome. Analysis of these data enabled the discovery of primate-specific gene regulatory elements and the demarcation of the exons of multiple genes. Much of the information content of the comprehensive primate sequence comparisons could be captured with a small subset of phylogenetically close primates. These results demonstrate the utility of intraprimate sequence comparisons to discover common mammalian as well as primate-specific functional elements in the human genome, which are unattainable through the evaluation of more evolutionarily distant species.

Effective Feature Selection for Classification of Promoter Sequences.

PubMed

K, Kouser; P G, Lavanya; Rangarajan, Lalitha; K, Acharya Kshitish

2016-01-01

Exploring novel computational methods in making sense of biological data has not only been a necessity, but also productive. A part of this trend is the search for more efficient in silico methods/tools for analysis of promoters, which are parts of DNA sequences that are involved in regulation of expression of genes into other functional molecules. Promoter regions vary greatly in their function based on the sequence of nucleotides and the arrangement of protein-binding short-regions called motifs. In fact, the regulatory nature of the promoters seems to be largely driven by the selective presence and/or the arrangement of these motifs. Here, we explore computational classification of promoter sequences based on the pattern of motif distributions, as such classification can pave a new way of functional analysis of promoters and to discover the functionally crucial motifs. We make use of Position Specific Motif Matrix (PSMM) features for exploring the possibility of accurately classifying promoter sequences using some of the popular classification techniques. The classification results on the complete feature set are low, perhaps due to the huge number of features. We propose two ways of reducing features. Our test results show improvement in the classification output after the reduction of features. The results also show that decision trees outperform SVM (Support Vector Machine), KNN (K Nearest Neighbor) and ensemble classifier LibD3C, particularly with reduced features. The proposed feature selection methods outperform some of the popular feature transformation methods such as PCA and SVD. Also, the methods proposed are as accurate as MRMR (feature selection method) but much faster than MRMR. Such methods could be useful to categorize new promoters and explore regulatory mechanisms of gene expressions in complex eukaryotic species.

PreCisIon: PREdiction of CIS-regulatory elements improved by gene's positION.

PubMed

Elati, Mohamed; Nicolle, Rémy; Junier, Ivan; Fernández, David; Fekih, Rim; Font, Julio; Képès, François

2013-02-01

Conventional approaches to predict transcriptional regulatory interactions usually rely on the definition of a shared motif sequence on the target genes of a transcription factor (TF). These efforts have been frustrated by the limited availability and accuracy of TF binding site motifs, usually represented as position-specific scoring matrices, which may match large numbers of sites and produce an unreliable list of target genes. To improve the prediction of binding sites, we propose to additionally use the unrelated knowledge of the genome layout. Indeed, it has been shown that co-regulated genes tend to be either neighbors or periodically spaced along the whole chromosome. This study demonstrates that respective gene positioning carries significant information. This novel type of information is combined with traditional sequence information by a machine learning algorithm called PreCisIon. To optimize this combination, PreCisIon builds a strong gene target classifier by adaptively combining weak classifiers based on either local binding sequence or global gene position. This strategy generically paves the way to the optimized incorporation of any future advances in gene target prediction based on local sequence, genome layout or on novel criteria. With the current state of the art, PreCisIon consistently improves methods based on sequence information only. This is shown by implementing a cross-validation analysis of the 20 major TFs from two phylogenetically remote model organisms. For Bacillus subtilis and Escherichia coli, respectively, PreCisIon achieves on average an area under the receiver operating characteristic curve of 70 and 60%, a sensitivity of 80 and 70% and a specificity of 60 and 56%. The newly predicted gene targets are demonstrated to be functionally consistent with previously known targets, as assessed by analysis of Gene Ontology enrichment or of the relevant literature and databases.

[The ENCODE project and functional genomics studies].

PubMed

Ding, Nan; Qu, Hongzhu; Fang, Xiangdong

2014-03-01

Upon the completion of the Human Genome Project, scientists have been trying to interpret the underlying genomic code for human biology. Since 2003, National Human Genome Research Institute (NHGRI) has invested nearly $0.3 billion and gathered over 440 scientists from more than 32 institutions in the United States, China, United Kingdom, Japan, Spain and Singapore to initiate the Encyclopedia of DNA Elements (ENCODE) project, aiming to identify and analyze all regulatory elements in the human genome. Taking advantage of the development of next-generation sequencing technologies and continuous improvement of experimental methods, ENCODE had made remarkable achievements: identified methylation and histone modification of DNA sequences and their regulatory effects on gene expression through altering chromatin structures, categorized binding sites of various transcription factors and constructed their regulatory networks, further revised and updated database for pseudogenes and non-coding RNA, and identified SNPs in regulatory sequences associated with diseases. These findings help to comprehensively understand information embedded in gene and genome sequences, the function of regulatory elements as well as the molecular mechanism underlying the transcriptional regulation by noncoding regions, and provide extensive data resource for life sciences, particularly for translational medicine. We re-viewed the contributions of high-throughput sequencing platform development and bioinformatical technology improve-ment to the ENCODE project, the association between epigenetics studies and the ENCODE project, and the major achievement of the ENCODE project. We also provided our prospective on the role of the ENCODE project in promoting the development of basic and clinical medicine.

Inverted repeats in the promoter as an autoregulatory sequence for TcrX in Mycobacterium tuberculosis

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

Bhattacharya, Monolekha; Das, Amit Kumar, E-mail: amitk@hijli.iitkgp.ernet.in

Highlights: Black-Right-Pointing-Pointer The regulatory sequences recognized by TcrX have been identified. Black-Right-Pointing-Pointer The regulatory region comprises of inverted repeats segregated by 30 bp region. Black-Right-Pointing-Pointer The mode of binding of TcrX with regulatory sequence is unique. Black-Right-Pointing-Pointer In silico TcrX-DNA docked model binds one of the inverted repeats. Black-Right-Pointing-Pointer Both phosphorylated and unphosphorylated TcrX binds regulatory sequence in vitro. -- Abstract: TcrY, a histidine kinase, and TcrX, a response regulator, constitute a two-component system in Mycobacterium tuberculosis. tcrX, which is expressed during iron scarcity, is instrumental in the survival of iron-dependent M. tuberculosis. However, the regulator of tcrX/Y has notmore » been fully characterized. Crosslinking studies of TcrX reveal that it can form oligomers in vitro. Electrophoretic mobility shift assays (EMSAs) show that TcrX recognizes two regions in the promoter that are comprised of inverted repeats separated by {approx}30 bp. The dimeric in silico model of TcrX predicts binding to one of these inverted repeat regions. Site-directed mutagenesis and radioactive phosphorylation indicate that D54 of TcrX is phosphorylated by H256 of TcrY. However, phosphorylated and unphosphorylated TcrX bind the regulatory sequence with equal efficiency, which was shown with an EMSA using the D54A TcrX mutant.« less

SPAR: small RNA-seq portal for analysis of sequencing experiments.

PubMed

Kuksa, Pavel P; Amlie-Wolf, Alexandre; Katanic, Živadin; Valladares, Otto; Wang, Li-San; Leung, Yuk Yee

2018-05-04

The introduction of new high-throughput small RNA sequencing protocols that generate large-scale genomics datasets along with increasing evidence of the significant regulatory roles of small non-coding RNAs (sncRNAs) have highlighted the urgent need for tools to analyze and interpret large amounts of small RNA sequencing data. However, it remains challenging to systematically and comprehensively discover and characterize sncRNA genes and specifically-processed sncRNA products from these datasets. To fill this gap, we present Small RNA-seq Portal for Analysis of sequencing expeRiments (SPAR), a user-friendly web server for interactive processing, analysis, annotation and visualization of small RNA sequencing data. SPAR supports sequencing data generated from various experimental protocols, including smRNA-seq, short total RNA sequencing, microRNA-seq, and single-cell small RNA-seq. Additionally, SPAR includes publicly available reference sncRNA datasets from our DASHR database and from ENCODE across 185 human tissues and cell types to produce highly informative small RNA annotations across all major small RNA types and other features such as co-localization with various genomic features, precursor transcript cleavage patterns, and conservation. SPAR allows the user to compare the input experiment against reference ENCODE/DASHR datasets. SPAR currently supports analyses of human (hg19, hg38) and mouse (mm10) sequencing data. SPAR is freely available at https://www.lisanwanglab.org/SPAR.

Massively parallel cis-regulatory analysis in the mammalian central nervous system

PubMed Central

Shen, Susan Q.; Myers, Connie A.; Hughes, Andrew E.O.; Byrne, Leah C.; Flannery, John G.; Corbo, Joseph C.

2016-01-01

Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell–derived organoids. PMID:26576614

CD4+ virtual memory: Antigen-inexperienced T cells reside in the naïve, regulatory, and memory T cell compartments at similar frequencies, implications for autoimmunity.

PubMed

Marusina, Alina I; Ono, Yoko; Merleev, Alexander A; Shimoda, Michiko; Ogawa, Hiromi; Wang, Elizabeth A; Kondo, Kayo; Olney, Laura; Luxardi, Guillaume; Miyamura, Yoshinori; Yilma, Tilahun D; Villalobos, Itzel Bustos; Bergstrom, Jennifer W; Kronenberg, Daniel G; Soulika, Athena M; Adamopoulos, Iannis E; Maverakis, Emanual

2017-02-01

It is widely accepted that central and effector memory CD4 + T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4 + T cell repertoire analysis of highly purified T cell populations from naive animals revealed that the HEL-specific clones displayed effector and central "memory" cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4 + T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4 + T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4 + T cell maturation in which a specific clone can undergo a differentiation process to exhibit a "memory" or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4 + T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones. Published by Elsevier Ltd.

A primer on thermodynamic-based models for deciphering transcriptional regulatory logic.

PubMed

Dresch, Jacqueline M; Richards, Megan; Ay, Ahmet

2013-09-01

A rigorous analysis of transcriptional regulation at the DNA level is crucial to the understanding of many biological systems. Mathematical modeling has offered researchers a new approach to understanding this central process. In particular, thermodynamic-based modeling represents the most biophysically informed approach aimed at connecting DNA level regulatory sequences to the expression of specific genes. The goal of this review is to give biologists a thorough description of the steps involved in building, analyzing, and implementing a thermodynamic-based model of transcriptional regulation. The data requirements for this modeling approach are described, the derivation for a specific regulatory region is shown, and the challenges and future directions for the quantitative modeling of gene regulation are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Sasquatch: predicting the impact of regulatory SNPs on transcription factor binding from cell- and tissue-specific DNase footprints.

PubMed

Schwessinger, Ron; Suciu, Maria C; McGowan, Simon J; Telenius, Jelena; Taylor, Stephen; Higgs, Doug R; Hughes, Jim R

2017-10-01

In the era of genome-wide association studies (GWAS) and personalized medicine, predicting the impact of single nucleotide polymorphisms (SNPs) in regulatory elements is an important goal. Current approaches to determine the potential of regulatory SNPs depend on inadequate knowledge of cell-specific DNA binding motifs. Here, we present Sasquatch, a new computational approach that uses DNase footprint data to estimate and visualize the effects of noncoding variants on transcription factor binding. Sasquatch performs a comprehensive k -mer-based analysis of DNase footprints to determine any k -mer's potential for protein binding in a specific cell type and how this may be changed by sequence variants. Therefore, Sasquatch uses an unbiased approach, independent of known transcription factor binding sites and motifs. Sasquatch only requires a single DNase-seq data set per cell type, from any genotype, and produces consistent predictions from data generated by different experimental procedures and at different sequence depths. Here we demonstrate the effectiveness of Sasquatch using previously validated functional SNPs and benchmark its performance against existing approaches. Sasquatch is available as a versatile webtool incorporating publicly available data, including the human ENCODE collection. Thus, Sasquatch provides a powerful tool and repository for prioritizing likely regulatory SNPs in the noncoding genome. © 2017 Schwessinger et al.; Published by Cold Spring Harbor Laboratory Press.

Parallel evolution of chordate cis-regulatory code for development.

PubMed

Doglio, Laura; Goode, Debbie K; Pelleri, Maria C; Pauls, Stefan; Frabetti, Flavia; Shimeld, Sebastian M; Vavouri, Tanya; Elgar, Greg

2013-11-01

Urochordates are the closest relatives of vertebrates and at the larval stage, possess a characteristic bilateral chordate body plan. In vertebrates, the genes that orchestrate embryonic patterning are in part regulated by highly conserved non-coding elements (CNEs), yet these elements have not been identified in urochordate genomes. Consequently the evolution of the cis-regulatory code for urochordate development remains largely uncharacterised. Here, we use genome-wide comparisons between C. intestinalis and C. savignyi to identify putative urochordate cis-regulatory sequences. Ciona conserved non-coding elements (ciCNEs) are associated with largely the same key regulatory genes as vertebrate CNEs. Furthermore, some of the tested ciCNEs are able to activate reporter gene expression in both zebrafish and Ciona embryos, in a pattern that at least partially overlaps that of the gene they associate with, despite the absence of sequence identity. We also show that the ability of a ciCNE to up-regulate gene expression in vertebrate embryos can in some cases be localised to short sub-sequences, suggesting that functional cross-talk may be defined by small regions of ancestral regulatory logic, although functional sub-sequences may also be dispersed across the whole element. We conclude that the structure and organisation of cis-regulatory modules is very different between vertebrates and urochordates, reflecting their separate evolutionary histories. However, functional cross-talk still exists because the same repertoire of transcription factors has likely guided their parallel evolution, exploiting similar sets of binding sites but in different combinations.

Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation

PubMed Central

Hall, Megan P.; Nagel, Roland J.; Fagg, W. Samuel; Shiue, Lily; Cline, Melissa S.; Perriman, Rhonda J.; Donohue, John Paul; Ares, Manuel

2013-01-01

Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA (“STAR” motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3′ UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation. PMID:23525800

De Novo Assembly and Characterization of the Transcriptome of the Chinese Medicinal Herb, Gentiana rigescens

PubMed Central

Zhang, Xiaodong; Allan, Andrew C.; Li, Caixia; Wang, Yuanzhong; Yao, Qiuyang

2015-01-01

Gentiana rigescens is an important medicinal herb in China. The main validated medicinal component gentiopicroside is synthesized in shoots, but is mainly found in the plant’s roots. The gentiopicroside biosynthetic pathway and its regulatory control remain to be elucidated. Genome resources of gentian are limited. Next-generation sequencing (NGS) technologies can aid in supplying global gene expression profiles. In this study we present sequence and transcript abundance data for the root and leaf transcriptome of G. rigescens, obtained using the Illumina Hiseq2000. Over fifty million clean reads were obtained from leaf and root libraries. This yields 76,717 unigenes with an average length of 753 bp. Among these, 33,855 unigenes were identified as putative homologs of annotated sequences in public protein and nucleotide databases. Digital abundance analysis identified 3306 unigenes differentially enriched between leaf and root. Unigenes found in both tissues were categorized according to their putative functional categories. Of the differentially expressed genes, over 130 were annotated as related to terpenoid biosynthesis. This work is the first study of global transcriptome analyses in gentian. These sequences and putative functional data comprise a resource for future investigation of terpenoid biosynthesis in Gentianaceae species and annotation of the gentiopicroside biosynthetic pathway and its regulatory mechanisms. PMID:26006235

CoryneRegNet: an ontology-based data warehouse of corynebacterial transcription factors and regulatory networks.

PubMed

Baumbach, Jan; Brinkrolf, Karina; Czaja, Lisa F; Rahmann, Sven; Tauch, Andreas

2006-02-14

The application of DNA microarray technology in post-genomic analysis of bacterial genome sequences has allowed the generation of huge amounts of data related to regulatory networks. This data along with literature-derived knowledge on regulation of gene expression has opened the way for genome-wide reconstruction of transcriptional regulatory networks. These large-scale reconstructions can be converted into in silico models of bacterial cells that allow a systematic analysis of network behavior in response to changing environmental conditions. CoryneRegNet was designed to facilitate the genome-wide reconstruction of transcriptional regulatory networks of corynebacteria relevant in biotechnology and human medicine. During the import and integration process of data derived from experimental studies or literature knowledge CoryneRegNet generates links to genome annotations, to identified transcription factors and to the corresponding cis-regulatory elements. CoryneRegNet is based on a multi-layered, hierarchical and modular concept of transcriptional regulation and was implemented by using the relational database management system MySQL and an ontology-based data structure. Reconstructed regulatory networks can be visualized by using the yFiles JAVA graph library. As an application example of CoryneRegNet, we have reconstructed the global transcriptional regulation of a cellular module involved in SOS and stress response of corynebacteria. CoryneRegNet is an ontology-based data warehouse that allows a pertinent data management of regulatory interactions along with the genome-scale reconstruction of transcriptional regulatory networks. These models can further be combined with metabolic networks to build integrated models of cellular function including both metabolism and its transcriptional regulation.

Genome-wide analysis of regulatory proteases sequences identified through bioinformatics data mining in Taenia solium.

PubMed

Yan, Hong-Bin; Lou, Zhong-Zi; Li, Li; Brindley, Paul J; Zheng, Yadong; Luo, Xuenong; Hou, Junling; Guo, Aijiang; Jia, Wan-Zhong; Cai, Xuepeng

2014-06-04

Cysticercosis remains a major neglected tropical disease of humanity in many regions, especially in sub-Saharan Africa, Central America and elsewhere. Owing to the emerging drug resistance and the inability of current drugs to prevent re-infection, identification of novel vaccines and chemotherapeutic agents against Taenia solium and related helminth pathogens is a public health priority. The T. solium genome and the predicted proteome were reported recently, providing a wealth of information from which new interventional targets might be identified. In order to characterize and classify the entire repertoire of protease-encoding genes of T. solium, which act fundamental biological roles in all life processes, we analyzed the predicted proteins of this cestode through a combination of bioinformatics tools. Functional annotation was performed to yield insights into the signaling processes relevant to the complex developmental cycle of this tapeworm and to highlight a suite of the proteases as potential intervention targets. Within the genome of this helminth parasite, we identified 200 open reading frames encoding proteases from five clans, which correspond to 1.68% of the 11,902 protein-encoding genes predicted to be present in its genome. These proteases include calpains, cytosolic, mitochondrial signal peptidases, ubiquitylation related proteins, and others. Many not only show significant similarity to proteases in the Conserved Domain Database but have conserved active sites and catalytic domains. KEGG Automatic Annotation Server (KAAS) analysis indicated that ~60% of these proteases share strong sequence identities with proteins of the KEGG database, which are involved in human disease, metabolic pathways, genetic information processes, cellular processes, environmental information processes and organismal systems. Also, we identified signal peptides and transmembrane helices through comparative analysis with classes of important regulatory proteases. Phylogenetic analysis using Bayes approach provided support for inferring functional divergence among regulatory cysteine and serine proteases. Numerous putative proteases were identified for the first time in T. solium, and important regulatory proteases have been predicted. This comprehensive analysis not only complements the growing knowledge base of proteolytic enzymes, but also provides a platform from which to expand knowledge of cestode proteases and to explore their biochemistry and potential as intervention targets.

Identification and comparative analysis of differential gene expression in soybean leaf tissue under drought and flooding stress revealed by RNA-Seq

USDA-ARS?s Scientific Manuscript database

Soybean is the second largest crop in the US. Its yield directly impacts US agricultural economics. Drought and flooding are two major causes for soybean yield loss. To better understand their underlying molecular regulatory mechanisms, we sequenced the transcriptomes of soybean grown in drought a...

Expression analysis of genes associated with sucrose accumulation in sugarcane (Saccharum spp. hybrids) varieties differing in content and time of peak sucrose storage

USDA-ARS?s Scientific Manuscript database

Sucrose synthesis/accumulation in sugarcane is a complex process involving many genes and regulatory sequences that control biochemical events in source-sink tissues. Among these, sucrose synthase (SuSy), sucrose-phosphate synthase (SPS), soluble acid (SAI) and cell-wall invertase (CWI) are importan...

Cis-regulatory landscapes of four cell types of the retina.

PubMed

Hartl, Dominik; Krebs, Arnaud R; Jüttner, Josephine; Roska, Botond; Schübeler, Dirk

2017-11-16

The retina is composed of ∼50 cell-types with specific functions for the process of vision. Identification of the cis-regulatory elements active in retinal cell-types is key to elucidate the networks controlling this diversity. Here, we combined transcriptome and epigenome profiling to map the regulatory landscape of four cell-types isolated from mouse retinas including rod and cone photoreceptors as well as rare inter-neuron populations such as horizontal and starburst amacrine cells. Integration of this information reveals sequence determinants and candidate transcription factors for controlling cellular specialization. Additionally, we refined parallel reporter assays to enable studying the transcriptional activity of large collection of sequences in individual cell-types isolated from a tissue. We provide proof of concept for this approach and its scalability by characterizing the transcriptional capacity of several hundred putative regulatory sequences within individual retinal cell-types. This generates a catalogue of cis-regulatory regions active in retinal cell types and we further demonstrate their utility as potential resource for cellular tagging and manipulation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Functional conservation of a forebrain enhancer from the elephant shark (Callorhinchus milii ) in zebrafish and mice.

PubMed

MacDonald, Ryan B; Debiais-Thibaud, Mélanie; Martin, Kyle; Poitras, Luc; Tay, Boon-Hui; Venkatesh, Byrappa; Ekker, Marc

2010-05-26

The phylogenetic position of the elephant shark (Callorhinchus milii ) is particularly relevant to study the evolution of genes and gene regulation in vertebrates. Here we examine the evolution of Dlx homeobox gene regulation during vertebrate embryonic development with a particular focus on the forebrain. We first identified the elephant shark sequence orthologous to the URE2 cis -regulatory element of the mouse Dlx1/Dlx2 locus (herein named CmURE2). We then conducted a comparative study of the sequence and enhancer activity of CmURE2 with that of orthologous regulatory sequences from zebrafish and mouse. The CmURE2 sequence shows a high percentage of identity with its mouse and zebrafish counterparts but is overall more similar to mouse URE2 (MmURE2) than to zebrafish URE2 (DrURE2). In transgenic zebrafish and mouse embryos, CmURE2 displayed enhancer activity in the forebrain that overlapped with that of DrURE2 and MmURE2. However, we detected notable differences in the activity of the three sequences in the diencephalon. Outside of the forebrain, CmURE2 shows enhancer activity in areas such as the pharyngeal arches and dorsal root ganglia where its' counterparts are also active. Our transgenic assays show that part of the URE2 enhancer activity is conserved throughout jawed vertebrates but also that new characteristics have evolved in the different groups. Our study demonstrates that the elephant shark is a useful outgroup to study the evolution of regulatory mechanisms in vertebrates and to address how changes in the sequence of cis -regulatory elements translate into changes in their regulatory activity.

The technology and biology of single-cell RNA sequencing.

PubMed

Kolodziejczyk, Aleksandra A; Kim, Jong Kyoung; Svensson, Valentine; Marioni, John C; Teichmann, Sarah A

2015-05-21

The differences between individual cells can have profound functional consequences, in both unicellular and multicellular organisms. Recently developed single-cell mRNA-sequencing methods enable unbiased, high-throughput, and high-resolution transcriptomic analysis of individual cells. This provides an additional dimension to transcriptomic information relative to traditional methods that profile bulk populations of cells. Already, single-cell RNA-sequencing methods have revealed new biology in terms of the composition of tissues, the dynamics of transcription, and the regulatory relationships between genes. Rapid technological developments at the level of cell capture, phenotyping, molecular biology, and bioinformatics promise an exciting future with numerous biological and medical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

The transcriptional terminator sequences downstream of the covR gene terminate covR/S operon transcription to generate covR monocistronic transcripts in Streptococcus pyogenes.

PubMed

Chiang-Ni, Chuan; Tsou, Chih-Cheng; Lin, Yee-Shin; Chuang, Woei-Jer; Lin, Ming-T; Liu, Ching-Chuan; Wu, Jiunn-Jong

2008-12-31

CovR/S is an important two component regulatory system, which regulates about 15% of the gene expression in Streptococcus pyogenes. The covR/S locus was identified as an operon generating an RNA transcript around 2.5-kb in size. In this study, we found the covR/S operon produced three RNA transcripts (around 2.5-, 1.0-, and 0.8-kb in size). Using RNA transcriptional terminator sequence prediction and transcriptional terminator analysis, we identified two atypical rho-independent terminator sequences downstream of the covR gene and showed these terminator sequences terminate RNA transcription efficiently. These results indicate that covR/S operon generates covR/S transcript and monocistronic covR transcripts.

A prior-based integrative framework for functional transcriptional regulatory network inference

PubMed Central

Siahpirani, Alireza F.

2017-01-01

Abstract Transcriptional regulatory networks specify regulatory proteins controlling the context-specific expression levels of genes. Inference of genome-wide regulatory networks is central to understanding gene regulation, but remains an open challenge. Expression-based network inference is among the most popular methods to infer regulatory networks, however, networks inferred from such methods have low overlap with experimentally derived (e.g. ChIP-chip and transcription factor (TF) knockouts) networks. Currently we have a limited understanding of this discrepancy. To address this gap, we first develop a regulatory network inference algorithm, based on probabilistic graphical models, to integrate expression with auxiliary datasets supporting a regulatory edge. Second, we comprehensively analyze our and other state-of-the-art methods on different expression perturbation datasets. Networks inferred by integrating sequence-specific motifs with expression have substantially greater agreement with experimentally derived networks, while remaining more predictive of expression than motif-based networks. Our analysis suggests natural genetic variation as the most informative perturbation for network inference, and, identifies core TFs whose targets are predictable from expression. Multiple reasons make the identification of targets of other TFs difficult, including network architecture and insufficient variation of TF mRNA level. Finally, we demonstrate the utility of our inference algorithm to infer stress-specific regulatory networks and for regulator prioritization. PMID:27794550

A survey of the sorghum transcriptome using single-molecule long reads

DOE PAGES

Abdel-Ghany, Salah E.; Hamilton, Michael; Jacobi, Jennifer L.; ...

2016-06-24

Alternative splicing and alternative polyadenylation (APA) of pre-mRNAs greatly contribute to transcriptome diversity, coding capacity of a genome and gene regulatory mechanisms in eukaryotes. Second-generation sequencing technologies have been extensively used to analyse transcriptomes. However, a major limitation of short-read data is that it is difficult to accurately predict full-length splice isoforms. Here we sequenced the sorghum transcriptome using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and APA sites. Our analysis reveals transcriptome-wide full-length isoforms at an unprecedented scale with over 11,000 novelmore » splice isoforms. Additionally, we uncover APA ofB11,000 expressed genes and more than 2,100 novel genes. Lastly, these results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop. The TAPIS pipeline will serve as a useful tool to analyse Iso-Seq data from any organism.« less

A survey of the sorghum transcriptome using single-molecule long reads

PubMed Central

Abdel-Ghany, Salah E.; Hamilton, Michael; Jacobi, Jennifer L.; Ngam, Peter; Devitt, Nicholas; Schilkey, Faye; Ben-Hur, Asa; Reddy, Anireddy S. N.

2016-01-01

Alternative splicing and alternative polyadenylation (APA) of pre-mRNAs greatly contribute to transcriptome diversity, coding capacity of a genome and gene regulatory mechanisms in eukaryotes. Second-generation sequencing technologies have been extensively used to analyse transcriptomes. However, a major limitation of short-read data is that it is difficult to accurately predict full-length splice isoforms. Here we sequenced the sorghum transcriptome using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and APA sites. Our analysis reveals transcriptome-wide full-length isoforms at an unprecedented scale with over 11,000 novel splice isoforms. Additionally, we uncover APA of ∼11,000 expressed genes and more than 2,100 novel genes. These results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop. The TAPIS pipeline will serve as a useful tool to analyse Iso-Seq data from any organism. PMID:27339290

Introduction to bioinformatics.

PubMed

Can, Tolga

2014-01-01

Bioinformatics is an interdisciplinary field mainly involving molecular biology and genetics, computer science, mathematics, and statistics. Data intensive, large-scale biological problems are addressed from a computational point of view. The most common problems are modeling biological processes at the molecular level and making inferences from collected data. A bioinformatics solution usually involves the following steps: Collect statistics from biological data. Build a computational model. Solve a computational modeling problem. Test and evaluate a computational algorithm. This chapter gives a brief introduction to bioinformatics by first providing an introduction to biological terminology and then discussing some classical bioinformatics problems organized by the types of data sources. Sequence analysis is the analysis of DNA and protein sequences for clues regarding function and includes subproblems such as identification of homologs, multiple sequence alignment, searching sequence patterns, and evolutionary analyses. Protein structures are three-dimensional data and the associated problems are structure prediction (secondary and tertiary), analysis of protein structures for clues regarding function, and structural alignment. Gene expression data is usually represented as matrices and analysis of microarray data mostly involves statistics analysis, classification, and clustering approaches. Biological networks such as gene regulatory networks, metabolic pathways, and protein-protein interaction networks are usually modeled as graphs and graph theoretic approaches are used to solve associated problems such as construction and analysis of large-scale networks.

Expansin polynucleotides, related polypeptides and methods of use

DOEpatents

Cosgrove, Daniel J.; Wu, Yajun

2006-02-21

The present invention relates to beta expansin polypeptides, nucleotide sequences encoding the same and regulatory elements and their use in altering cell wall structure in plants. Nucleic acid constructs comprising a beta expansin sequence operably linked to a promoter, or other regulatory sequence are disclosed as well as vectors, plant cells, plants, and transformed seeds containing such constructs are provided. Methods for the use of such constructs in repressing or inducing expression of a beta expansin sequences in a plant are also provided as well as methods for harvesting transgenic expansin proteins. In addition, methods are provided for inhibiting or improving cell wall structure in plants by repression or induction of expansin sequences in plants.

In silico modeling of epigenetic-induced changes in photoreceptor cis-regulatory elements.

PubMed

Hossain, Reafa A; Dunham, Nicholas R; Enke, Raymond A; Berndsen, Christopher E

2018-01-01

DNA methylation is a well-characterized epigenetic repressor of mRNA transcription in many plant and vertebrate systems. However, the mechanism of this repression is not fully understood. The process of transcription is controlled by proteins that regulate recruitment and activity of RNA polymerase by binding to specific cis-regulatory sequences. Cone-rod homeobox (CRX) is a well-characterized mammalian transcription factor that controls photoreceptor cell-specific gene expression. Although much is known about the functions and DNA binding specificity of CRX, little is known about how DNA methylation modulates CRX binding affinity to genomic cis-regulatory elements. We used bisulfite pyrosequencing of human ocular tissues to measure DNA methylation levels of the regulatory regions of RHO , PDE6B, PAX6 , and LINE1 retrotransposon repeats. To describe the molecular mechanism of repression, we used molecular modeling to illustrate the effect of DNA methylation on human RHO regulatory sequences. In this study, we demonstrate an inverse correlation between DNA methylation in regulatory regions adjacent to the human RHO and PDE6B genes and their subsequent transcription in human ocular tissues. Docking of CRX to the DNA models shows that CRX interacts with the grooves of these sequences, suggesting changes in groove structure could regulate binding. Molecular dynamics simulations of the RHO promoter and enhancer regions show changes in the flexibility and groove width upon epigenetic modification. Models also demonstrate changes in the local dynamics of CRX binding sites within RHO regulatory sequences which may account for the repression of CRX-dependent transcription. Collectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.

Curated collection of yeast transcription factor DNA binding specificity data reveals novel structural and gene regulatory insights

PubMed Central

2011-01-01

Background Transcription factors (TFs) play a central role in regulating gene expression by interacting with cis-regulatory DNA elements associated with their target genes. Recent surveys have examined the DNA binding specificities of most Saccharomyces cerevisiae TFs, but a comprehensive evaluation of their data has been lacking. Results We analyzed in vitro and in vivo TF-DNA binding data reported in previous large-scale studies to generate a comprehensive, curated resource of DNA binding specificity data for all characterized S. cerevisiae TFs. Our collection comprises DNA binding site motifs and comprehensive in vitro DNA binding specificity data for all possible 8-bp sequences. Investigation of the DNA binding specificities within the basic leucine zipper (bZIP) and VHT1 regulator (VHR) TF families revealed unexpected plasticity in TF-DNA recognition: intriguingly, the VHR TFs, newly characterized by protein binding microarrays in this study, recognize bZIP-like DNA motifs, while the bZIP TF Hac1 recognizes a motif highly similar to the canonical E-box motif of basic helix-loop-helix (bHLH) TFs. We identified several TFs with distinct primary and secondary motifs, which might be associated with different regulatory functions. Finally, integrated analysis of in vivo TF binding data with protein binding microarray data lends further support for indirect DNA binding in vivo by sequence-specific TFs. Conclusions The comprehensive data in this curated collection allow for more accurate analyses of regulatory TF-DNA interactions, in-depth structural studies of TF-DNA specificity determinants, and future experimental investigations of the TFs' predicted target genes and regulatory roles. PMID:22189060

A transcriptional blueprint for a spiral-cleaving embryo.

PubMed

Chou, Hsien-Chao; Pruitt, Margaret M; Bastin, Benjamin R; Schneider, Stephan Q

2016-08-05

The spiral cleavage mode of early development is utilized in over one-third of all animal phyla and generates embryonic cells of different size, position, and fate through a conserved set of stereotypic and invariant asymmetric cell divisions. Despite the widespread use of spiral cleavage, regulatory and molecular features for any spiral-cleaving embryo are largely uncharted. To address this gap we use RNA-sequencing on the spiralian model Platynereis dumerilii to capture and quantify the first complete genome-wide transcriptional landscape of early spiral cleavage. RNA-sequencing datasets from seven stages in early Platynereis development, from the zygote to the protrochophore, are described here including the de novo assembly and annotation of ~17,200 Platynereis genes. Depth and quality of the RNA-sequencing datasets allow the identification of the temporal onset and level of transcription for each annotated gene, even if the expression is restricted to a single cell. Over 4000 transcripts are maternally contributed and cleared by the end of the early spiral cleavage phase. Small early waves of zygotic expression are followed by major waves of thousands of genes, demarcating the maternal to zygotic transition shortly after the completion of spiral cleavages in this annelid species. Our comprehensive stage-specific transcriptional analysis of early embryonic stages in Platynereis elucidates the regulatory genome during early spiral embryogenesis and defines the maternal to zygotic transition in Platynereis embryos. This transcriptome assembly provides the first systems-level view of the transcriptional and regulatory landscape for a spiral-cleaving embryo.

Evaluating brief motivational and self-regulatory hand hygiene interventions: a cross-over longitudinal design.

PubMed

Lhakhang, Pempa; Lippke, Sonia; Knoll, Nina; Schwarzer, Ralf

2015-02-04

Frequent handwashing can prevent infections, but non-compliance to hand hygiene is pervasive. Few theory- and evidence-based interventions to improve regular handwashing are available. Therefore, two intervention modules, a motivational and a self-regulatory one, were designed and evaluated. In a longitudinal study, 205 young adults, aged 18 to 26 years, were randomized into two intervention groups. The Mot-SelfR group received first a motivational intervention (Mot; risk perception and outcome expectancies) followed by a self-regulatory intervention (SelfR; perceived self-efficacy and planning) 17 days later. The SelfR-Mot group received the same two intervention modules in the opposite order. Follow-up data were assessed 17 and 34 days after the baseline. Both intervention sequences led to an increase in handwashing frequency, intention, self-efficacy, and planning. Also, overall gains were found for the self-regulatory module (increased planning and self-efficacy levels) and the motivational module (intention). Within groups, the self-regulatory module appeared to be more effective than the motivational module, independent of sequence. Self-regulatory interventions can help individuals to exhibit more handwashing. Sequencing may be important as a motivation module (Mot) first helps to set the goal and a self-regulatory module (SelfR) then helps to translate this goal into actual behavior, but further research is needed to evaluate mechanisms.

Validation of Metagenomic Next-Generation Sequencing Tests for Universal Pathogen Detection.

PubMed

Schlaberg, Robert; Chiu, Charles Y; Miller, Steve; Procop, Gary W; Weinstock, George

2017-06-01

- Metagenomic sequencing can be used for detection of any pathogens using unbiased, shotgun next-generation sequencing (NGS), without the need for sequence-specific amplification. Proof-of-concept has been demonstrated in infectious disease outbreaks of unknown causes and in patients with suspected infections but negative results for conventional tests. Metagenomic NGS tests hold great promise to improve infectious disease diagnostics, especially in immunocompromised and critically ill patients. - To discuss challenges and provide example solutions for validating metagenomic pathogen detection tests in clinical laboratories. A summary of current regulatory requirements, largely based on prior guidance for NGS testing in constitutional genetics and oncology, is provided. - Examples from 2 separate validation studies are provided for steps from assay design, and validation of wet bench and bioinformatics protocols, to quality control and assurance. - Although laboratory and data analysis workflows are still complex, metagenomic NGS tests for infectious diseases are increasingly being validated in clinical laboratories. Many parallels exist to NGS tests in other fields. Nevertheless, specimen preparation, rapidly evolving data analysis algorithms, and incomplete reference sequence databases are idiosyncratic to the field of microbiology and often overlooked.

cGRNB: a web server for building combinatorial gene regulatory networks through integrated engineering of seed-matching sequence information and gene expression datasets.

PubMed

Xu, Huayong; Yu, Hui; Tu, Kang; Shi, Qianqian; Wei, Chaochun; Li, Yuan-Yuan; Li, Yi-Xue

2013-01-01

We are witnessing rapid progress in the development of methodologies for building the combinatorial gene regulatory networks involving both TFs (Transcription Factors) and miRNAs (microRNAs). There are a few tools available to do these jobs but most of them are not easy to use and not accessible online. A web server is especially needed in order to allow users to upload experimental expression datasets and build combinatorial regulatory networks corresponding to their particular contexts. In this work, we compiled putative TF-gene, miRNA-gene and TF-miRNA regulatory relationships from forward-engineering pipelines and curated them as built-in data libraries. We streamlined the R codes of our two separate forward-and-reverse engineering algorithms for combinatorial gene regulatory network construction and formalized them as two major functional modules. As a result, we released the cGRNB (combinatorial Gene Regulatory Networks Builder): a web server for constructing combinatorial gene regulatory networks through integrated engineering of seed-matching sequence information and gene expression datasets. The cGRNB enables two major network-building modules, one for MPGE (miRNA-perturbed gene expression) datasets and the other for parallel miRNA/mRNA expression datasets. A miRNA-centered two-layer combinatorial regulatory cascade is the output of the first module and a comprehensive genome-wide network involving all three types of combinatorial regulations (TF-gene, TF-miRNA, and miRNA-gene) are the output of the second module. In this article we propose cGRNB, a web server for building combinatorial gene regulatory networks through integrated engineering of seed-matching sequence information and gene expression datasets. Since parallel miRNA/mRNA expression datasets are rapidly accumulated by the advance of next-generation sequencing techniques, cGRNB will be very useful tool for researchers to build combinatorial gene regulatory networks based on expression datasets. The cGRNB web-server is free and available online at http://www.scbit.org/cgrnb.

Integrated transcriptome sequencing and dynamic analysis reveal carbon source partitioning between terpenoid and oil accumulation in developing Lindera glauca fruits.

PubMed

Niu, Jun; Chen, Yinlei; An, Jiyong; Hou, Xinyu; Cai, Jian; Wang, Jia; Zhang, Zhixiang; Lin, Shanzhi

2015-10-08

Lindera glauca fruits (LGF) with the abundance of terpenoid and oil has emerged as a novel specific material for industrial and medicinal application in China, but the complex regulatory mechanisms of carbon source partitioning into terpenoid biosynthetic pathway (TBP) and oil biosynthetic pathway (OBP) in developing LGF is still unknown. Here we perform the analysis of contents and compositions of terpenoid and oil from 7 stages of developing LGF to characterize a dramatic difference in temporal accumulative patterns. The resulting 3 crucial samples at 50, 125 and 150 days after flowering (DAF) were selected for comparative deep transcriptome analysis. By Illumina sequencing, the obtained approximately 81 million reads are assembled into 69,160 unigenes, among which 174, 71, 81 and 155 unigenes are implicated in glycolysis, pentose phosphate pathway (PPP), TBP and OBP, respectively. Integrated differential expression profiling and qRT-PCR, we specifically characterize the key enzymes and transcription factors (TFs) involved in regulating carbon allocation ratios for terpenoid or oil accumulation in developing LGF. These results contribute to our understanding of the regulatory mechanisms of carbon source partitioning between terpenoid and oil in developing LGF, and to the improvement of resource utilization and molecular breeding for L. glauca.

Recent advances in ChIP-seq analysis: from quality management to whole-genome annotation.

PubMed

Nakato, Ryuichiro; Shirahige, Katsuhiko

2017-03-01

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) analysis can detect protein/DNA-binding and histone-modification sites across an entire genome. Recent advances in sequencing technologies and analyses enable us to compare hundreds of samples simultaneously; such large-scale analysis has potential to reveal the high-dimensional interrelationship level for regulatory elements and annotate novel functional genomic regions de novo. Because many experimental considerations are relevant to the choice of a method in a ChIP-seq analysis, the overall design and quality management of the experiment are of critical importance. This review offers guiding principles of computation and sample preparation for ChIP-seq analyses, highlighting the validity and limitations of the state-of-the-art procedures at each step. We also discuss the latest challenges of single-cell analysis that will encourage a new era in this field. © The Author 2016. Published by Oxford University Press.

A regulatory sequence from the retinoid X receptor γ gene directs expression to horizontal cells and photoreceptors in the embryonic chicken retina.

PubMed

Blixt, Maria K E; Hallböök, Finn

2016-01-01

Combining techniques of episomal vector gene-specific Cre expression and genomic integration using the piggyBac transposon system enables studies of gene expression-specific cell lineage tracing in the chicken retina. In this work, we aimed to target the retinal horizontal cell progenitors. A 208 bp gene regulatory sequence from the chicken retinoid X receptor γ gene (RXRγ208) was used to drive Cre expression. RXRγ is expressed in progenitors and photoreceptors during development. The vector was combined with a piggyBac "donor" vector containing a floxed STOP sequence followed by enhanced green fluorescent protein (EGFP), as well as a piggyBac helper vector for efficient integration into the host cell genome. The vectors were introduced into the embryonic chicken retina with in ovo electroporation. Tissue electroporation targets specific developmental time points and in specific structures. Cells that drove Cre expression from the regulatory RXRγ208 sequence excised the floxed STOP-sequence and expressed GFP. The approach generated a stable lineage with robust expression of GFP in retinal cells that have activated transcription from the RXRγ208 sequence. Furthermore, GFP was expressed in cells that express horizontal or photoreceptor markers when electroporation was performed between developmental stages 22 and 28. Electroporation of a stage 12 optic cup gave multiple cell types in accordance with RXRγ gene expression in the early retina. In this study, we describe an easy, cost-effective, and time-efficient method for testing regulatory sequences in general. More specifically, our results open up the possibility for further studies of the RXRγ-gene regulatory network governing the formation of photoreceptor and horizontal cells. In addition, the method presents approaches to target the expression of effector genes, such as regulators of cell fate or cell cycle progression, to these cells and their progenitor.

Comparing anterior and posterior Hox complex formation reveals guidelines for predicting cis-regulatory elements

PubMed Central

Uhl, Juli D.; Cook, Tiffany A.; Gebelein, Brian

2010-01-01

Hox transcription factors specify numerous cell fates along the anterior-posterior axis by regulating the expression of downstream target genes. While expression analysis has uncovered large numbers of de-regulated genes in cells with altered Hox activity, determining which are direct versus indirect targets has remained a significant challenge. Here, we characterize the DNA binding activity of Hox transcription factor complexes on eight experimentally verified cis-regulatory elements. Hox factors regulate the activity of each element by forming protein complexes with two cofactor proteins, Extradenticle (Exd) and Homothorax (Hth). Using comparative DNA binding assays, we found that a number of flexible arrangements of Hox, Exd, and Hth binding sites mediate cooperative transcription factor complexes. Moreover, analysis of a Distal-less regulatory element (DMXR) that is repressed by abdominal Hox factors revealed that suboptimal binding sites can be combined to form high affinity transcription complexes. Lastly, we determined that the anterior Hox factors are more dependent upon Exd and Hth for complex formation than posterior Hox factors. Based upon these findings, we suggest a general set of guidelines to serve as a basis for designing bioinformatics algorithms aimed at identifying Hox regulatory elements using the wealth of recently sequenced genomes. PMID:20398649

Updated regulation curation model at the Saccharomyces Genome Database

PubMed Central

Engel, Stacia R; Skrzypek, Marek S; Hellerstedt, Sage T; Wong, Edith D; Nash, Robert S; Weng, Shuai; Binkley, Gail; Sheppard, Travis K; Karra, Kalpana; Cherry, J Michael

2018-01-01

Abstract The Saccharomyces Genome Database (SGD) provides comprehensive, integrated biological information for the budding yeast Saccharomyces cerevisiae, along with search and analysis tools to explore these data, enabling the discovery of functional relationships between sequence and gene products in fungi and higher organisms. We have recently expanded our data model for regulation curation to address regulation at the protein level in addition to transcription, and are presenting the expanded data on the ‘Regulation’ pages at SGD. These pages include a summary describing the context under which the regulator acts, manually curated and high-throughput annotations showing the regulatory relationships for that gene and a graphical visualization of its regulatory network and connected networks. For genes whose products regulate other genes or proteins, the Regulation page includes Gene Ontology enrichment analysis of the biological processes in which those targets participate. For DNA-binding transcription factors, we also provide other information relevant to their regulatory function, such as DNA binding site motifs and protein domains. As with other data types at SGD, all regulatory relationships and accompanying data are available through YeastMine, SGD’s data warehouse based on InterMine. Database URL: http://www.yeastgenome.org PMID:29688362

In silico analysis of cis-acting regulatory elements in 5' regulatory regions of sucrose transporter gene families in rice (Oryza sativa Japonica) and Arabidopsis thaliana.

PubMed

Ibraheem, Omodele; Botha, Christiaan E J; Bradley, Graeme

2010-12-01

The regulation of gene expression involves a multifarious regulatory system. Each gene contains a unique combination of cis-acting regulatory sequence elements in the 5' regulatory region that determines its temporal and spatial expression. Cis-acting regulatory elements are essential transcriptional gene regulatory units; they control many biological processes and stress responses. Thus a full understanding of the transcriptional gene regulation system will depend on successful functional analyses of cis-acting elements. Cis-acting regulatory elements present within the 5' regulatory region of the sucrose transporter gene families in rice (Oryza sativa Japonica cultivar-group) and Arabidopsis thaliana, were identified using a bioinformatics approach. The possible cis-acting regulatory elements were predicted by scanning 1.5kbp of 5' regulatory regions of the sucrose transporter genes translational start sites, using Plant CARE, PLACE and Genomatix Matinspector professional databases. Several cis-acting regulatory elements that are associated with plant development, plant hormonal regulation and stress response were identified, and were present in varying frequencies within the 1.5kbp of 5' regulatory region, among which are; A-box, RY, CAT, Pyrimidine-box, Sucrose-box, ABRE, ARF, ERE, GARE, Me-JA, ARE, DRE, GA-motif, GATA, GT-1, MYC, MYB, W-box, and I-box. This result reveals the probable cis-acting regulatory elements that possibly are involved in the expression and regulation of sucrose transporter gene families in rice and Arabidopsis thaliana during cellular development or environmental stress conditions. Copyright © 2010 Elsevier Ltd. All rights reserved.

Dcode.org anthology of comparative genomic tools.

PubMed

Loots, Gabriela G; Ovcharenko, Ivan

2005-07-01

Comparative genomics provides the means to demarcate functional regions in anonymous DNA sequences. The successful application of this method to identifying novel genes is currently shifting to deciphering the non-coding encryption of gene regulation across genomes. To facilitate the practical application of comparative sequence analysis to genetics and genomics, we have developed several analytical and visualization tools for the analysis of arbitrary sequences and whole genomes. These tools include two alignment tools, zPicture and Mulan; a phylogenetic shadowing tool, eShadow for identifying lineage- and species-specific functional elements; two evolutionary conserved transcription factor analysis tools, rVista and multiTF; a tool for extracting cis-regulatory modules governing the expression of co-regulated genes, Creme 2.0; and a dynamic portal to multiple vertebrate and invertebrate genome alignments, the ECR Browser. Here, we briefly describe each one of these tools and provide specific examples on their practical applications. All the tools are publicly available at the http://www.dcode.org/ website.

Early Evolution of Conserved Regulatory Sequences Associated with Development in Vertebrates

PubMed Central

McEwen, Gayle K.; Goode, Debbie K.; Parker, Hugo J.; Woolfe, Adam; Callaway, Heather; Elgar, Greg

2009-01-01

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA. PMID:20011110

Analysis of the putative regulatory region of the gastric inhibitory polypeptide receptor gene in food-dependent Cushing's syndrome.

PubMed

Antonini, S R; N'Diaye, N; Baldacchino, V; Hamet, P; Tremblay, J; Lacroix, A

2004-07-01

Gastric inhibitory polypeptide (GIP)-dependent Cushing's syndrome (CS) results from the ectopic expression of non-mutated GIP receptor (hGIPR) in the adrenal cortex. We evaluated whether mutations or polymorphisms in the regulatory region of the GIPR gene could lead to this aberrant expression. We studied 9.0kb upstream and 1.3kb downstream of the GIPR gene putative promoter (pProm) by sequencing leukocyte DNA from controls and from adrenal tissues of GIP- and non-GIP-dependent CS patients. The putative proximal promoter region (800 bp) and the first exon and intron of the hGIPR gene were sequenced on adrenal DNA from nine GIP-dependent CS, as well as on leukocyte DNA of nine normal controls. Three variations found in this region were found in all patients and controls; at position -4/-5, an insertion of a T was seen in four out of nine patients and in five out of nine controls. Transient transfection studies conducted in rat GC and mouse Y1 cells showed that the TT allele confers loss of 40% in the promoter activity. The analysis of the 8-kb distal pProm region revealed eight distal single nucleotide polymorphisms (SNPs) without probable association with the disease, since frequencies in patients and controls were very similar. In conclusion, mutations or SNPs in the regulatory region of the GIPR gene are unlikely to underlie GIP-dependent CS. Copyright 2004 Elsevier Ltd.

PAQ: Partition Analysis of Quasispecies.

PubMed

Baccam, P; Thompson, R J; Fedrigo, O; Carpenter, S; Cornette, J L

2001-01-01

The complexities of genetic data may not be accurately described by any single analytical tool. Phylogenetic analysis is often used to study the genetic relationship among different sequences. Evolutionary models and assumptions are invoked to reconstruct trees that describe the phylogenetic relationship among sequences. Genetic databases are rapidly accumulating large amounts of sequences. Newly acquired sequences, which have not yet been characterized, may require preliminary genetic exploration in order to build models describing the evolutionary relationship among sequences. There are clustering techniques that rely less on models of evolution, and thus may provide nice exploratory tools for identifying genetic similarities. Some of the more commonly used clustering methods perform better when data can be grouped into mutually exclusive groups. Genetic data from viral quasispecies, which consist of closely related variants that differ by small changes, however, may best be partitioned by overlapping groups. We have developed an intuitive exploratory program, Partition Analysis of Quasispecies (PAQ), which utilizes a non-hierarchical technique to partition sequences that are genetically similar. PAQ was used to analyze a data set of human immunodeficiency virus type 1 (HIV-1) envelope sequences isolated from different regions of the brain and another data set consisting of the equine infectious anemia virus (EIAV) regulatory gene rev. Analysis of the HIV-1 data set by PAQ was consistent with phylogenetic analysis of the same data, and the EIAV rev variants were partitioned into two overlapping groups. PAQ provides an additional tool which can be used to glean information from genetic data and can be used in conjunction with other tools to study genetic similarities and genetic evolution of viral quasispecies.

An Integrated Analysis of MicroRNA and mRNA Expression Profiles to Identify RNA Expression Signatures in Lambskin Hair Follicles in Hu Sheep

PubMed Central

Lv, Xiaoyang; Sun, Wei; Yin, Jinfeng; Ni, Rong; Su, Rui; Wang, Qingzeng; Gao, Wen; Bao, Jianjun; Yu, Jiarui; Wang, Lihong; Chen, Ling

2016-01-01

Wave patterns in lambskin hair follicles are an important factor determining the quality of sheep’s wool. Hair follicles in lambskin from Hu sheep, a breed unique to China, have 3 types of waves, designated as large, medium, and small. The quality of wool from small wave follicles is excellent, while the quality of large waves is considered poor. Because no molecular and biological studies on hair follicles of these sheep have been conducted to date, the molecular mechanisms underlying the formation of different wave patterns is currently unknown. The aim of this article was to screen the candidate microRNAs (miRNA) and genes for the development of hair follicles in Hu sheep. Two-day-old Hu lambs were selected from full-sib individuals that showed large, medium, and small waves. Integrated analysis of microRNA and mRNA expression profiles employed high-throughout sequencing technology. Approximately 13, 24, and 18 differentially expressed miRNAs were found between small and large waves, small and medium waves, and medium and large waves, respectively. A total of 54, 190, and 81 differentially expressed genes were found between small and large waves, small and medium waves, and medium and large waves, respectively, by RNA sequencing (RNA-seq) analysis. Differentially expressed genes were classified using gene ontology and pathway analyses. They were found to be mainly involved in cell differentiation, proliferation, apoptosis, growth, immune response, and ion transport, and were associated with MAPK and the Notch signaling pathway. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of differentially-expressed miRNA and genes were consistent with sequencing results. Integrated analysis of miRNA and mRNA expression indicated that, compared to small waves, large waves included 4 downregulated miRNAs that had regulatory effects on 8 upregulated genes and 3 upregulated miRNAs, which in turn influenced 13 downregulated genes. Compared to small waves, medium waves included 13 downregulated miRNAs that had regulatory effects on 64 upregulated genes and 4 upregulated miRNAs, which in turn had regulatory effects on 22 downregulated genes. Compared to medium waves, large waves consisted of 13 upregulated miRNAs that had regulatory effects on 48 downregulated genes. These differentially expressed miRNAs and genes may play a significant role in forming different patterns, and provide evidence for the molecular mechanisms underlying the formation of hair follicles of varying patterns. PMID:27404636

Complete genome sequence of an isolate of Potato virus X (PVX) infecting Cape gooseberry (Physalis peruviana) in Colombia.

PubMed

Gutiérrez, Pablo A; Alzate, Juan F; Montoya, Mauricio Marín

2015-06-01

Transcriptome analysis of a Cape gooseberry (Physalis peruviana) plant with leaf symptoms of a mild yellow mosaic typical of a viral disease revealed an infection with Potato virus X (PVX). The genome sequence of the PVX-Physalis isolate comprises 6435 nt and exhibits higher sequence similarity to members of the Eurasian group of PVX (~95 %) than to the American group (~77 %). Genome organization is similar to other PVX isolates with five open reading frames coding for proteins RdRp, TGBp1, TGBp2, TGBp3, and CP. 5' and 3' untranslated regions revealed all regulatory motifs typically found in PVX isolates. The PVX-Physalis genome is the only complete sequence available for a Potexvirus in Colombia and is a new addition to the restricted number of available sequences of PVX isolates infecting plant species different to potato.

Alu-mediated deletion of SOX10 regulatory elements in Waardenburg syndrome type 4

PubMed Central

Bondurand, Nadége; Fouquet, Virginie; Baral, Viviane; Lecerf, Laure; Loundon, Natalie; Goossens, Michel; Duriez, Benedicte; Labrune, Philippe; Pingault, Veronique

2012-01-01

Waardenburg syndrome type 4 (WS4) is a rare neural crest disorder defined by the combination of Waardenburg syndrome (sensorineural hearing loss and pigmentation defects) and Hirschsprung disease (intestinal aganglionosis). Three genes are known to be involved in this syndrome, that is, EDN3 (endothelin-3), EDNRB (endothelin receptor type B), and SOX10. However, 15–35% of WS4 remains unexplained at the molecular level, suggesting that other genes could be involved and/or that mutations within known genes may have escaped previous screenings. Here, we searched for deletions within recently identified SOX10 regulatory sequences and describe the first characterization of a WS4 patient presenting with a large deletion encompassing three of these enhancers. Analysis of the breakpoint region suggests a complex rearrangement involving three Alu sequences that could be mediated by a FosTes/MMBIR replication mechanism. Taken together with recent reports, our results demonstrate that the disruption of highly conserved non-coding elements located within or at a long distance from the coding sequences of key genes can result in several neurocristopathies. This opens up new routes to the molecular dissection of neural crest disorders. PMID:22378281

Generation of transgenic goats by pronuclear microinjection: a retrospective analysis of a commercial operation (1995-2012).

PubMed

Gavin, W; Blash, S; Buzzell, N; Pollock, D; Chen, L; Hawkins, N; Howe, J; Miner, K; Pollock, J; Porter, C; Schofield, M; Echelard, Y; Meade, H

2018-02-01

Production of transgenic founder goats involves introducing and stably integrating an engineered piece of DNA into the genome of the animal. At LFB USA, the ultimate use of these transgenic goats is for the production of recombinant human protein therapeutics in the milk of these dairy animals. The transgene or construct typically links a milk protein specific promoter sequence, the coding sequence for the gene of interest, and the necessary downstream regulatory sequences thereby directing expression of the recombinant protein in the milk during the lactation period. Over the time period indicated (1995-2012), pronuclear microinjection was used in a number of programs to insert transgenes into 18,120, 1- or 2- cell stage fertilized embryos. These embryos were transferred into 4180 synchronized recipient females with 1934 (47%) recipients becoming pregnant, 2594 offspring generated, and a 109 (4.2%) of those offspring determined to be transgenic. Even with new and improving genome editing tools now available, pronuclear microinjection is still the predominant and proven technology used in this commercial setting supporting regulatory filings and market authorizations when producing founder transgenic animals with large transgenes (> 10 kb) such as those necessary for directing monoclonal antibody production in milk.

Structural basis for regulation of rhizobial nodulation and symbiosis gene expression by the regulatory protein NolR.

PubMed

Lee, Soon Goo; Krishnan, Hari B; Jez, Joseph M

2014-04-29

The symbiosis between rhizobial microbes and host plants involves the coordinated expression of multiple genes, which leads to nodule formation and nitrogen fixation. As part of the transcriptional machinery for nodulation and symbiosis across a range of Rhizobium, NolR serves as a global regulatory protein. Here, we present the X-ray crystal structures of NolR in the unliganded form and complexed with two different 22-base pair (bp) double-stranded operator sequences (oligos AT and AA). Structural and biochemical analysis of NolR reveals protein-DNA interactions with an asymmetric operator site and defines a mechanism for conformational switching of a key residue (Gln56) to accommodate variation in target DNA sequences from diverse rhizobial genes for nodulation and symbiosis. This conformational switching alters the energetic contributions to DNA binding without changes in affinity for the target sequence. Two possible models for the role of NolR in the regulation of different nodulation and symbiosis genes are proposed. To our knowledge, these studies provide the first structural insight on the regulation of genes involved in the agriculturally and ecologically important symbiosis of microbes and plants that leads to nodule formation and nitrogen fixation.

Alu-mediated deletion of SOX10 regulatory elements in Waardenburg syndrome type 4.

PubMed

Bondurand, Nadége; Fouquet, Virginie; Baral, Viviane; Lecerf, Laure; Loundon, Natalie; Goossens, Michel; Duriez, Benedicte; Labrune, Philippe; Pingault, Veronique

2012-09-01

Waardenburg syndrome type 4 (WS4) is a rare neural crest disorder defined by the combination of Waardenburg syndrome (sensorineural hearing loss and pigmentation defects) and Hirschsprung disease (intestinal aganglionosis). Three genes are known to be involved in this syndrome, that is, EDN3 (endothelin-3), EDNRB (endothelin receptor type B), and SOX10. However, 15-35% of WS4 remains unexplained at the molecular level, suggesting that other genes could be involved and/or that mutations within known genes may have escaped previous screenings. Here, we searched for deletions within recently identified SOX10 regulatory sequences and describe the first characterization of a WS4 patient presenting with a large deletion encompassing three of these enhancers. Analysis of the breakpoint region suggests a complex rearrangement involving three Alu sequences that could be mediated by a FosTes/MMBIR replication mechanism. Taken together with recent reports, our results demonstrate that the disruption of highly conserved non-coding elements located within or at a long distance from the coding sequences of key genes can result in several neurocristopathies. This opens up new routes to the molecular dissection of neural crest disorders.

GREAT: a web portal for Genome Regulatory Architecture Tools

PubMed Central

Bouyioukos, Costas; Bucchini, François; Elati, Mohamed; Képès, François

2016-01-01

GREAT (Genome REgulatory Architecture Tools) is a novel web portal for tools designed to generate user-friendly and biologically useful analysis of genome architecture and regulation. The online tools of GREAT are freely accessible and compatible with essentially any operating system which runs a modern browser. GREAT is based on the analysis of genome layout -defined as the respective positioning of co-functional genes- and its relation with chromosome architecture and gene expression. GREAT tools allow users to systematically detect regular patterns along co-functional genomic features in an automatic way consisting of three individual steps and respective interactive visualizations. In addition to the complete analysis of regularities, GREAT tools enable the use of periodicity and position information for improving the prediction of transcription factor binding sites using a multi-view machine learning approach. The outcome of this integrative approach features a multivariate analysis of the interplay between the location of a gene and its regulatory sequence. GREAT results are plotted in web interactive graphs and are available for download either as individual plots, self-contained interactive pages or as machine readable tables for downstream analysis. The GREAT portal can be reached at the following URL https://absynth.issb.genopole.fr/GREAT and each individual GREAT tool is available for downloading. PMID:27151196

Primate-Specific Evolution of an LDLR Enhancer

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

Wang, Qian-fei; Prabhakar, Shyam; Wang, Qianben

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 leadingmore » to human.« less

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

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

Sternberg, E.A.; Spizz, G.; Perry, W.M.

1988-07-01

Terminal differentiation of skeletal myobalsts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzymte of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers.

The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4).

PubMed

Huntemann, Marcel; Ivanova, Natalia N; Mavromatis, Konstantinos; Tripp, H James; Paez-Espino, David; Palaniappan, Krishnaveni; Szeto, Ernest; Pillay, Manoj; Chen, I-Min A; Pati, Amrita; Nielsen, Torben; Markowitz, Victor M; Kyrpides, Nikos C

2015-01-01

The DOE-JGI Microbial Genome Annotation Pipeline performs structural and functional annotation of microbial genomes that are further included into the Integrated Microbial Genome comparative analysis system. MGAP is applied to assembled nucleotide sequence datasets that are provided via the IMG submission site. Dataset submission for annotation first requires project and associated metadata description in GOLD. The MGAP sequence data processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNA features, as well as CRISPR elements. Structural annotation is followed by assignment of protein product names and functions.

Identification, characterization and expression analysis of pigeonpea miRNAs in response to Fusarium wilt.

PubMed

Hussain, Khalid; Mungikar, Kanak; Kulkarni, Abhijeet; Kamble, Avinash

2018-05-05

Upon confrontation with unfavourable conditions, plants invoke a very complex set of biochemical and physiological reactions and alter gene expression patterns to combat the situations. MicroRNAs (miRNAs), a class of small non-coding RNA, contribute extensively in regulation of gene expression through translation inhibition or degradation of their target mRNAs during such conditions. Therefore, identification of miRNAs and their targets holds importance in understanding the regulatory networks triggered during stress. Structure and sequence similarity based in silico prediction of miRNAs in Cajanus cajan L. (Pigeonpea) draft genome sequence has been carried out earlier. These annotations also appear in related GenBank genome sequence entries. However, there are no reports available on context dependent miRNA expression and their targets in pigeonpea. Therefore, in the present study we addressed these questions computationally, using pigeonpea EST sequence information. We identified five novel pigeonpea miRNA precursors, their mature forms and targets. Interestingly, only one of these miRNAs (miR169i-3p) was identified earlier in draft genome sequence. We then validated expression of these miRNAs, experimentally. It was also observed that these miRNAs show differential expression patterns in response to Fusarium inoculation indicating their biotic stress responsive nature. Overall these results will help towards better understanding the regulatory network of defense during pigeonpea -pathogen interactions and role of miRNAs in the process. Copyright © 2018 Elsevier B.V. All rights reserved.

CoryneRegNet: An ontology-based data warehouse of corynebacterial transcription factors and regulatory networks

PubMed Central

Baumbach, Jan; Brinkrolf, Karina; Czaja, Lisa F; Rahmann, Sven; Tauch, Andreas

2006-01-01

Background The application of DNA microarray technology in post-genomic analysis of bacterial genome sequences has allowed the generation of huge amounts of data related to regulatory networks. This data along with literature-derived knowledge on regulation of gene expression has opened the way for genome-wide reconstruction of transcriptional regulatory networks. These large-scale reconstructions can be converted into in silico models of bacterial cells that allow a systematic analysis of network behavior in response to changing environmental conditions. Description CoryneRegNet was designed to facilitate the genome-wide reconstruction of transcriptional regulatory networks of corynebacteria relevant in biotechnology and human medicine. During the import and integration process of data derived from experimental studies or literature knowledge CoryneRegNet generates links to genome annotations, to identified transcription factors and to the corresponding cis-regulatory elements. CoryneRegNet is based on a multi-layered, hierarchical and modular concept of transcriptional regulation and was implemented by using the relational database management system MySQL and an ontology-based data structure. Reconstructed regulatory networks can be visualized by using the yFiles JAVA graph library. As an application example of CoryneRegNet, we have reconstructed the global transcriptional regulation of a cellular module involved in SOS and stress response of corynebacteria. Conclusion CoryneRegNet is an ontology-based data warehouse that allows a pertinent data management of regulatory interactions along with the genome-scale reconstruction of transcriptional regulatory networks. These models can further be combined with metabolic networks to build integrated models of cellular function including both metabolism and its transcriptional regulation. PMID:16478536

A high-resolution enhancer atlas of the developing telencephalon.

PubMed

Visel, Axel; Taher, Leila; Girgis, Hani; May, Dalit; Golonzhka, Olga; Hoch, Renee V; McKinsey, Gabriel L; Pattabiraman, Kartik; Silberberg, Shanni N; Blow, Matthew J; Hansen, David V; Nord, Alex S; Akiyama, Jennifer A; Holt, Amy; Hosseini, Roya; Phouanenavong, Sengthavy; Plajzer-Frick, Ingrid; Shoukry, Malak; Afzal, Veena; Kaplan, Tommy; Kriegstein, Arnold R; Rubin, Edward M; Ovcharenko, Ivan; Pennacchio, Len A; Rubenstein, John L R

2013-02-14

The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders. Copyright © 2013 Elsevier Inc. All rights reserved.

Genome-wide methylation analysis identified sexually dimorphic methylated regions in hybrid tilapia

PubMed Central

Wan, Zi Yi; Xia, Jun Hong; Lin, Grace; Wang, Le; Lin, Valerie C. L.; Yue, Gen Hua

2016-01-01

Sexual dimorphism is an interesting biological phenomenon. Previous studies showed that DNA methylation might play a role in sexual dimorphism. However, the overall picture of the genome-wide methylation landscape in sexually dimorphic species remains unclear. We analyzed the DNA methylation landscape and transcriptome in hybrid tilapia (Oreochromis spp.) using whole genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq). We found 4,757 sexually dimorphic differentially methylated regions (DMRs), with significant clusters of DMRs located on chromosomal regions associated with sex determination. CpG methylation in promoter regions was negatively correlated with the gene expression level. MAPK/ERK pathway was upregulated in male tilapia. We also inferred active cis-regulatory regions (ACRs) in skeletal muscle tissues from WGBS datasets, revealing sexually dimorphic cis-regulatory regions. These results suggest that DNA methylation contribute to sex-specific phenotypes and serve as resources for further investigation to analyze the functions of these regions and their contributions towards sexual dimorphisms. PMID:27782217

Mapping and analysis of Caenorhabditis elegans transcription factor sequence specificities

PubMed Central

Narasimhan, Kamesh; Lambert, Samuel A; Yang, Ally WH; Riddell, Jeremy; Mnaimneh, Sanie; Zheng, Hong; Albu, Mihai; Najafabadi, Hamed S; Reece-Hoyes, John S; Fuxman Bass, Juan I; Walhout, Albertha JM; Weirauch, Matthew T; Hughes, Timothy R

2015-01-01

Caenorhabditis elegans is a powerful model for studying gene regulation, as it has a compact genome and a wealth of genomic tools. However, identification of regulatory elements has been limited, as DNA-binding motifs are known for only 71 of the estimated 763 sequence-specific transcription factors (TFs). To address this problem, we performed protein binding microarray experiments on representatives of canonical TF families in C. elegans, obtaining motifs for 129 TFs. Additionally, we predict motifs for many TFs that have DNA-binding domains similar to those already characterized, increasing coverage of binding specificities to 292 C. elegans TFs (∼40%). These data highlight the diversification of binding motifs for the nuclear hormone receptor and C2H2 zinc finger families and reveal unexpected diversity of motifs for T-box and DM families. Motif enrichment in promoters of functionally related genes is consistent with known biology and also identifies putative regulatory roles for unstudied TFs. DOI: http://dx.doi.org/10.7554/eLife.06967.001 PMID:25905672

Functional analysis of Drosophila HSP70 promoter with different HSE numbers in human cells.

PubMed

Kust, Nadezda; Rybalkina, Ekaterina; Mertsalov, Ilya; Savchenko, Ekaterina; Revishchin, Alexander; Pavlova, Gali

2014-01-01

The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38 °C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.

Novel phytochrome sequences in Arabidopsis thaliana: Structure, evolution, and differential expression of a plant regulatory photoreceptor family

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

Sharrock, R.A.; Quail, P.H.

1989-01-01

Phytochrome is a plant regulatory photoreceptor that mediates red light effects on a wide variety of physiological and molecular responses. DNA blot analysis indicates that the Arabidopsis thaliana genome contains four to five phytochrome-related gene sequences. The authors have isolated and sequenced cDNA clones corresponding to three of these genes and have deduced the amino acid sequence of the full-length polypeptide encoded in each case. One of these proteins (phyA) shows 65-80% amino acid sequence identity with the major, etiolated-tissue phytochrome apoproteins described previously in other plant species. The other two polypeptides (phyB and phyC) are unique in that theymore » have low sequence identity with each other, with phyA, and with all previously described phytochromes. The phyA, phyB, and phyC proteins are of similar molecular mass, have related hydropathic profiles, and contain a conserved chromophore attachment region. However, the sequence comparison data indicate that the three phy genes diverged early in plant evolution, well before the divergence of the two major groups of angiosperms, the monocots and dicots. The steady-state level of the phyA transcript is high in dark-grown A. thaliana seedlings and is down-regulated by light. In contrast, the phyB and phyC transcripts are present at lower levels and are not strongly light-regulated. These findings indicate that the red/far red light-responsive phytochrome photoreceptor system in A. thaliana, and perhaps in all higher plants, consists of a family of chromoproteins that are heterogeneous in structure and regulation.« less

Integrative analysis of Arabidopsis thaliana transcriptomics reveals intuitive splicing mechanism for circular RNA.

PubMed

Sun, Xiaoyong; Wang, Lin; Ding, Jiechao; Wang, Yanru; Wang, Jiansheng; Zhang, Xiaoyang; Che, Yulei; Liu, Ziwei; Zhang, Xinran; Ye, Jiazhen; Wang, Jie; Sablok, Gaurav; Deng, Zhiping; Zhao, Hongwei

2016-10-01

A new regulatory class of small endogenous RNAs called circular RNAs (circRNAs) has been described as miRNA sponges in animals. Using 16 Arabidopsis thaliana RNA-Seq data sets, we identified 803 circRNAs in RNase R-/non-RNase R-treated samples. The results revealed the following features: Canonical and noncanonical splicing can generate circRNAs; chloroplasts are a hotspot for circRNA generation; furthermore, limited complementary sequences exist not only in introns, but also in the sequences flanking splice sites. The latter finding suggests that multiple combinations between complementary sequences may facilitate the formation of the circular structure. Our results contribute to a better understanding of this novel class of plant circRNAs. © 2016 Federation of European Biochemical Societies.

Estimation of pairwise sequence similarity of mammalian enhancers with word neighbourhood counts.

PubMed

Göke, Jonathan; Schulz, Marcel H; Lasserre, Julia; Vingron, Martin

2012-03-01

The identity of cells and tissues is to a large degree governed by transcriptional regulation. A major part is accomplished by the combinatorial binding of transcription factors at regulatory sequences, such as enhancers. Even though binding of transcription factors is sequence-specific, estimating the sequence similarity of two functionally similar enhancers is very difficult. However, a similarity measure for regulatory sequences is crucial to detect and understand functional similarities between two enhancers and will facilitate large-scale analyses like clustering, prediction and classification of genome-wide datasets. We present the standardized alignment-free sequence similarity measure N2, a flexible framework that is defined for word neighbourhoods. We explore the usefulness of adding reverse complement words as well as words including mismatches into the neighbourhood. On simulated enhancer sequences as well as functional enhancers in mouse development, N2 is shown to outperform previous alignment-free measures. N2 is flexible, faster than competing methods and less susceptible to single sequence noise and the occurrence of repetitive sequences. Experiments on the mouse enhancers reveal that enhancers active in different tissues can be separated by pairwise comparison using N2. N2 represents an improvement over previous alignment-free similarity measures without compromising speed, which makes it a good candidate for large-scale sequence comparison of regulatory sequences. The software is part of the open-source C++ library SeqAn (www.seqan.de) and a compiled version can be downloaded at http://www.seqan.de/projects/alf.html. Supplementary data are available at Bioinformatics online.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

PubMed Central

Hirsh, J; Morgan, B A; Scholnick, S B

1986-01-01

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity. Images PMID:3099170

OPAQUE11 Is a Central Hub of the Regulatory Network for Maize Endosperm Development and Nutrient Metabolism[OPEN

PubMed Central

Feng, Fan; Qi, Weiwei; Lv, Yuanda; Yan, Shumei; Xu, Liming; Yang, Wenyao; Yuan, Yue; Chen, Yihan

2018-01-01

Maize (Zea mays) endosperm is a primary tissue for nutrient storage and is highly differentiated during development. However, the regulatory networks of endosperm development and nutrient metabolism remain largely unknown. Maize opaque11 (o11) is a classic seed mutant with a small and opaque endosperm showing decreased starch and protein accumulation. We cloned O11 and found that it encodes an endosperm-specific bHLH transcription factor (TF). Loss of function of O11 significantly affected transcription of carbohydrate/amino acid metabolism and stress response genes. Genome-wide binding site analysis revealed 9885 O11 binding sites distributed over 6033 genes. Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with RNA sequencing (RNA-seq) assays, we identified 259 O11-modulated target genes. O11 was found to directly regulate key TFs in endosperm development (NKD2 and ZmDOF3) and nutrient metabolism (O2 and PBF). Moreover, O11 directly regulates cyPPDKs and multiple carbohydrate metabolic enzymes. O11 is an activator of ZmYoda, suggesting its regulatory function through the MAPK pathway in endosperm development. Many stress-response genes are also direct targets of O11. In addition, 11 O11-interacting proteins were identified, including ZmIce1, which coregulates stress response targets and ZmYoda with O11. Therefore, this study reveals an endosperm regulatory network centered around O11, which coordinates endosperm development, metabolism and stress responses. PMID:29436476

The G-Box Transcriptional Regulatory Code in Arabidopsis1[OPEN

PubMed Central

Shepherd, Samuel J.K.; Brestovitsky, Anna; Dickinson, Patrick; Biswas, Surojit

2017-01-01

Plants have significantly more transcription factor (TF) families than animals and fungi, and plant TF families tend to contain more genes; these expansions are linked to adaptation to environmental stressors. Many TF family members bind to similar or identical sequence motifs, such as G-boxes (CACGTG), so it is difficult to predict regulatory relationships. We determined that the flanking sequences near G-boxes help determine in vitro specificity but that this is insufficient to predict the transcription pattern of genes near G-boxes. Therefore, we constructed a gene regulatory network that identifies the set of bZIPs and bHLHs that are most predictive of the expression of genes downstream of perfect G-boxes. This network accurately predicts transcriptional patterns and reconstructs known regulatory subnetworks. Finally, we present Ara-BOX-cis (araboxcis.org), a Web site that provides interactive visualizations of the G-box regulatory network, a useful resource for generating predictions for gene regulatory relations. PMID:28864470

VIZARD: analysis of Affymetrix Arabidopsis GeneChip data

NASA Technical Reports Server (NTRS)

Moseyko, Nick; Feldman, Lewis J.

2002-01-01

SUMMARY: The Affymetrix GeneChip Arabidopsis genome array has proved to be a very powerful tool for the analysis of gene expression in Arabidopsis thaliana, the most commonly studied plant model organism. VIZARD is a Java program created at the University of California, Berkeley, to facilitate analysis of Arabidopsis GeneChip data. It includes several integrated tools for filtering, sorting, clustering and visualization of gene expression data as well as tools for the discovery of regulatory motifs in upstream sequences. VIZARD also includes annotation and upstream sequence databases for the majority of genes represented on the Affymetrix Arabidopsis GeneChip array. AVAILABILITY: VIZARD is available free of charge for educational, research, and not-for-profit purposes, and can be downloaded at http://www.anm.f2s.com/research/vizard/ CONTACT: moseyko@uclink4.berkeley.edu.

Transcription Factors Bind Thousands of Active and InactiveRegions in the Drosophila Blastoderm

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

Li, Xiao-Yong; MacArthur, Stewart; Bourgon, Richard

2008-01-10

Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. Here, we use whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior-posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched inmore » bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over forty well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal-ventral patterning genes, whose expression we show to be quantitatively modulated by anterior-posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly-bound regions are not involved in early-embryonic transcriptional regulation, and a significant proportion may be nonfunctional. Surprisingly, for five of the six factors, their recognition sites are not unambiguously more constrained evolutionarily than the immediate flanking DNA, even in more highly bound and presumably functional regions, indicating that comparative DNA sequence analysis is limited in its ability to identify functional transcription factor targets.« less

Identification and Potential Regulatory Properties of Evolutionary Conserved Regions (ECRs) at the Schizophrenia-Associated MIR137 Locus.

PubMed

Gianfrancesco, Olympia; Griffiths, Daniel; Myers, Paul; Collier, David A; Bubb, Vivien J; Quinn, John P

2016-10-01

Genome-wide association studies (GWAS) have identified a region at chromosome 1p21.3, containing the microRNA MIR137, to be among the most significant associations for schizophrenia. However, the mechanism by which genetic variation at this locus increases risk of schizophrenia is unknown. Identifying key regulatory regions around MIR137 is crucial to understanding the potential role of this gene in the aetiology of psychiatric disorders. Through alignment of vertebrate genomes, we identified seven non-coding regions at the MIR137 locus with conservation comparable to exons (>70 %). Bioinformatic analysis using the Psychiatric Genomics Consortium GWAS dataset for schizophrenia showed five of the ECRs to have genome-wide significant SNPs in or adjacent to their sequence. Analysis of available datasets on chromatin marks and histone modification data showed that three of the ECRs were predicted to be functional in the human brain, and three in development. In vitro analysis of ECR activity using reporter gene assays showed that all seven of the selected ECRs displayed transcriptional regulatory activity in the SH-SY5Y neuroblastoma cell line. This data suggests a regulatory role in the developing and adult brain for these highly conserved regions at the MIR137 schizophrenia-associated locus and further that these domains could act individually or synergistically to regulate levels of MIR137 expression.

Whole-Genome Sequencing and Comparative Genome Analysis of Bacillus subtilis Strains Isolated from Non-Salted Fermented Soybean Foods.

PubMed

Kamada, Mayumi; Hase, Sumitaka; Fujii, Kazushi; Miyake, Masato; Sato, Kengo; Kimura, Keitarou; Sakakibara, Yasubumi

2015-01-01

Bacillus subtilis is the main component in the fermentation of soybeans. To investigate the genetics of the soybean-fermenting B. subtilis strains and its relationship with the productivity of extracellular poly-γ-glutamic acid (γPGA), we sequenced the whole genome of eight B. subtilis stains isolated from non-salted fermented soybean foods in Southeast Asia. Assembled nucleotide sequences were compared with those of a natto (fermented soybean food) starter strain B. subtilis BEST195 and the laboratory standard strain B. subtilis 168 that is incapable of γPGA production. Detected variants were investigated in terms of insertion sequences, biotin synthesis, production of subtilisin NAT, and regulatory genes for γPGA synthesis, which were related to fermentation process. Comparing genome sequences, we found that the strains that produce γPGA have a deletion in a protein that constitutes the flagellar basal body, and this deletion was not found in the non-producing strains. We further identified diversity in variants of the bio operon, which is responsible for the biotin auxotrophism of the natto starter strains. Phylogenetic analysis using multilocus sequencing typing revealed that the B. subtilis strains isolated from the non-salted fermented soybeans were not clustered together, while the natto-fermenting strains were tightly clustered; this analysis also suggested that the strain isolated from "Tua Nao" of Thailand traces a different evolutionary process from other strains.

Identification of microRNAs differentially expressed involved in male flower development.

PubMed

Wang, Zhengjia; Huang, Jianqin; Sun, Zhichao; Zheng, Bingsong

2015-03-01

Hickory (Carya cathayensis Sarg.) is one of the most economically important woody trees in eastern China, but its long flowering phase delays yield. Our understanding of the regulatory roles of microRNAs (miRNAs) in male flower development in hickory remains poor. Using high-throughput sequencing technology, we have pyrosequenced two small RNA libraries from two male flower differentiation stages in hickory. Analysis of the sequencing data identified 114 conserved miRNAs that belonged to 23 miRNA families, five novel miRNAs including their corresponding miRNA*s, and 22 plausible miRNA candidates. Differential expression analysis revealed 12 miRNA sequences that were upregulated in the later (reproductive) stage of male flower development. Quantitative real-time PCR showed similar expression trends as that of the deep sequencing. Novel miRNAs and plausible miRNA candidates were predicted using bioinformatic analysis methods. The miRNAs newly identified in this study have increased the number of known miRNAs in hickory, and the identification of differentially expressed miRNAs will provide new avenues for studies into miRNAs involved in the process of male flower development in hickory and other related trees.

Making the Leap from Research Laboratory to Clinic: Challenges and Opportunities for Next-Generation Sequencing in Infectious Disease Diagnostics

PubMed Central

Goldberg, Brittany; Sichtig, Heike; Geyer, Chelsie; Ledeboer, Nathan

2015-01-01

ABSTRACT Next-generation DNA sequencing (NGS) has progressed enormously over the past decade, transforming genomic analysis and opening up many new opportunities for applications in clinical microbiology laboratories. The impact of NGS on microbiology has been revolutionary, with new microbial genomic sequences being generated daily, leading to the development of large databases of genomes and gene sequences. The ability to analyze microbial communities without culturing organisms has created the ever-growing field of metagenomics and microbiome analysis and has generated significant new insights into the relation between host and microbe. The medical literature contains many examples of how this new technology can be used for infectious disease diagnostics and pathogen analysis. The implementation of NGS in medical practice has been a slow process due to various challenges such as clinical trials, lack of applicable regulatory guidelines, and the adaptation of the technology to the clinical environment. In April 2015, the American Academy of Microbiology (AAM) convened a colloquium to begin to define these issues, and in this document, we present some of the concepts that were generated from these discussions. PMID:26646014

Comparative Genome Sequence Analysis of the Bpa/Str Region in Mouse and Man

PubMed Central

Mallon, A.-M.; Platzer, M.; Bate, R.; Gloeckner, G.; Botcherby, M.R.M.; Nordsiek, G.; Strivens, M.A.; Kioschis, P.; Dangel, A.; Cunningham, D.; Straw, R.N.A.; Weston, P.; Gilbert, M.; Fernando, S.; Goodall, K.; Hunter, G.; Greystrong, J.S.; Clarke, D.; Kimberley, C.; Goerdes, M.; Blechschmidt, K.; Rump, A.; Hinzmann, B.; Mundy, C.R.; Miller, W.; Poustka, A.; Herman, G.E.; Rhodes, M.; Denny, P.; Rosenthal, A.; Brown, S.D.M.

2000-01-01

The progress of human and mouse genome sequencing programs presages the possibility of systematic cross-species comparison of the two genomes as a powerful tool for gene and regulatory element identification. As the opportunities to perform comparative sequence analysis emerge, it is important to develop parameters for such analyses and to examine the outcomes of cross-species comparison. Our analysis used gene prediction and a database search of 430 kb of genomic sequence covering the Bpa/Str region of the mouse X chromosome, and 745 kb of genomic sequence from the homologous human X chromosome region. We identified 11 genes in mouse and 13 genes and two pseudogenes in human. In addition, we compared the mouse and human sequences using pairwise alignment and searches for evolutionary conserved regions (ECRs) exceeding a defined threshold of sequence identity. This approach aided the identification of at least four further putative conserved genes in the region. Comparative sequencing revealed that this region is a mosaic in evolutionary terms, with considerably more rearrangement between the two species than realized previously from comparative mapping studies. Surprisingly, this region showed an extremely high LINE and low SINE content, low G+C content, and yet a relatively high gene density, in contrast to the low gene density usually associated with such regions. [The sequence data described in this paper have been submitted to EMBL under the following accession nos.: Mouse Genomic Sequence: Mouse contig A (AL021127), Mouse contig B (AL049866), BAC41M10 (AL136328), PAC303O11(AL136329). Human Genomic Sequence: Human contig 1 (U82671, U82670), Human contig 2 (U82695).] PMID:10854409

Sounds of silence: synonymous nucleotides as a key to biological regulation and complexity

PubMed Central

Shabalina, Svetlana A.; Spiridonov, Nikolay A.; Kashina, Anna

2013-01-01

Messenger RNA is a key component of an intricate regulatory network of its own. It accommodates numerous nucleotide signals that overlap protein coding sequences and are responsible for multiple levels of regulation and generation of biological complexity. A wealth of structural and regulatory information, which mRNA carries in addition to the encoded amino acid sequence, raises the question of how these signals and overlapping codes are delineated along non-synonymous and synonymous positions in protein coding regions, especially in eukaryotes. Silent or synonymous codon positions, which do not determine amino acid sequences of the encoded proteins, define mRNA secondary structure and stability and affect the rate of translation, folding and post-translational modifications of nascent polypeptides. The RNA level selection is acting on synonymous sites in both prokaryotes and eukaryotes and is more common than previously thought. Selection pressure on the coding gene regions follows three-nucleotide periodic pattern of nucleotide base-pairing in mRNA, which is imposed by the genetic code. Synonymous positions of the coding regions have a higher level of hybridization potential relative to non-synonymous positions, and are multifunctional in their regulatory and structural roles. Recent experimental evidence and analysis of mRNA structure and interspecies conservation suggest that there is an evolutionary tradeoff between selective pressure acting at the RNA and protein levels. Here we provide a comprehensive overview of the studies that define the role of silent positions in regulating RNA structure and processing that exert downstream effects on proteins and their functions. PMID:23293005

Coordinate cytokine regulatory sequences

DOEpatents

Frazer, Kelly A.; Rubin, Edward M.; Loots, Gabriela G.

2005-05-10

The present invention provides CNS sequences that regulate the cytokine gene expression, expression cassettes and vectors comprising or lacking the CNS sequences, host cells and non-human transgenic animals comprising the CNS sequences or lacking the CNS sequences. The present invention also provides methods for identifying compounds that modulate the functions of CNS sequences as well as methods for diagnosing defects in the CNS sequences of patients.

Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene.

PubMed

Dale, Rodney M; Topczewski, Jacek

2011-09-15

Zebrafish (Danio rerio) is an excellent model organism for the study of vertebrate development including skeletogenesis. Studies of mammalian cartilage formation were greatly advanced through the use of a cartilage specific regulatory element of the Collagen type II alpha 1 (Col2a1) gene. In an effort to isolate such an element in zebrafish, we compared the expression of two col2a1 homologues and found that expression of col2a1b, a previously uncharacterized zebrafish homologue, only partially overlaps with col2a1a. We focused our analysis on col2a1a, as it is expressed in both the stacked chondrocytes and the perichondrium. By comparing the genomic sequence surrounding the predicted transcriptional start site of col2a1a among several species of teleosts we identified a small highly conserved sequence (R2) located 1.7 kb upstream of the presumptive transcriptional initiation site. Interestingly, neither the sequence nor location of this element is conserved between teleost and mammalian Col2a1. We generated transient and stable transgenic lines with just the R2 element or the entire 1.7 kb fragment 5' of the transcriptional initiation site. The identified regulatory elements enable the tracking of cellular development in various tissues by driving robust reporter expression in craniofacial cartilage, ear, notochord, floor plate, hypochord and fins in a pattern similar to the expression of endogenous col2a1a. Using a reporter gene driven by the R2 regulatory element, we analyzed the morphogenesis of the notochord sheath cells as they withdraw from the stack of initially uniform cells and encase the inflating vacuolated notochord cells. Finally, we show that like endogenous col2a1a, craniofacial expression of these reporter constructs depends on Sox9a transcription factor activity. At the same time, notochord expression is maintained after Sox9a knockdown, suggesting that other factors can activate expression through the identified regulatory element in this tissue. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene

PubMed Central

Dale, Rodney M.; Topczewski, Jacek

2011-01-01

Zebrafish (Danio rerio) is an excellent model organism for the study of vertebrate development including skeletogenesis. Studies of mammalian cartilage formation were greatly advanced through the use of a cartilage specific regulatory element of the Collagen type II alpha 1 (Col2a1) gene. In an effort to isolate such an element in zebrafish, we compared the expression of two col2a1 homologues and found that expression of col2a1b, a previously uncharacterized zebrafish homologue, only partially overlaps with col2a1a. We focused our analysis on col2a1a, as it is expressed in both the stacked chondrocytes and the perichondrium. By comparing the genomic sequence surrounding the predicted transcriptional start site of col2a1a among several species of teleosts we identified a small highly conserved sequence (R2) located 1.7 kb upstream of the presumptive transcriptional initiation site. Interestingly, neither the sequence nor location of this element is conserved between teleost and mammalian Col2a1. We generated transient and stable transgenic lines with just the R2 element or the entire 1.7 kb fragment 5’ of the transcriptional initiation site. The identified regulatory elements enable the tracking of cellular development in various tissues by driving robust reporter expression in craniofacial cartilage, ear, notochord, floor plate, hypochord and fins in a pattern similar to the expression of endogenous col2a1a. Using a reporter gene driven by the R2 regulatory element, we analyzed the morphogenesis of the notochord sheath cells as they withdraw from the stack of initially uniform cells and encase the inflating vacuolated notochord cells. Finally, we show that like endogenous col2a1a, craniofacial expression of these reporter constructs depends on Sox9a transcription factor activity. At the same time, notochord expression is maintained after Sox9a knockdown, suggesting that other factors can activate expression through the identified regulatory element in this tissue. PMID:21723274

Conserved and species-specific transcription factor co-binding patterns drive divergent gene regulation in human and mouse

PubMed Central

Diehl, Adam G

2018-01-01

Abstract The mouse is widely used as system to study human genetic mechanisms. However, extensive rewiring of transcriptional regulatory networks often confounds translation of findings between human and mouse. Site-specific gain and loss of individual transcription factor binding sites (TFBS) has caused functional divergence of orthologous regulatory loci, and so we must look beyond this positional conservation to understand common themes of regulatory control. Fortunately, transcription factor co-binding patterns shared across species often perform conserved regulatory functions. These can be compared to ‘regulatory sentences’ that retain the same meanings regardless of sequence and species context. By analyzing TFBS co-occupancy patterns observed in four human and mouse cell types, we learned a regulatory grammar: the rules by which TFBS are combined into meaningful regulatory sentences. Different parts of this grammar associate with specific sets of functional annotations regardless of sequence conservation and predict functional signatures more accurately than positional conservation. We further show that both species-specific and conserved portions of this grammar are involved in gene expression divergence and human disease risk. These findings expand our understanding of transcriptional regulatory mechanisms, suggesting that phenotypic divergence and disease risk are driven by a complex interplay between deeply conserved and species-specific transcriptional regulatory pathways. PMID:29361190

Evidence for Deep Regulatory Similarities in Early Developmental Programs across Highly Diverged Insects

PubMed Central

Zhang, Yinan; Samee, Md. Abul Hassan; Halfon, Marc S.; Sinha, Saurabh

2014-01-01

Many genes familiar from Drosophila development, such as the so-called gap, pair-rule, and segment polarity genes, play important roles in the development of other insects and in many cases appear to be deployed in a similar fashion, despite the fact that Drosophila-like “long germband” development is highly derived and confined to a subset of insect families. Whether or not these similarities extend to the regulatory level is unknown. Identification of regulatory regions beyond the well-studied Drosophila has been challenging as even within the Diptera (flies, including mosquitoes) regulatory sequences have diverged past the point of recognition by standard alignment methods. Here, we demonstrate that methods we previously developed for computational cis-regulatory module (CRM) discovery in Drosophila can be used effectively in highly diverged (250–350 Myr) insect species including Anopheles gambiae, Tribolium castaneum, Apis mellifera, and Nasonia vitripennis. In Drosophila, we have successfully used small sets of known CRMs as “training data” to guide the search for other CRMs with related function. We show here that although species-specific CRM training data do not exist, training sets from Drosophila can facilitate CRM discovery in diverged insects. We validate in vivo over a dozen new CRMs, roughly doubling the number of known CRMs in the four non-Drosophila species. Given the growing wealth of Drosophila CRM annotation, these results suggest that extensive regulatory sequence annotation will be possible in newly sequenced insects without recourse to costly and labor-intensive genome-scale experiments. We develop a new method, Regulus, which computes a probabilistic score of similarity based on binding site composition (despite the absence of nucleotide-level sequence alignment), and demonstrate similarity between functionally related CRMs from orthologous loci. Our work represents an important step toward being able to trace the evolutionary history of gene regulatory networks and defining the mechanisms underlying insect evolution. PMID:25173756

Evidence for deep regulatory similarities in early developmental programs across highly diverged insects.

PubMed

Kazemian, Majid; Suryamohan, Kushal; Chen, Jia-Yu; Zhang, Yinan; Samee, Md Abul Hassan; Halfon, Marc S; Sinha, Saurabh

2014-09-01

Many genes familiar from Drosophila development, such as the so-called gap, pair-rule, and segment polarity genes, play important roles in the development of other insects and in many cases appear to be deployed in a similar fashion, despite the fact that Drosophila-like "long germband" development is highly derived and confined to a subset of insect families. Whether or not these similarities extend to the regulatory level is unknown. Identification of regulatory regions beyond the well-studied Drosophila has been challenging as even within the Diptera (flies, including mosquitoes) regulatory sequences have diverged past the point of recognition by standard alignment methods. Here, we demonstrate that methods we previously developed for computational cis-regulatory module (CRM) discovery in Drosophila can be used effectively in highly diverged (250-350 Myr) insect species including Anopheles gambiae, Tribolium castaneum, Apis mellifera, and Nasonia vitripennis. In Drosophila, we have successfully used small sets of known CRMs as "training data" to guide the search for other CRMs with related function. We show here that although species-specific CRM training data do not exist, training sets from Drosophila can facilitate CRM discovery in diverged insects. We validate in vivo over a dozen new CRMs, roughly doubling the number of known CRMs in the four non-Drosophila species. Given the growing wealth of Drosophila CRM annotation, these results suggest that extensive regulatory sequence annotation will be possible in newly sequenced insects without recourse to costly and labor-intensive genome-scale experiments. We develop a new method, Regulus, which computes a probabilistic score of similarity based on binding site composition (despite the absence of nucleotide-level sequence alignment), and demonstrate similarity between functionally related CRMs from orthologous loci. Our work represents an important step toward being able to trace the evolutionary history of gene regulatory networks and defining the mechanisms underlying insect evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genomic analysis reveals major determinants of cis-regulatory variation in Capsella grandiflora

PubMed Central

Steige, Kim A.; Laenen, Benjamin; Reimegård, Johan; Slotte, Tanja

2017-01-01

Understanding the causes of cis-regulatory variation is a long-standing aim in evolutionary biology. Although cis-regulatory variation has long been considered important for adaptation, we still have a limited understanding of the selective importance and genomic determinants of standing cis-regulatory variation. To address these questions, we studied the prevalence, genomic determinants, and selective forces shaping cis-regulatory variation in the outcrossing plant Capsella grandiflora. We first identified a set of 1,010 genes with common cis-regulatory variation using analyses of allele-specific expression (ASE). Population genomic analyses of whole-genome sequences from 32 individuals showed that genes with common cis-regulatory variation (i) are under weaker purifying selection and (ii) undergo less frequent positive selection than other genes. We further identified genomic determinants of cis-regulatory variation. Gene body methylation (gbM) was a major factor constraining cis-regulatory variation, whereas presence of nearby transposable elements (TEs) and tissue specificity of expression increased the odds of ASE. Our results suggest that most common cis-regulatory variation in C. grandiflora is under weak purifying selection, and that gene-specific functional constraints are more important for the maintenance of cis-regulatory variation than genome-scale variation in the intensity of selection. Our results agree with previous findings that suggest TE silencing affects nearby gene expression, and provide evidence for a link between gbM and cis-regulatory constraint, possibly reflecting greater dosage sensitivity of body-methylated genes. Given the extensive conservation of gbM in flowering plants, this suggests that gbM could be an important predictor of cis-regulatory variation in a wide range of plant species. PMID:28096395

In-Silico Identification Of Micro-Loops In Myelodysplastic Syndromes

NASA Astrophysics Data System (ADS)

Beck, Dominik; Brandl, Miriam; Pham, Tuan D.; Chang, Chung-Che; Zhou, Xiaobo

2011-06-01

Micro-loops are regulatory network motifs that leverage transcriptional and posttranscriptional control to effectively regulate the transcriptome. In this paper a regulatory network for Myelodysplastic Syndromes (MDSs) was constructed from the literature and publicly available data sources. The network was filtered using data from deep-sequencing of small RNAs, exon and microarrays. Motif discovery showed that micro-loops might exist in MDS. We further used the identified micro-loops and performed basic network analysis to identify the known disease gene RUNX1/AML, as well as miRNA family hsa-mir-181. This suggested that the concept of micro-loops can be applied to enhance disease gene identification and biomarker discovery.

Functional analysis of the upstream regulatory region of chicken miR-17-92 cluster.

PubMed

Cheng, Min; Zhang, Wen-jian; Xing, Tian-yu; Yan, Xiao-hong; Li, Yu-mao; Li, Hui; Wang, Ning

2016-08-01

miR-17-92 cluster plays important roles in cell proliferation, differentiation, apoptosis, animal development and tumorigenesis. The transcriptional regulation of miR-17-92 cluster has been extensively studied in mammals, but not in birds. To date, avian miR-17-92 cluster genomic structure has not been fully determined. The promoter location and sequence of miR-17-92 cluster have not been determined, due to the existence of a genomic gap sequence upstream of miR-17-92 cluster in all the birds whose genomes have been sequenced. In this study, genome walking was used to close the genomic gap upstream of chicken miR-17-92 cluster. In addition, bioinformatics analysis, reporter gene assay and truncation mutagenesis were used to investigate functional role of the genomic gap sequence. Genome walking analysis showed that the gap region was 1704 bp long, and its GC content was 80.11%. Bioinformatics analysis showed that in the gap region, there was a 200 bp conserved sequence among the tested 10 species (Gallus gallus, Homo sapiens, Pan troglodytes, Bos taurus, Sus scrofa, Rattus norvegicus, Mus musculus, Possum, Danio rerio, Rana nigromaculata), which is core promoter region of mammalian miR-17-92 host gene (MIR17HG). Promoter luciferase reporter gene vector of the gap region was constructed and reporter assay was performed. The result showed that the promoter activity of pGL3-cMIR17HG (-4228/-2506) was 417 times than that of negative control (empty pGL3 basic vector), suggesting that chicken miR-17-92 cluster promoter exists in the gap region. To further gain insight into the promoter structure, two different truncations for the cloned gap sequence were generated by PCR. One had a truncation of 448 bp at the 5'-end and the other had a truncation of 894 bp at the 3'-end. Further reporter analysis showed that compared with the promoter activity of pGL3-cMIR17HG (-4228/-2506), the reporter activities of the 5'-end truncation and the 3'-end truncation were reduced by 19.82% and 60.14%, respectively. These data demonstrated that the important promoter region of chicken miR-17-92 cluster is located in the -3400/-2506 bp region. Our results lay the foundation for revealing the transcriptional regulatory mechanisms of chicken miR-17-92 cluster.

A cis-regulatory logic simulator.

PubMed

Zeigler, Robert D; Gertz, Jason; Cohen, Barak A

2007-07-27

A major goal of computational studies of gene regulation is to accurately predict the expression of genes based on the cis-regulatory content of their promoters. The development of computational methods to decode the interactions among cis-regulatory elements has been slow, in part, because it is difficult to know, without extensive experimental validation, whether a particular method identifies the correct cis-regulatory interactions that underlie a given set of expression data. There is an urgent need for test expression data in which the interactions among cis-regulatory sites that produce the data are known. The ability to rapidly generate such data sets would facilitate the development and comparison of computational methods that predict gene expression patterns from promoter sequence. We developed a gene expression simulator which generates expression data using user-defined interactions between cis-regulatory sites. The simulator can incorporate additive, cooperative, competitive, and synergistic interactions between regulatory elements. Constraints on the spacing, distance, and orientation of regulatory elements and their interactions may also be defined and Gaussian noise can be added to the expression values. The simulator allows for a data transformation that simulates the sigmoid shape of expression levels from real promoters. We found good agreement between sets of simulated promoters and predicted regulatory modules from real expression data. We present several data sets that may be useful for testing new methodologies for predicting gene expression from promoter sequence. We developed a flexible gene expression simulator that rapidly generates large numbers of simulated promoters and their corresponding transcriptional output based on specified interactions between cis-regulatory sites. When appropriate rule sets are used, the data generated by our simulator faithfully reproduces experimentally derived data sets. We anticipate that using simulated gene expression data sets will facilitate the direct comparison of computational strategies to predict gene expression from promoter sequence. The source code is available online and as additional material. The test sets are available as additional material.

A 3.0-kb deletion including an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene in an individual with the Bm phenotype.

PubMed

Sano, R; Kuboya, E; Nakajima, T; Takahashi, Y; Takahashi, K; Kubo, R; Kominato, Y; Takeshita, H; Yamao, H; Kishida, T; Isa, K; Ogasawara, K; Uchikawa, M

2015-04-01

We developed a sequence-specific primer PCR (SSP-PCR) for detection of a 5.8-kb deletion (B(m) 5.8) involving an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene. Using this SSP-PCR, we performed genetic analysis of 382 individuals with Bm or ABm. The 5.8-kb deletion was found in 380 individuals, and disruption of the GATA motif in the regulatory element was found in one individual. Furthermore, a novel 3.0-kb deletion involving the element (B(m) 3.0) was demonstrated in the remaining individual. Comparisons of single-nucleotide polymorphisms and microsatellites in intron 1 between B(m) 5.8 and B(m) 3.0 suggested that these deletions occurred independently. © 2014 International Society of Blood Transfusion.

Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis.

PubMed

Marvig, Rasmus Lykke; Sommer, Lea Mette; Molin, Søren; Johansen, Helle Krogh

2015-01-01

Little is known about how within-host evolution compares between genotypically different strains of the same pathogenic species. We sequenced the whole genomes of 474 longitudinally collected clinical isolates of Pseudomonas aeruginosa sampled from 34 children and young individuals with cystic fibrosis. Our analysis of 36 P. aeruginosa lineages identified convergent molecular evolution in 52 genes. This list of genes suggests a role in host adaptation for remodeling of regulatory networks and central metabolism, acquisition of antibiotic resistance and loss of extracellular virulence factors. Furthermore, we find an ordered succession of mutations in key regulatory networks. Accordingly, mutations in downstream transcriptional regulators were contingent upon mutations in upstream regulators, suggesting that remodeling of regulatory networks might be important in adaptation. The characterization of genes involved in host adaptation may help in predicting bacterial evolution in patients with cystic fibrosis and in the design of future intervention strategies.

Precision medicine in the age of big data: The present and future role of large-scale unbiased sequencing in drug discovery and development.

PubMed

Vicini, P; Fields, O; Lai, E; Litwack, E D; Martin, A-M; Morgan, T M; Pacanowski, M A; Papaluca, M; Perez, O D; Ringel, M S; Robson, M; Sakul, H; Vockley, J; Zaks, T; Dolsten, M; Søgaard, M

2016-02-01

High throughput molecular and functional profiling of patients is a key driver of precision medicine. DNA and RNA characterization has been enabled at unprecedented cost and scale through rapid, disruptive progress in sequencing technology, but challenges persist in data management and interpretation. We analyze the state-of-the-art of large-scale unbiased sequencing in drug discovery and development, including technology, application, ethical, regulatory, policy and commercial considerations, and discuss issues of LUS implementation in clinical and regulatory practice. © 2015 American Society for Clinical Pharmacology and Therapeutics.

Conserved regulatory elements of the promoter sequence of the gene rpoH of enteric bacteria

PubMed Central

Ramírez-Santos, Jesús; Collado-Vides, Julio; García-Varela, Martin; Gómez-Eichelmann, M. Carmen

2001-01-01

The rpoH regulatory region of different members of the enteric bacteria family was sequenced or downloaded from GenBank and compared. In addition, the transcriptional start sites of rpoH of Yersinia frederiksenii and Proteus mirabilis, two distant members of this family, were determined. Sequences similar to the σ70 promoters P1, P4 and P5, to the σE promoter P3 and to boxes DnaA1, DnaA2, cAMP receptor protein (CRP) boxes CRP1, CRP2 and box CytR present in Escherichia coli K12, were identified in sequences of closely related bacteria such as: E.coli, Shigella flexneri, Salmonella enterica serovar Typhimurium, Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae. In more distant bacteria, Y.frederiksenii and P.mirabilis, the rpoH regulatory region has a distal P1-like σ70 promoter and two proximal promoters: a heat-induced σE-like promoter and a σ70 promoter. Sequences similar to the regulatory boxes were not identified in these bacteria. This study suggests that the general pattern of transcription of the rpoH gene in enteric bacteria includes a distal σ70 promoter, >200 nt upstream of the initiation codon, and two proximal promoters: a heat-induced σE-like promoter and a σ70 promoter. A second proximal σ70 promoter under catabolite-regulation is probably present only in bacteria closely related to E.coli. PMID:11139607

GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation.

PubMed

Han, Kook; Tjaden, Brian; Lory, Stephen

2016-12-22

The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique, referred to as global small non-coding RNA target identification by ligation and sequencing (GRIL-seq), is based on preferential ligation of sRNAs to the ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimaeras. In addition to the RNA chaperone Hfq, the GRIL-seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrated that direct regulatory targets of this sRNA can readily be identified. Therefore, GRIL-seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but also for uncovering novel roles for sRNAs and their targets in complex regulatory networks.

Isolation and characterization of the promoter sequence of a cassava gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in storage roots.

PubMed

de Souza, C R; Aragão, F J; Moreira, E C O; Costa, C N M; Nascimento, S B; Carvalho, L J

2009-03-24

Cassava is one of the most important tropical food crops for more than 600 million people worldwide. Transgenic technologies can be useful for increasing its nutritional value and its resistance to viral diseases and insect pests. However, tissue-specific promoters that guarantee correct expression of transgenes would be necessary. We used inverse polymerase chain reaction to isolate a promoter sequence of the Mec1 gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in cassava storage roots. In silico analysis revealed putative cis-acting regulatory elements within this promoter sequence, including root-specific elements that may be required for its expression in vascular tissues. Transient expression experiments showed that the Mec1 promoter is functional, since this sequence was able to drive GUS expression in bean embryonic axes. Results from our computational analysis can serve as a guide for functional experiments to identify regions with tissue-specific Mec1 promoter activity. The DNA sequence that we identified is a new promoter that could be a candidate for genetic engineering of cassava roots.

DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1.

PubMed

Choudhary, M; Kaplan, S

2000-02-15

This paper describes the DNA sequence of the photosynthesis region of Rhodobacter sphaeroides 2.4.1 (T). The photosynthesis gene cluster is located within a approximately 73 kb Ase I genomic DNA fragment containing the puf, puhA, cycA and puc operons. A total of 65 open reading frames (ORFs) have been identified, of which 61 showed significant similarity to genes/proteins of other organisms while only four did not reveal any significant sequence similarity to any gene/protein sequences in the database. The data were compared with the corresponding genes/ORFs from a different strain of R.sphaeroides and Rhodobacter capsulatus, a close relative of R. sphaeroides. A detailed analysis of the gene organization in the photosynthesis region revealed a similar gene order in both species with some notable differences located to the pucBAC = cycA region. In addition, photosynthesis gene regulatory protein (PpsR, FNR, IHF) binding motifs in upstream sequences of a number of photosynthesis genes have been identified and shown to differ between these two species. The difference in gene organization relative to pucBAC and cycA suggests that this region originated independently of the photosynthesis gene cluster of R.sphaeroides.

[Isolation and function of genes regulating aphB expression in Vibrio cholerae].

PubMed

Chen, Haili; Zhu, Zhaoqin; Zhong, Zengtao; Zhu, Jun; Kan, Biao

2012-02-04

We identified genes that regulate the expression of aphB, the gene encoding a key virulence regulator in Vibrio cholerae O1 E1 Tor C6706(-). We constructed a transposon library in V. cholerae C6706 strain containing a P(aphB)-luxCDABE and P(aphB)-lacZ transcriptional reporter plasmids. Using a chemiluminescence imager system, we rapidly detected aphB promoter expression level at a large scale. We then sequenced the transposon insertion sites by arbitrary PCR and sequencing analysis. We obtained two candidate mutants T1 and T2 which displayed reduced aphB expression from approximately 40,000 transposon insertion mutants. Sequencing analysis shows that Tn inserted in vc1585 reading frame in the T1 mutant and Tn inserted in the end of coding sequence of vc1602 in the T2 mutant. By using a genetic screen, we identified two potential genes that may involve in regulation of the expression of the key virulence regulator AphB. This study sheds light on our further investigation to fully understand V. cholerae virulence gene regulatory cascades.

Molecular cloning and sequence analysis of the Anticarsia gemmatalis multicapsid nuclear polyhedrosis virus GP64 glycoprotein.

PubMed

Pilloff, Marcela Gabriela; Bilen, Marcos Fabián; Belaich, Mariano Nicolás; Lozano, Mario Enrique; Ghiringhelli, Pablo Daniel

2003-01-01

The gp64 locus of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate Santa Fe (AgMNPV-SF) was characterised molecularly in our laboratory. To this end, we have located and cloned a AgMNPV-SF genomic DNA fragment containing the gp64 gene and sequenced the complete gp64 locus. Nucleotide sequence analysis indicated that the AgMNPV gp64 gene consists of a 1500 nucleotide open reading frame (ORF), encoding a protein of 499 amino acids. Of the seven gp64 homologues identified to date, the AgMNPV gp64 ORF shared most sequence similarity with the gp64 gene of Orgyia pseudotsugata MNPV. The GP64 from AgMNPV is the smallest baculoviral envelope glycoprotein found to date, differing in 10 or more residues from the other group I nucleopolyhedroviruses. The biological activity of AgMNPV GP64 protein was assessed by cell fusion assays in UFL-AG-286 cells using the obtained recombinant plasmids. In the upstream and downstream regions, relative to the gp64 ORF, we found different conserved transcriptional and post-transcriptional regulatory elements, respectively.

The origins and evolutionary history of human non-coding RNA regulatory networks.

PubMed

Sherafatian, Masih; Mowla, Seyed Javad

2017-04-01

The evolutionary history and origin of the regulatory function of animal non-coding RNAs are not well understood. Lack of conservation of long non-coding RNAs and small sizes of microRNAs has been major obstacles in their phylogenetic analysis. In this study, we tried to shed more light on the evolution of ncRNA regulatory networks by changing our phylogenetic strategy to focus on the evolutionary pattern of their protein coding targets. We used available target databases of miRNAs and lncRNAs to find their protein coding targets in human. We were able to recognize evolutionary hallmarks of ncRNA targets by phylostratigraphic analysis. We found the conventional 3'-UTR and lesser known 5'-UTR targets of miRNAs to be enriched at three consecutive phylostrata. Firstly, in eukaryata phylostratum corresponding to the emergence of miRNAs, our study revealed that miRNA targets function primarily in cell cycle processes. Moreover, the same overrepresentation of the targets observed in the next two consecutive phylostrata, opisthokonta and eumetazoa, corresponded to the expansion periods of miRNAs in animals evolution. Coding sequence targets of miRNAs showed a delayed rise at opisthokonta phylostratum, compared to the 3' and 5' UTR targets of miRNAs. LncRNA regulatory network was the latest to evolve at eumetazoa.

CLIP-seq analysis of multi-mapped reads discovers novel functional RNA regulatory sites in the human transcriptome.

PubMed

Zhang, Zijun; Xing, Yi

2017-09-19

Crosslinking or RNA immunoprecipitation followed by sequencing (CLIP-seq or RIP-seq) allows transcriptome-wide discovery of RNA regulatory sites. As CLIP-seq/RIP-seq reads are short, existing computational tools focus on uniquely mapped reads, while reads mapped to multiple loci are discarded. We present CLAM (CLIP-seq Analysis of Multi-mapped reads). CLAM uses an expectation-maximization algorithm to assign multi-mapped reads and calls peaks combining uniquely and multi-mapped reads. To demonstrate the utility of CLAM, we applied it to a wide range of public CLIP-seq/RIP-seq datasets involving numerous splicing factors, microRNAs and m6A RNA methylation. CLAM recovered a large number of novel RNA regulatory sites inaccessible by uniquely mapped reads. The functional significance of these sites was demonstrated by consensus motif patterns and association with alternative splicing (splicing factors), transcript abundance (AGO2) and mRNA half-life (m6A). CLAM provides a useful tool to discover novel protein-RNA interactions and RNA modification sites from CLIP-seq and RIP-seq data, and reveals the significant contribution of repetitive elements to the RNA regulatory landscape of the human transcriptome. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

TSSer: an automated method to identify transcription start sites in prokaryotic genomes from differential RNA sequencing data.

PubMed

Jorjani, Hadi; Zavolan, Mihaela

2014-04-01

Accurate identification of transcription start sites (TSSs) is an essential step in the analysis of transcription regulatory networks. In higher eukaryotes, the capped analysis of gene expression technology enabled comprehensive annotation of TSSs in genomes such as those of mice and humans. In bacteria, an equivalent approach, termed differential RNA sequencing (dRNA-seq), has recently been proposed, but the application of this approach to a large number of genomes is hindered by the paucity of computational analysis methods. With few exceptions, when the method has been used, annotation of TSSs has been largely done manually. In this work, we present a computational method called 'TSSer' that enables the automatic inference of TSSs from dRNA-seq data. The method rests on a probabilistic framework for identifying both genomic positions that are preferentially enriched in the dRNA-seq data as well as preferentially captured relative to neighboring genomic regions. Evaluating our approach for TSS calling on several publicly available datasets, we find that TSSer achieves high consistency with the curated lists of annotated TSSs, but identifies many additional TSSs. Therefore, TSSer can accelerate genome-wide identification of TSSs in bacterial genomes and can aid in further characterization of bacterial transcription regulatory networks. TSSer is freely available under GPL license at http://www.clipz.unibas.ch/TSSer/index.php

[Expression, purification and activity analysis of GGDEF and EAL domain-containing proteins from Lactobacillus acidophilus].

PubMed

He, Jia-Hui; Sun, Jie-Li; Yan, Wen-Juan; Wang, Fang

2017-05-20

To identify the functions of the proteins containing the GGDEF or EAL domain in Lactobacillus acidophilus for investigation of the regulatory mechanism of c-di-GMP in this strain. The DNA fragments of NH13_07045-GGDEF, NH13_07050 and NH13_07055 from Lactobacillus acidophilus ATCC4356 were amplified by PCR and cloned into the expression vector pMAL-His-c2. After sequencing, the recombinant plasmids were transformed into competent Escherichia coli cells, which were induced by IPTG to express the recombinant proteins fused with maltose binding protein (MBP). The fusion proteins were purified using amylose resin column for diguanylate cyclase (DGC) or phosphodiesterase (PDE) activity assays in vitro followed by analysis with high-performance liquid chromatography (HPLC). The target DNA fragments were obtained by PCR, and their sequences were all identical to that in GenBank. The purified and concentrated fusion proteins, which were identified by SDS-PAGE and Western blotting, had relative molecular masses of 59 kD, 67 kD and 72 kD. HPLC analysis showed no DGC activity in NH13_07045-GGDEF, while PDE activity was found in NH13_07050 but not in NH13_07055. We obtained the protein encoded by NH13_07050 that possesses PDE activity in vitro. This protein may facilitate the evaluation of the regulatory function of c-di-GMP in Lactobacillus acidophilus.

The 'dark matter' in the plant genomes: non-coding and unannotated DNA sequences associated with open chromatin.

PubMed

Jiang, Jiming

2015-04-01

Sequencing of complete plant genomes has become increasingly more routine since the advent of the next-generation sequencing technology. Identification and annotation of large amounts of noncoding but functional DNA sequences, including cis-regulatory DNA elements (CREs), have become a new frontier in plant genome research. Genomic regions containing active CREs bound to regulatory proteins are hypersensitive to DNase I digestion and are called DNase I hypersensitive sites (DHSs). Several recent DHS studies in plants illustrate that DHS datasets produced by DNase I digestion followed by next-generation sequencing (DNase-seq) are highly valuable for the identification and characterization of CREs associated with plant development and responses to environmental cues. DHS-based genomic profiling has opened a door to identify and annotate the 'dark matter' in sequenced plant genomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modelling Human Regulatory Variation in Mouse: Finding the Function in Genome-Wide Association Studies and Whole-Genome Sequencing

PubMed Central

Schmouth, Jean-François; Bonaguro, Russell J.; Corso-Diaz, Ximena; Simpson, Elizabeth M.

2012-01-01

An increasing body of literature from genome-wide association studies and human whole-genome sequencing highlights the identification of large numbers of candidate regulatory variants of potential therapeutic interest in numerous diseases. Our relatively poor understanding of the functions of non-coding genomic sequence, and the slow and laborious process of experimental validation of the functional significance of human regulatory variants, limits our ability to fully benefit from this information in our efforts to comprehend human disease. Humanized mouse models (HuMMs), in which human genes are introduced into the mouse, suggest an approach to this problem. In the past, HuMMs have been used successfully to study human disease variants; e.g., the complex genetic condition arising from Down syndrome, common monogenic disorders such as Huntington disease and β-thalassemia, and cancer susceptibility genes such as BRCA1. In this commentary, we highlight a novel method for high-throughput single-copy site-specific generation of HuMMs entitled High-throughput Human Genes on the X Chromosome (HuGX). This method can be applied to most human genes for which a bacterial artificial chromosome (BAC) construct can be derived and a mouse-null allele exists. This strategy comprises (1) the use of recombineering technology to create a human variant–harbouring BAC, (2) knock-in of this BAC into the mouse genome using Hprt docking technology, and (3) allele comparison by interspecies complementation. We demonstrate the throughput of the HuGX method by generating a series of seven different alleles for the human NR2E1 gene at Hprt. In future challenges, we consider the current limitations of experimental approaches and call for a concerted effort by the genetics community, for both human and mouse, to solve the challenge of the functional analysis of human regulatory variation. PMID:22396661

New families of human regulatory RNA structures identified by comparative analysis of vertebrate genomes.

PubMed

Parker, Brian J; Moltke, Ida; Roth, Adam; Washietl, Stefan; Wen, Jiayu; Kellis, Manolis; Breaker, Ronald; Pedersen, Jakob Skou

2011-11-01

Regulatory RNA structures are often members of families with multiple paralogous instances across the genome. Family members share functional and structural properties, which allow them to be studied as a whole, facilitating both bioinformatic and experimental characterization. We have developed a comparative method, EvoFam, for genome-wide identification of families of regulatory RNA structures, based on primary sequence and secondary structure similarity. We apply EvoFam to a 41-way genomic vertebrate alignment. Genome-wide, we identify 220 human, high-confidence families outside protein-coding regions comprising 725 individual structures, including 48 families with known structural RNA elements. Known families identified include both noncoding RNAs, e.g., miRNAs and the recently identified MALAT1/MEN β lincRNA family; and cis-regulatory structures, e.g., iron-responsive elements. We also identify tens of new families supported by strong evolutionary evidence and other statistical evidence, such as GO term enrichments. For some of these, detailed analysis has led to the formulation of specific functional hypotheses. Examples include two hypothesized auto-regulatory feedback mechanisms: one involving six long hairpins in the 3'-UTR of MAT2A, a key metabolic gene that produces the primary human methyl donor S-adenosylmethionine; the other involving a tRNA-like structure in the intron of the tRNA maturation gene POP1. We experimentally validate the predicted MAT2A structures. Finally, we identify potential new regulatory networks, including large families of short hairpins enriched in immunity-related genes, e.g., TNF, FOS, and CTLA4, which include known transcript destabilizing elements. Our findings exemplify the diversity of post-transcriptional regulation and provide a resource for further characterization of new regulatory mechanisms and families of noncoding RNAs.

Genome-Wide Identification of Different Dormant Medicago sativa L. MicroRNAs in Response to Fall Dormancy

PubMed Central

Du, Hongqi; Sun, Xiaoge; Shi, Yinghua; Wang, Chengzhang

2014-01-01

Background MicroRNAs (miRNAs) are a class of regulatory small RNAs (sRNAs) that regulate gene post-transcriptional expression in plants and animals. High-throughput sequencing technology is capable of identifying small RNAs in plant species. Alfalfa (Medicago sativa L.) is one of the most widely cultivated perennial forage legumes worldwide, and fall dormancy is an adaptive characteristic related to the biomass production and winter survival in alfalfa. Here, we applied high-throughput sRNA sequencing to identify some miRNAs that were responsive to fall dormancy in standard variety (Maverick and CUF101) of alfalfa. Results Four sRNA libraries were generated and sequenced from alfalfa leaves in two typical varieties at distinct seasons. Through integrative analysis, we identified 51 novel miRNA candidates of 206 families. Additionally, we identified 28 miRNAs associated with fall dormancy in standard variety (Maverick and CUF101), including 20 known miRNAs and eight novel miRNAs. Both high-throughput sequencing and RT-qPCR confirmed that eight known miRNA members were up-regulated and six known miRNA members were down-regulated in response to fall dormancy in standard variety (Maverick and CUF101). Among the 51 novel miRNA candidates, five miRNAs were up-regulated and three miRNAs were down-regulated in response to fall dormancy in standard variety (Maverick and CUF101), and five of them were confirmed by Northern blot analysis. Conclusion We identified 20 known miRNAs and eight new miRNA candidates that were responsive to fall dormancy in standard variety (Maverick and CUF101) by high-throughput sequencing of small RNAs from Medicago sativa. Our data provide a useful resource for investigating miRNA-mediated regulatory mechanisms of fall dormancy in alfalfa, and these findings are important for our understanding of the roles played by miRNAs in the response of plants to abiotic stress in general and fall dormancy in alfalfa. PMID:25473944

Genome-wide identification of different dormant Medicago sativa L. MicroRNAs in response to fall dormancy.

PubMed

Fan, Wenna; Zhang, Senhao; Du, Hongqi; Sun, Xiaoge; Shi, Yinghua; Wang, Chengzhang

2014-01-01

MicroRNAs (miRNAs) are a class of regulatory small RNAs (sRNAs) that regulate gene post-transcriptional expression in plants and animals. High-throughput sequencing technology is capable of identifying small RNAs in plant species. Alfalfa (Medicago sativa L.) is one of the most widely cultivated perennial forage legumes worldwide, and fall dormancy is an adaptive characteristic related to the biomass production and winter survival in alfalfa. Here, we applied high-throughput sRNA sequencing to identify some miRNAs that were responsive to fall dormancy in standard variety (Maverick and CUF101) of alfalfa. Four sRNA libraries were generated and sequenced from alfalfa leaves in two typical varieties at distinct seasons. Through integrative analysis, we identified 51 novel miRNA candidates of 206 families. Additionally, we identified 28 miRNAs associated with fall dormancy in standard variety (Maverick and CUF101), including 20 known miRNAs and eight novel miRNAs. Both high-throughput sequencing and RT-qPCR confirmed that eight known miRNA members were up-regulated and six known miRNA members were down-regulated in response to fall dormancy in standard variety (Maverick and CUF101). Among the 51 novel miRNA candidates, five miRNAs were up-regulated and three miRNAs were down-regulated in response to fall dormancy in standard variety (Maverick and CUF101), and five of them were confirmed by Northern blot analysis. We identified 20 known miRNAs and eight new miRNA candidates that were responsive to fall dormancy in standard variety (Maverick and CUF101) by high-throughput sequencing of small RNAs from Medicago sativa. Our data provide a useful resource for investigating miRNA-mediated regulatory mechanisms of fall dormancy in alfalfa, and these findings are important for our understanding of the roles played by miRNAs in the response of plants to abiotic stress in general and fall dormancy in alfalfa.

Genetic Relatedness of Clostridium difficile Isolates from Various Origins Determined by Triple-Locus Sequence Analysis Based on Toxin Regulatory Genes tcdC, tcdR, and cdtR▿

PubMed Central

Bouvet, Philippe J. M.; Popoff, Michel R.

2008-01-01

A triple-locus nucleotide sequence analysis based on toxin regulatory genes tcdC, tcdR and cdtR was initiated to assess the sequence variability of these genes among Clostridium difficile isolates and to study the genetic relatedness between isolates. A preliminary investigation of the variability of the tcdC gene was done with 57 clinical and veterinary isolates. Twenty-three isolates representing nine main clusters were selected for tcdC, tcdR, and cdtR analysis. The numbers of alleles found for tcdC, tcdR and cdtR were nine, six, and five, respectively. All strains possessed the cdtR gene except toxin A-negative toxin B-positive variants. All but one binary toxin CDT-positive isolate harbored a deletion (>1 bp) in the tcdC gene. The combined analyses of the three genes allowed us to distinguish five lineages correlated with the different types of deletion in tcdC, i.e., 18 bp (associated or not with a deletion at position 117), 36 bp, 39 bp, and 54 bp, and with the wild-type tcdC (no deletion). The tcdR and tcdC genes, though located within the same pathogenicity locus, were found to have evolved separately. Coevolution of the three genes was noted only with strains harboring a 39-bp or a 54-bp deletion in tcdC that formed two homogeneous, separate divergent clusters. Our study supported the existence of the known clones (PCR ribotype 027 isolates and toxin A-negative toxin B-positive C. difficile variants) and evidence for clonality of isolates with a 39-bp deletion (toxinotype V, PCR ribotype 078) that are frequently isolated worldwide from human infections and from food animals. PMID:18832125

Promoter analysis reveals cis-regulatory motifs associated with the expression of the WRKY transcription factor CrWRKY1 in Catharanthus roseus.

PubMed

Yang, Zhirong; Patra, Barunava; Li, Runzhi; Pattanaik, Sitakanta; Yuan, Ling

2013-12-01

WRKY transcription factors (TFs) are emerging as an important group of regulators of plant secondary metabolism. However, the cis-regulatory elements associated with their regulation have not been well characterized. We have previously demonstrated that CrWRKY1, a member of subgroup III of the WRKY TF family, regulates biosynthesis of terpenoid indole alkaloids in the ornamental and medicinal plant, Catharanthus roseus. Here, we report the isolation and functional characterization of the CrWRKY1 promoter. In silico analysis of the promoter sequence reveals the presence of several potential TF binding motifs, indicating the involvement of additional TFs in the regulation of the TIA pathway. The CrWRKY1 promoter can drive the expression of a β-glucuronidase (GUS) reporter gene in native (C. roseus protoplasts and transgenic hairy roots) and heterologous (transgenic tobacco seedlings) systems. Analysis of 5'- or 3'-end deletions indicates that the sequence located between positions -140 to -93 bp and -3 to +113 bp, relative to the transcription start site, is critical for promoter activity. Mutation analysis shows that two overlapping as-1 elements and a CT-rich motif contribute significantly to promoter activity. The CrWRKY1 promoter is induced in response to methyl jasmonate (MJ) treatment and the promoter region between -230 and -93 bp contains a putative MJ-responsive element. The CrWRKY1 promoter can potentially be used as a tool to isolate novel TFs involved in the regulation of the TIA pathway.

MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing.

PubMed

Zackay, Arie; Steinhoff, Christine

2010-12-15

Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http://methvisual.molgen.mpg.de/ and from the Bioconductor Consortium http://www.bioconductor.org.

MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing

PubMed Central

2010-01-01

Background Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. Findings MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. Conclusions The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http://methvisual.molgen.mpg.de/ and from the Bioconductor Consortium http://www.bioconductor.org. PMID:21159174

Droplet barcoding for single cell transcriptomics applied to embryonic stem cells

PubMed Central

Klein, Allon M; Mazutis, Linas; Akartuna, Ilke; Tallapragada, Naren; Veres, Adrian; Li, Victor; Peshkin, Leonid; Weitz, David A; Kirschner, Marc W

2015-01-01

Summary It has long been the dream of biologists to map gene expression at the single cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after LIF withdrawal. The reproducibility of these high-throughput single cell data allowed us to deconstruct cell populations and infer gene expression relationships. PMID:26000487

Genome sequence of the model medicinal mushroom Ganoderma lucidum

PubMed Central

Chen, Shilin; Xu, Jiang; Liu, Chang; Zhu, Yingjie; Nelson, David R.; Zhou, Shiguo; Li, Chunfang; Wang, Lizhi; Guo, Xu; Sun, Yongzhen; Luo, Hongmei; Li, Ying; Song, Jingyuan; Henrissat, Bernard; Levasseur, Anthony; Qian, Jun; Li, Jianqin; Luo, Xiang; Shi, Linchun; He, Liu; Xiang, Li; Xu, Xiaolan; Niu, Yunyun; Li, Qiushi; Han, Mira V.; Yan, Haixia; Zhang, Jin; Chen, Haimei; Lv, Aiping; Wang, Zhen; Liu, Mingzhu; Schwartz, David C.; Sun, Chao

2012-01-01

Ganoderma lucidum is a widely used medicinal macrofungus in traditional Chinese medicine that creates a diverse set of bioactive compounds. Here we report its 43.3-Mb genome, encoding 16,113 predicted genes, obtained using next-generation sequencing and optical mapping approaches. The sequence analysis reveals an impressive array of genes encoding cytochrome P450s (CYPs), transporters and regulatory proteins that cooperate in secondary metabolism. The genome also encodes one of the richest sets of wood degradation enzymes among all of the sequenced basidiomycetes. In all, 24 physical CYP gene clusters are identified. Moreover, 78 CYP genes are coexpressed with lanosterol synthase, and 16 of these show high similarity to fungal CYPs that specifically hydroxylate testosterone, suggesting their possible roles in triterpenoid biosynthesis. The elucidation of the G. lucidum genome makes this organism a potential model system for the study of secondary metabolic pathways and their regulation in medicinal fungi. PMID:22735441

SuperSAGE analysis of the Nicotiana attenuata transcriptome after fatty acid-amino acid elicitation (FAC): identification of early mediators of insect responses

PubMed Central

2010-01-01

Background Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown. Results We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the M. sexta-N. attenuata interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated ≥ 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the M. sexta-N. attenuata interaction. Conclusions The analysis of the early changes in the transcriptome of N. attenuata after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process. PMID:20398280

SuperSAGE analysis of the Nicotiana attenuata transcriptome after fatty acid-amino acid elicitation (FAC): identification of early mediators of insect responses.

PubMed

Gilardoni, Paola A; Schuck, Stefan; Jüngling, Ruth; Rotter, Björn; Baldwin, Ian T; Bonaventure, Gustavo

2010-04-14

Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown. We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the M. sexta-N. attenuata interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated >or= 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the M. sexta-N. attenuata interaction. The analysis of the early changes in the transcriptome of N. attenuata after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process.

The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4)

DOE PAGES

Huntemann, Marcel; Ivanova, Natalia N.; Mavromatis, Konstantinos; ...

2015-10-26

The DOE-JGI Microbial Genome Annotation Pipeline performs structural and functional annotation of microbial genomes that are further included into the Integrated Microbial Genome comparative analysis system. MGAP is applied to assembled nucleotide sequence datasets that are provided via the IMG submission site. Dataset submission for annotation first requires project and associated metadata description in GOLD. The MGAP sequence data processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNA features, as well as CRISPR elements. In conclusion, structural annotation is followed by assignment of protein product names and functions.

The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4)

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

Huntemann, Marcel; Ivanova, Natalia N.; Mavromatis, Konstantinos

The DOE-JGI Microbial Genome Annotation Pipeline performs structural and functional annotation of microbial genomes that are further included into the Integrated Microbial Genome comparative analysis system. MGAP is applied to assembled nucleotide sequence datasets that are provided via the IMG submission site. Dataset submission for annotation first requires project and associated metadata description in GOLD. The MGAP sequence data processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNA features, as well as CRISPR elements. In conclusion, structural annotation is followed by assignment of protein product names and functions.

An Autonomous BMP2 Regulatory Element in Mesenchymal Cells

PubMed Central

Kruithof, Boudewijn P.T.; Fritz, David T.; Liu, Yijun; Garsetti, Diane E.; Frank, David B.; Pregizer, Steven K.; Gaussin, Vinciane; Mortlock, Douglas P.; Rogers, Melissa B.

2014-01-01

BMP2 is a morphogen that controls mesenchymal cell differentiation and behavior. For example, BMP2 concentration controls the differentiation of mesenchymal precursors into myocytes, adipocytes, chondrocytes, and osteoblasts. Sequences within the 3′untranslated region (UTR) of the Bmp2 mRNA mediate a post-transcriptional block of protein synthesis. Interaction of cell and developmental stage-specific trans-regulatory factors with the 3′UTR is a nimble and versatile mechanism for modulating this potent morphogen in different cell types. We show here, that an ultra-conserved sequence in the 3′UTR functions independently of promoter, coding region, and 3′UTR context in primary and immortalized tissue culture cells and in transgenic mice. Our findings indicate that the ultra-conserved sequence is an autonomously functioning post-transcriptional element that may be used to modulate the level of BMP2 and other proteins while retaining tissue specific regulatory elements. PMID:21268088

Principles of regulatory information conservation between mouse and human.

PubMed

Cheng, Yong; Ma, Zhihai; Kim, Bong-Hyun; Wu, Weisheng; Cayting, Philip; Boyle, Alan P; Sundaram, Vasavi; Xing, Xiaoyun; Dogan, Nergiz; Li, Jingjing; Euskirchen, Ghia; Lin, Shin; Lin, Yiing; Visel, Axel; Kawli, Trupti; Yang, Xinqiong; Patacsil, Dorrelyn; Keller, Cheryl A; Giardine, Belinda; Kundaje, Anshul; Wang, Ting; Pennacchio, Len A; Weng, Zhiping; Hardison, Ross C; Snyder, Michael P

2014-11-20

To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human-mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and with genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.

Transposable elements and G-quadruplexes.

PubMed

Kejnovsky, Eduard; Tokan, Viktor; Lexa, Matej

2015-09-01

A significant part of eukaryotic genomes is formed by transposable elements (TEs) containing not only genes but also regulatory sequences. Some of the regulatory sequences located within TEs can form secondary structures like hairpins or three-stranded (triplex DNA) and four-stranded (quadruplex DNA) conformations. This review focuses on recent evidence showing that G-quadruplex-forming sequences in particular are often present in specific parts of TEs in plants and humans. We discuss the potential role of these structures in the TE life cycle as well as the impact of G-quadruplexes on replication, transcription, translation, chromatin status, and recombination. The aim of this review is to emphasize that TEs may serve as vehicles for the genomic spread of G-quadruplexes. These non-canonical DNA structures and their conformational switches may constitute another regulatory system that, together with small and long non-coding RNA molecules and proteins, contribute to the complex cellular network resulting in the large diversity of eukaryotes.

GREAT: a web portal for Genome Regulatory Architecture Tools.

PubMed

Bouyioukos, Costas; Bucchini, François; Elati, Mohamed; Képès, François

2016-07-08

GREAT (Genome REgulatory Architecture Tools) is a novel web portal for tools designed to generate user-friendly and biologically useful analysis of genome architecture and regulation. The online tools of GREAT are freely accessible and compatible with essentially any operating system which runs a modern browser. GREAT is based on the analysis of genome layout -defined as the respective positioning of co-functional genes- and its relation with chromosome architecture and gene expression. GREAT tools allow users to systematically detect regular patterns along co-functional genomic features in an automatic way consisting of three individual steps and respective interactive visualizations. In addition to the complete analysis of regularities, GREAT tools enable the use of periodicity and position information for improving the prediction of transcription factor binding sites using a multi-view machine learning approach. The outcome of this integrative approach features a multivariate analysis of the interplay between the location of a gene and its regulatory sequence. GREAT results are plotted in web interactive graphs and are available for download either as individual plots, self-contained interactive pages or as machine readable tables for downstream analysis. The GREAT portal can be reached at the following URL https://absynth.issb.genopole.fr/GREAT and each individual GREAT tool is available for downloading. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

PubMed Central

Robinson, Lois; Panayiotakis, Alexandra; Papas, Takis S.; Kola, Ismail; Seth, Arun

1997-01-01

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene. PMID:9207063

Existence of a True Phosphofructokinase in Bacillus sphaericus: Cloning and Sequencing of the pfk Gene

PubMed Central

Alice, Alejandro F.; Pérez-Martínez, Gaspar; Sánchez-Rivas, Carmen

2002-01-01

Some strains of Bacillus sphaericus are entomopathogenic to mosquito larvae, which transmit diseases, such as filariasis and malaria, affecting millions of people worldwide. This species is unable to use hexoses and pentoses as unique carbon sources, which was proposed to be due to the lack of glycolytic enzymes, such as 6-phosphofructokinase (PFK). In this study, PFK activity was detected and the pfk gene was cloned and sequenced. Furthermore, this gene was shown to be present in strains belonging to all the homology groups of this heterogeneous species, in which PFK activity was also detected. A careful sequence analysis revealed the conservation of different catalytic and regulatory residues, as well as the enzyme's phylogenetic affiliation with the family of allosteric ATP-PFK enzymes. PMID:12450869

Comparative genomics of 9 novel Paenibacillus larvae bacteriophages

PubMed Central

Stamereilers, Casey; LeBlanc, Lucy; Yost, Diane; Amy, Penny S.; Tsourkas, Philippos K.

2016-01-01

ABSTRACT American Foulbrood Disease, caused by the bacterium Paenibacillus larvae, is one of the most destructive diseases of the honeybee, Apis mellifera. Our group recently published the sequences of 9 new phages with the ability to infect and lyse P. larvae. Here, we characterize the genomes of these P. larvae phages, compare them to each other and to other sequenced P. larvae phages, and putatively identify protein function. The phage genomes are 38–45 kb in size and contain 68–86 genes, most of which appear to be unique to P. larvae phages. We classify P. larvae phages into 2 main clusters and one singleton based on nucleotide sequence identity. Three of the new phages show sequence similarity to other sequenced P. larvae phages, while the remaining 6 do not. We identified functions for roughly half of the P. larvae phage proteins, including structural, assembly, host lysis, DNA replication/metabolism, regulatory, and host-related functions. Structural and assembly proteins are highly conserved among our phages and are located at the start of the genome. DNA replication/metabolism, regulatory, and host-related proteins are located in the middle and end of the genome, and are not conserved, with many of these genes found in some of our phages but not others. All nine phages code for a conserved N-acetylmuramoyl-L-alanine amidase. Comparative analysis showed the phages use the “cohesive ends with 3′ overhang” DNA packaging strategy. This work is the first in-depth study of P. larvae phage genomics, and serves as a marker for future work in this area. PMID:27738559

PlantTFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants.

PubMed

Jin, Jinpu; Tian, Feng; Yang, De-Chang; Meng, Yu-Qi; Kong, Lei; Luo, Jingchu; Gao, Ge

2017-01-04

With the goal of providing a comprehensive, high-quality resource for both plant transcription factors (TFs) and their regulatory interactions with target genes, we upgraded plant TF database PlantTFDB to version 4.0 (http://planttfdb.cbi.pku.edu.cn/). In the new version, we identified 320 370 TFs from 165 species, presenting a more comprehensive genomic TF repertoires of green plants. Besides updating the pre-existing abundant functional and evolutionary annotation for identified TFs, we generated three new types of annotation which provide more directly clues to investigate functional mechanisms underlying: (i) a set of high-quality, non-redundant TF binding motifs derived from experiments; (ii) multiple types of regulatory elements identified from high-throughput sequencing data; (iii) regulatory interactions curated from literature and inferred by combining TF binding motifs and regulatory elements. In addition, we upgraded previous TF prediction server, and set up four novel tools for regulation prediction and functional enrichment analyses. Finally, we set up a novel companion portal PlantRegMap (http://plantregmap.cbi.pku.edu.cn) for users to access the regulation resource and analysis tools conveniently. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis

PubMed Central

Moore, Michael; Zhang, Chaolin; Gantman, Emily Conn; Mele, Aldo; Darnell, Jennifer C.; Darnell, Robert B.

2014-01-01

Summary Identifying sites where RNA binding proteins (RNABPs) interact with target RNAs opens the door to understanding the vast complexity of RNA regulation. UV-crosslinking and immunoprecipitation (CLIP) is a transformative technology in which RNAs purified from in vivo cross-linked RNA-protein complexes are sequenced to reveal footprints of RNABP:RNA contacts. CLIP combined with high throughput sequencing (HITS-CLIP) is a generalizable strategy to produce transcriptome-wide RNA binding maps with higher accuracy and resolution than standard RNA immunoprecipitation (RIP) profiling or purely computational approaches. Applying CLIP to Argonaute proteins has expanded the utility of this approach to mapping binding sites for microRNAs and other small regulatory RNAs. Finally, recent advances in data analysis take advantage of crosslinked-induced mutation sites (CIMS) to refine RNA-binding maps to single-nucleotide resolution. Once IP conditions are established, HITS-CLIP takes approximately eight days to prepare RNA for sequencing. Established pipelines for data analysis, including for CIMS, take 3-4 days. PMID:24407355

Pyicos: a versatile toolkit for the analysis of high-throughput sequencing data.

PubMed

Althammer, Sonja; González-Vallinas, Juan; Ballaré, Cecilia; Beato, Miguel; Eyras, Eduardo

2011-12-15

High-throughput sequencing (HTS) has revolutionized gene regulation studies and is now fundamental for the detection of protein-DNA and protein-RNA binding, as well as for measuring RNA expression. With increasing variety and sequencing depth of HTS datasets, the need for more flexible and memory-efficient tools to analyse them is growing. We describe Pyicos, a powerful toolkit for the analysis of mapped reads from diverse HTS experiments: ChIP-Seq, either punctuated or broad signals, CLIP-Seq and RNA-Seq. We prove the effectiveness of Pyicos to select for significant signals and show that its accuracy is comparable and sometimes superior to that of methods specifically designed for each particular type of experiment. Pyicos facilitates the analysis of a variety of HTS datatypes through its flexibility and memory efficiency, providing a useful framework for data integration into models of regulatory genomics. Open-source software, with tutorials and protocol files, is available at http://regulatorygenomics.upf.edu/pyicos or as a Galaxy server at http://regulatorygenomics.upf.edu/galaxy eduardo.eyras@upf.edu Supplementary data are available at Bioinformatics online.

Biophysics and bioinformatics of transcription regulation in bacteria and bacteriophages

NASA Astrophysics Data System (ADS)

Djordjevic, Marko

2005-11-01

Due to rapid accumulation of biological data, bioinformatics has become a very important branch of biological research. In this thesis, we develop novel bioinformatic approaches and aid design of biological experiments by using ideas and methods from statistical physics. Identification of transcription factor binding sites within the regulatory segments of genomic DNA is an important step towards understanding of the regulatory circuits that control expression of genes. We propose a novel, biophysics based algorithm, for the supervised detection of transcription factor (TF) binding sites. The method classifies potential binding sites by explicitly estimating the sequence-specific binding energy and the chemical potential of a given TF. In contrast with the widely used information theory based weight matrix method, our approach correctly incorporates saturation in the transcription factor/DNA binding probability. This results in a significant reduction in the number of expected false positives, and in the explicit appearance---and determination---of a binding threshold. The new method was used to identify likely genomic binding sites for the Escherichia coli TFs, and to examine the relationship between TF binding specificity and degree of pleiotropy (number of regulatory targets). We next address how parameters of protein-DNA interactions can be obtained from data on protein binding to random oligos under controlled conditions (SELEX experiment data). We show that 'robust' generation of an appropriate data set is achieved by a suitable modification of the standard SELEX procedure, and propose a novel bioinformatic algorithm for analysis of such data. Finally, we use quantitative data analysis, bioinformatic methods and kinetic modeling to analyze gene expression strategies of bacterial viruses. We study bacteriophage Xp10 that infects rice pathogen Xanthomonas oryzae. Xp10 is an unusual bacteriophage, which has morphology and genome organization that most closely resembles temperate phages, such as lambda. It, however, encodes its own T7-like RNA polymerase (characteristic of virulent phages), whose role in gene expression was unclear. Our analysis resulted in quantitative understanding of the role of both host and phage RNA polymerase, and in the identification of the previously unknown promoter sequence for Xp10 RNA polymerase. More generally, an increasing number of phage genomes are being sequenced every year, and we expect that methods of quantitative data analysis that we introduced will provide an efficient way to study gene expression strategies of novel bacterial viruses.

Simple Shared Motifs (SSM) in conserved region of promoters: a new approach to identify co-regulation patterns.

PubMed

Gruel, Jérémy; LeBorgne, Michel; LeMeur, Nolwenn; Théret, Nathalie

2011-09-12

Regulation of gene expression plays a pivotal role in cellular functions. However, understanding the dynamics of transcription remains a challenging task. A host of computational approaches have been developed to identify regulatory motifs, mainly based on the recognition of DNA sequences for transcription factor binding sites. Recent integration of additional data from genomic analyses or phylogenetic footprinting has significantly improved these methods. Here, we propose a different approach based on the compilation of Simple Shared Motifs (SSM), groups of sequences defined by their length and similarity and present in conserved sequences of gene promoters. We developed an original algorithm to search and count SSM in pairs of genes. An exceptional number of SSM is considered as a common regulatory pattern. The SSM approach is applied to a sample set of genes and validated using functional gene-set enrichment analyses. We demonstrate that the SSM approach selects genes that are over-represented in specific biological categories (Ontology and Pathways) and are enriched in co-expressed genes. Finally we show that genes co-expressed in the same tissue or involved in the same biological pathway have increased SSM values. Using unbiased clustering of genes, Simple Shared Motifs analysis constitutes an original contribution to provide a clearer definition of expression networks.

Simple Shared Motifs (SSM) in conserved region of promoters: a new approach to identify co-regulation patterns

PubMed Central

2011-01-01

Background Regulation of gene expression plays a pivotal role in cellular functions. However, understanding the dynamics of transcription remains a challenging task. A host of computational approaches have been developed to identify regulatory motifs, mainly based on the recognition of DNA sequences for transcription factor binding sites. Recent integration of additional data from genomic analyses or phylogenetic footprinting has significantly improved these methods. Results Here, we propose a different approach based on the compilation of Simple Shared Motifs (SSM), groups of sequences defined by their length and similarity and present in conserved sequences of gene promoters. We developed an original algorithm to search and count SSM in pairs of genes. An exceptional number of SSM is considered as a common regulatory pattern. The SSM approach is applied to a sample set of genes and validated using functional gene-set enrichment analyses. We demonstrate that the SSM approach selects genes that are over-represented in specific biological categories (Ontology and Pathways) and are enriched in co-expressed genes. Finally we show that genes co-expressed in the same tissue or involved in the same biological pathway have increased SSM values. Conclusions Using unbiased clustering of genes, Simple Shared Motifs analysis constitutes an original contribution to provide a clearer definition of expression networks. PMID:21910886

Functional annotation of HOT regions in the human genome: implications for human disease and cancer

PubMed Central

Li, Hao; Chen, Hebing; Liu, Feng; Ren, Chao; Wang, Shengqi; Bo, Xiaochen; Shu, Wenjie

2015-01-01

Advances in genome-wide association studies (GWAS) and large-scale sequencing studies have resulted in an impressive and growing list of disease- and trait-associated genetic variants. Most studies have emphasised the discovery of genetic variation in coding sequences, however, the noncoding regulatory effects responsible for human disease and cancer biology have been substantially understudied. To better characterise the cis-regulatory effects of noncoding variation, we performed a comprehensive analysis of the genetic variants in HOT (high-occupancy target) regions, which are considered to be one of the most intriguing findings of recent large-scale sequencing studies. We observed that GWAS variants that map to HOT regions undergo a substantial net decrease and illustrate development-specific localisation during haematopoiesis. Additionally, genetic risk variants are disproportionally enriched in HOT regions compared with LOT (low-occupancy target) regions in both disease-relevant and cancer cells. Importantly, this enrichment is biased toward disease- or cancer-specific cell types. Furthermore, we observed that cancer cells generally acquire cancer-specific HOT regions at oncogenes through diverse mechanisms of cancer pathogenesis. Collectively, our findings demonstrate the key roles of HOT regions in human disease and cancer and represent a critical step toward further understanding disease biology, diagnosis, and therapy. PMID:26113264

Functional annotation of HOT regions in the human genome: implications for human disease and cancer.

PubMed

Li, Hao; Chen, Hebing; Liu, Feng; Ren, Chao; Wang, Shengqi; Bo, Xiaochen; Shu, Wenjie

2015-06-26

Advances in genome-wide association studies (GWAS) and large-scale sequencing studies have resulted in an impressive and growing list of disease- and trait-associated genetic variants. Most studies have emphasised the discovery of genetic variation in coding sequences, however, the noncoding regulatory effects responsible for human disease and cancer biology have been substantially understudied. To better characterise the cis-regulatory effects of noncoding variation, we performed a comprehensive analysis of the genetic variants in HOT (high-occupancy target) regions, which are considered to be one of the most intriguing findings of recent large-scale sequencing studies. We observed that GWAS variants that map to HOT regions undergo a substantial net decrease and illustrate development-specific localisation during haematopoiesis. Additionally, genetic risk variants are disproportionally enriched in HOT regions compared with LOT (low-occupancy target) regions in both disease-relevant and cancer cells. Importantly, this enrichment is biased toward disease- or cancer-specific cell types. Furthermore, we observed that cancer cells generally acquire cancer-specific HOT regions at oncogenes through diverse mechanisms of cancer pathogenesis. Collectively, our findings demonstrate the key roles of HOT regions in human disease and cancer and represent a critical step toward further understanding disease biology, diagnosis, and therapy.

Isolation and Characterization of the PKAr Gene From a Plant Pathogen, Curvularia lunata.

PubMed

Liu, T; Ma, B C; Hou, J M; Zuo, Y H

2014-09-01

By using EST database from a full-length cDNA library of Curvularia lunata, we have isolated a 2.9 kb cDNA, termed PKAr. An ORF of 1,383 bp encoding a polypeptide of 460 amino acids with molecular weight 50.1 kDa, (GeneBank Acc. No. KF675744) was cloned. The deduced amino acid sequence of the PKAr shows 90 and 88 % identity with cAMP-dependent protein kinase A regulatory subunit from Alternaria alternate and Pyrenophora tritici-repentis Pt-1C-BFP, respectively. Database analysis revealed that the deduced amino acid sequence of PKAr shares considerable similarity with that of PKA regulatory subunits in other organisms, particularly in the conserved regions. No introns were identified within the 1,383 bp of ORF compared with PKAr genomic DNA sequence. Southern blot indicated that PKAr existed as a single copy per genome. The mRNA expression level of PKAr in different development stages were demonstrated using real-time quantitative PCR. The results showed that the level of PKAr expression was highest in vegetative growth mycelium, which indicated it might play an important role in the vegetative growth of C. lunata. These results provided a fundamental supporting research on the function of PKAr in plant pathogen, C. lunata.

K2 and K2*: efficient alignment-free sequence similarity measurement based on Kendall statistics.

PubMed

Lin, Jie; Adjeroh, Donald A; Jiang, Bing-Hua; Jiang, Yue

2018-05-15

Alignment-free sequence comparison methods can compute the pairwise similarity between a huge number of sequences much faster than sequence-alignment based methods. We propose a new non-parametric alignment-free sequence comparison method, called K2, based on the Kendall statistics. Comparing to the other state-of-the-art alignment-free comparison methods, K2 demonstrates competitive performance in generating the phylogenetic tree, in evaluating functionally related regulatory sequences, and in computing the edit distance (similarity/dissimilarity) between sequences. Furthermore, the K2 approach is much faster than the other methods. An improved method, K2*, is also proposed, which is able to determine the appropriate algorithmic parameter (length) automatically, without first considering different values. Comparative analysis with the state-of-the-art alignment-free sequence similarity methods demonstrates the superiority of the proposed approaches, especially with increasing sequence length, or increasing dataset sizes. The K2 and K2* approaches are implemented in the R language as a package and is freely available for open access (http://community.wvu.edu/daadjeroh/projects/K2/K2_1.0.tar.gz). yueljiang@163.com. Supplementary data are available at Bioinformatics online.

Genome-wide identification of Hfq-regulated small RNAs in the fire blight pathogen Erwinia amylovora discovered small RNAs with virulence regulatory function.

PubMed

Zeng, Quan; Sundin, George W

2014-05-31

Erwinia amylovora is a phytopathogenic bacterium and causal agent of fire blight disease in apples and pears. Although many virulence factors have been characterized, the coordination of expression of these virulence factors in E. amylovora is still not clear. Regulatory small RNAs (sRNAs) are important post-transcriptional regulatory components in bacteria. A large number of sRNAs require the RNA chaperone Hfq for both stability and functional activation. In E. amylovora, Hfq was identified as a major regulator of virulence and various virulence traits. However, information is still lacking about Hfq-dependent sRNAs on a genome scale, including the virulence regulatory functions of these sRNAs in E. amylovora. Using both an RNA-seq analysis and a Rho-independent terminator search, 40 candidate Hfq-dependent sRNAs were identified in E. amylovora. The expression and sizes of 12 sRNAs and the sequence boundaries of seven sRNAs were confirmed by Northern blot and 5' RACE assay respectively. Sequence conservation analysis identified sRNAs conserved only in the Erwinia genus as well as E. amylovora species-specific sRNAs. In addition, a dynamic re-patterning of expression of Hfq-dependent sRNAs was observed at 6 and 12 hours after induction in Hrp-inducing minimal medium. Furthermore, sRNAs that control virulence traits were characterized, among which ArcZ positively controls the type III secretion system (T3SS), amylovoran exopolysaccahride production, biofilm formation, and motility, and negatively modulates attachment while RmaA (Hrs6) and OmrAB both negatively regulate amylovoran production and positively regulate motility. This study has significantly enhanced our understanding of the Hfq-dependent sRNAs in E. amylovora at the genome level. The identification of multiple virulence-regulating sRNAs also suggests that post-transcriptional regulation by sRNAs may play a role in the deployment of virulence factors needed during varying stages of pathogenesis during host invasion by E. amylovora.

Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation.

PubMed

Tao, Wenjing; Sun, Lina; Shi, Hongjuan; Cheng, Yunying; Jiang, Dongneng; Fu, Beide; Conte, Matthew A; Gammerdinger, William J; Kocher, Thomas D; Wang, Deshou

2016-05-04

MicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia. We identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish. The sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.

Large-Scale Biomonitoring of Remote and Threatened Ecosystems via High-Throughput Sequencing

PubMed Central

Gibson, Joel F.; Shokralla, Shadi; Curry, Colin; Baird, Donald J.; Monk, Wendy A.; King, Ian; Hajibabaei, Mehrdad

2015-01-01

Biodiversity metrics are critical for assessment and monitoring of ecosystems threatened by anthropogenic stressors. Existing sorting and identification methods are too expensive and labour-intensive to be scaled up to meet management needs. Alternately, a high-throughput DNA sequencing approach could be used to determine biodiversity metrics from bulk environmental samples collected as part of a large-scale biomonitoring program. Here we show that both morphological and DNA sequence-based analyses are suitable for recovery of individual taxonomic richness, estimation of proportional abundance, and calculation of biodiversity metrics using a set of 24 benthic samples collected in the Peace-Athabasca Delta region of Canada. The high-throughput sequencing approach was able to recover all metrics with a higher degree of taxonomic resolution than morphological analysis. The reduced cost and increased capacity of DNA sequence-based approaches will finally allow environmental monitoring programs to operate at the geographical and temporal scale required by industrial and regulatory end-users. PMID:26488407

APPLaUD: access for patients and participants to individual level uninterpreted genomic data.

PubMed

Thorogood, Adrian; Bobe, Jason; Prainsack, Barbara; Middleton, Anna; Scott, Erick; Nelson, Sarah; Corpas, Manuel; Bonhomme, Natasha; Rodriguez, Laura Lyman; Murtagh, Madeleine; Kleiderman, Erika

2018-02-17

There is a growing support for the stance that patients and research participants should have better and easier access to their raw (uninterpreted) genomic sequence data in both clinical and research contexts. We review legal frameworks and literature on the benefits, risks, and practical barriers of providing individuals access to their data. We also survey genomic sequencing initiatives that provide or plan to provide individual access. Many patients and research participants expect to be able to access their health and genomic data. Individuals have a legal right to access their genomic data in some countries and contexts. Moreover, increasing numbers of participatory research projects, direct-to-consumer genetic testing companies, and now major national sequencing initiatives grant individuals access to their genomic sequence data upon request. Drawing on current practice and regulatory analysis, we outline legal, ethical, and practical guidance for genomic sequencing initiatives seeking to offer interested patients and participants access to their raw genomic data.

Mapping cis-Regulatory Domains in the Human Genome UsingMulti-Species Conservation of Synteny

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

Ahituv, Nadav; Prabhakar, Shyam; Poulin, Francis

2005-06-13

Our inability to associate distant regulatory elements with the genes that they regulate has largely precluded their examination for sequence alterations contributing to human disease. One major obstacle is the large genomic space surrounding targeted genes in which such elements could potentially reside. In order to delineate gene regulatory boundaries we used whole-genome human-mouse-chicken (HMC) and human-mouse-frog (HMF) multiple alignments to compile conserved blocks of synteny (CBS), under the hypothesis that these blocks have been kept intact throughout evolution at least in part by the requirement of regulatory elements to stay linked to the genes that they regulate. A totalmore » of 2,116 and 1,942 CBS>200 kb were assembled for HMC and HMF respectively, encompassing 1.53 and 0.86 Gb of human sequence. To support the existence of complex long-range regulatory domains within these CBS we analyzed the prevalence and distribution of chromosomal aberrations leading to position effects (disruption of a genes regulatory environment), observing a clear bias not only for mapping onto CBS but also for longer CBS size. Our results provide a genome wide data set characterizing the regulatory domains of genes and the conserved regulatory elements within them.« less

Integrative analysis of 111 reference human epigenomes

PubMed Central

Kundaje, Anshul; Meuleman, Wouter; Ernst, Jason; Bilenky, Misha; Yen, Angela; Kheradpour, Pouya; Zhang, Zhizhuo; Heravi-Moussavi, Alireza; Liu, Yaping; Amin, Viren; Ziller, Michael J; Whitaker, John W; Schultz, Matthew D; Sandstrom, Richard S; Eaton, Matthew L; Wu, Yi-Chieh; Wang, Jianrong; Ward, Lucas D; Sarkar, Abhishek; Quon, Gerald; Pfenning, Andreas; Wang, Xinchen; Claussnitzer, Melina; Coarfa, Cristian; Harris, R Alan; Shoresh, Noam; Epstein, Charles B; Gjoneska, Elizabeta; Leung, Danny; Xie, Wei; Hawkins, R David; Lister, Ryan; Hong, Chibo; Gascard, Philippe; Mungall, Andrew J; Moore, Richard; Chuah, Eric; Tam, Angela; Canfield, Theresa K; Hansen, R Scott; Kaul, Rajinder; Sabo, Peter J; Bansal, Mukul S; Carles, Annaick; Dixon, Jesse R; Farh, Kai-How; Feizi, Soheil; Karlic, Rosa; Kim, Ah-Ram; Kulkarni, Ashwinikumar; Li, Daofeng; Lowdon, Rebecca; Mercer, Tim R; Neph, Shane J; Onuchic, Vitor; Polak, Paz; Rajagopal, Nisha; Ray, Pradipta; Sallari, Richard C; Siebenthall, Kyle T; Sinnott-Armstrong, Nicholas; Stevens, Michael; Thurman, Robert E; Wu, Jie; Zhang, Bo; Zhou, Xin; Beaudet, Arthur E; Boyer, Laurie A; De Jager, Philip; Farnham, Peggy J; Fisher, Susan J; Haussler, David; Jones, Steven; Li, Wei; Marra, Marco; McManus, Michael T; Sunyaev, Shamil; Thomson, James A; Tlsty, Thea D; Tsai, Li-Huei; Wang, Wei; Waterland, Robert A; Zhang, Michael; Chadwick, Lisa H; Bernstein, Bradley E; Costello, Joseph F; Ecker, Joseph R; Hirst, Martin; Meissner, Alexander; Milosavljevic, Aleksandar; Ren, Bing; Stamatoyannopoulos, John A; Wang, Ting; Kellis, Manolis

2015-01-01

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but a similar reference has lacked for epigenomic studies. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection to-date of human epigenomes for primary cells and tissues. Here, we describe the integrative analysis of 111 reference human epigenomes generated as part of the program, profiled for histone modification patterns, DNA accessibility, DNA methylation, and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically-relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation, and human disease. PMID:25693563

Integrative analysis of 111 reference human epigenomes.

PubMed

Kundaje, Anshul; Meuleman, Wouter; Ernst, Jason; Bilenky, Misha; Yen, Angela; Heravi-Moussavi, Alireza; Kheradpour, Pouya; Zhang, Zhizhuo; Wang, Jianrong; Ziller, Michael J; Amin, Viren; Whitaker, John W; Schultz, Matthew D; Ward, Lucas D; Sarkar, Abhishek; Quon, Gerald; Sandstrom, Richard S; Eaton, Matthew L; Wu, Yi-Chieh; Pfenning, Andreas R; Wang, Xinchen; Claussnitzer, Melina; Liu, Yaping; Coarfa, Cristian; Harris, R Alan; Shoresh, Noam; Epstein, Charles B; Gjoneska, Elizabeta; Leung, Danny; Xie, Wei; Hawkins, R David; Lister, Ryan; Hong, Chibo; Gascard, Philippe; Mungall, Andrew J; Moore, Richard; Chuah, Eric; Tam, Angela; Canfield, Theresa K; Hansen, R Scott; Kaul, Rajinder; Sabo, Peter J; Bansal, Mukul S; Carles, Annaick; Dixon, Jesse R; Farh, Kai-How; Feizi, Soheil; Karlic, Rosa; Kim, Ah-Ram; Kulkarni, Ashwinikumar; Li, Daofeng; Lowdon, Rebecca; Elliott, GiNell; Mercer, Tim R; Neph, Shane J; Onuchic, Vitor; Polak, Paz; Rajagopal, Nisha; Ray, Pradipta; Sallari, Richard C; Siebenthall, Kyle T; Sinnott-Armstrong, Nicholas A; Stevens, Michael; Thurman, Robert E; Wu, Jie; Zhang, Bo; Zhou, Xin; Beaudet, Arthur E; Boyer, Laurie A; De Jager, Philip L; Farnham, Peggy J; Fisher, Susan J; Haussler, David; Jones, Steven J M; Li, Wei; Marra, Marco A; McManus, Michael T; Sunyaev, Shamil; Thomson, James A; Tlsty, Thea D; Tsai, Li-Huei; Wang, Wei; Waterland, Robert A; Zhang, Michael Q; Chadwick, Lisa H; Bernstein, Bradley E; Costello, Joseph F; Ecker, Joseph R; Hirst, Martin; Meissner, Alexander; Milosavljevic, Aleksandar; Ren, Bing; Stamatoyannopoulos, John A; Wang, Ting; Kellis, Manolis

2015-02-19

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Genome-wide DNase hypersensitivity, and occupancy of RUNX2 and CTCF reveal a highly dynamic gene regulome during MC3T3 pre-osteoblast differentiation.

PubMed

Tai, Phillip W L; Wu, Hai; van Wijnen, André J; Stein, Gary S; Stein, Janet L; Lian, Jane B

2017-01-01

The ability to discover regulatory sequences that control bone-related genes during development has been greatly improved by massively parallel sequencing methodologies. To expand our understanding of cis-regulatory regions critical to the control of gene expression during osteoblastogenesis, we probed the presence of open chromatin states across the osteoblast genome using global DNase hypersensitivity (DHS) mapping. Our profiling of MC3T3 mouse pre-osteoblasts during differentiation has identified more than 224,000 unique DHS sites. Approximately 65% of these sites are dynamic during temporal stages of osteoblastogenesis, and a majority of them are located within non-promoter (intergenic and intronic) regions. Nearly half of all DHS sites (both constitutive and dynamic) overlap binding events of the bone-essential RUNX2 and/or the chromatin-related CTCF transcription factors. This finding reinforces the role of these regulatory proteins as essential components of the bone gene regulome. We observe a reduction in chromatin accessibility throughout the genome between pre-osteoblast and early osteoblasts. Our analysis also defined a class of differentially expressed genes that harbor DHS peaks centered within 1 kb downstream of transcriptional end sites (TES). These DHSs at the 3'-flanks of genes exhibit dynamic changes during differentiation that may impact regulation of the osteoblast genome. Taken together, the distribution of DHS regions within non-promoter locations harboring osteoblast and chromatin related transcription factor binding motifs, reflect novel cis-regulatory requirements to support temporal gene expression in differentiating osteoblasts.

Cloning and Characterization of 5′ Flanking Regulatory Sequences of AhLEC1B Gene from Arachis Hypogaea L.

PubMed Central

Tang, Guiying; Xu, Pingli; Liu, Wei; Liu, Zhanji; Shan, Lei

2015-01-01

LEAFY COTYLEDON1 (LEC1) is a B subunit of Nuclear Factor Y (NF-YB) transcription factor that mainly accumulates during embryo development. We cloned the 5′ flanking regulatory sequence of AhLEC1B gene, a homolog of Arabidopsis LEC1, and analyzed its regulatory elements using online software. To identify the crucial regulatory region, we generated a series of GUS expression frameworks driven by different length promoters with 5′ terminal and/or 3′ terminal deletion. We further characterized the GUS expression patterns in the transgenic Arabidopsis lines. Our results show that both the 65bp proximal promoter region and the 52bp 5′ UTR of AhLEC1B contain the key motifs required for the essential promoting activity. Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements. PMID:26426444

Linking disease-associated genes to regulatory networks via promoter organization

PubMed Central

Döhr, S.; Klingenhoff, A.; Maier, H.; de Angelis, M. Hrabé; Werner, T.; Schneider, R.

2005-01-01

Pathway- or disease-associated genes may participate in more than one transcriptional co-regulation network. Such gene groups can be readily obtained by literature analysis or by high-throughput techniques such as microarrays or protein-interaction mapping. We developed a strategy that defines regulatory networks by in silico promoter analysis, finding potentially co-regulated subgroups without a priori knowledge. Pairs of transcription factor binding sites conserved in orthologous genes (vertically) as well as in promoter sequences of co-regulated genes (horizontally) were used as seeds for the development of promoter models representing potential co-regulation. This approach was applied to a Maturity Onset Diabetes of the Young (MODY)-associated gene list, which yielded two models connecting functionally interacting genes within MODY-related insulin/glucose signaling pathways. Additional genes functionally connected to our initial gene list were identified by database searches with these promoter models. Thus, data-driven in silico promoter analysis allowed integrating molecular mechanisms with biological functions of the cell. PMID:15701758

Multiple microRNAs regulate human FOXP2 gene expression by targeting sequences in its 3' untranslated region.

PubMed

Fu, Lijuan; Shi, Zhimin; Luo, Guanzheng; Tu, Weihong; Wang, XiuJie; Fang, Zhide; Li, XiaoChing

2014-10-01

Mutations in the human FOXP2 gene cause speech and language impairments. The FOXP2 protein is a transcription factor that regulates the expression of many downstream genes, which may have important roles in nervous system development and function. An adequate amount of functional FOXP2 protein is thought to be critical for the proper development of the neural circuitry underlying speech and language. However, how FOXP2 gene expression is regulated is not clearly understood. The FOXP2 mRNA has an approximately 4-kb-long 3' untranslated region (3' UTR), twice as long as its protein coding region, indicating that FOXP2 can be regulated by microRNAs (miRNAs). We identified multiple miRNAs that regulate the expression of the human FOXP2 gene using sequence analysis and in vitro cell systems. Focusing on let-7a, miR-9, and miR-129-5p, three brain-enriched miRNAs, we show that these miRNAs regulate human FOXP2 expression in a dosage-dependent manner and target specific sequences in the FOXP2 3' UTR. We further show that these three miRNAs are expressed in the cerebellum of the human fetal brain, where FOXP2 is known to be expressed. Our results reveal novel regulatory functions of the human FOXP2 3' UTR sequence and regulatory interactions between multiple miRNAs and the human FOXP2 gene. The expression of let-7a, miR-9, and miR-129-5p in the human fetal cerebellum is consistent with their roles in regulating FOXP2 expression during early cerebellum development. These results suggest that various genetic and environmental factors may contribute to speech and language development and related neural developmental disorders via the miRNA-FOXP2 regulatory network.

A novel approach to identifying regulatory motifs in distantly related genomes

PubMed Central

Van Hellemont, Ruth; Monsieurs, Pieter; Thijs, Gert; De Moor, Bart; Van de Peer, Yves; Marchal, Kathleen

2005-01-01

Although proven successful in the identification of regulatory motifs, phylogenetic footprinting methods still show some shortcomings. To assess these difficulties, most apparent when applying phylogenetic footprinting to distantly related organisms, we developed a two-step procedure that combines the advantages of sequence alignment and motif detection approaches. The results on well-studied benchmark datasets indicate that the presented method outperforms other methods when the sequences become either too long or too heterogeneous in size. PMID:16420672

m6aViewer: software for the detection, analysis, and visualization of N6-methyladenosine peaks from m6A-seq/ME-RIP sequencing data.

PubMed

Antanaviciute, Agne; Baquero-Perez, Belinda; Watson, Christopher M; Harrison, Sally M; Lascelles, Carolina; Crinnion, Laura; Markham, Alexander F; Bonthron, David T; Whitehouse, Adrian; Carr, Ian M

2017-10-01

Recent methods for transcriptome-wide N 6 -methyladenosine (m 6 A) profiling have facilitated investigations into the RNA methylome and established m 6 A as a dynamic modification that has critical regulatory roles in gene expression and may play a role in human disease. However, bioinformatics resources available for the analysis of m 6 A sequencing data are still limited. Here, we describe m6aViewer-a cross-platform application for analysis and visualization of m 6 A peaks from sequencing data. m6aViewer implements a novel m 6 A peak-calling algorithm that identifies high-confidence methylated residues with more precision than previously described approaches. The application enables data analysis through a graphical user interface, and thus, in contrast to other currently available tools, does not require the user to be skilled in computer programming. m6aViewer and test data can be downloaded here: http://dna2.leeds.ac.uk/m6a. © 2017 Antanaviciute et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

LexA Binds to Transcription Regulatory Site of Cell Division Gene ftsZ in Toxic Cyanobacterium Microcystis aeruginosa.

PubMed

Honda, Takashi; Morimoto, Daichi; Sako, Yoshihiko; Yoshida, Takashi

2018-05-17

Previously, we showed that DNA replication and cell division in toxic cyanobacterium Microcystis aeruginosa are coordinated by transcriptional regulation of cell division gene ftsZ and that an unknown protein specifically bound upstream of ftsZ (BpFz; DNA-binding protein to an upstream site of ftsZ) during successful DNA replication and cell division. Here, we purified BpFz from M. aeruginosa strain NIES-298 using DNA-affinity chromatography and gel-slicing combined with gel electrophoresis mobility shift assay (EMSA). The N-terminal amino acid sequence of BpFz was identified as TNLESLTQ, which was identical to that of transcription repressor LexA from NIES-843. EMSA analysis using mutant probes showed that the sequence GTACTAN 3 GTGTTC was important in LexA binding. Comparison of the upstream regions of lexA in the genomes of closely related cyanobacteria suggested that the sequence TASTRNNNNTGTWC could be a putative LexA recognition sequence (LexA box). Searches for TASTRNNNNTGTWC as a transcriptional regulatory site (TRS) in the genome of M. aeruginosa NIES-843 showed that it was present in genes involved in cell division, photosynthesis, and extracellular polysaccharide biosynthesis. Considering that BpFz binds to the TRS of ftsZ during normal cell division, LexA may function as a transcriptional activator of genes related to cell reproduction in M. aeruginosa, including ftsZ. This may be an example of informality in the control of bacterial cell division.

College of American Pathologists' laboratory standards for next-generation sequencing clinical tests.

PubMed

Aziz, Nazneen; Zhao, Qin; Bry, Lynn; Driscoll, Denise K; Funke, Birgit; Gibson, Jane S; Grody, Wayne W; Hegde, Madhuri R; Hoeltge, Gerald A; Leonard, Debra G B; Merker, Jason D; Nagarajan, Rakesh; Palicki, Linda A; Robetorye, Ryan S; Schrijver, Iris; Weck, Karen E; Voelkerding, Karl V

2015-04-01

The higher throughput and lower per-base cost of next-generation sequencing (NGS) as compared to Sanger sequencing has led to its rapid adoption in clinical testing. The number of laboratories offering NGS-based tests has also grown considerably in the past few years, despite the fact that specific Clinical Laboratory Improvement Amendments of 1988/College of American Pathologists (CAP) laboratory standards had not yet been developed to regulate this technology. To develop a checklist for clinical testing using NGS technology that sets standards for the analytic wet bench process and for bioinformatics or "dry bench" analyses. As NGS-based clinical tests are new to diagnostic testing and are of much greater complexity than traditional Sanger sequencing-based tests, there is an urgent need to develop new regulatory standards for laboratories offering these tests. To develop the necessary regulatory framework for NGS and to facilitate appropriate adoption of this technology for clinical testing, CAP formed a committee in 2011, the NGS Work Group, to deliberate upon the contents to be included in the checklist. Results . -A total of 18 laboratory accreditation checklist requirements for the analytic wet bench process and bioinformatics analysis processes have been included within CAP's molecular pathology checklist (MOL). This report describes the important issues considered by the CAP committee during the development of the new checklist requirements, which address documentation, validation, quality assurance, confirmatory testing, exception logs, monitoring of upgrades, variant interpretation and reporting, incidental findings, data storage, version traceability, and data transfer confidentiality.

Whole-Genome Sequencing and Comparative Genome Analysis of Bacillus subtilis Strains Isolated from Non-Salted Fermented Soybean Foods

PubMed Central

Kamada, Mayumi; Hase, Sumitaka; Fujii, Kazushi; Miyake, Masato; Sato, Kengo; Kimura, Keitarou; Sakakibara, Yasubumi

2015-01-01

Bacillus subtilis is the main component in the fermentation of soybeans. To investigate the genetics of the soybean-fermenting B. subtilis strains and its relationship with the productivity of extracellular poly-γ-glutamic acid (γPGA), we sequenced the whole genome of eight B. subtilis stains isolated from non-salted fermented soybean foods in Southeast Asia. Assembled nucleotide sequences were compared with those of a natto (fermented soybean food) starter strain B. subtilis BEST195 and the laboratory standard strain B. subtilis 168 that is incapable of γPGA production. Detected variants were investigated in terms of insertion sequences, biotin synthesis, production of subtilisin NAT, and regulatory genes for γPGA synthesis, which were related to fermentation process. Comparing genome sequences, we found that the strains that produce γPGA have a deletion in a protein that constitutes the flagellar basal body, and this deletion was not found in the non-producing strains. We further identified diversity in variants of the bio operon, which is responsible for the biotin auxotrophism of the natto starter strains. Phylogenetic analysis using multilocus sequencing typing revealed that the B. subtilis strains isolated from the non-salted fermented soybeans were not clustered together, while the natto-fermenting strains were tightly clustered; this analysis also suggested that the strain isolated from “Tua Nao” of Thailand traces a different evolutionary process from other strains. PMID:26505996

Identification and nucleotide sequence analysis of the repetitive DNA element in the genome of fish lymphocystis disease virus.

PubMed

Schnitzler, P; Delius, H; Scholz, J; Touray, M; Orth, E; Darai, G

1987-12-01

The genome of the fish lymphocystis disease virus (FLDV) was screened for the existence of repetitive DNA sequences using a defined and complete gene library of the viral genome (98 kbp) by DNA-DNA hybridization, heteroduplex analysis, and restriction fine mapping. A repetitive DNA sequence was detected at the coordinates 0.034 to 0.057 and 0.718 to 0.736 map units (m.u.) of the FLDV genome. The first region (0.034 to 0.057 m.u.) corresponds to the 5' terminus of the EcoRI FLDV DNA fragment B (0.034 to 0.165 m.u.) and the second region (0.718 to 0.736 m.u.) is identical to the EcoRI DNA fragment M of the viral genome. The DNA nucleotide sequence of the EcoRI FLDV DNA fragment M was determined. This analysis revealed the presence of many short direct and inverted repetitions, e.g., a 18-mer direct repetition (TTTAAAATTTAATTAA) that started at nucleotide positions 812 and 942 and a 14-mer inverted repeat (TTAAATTTAAATTT) at nucleotide positions 820 and 959. Only short open reading frames were detected within this region. The DNA repetitions are discussed as sequences that play a possible regulatory role for virus replication. Furthermore, hybridization experiments revealed that the repetitive DNA sequences are conserved in the genome of different strains of fish lymphocystis disease virus isolated from two species of Pleuronectidae (flounder and dab).

The FDA's Experience with Emerging Genomics Technologies-Past, Present, and Future.

PubMed

Xu, Joshua; Thakkar, Shraddha; Gong, Binsheng; Tong, Weida

2016-07-01

The rapid advancement of emerging genomics technologies and their application for assessing safety and efficacy of FDA-regulated products require a high standard of reliability and robustness supporting regulatory decision-making in the FDA. To facilitate the regulatory application, the FDA implemented a novel data submission program, Voluntary Genomics Data Submission (VGDS), and also to engage the stakeholders. As part of the endeavor, for the past 10 years, the FDA has led an international consortium of regulatory agencies, academia, pharmaceutical companies, and genomics platform providers, which was named MicroArray Quality Control Consortium (MAQC), to address issues such as reproducibility, precision, specificity/sensitivity, and data interpretation. Three projects have been completed so far assessing these genomics technologies: gene expression microarrays, whole genome genotyping arrays, and whole transcriptome sequencing (i.e., RNA-seq). The resultant studies provide the basic parameters for fit-for-purpose application of these new data streams in regulatory environments, and the solutions have been made available to the public through peer-reviewed publications. The latest MAQC project is also called the SEquencing Quality Control (SEQC) project focused on next-generation sequencing. Using reference samples with built-in controls, SEQC studies have demonstrated that relative gene expression can be measured accurately and reliably across laboratories and RNA-seq platforms. Besides prediction performance comparable to microarrays in clinical settings and safety assessments, RNA-seq is shown to have better sensitivity for low expression and reveal novel transcriptomic features. Future effort of MAQC will be focused on quality control of whole genome sequencing and targeted sequencing.

The FDA’s Experience with Emerging Genomics Technologies—Past, Present, and Future

PubMed Central

Xu, Joshua; Thakkar, Shraddha; Gong, Binsheng; Tong, Weida

2016-01-01

The rapid advancement of emerging genomics technologies and their application for assessing safety and efficacy of FDA-regulated products require a high standard of reliability and robustness supporting regulatory decision-making in the FDA. To facilitate the regulatory application, the FDA implemented a novel data submission program, Voluntary Genomics Data Submission (VGDS), and also to engage the stakeholders. As part of the endeavor, for the past 10 years, the FDA has led an international consortium of regulatory agencies, academia, pharmaceutical companies, and genomics platform providers, which was named MicroArray Quality Control Consortium (MAQC), to address issues such as reproducibility, precision, specificity/sensitivity, and data interpretation. Three projects have been completed so far assessing these genomics technologies: gene expression microarrays, whole genome genotyping arrays, and whole transcriptome sequencing (i.e., RNA-seq). The resultant studies provide the basic parameters for fit-for-purpose application of these new data streams in regulatory environments, and the solutions have been made available to the public through peer-reviewed publications. The latest MAQC project is also called the SEquencing Quality Control (SEQC) project focused on next-generation sequencing. Using reference samples with built-in controls, SEQC studies have demonstrated that relative gene expression can be measured accurately and reliably across laboratories and RNA-seq platforms. Besides prediction performance comparable to microarrays in clinical settings and safety assessments, RNA-seq is shown to have better sensitivity for low expression and reveal novel transcriptomic features. Future effort of MAQC will be focused on quality control of whole genome sequencing and targeted sequencing. PMID:27116022

Quantitative DNA Methylation Analysis Identifies a Single CpG Dinucleotide Important for ZAP-70 Expression and Predictive of Prognosis in Chronic Lymphocytic Leukemia

PubMed Central

Claus, Rainer; Lucas, David M.; Stilgenbauer, Stephan; Ruppert, Amy S.; Yu, Lianbo; Zucknick, Manuela; Mertens, Daniel; Bühler, Andreas; Oakes, Christopher C.; Larson, Richard A.; Kay, Neil E.; Jelinek, Diane F.; Kipps, Thomas J.; Rassenti, Laura Z.; Gribben, John G.; Döhner, Hartmut; Heerema, Nyla A.; Marcucci, Guido; Plass, Christoph; Byrd, John C.

2012-01-01

Purpose Increased ZAP-70 expression predicts poor prognosis in chronic lymphocytic leukemia (CLL). Current methods for accurately measuring ZAP-70 expression are problematic, preventing widespread application of these tests in clinical decision making. We therefore used comprehensive DNA methylation profiling of the ZAP-70 regulatory region to identify sites important for transcriptional control. Patients and Methods High-resolution quantitative DNA methylation analysis of the entire ZAP-70 gene regulatory regions was conducted on 247 samples from patients with CLL from four independent clinical studies. Results Through this comprehensive analysis, we identified a small area in the 5′ regulatory region of ZAP-70 that showed large variability in methylation in CLL samples but was universally methylated in normal B cells. High correlation with mRNA and protein expression, as well as activity in promoter reporter assays, revealed that within this differentially methylated region, a single CpG dinucleotide and neighboring nucleotides are particularly important in ZAP-70 transcriptional regulation. Furthermore, by using clustering approaches, we identified a prognostic role for this site in four independent data sets of patients with CLL using time to treatment, progression-free survival, and overall survival as clinical end points. Conclusion Comprehensive quantitative DNA methylation analysis of the ZAP-70 gene in CLL identified important regions responsible for transcriptional regulation. In addition, loss of methylation at a specific single CpG dinucleotide in the ZAP-70 5′ regulatory sequence is a highly predictive and reproducible biomarker of poor prognosis in this disease. This work demonstrates the feasibility of using quantitative specific ZAP-70 methylation analysis as a relevant clinically applicable prognostic test in CLL. PMID:22564988

Massive contribution of transposable elements to mammalian regulatory sequences.

PubMed

Rayan, Nirmala Arul; Del Rosario, Ricardo C H; Prabhakar, Shyam

2016-09-01

Barbara McClintock discovered the existence of transposable elements (TEs) in the late 1940s and initially proposed that they contributed to the gene regulatory program of higher organisms. This controversial idea gained acceptance only much later in the 1990s, when the first examples of TE-derived promoter sequences were uncovered. It is now known that half of the human genome is recognizably derived from TEs. It is thus important to understand the scope and nature of their contribution to gene regulation. Here, we provide a timeline of major discoveries in this area and discuss how transposons have revolutionized our understanding of mammalian genomes, with a special emphasis on the massive contribution of TEs to primate evolution. Our analysis of primate-specific functional elements supports a simple model for the rate at which new functional elements arise in unique and TE-derived DNA. Finally, we discuss some of the challenges and unresolved questions in the field, which need to be addressed in order to fully characterize the impact of TEs on gene regulation, evolution and disease processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Isolation of Persicaria minor sesquiterpene synthase promoter and its deletions for transgenic Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Omar, Aimi Farehah; Ismail, Ismanizan

2016-11-01

Sesquiterpene synthase (SS) catalyzes the formation of sesquiterpenes from farnesyl diphosphate (FDP) via carbocation intermediates. In this study, the promoter region of sesquiterpene synthase was isolated from Persicaria minor to identify possible cis-acting elements in the promoter. The full-length PmSS promoter of P. minor is 1824-bp sequences. The sequence was analyzed and several putative cis-acting regulatory elements were identified. Three cis-acting regulatory elements were selected for deletion analysis which are cis-acting element involved in wound responsiveness (WUN), cis - acting element involved in defense and stress responsiveness (TC) and cis-acting element involved in ABA responsiveness (ABRE). Series of deletions were conducted to assess the promoter activity producing three truncated fragments promoter; Prom 2 1606-bp, Prom 3 1144- bp, and Prom 4 921-bp. The full-length promoter and its deletion series were cloned into the pBGWFS7 vector which contain β-glucuronidase (GUS) gene and green fluorescent protein (GFP) as the reporter gene. All constructs were successfully transformed into Arabidopsis thaliana based on PCR of positive BASTA resistance plants.

Murine homeobox-containing gene, Msx-1: analysis of genomic organization, promoter structure, and potential autoregulatory cis-acting elements.

PubMed

Kuzuoka, M; Takahashi, T; Guron, C; Raghow, R

1994-05-01

Detailed molecular organization of the coding and upstream regulatory regions of the murine homeodomain-containing gene, Msx-1, is reported. The protein-encoding portion of the gene is contained in two exons, 590 and 1214 bp in length, separated by a 2107-bp intron; the homeodomain is located in the second exon. The two-exon organization of the murine Msx-1 gene resembles a number of other homeodomain-containing genes. The 5'-(GTAAGT) and 3'-(CCCTAG) splicing junctions and the mRNA polyadenylation signal (UAUAA) of the murine Msx-1 gene are also characteristic of other vertebrate genes. By nuclease protection and primer extension assays, the start of transcription of the Msx-1 gene was located 256 bp upstream of the first AUG. Computer analysis of the promoter proximal 1280-bp sequence revealed a number of potentially important cis-regulatory sequences; these include the recognition elements for Ap-1, Ap-2, Ap-3, Sp-1, a possible binding site for RAR:RXR, and a number of TCF-1 consensus motifs. Importantly, a perfect reverse complement of (C/G)TTAATTG, which was recently shown to be an optimal binding sequence for the homeodomain of Msx-1 protein (K.M. Catron, N. Iler, and C. Abate (1993) Mol. Cell. Biol. 13:2354-2365), was also located in the murine Msx-1 promoter. Binding of bacterially expressed Msx-1 homeodomain polypeptide to Msx-1-specific oligonucleotide was experimentally demonstrated, raising a distinct possibility of autoregulation of this developmentally regulated gene.

Tissue expression analysis, cloning and characterization of the 5'-regulatory region of the bovine FABP3 gene.

PubMed

Li, Anning; Wu, Lijuan; Wang, Xiaoyu; Xin, Yaping; Zan, Linsen

2016-09-01

Fatty acid binding protein 3 (FABP3) is a member of the FABP family which bind fatty acids and have an important role in fatty acid metabolism. A large number of studies have shown that the genetic polymorphisms of FABP3 are positively correlated with intramuscular fat (IMF) content in domestic animals, however, the function and transcriptional characteristics of FABP3 in cattle remain unclear. Real-time PCR analysis revealed that bovine FABP3 was highly expressed in cardiac tissue. The 5'-regulatory region of bovine FABP3 was cloned and its transcription initiation sites were identified. Sequence analysis showed that many transcriptional factor binding sites including TATA-box and CCAAT-box were present on the 5'-flanking region of bovine FABP3, and four CpG islands were found on nucleotides from -891 to +118. Seven serial deletion constructs of the 5'-regulatory region evaluated in dual-luciferase reporter assay indicated that its core promoter was 384 base pairs upstream from the transcription initiation site. The transcriptional factor binding sites RXRα, KLF15, CREB and Sp1 were conserved in the core promoter of cattle, sheep, pigs and dogs. These results provide further understanding of the function and regulation mechanism of bovine FABP3.

Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells

PubMed Central

Camp, J. Gray; Weiser, Matthew; Cocchiaro, Jordan L.; Kingsley, David M.; Furey, Terrence S.; Sheikh, Shehzad Z.; Rawls, John F.

2017-01-01

The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology. PMID:28850571

Translating natural genetic variation to gene expression in a computational model of the Drosophila gap gene regulatory network

PubMed Central

Kozlov, Konstantin N.; Kulakovskiy, Ivan V.; Zubair, Asif; Marjoram, Paul; Lawrie, David S.; Nuzhdin, Sergey V.; Samsonova, Maria G.

2017-01-01

Annotating the genotype-phenotype relationship, and developing a proper quantitative description of the relationship, requires understanding the impact of natural genomic variation on gene expression. We apply a sequence-level model of gap gene expression in the early development of Drosophila to analyze single nucleotide polymorphisms (SNPs) in a panel of natural sequenced D. melanogaster lines. Using a thermodynamic modeling framework, we provide both analytical and computational descriptions of how single-nucleotide variants affect gene expression. The analysis reveals that the sequence variants increase (decrease) gene expression if located within binding sites of repressors (activators). We show that the sign of SNP influence (activation or repression) may change in time and space and elucidate the origin of this change in specific examples. The thermodynamic modeling approach predicts non-local and non-linear effects arising from SNPs, and combinations of SNPs, in individual fly genotypes. Simulation of individual fly genotypes using our model reveals that this non-linearity reduces to almost additive inputs from multiple SNPs. Further, we see signatures of the action of purifying selection in the gap gene regulatory regions. To infer the specific targets of purifying selection, we analyze the patterns of polymorphism in the data at two phenotypic levels: the strengths of binding and expression. We find that combinations of SNPs show evidence of being under selective pressure, while individual SNPs do not. The model predicts that SNPs appear to accumulate in the genotypes of the natural population in a way biased towards small increases in activating action on the expression pattern. Taken together, these results provide a systems-level view of how genetic variation translates to the level of gene regulatory networks via combinatorial SNP effects. PMID:28898266

Target mimics: an embedded layer of microRNA-involved gene regulatory networks in plants.

PubMed

Meng, Yijun; Shao, Chaogang; Wang, Huizhong; Jin, Yongfeng

2012-05-21

MicroRNAs (miRNAs) play an essential role in gene regulation in plants. At the same time, the expression of miRNA genes is also tightly controlled. Recently, a novel mechanism called "target mimicry" was discovered, providing another layer for modulating miRNA activities. However, except for the artificial target mimics manipulated for functional studies on certain miRNA genes, only one example, IPS1 (Induced by Phosphate Starvation 1)-miR399 was experimentally confirmed in planta. To date, few analyses for comprehensive identification of natural target mimics have been performed in plants. Thus, limited evidences are available to provide detailed information for interrogating the questionable issue whether target mimicry was widespread in planta, and implicated in certain biological processes. In this study, genome-wide computational prediction of endogenous miRNA mimics was performed in Arabidopsis and rice, and dozens of target mimics were identified. In contrast to a recent report, the densities of target mimic sites were found to be much higher within the untranslated regions (UTRs) when compared to those within the coding sequences (CDSs) in both plants. Some novel sequence characteristics were observed for the miRNAs that were potentially regulated by the target mimics. GO (Gene Ontology) term enrichment analysis revealed some functional insights into the predicted mimics. After degradome sequencing data-based identification of miRNA targets, the regulatory networks constituted by target mimics, miRNAs and their downstream targets were constructed, and some intriguing subnetworks were further exploited. These results together suggest that target mimicry may be widely implicated in regulating miRNA activities in planta, and we hope this study could expand the current understanding of miRNA-involved regulatory networks.

Cis-regulatory somatic mutations and gene-expression alteration in B-cell lymphomas.

PubMed

Mathelier, Anthony; Lefebvre, Calvin; Zhang, Allen W; Arenillas, David J; Ding, Jiarui; Wasserman, Wyeth W; Shah, Sohrab P

2015-04-23

With the rapid increase of whole-genome sequencing of human cancers, an important opportunity to analyze and characterize somatic mutations lying within cis-regulatory regions has emerged. A focus on protein-coding regions to identify nonsense or missense mutations disruptive to protein structure and/or function has led to important insights; however, the impact on gene expression of mutations lying within cis-regulatory regions remains under-explored. We analyzed somatic mutations from 84 matched tumor-normal whole genomes from B-cell lymphomas with accompanying gene expression measurements to elucidate the extent to which these cancers are disrupted by cis-regulatory mutations. We characterize mutations overlapping a high quality set of well-annotated transcription factor binding sites (TFBSs), covering a similar portion of the genome as protein-coding exons. Our results indicate that cis-regulatory mutations overlapping predicted TFBSs are enriched in promoter regions of genes involved in apoptosis or growth/proliferation. By integrating gene expression data with mutation data, our computational approach culminates with identification of cis-regulatory mutations most likely to participate in dysregulation of the gene expression program. The impact can be measured along with protein-coding mutations to highlight key mutations disrupting gene expression and pathways in cancer. Our study yields specific genes with disrupted expression triggered by genomic mutations in either the coding or the regulatory space. It implies that mutated regulatory components of the genome contribute substantially to cancer pathways. Our analyses demonstrate that identifying genomically altered cis-regulatory elements coupled with analysis of gene expression data will augment biological interpretation of mutational landscapes of cancers.

Antitumor Effects of Epidrug/IFNα Combination Driven by Modulated Gene Signatures in Both Colorectal Cancer and Dendritic Cells.

PubMed

Fragale, Alessandra; Romagnoli, Giulia; Licursi, Valerio; Buoncervello, Maria; Del Vecchio, Giorgia; Giuliani, Caterina; Parlato, Stefania; Leone, Celeste; De Angelis, Marta; Canini, Irene; Toschi, Elena; Belardelli, Filippo; Negri, Rodolfo; Capone, Imerio; Presutti, Carlo; Gabriele, Lucia

2017-07-01

Colorectal cancer results from the progressive accumulation of genetic and epigenetic alterations. IFN signaling defects play an important role in the carcinogenesis process, in which the inability of IFN transcription regulatory factors (IRF) to access regulatory sequences in IFN-stimulated genes (ISG) in tumors and in immune cells may be pivotal. We reported that low-dose combination of two FDA-approved epidrugs, azacytidine (A) and romidepsin (R), with IFNα2 (ARI) hampers the aggressiveness of both colorectal cancer metastatic and stem cells in vivo and triggers immunogenic cell death signals that stimulate dendritic cell (DC) function. Here, we investigated the molecular signals induced by ARI treatment and found that this drug combination increased the accessibility to regulatory sequences of ISGs and IRFs that were epigenetically silenced in both colorectal cancer cells and DCs. Likewise, specific ARI-induced histone methylation and acetylation changes marked epigenetically affected ISG promoters in both metastatic cancer cells and DCs. Analysis by ChIP-seq confirmed such ARI-induced epigenetically regulated IFN signature. The activation of this signal endowed DCs with a marked migratory capability. Our results establish a direct correlation between reexpression of silenced ISGs by epigenetic control and ARI anticancer activity and provide new knowledge for the development of innovative combined therapeutic strategies for colorectal cancer. Cancer Immunol Res; 5(7); 604-16. ©2017 AACR . ©2017 American Association for Cancer Research.

Comparison of Five Major Trichome Regulatory Genes in Brassica villosa with Orthologues within the Brassicaceae

PubMed Central

Nayidu, Naghabushana K.; Kagale, Sateesh; Taheri, Ali; Withana-Gamage, Thushan S.; Parkin, Isobel A. P.; Sharpe, Andrew G.; Gruber, Margaret Y.

2014-01-01

Coding sequences for major trichome regulatory genes, including the positive regulators GLABRA 1(GL1), GLABRA 2 (GL2), ENHANCER OF GLABRA 3 (EGL3), and TRANSPARENT TESTA GLABRA 1 (TTG1) and the negative regulator TRIPTYCHON (TRY), were cloned from wild Brassica villosa, which is characterized by dense trichome coverage over most of the plant. Transcript (FPKM) levels from RNA sequencing indicated much higher expression of the GL2 and TTG1 regulatory genes in B. villosa leaves compared with expression levels of GL1 and EGL3 genes in either B. villosa or the reference genome species, glabrous B. oleracea; however, cotyledon TTG1 expression was high in both species. RNA sequencing and Q-PCR also revealed an unusual expression pattern for the negative regulators TRY and CPC, which were much more highly expressed in trichome-rich B. villosa leaves than in glabrous B. oleracea leaves and in glabrous cotyledons from both species. The B. villosa TRY expression pattern also contrasted with TRY expression patterns in two diploid Brassica species, and with the Arabidopsis model for expression of negative regulators of trichome development. Further unique sequence polymorphisms, protein characteristics, and gene evolution studies highlighted specific amino acids in GL1 and GL2 coding sequences that distinguished glabrous species from hairy species and several variants that were specific for each B. villosa gene. Positive selection was observed for GL1 between hairy and non-hairy plants, and as expected the origin of the four expressed positive trichome regulatory genes in B. villosa was predicted to be from B. oleracea. In particular the unpredicted expression patterns for TRY and CPC in B. villosa suggest additional characterization is needed to determine the function of the expanded families of trichome regulatory genes in more complex polyploid species within the Brassicaceae. PMID:24755905

Replicative Intermediates of Human Papillomavirus Type 11 in Laryngeal Papillomas: Site of Replication Initiation and Direction of Replication

NASA Astrophysics Data System (ADS)

Auborn, K. J.; Little, R. D.; Platt, T. H. K.; Vaccariello, M. A.; Schildkraut, C. L.

1994-07-01

We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180circ opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.

TEtools facilitates big data expression analysis of transposable elements and reveals an antagonism between their activity and that of piRNA genes

PubMed Central

Lerat, Emmanuelle; Fablet, Marie; Modolo, Laurent; Lopez-Maestre, Hélène

2017-01-01

Abstract Over recent decades, substantial efforts have been made to understand the interactions between host genomes and transposable elements (TEs). The impact of TEs on the regulation of host genes is well known, with TEs acting as platforms of regulatory sequences. Nevertheless, due to their repetitive nature it is considerably hard to integrate TE analysis into genome-wide studies. Here, we developed a specific tool for the analysis of TE expression: TEtools. This tool takes into account the TE sequence diversity of the genome, it can be applied to unannotated or unassembled genomes and is freely available under the GPL3 (https://github.com/l-modolo/TEtools). TEtools performs the mapping of RNA-seq data obtained from classical mRNAs or small RNAs onto a list of TE sequences and performs differential expression analyses with statistical relevance. Using this tool, we analyzed TE expression from five Drosophila wild-type strains. Our data show for the first time that the activity of TEs is strictly linked to the activity of the genes implicated in the piwi-interacting RNA biogenesis and therefore fits an arms race scenario between TE sequences and host control genes. PMID:28204592

A synthetic biology approach to the development of transcriptional regulatory models and custom enhancer design☆,☆☆

PubMed Central

Martinez, Carlos A.; Barr, Kenneth; Kim, Ah-Ram; Reinitz, John

2013-01-01

Synthetic biology offers novel opportunities for elucidating transcriptional regulatory mechanisms and enhancer logic. Complex cis-regulatory sequences—like the ones driving expression of the Drosophila even-skipped gene—have proven difficult to design from existing knowledge, presumably due to the large number of protein-protein interactions needed to drive the correct expression patterns of genes in multicellular organisms. This work discusses two novel computational methods for the custom design of enhancers that employ a sophisticated, empirically validated transcriptional model, optimization algorithms, and synthetic biology. These synthetic elements have both utilitarian and academic value, including improving existing regulatory models as well as evolutionary questions. The first method involves the use of simulated annealing to explore the sequence space for synthetic enhancers whose expression output fit a given search criterion. The second method uses a novel optimization algorithm to find functionally accessible pathways between two enhancer sequences. These paths describe a set of mutations wherein the predicted expression pattern does not significantly vary at any point along the path. Both methods rely on a predictive mathematical framework that maps the enhancer sequence space to functional output. PMID:23732772

Functional Assessment of Disease-Associated Regulatory Variants In Vivo Using a Versatile Dual Colour Transgenesis Strategy in Zebrafish

PubMed Central

Bhatia, Shipra; Gordon, Christopher T.; Foster, Robert G.; Melin, Lucie; Abadie, Véronique; Baujat, Geneviève; Vazquez, Marie-Paule; Amiel, Jeanne; Lyonnet, Stanislas; van Heyningen, Veronica; Kleinjan, Dirk A.

2015-01-01

Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function. PMID:26030420

On the way toward systems biology of Aspergillus fumigatus infection.

PubMed

Albrecht, Daniela; Kniemeyer, Olaf; Mech, Franziska; Gunzer, Matthias; Brakhage, Axel; Guthke, Reinhard

2011-06-01

Pathogenicity of Aspergillus fumigatus is multifactorial. Thus, global studies are essential for the understanding of the infection process. Therefore, a data warehouse was established where genome sequence, transcriptome and proteome data are stored. These data are analyzed for the elucidation of virulence determinants. The data analysis workflow starts with pre-processing including imputing of missing values and normalization. Last step is the identification of differentially expressed genes/proteins as interesting candidates for further analysis, in particular for functional categorization and correlation studies. Sequence data and other prior knowledge extracted from databases are integrated to support the inference of gene regulatory networks associated with pathogenicity. This knowledge-assisted data analysis aims at establishing mathematical models with predictive strength to assist further experimental work. Recently, first steps were done to extend the integrative data analysis and computational modeling by evaluating spatio-temporal data (movies) that monitor interactions of A. fumigatus morphotypes (e.g. conidia) with host immune cells. Copyright © 2011 Elsevier GmbH. All rights reserved.

Location analysis for the estrogen receptor-α reveals binding to diverse ERE sequences and widespread binding within repetitive DNA elements

PubMed Central

Mason, Christopher E.; Shu, Feng-Jue; Wang, Cheng; Session, Ryan M.; Kallen, Roland G.; Sidell, Neil; Yu, Tianwei; Liu, Mei Hui; Cheung, Edwin; Kallen, Caleb B.

2010-01-01

Location analysis for estrogen receptor-α (ERα)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here, we present the estrogen response element (ERE) sequences that were identified at ERα-bound loci and quantify the incidence of ERE sequences under two stringencies of detection: <10% and 10–20% nucleotide deviation from the canonical ERE sequence. We demonstrate that ∼50% of all ERα-bound loci do not have a discernable ERE and show that most ERα-bound EREs are not perfect consensus EREs. Approximately one-third of all ERα-bound ERE sequences reside within repetitive DNA sequences, most commonly of the AluS family. In addition, the 3-bp spacer between the inverted ERE half-sites, rather than being random nucleotides, is C(A/T)G-enriched at bona fide receptor targets. Diverse ERα-bound loci were validated using electrophoretic mobility shift assay and ChIP-polymerase chain reaction (PCR). The functional significance of receptor-bound loci was demonstrated using luciferase reporter assays which proved that repetitive element ERE sequences contribute to enhancer function. ChIP-PCR demonstrated estrogen-dependent recruitment of the coactivator SRC3 to these loci in vivo. Our data demonstrate that ERα binds to widely variant EREs with less sequence specificity than had previously been suspected and that binding at repetitive and nonrepetitive genomic targets is favored by specific trinucleotide spacers. PMID:20047966

Location analysis for the estrogen receptor-alpha reveals binding to diverse ERE sequences and widespread binding within repetitive DNA elements.

PubMed

Mason, Christopher E; Shu, Feng-Jue; Wang, Cheng; Session, Ryan M; Kallen, Roland G; Sidell, Neil; Yu, Tianwei; Liu, Mei Hui; Cheung, Edwin; Kallen, Caleb B

2010-04-01

Location analysis for estrogen receptor-alpha (ERalpha)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here, we present the estrogen response element (ERE) sequences that were identified at ERalpha-bound loci and quantify the incidence of ERE sequences under two stringencies of detection: <10% and 10-20% nucleotide deviation from the canonical ERE sequence. We demonstrate that approximately 50% of all ERalpha-bound loci do not have a discernable ERE and show that most ERalpha-bound EREs are not perfect consensus EREs. Approximately one-third of all ERalpha-bound ERE sequences reside within repetitive DNA sequences, most commonly of the AluS family. In addition, the 3-bp spacer between the inverted ERE half-sites, rather than being random nucleotides, is C(A/T)G-enriched at bona fide receptor targets. Diverse ERalpha-bound loci were validated using electrophoretic mobility shift assay and ChIP-polymerase chain reaction (PCR). The functional significance of receptor-bound loci was demonstrated using luciferase reporter assays which proved that repetitive element ERE sequences contribute to enhancer function. ChIP-PCR demonstrated estrogen-dependent recruitment of the coactivator SRC3 to these loci in vivo. Our data demonstrate that ERalpha binds to widely variant EREs with less sequence specificity than had previously been suspected and that binding at repetitive and nonrepetitive genomic targets is favored by specific trinucleotide spacers.

Integration of Bioinformatics and Synthetic Promoters Leads to the Discovery of Novel Elicitor-Responsive cis-Regulatory Sequences in Arabidopsis1[C][W][OA

PubMed Central

Koschmann, Jeannette; Machens, Fabian; Becker, Marlies; Niemeyer, Julia; Schulze, Jutta; Bülow, Lorenz; Stahl, Dietmar J.; Hehl, Reinhard

2012-01-01

A combination of bioinformatic tools, high-throughput gene expression profiles, and the use of synthetic promoters is a powerful approach to discover and evaluate novel cis-sequences in response to specific stimuli. With Arabidopsis (Arabidopsis thaliana) microarray data annotated to the PathoPlant database, 732 different queries with a focus on fungal and oomycete pathogens were performed, leading to 510 up-regulated gene groups. Using the binding site estimation suite of tools, BEST, 407 conserved sequence motifs were identified in promoter regions of these coregulated gene sets. Motif similarities were determined with STAMP, classifying the 407 sequence motifs into 37 families. A comparative analysis of these 37 families with the AthaMap, PLACE, and AGRIS databases revealed similarities to known cis-elements but also led to the discovery of cis-sequences not yet implicated in pathogen response. Using a parsley (Petroselinum crispum) protoplast system and a modified reporter gene vector with an internal transformation control, 25 elicitor-responsive cis-sequences from 10 different motif families were identified. Many of the elicitor-responsive cis-sequences also drive reporter gene expression in an Agrobacterium tumefaciens infection assay in Nicotiana benthamiana. This work significantly increases the number of known elicitor-responsive cis-sequences and demonstrates the successful integration of a diverse set of bioinformatic resources combined with synthetic promoter analysis for data mining and functional screening in plant-pathogen interaction. PMID:22744985

Comparing a motivational and a self-regulatory intervention to adopt an oral self-care regimen: a two-sequential randomized crossover trial.

PubMed

Lhakhang, Pempa; Gholami, Maryam; Knoll, Nina; Schwarzer, Ralf

2015-01-01

A sequential intervention to facilitate the adoption and maintenance of dental flossing was conducted among 205 students in India, aged 18-26 years. Two experimental groups received different treatment sequences and were observed at three assessment points, 34 days apart. One group received first a motivational intervention (intention, outcome expectancies, and risk perception, followed by a self-regulatory intervention (planning, self-efficacy, and action control). The second group received the same intervention in the opposite order. Both intervention sequences yielded gains in terms of flossing, planning, self-efficacy, and action control. However, at Time 2, those who had received the self-regulatory intervention first, were superior to their counterparts who had received the motivational intervention first. At Time 3, differences vanished as everyone had then received both interventions. Thus, findings highlight the benefits of a self-regulatory compared to a mere motivational intervention.

Sheep genome functional annotation reveals proximal regulatory elements contributed to the evolution of modern breeds.

PubMed

Naval-Sanchez, Marina; Nguyen, Quan; McWilliam, Sean; Porto-Neto, Laercio R; Tellam, Ross; Vuocolo, Tony; Reverter, Antonio; Perez-Enciso, Miguel; Brauning, Rudiger; Clarke, Shannon; McCulloch, Alan; Zamani, Wahid; Naderi, Saeid; Rezaei, Hamid Reza; Pompanon, Francois; Taberlet, Pierre; Worley, Kim C; Gibbs, Richard A; Muzny, Donna M; Jhangiani, Shalini N; Cockett, Noelle; Daetwyler, Hans; Kijas, James

2018-02-28

Domestication fundamentally reshaped animal morphology, physiology and behaviour, offering the opportunity to investigate the molecular processes driving evolutionary change. Here we assess sheep domestication and artificial selection by comparing genome sequence from 43 modern breeds (Ovis aries) and their Asian mouflon ancestor (O. orientalis) to identify selection sweeps. Next, we provide a comparative functional annotation of the sheep genome, validated using experimental ChIP-Seq of sheep tissue. Using these annotations, we evaluate the impact of selection and domestication on regulatory sequences and find that sweeps are significantly enriched for protein coding genes, proximal regulatory elements of genes and genome features associated with active transcription. Finally, we find individual sites displaying strong allele frequency divergence are enriched for the same regulatory features. Our data demonstrate that remodelling of gene expression is likely to have been one of the evolutionary forces that drove phenotypic diversification of this common livestock species.

TranslatomeDB: a comprehensive database and cloud-based analysis platform for translatome sequencing data

PubMed Central

Liu, Wanting; Xiang, Lunping; Zheng, Tingkai; Jin, Jingjie

2018-01-01

Abstract Translation is a key regulatory step, linking transcriptome and proteome. Two major methods of translatome investigations are RNC-seq (sequencing of translating mRNA) and Ribo-seq (ribosome profiling). To facilitate the investigation of translation, we built a comprehensive database TranslatomeDB (http://www.translatomedb.net/) which provides collection and integrated analysis of published and user-generated translatome sequencing data. The current version includes 2453 Ribo-seq, 10 RNC-seq and their 1394 corresponding mRNA-seq datasets in 13 species. The database emphasizes the analysis functions in addition to the dataset collections. Differential gene expression (DGE) analysis can be performed between any two datasets of same species and type, both on transcriptome and translatome levels. The translation indices translation ratios, elongation velocity index and translational efficiency can be calculated to quantitatively evaluate translational initiation efficiency and elongation velocity, respectively. All datasets were analyzed using a unified, robust, accurate and experimentally-verifiable pipeline based on the FANSe3 mapping algorithm and edgeR for DGE analyzes. TranslatomeDB also allows users to upload their own datasets and utilize the identical unified pipeline to analyze their data. We believe that our TranslatomeDB is a comprehensive platform and knowledgebase on translatome and proteome research, releasing the biologists from complex searching, analyzing and comparing huge sequencing data without needing local computational power. PMID:29106630

Constraints on the evolution of a doublesex target gene arising from doublesex’s pleiotropic deployment

PubMed Central

Luo, Shengzhan D.; Baker, Bruce S.

2015-01-01

“Regulatory evolution,” that is, changes in a gene’s expression pattern through changes at its regulatory sequence, rather than changes at the coding sequence of the gene or changes of the upstream transcription factors, has been increasingly recognized as a pervasive evolution mechanism. Many somatic sexually dimorphic features of Drosophila melanogaster are the results of gene expression regulated by the doublesex (dsx) gene, which encodes sex-specific transcription factors (DSXF in females and DSXM in males). Rapid changes in such sexually dimorphic features are likely a result of changes at the regulatory sequence of the target genes. We focused on the Flavin-containing monooxygenase-2 (Fmo-2) gene, a likely direct dsx target, to elucidate how sexually dimorphic expression and its evolution are brought about. We found that dsx is deployed to regulate the Fmo-2 transcription both in the midgut and in fat body cells of the spermatheca (a female-specific tissue), through a canonical DSX-binding site in the Fmo-2 regulatory sequence. In the melanogaster group, Fmo-2 transcription in the midgut has evolved rapidly, in contrast to the conserved spermathecal transcription. We identified two cis-regulatory modules (CRM-p and CRM-d) that direct sexually monomorphic or dimorphic Fmo-2 transcription, respectively, in the midguts of these species. Changes of Fmo-2 transcription in the midgut from sexually dimorphic to sexually monomorphic in some species are caused by the loss of CRM-d function, but not the loss of the canonical DSX-binding site. Thus, conferring transcriptional regulation on a CRM level allows the regulation to evolve rapidly in one tissue while evading evolutionary constraints posed by other tissues. PMID:25675536

Bacillus Anthracis Comparative Genome Analysis in Support of the Amerithrax Investigation

DTIC Science & Technology

2011-02-02

ability to sporulate . The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of...mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F...a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were

Sequence-based model of gap gene regulatory network.

PubMed

Kozlov, Konstantin; Gursky, Vitaly; Kulakovskiy, Ivan; Samsonova, Maria

2014-01-01

The detailed analysis of transcriptional regulation is crucially important for understanding biological processes. The gap gene network in Drosophila attracts large interest among researches studying mechanisms of transcriptional regulation. It implements the most upstream regulatory layer of the segmentation gene network. The knowledge of molecular mechanisms involved in gap gene regulation is far less complete than that of genetics of the system. Mathematical modeling goes beyond insights gained by genetics and molecular approaches. It allows us to reconstruct wild-type gene expression patterns in silico, infer underlying regulatory mechanism and prove its sufficiency. We developed a new model that provides a dynamical description of gap gene regulatory systems, using detailed DNA-based information, as well as spatial transcription factor concentration data at varying time points. We showed that this model correctly reproduces gap gene expression patterns in wild type embryos and is able to predict gap expression patterns in Kr mutants and four reporter constructs. We used four-fold cross validation test and fitting to random dataset to validate the model and proof its sufficiency in data description. The identifiability analysis showed that most model parameters are well identifiable. We reconstructed the gap gene network topology and studied the impact of individual transcription factor binding sites on the model output. We measured this impact by calculating the site regulatory weight as a normalized difference between the residual sum of squares error for the set of all annotated sites and for the set with the site of interest excluded. The reconstructed topology of the gap gene network is in agreement with previous modeling results and data from literature. We showed that 1) the regulatory weights of transcription factor binding sites show very weak correlation with their PWM score; 2) sites with low regulatory weight are important for the model output; 3) functional important sites are not exclusively located in cis-regulatory elements, but are rather dispersed through regulatory region. It is of importance that some of the sites with high functional impact in hb, Kr and kni regulatory regions coincide with strong sites annotated and verified in Dnase I footprint assays.

Positive and negative regulatory regions control the spatial distribution of polygalacturonase transcription in tomato fruit pericarp.

PubMed Central

Montgomery, J; Pollard, V; Deikman, J; Fischer, R L

1993-01-01

The tomato fruit consists of a thick, fleshy pericarp composed predominantly of highly vacuolated parenchymatous cells, which surrounds the seeds. During ripening, the activation of gene expression results in dramatic biochemical and physiological changes in the pericarp. The polygalacturonase (PG) gene, unlike many fruit ripening-induced genes, is not activated by the increase in ethylene hormone concentration associated with the onset of ripening. To investigate ethylene concentration-independent gene transcription in ripe tomato fruit, we analyzed the expression of chimeric PG promoter-beta-glucuronidase (GUS) reporter gene fusions in transgenic tomato plants. We determined that a 1.4-kb PG promoter directs ripening-regulated transcription in outer pericarp but not in inner pericarp cells, with a sharp boundary of PG promoter activity located midway through the pericarp. Promoter deletion analysis indicated that a minimum of three promoter regions influence the spatial regulation of PG transcription. A positive regulatory region from -231 to -134 promotes gene transcription in the outer pericarp of ripe fruit. A second positive regulatory region from -806 to -443 extends gene activity to the inner pericarp. However, a negative regulatory region from -1411 to -1150 inhibits gene transcription in the inner pericarp. DNase I footprint analysis showed that nuclear proteins in unripe and ripe fruit interact with DNA sequences within each of these three regulatory regions. Thus, temporal and spatial control of PG transcription is mediated by the interaction of negative and positive regulatory promoter elements, resulting in gene activity in the outer pericarp but not the inner pericarp of ripe tomato fruit. The expression pattern of PG suggests that, although they are morphologically similar, there is a fundamental difference between the parenchymatous cells within the inner and outer pericarp. PMID:8400876

Revealing cell cycle control by combining model-based detection of periodic expression with novel cis-regulatory descriptors

PubMed Central

Andersson, Claes R; Hvidsten, Torgeir R; Isaksson, Anders; Gustafsson, Mats G; Komorowski, Jan

2007-01-01

Background We address the issue of explaining the presence or absence of phase-specific transcription in budding yeast cultures under different conditions. To this end we use a model-based detector of gene expression periodicity to divide genes into classes depending on their behavior in experiments using different synchronization methods. While computational inference of gene regulatory circuits typically relies on expression similarity (clustering) in order to find classes of potentially co-regulated genes, this method instead takes advantage of known time profile signatures related to the studied process. Results We explain the regulatory mechanisms of the inferred periodic classes with cis-regulatory descriptors that combine upstream sequence motifs with experimentally determined binding of transcription factors. By systematic statistical analysis we show that periodic classes are best explained by combinations of descriptors rather than single descriptors, and that different combinations correspond to periodic expression in different classes. We also find evidence for additive regulation in that the combinations of cis-regulatory descriptors associated with genes periodically expressed in fewer conditions are frequently subsets of combinations associated with genes periodically expression in more conditions. Finally, we demonstrate that our approach retrieves combinations that are more specific towards known cell-cycle related regulators than the frequently used clustering approach. Conclusion The results illustrate how a model-based approach to expression analysis may be particularly well suited to detect biologically relevant mechanisms. Our new approach makes it possible to provide more refined hypotheses about regulatory mechanisms of the cell cycle and it can easily be adjusted to reveal regulation of other, non-periodic, cellular processes. PMID:17939860

Functional analysis of a large set of BRCA2 exon 7 variants highlights the predictive value of hexamer scores in detecting alterations of exonic splicing regulatory elements.

PubMed

Di Giacomo, Daniela; Gaildrat, Pascaline; Abuli, Anna; Abdat, Julie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

2013-11-01

Exonic variants can alter pre-mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon 7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5' splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon 7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n = 36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers' scores recently established by Ke et al. (). Comparisons of hexamer-based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high-throughput sequencing. © 2013 WILEY PERIODICALS, INC.

A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region.

PubMed

Bellacosa, A; Testa, J R; Staal, S P; Tsichlis, P N

1991-10-11

The v-akt oncogene codes for a 105-kilodalton fusion phosphoprotein containing Gag sequences at its amino terminus. Sequence analysis of v-akt and biochemical characterization of its product revealed that it codes for a protein kinase C-related serine-threonine kinase whose cellular homolog is expressed in most tissues, with the highest amount found in thymus. Although Akt is a serine-threonine kinase, part of its regulatory region is similar to the Src homology-2 domain, a structural motif characteristic of cytoplasmic tyrosine kinases that functions in protein-protein interactions. This suggests that Akt may form a functional link between tyrosine and serine-threonine phosphorylation pathways.

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

Kerr, J.M.; Fisher, L.W.; Termine, J.D.

The authors have isolated and partially sequenced the human bone sialoprotein gene (IBSP). IBSP has been sublocalized by in situ hybridization to chromosome 4q38-q31 and is composed of six small exons (51 to 159 bp) and 1 large exon ([approximately]2.6 kb). The intron/exon junctions defined by sequence analysis are of class O, retaining an intact coding triplet. Sequence analysis of the 5[prime] upstream region revealed a TATAA (nucleotides -30 to-25 from the transcriptional start point) and a CCAAT (nucleotides -56 to-52) box, both in the reverse orientation. Intron 1 contains interesting structural elements composed of polypyrimidine repeats followed by amore » poly(AC)[sub n] tract. Both types of structural elements have been detected in promoter regions of other genes and have been implicated in transcriptional regulation. Several differences between the previously published cDNA sequence and the authors' sequence have been identified, most of which are contained within the untranslated exon 1. Three base revisions in the coding region include a G to T (Gly to Val, amino acid 195), T to C (Val to Ala, amino acid 268), and T to A (Glu to Asp, amino acid 270). In conclusion, the genomic organization and potential regulatory elements of human IBSP have been elucidated. 42 refs., 4 figs., 1 tab.« less

Nucleotide sequence and regulatory studies of VGF, a nervous system-specific mRNA that is rapidly and relatively selectively induced by nerve growth factor.

PubMed

Salton, S R

1991-09-01

A nervous system-specific mRNA that is rapidly induced in PC12 cells to a greater extent by nerve growth factor (NGF) than by epidermal growth factor treatment has been cloned. The polypeptide deduced from the nucleic acid sequence of the NGF33.1 cDNA clone contains regions of amino acid sequence identity with that predicted by the cDNA clone VGF, and further analysis suggests that both NGF33.1 and VGF cDNA clones very likely correspond to the same mRNA (VGF). In this report both the nucleic acid sequence that corresponds to VGF mRNA and the polypeptide predicted by the NGF33.1 cDNA clone are presented. Genomic Southern analysis and database comparison did not detect additional sequences with high homology to the VGF gene. Induction of VGF mRNA by depolarization and phorbol 12-myristate 13-acetate treatment was greater than by serum stimulation or protein kinase A pathway activation. These studies suggest that VGF mRNA is induced to the greatest extent by NGF treatment and that VGF is one of the most rapidly regulated neuronal mRNAs identified in PC12 cells.

Principles of regulatory information conservation between mouse and human

DOE PAGES

Cheng, Yong; Ma, Zhihai; Kim, Bong-Hyun; ...

2014-11-19

To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human–mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and withmore » genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Lastly, single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.« less

FARME DB: a functional antibiotic resistance element database

PubMed Central

Wallace, James C.; Port, Jesse A.; Smith, Marissa N.; Faustman, Elaine M.

2017-01-01

Antibiotic resistance (AR) is a major global public health threat but few resources exist that catalog AR genes outside of a clinical context. Current AR sequence databases are assembled almost exclusively from genomic sequences derived from clinical bacterial isolates and thus do not include many microbial sequences derived from environmental samples that confer resistance in functional metagenomic studies. These environmental metagenomic sequences often show little or no similarity to AR sequences from clinical isolates using standard classification criteria. In addition, existing AR databases provide no information about flanking sequences containing regulatory or mobile genetic elements. To help address this issue, we created an annotated database of DNA and protein sequences derived exclusively from environmental metagenomic sequences showing AR in laboratory experiments. Our Functional Antibiotic Resistant Metagenomic Element (FARME) database is a compilation of publically available DNA sequences and predicted protein sequences conferring AR as well as regulatory elements, mobile genetic elements and predicted proteins flanking antibiotic resistant genes. FARME is the first database to focus on functional metagenomic AR gene elements and provides a resource to better understand AR in the 99% of bacteria which cannot be cultured and the relationship between environmental AR sequences and antibiotic resistant genes derived from cultured isolates. Database URL: http://staff.washington.edu/jwallace/farme PMID:28077567

Genomic organization and sequence of the Gus-s/sup a/ allele of the murine. beta. -glucuronidase gene

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

Funkenstein, B.; Leary, S.L.; Stein, J.C.

1988-03-01

The Gus-s/sup ..cap alpha../ allele of the mouse ..beta..-glucuronidase gene exhibits a high degree of inducibility by androgens due to its linkage with the Gus-r/sup ..cap alpha../ regulatory locus. The authors isolated Gus-s/sup ..cap alpha../ on a 28-kilobase pair fragment of mouse chromosome 5 and found that it contains 12 exons and 11 intervening sequences spanning 14 kilobase pairs of this genomic segment. The mRNA cap site was identified by ribonuclease protection and primer extension analyses which revealed an unusually short 5' noncoding sequence of 12 nucleotides. Proximal regulatory sequences in the 5'-flanking DNA and the complete sequence of themore » Gus-s/sup ..cap alpha../ mRNA transcript were also determined. Comparison of the amino acid sequence determined from the Gus-s/sup ..cap alpha../ nucleotide sequence with that of human ..beta..-glucuronidase indicated that the two human mRNA species differ due to alternate splicing of an exon homologous to exon 6 of the mouse gene.« less

Genomic deletion of a long-range bone enhancer misregulatessclerostin in Van Buchem disease

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

Loots, Gabriela G.; Kneissel, Michaela; Keller, Hansjoerg

2005-04-15

Mutations in distant regulatory elements can negatively impact human development and health, yet due to the difficulty of detecting these critical sequences we predominantly focus on coding sequences for diagnostic purposes. We have undertaken a comparative sequence-based approach to characterize a large noncoding region deleted in patients affected by Van Buchem disease (VB), a severe sclerosing bone dysplasia. Using BAC recombination and transgenesis we characterized the expression of human sclerostin (sost) from normal (hSOSTwt) or Van Buchem(hSOSTvb D) alleles. Only the hSOSTwt allele faithfully expressed high levels of human sost in the adult bone and impacted bone metabolism, consistent withmore » the model that the VB noncoding deletion removes a sost specific regulatory element. By exploiting cross-species sequence comparisons with in vitro and in vivo enhancer assays we were able to identify a candidate enhancer element that drives human sost expression in osteoblast-like cell lines in vitro and in the skeletal anlage of the E14.5 mouse embryo, and discovered a novel function for sclerostin during limb development. Our approach represents a framework for characterizing distant regulatory elements associated with abnormal human phenotypes.« less

RNA-Sequencing Analysis Reveals a Regulatory Role for Transcription Factor Fezf2 in the Mature Motor Cortex

PubMed Central

Clare, Alison J.; Wicky, Hollie E.; Empson, Ruth M.; Hughes, Stephanie M.

2017-01-01

Forebrain embryonic zinc finger (Fezf2) encodes a transcription factor essential for the specification of layer 5 projection neurons (PNs) in the developing cerebral cortex. As with many developmental transcription factors, Fezf2 continues to be expressed into adulthood, suggesting it remains crucial to the maintenance of neuronal phenotypes. Despite the continued expression, a function has yet to be explored for Fezf2 in the PNs of the developed cortex. Here, we investigated the role of Fezf2 in mature neurons, using lentiviral-mediated delivery of a shRNA to conditionally knockdown the expression of Fezf2 in the mouse primary motor cortex (M1). RNA-sequencing analysis of Fezf2-reduced M1 revealed significant changes to the transcriptome, identifying a regulatory role for Fezf2 in the mature M1. Kyoto Encyclopedia Genes and Genomes (KEGG) pathway analyses of Fezf2-regulated genes indicated a role in neuronal signaling and plasticity, with significant enrichment of neuroactive ligand-receptor interaction, cell adhesion molecules and calcium signaling pathways. Gene Ontology analysis supported a functional role for Fezf2-regulated genes in neuronal transmission and additionally indicated an importance in the regulation of behavior. Using the mammalian phenotype ontology database, we identified a significant overrepresentation of Fezf2-regulated genes associated with specific behavior phenotypes, including associative learning, social interaction, locomotor activation and hyperactivity. These roles were distinct from that of Fezf2-regulated genes identified in development, indicating a dynamic transition in Fezf2 function. Together our findings demonstrate a regulatory role for Fezf2 in the mature brain, with Fezf2-regulated genes having functional roles in sustaining normal neuronal and behavioral phenotypes. These results support the hypothesis that developmental transcription factors are important for maintaining neuron transcriptomes and that disruption of their expression could contribute to the progression of disease phenotypes. PMID:28936162

Structural analysis of a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase with an N-terminal chorismate mutase-like regulatory domain

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

Light, Samuel H.; Halavaty, Andrei S.; Minasov, George

2012-06-27

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase - an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis - and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn{sup 2+}more » and Mn{sup 2+} + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.« less

Discrete dynamical system modelling for gene regulatory networks of 5-hydroxymethylfurfural tolerance for ethanologenic yeast.

PubMed

Song, M; Ouyang, Z; Liu, Z L

2009-05-01

Composed of linear difference equations, a discrete dynamical system (DDS) model was designed to reconstruct transcriptional regulations in gene regulatory networks (GRNs) for ethanologenic yeast Saccharomyces cerevisiae in response to 5-hydroxymethylfurfural (HMF), a bioethanol conversion inhibitor. The modelling aims at identification of a system of linear difference equations to represent temporal interactions among significantly expressed genes. Power stability is imposed on a system model under the normal condition in the absence of the inhibitor. Non-uniform sampling, typical in a time-course experimental design, is addressed by a log-time domain interpolation. A statistically significant DDS model of the yeast GRN derived from time-course gene expression measurements by exposure to HMF, revealed several verified transcriptional regulation events. These events implicate Yap1 and Pdr3, transcription factors consistently known for their regulatory roles by other studies or postulated by independent sequence motif analysis, suggesting their involvement in yeast tolerance and detoxification of the inhibitor.

Potential Novel Mechanism for Axenfeld-Rieger Syndrome: Deletion of a Distant Region Containing Regulatory Elements of PITX2

PubMed Central

Volkmann, Bethany A.; Zinkevich, Natalya S.; Mustonen, Aki; Schilter, Kala F.; Bosenko, Dmitry V.; Reis, Linda M.; Broeckel, Ulrich; Link, Brian A.

2011-01-01

Purpose. Mutations in PITX2 are associated with Axenfeld-Rieger syndrome (ARS), which involves ocular, dental, and umbilical abnormalities. Identification of cis-regulatory elements of PITX2 is important to better understand the mechanisms of disease. Methods. Conserved noncoding elements surrounding PITX2/pitx2 were identified and examined through transgenic analysis in zebrafish; expression pattern was studied by in situ hybridization. Patient samples were screened for deletion/duplication of the PITX2 upstream region using arrays and probes. Results. Zebrafish pitx2 demonstrates conserved expression during ocular and craniofacial development. Thirteen conserved noncoding sequences positioned within a gene desert as far as 1.1 Mb upstream of the human PITX2 gene were identified; 11 have enhancer activities consistent with pitx2 expression. Ten elements mediated expression in the developing brain, four regions were active during eye formation, and two sequences were associated with craniofacial expression. One region, CE4, located approximately 111 kb upstream of PITX2, directed a complex pattern including expression in the developing eye and craniofacial region, the classic sites affected in ARS. Screening of ARS patients identified an approximately 7600-kb deletion that began 106 to 108 kb upstream of the PITX2 gene, leaving PITX2 intact while removing regulatory elements CE4 to CE13. Conclusions. These data suggest the presence of a complex distant regulatory matrix within the gene desert located upstream of PITX2 with an essential role in its activity and provides a possible mechanism for the previous reports of ARS in patients with balanced translocations involving the 4q25 region upstream of PITX2 and the current patient with an upstream deletion. PMID:20881290

Analysis of functional importance of binding sites in the Drosophila gap gene network model.

PubMed

Kozlov, Konstantin; Gursky, Vitaly V; Kulakovskiy, Ivan V; Dymova, Arina; Samsonova, Maria

2015-01-01

The statistical thermodynamics based approach provides a promising framework for construction of the genotype-phenotype map in many biological systems. Among important aspects of a good model connecting the DNA sequence information with that of a molecular phenotype (gene expression) is the selection of regulatory interactions and relevant transcription factor bindings sites. As the model may predict different levels of the functional importance of specific binding sites in different genomic and regulatory contexts, it is essential to formulate and study such models under different modeling assumptions. We elaborate a two-layer model for the Drosophila gap gene network and include in the model a combined set of transcription factor binding sites and concentration dependent regulatory interaction between gap genes hunchback and Kruppel. We show that the new variants of the model are more consistent in terms of gene expression predictions for various genetic constructs in comparison to previous work. We quantify the functional importance of binding sites by calculating their impact on gene expression in the model and calculate how these impacts correlate across all sites under different modeling assumptions. The assumption about the dual interaction between hb and Kr leads to the most consistent modeling results, but, on the other hand, may obscure existence of indirect interactions between binding sites in regulatory regions of distinct genes. The analysis confirms the previously formulated regulation concept of many weak binding sites working in concert. The model predicts a more or less uniform distribution of functionally important binding sites over the sets of experimentally characterized regulatory modules and other open chromatin domains.

Identification of Small RNAs in Desulfovibrio vulgaris Hildenborough

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

Burns, Andrew; Joachimiak, Marcin; Deutschbauer, Adam

2010-05-17

Desulfovibrio vulgaris is an anaerobic sulfate-reducing bacterium capable of facilitating the removal of toxic metals such as uranium from contaminated sites via reduction. As such, it is essential to understand the intricate regulatory cascades involved in how D. vulgaris and its relatives respond to stressors in such sites. One approach is the identification and analysis of small non-coding RNAs (sRNAs); molecules ranging in size from 20-200 nucleotides that predominantly affect gene regulation by binding to complementary mRNA in an anti-sense fashion and therefore provide an immediate regulatory response. To identify sRNAs in D. vulgaris, a bacterium that does not possessmore » an annotated hfq gene, RNA was pooled from stationary and exponential phases, nitrate exposure, and biofilm conditions. The subsequent RNA was size fractionated, modified, and converted to cDNA for high throughput transcriptomic deep sequencing. A computational approach to identify sRNAs via the alignment of seven separate Desulfovibrio genomes was also performed. From the deep sequencing analysis, 2,296 reads between 20 and 250 nt were identified with expression above genome background. Analysis of those reads limited the number of candidates to ~;;87 intergenic, while ~;;140 appeared to be antisense to annotated open reading frames (ORFs). Further BLAST analysis of the intergenic candidates and other Desulfovibrio genomes indicated that eight candidates were likely portions of ORFs not previously annotated in the D. vulgaris genome. Comparison of the intergenic and antisense data sets to the bioinformatical predicted candidates, resulted in ~;;54 common candidates. Current approaches using Northern analysis and qRT-PCR are being used toverify expression of the candidates and to further develop the role these sRNAs play in D. vulgaris regulation.« less

RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes.

PubMed

Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle

2016-01-01

Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

Foxl2 function in ovarian development.

PubMed

Uhlenhaut, Nina Henriette; Treier, Mathias

2006-07-01

Foxl2 is a forkhead transcription factor essential for proper reproductive function in females. Human patients carrying mutations in the FOXL2 gene display blepharophimosis/ptosis/epicanthus inversus syndrome (BPES), an autosomal dominant disease associated with eyelid defects and premature ovarian failure in females. Recently, animal models for BPES have been developed that in combination with a catalogue of human FOXL2 mutations provide further insight into its molecular function. Mice homozygous mutant for Foxl2 display craniofacial malformations and female infertility. The analysis of the murine phenotype has revealed that Foxl2 is required for granulosa cell function. These ovarian somatic cells surround and nourish the oocyte and play an important role in follicle formation and activation. Mutations upstream of FOXL2 in humans, not affecting the coding sequence itself, have also been shown to cause BPES, which points to the existence of a distant regulatory element necessary for proper gene expression. The same regulatory sequences may be deleted in the goat polled intersex syndrome (PIS), in which FoxL2 expression is severely reduced. Sequence comparison of FoxL2 from several vertebrate species has shown that it is a highly conserved gene involved in ovary development. Thus, the detailed understanding of Foxl2 function and regulation and the identification of its transcriptional targets may open new avenues for the treatment of female infertility in the future.

Pyicos: a versatile toolkit for the analysis of high-throughput sequencing data

PubMed Central

Althammer, Sonja; González-Vallinas, Juan; Ballaré, Cecilia; Beato, Miguel; Eyras, Eduardo

2011-01-01

Motivation: High-throughput sequencing (HTS) has revolutionized gene regulation studies and is now fundamental for the detection of protein–DNA and protein–RNA binding, as well as for measuring RNA expression. With increasing variety and sequencing depth of HTS datasets, the need for more flexible and memory-efficient tools to analyse them is growing. Results: We describe Pyicos, a powerful toolkit for the analysis of mapped reads from diverse HTS experiments: ChIP-Seq, either punctuated or broad signals, CLIP-Seq and RNA-Seq. We prove the effectiveness of Pyicos to select for significant signals and show that its accuracy is comparable and sometimes superior to that of methods specifically designed for each particular type of experiment. Pyicos facilitates the analysis of a variety of HTS datatypes through its flexibility and memory efficiency, providing a useful framework for data integration into models of regulatory genomics. Availability: Open-source software, with tutorials and protocol files, is available at http://regulatorygenomics.upf.edu/pyicos or as a Galaxy server at http://regulatorygenomics.upf.edu/galaxy Contact: eduardo.eyras@upf.edu Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:21994224

BioLayout(Java): versatile network visualisation of structural and functional relationships.

PubMed

Goldovsky, Leon; Cases, Ildefonso; Enright, Anton J; Ouzounis, Christos A

2005-01-01

Visualisation of biological networks is becoming a common task for the analysis of high-throughput data. These networks correspond to a wide variety of biological relationships, such as sequence similarity, metabolic pathways, gene regulatory cascades and protein interactions. We present a general approach for the representation and analysis of networks of variable type, size and complexity. The application is based on the original BioLayout program (C-language implementation of the Fruchterman-Rheingold layout algorithm), entirely re-written in Java to guarantee portability across platforms. BioLayout(Java) provides broader functionality, various analysis techniques, extensions for better visualisation and a new user interface. Examples of analysis of biological networks using BioLayout(Java) are presented.

Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.

PubMed

Klein, Allon M; Mazutis, Linas; Akartuna, Ilke; Tallapragada, Naren; Veres, Adrian; Li, Victor; Peshkin, Leonid; Weitz, David A; Kirschner, Marc W

2015-05-21

It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships. VIDEO ABSTRACT. Copyright © 2015 Elsevier Inc. All rights reserved.

The BaMM web server for de-novo motif discovery and regulatory sequence analysis.

PubMed

Kiesel, Anja; Roth, Christian; Ge, Wanwan; Wess, Maximilian; Meier, Markus; Söding, Johannes

2018-05-28

The BaMM web server offers four tools: (i) de-novo discovery of enriched motifs in a set of nucleotide sequences, (ii) scanning a set of nucleotide sequences with motifs to find motif occurrences, (iii) searching with an input motif for similar motifs in our BaMM database with motifs for >1000 transcription factors, trained from the GTRD ChIP-seq database and (iv) browsing and keyword searching the motif database. In contrast to most other servers, we represent sequence motifs not by position weight matrices (PWMs) but by Bayesian Markov Models (BaMMs) of order 4, which we showed previously to perform substantially better in ROC analyses than PWMs or first order models. To address the inadequacy of P- and E-values as measures of motif quality, we introduce the AvRec score, the average recall over the TP-to-FP ratio between 1 and 100. The BaMM server is freely accessible without registration at https://bammmotif.mpibpc.mpg.de.

Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation.

PubMed Central

Chang, D D; Clayton, D A

1986-01-01

Transcription of the heavy strand of mouse mitochondrial DNA starts from two closely spaced, distinct sites located in the displacement loop region of the genome. We report here an analysis of regulatory sequences required for faithful transcription from these two sites. Data obtained from in vitro assays demonstrated that a 51-base-pair region, encompassing nucleotides -40 to +11 of the downstream start site, contains sufficient information for accurate transcription from both start sites. Deletion of the 3' flanking sequences, including one or both start sites to -17, resulted in the initiation of transcription by the mitochondrial RNA polymerase from alternative sites within vector DNA sequences. This feature places the mouse heavy-strand promoter uniquely among other known mitochondrial promoters, all of which absolutely require cognate start sites for transcription. Comparison of the heavy-strand promoter with those of other vertebrate mitochondrial DNAs revealed a remarkably high rate of sequence divergence among species. Images PMID:3785226

Interpreting Mammalian Evolution using Fugu Genome Comparisons

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

Stubbs, L; Ovcharenko, I; Loots, G G

2004-04-02

Comparative sequence analysis of the human and the pufferfish Fugu rubripes (fugu) genomes has revealed several novel functional coding and noncoding regions in the human genome. In particular, the fugu genome has been extremely valuable for identifying transcriptional regulatory elements in human loci harboring unusually high levels of evolutionary conservation to rodent genomes. In such regions, the large evolutionary distance between human and fishes provides an additional filter through which functional noncoding elements can be detected with high efficiency.

Premature terminator analysis sheds light on a hidden world of bacterial transcriptional attenuation.

PubMed

Naville, Magali; Gautheret, Daniel

2010-01-01

Bacterial transcription attenuation occurs through a variety of cis-regulatory elements that control gene expression in response to a wide range of signals. The signal-sensing structures in attenuators are so diverse and rapidly evolving that only a small fraction have been properly annotated and characterized to date. Here we apply a broad-spectrum detection tool in order to achieve a more complete view of the transcriptional attenuation complement of key bacterial species. Our protocol seeks gene families with an unusual frequency of 5' terminators found across multiple species. Many of the detected attenuators are part of annotated elements, such as riboswitches or T-boxes, which often operate through transcriptional attenuation. However, a significant fraction of candidates were not previously characterized in spite of their unmistakable footprint. We further characterized some of these new elements using sequence and secondary structure analysis. We also present elements that may control the expression of several non-homologous genes, suggesting co-transcription and response to common signals. An important class of such elements, which we called mobile attenuators, is provided by 3' terminators of insertion sequences or prophages that may be exapted as 5' regulators when inserted directly upstream of a cellular gene. We show here that attenuators involve a complex landscape of signal-detection structures spanning the entire bacterial domain. We discuss possible scenarios through which these diverse 5' regulatory structures may arise or evolve.

Differential expression analysis of Paralichthys olivaceus microRNAs in adult ovary and testis by deep sequencing.

PubMed

Gu, Yifeng; Zhang, Lei; Chen, Xiaowu

2014-08-01

MicroRNAs (miRNAs) play an important role in gonadal development and differentiation in fish. However, understanding of the mechanism of this process is hindered by our poor knowledge of miRNA expression patterns in fish gonads. In this study, miRNA libraries derived from adult gonads of Paralichthys olivaceus were generated by using next-generation sequencing (NGS) technology. Bioinformatics analysis was performed to distinguish mature miRNA sequences from two classes of small RNAs represented in the sequencing data. A total of 141 mature miRNAs were identified, in which 21 miRNAs were found in P. olivaceus for the first time. Variance and preference of miRNAs expression were concluded from the deep sequencing reads. Some miRNAs, such as pol-miR-143, pol-miR-26a and pol-let-7a were found with quite high expression levels in both gonads, while some exhibited a clear sex-biased expression in different gonad. Approximate 20.0% and 13.1% of the isolated miRNAs were preferentially expressed in the testis (FC<0.5) or ovary (FC>2), respectively. The identification and the preliminary analysis of the sex-biased expression of miRNAs in P. olivaceus gonads in our work by using NGS will provide us a basic catalog of miRNAs to facilitate future improvement and exploitation of sexual regulatory mechanisms in P. olivaceus. Copyright © 2014. Published by Elsevier Inc.

Identification of single nucleotide polymorphism in protein phosphatase 1 regulatory subunit 11 gene in Murrah bulls

PubMed Central

Jain, Varsha; Patel, Brijesh; Umar, Farhat Paul; Ajithakumar, H. M.; Gurjar, Suraj K.; Gupta, I. D.; Verma, Archana

2017-01-01

Aim: This study was conducted with the objective to identify single nucleotide polymorphism (SNP) in protein phosphatase 1 regulatory subunit 11 (PPP1R11) gene in Murrah bulls. Materials and Methods: Genomic DNA was isolated by phenol–chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal. The quality and concentration of DNA was checked by spectrophotometer reading and agarose gel electrophoresis. The target region of PPP1R11 gene was amplified using four sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced and aligned using Clustal Omega for identification of SNPs. Animals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using EcoNI restriction enzyme. Results: The sequences in the NCBI accession number NW_005785016.1 for Bubalus bubalis were compared and aligned with the edited sequences of Murrah bulls with Clustal Omega software. A total of 10 SNPs were found, out of which 1 at 5’UTR, 3 at intron 1, and 6 at intron 2 region. PCR-RFLP using restriction enzyme EcoNI revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study. Conclusion: A total of 10 SNPs were found. PCR-RFLP revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study, due to which association analysis with conception rate was not feasible. PMID:28344410

Genome-scale cold stress response regulatory networks in ten Arabidopsis thaliana ecotypes

PubMed Central

2013-01-01

Background Low temperature leads to major crop losses every year. Although several studies have been conducted focusing on diversity of cold tolerance level in multiple phenotypically divergent Arabidopsis thaliana (A. thaliana) ecotypes, genome-scale molecular understanding is still lacking. Results In this study, we report genome-scale transcript response diversity of 10 A. thaliana ecotypes originating from different geographical locations to non-freezing cold stress (10°C). To analyze the transcriptional response diversity, we initially compared transcriptome changes in all 10 ecotypes using Arabidopsis NimbleGen ATH6 microarrays. In total 6061 transcripts were significantly cold regulated (p < 0.01) in 10 ecotypes, including 498 transcription factors and 315 transposable elements. The majority of the transcripts (75%) showed ecotype specific expression pattern. By using sequence data available from Arabidopsis thaliana 1001 genome project, we further investigated sequence polymorphisms in the core cold stress regulon genes. Significant numbers of non-synonymous amino acid changes were observed in the coding region of the CBF regulon genes. Considering the limited knowledge about regulatory interactions between transcription factors and their target genes in the model plant A. thaliana, we have adopted a powerful systems genetics approach- Network Component Analysis (NCA) to construct an in-silico transcriptional regulatory network model during response to cold stress. The resulting regulatory network contained 1,275 nodes and 7,720 connections, with 178 transcription factors and 1,331 target genes. Conclusions A. thaliana ecotypes exhibit considerable variation in transcriptome level responses to non-freezing cold stress treatment. Ecotype specific transcripts and related gene ontology (GO) categories were identified to delineate natural variation of cold stress regulated differential gene expression in the model plant A. thaliana. The predicted regulatory network model was able to identify new ecotype specific transcription factors and their regulatory interactions, which might be crucial for their local geographic adaptation to cold temperature. Additionally, since the approach presented here is general, it could be adapted to study networks regulating biological process in any biological systems. PMID:24148294

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

Sattison, M.B.; Thatcher, T.A.; Knudsen, J.K.

The US Nuclear Regulatory Commission (NRC) has been using full-power. Level 1, limited-scope risk models for the Accident Sequence Precursor (ASP) program for over fifteen years. These models have evolved and matured over the years, as have probabilistic risk assessment (PRA) and computer technologies. Significant upgrading activities have been undertaken over the past three years, with involvement from the Offices of Nuclear Reactor Regulation (NRR), Analysis and Evaluation of Operational Data (AEOD), and Nuclear Regulatory Research (RES), and several national laboratories. Part of these activities was an RES-sponsored feasibility study investigating the ability to extend the ASP models to includemore » contributors to core damage from events initiated with the reactor at low power or shutdown (LP/SD), both internal events and external events. This paper presents only the LP/SD internal event modeling efforts.« less

Syndromes associated with Homo sapiens pol II regulatory genes.

PubMed

Bina, M; Demmon, S; Pares-Matos, E I

2000-01-01

The molecular basis of human characteristics is an intriguing but an unresolved problem. Human characteristics cover a broad spectrum, from the obvious to the abstract. Obvious characteristics may include morphological features such as height, shape, and facial form. Abstract characteristics may be hidden in processes that are controlled by hormones and the human brain. In this review we examine exaggerated characteristics presented as syndromes. Specifically, we focus on human genes that encode transcription factors to examine morphological, immunological, and hormonal anomalies that result from deletion, insertion, or mutation of genes that regulate transcription by RNA polymerase II (the Pol II genes). A close analysis of abnormal phenotypes can give clues into how sequence variations in regulatory genes and changes in transcriptional control may give rise to characteristics defined as complex traits.

Identification of novel craniofacial regulatory domains located far upstream of SOX9 and disrupted in Pierre Robin sequence

PubMed Central

Gordon, Christopher T.; Attanasio, Catia; Bhatia, Shipra; Benko, Sabina; Ansari, Morad; Tan, Tiong Y.; Munnich, Arnold; Pennacchio, Len A.; Abadie, Véronique; Temple, I. Karen; Goldenberg, Alice; van Heyningen, Veronica; Amiel, Jeanne; FitzPatrick, David; Kleinjan, Dirk A.; Visel, Axel; Lyonnet, Stanislas

2015-01-01

Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions and duplications within a ~2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ~1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harbouring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple non-coding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS. PMID:24934569

A liver enhancer in the fibrinogen gene cluster.

PubMed

Fort, Alexandre; Fish, Richard J; Attanasio, Catia; Dosch, Roland; Visel, Axel; Neerman-Arbez, Marguerite

2011-01-06

The plasma concentration of fibrinogen varies in the healthy human population between 1.5 and 3.5 g/L. Understanding the basis of this variability has clinical importance because elevated fibrinogen levels are associated with increased cardiovascular disease risk. To identify novel regulatory elements involved in the control of fibrinogen expression, we used sequence conservation and in silico-predicted regulatory potential to select 14 conserved noncoding sequences (CNCs) within the conserved block of synteny containing the fibrinogen locus. The regulatory potential of each CNC was tested in vitro using a luciferase reporter gene assay in fibrinogen-expressing hepatoma cell lines (HuH7 and HepG2). 4 potential enhancers were tested for their ability to direct enhanced green fluorescent protein expression in zebrafish embryos. CNC12, a sequence equidistant from the human fibrinogen alpha and beta chain genes, activates strong liver enhanced green fluorescent protein expression in injected embryos and their transgenic progeny. A transgenic assay in embryonic day 14.5 mouse embryos confirmed the ability of CNC12 to activate transcription in the liver. While additional experiments are necessary to prove the role of CNC12 in the regulation of fibrinogen, our study reveals a novel regulatory element in the fibrinogen locus that is active in the liver and may contribute to variable fibrinogen expression in humans.

Transcription Factor Map Alignment of Promoter Regions

PubMed Central

Blanco, Enrique; Messeguer, Xavier; Smith, Temple F; Guigó, Roderic

2006-01-01

We address the problem of comparing and characterizing the promoter regions of genes with similar expression patterns. This remains a challenging problem in sequence analysis, because often the promoter regions of co-expressed genes do not show discernible sequence conservation. In our approach, thus, we have not directly compared the nucleotide sequence of promoters. Instead, we have obtained predictions of transcription factor binding sites, annotated the predicted sites with the labels of the corresponding binding factors, and aligned the resulting sequences of labels—to which we refer here as transcription factor maps (TF-maps). To obtain the global pairwise alignment of two TF-maps, we have adapted an algorithm initially developed to align restriction enzyme maps. We have optimized the parameters of the algorithm in a small, but well-curated, collection of human–mouse orthologous gene pairs. Results in this dataset, as well as in an independent much larger dataset from the CISRED database, indicate that TF-map alignments are able to uncover conserved regulatory elements, which cannot be detected by the typical sequence alignments. PMID:16733547

Gene and domain duplication in the chordate Otx gene family: insights from amphioxus Otx.

PubMed

Williams, N A; Holland, P W

1998-05-01

We report the genomic organization and deduced protein sequence of a cephalochordate member of the Otx homeobox gene family (AmphiOtx) and show its probable single-copy state in the genome. We also present molecular phylogenetic analysis indicating that there was single ancestral Otx gene in the first chordates which was duplicated in the vertebrate lineage after it had split from the lineage leading to the cephalochordates. Duplication of a C-terminal protein domain has occurred specifically in the vertebrate lineage, strengthening the case for a single Otx gene in an ancestral chordate whose gene structure has been retained in an extant cephalochordate. Comparative analysis of protein sequences and published gene expression patterns suggest that the ancestral chordate Otx gene had roles in patterning the anterior mesendoderm and central nervous system. These roles were elaborated following Otx gene duplication in vertebrates, accompanied by regulatory and structural divergence, particularly of Otx1 descendant genes.

Analyzing multiple data sets by interconnecting RSAT programs via SOAP Web services: an example with ChIP-chip data.

PubMed

Sand, Olivier; Thomas-Chollier, Morgane; Vervisch, Eric; van Helden, Jacques

2008-01-01

This protocol shows how to access the Regulatory Sequence Analysis Tools (RSAT) via a programmatic interface in order to automate the analysis of multiple data sets. We describe the steps for writing a Perl client that connects to the RSAT Web services and implements a workflow to discover putative cis-acting elements in promoters of gene clusters. In the presented example, we apply this workflow to lists of transcription factor target genes resulting from ChIP-chip experiments. For each factor, the protocol predicts the binding motifs by detecting significantly overrepresented hexanucleotides in the target promoters and generates a feature map that displays the positions of putative binding sites along the promoter sequences. This protocol is addressed to bioinformaticians and biologists with programming skills (notions of Perl). Running time is approximately 6 min on the example data set.

Transcriptome analysis and related databases of Lactococcus lactis.

PubMed

Kuipers, Oscar P; de Jong, Anne; Baerends, Richard J S; van Hijum, Sacha A F T; Zomer, Aldert L; Karsens, Harma A; den Hengst, Chris D; Kramer, Naomi E; Buist, Girbe; Kok, Jan

2002-08-01

Several complete genome sequences of Lactococcus lactis and their annotations will become available in the near future, next to the already published genome sequence of L. lactis ssp. lactis IL 1403. This will allow intraspecies comparative genomics studies as well as functional genomics studies aimed at a better understanding of physiological processes and regulatory networks operating in lactococci. This paper describes the initial set-up of a DNA-microarray facility in our group, to enable transcriptome analysis of various Gram-positive bacteria, including a ssp. lactis and a ssp. cremoris strain of Lactococcus lactis. Moreover a global description will be given of the hardware and software requirements for such a set-up, highlighting the crucial integration of relevant bioinformatics tools and methods. This includes the development of MolGenIS, an information system for transcriptome data storage and retrieval, and LactococCye, a metabolic pathway/genome database of Lactococcus lactis.

Genome-wide prediction of cis-regulatory regions using supervised deep learning methods.

PubMed

Li, Yifeng; Shi, Wenqiang; Wasserman, Wyeth W

2018-05-31

In the human genome, 98% of DNA sequences are non-protein-coding regions that were previously disregarded as junk DNA. In fact, non-coding regions host a variety of cis-regulatory regions which precisely control the expression of genes. Thus, Identifying active cis-regulatory regions in the human genome is critical for understanding gene regulation and assessing the impact of genetic variation on phenotype. The developments of high-throughput sequencing and machine learning technologies make it possible to predict cis-regulatory regions genome wide. Based on rich data resources such as the Encyclopedia of DNA Elements (ENCODE) and the Functional Annotation of the Mammalian Genome (FANTOM) projects, we introduce DECRES based on supervised deep learning approaches for the identification of enhancer and promoter regions in the human genome. Due to their ability to discover patterns in large and complex data, the introduction of deep learning methods enables a significant advance in our knowledge of the genomic locations of cis-regulatory regions. Using models for well-characterized cell lines, we identify key experimental features that contribute to the predictive performance. Applying DECRES, we delineate locations of 300,000 candidate enhancers genome wide (6.8% of the genome, of which 40,000 are supported by bidirectional transcription data), and 26,000 candidate promoters (0.6% of the genome). The predicted annotations of cis-regulatory regions will provide broad utility for genome interpretation from functional genomics to clinical applications. The DECRES model demonstrates potentials of deep learning technologies when combined with high-throughput sequencing data, and inspires the development of other advanced neural network models for further improvement of genome annotations.

Diagnostic screening identifies a wide range of mutations involving the SHOX gene, including a common 47.5 kb deletion 160 kb downstream with a variable phenotypic effect.

PubMed

Bunyan, David J; Baker, Kevin R; Harvey, John F; Thomas, N Simon

2013-06-01

Léri-Weill dyschondrosteosis (LWD) results from heterozygous mutations of the SHOX gene, with homozygosity or compound heterozygosity resulting in the more severe form, Langer mesomelic dysplasia (LMD). These mutations typically take the form of whole or partial gene deletions, point mutations within the coding sequence, or large (>100 kb) 3' deletions of downstream regulatory elements. We have analyzed the coding sequence of the SHOX gene and its downstream regulatory regions in a cohort of 377 individuals referred with symptoms of LWD, LMD or short stature. A causative mutation was identified in 68% of the probands with LWD or LMD (91/134). In addition, a 47.5 kb deletion was found 160 kb downstream of the SHOX gene in 17 of the 377 patients (12% of the LWD referrals, 4.5% of all referrals). In 14 of these 17 patients, this was the only potentially causative abnormality detected (13 had symptoms consistent with LWD and one had short stature only), but the other three 47.5 kb deletions were found in patients with an additional causative SHOX mutation (with symptoms of LWD rather than LMD). Parental samples were available on 14/17 of these families, and analysis of these showed a more variable phenotype ranging from apparently unaffected to LWD. Breakpoint sequence analysis has shown that the 47.5 kb deletion is identical in all 17 patients, most likely due to an ancient founder mutation rather than recurrence. This deletion was not seen in 471 normal controls (P<0.0001), providing further evidence for a phenotypic effect, albeit one with variable penetration. Copyright © 2013 Wiley Periodicals, Inc.

Genetic Architecture of Flowering Phenology in Cereals and Opportunities for Crop Improvement

PubMed Central

Hill, Camilla B.; Li, Chengdao

2016-01-01

Cereal crop species including bread wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), rice (Oryza sativa L.), and maize (Zea mays L.) provide the bulk of human nutrition and agricultural products for industrial use. These four cereals are central to meet future demands of food supply for an increasing world population under a changing climate. A prerequisite for cereal crop production is the transition from vegetative to reproductive and grain-filling phases starting with flower initiation, a key developmental switch tightly regulated in all flowering plants. Although studies in the dicotyledonous model plant Arabidopsis thaliana build the foundations of our current understanding of plant phenology genes and regulation, the availability of genome assemblies with high-confidence sequences for rice, maize, and more recently bread wheat and barley, now allow the identification of phenology-associated gene orthologs in monocots. Together with recent advances in next-generation sequencing technologies, QTL analysis, mutagenesis, complementation analysis, and RNA interference, many phenology genes have been functionally characterized in cereal crops and conserved as well as functionally divergent genes involved in flowering were found. Epigenetic and other molecular regulatory mechanisms that respond to environmental and endogenous triggers create an enormous plasticity in flowering behavior among cereal crops to ensure flowering is only induced under optimal conditions. In this review, we provide a summary of recent discoveries of flowering time regulators with an emphasis on four cereal crop species (bread wheat, barley, rice, and maize), in particular, crop-specific regulatory mechanisms and genes. In addition, pleiotropic effects on agronomically important traits such as grain yield, impact on adaptation to new growing environments and conditions, genetic sequence-based selection and targeted manipulation of phenology genes, as well as crop growth simulation models for predictive crop breeding, are discussed. PMID:28066466

Small RNA profiling and degradome analysis reveal regulation of microRNA in peanut embryogenesis and early pod development.

PubMed

Gao, Chao; Wang, Pengfei; Zhao, Shuzhen; Zhao, Chuanzhi; Xia, Han; Hou, Lei; Ju, Zheng; Zhang, Ye; Li, Changsheng; Wang, Xingjun

2017-03-02

As a typical geocarpic plant, peanut embryogenesis and pod development are complex processes involving many gene regulatory pathways and controlled by appropriate hormone level. MicroRNAs (miRNAs) are small non-coding RNAs that play indispensable roles in post-transcriptional gene regulation. Recently, identification and characterization of peanut miRNAs has been described. However, whether miRNAs participate in the regulation of peanut embryogenesis and pod development has yet to be explored. In this study, small RNA and degradome libraries from peanut early pod of different developmental stages were constructed and sequenced. A total of 70 known and 24 novel miRNA families were discovered. Among them, 16 miRNA families were legume-specific and 12 families were peanut-specific. 30 known and 10 novel miRNA families were differentially expressed during pod development. In addition, 115 target genes were identified for 47 miRNA families by degradome sequencing. Several new targets that might be specific to peanut were found and further validated by RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM 5'-RACE). Furthermore, we performed profiling analysis of intact and total transcripts of several target genes, demonstrating that SPL (miR156/157), NAC (miR164), PPRP (miR167 and miR1088), AP2 (miR172) and GRF (miR396) are actively modulated during early pod development, respectively. Large numbers of miRNAs and their related target genes were identified through deep sequencing. These findings provided new information on miRNA-mediated regulatory pathways in peanut pod, which will contribute to the comprehensive understanding of the molecular mechanisms that governing peanut embryo and early pod development.

CoryneRegNet 3.0--an interactive systems biology platform for the analysis of gene regulatory networks in corynebacteria and Escherichia coli.

PubMed

Baumbach, Jan; Wittkop, Tobias; Rademacher, Katrin; Rahmann, Sven; Brinkrolf, Karina; Tauch, Andreas

2007-04-30

CoryneRegNet is an ontology-based data warehouse for the reconstruction and visualization of transcriptional regulatory interactions in prokaryotes. To extend the biological content of CoryneRegNet, we added comprehensive data on transcriptional regulations in the model organism Escherichia coli K-12, originally deposited in the international reference database RegulonDB. The enhanced web interface of CoryneRegNet offers several types of search options. The results of a search are displayed in a table-based style and include a visualization of the genetic organization of the respective gene region. Information on DNA binding sites of transcriptional regulators is depicted by sequence logos. The results can also be displayed by several layouters implemented in the graphical user interface GraphVis, allowing, for instance, the visualization of genome-wide network reconstructions and the homology-based inter-species comparison of reconstructed gene regulatory networks. In an application example, we compare the composition of the gene regulatory networks involved in the SOS response of E. coli and Corynebacterium glutamicum. CoryneRegNet is available at the following URL: http://www.cebitec.uni-bielefeld.de/groups/gi/software/coryneregnet/.

Explaining the disease phenotype of intergenic SNP through predicted long range regulation

PubMed Central

Chen, Jingqi; Tian, Weidong

2016-01-01

Thousands of disease-associated SNPs (daSNPs) are located in intergenic regions (IGR), making it difficult to understand their association with disease phenotypes. Recent analysis found that non-coding daSNPs were frequently located in or approximate to regulatory elements, inspiring us to try to explain the disease phenotypes of IGR daSNPs through nearby regulatory sequences. Hence, after locating the nearest distal regulatory element (DRE) to a given IGR daSNP, we applied a computational method named INTREPID to predict the target genes regulated by the DRE, and then investigated their functional relevance to the IGR daSNP's disease phenotypes. 36.8% of all IGR daSNP-disease phenotype associations investigated were possibly explainable through the predicted target genes, which were enriched with, were functionally relevant to, or consisted of the corresponding disease genes. This proportion could be further increased to 60.5% if the LD SNPs of daSNPs were also considered. Furthermore, the predicted SNP-target gene pairs were enriched with known eQTL/mQTL SNP-gene relationships. Overall, it's likely that IGR daSNPs may contribute to disease phenotypes by interfering with the regulatory function of their nearby DREs and causing abnormal expression of disease genes. PMID:27280978

Murine endogenous retroviruses

PubMed Central

2016-01-01

Up to 10% of the mouse genome is comprised of endogenous retrovirus (ERV) sequences, and most represent the remains of ancient germ line infections. Our knowledge of the three distinct classes of ERVs is inversely correlated with their copy number, and their characterization has benefited from the availability of divergent wild mouse species and subspecies, and from ongoing analysis of the Mus genome sequence. In contrast to human ERVs, which are nearly all extinct, active mouse ERVs can still be found in all three ERV classes. The distribution and diversity of ERVs has been shaped by host-virus interactions over the course of evolution, but ERVs have also been pivotal in shaping the mouse genome by altering host genes through insertional mutagenesis, by adding novel regulatory and coding sequences, and by their co-option by host cells as retroviral resistance genes. We review mechanisms by which an adaptive coexistence has evolved. (Part of a Multi-author Review) PMID:18818872

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells.

PubMed

Malina, Halina Z

2011-01-19

The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases.

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells

PubMed Central

2011-01-01

Background The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Results Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. Conclusions The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases. PMID:21247434

MicroRNA and Transcription Factor: Key Players in Plant Regulatory Network.

PubMed

Samad, Abdul F A; Sajad, Muhammad; Nazaruddin, Nazaruddin; Fauzi, Izzat A; Murad, Abdul M A; Zainal, Zamri; Ismail, Ismanizan

2017-01-01

Recent achievements in plant microRNA (miRNA), a large class of small and non-coding RNAs, are very exciting. A wide array of techniques involving forward genetic, molecular cloning, bioinformatic analysis, and the latest technology, deep sequencing have greatly advanced miRNA discovery. A tiny miRNA sequence has the ability to target single/multiple mRNA targets. Most of the miRNA targets are transcription factors (TFs) which have paramount importance in regulating the plant growth and development. Various families of TFs, which have regulated a range of regulatory networks, may assist plants to grow under normal and stress environmental conditions. This present review focuses on the regulatory relationships between miRNAs and different families of TFs like; NF-Y, MYB, AP2, TCP, WRKY, NAC, GRF, and SPL. For instance NF-Y play important role during drought tolerance and flower development, MYB are involved in signal transduction and biosynthesis of secondary metabolites, AP2 regulate the floral development and nodule formation, TCP direct leaf development and growth hormones signaling. WRKY have known roles in multiple stress tolerances, NAC regulate lateral root formation, GRF are involved in root growth, flower, and seed development, and SPL regulate plant transition from juvenile to adult. We also studied the relation between miRNAs and TFs by consolidating the research findings from different plant species which will help plant scientists in understanding the mechanism of action and interaction between these regulators in the plant growth and development under normal and stress environmental conditions.

Inferring Selective Constraint from Population Genomic Data Suggests Recent Regulatory Turnover in the Human Brain

PubMed Central

Schrider, Daniel R.; Kern, Andrew D.

2015-01-01

The comparative genomics revolution of the past decade has enabled the discovery of functional elements in the human genome via sequence comparison. While that is so, an important class of elements, those specific to humans, is entirely missed by searching for sequence conservation across species. Here we present an analysis based on variation data among human genomes that utilizes a supervised machine learning approach for the identification of human-specific purifying selection in the genome. Using only allele frequency information from the complete low-coverage 1000 Genomes Project data set in conjunction with a support vector machine trained from known functional and nonfunctional portions of the genome, we are able to accurately identify portions of the genome constrained by purifying selection. Our method identifies previously known human-specific gains or losses of function and uncovers many novel candidates. Candidate targets for gain and loss of function along the human lineage include numerous putative regulatory regions of genes essential for normal development of the central nervous system, including a significant enrichment of gain of function events near neurotransmitter receptor genes. These results are consistent with regulatory turnover being a key mechanism in the evolution of human-specific characteristics of brain development. Finally, we show that the majority of the genome is unconstrained by natural selection currently, in agreement with what has been estimated from phylogenetic methods but in sharp contrast to estimates based on transcriptomics or other high-throughput functional methods. PMID:26590212

Bayesian Inference of Allele-Specific Gene Expression Indicates Abundant Cis-Regulatory Variation in Natural Flycatcher Populations

PubMed Central

Wang, Mi

2017-01-01

Abstract Polymorphism in cis-regulatory sequences can lead to different levels of expression for the two alleles of a gene, providing a starting point for the evolution of gene expression. Little is known about the genome-wide abundance of genetic variation in gene regulation in natural populations but analysis of allele-specific expression (ASE) provides a means for investigating such variation. We performed RNA-seq of multiple tissues from population samples of two closely related flycatcher species and developed a Bayesian algorithm that maximizes data usage by borrowing information from the whole data set and combines several SNPs per transcript to detect ASE. Of 2,576 transcripts analyzed in collared flycatcher, ASE was detected in 185 (7.2%) and a similar frequency was seen in the pied flycatcher. Transcripts with statistically significant ASE commonly showed the major allele in >90% of the reads, reflecting that power was highest when expression was heavily biased toward one of the alleles. This would suggest that the observed frequencies of ASE likely are underestimates. The proportion of ASE transcripts varied among tissues, being lowest in testis and highest in muscle. Individuals often showed ASE of particular transcripts in more than one tissue (73.4%), consistent with a genetic basis for regulation of gene expression. The results suggest that genetic variation in regulatory sequences commonly affects gene expression in natural populations and that it provides a seedbed for phenotypic evolution via divergence in gene expression. PMID:28453623

Systems analysis of cis-regulatory motifs in C4 photosynthesis genes using maize and rice leaf transcriptomic data during a process of de-etiolation

PubMed Central

Xu, Jiajia; Bräutigam, Andrea; Weber, Andreas P. M.; Zhu, Xin-Guang

2016-01-01

Identification of potential cis-regulatory motifs controlling the development of C4 photosynthesis is a major focus of current research. In this study, we used time-series RNA-seq data collected from etiolated maize and rice leaf tissues sampled during a de-etiolation process to systematically characterize the expression patterns of C4-related genes and to further identify potential cis elements in five different genomic regions (i.e. promoter, 5′UTR, 3′UTR, intron, and coding sequence) of C4 orthologous genes. The results demonstrate that although most of the C4 genes show similar expression patterns, a number of them, including chloroplast dicarboxylate transporter 1, aspartate aminotransferase, and triose phosphate transporter, show shifted expression patterns compared with their C3 counterparts. A number of conserved short DNA motifs between maize C4 genes and their rice orthologous genes were identified not only in the promoter, 5′UTR, 3′UTR, and coding sequences, but also in the introns of core C4 genes. We also identified cis-regulatory motifs that exist in maize C4 genes and also in genes showing similar expression patterns as maize C4 genes but that do not exist in rice C3 orthologs, suggesting a possible recruitment of pre-existing cis-elements from genes unrelated to C4 photosynthesis into C4 photosynthesis genes during C4 evolution. PMID:27436282

Automated conserved non-coding sequence (CNS) discovery reveals differences in gene content and promoter evolution among grasses

PubMed Central

Turco, Gina; Schnable, James C.; Pedersen, Brent; Freeling, Michael

2013-01-01

Conserved non-coding sequences (CNS) are islands of non-coding sequence that, like protein coding exons, show less divergence in sequence between related species than functionless DNA. Several CNSs have been demonstrated experimentally to function as cis-regulatory regions. However, the specific functions of most CNSs remain unknown. Previous searches for CNS in plants have either anchored on exons and only identified nearby sequences or required years of painstaking manual annotation. Here we present an open source tool that can accurately identify CNSs between any two related species with sequenced genomes, including both those immediately adjacent to exons and distal sequences separated by >12 kb of non-coding sequence. We have used this tool to characterize new motifs, associate CNSs with additional functions, and identify previously undetected genes encoding RNA and protein in the genomes of five grass species. We provide a list of 15,363 orthologous CNSs conserved across all grasses tested. We were also able to identify regulatory sequences present in the common ancestor of grasses that have been lost in one or more extant grass lineages. Lists of orthologous gene pairs and associated CNSs are provided for reference inbred lines of arabidopsis, Japonica rice, foxtail millet, sorghum, brachypodium, and maize. PMID:23874343

Integrated Regulatory and Metabolic Networks of the Marine Diatom Phaeodactylum tricornutum Predict the Response to Rising CO 2 Levels

DOE PAGES

Levering, Jennifer; Dupont, Christopher L.; Allen, Andrew E.; ...

2017-02-14

Diatoms are eukaryotic microalgae that are responsible for up to 40% of the ocean’s primary productivity. How diatoms respond to environmental perturbations such as elevated carbon concentrations in the atmosphere is currently poorly understood. We developed a transcriptional regulatory network based on various transcriptome sequencing expression libraries for different environmental responses to gain insight into the marine diatom’s metabolic and regulatory interactions and provide a comprehensive framework of responses to increasing atmospheric carbon levels. This transcriptional regulatory network was integrated with a recently published genome-scale metabolic model of Phaeodactylum tricornutum to explore the connectivity of the regulatory network and sharedmore » metabolites. The integrated regulatory and metabolic model revealed highly connected modules within carbon and nitrogen metabolism. P. tricornutum’s response to rising carbon levels was analyzed by using the recent genome-scale metabolic model with cross comparison to experimental manipulations of carbon dioxide. Using a systems biology approach, we studied the response of the marine diatom Phaeodactylum tricornutum to changing atmospheric carbon concentrations on an ocean-wide scale. By integrating an available genome-scale metabolic model and a newly developed transcriptional regulatory network inferred from transcriptome sequencing expression data, we demonstrate that carbon metabolism and nitrogen metabolism are strongly connected and the genes involved are coregulated in this model diatom. These tight regulatory constraints could play a major role during the adaptation of P. tricornutum to increasing carbon levels. The transcriptional regulatory network developed can be further used to study the effects of different environmental perturbations on P. tricornutum’s metabolism.« less

Integrated Regulatory and Metabolic Networks of the Marine Diatom Phaeodactylum tricornutum Predict the Response to Rising CO 2 Levels

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

Levering, Jennifer; Dupont, Christopher L.; Allen, Andrew E.

Diatoms are eukaryotic microalgae that are responsible for up to 40% of the ocean’s primary productivity. How diatoms respond to environmental perturbations such as elevated carbon concentrations in the atmosphere is currently poorly understood. We developed a transcriptional regulatory network based on various transcriptome sequencing expression libraries for different environmental responses to gain insight into the marine diatom’s metabolic and regulatory interactions and provide a comprehensive framework of responses to increasing atmospheric carbon levels. This transcriptional regulatory network was integrated with a recently published genome-scale metabolic model of Phaeodactylum tricornutum to explore the connectivity of the regulatory network and sharedmore » metabolites. The integrated regulatory and metabolic model revealed highly connected modules within carbon and nitrogen metabolism. P. tricornutum’s response to rising carbon levels was analyzed by using the recent genome-scale metabolic model with cross comparison to experimental manipulations of carbon dioxide. Using a systems biology approach, we studied the response of the marine diatom Phaeodactylum tricornutum to changing atmospheric carbon concentrations on an ocean-wide scale. By integrating an available genome-scale metabolic model and a newly developed transcriptional regulatory network inferred from transcriptome sequencing expression data, we demonstrate that carbon metabolism and nitrogen metabolism are strongly connected and the genes involved are coregulated in this model diatom. These tight regulatory constraints could play a major role during the adaptation of P. tricornutum to increasing carbon levels. The transcriptional regulatory network developed can be further used to study the effects of different environmental perturbations on P. tricornutum’s metabolism.« less

Sequence divergence in the 3'-untranslated region has an effect on the subfunctionalization of duplicate genes.

PubMed

Tong, Ying; Zheng, Kang; Zhao, Shufang; Xiao, Guanxiu; Luo, Chen

2012-11-01

Recent studies demonstrated that sequence divergence in both transcriptional regulatory region and coding region contributes to the subfunctionalization of duplicate gene. However, whether sequence divergence in the 3'-untranslated region (3'-UTR) has an impact on the subfunctionalization of duplicate genes remains unclear. Here, we identified two diverging duplicate vsx1 (visual system homeobox-1) loci in goldfish, named vsx1A1 and vsx1A2. Phylogenetic analysis suggests that vsx1A1 and vsx1A2 may arise from a duplication of vsx1 after the separation of goldfish and zebrafish. Sequence comparison revealed that divergence in both transcriptional and translational regulatory regions is higher than divergence in the introns. vsx1A2 expresses during blastula and gastrula stages and in adult retina but silences from segmentation stage to hatching stage, vsx1A1 starts expression from segmentation onward. Comparing to that zebrafish vsx1 expresses in all the developmental stages and in the adult retina, it appears that goldfish vsx1A1 and vsx1A2 are under going to share the functions of ancestral vsx1. The different but overlapping temporal expression patterns of vsx1A1 and vsx1A2 suggest that sequence divergence in the promoter region of duplicate vsx1 is not sufficient for partitioning the functions of ancestral vsx1. By comparing vsx1A1 and vsx1A2 3'-UTR-linked green fluorescent protein gene expression patterns, we demonstrated that the 3'-UTR of vsx1A1 remains but the 3'-UTR of vsx1A2 has lost the capability of mediating bipolar cell specific expression during retina development. These results indicate that sequence divergence in the 3'-UTRs has a clear effect on subfunctionalization of the duplicate genes. © 2012 WILEY PERIODICALS, INC.

Chromatin accessibility prediction via a hybrid deep convolutional neural network.

PubMed

Liu, Qiao; Xia, Fei; Yin, Qijin; Jiang, Rui

2018-03-01

A majority of known genetic variants associated with human-inherited diseases lie in non-coding regions that lack adequate interpretation, making it indispensable to systematically discover functional sites at the whole genome level and precisely decipher their implications in a comprehensive manner. Although computational approaches have been complementing high-throughput biological experiments towards the annotation of the human genome, it still remains a big challenge to accurately annotate regulatory elements in the context of a specific cell type via automatic learning of the DNA sequence code from large-scale sequencing data. Indeed, the development of an accurate and interpretable model to learn the DNA sequence signature and further enable the identification of causative genetic variants has become essential in both genomic and genetic studies. We proposed Deopen, a hybrid framework mainly based on a deep convolutional neural network, to automatically learn the regulatory code of DNA sequences and predict chromatin accessibility. In a series of comparison with existing methods, we show the superior performance of our model in not only the classification of accessible regions against background sequences sampled at random, but also the regression of DNase-seq signals. Besides, we further visualize the convolutional kernels and show the match of identified sequence signatures and known motifs. We finally demonstrate the sensitivity of our model in finding causative noncoding variants in the analysis of a breast cancer dataset. We expect to see wide applications of Deopen with either public or in-house chromatin accessibility data in the annotation of the human genome and the identification of non-coding variants associated with diseases. Deopen is freely available at https://github.com/kimmo1019/Deopen. ruijiang@tsinghua.edu.cn. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

77 FR 7942 - Semiannual Regulatory Agenda

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-13

... Sequence No. Title Identifier No. 315 Energy Efficiency 1904-AB57 Standards for Battery Chargers and... Renewable Energy (EE) Proposed Rule Stage 315. Energy Efficiency Standards for Battery Chargers and External... regulatory flexibility agenda is made up of six rulemakings setting energy efficiency standards for the...

The first FDA marketing authorizations of next-generation sequencing technology and tests: challenges, solutions and impact for future assays.

PubMed

Bijwaard, Karen; Dickey, Jennifer S; Kelm, Kellie; Težak, Živana

2015-01-01

The rapid emergence and clinical translation of novel high-throughput sequencing technologies created a need to clarify the regulatory pathway for the evaluation and authorization of these unique technologies. Recently, the US FDA authorized for marketing four next generation sequencing (NGS)-based diagnostic devices which consisted of two heritable disease-specific assays, library preparation reagents and a NGS platform that are intended for human germline targeted sequencing from whole blood. These first authorizations can serve as a case study in how different types of NGS-based technology are reviewed by the FDA. In this manuscript we describe challenges associated with the evaluation of these novel technologies and provide an overview of what was reviewed. Besides making validated NGS-based devices available for in vitro diagnostic use, these first authorizations create a regulatory path for similar future instruments and assays.

RNA-based regulation of genes of tryptophan synthesis and degradation, in bacteria

PubMed Central

Yanofsky, Charles

2007-01-01

We are now aware that RNA-based regulatory mechanisms are commonly used to control gene expression in many organisms. These mechanisms offer the opportunity to exploit relatively short, unique RNA sequences, in altering transcription, translation, and/or mRNA stability, in response to the presence of a small or large signal molecule. The ability of an RNA segment to fold and form alternative hairpin secondary structures—each dedicated to a different regulatory function—permits selection of specific sequences that can affect transcription and/or translation. In the present paper I will focus on our current understanding of the RNA-based regulatory mechanisms used by Escherichia coli and Bacillus subtilis in controlling expression of the tryptophan biosynthetic operon. The regulatory mechanisms they use for this purpose differ, suggesting that these organisms, or their ancestors, adopted different strategies during their evolution. I will also describe the RNA-based mechanism used by E. coli in regulating expression of its operon responsible for tryptophan degradation, the tryptophanase operon. PMID:17601995

MicroRNAs play critical roles during plant development and in response to abiotic stresses.

PubMed

de Lima, Júlio César; Loss-Morais, Guilherme; Margis, Rogerio

2012-12-01

MicroRNAs (miRNAs) have been identified as key molecules in regulatory networks. The fine-tuning role of miRNAs in addition to the regulatory role of transcription factors has shown that molecular events during development are tightly regulated. In addition, several miRNAs play crucial roles in the response to abiotic stress induced by drought, salinity, low temperatures, and metals such as aluminium. Interestingly, several miRNAs have overlapping roles with regard to development, stress responses, and nutrient homeostasis. Moreover, in response to the same abiotic stresses, different expression patterns for some conserved miRNA families among different plant species revealed different metabolic adjustments. The use of deep sequencing technologies for the characterisation of miRNA frequency and the identification of new miRNAs adds complexity to regulatory networks in plants. In this review, we consider the regulatory role of miRNAs in plant development and abiotic stresses, as well as the impact of deep sequencing technologies on the generation of miRNA data.

SPIRE, a modular pipeline for eQTL analysis of RNA-Seq data, reveals a regulatory hotspot controlling miRNA expression in C. elegans.

PubMed

Kel, Ivan; Chang, Zisong; Galluccio, Nadia; Romeo, Margherita; Beretta, Stefano; Diomede, Luisa; Mezzelani, Alessandra; Milanesi, Luciano; Dieterich, Christoph; Merelli, Ivan

2016-10-18

The interpretation of genome-wide association study is difficult, as it is hard to understand how polymorphisms can affect gene regulation, in particular for trans-regulatory elements located far from their controlling gene. Using RNA or protein expression data as phenotypes, it is possible to correlate their variations with specific genotypes. This technique is usually referred to as expression Quantitative Trait Loci (eQTLs) analysis and only few packages exist for the integration of genotype patterns and expression profiles. In particular, tools are needed for the analysis of next-generation sequencing (NGS) data on a genome-wide scale, which is essential to identify eQTLs able to control a large number of genes (hotspots). Here we present SPIRE (Software for Polymorphism Identification Regulating Expression), a generic, modular and functionally highly flexible pipeline for eQTL processing. SPIRE integrates different univariate and multivariate approaches for eQTL analysis, paying particular attention to the scalability of the procedure in order to support cis- as well as trans-mapping, thus allowing the identification of hotspots in NGS data. In particular, we demonstrated how SPIRE can handle big association study datasets, reproducing published results and improving the identification of trans-eQTLs. Furthermore, we employed the pipeline to analyse novel data concerning the genotypes of two different C. elegans strains (N2 and Hawaii) and related miRNA expression data, obtained using RNA-Seq. A miRNA regulatory hotspot was identified in chromosome 1, overlapping the transcription factor grh-1, known to be involved in the early phases of embryonic development of C. elegans. In a follow-up qPCR experiment we were able to verify most of the predicted eQTLs, as well as to show, for a novel miRNA, a significant difference in the sequences of the two analysed strains of C. elegans. SPIRE is publicly available as open source software at , together with some example data, a readme file, supplementary material and a short tutorial.

TranslatomeDB: a comprehensive database and cloud-based analysis platform for translatome sequencing data.

PubMed

Liu, Wanting; Xiang, Lunping; Zheng, Tingkai; Jin, Jingjie; Zhang, Gong

2018-01-04

Translation is a key regulatory step, linking transcriptome and proteome. Two major methods of translatome investigations are RNC-seq (sequencing of translating mRNA) and Ribo-seq (ribosome profiling). To facilitate the investigation of translation, we built a comprehensive database TranslatomeDB (http://www.translatomedb.net/) which provides collection and integrated analysis of published and user-generated translatome sequencing data. The current version includes 2453 Ribo-seq, 10 RNC-seq and their 1394 corresponding mRNA-seq datasets in 13 species. The database emphasizes the analysis functions in addition to the dataset collections. Differential gene expression (DGE) analysis can be performed between any two datasets of same species and type, both on transcriptome and translatome levels. The translation indices translation ratios, elongation velocity index and translational efficiency can be calculated to quantitatively evaluate translational initiation efficiency and elongation velocity, respectively. All datasets were analyzed using a unified, robust, accurate and experimentally-verifiable pipeline based on the FANSe3 mapping algorithm and edgeR for DGE analyzes. TranslatomeDB also allows users to upload their own datasets and utilize the identical unified pipeline to analyze their data. We believe that our TranslatomeDB is a comprehensive platform and knowledgebase on translatome and proteome research, releasing the biologists from complex searching, analyzing and comparing huge sequencing data without needing local computational power. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cnidarian Cell Type Diversity and Regulation Revealed by Whole-Organism Single-Cell RNA-Seq.

PubMed

Sebé-Pedrós, Arnau; Saudemont, Baptiste; Chomsky, Elad; Plessier, Flora; Mailhé, Marie-Pierre; Renno, Justine; Loe-Mie, Yann; Lifshitz, Aviezer; Mukamel, Zohar; Schmutz, Sandrine; Novault, Sophie; Steinmetz, Patrick R H; Spitz, François; Tanay, Amos; Marlow, Heather

2018-05-31

The emergence and diversification of cell types is a leading factor in animal evolution. So far, systematic characterization of the gene regulatory programs associated with cell type specificity was limited to few cell types and few species. Here, we perform whole-organism single-cell transcriptomics to map adult and larval cell types in the cnidarian Nematostella vectensis, a non-bilaterian animal with complex tissue-level body-plan organization. We uncover eight broad cell classes in Nematostella, including neurons, cnidocytes, and digestive cells. Each class comprises different subtypes defined by the expression of multiple specific markers. In particular, we characterize a surprisingly diverse repertoire of neurons, which comparative analysis suggests are the result of lineage-specific diversification. By integrating transcription factor expression, chromatin profiling, and sequence motif analysis, we identify the regulatory codes that underlie Nematostella cell-specific expression. Our study reveals cnidarian cell type complexity and provides insights into the evolution of animal cell-specific genomic regulation. Copyright © 2018 Elsevier Inc. All rights reserved.

Systems genetics identifies Sestrin 3 as a regulator of a proconvulsant gene network in human epileptic hippocampus

PubMed Central

Johnson, Michael R.; Rossetti, Tiziana; Speed, Doug; Srivastava, Prashant K.; Chadeau-Hyam, Marc; Hajji, Nabil; Dabrowska, Aleksandra; Rotival, Maxime; Razzaghi, Banafsheh; Kovac, Stjepana; Wanisch, Klaus; Grillo, Federico W.; Slaviero, Anna; Langley, Sarah R.; Shkura, Kirill; Roncon, Paolo; De, Tisham; Mattheisen, Manuel; Niehusmann, Pitt; O’Brien, Terence J.; Petrovski, Slave; von Lehe, Marec; Hoffmann, Per; Eriksson, Johan; Coffey, Alison J.; Cichon, Sven; Walker, Matthew; Simonato, Michele; Danis, Bénédicte; Mazzuferi, Manuela; Foerch, Patrik; Schoch, Susanne; De Paola, Vincenzo; Kaminski, Rafal M.; Cunliffe, Vincent T.; Becker, Albert J.; Petretto, Enrico

2015-01-01

Gene-regulatory network analysis is a powerful approach to elucidate the molecular processes and pathways underlying complex disease. Here we employ systems genetics approaches to characterize the genetic regulation of pathophysiological pathways in human temporal lobe epilepsy (TLE). Using surgically acquired hippocampi from 129 TLE patients, we identify a gene-regulatory network genetically associated with epilepsy that contains a specialized, highly expressed transcriptional module encoding proconvulsive cytokines and Toll-like receptor signalling genes. RNA sequencing analysis in a mouse model of TLE using 100 epileptic and 100 control hippocampi shows the proconvulsive module is preserved across-species, specific to the epileptic hippocampus and upregulated in chronic epilepsy. In the TLE patients, we map the trans-acting genetic control of this proconvulsive module to Sestrin 3 (SESN3), and demonstrate that SESN3 positively regulates the module in macrophages, microglia and neurons. Morpholino-mediated Sesn3 knockdown in zebrafish confirms the regulation of the transcriptional module, and attenuates chemically induced behavioural seizures in vivo. PMID:25615886

TrawlerWeb: an online de novo motif discovery tool for next-generation sequencing datasets.

PubMed

Dang, Louis T; Tondl, Markus; Chiu, Man Ho H; Revote, Jerico; Paten, Benedict; Tano, Vincent; Tokolyi, Alex; Besse, Florence; Quaife-Ryan, Greg; Cumming, Helen; Drvodelic, Mark J; Eichenlaub, Michael P; Hallab, Jeannette C; Stolper, Julian S; Rossello, Fernando J; Bogoyevitch, Marie A; Jans, David A; Nim, Hieu T; Porrello, Enzo R; Hudson, James E; Ramialison, Mirana

2018-04-05

A strong focus of the post-genomic era is mining of the non-coding regulatory genome in order to unravel the function of regulatory elements that coordinate gene expression (Nat 489:57-74, 2012; Nat 507:462-70, 2014; Nat 507:455-61, 2014; Nat 518:317-30, 2015). Whole-genome approaches based on next-generation sequencing (NGS) have provided insight into the genomic location of regulatory elements throughout different cell types, organs and organisms. These technologies are now widespread and commonly used in laboratories from various fields of research. This highlights the need for fast and user-friendly software tools dedicated to extracting cis-regulatory information contained in these regulatory regions; for instance transcription factor binding site (TFBS) composition. Ideally, such tools should not require prior programming knowledge to ensure they are accessible for all users. We present TrawlerWeb, a web-based version of the Trawler_standalone tool (Nat Methods 4:563-5, 2007; Nat Protoc 5:323-34, 2010), to allow for the identification of enriched motifs in DNA sequences obtained from next-generation sequencing experiments in order to predict their TFBS composition. TrawlerWeb is designed for online queries with standard options common to web-based motif discovery tools. In addition, TrawlerWeb provides three unique new features: 1) TrawlerWeb allows the input of BED files directly generated from NGS experiments, 2) it automatically generates an input-matched biologically relevant background, and 3) it displays resulting conservation scores for each instance of the motif found in the input sequences, which assists the researcher in prioritising the motifs to validate experimentally. Finally, to date, this web-based version of Trawler_standalone remains the fastest online de novo motif discovery tool compared to other popular web-based software, while generating predictions with high accuracy. TrawlerWeb provides users with a fast, simple and easy-to-use web interface for de novo motif discovery. This will assist in rapidly analysing NGS datasets that are now being routinely generated. TrawlerWeb is freely available and accessible at: http://trawler.erc.monash.edu.au .

The role of heterologous chloroplast sequence elements in transgene integration and expression.

PubMed

Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

2010-04-01

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5' untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5' UTR and 3' UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5' UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5' UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation.

The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression1[W][OA

PubMed Central

Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

2010-01-01

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5′ untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5′ UTR and 3′ UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5′ UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5′ UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation. PMID:20130101

Phylogenetic analysis reveals conservation and diversification of micro RNA166 genes among diverse plant species.

PubMed

Barik, Suvakanta; SarkarDas, Shabari; Singh, Archita; Gautam, Vibhav; Kumar, Pramod; Majee, Manoj; Sarkar, Ananda K

2014-01-01

Similar to the majority of the microRNAs, mature miR166s are derived from multiple members of MIR166 genes (precursors) and regulate various aspects of plant development by negatively regulating their target genes (Class III HD-ZIP). The evolutionary conservation or functional diversification of miRNA166 family members remains elusive. Here, we show the phylogenetic relationships among MIR166 precursor and mature sequences from three diverse model plant species. Despite strong conservation, some mature miR166 sequences, such as ppt-miR166m, have undergone sequence variation. Critical sequence variation in ppt-miR166m has led to functional diversification, as it targets non-HD-ZIPIII gene transcript (s). MIR166 precursor sequences have diverged in a lineage specific manner, and both precursors and mature osa-miR166i/j are highly conserved. Interestingly, polycistronic MIR166s were present in Physcomitrella and Oryza but not in Arabidopsis. The nature of cis-regulatory motifs on the upstream promoter sequences of MIR166 genes indicates their possible contribution to the functional variation observed among miR166 species. Copyright © 2013 Elsevier Inc. All rights reserved.

General distribution of the nitrogen control gene ntcA in cyanobacteria.

PubMed Central

Frías, J E; Mérida, A; Herrero, A; Martín-Nieto, J; Flores, E

1993-01-01

The ntcA gene from Synechococcus sp. strain PCC 7942 encodes a regulatory protein which is required for the expression of all of the genes known to be subject to repression by ammonium in that cyanobacterium. Homologs to ntcA have now been cloned by hybridization from the cyanobacteria Synechocystis sp. strain PCC 6803 and Anabaena sp. strain PCC 7120. Sequence analysis has shown that these ntcA genes would encode polypeptides strongly similar (77 to 79% identity) to the Synechococcus NtcA protein. Sequences hybridizing to ntcA have been detected in the genomes of nine other cyanobacteria that were tested, including strains of the genera Anabaena, Calothrix, Fischerella, Nostoc, Pseudoanabaena, Synechococcus, and Synechocystis. Images PMID:8366058

Physiological and molecular characterization of genetic competence in Streptococcus sanguinis.

PubMed

Rodriguez, A M; Callahan, J E; Fawcett, P; Ge, X; Xu, P; Kitten, T

2011-04-01

Streptococcus sanguinis is a major component of the oral flora and an important cause of infective endocarditis. Although S. sanguinis is naturally competent, genome sequencing has suggested significant differences in the S. sanguinis competence system relative to those of other streptococci. An S. sanguinis mutant possessing an in-frame deletion in the comC gene, which encodes competence-stimulating peptide (CSP), was created. Addition of synthetic CSP induced competence in this strain. Gene expression in this strain was monitored by microarray analysis at multiple time-points from 2.5 to 30 min after CSP addition, and verified by quantitative reverse transcription-polymerase chain reaction. Over 200 genes were identified whose expression was altered at least two-fold in at least one time point, with the majority upregulated. The 'late' response was typical of that seen in previous studies. However, comparison of the 'early' response in S. sanguinis with that of other oral streptococci revealed unexpected differences with regard to the number of genes induced, the nature of those genes, and their putative upstream regulatory sequences. Streptococcus sanguinis possesses a comparatively limited early response, which may define a minimal streptococcal competence regulatory circuit. © 2011 John Wiley & Sons A/S.

Physiological and molecular characterization of genetic competence in Streptococcus sanguinis

PubMed Central

Rodriguez, Alejandro Miguel; Callahan, Jill E.; Fawcett, Paul; Ge, Xiuchun; Xu, Ping; Kitten, Todd

2011-01-01

SUMMARY Streptococcus sanguinis is a major component of the oral flora and an important cause of infective endocarditis. Although S. sanguinis is naturally competent, genome sequencing has suggested significant differences in the S. sanguinis competence system relative to those of other streptococci. An S. sanguinis mutant possessing an in-frame deletion in the comC gene, which encodes competence-stimulating peptide (CSP), was created. Addition of synthetic CSP induced competence in this strain. Gene expression in this strain was monitored by microarray analysis at multiple time points from 2.5 to 30 min after CSP addition, and verified by quantitative RT-PCR. Over 200 genes were identified whose expression was altered at least two-fold in at least one time point, with the majority upregulated. The “late” response was typical of that seen in previous studies. However, comparison of the “early” response in S. sanguinis with that of other oral streptococci revealed unexpected differences with regard to the number of genes induced, the nature of these genes, and their putative upstream regulatory sequences. S. sanguinis possesses a comparatively limited early response, which may define a minimal streptococcal competence regulatory circuit. PMID:21375701

The twilight zone of cis element alignments.

PubMed

Sebastian, Alvaro; Contreras-Moreira, Bruno

2013-02-01

Sequence alignment of proteins and nucleic acids is a routine task in bioinformatics. Although the comparison of complete peptides, genes or genomes can be undertaken with a great variety of tools, the alignment of short DNA sequences and motifs entails pitfalls that have not been fully addressed yet. Here we confront the structural superposition of transcription factors with the sequence alignment of their recognized cis elements. Our goals are (i) to test TFcompare (http://floresta.eead.csic.es/tfcompare), a structural alignment method for protein-DNA complexes; (ii) to benchmark the pairwise alignment of regulatory elements; (iii) to define the confidence limits and the twilight zone of such alignments and (iv) to evaluate the relevance of these thresholds with elements obtained experimentally. We find that the structure of cis elements and protein-DNA interfaces is significantly more conserved than their sequence and measures how this correlates with alignment errors when only sequence information is considered. Our results confirm that DNA motifs in the form of matrices produce better alignments than individual sequences. Finally, we report that empirical and theoretically derived twilight thresholds are useful for estimating the natural plasticity of regulatory sequences, and hence for filtering out unreliable alignments.

The twilight zone of cis element alignments

PubMed Central

Sebastian, Alvaro; Contreras-Moreira, Bruno

2013-01-01

Sequence alignment of proteins and nucleic acids is a routine task in bioinformatics. Although the comparison of complete peptides, genes or genomes can be undertaken with a great variety of tools, the alignment of short DNA sequences and motifs entails pitfalls that have not been fully addressed yet. Here we confront the structural superposition of transcription factors with the sequence alignment of their recognized cis elements. Our goals are (i) to test TFcompare (http://floresta.eead.csic.es/tfcompare), a structural alignment method for protein–DNA complexes; (ii) to benchmark the pairwise alignment of regulatory elements; (iii) to define the confidence limits and the twilight zone of such alignments and (iv) to evaluate the relevance of these thresholds with elements obtained experimentally. We find that the structure of cis elements and protein–DNA interfaces is significantly more conserved than their sequence and measures how this correlates with alignment errors when only sequence information is considered. Our results confirm that DNA motifs in the form of matrices produce better alignments than individual sequences. Finally, we report that empirical and theoretically derived twilight thresholds are useful for estimating the natural plasticity of regulatory sequences, and hence for filtering out unreliable alignments. PMID:23268451

MINER: exploratory analysis of gene interaction networks by machine learning from expression data.

PubMed

Kadupitige, Sidath Randeni; Leung, Kin Chun; Sellmeier, Julia; Sivieng, Jane; Catchpoole, Daniel R; Bain, Michael E; Gaëta, Bruno A

2009-12-03

The reconstruction of gene regulatory networks from high-throughput "omics" data has become a major goal in the modelling of living systems. Numerous approaches have been proposed, most of which attempt only "one-shot" reconstruction of the whole network with no intervention from the user, or offer only simple correlation analysis to infer gene dependencies. We have developed MINER (Microarray Interactive Network Exploration and Representation), an application that combines multivariate non-linear tree learning of individual gene regulatory dependencies, visualisation of these dependencies as both trees and networks, and representation of known biological relationships based on common Gene Ontology annotations. MINER allows biologists to explore the dependencies influencing the expression of individual genes in a gene expression data set in the form of decision, model or regression trees, using their domain knowledge to guide the exploration and formulate hypotheses. Multiple trees can then be summarised in the form of a gene network diagram. MINER is being adopted by several of our collaborators and has already led to the discovery of a new significant regulatory relationship with subsequent experimental validation. Unlike most gene regulatory network inference methods, MINER allows the user to start from genes of interest and build the network gene-by-gene, incorporating domain expertise in the process. This approach has been used successfully with RNA microarray data but is applicable to other quantitative data produced by high-throughput technologies such as proteomics and "next generation" DNA sequencing.

Unraveling transcriptional control and cis-regulatory codes using the software suite GeneACT

PubMed Central

Cheung, Tom Hiu; Kwan, Yin Lam; Hamady, Micah; Liu, Xuedong

2006-01-01

Deciphering gene regulatory networks requires the systematic identification of functional cis-acting regulatory elements. We present a suite of web-based bioinformatics tools, called GeneACT , that can rapidly detect evolutionarily conserved transcription factor binding sites or microRNA target sites that are either unique or over-represented in differentially expressed genes from DNA microarray data. GeneACT provides graphic visualization and extraction of common regulatory sequence elements in the promoters and 3'-untranslated regions that are conserved across multiple mammalian species. PMID:17064417

cis-Regulatory Mutations Are a Genetic Cause of Human Limb Malformations

PubMed Central

VanderMeer, Julia E.; Ahituv, Nadav

2011-01-01

The underlying mutations that cause human limb malformations are often difficult to determine, particularly for limb malformations that occur as isolated traits. Evidence from a variety of studies shows that cis-regulatory mutations, specifically in enhancers, can lead to some of these isolated limb malformations. Here, we provide a review of human limb malformations that have been shown to be caused by enhancer mutations and propose that cis-regulatory mutations will continue to be identified as the cause of additional human malformations as our understanding of regulatory sequences improves. PMID:21509892

75 FR 21963 - Regulatory Flexibility Agenda

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-26

... Materials 3235-AK25 DIVISION OF INVESTMENT MANAGEMENT--Proposed Rule Stage Regulation Sequence Title... 3235-AI17 DIVISION OF INVESTMENT MANAGEMENT--Completed Actions Regulation Sequence Title Identifier... Management Investment Company 3235-AJ11 Shares, Unit Investment Trust Interests, and Municipal Fund...

75 FR 79763 - Regulatory Agenda

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2010-12-20

... Current Food Good Manufacturing Practices Regulation 0910-AG36 Food and Drug Administration--Completed... Sequence Title Identifier Number Number 310 Quality Assurance Requirements for Respirators 0920-AA04 Food and Drug Administration--Prerule Stage Regulation Sequence Title Identifier Number Number 311 Food...

LDsplit: screening for cis-regulatory motifs stimulating meiotic recombination hotspots by analysis of DNA sequence polymorphisms.

PubMed

Yang, Peng; Wu, Min; Guo, Jing; Kwoh, Chee Keong; Przytycka, Teresa M; Zheng, Jie

2014-02-17

As a fundamental genomic element, meiotic recombination hotspot plays important roles in life sciences. Thus uncovering its regulatory mechanisms has broad impact on biomedical research. Despite the recent identification of the zinc finger protein PRDM9 and its 13-mer binding motif as major regulators for meiotic recombination hotspots, other regulators remain to be discovered. Existing methods for finding DNA sequence motifs of recombination hotspots often rely on the enrichment of co-localizations between hotspots and short DNA patterns, which ignore the cross-individual variation of recombination rates and sequence polymorphisms in the population. Our objective in this paper is to capture signals encoded in genetic variations for the discovery of recombination-associated DNA motifs. Recently, an algorithm called "LDsplit" has been designed to detect the association between single nucleotide polymorphisms (SNPs) and proximal meiotic recombination hotspots. The association is measured by the difference of population recombination rates at a hotspot between two alleles of a candidate SNP. Here we present an open source software tool of LDsplit, with integrative data visualization for recombination hotspots and their proximal SNPs. Applying LDsplit on SNPs inside an established 7-mer motif bound by PRDM9 we observed that SNP alleles preserving the original motif tend to have higher recombination rates than the opposite alleles that disrupt the motif. Running on SNP windows around hotspots each containing an occurrence of the 7-mer motif, LDsplit is able to guide the established motif finding algorithm of MEME to recover the 7-mer motif. In contrast, without LDsplit the 7-mer motif could not be identified. LDsplit is a software tool for the discovery of cis-regulatory DNA sequence motifs stimulating meiotic recombination hotspots by screening and narrowing down to hotspot associated SNPs. It is the first computational method that utilizes the genetic variation of recombination hotspots among individuals, opening a new avenue for motif finding. Tested on an established motif and simulated datasets, LDsplit shows promise to discover novel DNA motifs for meiotic recombination hotspots.

LDsplit: screening for cis-regulatory motifs stimulating meiotic recombination hotspots by analysis of DNA sequence polymorphisms

PubMed Central

2014-01-01

Background As a fundamental genomic element, meiotic recombination hotspot plays important roles in life sciences. Thus uncovering its regulatory mechanisms has broad impact on biomedical research. Despite the recent identification of the zinc finger protein PRDM9 and its 13-mer binding motif as major regulators for meiotic recombination hotspots, other regulators remain to be discovered. Existing methods for finding DNA sequence motifs of recombination hotspots often rely on the enrichment of co-localizations between hotspots and short DNA patterns, which ignore the cross-individual variation of recombination rates and sequence polymorphisms in the population. Our objective in this paper is to capture signals encoded in genetic variations for the discovery of recombination-associated DNA motifs. Results Recently, an algorithm called “LDsplit” has been designed to detect the association between single nucleotide polymorphisms (SNPs) and proximal meiotic recombination hotspots. The association is measured by the difference of population recombination rates at a hotspot between two alleles of a candidate SNP. Here we present an open source software tool of LDsplit, with integrative data visualization for recombination hotspots and their proximal SNPs. Applying LDsplit on SNPs inside an established 7-mer motif bound by PRDM9 we observed that SNP alleles preserving the original motif tend to have higher recombination rates than the opposite alleles that disrupt the motif. Running on SNP windows around hotspots each containing an occurrence of the 7-mer motif, LDsplit is able to guide the established motif finding algorithm of MEME to recover the 7-mer motif. In contrast, without LDsplit the 7-mer motif could not be identified. Conclusions LDsplit is a software tool for the discovery of cis-regulatory DNA sequence motifs stimulating meiotic recombination hotspots by screening and narrowing down to hotspot associated SNPs. It is the first computational method that utilizes the genetic variation of recombination hotspots among individuals, opening a new avenue for motif finding. Tested on an established motif and simulated datasets, LDsplit shows promise to discover novel DNA motifs for meiotic recombination hotspots. PMID:24533858

Transcriptome Analysis of Manganese-deficient Chlamydomonas reinhardtii Provides Insight on the Chlorophyll Biosynthesis Pathway

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

Lockhart, Ainsley; Zvenigorodsky, Natasha; Pedraza, Mary Ann

2011-08-11

The biosynthesis of chlorophyll and other tetrapyrroles is a vital but poorly understood process. Recent genomic advances with the unicellular green algae Chlamydomonas reinhardtii have created opportunity to more closely examine the mechanisms of the chlorophyll biosynthesis pathway via transcriptome analysis. Manganese is a nutrient of interest for complex reactions because of its multiple stable oxidation states and role in molecular oxygen coordination. C. reinhardtii was cultured in Manganese-deplete Tris-acetate-phosphate (TAP) media for 24 hours and used to create cDNA libraries for sequencing using Illumina TruSeq technology. Transcriptome analysis provided intriguing insight on possible regulatory mechanisms in the pathway. Evidencemore » supports similarities of GTR (Glutamyl-tRNA synthase) to its Chlorella vulgaris homolog in terms of Mn requirements. Data was also suggestive of Mn-related compensatory up-regulation for pathway proteins CHLH1 (Manganese Chelatase), GUN4 (Magnesium chelatase activating protein), and POR1 (Light-dependent protochlorophyllide reductase). Intriguingly, data suggests possible reciprocal expression of oxygen dependent CPX1 (coproporphyrinogen III oxidase) and oxygen independent CPX2. Further analysis using RT-PCR could provide compelling evidence for several novel regulatory mechanisms in the chlorophyll biosynthesis pathway.« less

Regulatory RNA binding proteins contribute to the transcriptome-wide splicing alterations in human cellular senescence.

PubMed

Dong, Qiongye; Wei, Lei; Zhang, Michael Q; Wang, Xiaowo

2018-06-24

Dysregulation of mRNA splicing has been observed in certain cellular senescence process. However, the common splicing alterations on the whole transcriptome shared by various types of senescence are poorly understood. In order to systematically identify senescence-associated transcriptomic changes in genome-wide scale, we collected RNA sequencing datasets of different human cell types with a variety of senescence-inducing methods from public databases and performed meta-analysis. First, we discovered that a group of RNA binding proteins were consistently down-regulated in diverse senescent samples and identified 406 senescence-associated common differential splicing events. Then, eight differentially expressed RNA binding proteins were predicted to regulate these senescence-associated splicing alterations through an enrichment analysis of their RNA binding information, including motif scanning and enhanced cross-linking immunoprecipitation data. In addition, we constructed the splicing regulatory modules that might contribute to senescence-associated biological processes. Finally, it was confirmed that knockdown of the predicted senescence-associated potential splicing regulators through shRNAs in HepG2 cell line could result in senescence-like splicing changes. Taken together, our work demonstrated a broad range of common changes in mRNA splicing switches and detected their central regulatory RNA binding proteins during senescence. These findings would help to better understand the coordinating splicing alterations in cellular senescence.

Evolution of EF-hand calcium-modulated proteins. IV. Exon shuffling did not determine the domain compositions of EF-hand proteins

NASA Technical Reports Server (NTRS)

Kretsinger, R. H.; Nakayama, S.

1993-01-01

In the previous three reports in this series we demonstrated that the EF-hand family of proteins evolved by a complex pattern of gene duplication, transposition, and splicing. The dendrograms based on exon sequences are nearly identical to those based on protein sequences for troponin C, the essential light chain myosin, the regulatory light chain, and calpain. This validates both the computational methods and the dendrograms for these subfamilies. The proposal of congruence for calmodulin, troponin C, essential light chain, and regulatory light chain was confirmed. There are, however, significant differences in the calmodulin dendrograms computed from DNA and from protein sequences. In this study we find that introns are distributed throughout the EF-hand domain and the interdomain regions. Further, dendrograms based on intron type and distribution bear little resemblance to those based on protein or on DNA sequences. We conclude that introns are inserted, and probably deleted, with relatively high frequency. Further, in the EF-hand family exons do not correspond to structural domains and exon shuffling played little if any role in the evolution of this widely distributed homolog family. Calmodulin has had a turbulent evolution. Its dendrograms based on protein sequence, exon sequence, 3'-tail sequence, intron sequences, and intron positions all show significant differences.

Automated selection of synthetic biology parts for genetic regulatory networks.

PubMed

Yaman, Fusun; Bhatia, Swapnil; Adler, Aaron; Densmore, Douglas; Beal, Jacob

2012-08-17

Raising the level of abstraction for synthetic biology design requires solving several challenging problems, including mapping abstract designs to DNA sequences. In this paper we present the first formalism and algorithms to address this problem. The key steps of this transformation are feature matching, signal matching, and part matching. Feature matching ensures that the mapping satisfies the regulatory relationships in the abstract design. Signal matching ensures that the expression levels of functional units are compatible. Finally, part matching finds a DNA part sequence that can implement the design. Our software tool MatchMaker implements these three steps.

A conserved RNA structural element within the hepatitis B virus post-transcriptional regulatory element enhance nuclear export of intronless transcripts and repress the splicing mechanism.

PubMed

Visootsat, Akasit; Payungporn, Sunchai; T-Thienprasert, Nattanan P

2015-12-01

Hepatitis B virus (HBV) infection is a primary cause of hepatocellular carcinoma and liver cirrhosis worldwide. To develop novel antiviral drugs, a better understanding of HBV gene expression regulation is vital. One important aspect is to understand how HBV hijacks the cellular machinery to export unspliced RNA from the nucleus. The HBV post-transcriptional regulatory element (HBV PRE) has been proposed to be the HBV RNA nuclear export element. However, the function remains controversial, and the core element is unclear. This study, therefore, aimed to identify functional regulatory elements within the HBV PRE and investigate their functions. Using bioinformatics programs based on sequence conservation and conserved RNA secondary structures, three regulatory elements were predicted, namely PRE 1151-1410, PRE 1520-1620 and PRE 1650-1684. PRE 1151-1410 significantly increased intronless and unspliced luciferase activity in both HepG2 and COS-7 cells. Likewise, PRE 1151-1410 significantly elevated intronless and unspliced HBV surface transcripts in liver cancer cells. Moreover, motif analysis predicted that PRE 1151-1410 contains several regulatory motifs. This study reported the roles of PRE 1151-1410 in intronless transcript nuclear export and the splicing mechanism. Additionally, these results provide knowledge in the field of HBV RNA regulation. Moreover, PRE 1151-1410 may be used to enhance the expression of other mRNAs in intronless reporter plasmids.

75 FR 79937 - Regulatory Flexibility Agenda

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-20

... INVESTMENT MANAGEMENT--Final Rule Stage Regulation Sequence Title Identifier Number Number 617 Temporary Rule Regarding Principal Trades With Certain Advisory Clients 3235-AJ96 DIVISION OF INVESTMENT MANAGEMENT... Sequence Title Identifier Number Number 626 Confirmation of Transactions in Open-End Management Investment...

Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus.

PubMed Central

Raibaud, A; Zalacain, M; Holt, T G; Tizard, R; Thompson, C J

1991-01-01

Nucleotide sequence analysis of a 5,000-bp region of the bialaphos antibiotic production (bap) gene cluster defined five open reading frames (ORFs) which predicted structural genes in the order bah, ORF1, ORF2, and ORF3 followed by the regulatory gene, brpA (H. Anzai, T. Murakami, S. Imai, A. Satoh, K. Nagaoka, and C.J. Thompson, J. Bacteriol. 169:3482-3488, 1987). The four structural genes were translationally coupled and apparently cotranscribed from an undefined promoter(s) under the positive control of the brpA gene product. S1 mapping experiments indicated that brpA was transcribed by two promoters (brpAp1 and brpAp2) which initiate transcription 150 and 157 bp upstream of brp A within an intergenic region and at least one promoter further upstream within the bap gene cluster (brpAp3). All three transcripts were present at low levels during exponential growth and increased just before the stationary phase. The levels of the brpAp3 band continued to increase at the onset of stationary phase, whereas brpAp1-and brpAp2-protected fragments showed no further change. BrpA contained a possible helix-turn-helix motif at its C terminus which was similar to the C-terminal regulatory motif found in the receiver component of a family of two-component transcriptional activator proteins. This motif was not associated with the N-terminal domain conserved in other members of the family. The structural gene cluster sequenced began with bah, encoding a bialaphos acetylhydrolase which removes the N-acetyl group from bialaphos as one of the final steps in the biosynthetic pathway. The observation that Bah was similar to a rat and to a bacterial (Acinetobacter calcoaceticus) lipase probably reflects the fact that the ester bonds of triglycerides and the amide bond linking acetate to phosphinothricin are similar and hydrolysis is catalyzed by structurally related enzymes. This was followed by two regions encoding ORF1 and ORF2 which were similar to each other (48% nucleotide identity, 31% amino acid identity), as well as to GrsT, a protein encoded by a gene located adjacent to gramicidin S synthetase in Bacillus brevis, and to vertebrate (mallard duck and rat) thioesterases. The amino acid sequence and hydrophobicity profile of ORF3 indicated that it was related to a family of membrane transport proteins. It was strikingly similar to the citrate uptake protein encoded by the transposon Tn3411. Images PMID:2066341

A role for circadian evening elements in cold-regulated gene expression in Arabidopsis.

PubMed

Mikkelsen, Michael D; Thomashow, Michael F

2009-10-01

The plant transcriptome is dramatically altered in response to low temperature. The cis-acting DNA regulatory elements and trans-acting factors that regulate the majority of cold-regulated genes are unknown. Previous bioinformatic analysis has indicated that the promoters of cold-induced genes are enriched in the Evening Element (EE), AAAATATCT, a DNA regulatory element that has a role in circadian-regulated gene expression. Here we tested the role of EE and EE-like (EEL) elements in cold-induced expression of two Arabidopsis genes, CONSTANS-like 1 (COL1; At5g54470) and a gene encoding a 27-kDa protein of unknown function that we designated COLD-REGULATED GENE 27 (COR27; At5g42900). Mutational analysis indicated that the EE/EEL elements were required for cold induction of COL1 and COR27, and that their action was amplified through coupling with ABA response element (ABRE)-like (ABREL) motifs. An artificial promoter consisting solely of four EE motifs interspersed with three ABREL motifs was sufficient to impart cold-induced gene expression. Both COL1 and COR27 were found to be regulated by the circadian clock at warm growth temperatures and cold-induction of COR27 was gated by the clock. These results suggest that cold- and clock-regulated gene expression are integrated through regulatory proteins that bind to EE and EEL elements supported by transcription factors acting at ABREL sequences. Bioinformatic analysis indicated that the coupling of EE and EEL motifs with ABREL motifs is highly enriched in cold-induced genes and thus may constitute a DNA regulatory element pair with a significant role in configuring the low-temperature transcriptome.

Disease-Causing 7.4 kb Cis-Regulatory Deletion Disrupting Conserved Non-Coding Sequences and Their Interaction with the FOXL2 Promotor: Implications for Mutation Screening

PubMed Central

Dostie, Josée; Lemire, Edmond; Bouchard, Philippe; Field, Michael; Jones, Kristie; Lorenz, Birgit; Menten, Björn; Buysse, Karen; Pattyn, Filip; Friedli, Marc; Ucla, Catherine; Rossier, Colette; Wyss, Carine; Speleman, Frank; De Paepe, Anne; Dekker, Job; Antonarakis, Stylianos E.; De Baere, Elfride

2009-01-01

To date, the contribution of disrupted potentially cis-regulatory conserved non-coding sequences (CNCs) to human disease is most likely underestimated, as no systematic screens for putative deleterious variations in CNCs have been conducted. As a model for monogenic disease we studied the involvement of genetic changes of CNCs in the cis-regulatory domain of FOXL2 in blepharophimosis syndrome (BPES). Fifty-seven molecularly unsolved BPES patients underwent high-resolution copy number screening and targeted sequencing of CNCs. Apart from three larger distant deletions, a de novo deletion as small as 7.4 kb was found at 283 kb 5′ to FOXL2. The deletion appeared to be triggered by an H-DNA-induced double-stranded break (DSB). In addition, it disrupts a novel long non-coding RNA (ncRNA) PISRT1 and 8 CNCs. The regulatory potential of the deleted CNCs was substantiated by in vitro luciferase assays. Interestingly, Chromosome Conformation Capture (3C) of a 625 kb region surrounding FOXL2 in expressing cellular systems revealed physical interactions of three upstream fragments and the FOXL2 core promoter. Importantly, one of these contains the 7.4 kb deleted fragment. Overall, this study revealed the smallest distant deletion causing monogenic disease and impacts upon the concept of mutation screening in human disease and developmental disorders in particular. PMID:19543368

A genome-wide SNP scan accelerates trait-regulatory genomic loci identification in chickpea

PubMed Central

Kujur, Alice; Bajaj, Deepak; Upadhyaya, Hari D.; Das, Shouvik; Ranjan, Rajeev; Shree, Tanima; Saxena, Maneesha S.; Badoni, Saurabh; Kumar, Vinod; Tripathi, Shailesh; Gowda, C.L.L.; Sharma, Shivali; Singh, Sube; Tyagi, Akhilesh K.; Parida, Swarup K.

2015-01-01

We identified 44844 high-quality SNPs by sequencing 92 diverse chickpea accessions belonging to a seed and pod trait-specific association panel using reference genome- and de novo-based GBS (genotyping-by-sequencing) assays. A GWAS (genome-wide association study) in an association panel of 211, including the 92 sequenced accessions, identified 22 major genomic loci showing significant association (explaining 23–47% phenotypic variation) with pod and seed number/plant and 100-seed weight. Eighteen trait-regulatory major genomic loci underlying 13 robust QTLs were validated and mapped on an intra-specific genetic linkage map by QTL mapping. A combinatorial approach of GWAS, QTL mapping and gene haplotype-specific LD mapping and transcript profiling uncovered one superior haplotype and favourable natural allelic variants in the upstream regulatory region of a CesA-type cellulose synthase (Ca_Kabuli_CesA3) gene regulating high pod and seed number/plant (explaining 47% phenotypic variation) in chickpea. The up-regulation of this superior gene haplotype correlated with increased transcript expression of Ca_Kabuli_CesA3 gene in the pollen and pod of high pod/seed number accession, resulting in higher cellulose accumulation for normal pollen and pollen tube growth. A rapid combinatorial genome-wide SNP genotyping-based approach has potential to dissect complex quantitative agronomic traits and delineate trait-regulatory genomic loci (candidate genes) for genetic enhancement in crop plants, including chickpea. PMID:26058368

The DNA Methylome of Human Peripheral Blood Mononuclear Cells

PubMed Central

Ye, Mingzhi; Zheng, Hancheng; Yu, Jian; Wu, Honglong; Sun, Jihua; Zhang, Hongyu; Chen, Quan; Luo, Ruibang; Chen, Minfeng; He, Yinghua; Jin, Xin; Zhang, Qinghui; Yu, Chang; Zhou, Guangyu; Sun, Jinfeng; Huang, Yebo; Zheng, Huisong; Cao, Hongzhi; Zhou, Xiaoyu; Guo, Shicheng; Hu, Xueda; Li, Xin; Kristiansen, Karsten; Bolund, Lars; Xu, Jiujin; Wang, Wen; Yang, Huanming; Wang, Jian; Li, Ruiqiang; Beck, Stephan; Wang, Jun; Zhang, Xiuqing

2010-01-01

DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and <0.2% of non-CpG sites were methylated, demonstrating that non-CpG cytosine methylation is minor in human PBMC. Analysis of the PBMC methylome revealed a rich epigenomic landscape for 20 distinct genomic features, including regulatory, protein-coding, non-coding, RNA-coding, and repeat sequences. Integration of our methylome data with the YH genome sequence enabled a first comprehensive assessment of allele-specific methylation (ASM) between the two haploid methylomes of any individual and allowed the identification of 599 haploid differentially methylated regions (hDMRs) covering 287 genes. Of these, 76 genes had hDMRs within 2 kb of their transcriptional start sites of which >80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies. PMID:21085693

RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes

PubMed Central

Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle

2016-01-01

ABSTRACT Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation. PMID:26580233

Improved PCR assay for the specific detection and quantitation of Escherichia coli serotype O157 in water.

PubMed

Cho, Min Seok; Joh, Kiseong; Ahn, Tae-Young; Park, Dong Suk

2014-09-01

Escherichia coli serotype O157 is still a major global healthcare problem. However, only limited information is now available on the molecular and serological detection of pathogenic bacteria. Therefore, the development of appropriate strategies for their rapid identification and monitoring is still needed. In general, the sequence analysis based on stx, slt, eae, hlyA, rfb, and fliCh7 genes is widely employed for the identification of E. coli serotype O157; but there have been critical defects in the diagnosis and identification of E. coli serotype O157, in that they are also present in other E. coli serogroups. In this study, NCBI-BLAST searches using the nucleotide sequences of the putative regulatory protein gene from E. coli O157:H7 str. Sakai found sequence difference at the serotype level. The specific primers from the putative regulatory protein gene were designed and investigated for their sensitivity and specificity for detecting the pathogen in environment water samples. The specificity of the primer set was evaluated using genomic DNA from 8 isolates of E. coli serotype O157 and 32 other reference strains. In addition, the sensitivity and specificity of this assay were confirmed by successful identification of E. coli serotype O157 in environmental water samples. In conclusion, this study showed that the newly developed quantitative serotype-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk E. coli serotype O157.

Bat Accelerated Regions Identify a Bat Forelimb Specific Enhancer in the HoxD Locus

PubMed Central

Mason, Mandy K.; VanderMeer, Julia E.; Zhao, Jingjing; Eckalbar, Walter L.; Logan, Malcolm; Illing, Nicola; Pollard, Katherine S.; Ahituv, Nadav

2016-01-01

The molecular events leading to the development of the bat wing remain largely unknown, and are thought to be caused, in part, by changes in gene expression during limb development. These expression changes could be instigated by variations in gene regulatory enhancers. Here, we used a comparative genomics approach to identify regions that evolved rapidly in the bat ancestor, but are highly conserved in other vertebrates. We discovered 166 bat accelerated regions (BARs) that overlap H3K27ac and p300 ChIP-seq peaks in developing mouse limbs. Using a mouse enhancer assay, we show that five Myotis lucifugus BARs drive gene expression in the developing mouse limb, with the majority showing differential enhancer activity compared to the mouse orthologous BAR sequences. These include BAR116, which is located telomeric to the HoxD cluster and had robust forelimb expression for the M. lucifugus sequence and no activity for the mouse sequence at embryonic day 12.5. Developing limb expression analysis of Hoxd10-Hoxd13 in Miniopterus natalensis bats showed a high-forelimb weak-hindlimb expression for Hoxd10-Hoxd11, similar to the expression trend observed for M. lucifugus BAR116 in mice, suggesting that it could be involved in the regulation of the bat HoxD complex. Combined, our results highlight novel regulatory regions that could be instrumental for the morphological differences leading to the development of the bat wing. PMID:27019019

Cronobacter, the emergent bacterial pathogen Enterobacter sakazakii comes of age; MLST and whole genome sequence analysis.

PubMed

Forsythe, Stephen J; Dickins, Benjamin; Jolley, Keith A

2014-12-16

Following the association of Cronobacter spp. to several publicized fatal outbreaks in neonatal intensive care units of meningitis and necrotising enterocolitis, the World Health Organization (WHO) in 2004 requested the establishment of a molecular typing scheme to enable the international control of the organism. This paper presents the application of Next Generation Sequencing (NGS) to Cronobacter which has led to the establishment of the Cronobacter PubMLST genome and sequence definition database (http://pubmlst.org/cronobacter/) containing over 1000 isolates with metadata along with the recognition of specific clonal lineages linked to neonatal meningitis and adult infections Whole genome sequencing and multilocus sequence typing (MLST) has supports the formal recognition of the genus Cronobacter composed of seven species to replace the former single species Enterobacter sakazakii. Applying the 7-loci MLST scheme to 1007 strains revealed 298 definable sequence types, yet only C. sakazakii clonal complex 4 (CC4) was principally associated with neonatal meningitis. This clonal lineage has been confirmed using ribosomal-MLST (51-loci) and whole genome-MLST (1865 loci) to analyse 107 whole genomes via the Cronobacter PubMLST database. This database has enabled the retrospective analysis of historic cases and outbreaks following re-identification of those strains. The Cronobacter PubMLST database offers a central, open access, reliable sequence-based repository for researchers. It has the capacity to create new analysis schemes 'on the fly', and to integrate metadata (source, geographic distribution, clinical presentation). It is also expandable and adaptable to changes in taxonomy, and able to support the development of reliable detection methods of use to industry and regulatory authorities. Therefore it meets the WHO (2004) request for the establishment of a typing scheme for this emergent bacterial pathogen. Whole genome sequencing has additionally shown a range of potential virulence and environmental fitness traits which may account for the association of C. sakazakii CC4 pathogenicity, and propensity for neonatal CNS.

Genetic characterization of the oxytocin-neurophysin I gene (OXT) and its regulatory regions analysis in domestic Old and New World camelids

PubMed Central

Ogah, Danlami Moses; Iannaccone, Marco; Erhardt, Georg; Di Stasio, Liliana; Cosenza, Gianfranco

2018-01-01

Oxytocin is a neurohypophysial peptide linked to a wide range of biological functions, including milk ejection, temperament and reproduction. Aims of the present study were a) the characterization of the OXT (Oxytocin-neurophysin I) gene and its regulatory regions in Old and New world camelids; b) the investigation of the genetic diversity and the discovery of markers potentially affecting the gene regulation. On average, the gene extends over 814 bp, ranging between 825 bp in dromedary, 811 bp in Bactrian and 810 bp in llama and alpaca. Such difference in size is due to a duplication event of 21 bp in dromedary. The main regulatory elements, including the composite hormone response elements (CHREs), were identified in the promoter, whereas the presence of mature microRNAs binding sequences in the 3’UTR improves the knowledge on the factors putatively involved in the OXT gene regulation, although their specific biological effect needs to be still elucidated. The sequencing of genomic DNA allowed the identification of 17 intraspecific polymorphisms and 69 nucleotide differences among the four species. One of these (MF464535:g.622C>G) is responsible, in alpaca, for the loss of a consensus sequence for the transcription factor SP1. Furthermore, the same SNP falls within a CpG island and it creates a new methylation site, thus opening future possibilities of investigation to verify the influence of the novel allelic variant in the OXT gene regulation. A PCR-RFLP method was setup for the genotyping and the frequency of the allele C was 0.93 in a population of 71 alpacas. The obtained data clarify the structure of OXT gene in domestic camelids and add knowledge to the genetic variability of a genomic region, which has received little investigation so far. These findings open the opportunity for new investigations, including association studies with productive and reproductive traits. PMID:29608621

Transcriptome and Biochemical Analysis of a Flower Color Polymorphism in Silene littorea (Caryophyllaceae)

PubMed Central

Casimiro-Soriguer, Inés; Narbona, Eduardo; Buide, M. L.; del Valle, José C.; Whittall, Justen B.

2016-01-01

Flower color polymorphisms are widely used as model traits from genetics to ecology, yet determining the biochemical and molecular basis can be challenging. Anthocyanin-based flower color variations can be caused by at least 12 structural and three regulatory genes in the anthocyanin biosynthetic pathway (ABP). We use mRNA-Seq to simultaneously sequence and estimate expression of these candidate genes in nine samples of Silene littorea representing three color morphs (dark pink, light pink and white) across three developmental stages in hopes of identifying the cause of flower color variation. We identified 29 putative paralogs for the 15 candidate genes in the ABP. We assembled complete coding sequences for 16 structural loci and nine of ten regulatory loci. Among these 29 putative paralogs, we identified 622 SNPs, yet only nine synonymous SNPs in Ans had allele frequencies that differentiated pigmented petals (dark pink and light pink) from white petals. These Ans allele frequency differences were further investigated with an expanded sequencing survey of 38 individuals, yet no SNPs consistently differentiated the color morphs. We also found one locus, F3h1, with strong differential expression between pigmented and white samples (>42x). This may be caused by decreased expression of Myb1a in white petal buds. Myb1a in S. littorea is a regulatory locus closely related to Subgroup 7 Mybs known to regulate F3h and other loci in the first half of the ABP in model species. We then compare the mRNA-Seq results with petal biochemistry which revealed cyanidin as the primary anthocyanin and five flavonoid intermediates. Concentrations of three of the flavonoid intermediates were significantly lower in white petals than in pigmented petals (rutin, quercetin and isovitexin). The biochemistry results for rutin, quercetin, luteolin and apigenin are consistent with the transcriptome results suggesting a blockage at F3h, possibly caused by downregulation of Myb1a. PMID:26973662

Genetic characterization of the oxytocin-neurophysin I gene (OXT) and its regulatory regions analysis in domestic Old and New World camelids.

PubMed

Pauciullo, Alfredo; Ogah, Danlami Moses; Iannaccone, Marco; Erhardt, Georg; Di Stasio, Liliana; Cosenza, Gianfranco

2018-01-01

Oxytocin is a neurohypophysial peptide linked to a wide range of biological functions, including milk ejection, temperament and reproduction. Aims of the present study were a) the characterization of the OXT (Oxytocin-neurophysin I) gene and its regulatory regions in Old and New world camelids; b) the investigation of the genetic diversity and the discovery of markers potentially affecting the gene regulation. On average, the gene extends over 814 bp, ranging between 825 bp in dromedary, 811 bp in Bactrian and 810 bp in llama and alpaca. Such difference in size is due to a duplication event of 21 bp in dromedary. The main regulatory elements, including the composite hormone response elements (CHREs), were identified in the promoter, whereas the presence of mature microRNAs binding sequences in the 3'UTR improves the knowledge on the factors putatively involved in the OXT gene regulation, although their specific biological effect needs to be still elucidated. The sequencing of genomic DNA allowed the identification of 17 intraspecific polymorphisms and 69 nucleotide differences among the four species. One of these (MF464535:g.622C>G) is responsible, in alpaca, for the loss of a consensus sequence for the transcription factor SP1. Furthermore, the same SNP falls within a CpG island and it creates a new methylation site, thus opening future possibilities of investigation to verify the influence of the novel allelic variant in the OXT gene regulation. A PCR-RFLP method was setup for the genotyping and the frequency of the allele C was 0.93 in a population of 71 alpacas. The obtained data clarify the structure of OXT gene in domestic camelids and add knowledge to the genetic variability of a genomic region, which has received little investigation so far. These findings open the opportunity for new investigations, including association studies with productive and reproductive traits.

Contrasting cDNA-AFLP profiles between crown and leaf tissues of cold-acclimated wheat plants indicate differing regulatory circuitries for low temperature tolerance.

PubMed

Ganeshan, Seedhabadee; Sharma, Pallavi; Young, Lester; Kumar, Ashwani; Fowler, D Brian; Chibbar, Ravindra N

2011-03-01

Low-temperature (LT) tolerance in winter wheat (Triticum aestivum L.) is an economically important but complex trait. Four selected wheat genotypes, a winter hardy cultivar, Norstar, a tender spring cultivar, Manitou and two near-isogenic lines with Vrn-A1 (spring Norstar) and vrn-A1 (winter Manitou) alleles of Manitou and Norstar were cold-acclimated at 6°C and crown and leaf tissues were collected at 0, 2, 14, 21, 35, 42, 56 and 70 days of cold acclimation. cDNA-AFLP profiling was used to determine temporal expression profiles of transcripts during cold-acclimation in crown and leaf tissues, separately to determine if LT regulatory circuitries in crown and leaf tissues could be delineated using this approach. Screening 64 primer combinations identified 4,074 and 2,757 differentially expressed transcript-derived fragments (TDFs) out of which 38 and 16% were up-regulated as compared to 3 and 6% that were down-regulated in crown and leaf tissues, respectively. DNA sequencing of TDFs revealed sequences common to both tissues including genes coding for DEAD-box RNA helicase, choline-phosphate cytidylyltransferase and delta-1-pyrroline carboxylate synthetase. TDF specific to crown tissues included genes coding for phospahtidylinositol kinase, auxin response factor protein and brassinosteroid insensitive 1-associated receptor kinase. In leaf, genes such as methylene tetrahydrofolate reductase, NADH-cytochrome b5 reductase and malate dehydrogenase were identified. However, 30 and 14% of the DNA sequences from the crown and leaf tissues, respectively, were hypothetical or unknown proteins. Cluster analysis of up-, down-regulated and unique TDFs, DNA sequence and real-time PCR validation, infer that mechanisms operating in crown and leaf tissue in response to LT are differently regulated and warrant further studies.

Identification of the Regulator Gene Responsible for the Acetone-Responsive Expression of the Binuclear Iron Monooxygenase Gene Cluster in Mycobacteria ▿

PubMed Central

Furuya, Toshiki; Hirose, Satomi; Semba, Hisashi; Kino, Kuniki

2011-01-01

The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the σ54 factor, a gene encoding the σ54 factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria. PMID:21856847

Comparative transgenic analysis of enhancers from the human SHOX and mouse Shox2 genomic regions.

PubMed

Rosin, Jessica M; Abassah-Oppong, Samuel; Cobb, John

2013-08-01

Disruption of presumptive enhancers downstream of the human SHOX gene (hSHOX) is a frequent cause of the zeugopodal limb defects characteristic of Léri-Weill dyschondrosteosis (LWD). The closely related mouse Shox2 gene (mShox2) is also required for limb development, but in the more proximal stylopodium. In this study, we used transgenic mice in a comparative approach to characterize enhancer sequences in the hSHOX and mShox2 genomic regions. Among conserved noncoding elements (CNEs) that function as enhancers in vertebrate genomes, those that are maintained near paralogous genes are of particular interest given their ancient origins. Therefore, we first analyzed the regulatory potential of a genomic region containing one such duplicated CNE (dCNE) downstream of mShox2 and hSHOX. We identified a strong limb enhancer directly adjacent to the mShox2 dCNE that recapitulates the expression pattern of the endogenous gene. Interestingly, this enhancer requires sequences only conserved in the mammalian lineage in order to drive strong limb expression, whereas the more deeply conserved sequences of the dCNE function as a neural enhancer. Similarly, we found that a conserved element downstream of hSHOX (CNE9) also functions as a neural enhancer in transgenic mice. However, when the CNE9 transgenic construct was enlarged to include adjacent, non-conserved sequences frequently deleted in LWD patients, the transgene drove expression in the zeugopodium of the limbs. Therefore, both hSHOX and mShox2 limb enhancers are coupled to distinct neural enhancers. This is the first report demonstrating the activity of cis-regulatory elements from the hSHOX and mShox2 genomic regions in mammalian embryos.

MOCCS: Clarifying DNA-binding motif ambiguity using ChIP-Seq data.

PubMed

Ozaki, Haruka; Iwasaki, Wataru

2016-08-01

As a key mechanism of gene regulation, transcription factors (TFs) bind to DNA by recognizing specific short sequence patterns that are called DNA-binding motifs. A single TF can accept ambiguity within its DNA-binding motifs, which comprise both canonical (typical) and non-canonical motifs. Clarification of such DNA-binding motif ambiguity is crucial for revealing gene regulatory networks and evaluating mutations in cis-regulatory elements. Although chromatin immunoprecipitation sequencing (ChIP-seq) now provides abundant data on the genomic sequences to which a given TF binds, existing motif discovery methods are unable to directly answer whether a given TF can bind to a specific DNA-binding motif. Here, we report a method for clarifying the DNA-binding motif ambiguity, MOCCS. Given ChIP-Seq data of any TF, MOCCS comprehensively analyzes and describes every k-mer to which that TF binds. Analysis of simulated datasets revealed that MOCCS is applicable to various ChIP-Seq datasets, requiring only a few minutes per dataset. Application to the ENCODE ChIP-Seq datasets proved that MOCCS directly evaluates whether a given TF binds to each DNA-binding motif, even if known position weight matrix models do not provide sufficient information on DNA-binding motif ambiguity. Furthermore, users are not required to provide numerous parameters or background genomic sequence models that are typically unavailable. MOCCS is implemented in Perl and R and is freely available via https://github.com/yuifu/moccs. By complementing existing motif-discovery software, MOCCS will contribute to the basic understanding of how the genome controls diverse cellular processes via DNA-protein interactions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multidrug Resistant Pseudomonas aeruginosa Causing Prosthetic Valve Endocarditis: A Genetic-Based Chronicle of Evolving Antibiotic Resistance.

PubMed

Domitrovic, T Nicholas; Hujer, Andrea M; Perez, Federico; Marshall, Steven H; Hujer, Kristine M; Woc-Colburn, Laila E; Parta, Mark; Bonomo, Robert A

2016-10-01

Background.  Successful treatment of infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa is thwarted by the emergence of antibiotic resistance and biofilm formation on prosthetic devices. Our aims were to decipher the molecular basis of resistance in a unique case of prosthetic valve endocarditis (PVE) caused by MDR P. aeruginosa . Methods.  Five sequential MDR P. aeruginosa blood isolates collected during a 7-month period were recovered from a patient suffering from PVE previously exposed to β-lactam antibiotics. Minimum inhibitory concentrations (MICs) of several classes of antibiotics were used to indicate clinical resistance characteristics; relatedness of the isolates was determined using multilocus sequence typing and repetitive sequence-based polymerase chain reaction. Amplification and sequencing of regulatory and resistance genes was performed. Results.  All isolates belonged to ST 298, possessed bla PDC-16 , and were resistant to fluoroquinolones and carbapenems. In the course of therapy, we observed a >2-fold increase in cephalosporin resistance (4 µg/mL to >16 µg/mL). Sequencing of the AmpC regulator, amp R, revealed a D135N point mutation in cephalosporin-resistant isolates. Common carbapenemase genes were not identified. All isolates demonstrated a premature stop codon at amino acid 79 of the outer membrane protein OprD and mutations in the quinolone resistance-determining regions of gyr A and par C. Point mutations in nal C, an efflux pump regulator, were also observed. Conclusions.  In this analysis, we chart the molecular evolution of β-lactam resistance in a case of PVE. We show that mutations in regulatory genes controlling efflux and cephalosporinase production contributed to the MDR phenotype.

The Metarhizium anisopliae trp1 gene: cloning and regulatory analysis.

PubMed

Staats, Charley Christian; Silva, Marcia Suzana Nunes; Pinto, Paulo Marcos; Vainstein, Marilene Henning; Schrank, Augusto

2004-07-01

The trp1 gene from the entomopathogenic fungus Metarhizium anisopliae, cloned by heterologous hybridization with the plasmid carrying the trpC gene from Aspergillus nidulans, was sequence characterized. The predicted translation product has the conserved catalytic domains of glutamine amidotransferase (G domain), indoleglycerolphosphate synthase (C domain), and phosphoribosyl anthranilate isomerase (F domain) organized as NH2-G-C-F-COOH. The ORF is interrupted by a single intron of 60 nt that is position conserved in relation to trp genes from Ascomycetes and length conserved in relation to Basidiomycetes species. RT-PCR analysis suggests constitutive expression of trp1 gene in M. anisopliae.

Secondary structural entropy in RNA switch (Riboswitch) identification.

PubMed

Manzourolajdad, Amirhossein; Arnold, Jonathan

2015-04-28

RNA regulatory elements play a significant role in gene regulation. Riboswitches, a widespread group of regulatory RNAs, are vital components of many bacterial genomes. These regulatory elements generally function by forming a ligand-induced alternative fold that controls access to ribosome binding sites or other regulatory sites in RNA. Riboswitch-mediated mechanisms are ubiquitous across bacterial genomes. A typical class of riboswitch has its own unique structural and biological complexity, making de novo riboswitch identification a formidable task. Traditionally, riboswitches have been identified through comparative genomics based on sequence and structural homology. The limitations of structural-homology-based approaches, coupled with the assumption that there is a great diversity of undiscovered riboswitches, suggests the need for alternative methods for riboswitch identification, possibly based on features intrinsic to their structure. As of yet, no such reliable method has been proposed. We used structural entropy of riboswitch sequences as a measure of their secondary structural dynamics. Entropy values of a diverse set of riboswitches were compared to that of their mutants, their dinucleotide shuffles, and their reverse complement sequences under different stochastic context-free grammar folding models. Significance of our results was evaluated by comparison to other approaches, such as the base-pairing entropy and energy landscapes dynamics. Classifiers based on structural entropy optimized via sequence and structural features were devised as riboswitch identifiers and tested on Bacillus subtilis, Escherichia coli, and Synechococcus elongatus as an exploration of structural entropy based approaches. The unusually long untranslated region of the cotH in Bacillus subtilis, as well as upstream regions of certain genes, such as the sucC genes were associated with significant structural entropy values in genome-wide examinations. Various tests show that there is in fact a relationship between higher structural entropy and the potential for the RNA sequence to have alternative structures, within the limitations of our methodology. This relationship, though modest, is consistent across various tests. Understanding the behavior of structural entropy as a fairly new feature for RNA conformational dynamics, however, may require extensive exploratory investigation both across RNA sequences and folding models.

Neocortical malformation as consequence of nonadaptive regulation of neuronogenetic sequence

NASA Technical Reports Server (NTRS)

Caviness, V. S. Jr; Takahashi, T.; Nowakowski, R. S.

2000-01-01

Variations in the structure of the neocortex induced by single gene mutations may be extreme or subtle. They differ from variations in neocortical structure encountered across and within species in that these "normal" structural variations are adaptive (both structurally and behaviorally), whereas those associated with disorders of development are not. Here we propose that they also differ in principle in that they represent disruptions of molecular mechanisms that are not normally regulatory to variations in the histogenetic sequence. We propose an algorithm for the operation of the neuronogenetic sequence in relation to the overall neocortical histogenetic sequence and highlight the restriction point of the G1 phase of the cell cycle as the master regulatory control point for normal coordinate structural variation across species and importantly within species. From considerations based on the anatomic evidence from neocortical malformation in humans, we illustrate in principle how this overall sequence appears to be disrupted by molecular biological linkages operating principally outside the control mechanisms responsible for the normal structural variation of the neocortex. MRDD Research Reviews 6:22-33, 2000. Copyright 2000 Wiley-Liss, Inc.

Artificial Intelligence, DNA Mimicry, and Human Health.

PubMed

Stefano, George B; Kream, Richard M

2017-08-14

The molecular evolution of genomic DNA across diverse plant and animal phyla involved dynamic registrations of sequence modifications to maintain existential homeostasis to increasingly complex patterns of environmental stressors. As an essential corollary, driver effects of positive evolutionary pressure are hypothesized to effect concerted modifications of genomic DNA sequences to meet expanded platforms of regulatory controls for successful implementation of advanced physiological requirements. It is also clearly apparent that preservation of updated registries of advantageous modifications of genomic DNA sequences requires coordinate expansion of convergent cellular proofreading/error correction mechanisms that are encoded by reciprocally modified genomic DNA. Computational expansion of operationally defined DNA memory extends to coordinate modification of coding and previously under-emphasized noncoding regions that now appear to represent essential reservoirs of untapped genetic information amenable to evolutionary driven recruitment into the realm of biologically active domains. Additionally, expansion of DNA memory potential via chemical modification and activation of noncoding sequences is targeted to vertical augmentation and integration of an expanded cadre of transcriptional and epigenetic regulatory factors affecting linear coding of protein amino acid sequences within open reading frames.

NMR studies on the structure and dynamics of lac operator DNA

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

Lee, S.C.

Nuclear Magnetic Resonance spectroscopy was used to elucidate the relationships between structure, dynamics and function of the gene regulatory sequence corresponding to the lactose operon operator of Escherichia coli. The length of the DNA fragments examined varied from 13 to 36 base pair, containing all or part of the operator sequence. These DNA fragments are either derived genetically or synthesized chemically. Resonances of the imino protons were assigned by one dimensional inter-base pair nuclear Overhauser enhancement (NOE) measurements. Imino proton exchange rates were measured by saturation recovery methods. Results from the kinetic measurements show an interesting dynamic heterogeneity with amore » maximum opening rate centered about a GTG/CAC sequence which correlates with the biological function of the operator DNA. This particular three base pair sequence occurs frequently and often symmetrically in prokaryotic nd eukaryotic DNA sites where one anticipates specific protein interaction for gene regulation. The observed sequence dependent imino proton exchange rate may be a reflection of variation of the local structure of regulatory DNA. The results also indicate that the observed imino proton exchange rates are length dependent.« less

Integrated Approach to Reconstruction of Microbial Regulatory Networks

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

Rodionov, Dmitry A; Novichkov, Pavel S

2013-11-04

This project had the goal(s) of development of integrated bioinformatics platform for genome-scale inference and visualization of transcriptional regulatory networks (TRNs) in bacterial genomes. The work was done in Sanford-Burnham Medical Research Institute (SBMRI, P.I. D.A. Rodionov) and Lawrence Berkeley National Laboratory (LBNL, co-P.I. P.S. Novichkov). The developed computational resources include: (1) RegPredict web-platform for TRN inference and regulon reconstruction in microbial genomes, and (2) RegPrecise database for collection, visualization and comparative analysis of transcriptional regulons reconstructed by comparative genomics. These analytical resources were selected as key components in the DOE Systems Biology KnowledgeBase (SBKB). The high-quality data accumulated inmore » RegPrecise will provide essential datasets of reference regulons in diverse microbes to enable automatic reconstruction of draft TRNs in newly sequenced genomes. We outline our progress toward the three aims of this grant proposal, which were: Develop integrated platform for genome-scale regulon reconstruction; Infer regulatory annotations in several groups of bacteria and building of reference collections of microbial regulons; and Develop KnowledgeBase on microbial transcriptional regulation.« less

Gene network polymorphism is the raw material of natural selection: the selfish gene network hypothesis.

PubMed

Boldogköi, Zsolt

2004-09-01

Population genetics, the mathematical theory of modern evolutionary biology, defines evolution as the alteration of the frequency of distinct gene variants (alleles) differing in fitness over the time. The major problem with this view is that in gene and protein sequences we can find little evidence concerning the molecular basis of phenotypic variance, especially those that would confer adaptive benefit to the bearers. Some novel data, however, suggest that a large amount of genetic variation exists in the regulatory region of genes within populations. In addition, comparison of homologous DNA sequences of various species shows that evolution appears to depend more strongly on gene expression than on the genes themselves. Furthermore, it has been demonstrated in several systems that genes form functional networks, whose products exhibit interrelated expression profiles. Finally, it has been found that regulatory circuits of development behave as evolutionary units. These data demonstrate that our view of evolution calls for a new synthesis. In this article I propose a novel concept, termed the selfish gene network hypothesis, which is based on an overall consideration of the above findings. The major statements of this hypothesis are as follows. (1) Instead of individual genes, gene networks (GNs) are responsible for the determination of traits and behaviors. (2) The primary source of microevolution is the intraspecific polymorphism in GNs and not the allelic variation in either the coding or the regulatory sequences of individual genes. (3) GN polymorphism is generated by the variation in the regulatory regions of the component genes and not by the variance in their coding sequences. (4) Evolution proceeds through continuous restructuring of the composition of GNs rather than fixing of specific alleles or GN variants.

Regulatory versus coding signatures of natural selection in a candidate gene involved in the adaptive divergence of whitefish species pairs (Coregonus spp.)

PubMed Central

Jeukens, Julie; Bernatchez, Louis

2012-01-01

While gene expression divergence is known to be involved in adaptive phenotypic divergence and speciation, the relative importance of regulatory and structural evolution of genes is poorly understood. A recent next-generation sequencing experiment allowed identifying candidate genes potentially involved in the ongoing speciation of sympatric dwarf and normal lake whitefish (Coregonus clupeaformis), such as cytosolic malate dehydrogenase (MDH1), which showed both significant expression and sequence divergence. The main goal of this study was to investigate into more details the signatures of natural selection in the regulatory and coding sequences of MDH1 in lake whitefish and test for parallelism of these signatures with other coregonine species. Sequencing of the two regions in 118 fish from four sympatric pairs of whitefish and two cisco species revealed a total of 35 single nucleotide polymorphisms (SNPs), with more genetic diversity in European compared to North American coregonine species. While the coding region was found to be under purifying selection, an SNP in the proximal promoter exhibited significant allele frequency divergence in a parallel manner among independent sympatric pairs of North American lake whitefish and European whitefish (C. lavaretus). According to transcription factor binding simulation for 22 regulatory haplotypes of MDH1, putative binding profiles were fairly conserved among species, except for the region around this SNP. Moreover, we found evidence for the role of this SNP in the regulation of MDH1 expression level. Overall, these results provide further evidence for the role of natural selection in gene regulation evolution among whitefish species pairs and suggest its possible link with patterns of phenotypic diversity observed in coregonine species. PMID:22408741

Regulatory versus coding signatures of natural selection in a candidate gene involved in the adaptive divergence of whitefish species pairs (Coregonus spp.).

PubMed

Jeukens, Julie; Bernatchez, Louis

2012-01-01

While gene expression divergence is known to be involved in adaptive phenotypic divergence and speciation, the relative importance of regulatory and structural evolution of genes is poorly understood. A recent next-generation sequencing experiment allowed identifying candidate genes potentially involved in the ongoing speciation of sympatric dwarf and normal lake whitefish (Coregonus clupeaformis), such as cytosolic malate dehydrogenase (MDH1), which showed both significant expression and sequence divergence. The main goal of this study was to investigate into more details the signatures of natural selection in the regulatory and coding sequences of MDH1 in lake whitefish and test for parallelism of these signatures with other coregonine species. Sequencing of the two regions in 118 fish from four sympatric pairs of whitefish and two cisco species revealed a total of 35 single nucleotide polymorphisms (SNPs), with more genetic diversity in European compared to North American coregonine species. While the coding region was found to be under purifying selection, an SNP in the proximal promoter exhibited significant allele frequency divergence in a parallel manner among independent sympatric pairs of North American lake whitefish and European whitefish (C. lavaretus). According to transcription factor binding simulation for 22 regulatory haplotypes of MDH1, putative binding profiles were fairly conserved among species, except for the region around this SNP. Moreover, we found evidence for the role of this SNP in the regulation of MDH1 expression level. Overall, these results provide further evidence for the role of natural selection in gene regulation evolution among whitefish species pairs and suggest its possible link with patterns of phenotypic diversity observed in coregonine species.

Adeno-associated virus type 2 rep gene-mediated inhibition of basal gene expression of human immunodeficiency virus type 1 involves its negative regulatory functions.

PubMed Central

Oelze, I; Rittner, K; Sczakiel, G

1994-01-01

Adeno-associated virus type 2 (AAV-2), a human parvovirus which is apathogenic in adults, inhibits replication and gene expression of human immunodeficiency virus type 1 (HIV-1) in human cells. The rep gene of AAV-2, which was shown earlier to be sufficient for this negative interference, also down-regulated the expression of heterologous sequences driven by the long terminal repeat (LTR) of HIV-1. This effect was observed in the absence of the HIV-1 transactivator Tat, i.e., at basal levels of LTR-driven transcription. In this work, we studied the involvement of functional subsequences of the HIV-1 LTR in rep-mediated inhibition in the absence of Tat. Mutated LTRs driving an indicator gene (cat) were cointroduced into human SW480 cells together with rep alone or with double-stranded DNA fragments or RNA containing sequences of the HIV-1 LTR. The results indicate that rep strongly enhances the function of negative regulatory elements of the LTR. In addition, the experiments revealed a transcribed sequence element located within the TAR-coding sequence termed AHHH (AAV-HIV homology element derived from HIV-1) which is involved in rep-mediated inhibition. The AHHH element is also involved in down-regulation of basal expression levels in the absence of rep, suggesting that AHHH also contributes to negative regulatory functions of the LTR of HIV-1. In contrast, positive regulatory elements of the HIV-1 LTR such as the NF kappa B and SP1 binding sites have no significant influence on the rep-mediated inhibition. Images PMID:8289357

DNaseI Hypersensitivity and Ultraconservation Reveal Novel, Interdependent Long-Range Enhancers at the Complex Pax6 Cis-Regulatory Region

PubMed Central

McBride, David J.; Buckle, Adam; van Heyningen, Veronica; Kleinjan, Dirk A.

2011-01-01

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene. The minimal essential region required for proper regulation of this complex locus has been defined through analysis of human aniridia-associated breakpoints and YAC transgenic rescue studies of the mouse smalleye mutant. We have carried out a systematic DNase I hypersensitive site (HS) analysis across 200 kb of this critical region of mouse chromosome 2E3 to identify putative regulatory elements. Mapping the identified HSs onto a percent identity plot (PIP) shows many HSs correspond to recognisable genomic features such as evolutionarily conserved sequences, CpG islands and retrotransposon derived repeats. We then focussed on a region previously shown to contain essential long range cis-regulatory information, the Pax6 downstream regulatory region (DRR), allowing comparison of mouse HS data with previous human HS data for this region. Reporter transgenic mice for two of the HS sites, HS5 and HS6, show that they function as tissue specific regulatory elements. In addition we have characterised enhancer activity of an ultra-conserved cis-regulatory region located near Pax6, termed E60. All three cis-elements exhibit multiple spatio-temporal activities in the embryo that overlap between themselves and other elements in the locus. Using a deletion set of YAC reporter transgenic mice we demonstrate functional interdependence of the elements. Finally, we use the HS6 enhancer as a marker for the migration of precerebellar neuro-epithelium cells to the hindbrain precerebellar nuclei along the posterior and anterior extramural streams allowing visualisation of migratory defects in both pathways in Pax6Sey/Sey mice. PMID:22220192

Highly accessible AU-rich regions in 3' untranslated regions are hotspots for binding of regulatory factors.

PubMed

Plass, Mireya; Rasmussen, Simon H; Krogh, Anders

2017-04-01

Post-transcriptional regulation is regarded as one of the major processes involved in the regulation of gene expression. It is mainly performed by RNA binding proteins and microRNAs, which target RNAs and typically affect their stability. Recent efforts from the scientific community have aimed at understanding post-transcriptional regulation at a global scale by using high-throughput sequencing techniques such as cross-linking and immunoprecipitation (CLIP), which facilitates identification of binding sites of these regulatory factors. However, the diversity in the experimental procedures and bioinformatics analyses has hindered the integration of multiple datasets and thus limited the development of an integrated view of post-transcriptional regulation. In this work, we have performed a comprehensive analysis of 107 CLIP datasets from 49 different RBPs in HEK293 cells to shed light on the complex interactions that govern post-transcriptional regulation. By developing a more stringent CLIP analysis pipeline we have discovered the existence of conserved regulatory AU-rich regions in the 3'UTRs where miRNAs and RBPs that regulate several processes such as polyadenylation or mRNA stability bind. Analogous to promoters, many factors have binding sites overlapping or in close proximity in these hotspots and hence the regulation of the mRNA may depend on their relative concentrations. This hypothesis is supported by RBP knockdown experiments that alter the relative concentration of RBPs in the cell. Upon AGO2 knockdown (KD), transcripts containing "free" target sites show increased expression levels compared to those containing target sites in hotspots, which suggests that target sites within hotspots are less available for miRNAs to bind. Interestingly, these hotspots appear enriched in genes with regulatory functions such as DNA binding and RNA binding. Taken together, our results suggest that hotspots are functional regulatory elements that define an extra layer of regulation of post-transcriptional regulatory networks.

Highly accessible AU-rich regions in 3’ untranslated regions are hotspots for binding of regulatory factors

PubMed Central

2017-01-01

Post-transcriptional regulation is regarded as one of the major processes involved in the regulation of gene expression. It is mainly performed by RNA binding proteins and microRNAs, which target RNAs and typically affect their stability. Recent efforts from the scientific community have aimed at understanding post-transcriptional regulation at a global scale by using high-throughput sequencing techniques such as cross-linking and immunoprecipitation (CLIP), which facilitates identification of binding sites of these regulatory factors. However, the diversity in the experimental procedures and bioinformatics analyses has hindered the integration of multiple datasets and thus limited the development of an integrated view of post-transcriptional regulation. In this work, we have performed a comprehensive analysis of 107 CLIP datasets from 49 different RBPs in HEK293 cells to shed light on the complex interactions that govern post-transcriptional regulation. By developing a more stringent CLIP analysis pipeline we have discovered the existence of conserved regulatory AU-rich regions in the 3’UTRs where miRNAs and RBPs that regulate several processes such as polyadenylation or mRNA stability bind. Analogous to promoters, many factors have binding sites overlapping or in close proximity in these hotspots and hence the regulation of the mRNA may depend on their relative concentrations. This hypothesis is supported by RBP knockdown experiments that alter the relative concentration of RBPs in the cell. Upon AGO2 knockdown (KD), transcripts containing “free” target sites show increased expression levels compared to those containing target sites in hotspots, which suggests that target sites within hotspots are less available for miRNAs to bind. Interestingly, these hotspots appear enriched in genes with regulatory functions such as DNA binding and RNA binding. Taken together, our results suggest that hotspots are functional regulatory elements that define an extra layer of regulation of post-transcriptional regulatory networks. PMID:28410363

Whole-Transcriptome Analysis of CD133+CD144+ Cancer Stem Cells Derived from Human Laryngeal Squamous Cell Carcinoma Cells.

PubMed

Wu, Yongyan; Zhang, Yuliang; Niu, Min; Shi, Yong; Liu, Hongliang; Yang, Dongli; Li, Fei; Lu, Yan; Bo, Yunfeng; Zhang, Ruiping; Li, Zhenyu; Luo, Hongjie; Cui, Jiajia; Sang, Jiangwei; Xiang, Caixia; Gao, Wei; Wen, Shuxin

2018-06-27

CD133+CD44+ cancer stem cells previously isolated from laryngeal squamous cell carcinoma (LSCC) cell lines showed strong malignancy and tumorigenicity. However, the molecular mechanism underlying the enhanced malignancy remained unclear. Cell proliferation assay, spheroid-formation experiment, RNA sequencing (RNA-seq), miRNA-seq, bioinformatic analysis, quantitative real-time PCR, migration assay, invasion assay, and luciferase reporter assay were used to identify differentially expressed mRNAs, lncRNAs, circRNAs and miRNAs, construct transcription regulatory network, and investigate functional roles and mechanism of circRNA in CD133+CD44+ laryngeal cancer stem cells. Differentially expressed genes in TDP cells were mainly enriched in the biological processes of cell differentiation, regulation of autophagy, negative regulation of cell death, regulation of cell growth, response to hypoxia, telomere maintenance, cellular response to gamma radiation, and regulation of apoptotic signaling, which are closely related to the malignant features of tumor cells. We constructed the regulatory network of differentially expressed circRNAs, miRNAs and mRNAs. qPCR findings for the expression of key genes in the network were consistent with the sequencing data. Moreover, our data revealed that circRNA hg19_circ_0005033 promotes proliferation, migration, invasion, and chemotherapy resistance of laryngeal cancer stem cells. This study provides potential biomarkers and targets for LSCC diagnosis and therapy, and provide important evidences for the heterogeneity of LSCC cells at the transcription level. © 2018 The Author(s). Published by S. Karger AG, Basel.

Integrated analysis of microRNA and gene expression profiles reveals a functional regulatory module associated with liver fibrosis.

PubMed

Chen, Wei; Zhao, Wenshan; Yang, Aiting; Xu, Anjian; Wang, Huan; Cong, Min; Liu, Tianhui; Wang, Ping; You, Hong

2017-12-15

Liver fibrosis, characterized with the excessive accumulation of extracellular matrix (ECM) proteins, represents the final common pathway of chronic liver inflammation. Ever-increasing evidence indicates microRNAs (miRNAs) dysregulation has important implications in the different stages of liver fibrosis. However, our knowledge of miRNA-gene regulation details pertaining to such disease remains unclear. The publicly available Gene Expression Omnibus (GEO) datasets of patients suffered from cirrhosis were extracted for integrated analysis. Differentially expressed miRNAs (DEMs) and genes (DEGs) were identified using GEO2R web tool. Putative target gene prediction of DEMs was carried out using the intersection of five major algorithms: DIANA-microT, TargetScan, miRanda, PICTAR5 and miRWalk. Functional miRNA-gene regulatory network (FMGRN) was constructed based on the computational target predictions at the sequence level and the inverse expression relationships between DEMs and DEGs. DAVID web server was selected to perform KEGG pathway enrichment analysis. Functional miRNA-gene regulatory module was generated based on the biological interpretation. Internal connections among genes in liver fibrosis-related module were determined using String database. MiRNA-gene regulatory modules related to liver fibrosis were experimentally verified in recombinant human TGFβ1 stimulated and specific miRNA inhibitor treated LX-2 cells. We totally identified 85 and 923 dysregulated miRNAs and genes in liver cirrhosis biopsy samples compared to their normal controls. All evident miRNA-gene pairs were identified and assembled into FMGRN which consisted of 990 regulations between 51 miRNAs and 275 genes, forming two big sub-networks that were defined as down-network and up-network, respectively. KEGG pathway enrichment analysis revealed that up-network was prominently involved in several KEGG pathways, in which "Focal adhesion", "PI3K-Akt signaling pathway" and "ECM-receptor interaction" were remarked significant (adjusted p<0.001). Genes enriched in these pathways coupled with their regulatory miRNAs formed a functional miRNA-gene regulatory module that contains 7 miRNAs, 22 genes and 42 miRNA-gene connections. Gene interaction analysis based on String database revealed that 8 out of 22 genes were highly clustered. Finally, we experimentally confirmed a functional regulatory module containing 5 miRNAs (miR-130b-3p, miR-148a-3p, miR-345-5p, miR-378a-3p, and miR-422a) and 6 genes (COL6A1, COL6A2, COL6A3, PIK3R3, COL1A1, CCND2) associated with liver fibrosis. Our integrated analysis of miRNA and gene expression profiles highlighted a functional miRNA-gene regulatory module associated with liver fibrosis, which, to some extent, may provide important clues to better understand the underlying pathogenesis of liver fibrosis. Copyright © 2017. Published by Elsevier B.V.

Detection and Analysis of Circular RNAs by RT-PCR.

PubMed

Panda, Amaresh C; Gorospe, Myriam

2018-03-20

Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al ., 2017b; Panda et al ., 2017c). However, the validation of true circRNAs identified by RNA sequencing requires other molecular biology techniques including reverse transcription (RT) followed by conventional or quantitative (q) polymerase chain reaction (PCR), and Northern blot analysis (Jeck and Sharpless, 2014). RT-qPCR analysis of circular RNAs using divergent primers has been widely used for the detection, validation, and sometimes quantification of circRNAs (Abdelmohsen et al ., 2015 and 2017; Panda et al ., 2017b). As detailed here, divergent primers designed to span the circRNA backsplice junction sequence can specifically amplify the circRNAs and not the counterpart linear RNA. In sum, RT-PCR analysis using divergent primers allows direct detection and quantification of circRNAs.

Process Mining Techniques for Analysing Patterns and Strategies in Students' Self-Regulated Learning

ERIC Educational Resources Information Center

Bannert, Maria; Reimann, Peter; Sonnenberg, Christoph

2014-01-01

Referring to current research on self-regulated learning, we analyse individual regulation in terms of a set of specific sequences of regulatory activities. Successful students perform regulatory activities such as analysing, planning, monitoring and evaluating cognitive and motivational aspects during learning not only with a higher frequency…

75 FR 69491 - Self-Regulatory Organizations; Order Approving Proposed Rule Change by New York Stock Exchange...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-12

... SECURITIES AND EXCHANGE COMMISSION [Release No. 34-63266; File No. SR-NYSE-2010-67] Self-Regulatory Organizations; Order Approving Proposed Rule Change by New York Stock Exchange LLC Changing the NYBX Order Execution Sequence November 5, 2010. I. Introduction On September 9, 2010, the New York...

77 FR 8034 - Semiannual Regulatory Agenda and Fiscal Year 2011 Regulatory Plan

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-13

... ``no'' for such fields. Because the CFPB is at an early stage of its operations, it is still in the... Financial Protection Bureau--Prerule Stage Regulation Sequence No. Title Identifier No. 492 Business Lending Data 3170-AA09 (Regulation B). Consumer Financial Protection Bureau--Proposed Rule Stage Regulation...

Genomic Organization and Identification of Promoter Regions for the BDNF Gene in the Pond Turtle Trachemys scripta elegans

PubMed Central

Zheng, Zhaoqing; Keifer, Joyce

2014-01-01

Brain-derived neurotrophic factor (BDNF) is an important regulator of neuronal development and synaptic function. The BDNF gene undergoes significant activity-dependent regulation during learning. Here, we identified the BDNF promoter regions, transcription start sites, and potential regulatory sequences for BDNF exons I–III that may contribute to activity-dependent gene and protein expression in the pond turtle Trachemys scripta elegans (tBDNF). By using transfection of BDNF promoter/luciferase plasmid constructs into human neuroblastoma SHSY5Y cells and mouse embryonic fibroblast NIH3T3 cells, we identified the basal regulatory activity of promoter sequences located upstream of each tBDNF exon, designated as pBDNFI–III. Further, through chromatin immunoprecipitation (ChIP) assays, we detected CREB binding directly to exon I and exon III promoters, while BHLHB2, but not CREB, binds within the exon II promoter. Elucidation of the promoter regions and regulatory protein binding sites in the tBDNF gene is essential for understanding the regulatory mechanisms that control tBDNF gene expression. PMID:24443176

Genomic organization and identification of promoter regions for the BDNF gene in the pond turtle Trachemys scripta elegans.

PubMed

Ambigapathy, Ganesh; Zheng, Zhaoqing; Keifer, Joyce

2014-08-01

Brain-derived neurotrophic factor (BDNF) is an important regulator of neuronal development and synaptic function. The BDNF gene undergoes significant activity-dependent regulation during learning. Here, we identified the BDNF promoter regions, transcription start sites, and potential regulatory sequences for BDNF exons I-III that may contribute to activity-dependent gene and protein expression in the pond turtle Trachemys scripta elegans (tBDNF). By using transfection of BDNF promoter/luciferase plasmid constructs into human neuroblastoma SHSY5Y cells and mouse embryonic fibroblast NIH3T3 cells, we identified the basal regulatory activity of promoter sequences located upstream of each tBDNF exon, designated as pBDNFI-III. Further, through chromatin immunoprecipitation (ChIP) assays, we detected CREB binding directly to exon I and exon III promoters, while BHLHB2, but not CREB, binds within the exon II promoter. Elucidation of the promoter regions and regulatory protein binding sites in the tBDNF gene is essential for understanding the regulatory mechanisms that control tBDNF gene expression.

GRIL-Seq, a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

PubMed Central

Han, Kook; Tjaden, Brian; Lory, Stephen

2017-01-01

The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base-pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique (referred to as GRIL-Seq) is based on preferential ligation of sRNAs to ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimeras. In addition to the RNA chaperone Hfq, the GRIL-Seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrate that direct regulatory targets of this sRNA can be readily identified. Therefore, GRIL-Seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but can also result in uncovering novel roles for sRNAs and their targets in complex regulatory networks. PMID:28005055

BiRen: predicting enhancers with a deep-learning-based model using the DNA sequence alone.

PubMed

Yang, Bite; Liu, Feng; Ren, Chao; Ouyang, Zhangyi; Xie, Ziwei; Bo, Xiaochen; Shu, Wenjie

2017-07-01

Enhancer elements are noncoding stretches of DNA that play key roles in controlling gene expression programmes. Despite major efforts to develop accurate enhancer prediction methods, identifying enhancer sequences continues to be a challenge in the annotation of mammalian genomes. One of the major issues is the lack of large, sufficiently comprehensive and experimentally validated enhancers for humans or other species. Thus, the development of computational methods based on limited experimentally validated enhancers and deciphering the transcriptional regulatory code encoded in the enhancer sequences is urgent. We present a deep-learning-based hybrid architecture, BiRen, which predicts enhancers using the DNA sequence alone. Our results demonstrate that BiRen can learn common enhancer patterns directly from the DNA sequence and exhibits superior accuracy, robustness and generalizability in enhancer prediction relative to other state-of-the-art enhancer predictors based on sequence characteristics. Our BiRen will enable researchers to acquire a deeper understanding of the regulatory code of enhancer sequences. Our BiRen method can be freely accessed at https://github.com/wenjiegroup/BiRen . shuwj@bmi.ac.cn or boxc@bmi.ac.cn. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequencing Quality Control consortium

PubMed Central

2014-01-01

We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the United States Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for junction discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed, for these and qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcript-level profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings. PMID:25150838

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

PubMed

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

2017-04-15

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

Interim Reliability Evaluation Program: analysis of the Browns Ferry, Unit 1, nuclear plant. Main report

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

Mays, S.E.; Poloski, J.P.; Sullivan, W.H.

1982-07-01

A probabilistic risk assessment (PRA) was made of the Browns Ferry, Unit 1, nuclear plant as part of the Nuclear Regulatory Commission's Interim Reliability Evaluation Program (IREP). Specific goals of the study were to identify the dominant contributors to core melt, develop a foundation for more extensive use of PRA methods, expand the cadre of experienced PRA practitioners, and apply procedures for extension of IREP analyses to other domestic light water reactors. Event tree and fault tree analyses were used to estimate the frequency of accident sequences initiated by transients and loss of coolant accidents. External events such as floods,more » fires, earthquakes, and sabotage were beyond the scope of this study and were, therefore, excluded. From these sequences, the dominant contributors to probable core melt frequency were chosen. Uncertainty and sensitivity analyses were performed on these sequences to better understand the limitations associated with the estimated sequence frequencies. Dominant sequences were grouped according to common containment failure modes and corresponding release categories on the basis of comparison with analyses of similar designs rather than on the basis of detailed plant-specific calculations.« less

MicroRNA and Transcription Factor: Key Players in Plant Regulatory Network

PubMed Central

Samad, Abdul F. A.; Sajad, Muhammad; Nazaruddin, Nazaruddin; Fauzi, Izzat A.; Murad, Abdul M. A.; Zainal, Zamri; Ismail, Ismanizan

2017-01-01

Recent achievements in plant microRNA (miRNA), a large class of small and non-coding RNAs, are very exciting. A wide array of techniques involving forward genetic, molecular cloning, bioinformatic analysis, and the latest technology, deep sequencing have greatly advanced miRNA discovery. A tiny miRNA sequence has the ability to target single/multiple mRNA targets. Most of the miRNA targets are transcription factors (TFs) which have paramount importance in regulating the plant growth and development. Various families of TFs, which have regulated a range of regulatory networks, may assist plants to grow under normal and stress environmental conditions. This present review focuses on the regulatory relationships between miRNAs and different families of TFs like; NF-Y, MYB, AP2, TCP, WRKY, NAC, GRF, and SPL. For instance NF-Y play important role during drought tolerance and flower development, MYB are involved in signal transduction and biosynthesis of secondary metabolites, AP2 regulate the floral development and nodule formation, TCP direct leaf development and growth hormones signaling. WRKY have known roles in multiple stress tolerances, NAC regulate lateral root formation, GRF are involved in root growth, flower, and seed development, and SPL regulate plant transition from juvenile to adult. We also studied the relation between miRNAs and TFs by consolidating the research findings from different plant species which will help plant scientists in understanding the mechanism of action and interaction between these regulators in the plant growth and development under normal and stress environmental conditions. PMID:28446918

Systems analysis of cis-regulatory motifs in C4 photosynthesis genes using maize and rice leaf transcriptomic data during a process of de-etiolation.

PubMed

Xu, Jiajia; Bräutigam, Andrea; Weber, Andreas P M; Zhu, Xin-Guang

2016-09-01

Identification of potential cis-regulatory motifs controlling the development of C4 photosynthesis is a major focus of current research. In this study, we used time-series RNA-seq data collected from etiolated maize and rice leaf tissues sampled during a de-etiolation process to systematically characterize the expression patterns of C4-related genes and to further identify potential cis elements in five different genomic regions (i.e. promoter, 5'UTR, 3'UTR, intron, and coding sequence) of C4 orthologous genes. The results demonstrate that although most of the C4 genes show similar expression patterns, a number of them, including chloroplast dicarboxylate transporter 1, aspartate aminotransferase, and triose phosphate transporter, show shifted expression patterns compared with their C3 counterparts. A number of conserved short DNA motifs between maize C4 genes and their rice orthologous genes were identified not only in the promoter, 5'UTR, 3'UTR, and coding sequences, but also in the introns of core C4 genes. We also identified cis-regulatory motifs that exist in maize C4 genes and also in genes showing similar expression patterns as maize C4 genes but that do not exist in rice C3 orthologs, suggesting a possible recruitment of pre-existing cis-elements from genes unrelated to C4 photosynthesis into C4 photosynthesis genes during C4 evolution. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Decoding the complex genetic causes of heart diseases using systems biology.

PubMed

Djordjevic, Djordje; Deshpande, Vinita; Szczesnik, Tomasz; Yang, Andrian; Humphreys, David T; Giannoulatou, Eleni; Ho, Joshua W K

2015-03-01

The pace of disease gene discovery is still much slower than expected, even with the use of cost-effective DNA sequencing and genotyping technologies. It is increasingly clear that many inherited heart diseases have a more complex polygenic aetiology than previously thought. Understanding the role of gene-gene interactions, epigenetics, and non-coding regulatory regions is becoming increasingly critical in predicting the functional consequences of genetic mutations identified by genome-wide association studies and whole-genome or exome sequencing. A systems biology approach is now being widely employed to systematically discover genes that are involved in heart diseases in humans or relevant animal models through bioinformatics. The overarching premise is that the integration of high-quality causal gene regulatory networks (GRNs), genomics, epigenomics, transcriptomics and other genome-wide data will greatly accelerate the discovery of the complex genetic causes of congenital and complex heart diseases. This review summarises state-of-the-art genomic and bioinformatics techniques that are used in accelerating the pace of disease gene discovery in heart diseases. Accompanying this review, we provide an interactive web-resource for systems biology analysis of mammalian heart development and diseases, CardiacCode ( http://CardiacCode.victorchang.edu.au/ ). CardiacCode features a dataset of over 700 pieces of manually curated genetic or molecular perturbation data, which enables the inference of a cardiac-specific GRN of 280 regulatory relationships between 33 regulator genes and 129 target genes. We believe this growing resource will fill an urgent unmet need to fully realise the true potential of predictive and personalised genomic medicine in tackling human heart disease.

StarScan: a web server for scanning small RNA targets from degradome sequencing data.

PubMed

Liu, Shun; Li, Jun-Hao; Wu, Jie; Zhou, Ke-Ren; Zhou, Hui; Yang, Jian-Hua; Qu, Liang-Hu

2015-07-01

Endogenous small non-coding RNAs (sRNAs), including microRNAs, PIWI-interacting RNAs and small interfering RNAs, play important gene regulatory roles in animals and plants by pairing to the protein-coding and non-coding transcripts. However, computationally assigning these various sRNAs to their regulatory target genes remains technically challenging. Recently, a high-throughput degradome sequencing method was applied to identify biologically relevant sRNA cleavage sites. In this study, an integrated web-based tool, StarScan (sRNA target Scan), was developed for scanning sRNA targets using degradome sequencing data from 20 species. Given a sRNA sequence from plants or animals, our web server performs an ultrafast and exhaustive search for potential sRNA-target interactions in annotated and unannotated genomic regions. The interactions between small RNAs and target transcripts were further evaluated using a novel tool, alignScore. A novel tool, degradomeBinomTest, was developed to quantify the abundance of degradome fragments located at the 9-11th nucleotide from the sRNA 5' end. This is the first web server for discovering potential sRNA-mediated RNA cleavage events in plants and animals, which affords mechanistic insights into the regulatory roles of sRNAs. The StarScan web server is available at http://mirlab.sysu.edu.cn/starscan/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

LncRNAs in Secondary Hair Follicle of Cashmere Goat: Identification, Expression, and Their Regulatory Network in Wnt Signaling Pathway.

PubMed

Bai, Wen L; Zhao, Su J; Wang, Ze Y; Zhu, Yu B; Dang, Yun L; Cong, Yu Y; Xue, Hui L; Wang, Wei; Deng, Liang; Guo, Dan; Wang, Shi Q; Zhu, Yan X; Yin, Rong H

2018-07-03

Long noncoding RNAs (lncRNAs) are a novel class of eukaryotic transcripts. They are thought to act as a critical regulator of protein-coding gene expression. Herein, we identified and characterized 13 putative lncRNAs from the expressed sequence tags from secondary hair follicle of Cashmere goat. Furthermore, we investigated their transcriptional pattern in secondary hair follicle of Liaoning Cashmere goat during telogen and anagen phases. Also, we generated intracellular regulatory networks of upregulated lncRNAs at anagen in Wnt signaling pathway based on bioinformatics analysis. The relative expression of six putative lncRNAs (lncRNA-599618, -599556, -599554, -599547, -599531, and -599509) at the anagen phase is significantly higher than that at telogen. Compared with anagen, the relative expression of four putative lncRNAs (lncRNA-599528, -599518, -599511, and -599497) was found to be significantly upregulated at telogen phase. The network generated showed that a rich and complex regulatory relationship of the putative lncRNAs and related miRNAs with their target genes in Wnt signaling pathway. Our results from the present study provided a foundation for further elucidating the functional and regulatory mechanisms of these putative lncRNAs in the development of secondary hair follicle and cashmere fiber growth of Cashmere goat.