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Sample records for conserved sequence motif

  1. Conserved sequence motifs among bacterial, eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily.

    PubMed Central

    Thaller, M. C.; Schippa, S.; Rossolini, G. M.

    1998-01-01

    Members of a new molecular family of bacterial nonspecific acid phosphatases (NSAPs), indicated as class C, were found to share significant sequence similarities to bacterial class B NSAPs and to some plant acid phosphatases, representing the first example of a family of bacterial NSAPs that has a relatively close eukaryotic counterpart. Despite the lack of an overall similarity, conserved sequence motifs were also identified among the above enzyme families (class B and class C bacterial NSAPs, and related plant phosphatases) and several other families of phosphohydrolases, including bacterial phosphoglycolate phosphatases, histidinol-phosphatase domains of the bacterial bifunctional enzymes imidazole-glycerolphosphate dehydratases, and bacterial, eukaryotic, and archaeal phosphoserine phosphatases and threalose-6-phosphatases. These conserved motifs are clustered within two domains, separated by a variable spacer region, according to the pattern [FILMAVT]-D-[ILFRMVY]-D-[GSNDE]-[TV]-[ILVAM]-[AT S VILMC]-X-¿YFWHKR)-X-¿YFWHNQ¿-X( 102,191)-¿KRHNQ¿-G-D-¿FYWHILVMC¿-¿QNH¿-¿FWYGP¿-D -¿PSNQYW¿. The dephosphorylating activity common to all these proteins supports the definition of this phosphatase motif and the inclusion of these enzymes into a superfamily of phosphohydrolases that we propose to indicate as "DDDD" after the presence of the four invariant aspartate residues. Database searches retrieved various hypothetical proteins of unknown function containing this or similar motifs, for which a phosphohydrolase activity could be hypothesized. PMID:9684901

  2. Computational Prediction of Phylogenetically Conserved Sequence Motifs for Five Different Candidate Genes in Type II Diabetic Nephropathy

    PubMed Central

    Sindhu, T; Rajamanikandan, S; Srinivasan, P

    2012-01-01

    Background: Computational identification of phylogenetic motifs helps to understand the knowledge about known functional features that includes catalytic site, substrate binding epitopes, and protein-protein interfaces. Furthermore, they are strongly conserved among orthologs, indicating their evolutionary importance. The study aimed to analyze five candidate genes involved in type II diabetic nephropathy and to predict phylogenetic motifs from their corresponding orthologous protein sequences. Methods: AKR1B1, APOE, ENPP1, ELMO1 and IGFBP1 are the genes that have been identified as an important target for type II diabetic nephropathy through experimental studies. Their corresponding protein sequences, structures, orthologous sequences were retrieved from UniprotKB, PDB, and PHOG database respectively. Multiple sequence alignments were constructed using ClustalW and phylogenetic motifs were identified using MINER. The occurrence of amino acids in the obtained phylogenetic motifs was generated using WebLogo and false positive expectations were calculated against phylogenetic similarity. Results: In total, 17 phylogenetic motifs were identified from the five proteins and the residues such as glycine, leucine, tryptophan, aspartic acid were found in appreciable frequency whereas arginine identified in all the predicted PMs. The result implies that these residues can be important to the functional and structural role of the proteins and calculated false positive expectations implies that they were generally conserved in traditional sense. Conclusion: The prediction of phylogenetic motifs is an accurate method for detecting functionally important conserved residues. The conserved motifs can be used as a potential drug target for type II diabetic nephropathy. PMID:23113206

  3. Detecting Remote Sequence Homology in Disordered Proteins: Discovery of Conserved Motifs in the N-Termini of Mononegavirales phosphoproteins

    PubMed Central

    Karlin, David; Belshaw, Robert

    2012-01-01

    Paramyxovirinae are a large group of viruses that includes measles virus and parainfluenza viruses. The viral Phosphoprotein (P) plays a central role in viral replication. It is composed of a highly variable, disordered N-terminus and a conserved C-terminus. A second viral protein alternatively expressed, the V protein, also contains the N-terminus of P, fused to a zinc finger. We suspected that, despite their high variability, the N-termini of P/V might all be homologous; however, using standard approaches, we could previously identify sequence conservation only in some Paramyxovirinae. We now compared the N-termini using sensitive sequence similarity search programs, able to detect residual similarities unnoticeable by conventional approaches. We discovered that all Paramyxovirinae share a short sequence motif in their first 40 amino acids, which we called soyuz1. Despite its short length (11–16aa), several arguments allow us to conclude that soyuz1 probably evolved by homologous descent, unlike linear motifs. Conservation across such evolutionary distances suggests that soyuz1 plays a crucial role and experimental data suggest that it binds the viral nucleoprotein to prevent its illegitimate self-assembly. In some Paramyxovirinae, the N-terminus of P/V contains a second motif, soyuz2, which might play a role in blocking interferon signaling. Finally, we discovered that the P of related Mononegavirales contain similarly overlooked motifs in their N-termini, and that their C-termini share a previously unnoticed structural similarity suggesting a common origin. Our results suggest several testable hypotheses regarding the replication of Mononegavirales and suggest that disordered regions with little overall sequence similarity, common in viral and eukaryotic proteins, might contain currently overlooked motifs (intermediate in length between linear motifs and disordered domains) that could be detected simply by comparing orthologous proteins. PMID:22403617

  4. WeederH: an algorithm for finding conserved regulatory motifs and regions in homologous sequences

    PubMed Central

    Pavesi, Giulio; Zambelli, Federico; Pesole, Graziano

    2007-01-01

    Background This work addresses the problem of detecting conserved transcription factor binding sites and in general regulatory regions through the analysis of sequences from homologous genes, an approach that is becoming more and more widely used given the ever increasing amount of genomic data available. Results We present an algorithm that identifies conserved transcription factor binding sites in a given sequence by comparing it to one or more homologs, adapting a framework we previously introduced for the discovery of sites in sequences from co-regulated genes. Differently from the most commonly used methods, the approach we present does not need or compute an alignment of the sequences investigated, nor resorts to descriptors of the binding specificity of known transcription factors. The main novel idea we introduce is a relative measure of conservation, assuming that true functional elements should present a higher level of conservation with respect to the rest of the sequence surrounding them. We present tests where we applied the algorithm to the identification of conserved annotated sites in homologous promoters, as well as in distal regions like enhancers. Conclusion Results of the tests show how the algorithm can provide fast and reliable predictions of conserved transcription factor binding sites regulating the transcription of a gene, with better performances than other available methods for the same task. We also show examples on how the algorithm can be successfully employed when promoter annotations of the genes investigated are missing, or when regulatory sites and regions are located far away from the genes. PMID:17286865

  5. The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

    PubMed Central

    Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

    2013-01-01

    AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5′-NNCCAC-3′ and 5′-GCGMGN′N′-3′ (M:A or C; N and N′ form Watson–Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences. PMID:23709277

  6. Analysis of Cytochrome P450 Conserved Sequence Motifs between Helices E and H: Prediction of Critical Motifs and Residues in Enzyme Functions

    PubMed Central

    Oezguen, Numan; Kumar, Santosh

    2014-01-01

    Rational approaches have been extensively used to investigate the role of active site residues in cytochrome P450 (CYP) functions. However, recent studies using random mutagenesis suggest an important role for non-active site residues in CYP functions. Meta-analysis of the random mutants showed that 75% of the functionally important non-active site residues are present in 20% of the entire protein between helices E and H (E-H) and conserved sequence motif (CSM) between 7 and 11. The CSM approach was developed recently to investigate the functional role of non-active site residues in CYP2B4. Furthermore, we identified and analyzed the CSM in multiple CYP families and subfamilies in the E-H region. Results from CSM analysis showed that CSM 7, 8, 10, and 11 are conserved in CYP1, CYP2, and CYP3 families, while CSM 9 is conserved only in CYP2 family. Analysis of different CYP2 subfamilies showed that CYP2B and CYP2C have similar characteristics in the CSM, while the characteristics of CYP2A and CYP2D subfamilies are different. Finally, we analyzed CSM 7, 8, 10, and 11, which are common in all the CYP families/subfamilies analyzed, in fifteen important drug-metabolizing CYPs. The results showed that while CSM 8 is most conserved among these CYPs, CSM 7, 9, and 10 have significant variations. We suggest that CSM8 has a common role in all the CYPs that have been analyzed, while CSM 7, 10, and 11 may have relatively specific role within the subfamily. We further suggest that these CSM play important role in opening and closing of the substrate access/egress channel by modulating the flexible/plastic region of the protein. Thus, site-directed mutagenesis of these CSM can be used to study structure-function and dynamic/plasticity-function relationships and to design CYP biocatalysts. PMID:25426333

  7. Alignment of U3 region sequences of mammalian type C viruses: identification of highly conserved motifs and implications for enhancer design.

    PubMed Central

    Golemis, E A; Speck, N A; Hopkins, N

    1990-01-01

    We aligned published sequences for the U3 region of 35 type C mammalian retroviruses. The alignment reveals that certain sequence motifs within the U3 region are strikingly conserved. A number of these motifs correspond to previously identified sites. In particular, we found that the enhancer region of most of the viruses examined contains a binding site for leukemia virus factor b, a viral corelike element, the consensus motif for nuclear factor 1, and the glucocorticoid response element. Most viruses containing more than one copy of enhancer sequences include these binding sites in both copies of the repeat. We consider this set of binding sites to constitute a framework for the enhancers of this set of viruses. Other highly conserved motifs in the U3 region include the retrovirus inverted repeat sequence, a negative regulatory element, and the CCAAT and TATA boxes. In addition, we identified two novel motifs in the promoter region that were exceptionally highly conserved but have not been previously described. PMID:2153223

  8. Linear array of conserved sequence motifs to discriminate protein subfamilies: study on pyridine nucleotide-disulfide reductases

    PubMed Central

    Avila, César L; Rapisarda, Viviana A; Farías, Ricardo N; De Las Rivas, Javier; Chehín, Rosana

    2007-01-01

    Background The pyridine nucleotide disulfide reductase (PNDR) is a large and heterogeneous protein family divided into two classes (I and II), which reflect the divergent evolution of its characteristic disulfide redox active site. However, not all the PNDR members fit into these categories and this suggests the need of further studies to achieve a more comprehensive classification of this complex family. Results A workflow to improve the clusterization of protein families based on the array of linear conserved motifs is designed. The method is applied to the PNDR large family finding two main groups, which correspond to PNDR classes I and II. However, two other separate protein clusters, previously classified as class I in most databases, are outgrouped: the peroxide reductases (NAOX, NAPE) and the type II NADH dehydrogenases (NDH-2). In this way, two novel PNDR classes III and IV for NAOX/NAPE and NDH-2 respectively are proposed. By knowledge-driven biochemical and functional data analyses done on the new class IV, a linear array of motifs putatively related to Cu(II)-reductase activity is detected in a specific subset of NDH-2. Conclusion The results presented are a novel contribution to the classification of the complex and large PNDR protein family, supporting its reclusterization into four classes. The linear array of motifs detected within the class IV PNDR subfamily could be useful as a signature for a particular subgroup of NDH-2. PMID:17367536

  9. Conserved sequence motifs upstream from the co-ordinately expressed vitellogenin and apoVLDLII genes of chicken.

    PubMed

    van het Schip, F; Strijker, R; Samallo, J; Gruber, M; Geert, A B

    1986-11-11

    The vitellogenin and apoVLDLII yolk protein genes of chicken are transcribed in the liver upon estrogenization. To get information on putative regulatory elements, we compared more than 2 kb of their 5' flanking DNA sequences. Common sequence motifs were found in regions exhibiting estrogen-induced changes in chromatin structure. Stretches of alternating pyrimidines and purines of about 30-nucleotides long are present at roughly similar positions. A distinct box of sequence homology in the chicken genes also appears to be present at a similar position in front of the vitellogenin genes of Xenopus laevis, but is absent from the estrogen-responsive egg-white protein genes expressed in the oviduct. In front of the vitellogenin (position -595) and the VLDLII gene (position -548), a DNA element of about 300 base-pairs was found, which possesses structural characteristics of a mobile genetic element and bears homology to the transposon-like Vi element of Xenopus laevis.

  10. Short sequence motifs, overrepresented in mammalian conservednon-coding sequences

    SciTech Connect

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

    2007-02-21

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

  11. ICAP-1, a Novel β1 Integrin Cytoplasmic Domain–associated Protein, Binds to a Conserved and Functionally Important NPXY Sequence Motif of β1 Integrin

    PubMed Central

    Chang, David D.; Wong, Carol; Smith, Healy; Liu, Jenny

    1997-01-01

    The cytoplasmic domains of integrins are essential for cell adhesion. We report identification of a novel protein, ICAP-1 (integrin cytoplasmic domain– associated protein-1), which binds to the β1 integrin cytoplasmic domain. The interaction between ICAP-1 and β1 integrins is highly specific, as demonstrated by the lack of interaction between ICAP-1 and the cytoplasmic domains of other β integrins, and requires a conserved and functionally important NPXY sequence motif found in the COOH-terminal region of the β1 integrin cytoplasmic domain. Mutational studies reveal that Asn and Tyr of the NPXY motif and a Val residue located NH2-terminal to this motif are critical for the ICAP-1 binding. Two isoforms of ICAP-1, a 200–amino acid protein (ICAP-1α) and a shorter 150–amino acid protein (ICAP-1β), derived from alternatively spliced mRNA, are expressed in most cells. ICAP-1α is a phosphoprotein and the extent of its phosphorylation is regulated by the cell–matrix interaction. First, an enhancement of ICAP-1α phosphorylation is observed when cells were plated on fibronectin-coated but not on nonspecific poly-l-lysine–coated surface. Second, the expression of a constitutively activated RhoA protein that disrupts the cell–matrix interaction results in dephosphorylation of ICAP-1α. The regulation of ICAP-1α phosphorylation by the cell–matrix interaction suggests an important role of ICAP-1 during integrin-dependent cell adhesion. PMID:9281591

  12. A conserved sequence extending motif III of the motor domain in the Snf2-family DNA translocase Rad54 is critical for ATPase activity.

    PubMed

    Zhang, Xiao-Ping; Janke, Ryan; Kingsley, James; Luo, Jerry; Fasching, Clare; Ehmsen, Kirk T; Heyer, Wolf-Dietrich

    2013-01-01

    Rad54 is a dsDNA-dependent ATPase that translocates on duplex DNA. Its ATPase function is essential for homologous recombination, a pathway critical for meiotic chromosome segregation, repair of complex DNA damage, and recovery of stalled or broken replication forks. In recombination, Rad54 cooperates with Rad51 protein and is required to dissociate Rad51 from heteroduplex DNA to allow access by DNA polymerases for recombination-associated DNA synthesis. Sequence analysis revealed that Rad54 contains a perfect match to the consensus PIP box sequence, a widely spread PCNA interaction motif. Indeed, Rad54 interacts directly with PCNA, but this interaction is not mediated by the Rad54 PIP box-like sequence. This sequence is located as an extension of motif III of the Rad54 motor domain and is essential for full Rad54 ATPase activity. Mutations in this motif render Rad54 non-functional in vivo and severely compromise its activities in vitro. Further analysis demonstrated that such mutations affect dsDNA binding, consistent with the location of this sequence motif on the surface of the cleft formed by two RecA-like domains, which likely forms the dsDNA binding site of Rad54. Our study identified a novel sequence motif critical for Rad54 function and showed that even perfect matches to the PIP box consensus may not necessarily identify PCNA interaction sites.

  13. Motif Yggdrasil: sampling sequence motifs from a tree mixture model.

    PubMed

    Andersson, Samuel A; Lagergren, Jens

    2007-06-01

    In phylogenetic foot-printing, putative regulatory elements are found in upstream regions of orthologous genes by searching for common motifs. Motifs in different upstream sequences are subject to mutations along the edges of the corresponding phylogenetic tree, consequently taking advantage of the tree in the motif search is an appealing idea. We describe the Motif Yggdrasil sampler; the first Gibbs sampler based on a general tree that uses unaligned sequences. Previous tree-based Gibbs samplers have assumed a star-shaped tree or partially aligned upstream regions. We give a probabilistic model (MY model) describing upstream sequences with regulatory elements and build a Gibbs sampler with respect to this model. The model allows toggling, i.e., the restriction of a position to a subset of nucleotides, but does not require aligned sequences nor edge lengths, which may be difficult to come by. We apply the collapsing technique to eliminate the need to sample nuisance parameters, and give a derivation of the predictive update formula. We show that the MY model improves the modeling of difficult motif instances and that the use of the tree achieves a substantial increase in nucleotide level correlation coefficient both for synthetic data and 37 bacterial lexA genes. We investigate the sensitivity to errors in the tree and show that using random trees MY sampler still has a performance similar to the original version.

  14. Genomic analysis of membrane protein families: abundance and conserved motifs

    PubMed Central

    Liu, Yang; Engelman, Donald M; Gerstein, Mark

    2002-01-01

    Background Polytopic membrane proteins can be related to each other on the basis of the number of transmembrane helices and sequence similarities. Building on the Pfam classification of protein domain families, and using transmembrane-helix prediction and sequence-similarity searching, we identified a total of 526 well-characterized membrane protein families in 26 recently sequenced genomes. To this we added a clustering of a number of predicted but unclassified membrane proteins, resulting in a total of 637 membrane protein families. Results Analysis of the occurrence and composition of these families revealed several interesting trends. The number of assigned membrane protein domains has an approximately linear relationship to the total number of open reading frames (ORFs) in 26 genomes studied. Caenorhabditis elegans is an apparent outlier, because of its high representation of seven-span transmembrane (7-TM) chemoreceptor families. In all genomes, including that of C. elegans, the number of distinct membrane protein families has a logarithmic relation to the number of ORFs. Glycine, proline, and tyrosine locations tend to be conserved in transmembrane regions within families, whereas isoleucine, valine, and methionine locations are relatively mutable. Analysis of motifs in putative transmembrane helices reveals that GxxxG and GxxxxxxG (which can be written GG4 and GG7, respectively; see Materials and methods) are among the most prevalent. This was noted in earlier studies; we now find these motifs are particularly well conserved in families, however, especially those corresponding to transporters, symporters, and channels. Conclusions We carried out a genome-wide analysis on patterns of the classified polytopic membrane protein families and analyzed the distribution of conserved amino acids and motifs in the transmembrane helix regions in these families. PMID:12372142

  15. Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated

    PubMed Central

    2010-01-01

    Background DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS") but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) not to be biological transcription factor binding sites ("empirical TFBS"). We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. Results Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. Conclusions Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation. PMID:20875111

  16. Improved K-means clustering algorithm for exploring local protein sequence motifs representing common structural property.

    PubMed

    Zhong, Wei; Altun, Gulsah; Harrison, Robert; Tai, Phang C; Pan, Yi

    2005-09-01

    Information about local protein sequence motifs is very important to the analysis of biologically significant conserved regions of protein sequences. These conserved regions can potentially determine the diverse conformation and activities of proteins. In this work, recurring sequence motifs of proteins are explored with an improved K-means clustering algorithm on a new dataset. The structural similarity of these recurring sequence clusters to produce sequence motifs is studied in order to evaluate the relationship between sequence motifs and their structures. To the best of our knowledge, the dataset used by our research is the most updated dataset among similar studies for sequence motifs. A new greedy initialization method for the K-means algorithm is proposed to improve traditional K-means clustering techniques. The new initialization method tries to choose suitable initial points, which are well separated and have the potential to form high-quality clusters. Our experiments indicate that the improved K-means algorithm satisfactorily increases the percentage of sequence segments belonging to clusters with high structural similarity. Careful comparison of sequence motifs obtained by the improved and traditional algorithms also suggests that the improved K-means clustering algorithm may discover some relatively weak and subtle sequence motifs, which are undetectable by the traditional K-means algorithms. Many biochemical tests reported in the literature show that these sequence motifs are biologically meaningful. Experimental results also indicate that the improved K-means algorithm generates more detailed sequence motifs representing common structures than previous research. Furthermore, these motifs are universally conserved sequence patterns across protein families, overcoming some weak points of other popular sequence motifs. The satisfactory result of the experiment suggests that this new K-means algorithm may be applied to other areas of bioinformatics

  17. The complete sequence of a Spanish isolate of Broad bean wilt virus 1 (BBWV-1) reveals a high variability and conserved motifs in the genus Fabavirus.

    PubMed

    Ferrer, R M; Guerri, J; Luis-Arteaga, M S; Moreno, P; Rubio, L

    2005-10-01

    The genome of a Spanish isolate of Broad bean wilt virus-1 (BBWV-1) was completely sequenced and compared with available sequences of other isolates of the genus Fabavirus (BBWV-1 and BBWV-2). This consisted of two RNAs of 5814 and 3431 nucleotides, respectively, and their organization was similar to that of other members of the family Comoviridae. Its mean nucleotide identity with a BBWV-1 American isolate was 81.5%, and between 59.8 and 63.5% with seven BBWV-2 isolates. Our analysis showed sequence stretches in the 5' non-coding regions which are conserved in both genomic RNAs and in BBWV-1 and BBWV-2 isolates.

  18. Localization of the labile disulfide bond between SU and TM of the murine leukemia virus envelope protein complex to a highly conserved CWLC motif in SU that resembles the active-site sequence of thiol-disulfide exchange enzymes.

    PubMed Central

    Pinter, A; Kopelman, R; Li, Z; Kayman, S C; Sanders, D A

    1997-01-01

    Previous studies have indicated that the surface (SU) and transmembrane (TM) subunits of the envelope protein (Env) of murine leukemia viruses (MuLVs) are joined by a labile disulfide bond that can be stabilized by treatment of virions with thiol-specific reagents. In the present study this observation was extended to the Envs of additional classes of MuLV, and the cysteines of SU involved in this linkage were mapped by proteolytic fragmentation analyses to the CWLC sequence present at the beginning of the C-terminal domain of SU. This sequence is highly conserved across a broad range of distantly related retroviruses and resembles the CXXC motif present at the active site of thiol-disulfide exchange enzymes. A model is proposed in which rearrangements of the SU-TM intersubunit disulfide linkage, mediated by the CWLC sequence, play roles in the assembly and function of the Env complex. PMID:9311907

  19. Finding sequence motifs in groups of functionally related proteins.

    PubMed

    Smith, H O; Annau, T M; Chandrasegaran, S

    1990-01-01

    We have developed a method for rapidly finding patterns of conserved amino acid residues (motifs) in groups of functionally related proteins. All 3-amino acid patterns in a group of proteins of the type aa1 d1 aa2 d2 aa3, where d1 and d2 are distances that can be varied in a range up to 24 residues, are accumulated into an array. Segments of the proteins containing those patterns that occur most frequently are aligned on each other by a scoring method that obtains an average relatedness value for all the amino acids in each column of the aligned sequence block based on the Dayhoff relatedness odds matrix. The automated method successfully finds and displays nearly all of the sequence motifs that have been previously reported to occur in 33 reverse transcriptases, 18 DNA integrases, and 30 DNA methyltransferases.

  20. WildSpan: mining structured motifs from protein sequences

    PubMed Central

    2011-01-01

    Background Automatic extraction of motifs from biological sequences is an important research problem in study of molecular biology. For proteins, it is desired to discover sequence motifs containing a large number of wildcard symbols, as the residues associated with functional sites are usually largely separated in sequences. Discovering such patterns is time-consuming because abundant combinations exist when long gaps (a gap consists of one or more successive wildcards) are considered. Mining algorithms often employ constraints to narrow down the search space in order to increase efficiency. However, improper constraint models might degrade the sensitivity and specificity of the motifs discovered by computational methods. We previously proposed a new constraint model to handle large wildcard regions for discovering functional motifs of proteins. The patterns that satisfy the proposed constraint model are called W-patterns. A W-pattern is a structured motif that groups motif symbols into pattern blocks interleaved with large irregular gaps. Considering large gaps reflects the fact that functional residues are not always from a single region of protein sequences, and restricting motif symbols into clusters corresponds to the observation that short motifs are frequently present within protein families. To efficiently discover W-patterns for large-scale sequence annotation and function prediction, this paper first formally introduces the problem to solve and proposes an algorithm named WildSpan (sequential pattern mining across large wildcard regions) that incorporates several pruning strategies to largely reduce the mining cost. Results WildSpan is shown to efficiently find W-patterns containing conserved residues that are far separated in sequences. We conducted experiments with two mining strategies, protein-based and family-based mining, to evaluate the usefulness of W-patterns and performance of WildSpan. The protein-based mining mode of WildSpan is developed for

  1. Sequence-Based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families

    PubMed Central

    Maimanakos, Janine; Chow, Jennifer; Gaßmeyer, Sarah K.; Güllert, Simon; Busch, Florian; Kourist, Robert; Streit, Wolfgang R.

    2016-01-01

    Arylmalonate Decarboxylases (AMDases, EC 4.1.1.76) are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica’s prototype appeared to be limited to the classes of Alpha-, Beta-, and Gamma-proteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the tripartite tricarboxylate transporters family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99%) of the (R)-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes. PMID:27610105

  2. Identification of imine reductase-specific sequence motifs.

    PubMed

    Fademrecht, Silvia; Scheller, Philipp N; Nestl, Bettina M; Hauer, Bernhard; Pleiss, Jürgen

    2016-05-01

    Chiral amines are valuable building blocks for the production of a variety of pharmaceuticals, agrochemicals and other specialty chemicals. Only recently, imine reductases (IREDs) were discovered which catalyze the stereoselective reduction of imines to chiral amines. Although several IREDs were biochemically characterized in the last few years, knowledge of the reaction mechanism and the molecular basis of substrate specificity and stereoselectivity is limited. To gain further insights into the sequence-function relationships, the Imine Reductase Engineering Database (www.IRED.BioCatNet.de) was established and a systematic analysis of 530 putative IREDs was performed. A standard numbering scheme based on R-IRED-Sk was introduced to facilitate the identification and communication of structurally equivalent positions in different proteins. A conservation analysis revealed a highly conserved cofactor binding region and a predominantly hydrophobic substrate binding cleft. Two IRED-specific motifs were identified, the cofactor binding motif GLGxMGx(5 )[ATS]x(4) Gx(4) [VIL]WNR[TS]x(2) [KR] and the active site motif Gx[DE]x[GDA]x[APS]x(3){K}x[ASL]x[LMVIAG]. Our results indicate a preference toward NADPH for all IREDs and explain why, despite their sequence similarity to β-hydroxyacid dehydrogenases (β-HADs), no conversion of β-hydroxyacids has been observed. Superfamily-specific conservations were investigated to explore the molecular basis of their stereopreference. Based on our analysis and previous experimental results on IRED mutants, an exclusive role of standard position 187 for stereoselectivity is excluded. Alternatively, two standard positions 139 and 194 were identified which are superfamily-specifically conserved and differ in R- and S-selective enzymes.

  3. Conservation defines functional motifs in the squint/nodal-related 1 RNA dorsal localization element

    PubMed Central

    Gilligan, Patrick C.; Kumari, Pooja; Lim, Shimin; Cheong, Albert; Chang, Alex; Sampath, Karuna

    2011-01-01

    RNA localization is emerging as a general principle of sub-cellular protein localization and cellular organization. However, the sequence and structural requirements in many RNA localization elements remain poorly understood. Whereas transcription factor-binding sites in DNA can be recognized as short degenerate motifs, and consensus binding sites readily inferred, protein-binding sites in RNA often contain structural features, and can be difficult to infer. We previously showed that zebrafish squint/nodal-related 1 (sqt/ndr1) RNA localizes to the future dorsal side of the embryo. Interestingly, mammalian nodal RNA can also localize to dorsal when injected into zebrafish embryos, suggesting that the sequence motif(s) may be conserved, even though the fish and mammal UTRs cannot be aligned. To define potential sequence and structural features, we obtained ndr1 3′-UTR sequences from approximately 50 fishes that are closely, or distantly, related to zebrafish, for high-resolution phylogenetic footprinting. We identify conserved sequence and structural motifs within the zebrafish/carp family and catfish. We find that two novel motifs, a single-stranded AGCAC motif and a small stem-loop, are required for efficient sqt RNA localization. These findings show that comparative sequencing in the zebrafish/carp family is an efficient approach for identifying weak consensus binding sites for RNA regulatory proteins. PMID:21149265

  4. The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization.

    PubMed Central

    Bernad, A; Lázaro, J M; Salas, M; Blanco, L

    1990-01-01

    The alpha-like DNA polymerases from bacteriophage phi 29 and other viruses, prokaryotes and eukaryotes contain an amino acid consensus sequence that has been proposed to form part of the dNTP binding site. We have used site-directed mutants to study five of the six highly conserved consecutive amino acids corresponding to the most conserved C-terminal segment (Tyr-Gly-Asp-Thr-Asp-Ser). Our results indicate that in phi 29 DNA polymerase this consensus sequence, although irrelevant for the 3'----5' exonuclease activity, is essential for initiation and elongation. Based on these results and on its homology with known or putative metal-binding amino acid sequences, we propose that in phi 29 DNA polymerase the Tyr-Gly-Asp-Thr-Asp-Ser consensus motif is part of the dNTP binding site, involved in the synthetic activities of the polymerase (i.e., initiation and polymerization), and that it is involved particularly in the metal binding associated with the dNTP site. Images PMID:2191296

  5. Occurrence probability of structured motifs in random sequences.

    PubMed

    Robin, S; Daudin, J-J; Richard, H; Sagot, M-F; Schbath, S

    2002-01-01

    The problem of extracting from a set of nucleic acid sequences motifs which may have biological function is more and more important. In this paper, we are interested in particular motifs that may be implicated in the transcription process. These motifs, called structured motifs, are composed of two ordered parts separated by a variable distance and allowing for substitutions. In order to assess their statistical significance, we propose approximations of the probability of occurrences of such a structured motif in a given sequence. An application of our method to evaluate candidate promoters in E. coli and B. subtilis is presented. Simulations show the goodness of the approximations.

  6. Probabilistic models for semisupervised discriminative motif discovery in DNA sequences.

    PubMed

    Kim, Jong Kyoung; Choi, Seungjin

    2011-01-01

    Methods for discriminative motif discovery in DNA sequences identify transcription factor binding sites (TFBSs), searching only for patterns that differentiate two sets (positive and negative sets) of sequences. On one hand, discriminative methods increase the sensitivity and specificity of motif discovery, compared to generative models. On the other hand, generative models can easily exploit unlabeled sequences to better detect functional motifs when labeled training samples are limited. In this paper, we develop a hybrid generative/discriminative model which enables us to make use of unlabeled sequences in the framework of discriminative motif discovery, leading to semisupervised discriminative motif discovery. Numerical experiments on yeast ChIP-chip data for discovering DNA motifs demonstrate that the best performance is obtained between the purely-generative and the purely-discriminative and the semisupervised learning improves the performance when labeled sequences are limited.

  7. iMotifs: an integrated sequence motif visualization and analysis environment

    PubMed Central

    Piipari, Matias; Down, Thomas A.; Saini, Harpreet; Enright, Anton; Hubbard, Tim J.P.

    2010-01-01

    Motivation: Short sequence motifs are an important class of models in molecular biology, used most commonly for describing transcription factor binding site specificity patterns. High-throughput methods have been recently developed for detecting regulatory factor binding sites in vivo and in vitro and consequently high-quality binding site motif data are becoming available for increasing number of organisms and regulatory factors. Development of intuitive tools for the study of sequence motifs is therefore important. iMotifs is a graphical motif analysis environment that allows visualization of annotated sequence motifs and scored motif hits in sequences. It also offers motif inference with the sensitive NestedMICA algorithm, as well as overrepresentation and pairwise motif matching capabilities. All of the analysis functionality is provided without the need to convert between file formats or learn different command line interfaces. The application includes a bundled and graphically integrated version of the NestedMICA motif inference suite that has no outside dependencies. Problems associated with local deployment of software are therefore avoided. Availability: iMotifs is licensed with the GNU Lesser General Public License v2.0 (LGPL 2.0). The software and its source is available at http://wiki.github.com/mz2/imotifs and can be run on Mac OS X Leopard (Intel/PowerPC). We also provide a cross-platform (Linux, OS X, Windows) LGPL 2.0 licensed library libxms for the Perl, Ruby, R and Objective-C programming languages for input and output of XMS formatted annotated sequence motif set files. Contact: matias.piipari@gmail.com; imotifs@googlegroups.com PMID:20106815

  8. Conservation of sequence in recombination signal sequence spacers.

    PubMed Central

    Ramsden, D A; Baetz, K; Wu, G E

    1994-01-01

    The variable domains of immunoglobulins and T cell receptors are assembled through the somatic, site specific recombination of multiple germline segments (V, D, and J segments) or V(D)J rearrangement. The recombination signal sequence (RSS) is necessary and sufficient for cell type specific targeting of the V(D)J rearrangement machinery to these germline segments. Previously, the RSS has been described as possessing both a conserved heptamer and a conserved nonamer motif. The heptamer and nonamer motifs are separated by a 'spacer' that was not thought to possess significant sequence conservation, however the length of the spacer could be either 12 +/- 1 bp or 23 +/- 1 bp long. In this report we have assembled and analyzed an extensive data base of published RSS. We have derived, through extensive consensus comparison, a more detailed description of the RSS than has previously been reported. Our analysis indicates that RSS spacers possess significant conservation of sequence, and that the conserved sequence in 12 bp spacers is similar to the conserved sequence in the first half of 23 bp spacers. PMID:8208601

  9. A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication

    PubMed Central

    Gardiner, Timothy J.; Christov, Christo P.; Langley, Alexander R.; Krude, Torsten

    2009-01-01

    Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication. PMID:19474146

  10. Structural assessment of glycyl mutations in invariantly conserved motifs.

    PubMed

    Prakash, Tulika; Sandhu, Kuljeet Singh; Singh, Nitin Kumar; Bhasin, Yasha; Ramakrishnan, C; Brahmachari, Samir K

    2007-11-15

    Motifs that are evolutionarily conserved in proteins are crucial to their structure and function. In one of our earlier studies, we demonstrated that the conserved motifs occurring invariantly across several organisms could act as structural determinants of the proteins. We observed the abundance of glycyl residues in these invariantly conserved motifs. The role of glycyl residues in highly conserved motifs has not been studied extensively. Thus, it would be interesting to examine the structural perturbations induced by mutation in these conserved glycyl sites. In this work, we selected a representative set of invariant signature (IS) peptides for which both the PDB structure and mutation information was available. We thoroughly analyzed the conformational features of the glycyl sites and their local interactions with the surrounding residues. Using Ramachandran angles, we showed that the glycyl residues occurring in these IS peptides, which have undergone mutation, occurred more often in the L-disallowed as compared with the L-allowed region of the Ramachandran plot. Short range contacts around the mutation site were analyzed to study the steric effects. With the results obtained from our analysis, we hypothesize that any change of activity arising because of such mutations must be attributed to the long-range interaction(s) of the new residue if the glycyl residue in the IS peptide occurred in the L-allowed region of the Ramachandran plot. However, the mutation of those conserved glycyl residues that occurred in the L-disallowed region of the Ramachandran plot might lead to an altered activity of the protein as a result of an altered conformation of the backbone in the immediate vicinity of the glycyl residue, in addition to long range effects arising from the long side chains of the new residue. Thus, the loss of activity because of mutation in the conserved glycyl site might either relate to long range interactions or to local perturbations around the site

  11. [Conserved motifs in the primary and secondary ITS1 structures in bryophytes].

    PubMed

    Milyutina, I A; Ignatov, M S

    2015-01-01

    A study of the ITS1 nucleotide sequences of 1000 moss species of 62 families, 11 liverwort species from five orders, and one hornwort Anthoceros agrestis identified five highly conserved motifs (CM1-CM5), which are presumably involved in pre-rRNA processing. Although the ITS1 sequences substantially differ in length and the extent of divergence, the conserved motifs are found in all of them. ITS1 secondary structures were constructed for 76 mosses, and main regularities at conserved motif positioning were observed. The positions of processing sites in the ITS1 secondary structure of the yeast Saccharomyces cerevisiae were found to be similar to the positions of the conserved motifs in the ITS1 secondary structures of mosses and liverworts. In addition, a potential hairpin formation in the putative secondary structure of a pre-rRNA fragment was considered for the region between ITS1 CM4-CM5 and a highly conserved region between hairpins 49 and 50 (H49 and H50) of the 18S rRNA.

  12. AliBiMotif: integrating alignment and biclustering to unravel transcription factor binding sites in DNA sequences.

    PubMed

    Gonçalves, Joana P; Moreau, Yves; Madeira, Sara C

    2012-01-01

    Transcription Factors (TFs) control transcription by binding to specific sites in the promoter regions of the target genes, which can be modelled by structured motifs. In this paper we propose AliBiMotif, a method combining sequence alignment and a biclustering approach based on efficient string matching techniques using suffix trees to unravel approximately conserved sets of blocks (structured motifs) while straightforwardly disregarding non-conserved stretches in-between. The ability to ignore the width of non-conserved regions is a major advantage of the proposed method over other motif finders, as the lengths of the binding sites are usually easier to estimate than the separating distances.

  13. Dipeptide frequency/bias analysis identifies conserved sites of nonrandomness shared by cysteine-rich motifs.

    PubMed

    Campion, S R; Ameen, A S; Lai, L; King, J M; Munzenmaier, T N

    2001-08-15

    This report describes the application of a simple computational tool, AAPAIR.TAB, for the systematic analysis of the cysteine-rich EGF, Sushi, and Laminin motif/sequence families at the two-amino acid level. Automated dipeptide frequency/bias analysis detects preferences in the distribution of amino acids in established protein families, by determining which "ordered dipeptides" occur most frequently in comprehensive motif-specific sequence data sets. Graphic display of the dipeptide frequency/bias data revealed family-specific preferences for certain dipeptides, but more importantly detected a shared preference for employment of the ordered dipeptides Gly-Tyr (GY) and Gly-Phe (GF) in all three protein families. The dipeptide Asn-Gly (NG) also exhibited high-frequency and bias in the EGF and Sushi motif families, whereas Asn-Thr (NT) was distinguished in the Laminin family. Evaluation of the distribution of dipeptides identified by frequency/bias analysis subsequently revealed the highly restricted localization of the G(F/Y) and N(G/T) sequence elements at two separate sites of extreme conservation in the consensus sequence of all three sequence families. The similar employment of the high-frequency/bias dipeptides in three distinct protein sequence families was further correlated with the concurrence of these shared molecular determinants at similar positions within the distinctive scaffolds of three structurally divergent, but similarly employed, motif modules.

  14. Fast and Accurate Discovery of Degenerate Linear Motifs in Protein Sequences

    PubMed Central

    Levy, Emmanuel D.; Michnick, Stephen W.

    2014-01-01

    Linear motifs mediate a wide variety of cellular functions, which makes their characterization in protein sequences crucial to understanding cellular systems. However, the short length and degenerate nature of linear motifs make their discovery a difficult problem. Here, we introduce MotifHound, an algorithm particularly suited for the discovery of small and degenerate linear motifs. MotifHound performs an exact and exhaustive enumeration of all motifs present in proteins of interest, including all of their degenerate forms, and scores the overrepresentation of each motif based on its occurrence in proteins of interest relative to a background (e.g., proteome) using the hypergeometric distribution. To assess MotifHound, we benchmarked it together with state-of-the-art algorithms. The benchmark consists of 11,880 sets of proteins from S. cerevisiae; in each set, we artificially spiked-in one motif varying in terms of three key parameters, (i) number of occurrences, (ii) length and (iii) the number of degenerate or “wildcard” positions. The benchmark enabled the evaluation of the impact of these three properties on the performance of the different algorithms. The results showed that MotifHound and SLiMFinder were the most accurate in detecting degenerate linear motifs. Interestingly, MotifHound was 15 to 20 times faster at comparable accuracy and performed best in the discovery of highly degenerate motifs. We complemented the benchmark by an analysis of proteins experimentally shown to bind the FUS1 SH3 domain from S. cerevisiae. Using the full-length protein partners as sole information, MotifHound recapitulated most experimentally determined motifs binding to the FUS1 SH3 domain. Moreover, these motifs exhibited properties typical of SH3 binding peptides, e.g., high intrinsic disorder and evolutionary conservation, despite the fact that none of these properties were used as prior information. MotifHound is available (http://michnick.bcm.umontreal.ca or http

  15. Predicting candidate genomic sequences that correspond to synthetic functional RNA motifs

    PubMed Central

    Laserson, Uri; Gan, Hin Hark; Schlick, Tamar

    2005-01-01

    Riboswitches and RNA interference are important emerging mechanisms found in many organisms to control gene expression. To enhance our understanding of such RNA roles, finding small regulatory motifs in genomes presents a challenge on a wide scale. Many simple functional RNA motifs have been found by in vitro selection experiments, which produce synthetic target-binding aptamers as well as catalytic RNAs, including the hammerhead ribozyme. Motivated by the prediction of Piganeau and Schroeder [(2003) Chem. Biol., 10, 103–104] that synthetic RNAs may have natural counterparts, we develop and apply an efficient computational protocol for identifying aptamer-like motifs in genomes. We define motifs from the sequence and structural information of synthetic aptamers, search for sequences in genomes that will produce motif matches, and then evaluate the structural stability and statistical significance of the potential hits. Our application to aptamers for streptomycin, chloramphenicol, neomycin B and ATP identifies 37 candidate sequences (in coding and non-coding regions) that fold to the target aptamer structures in bacterial and archaeal genomes. Further energetic screening reveals that several candidates exhibit energetic properties and sequence conservation patterns that are characteristic of functional motifs. Besides providing candidates for experimental testing, our computational protocol offers an avenue for expanding natural RNA's functional repertoire. PMID:16254081

  16. GOmotif: A web server for investigating the biological role of protein sequence motifs

    PubMed Central

    2011-01-01

    Background Many proteins contain conserved sequence patterns (motifs) that contribute to their functionality. The process of experimentally identifying and validating novel protein motifs can be difficult, expensive, and time consuming. A means for helping to identify in advance the possible function of a novel motif is important to test hypotheses concerning the biological relevance of these motifs, thus reducing experimental trial-and-error. Results GOmotif accepts PROSITE and regular expression formatted motifs as input and searches a Gene Ontology annotated protein database using motif search tools. The search returns the set of proteins containing matching motifs and their associated Gene Ontology terms. These results are presented as: 1) a hierarchical, navigable tree separated into the three Gene Ontology biological domains - biological process, cellular component, and molecular function; 2) corresponding pie charts indicating raw and statistically adjusted distributions of the results, and 3) an interactive graphical network view depicting the location of the results in the Gene Ontology. Conclusions GOmotif is a web-based tool designed to assist researchers in investigating the biological role of novel protein motifs. GOmotif can be freely accessed at http://www.gomotif.ca PMID:21943350

  17. A conserved heptamer motif for ribosomal RNA transcription termination in animal mitochondria.

    PubMed Central

    Valverde, J R; Marco, R; Garesse, R

    1994-01-01

    A search of sequence data bases for a tridecamer transcription termination signal, previously described in human mtDNA as being responsible for the accumulation of mitochondrial ribosomal RNAs (rRNAs) in excess over the rest of mitochondrial genes, has revealed that this termination signal occurs in equivalent positions in a wide variety of organisms from protozoa to mammals. Due to the compact organization of the mtDNA, the tridecamer motif usually appears as part of the 3' adjacent gene sequence. Because in phylogenetically widely separated organisms the mitochondrial genome has experienced many rearrangements, it is interesting that its occurrence near the 3' end of the large rRNA is independent of the adjacent gene. The tridecamer sequence has diverged in phylogenetically widely separated organisms. Nevertheless, a well-conserved heptamer--TGGCAGA, the mitochondrial rRNA termination box--can be defined. Although extending the experimental evidence of its role as a transcription termination signal in humans will be of great interest, its evolutionary conservation strongly suggests that mitochondrial rRNA transcription termination could be a widely conserved mechanism in animals. Furthermore, the conservation of a homologous tridecamer motif in one of the last 3' secondary loops of nonmitochondrial 23S-like rRNAs suggests that the role of the sequence has changed during mitochondrial evolution. PMID:7515499

  18. The distribution of RNA motifs in natural sequences.

    PubMed

    Bourdeau, V; Ferbeyre, G; Pageau, M; Paquin, B; Cedergren, R

    1999-11-15

    Functional analysis of genome sequences has largely ignored RNA genes and their structures. We introduce here the notion of 'ribonomics' to describe the search for the distribution of and eventually the determination of the physiological roles of these RNA structures found in the sequence databases. The utility of this approach is illustrated here by the identification in the GenBank database of RNA motifs having known binding or chemical activity. The frequency of these motifs indicates that most have originated from evolutionary drift and are selectively neutral. On the other hand, their distribution among species and their location within genes suggest that the destiny of these motifs may be more elaborate. For example, the hammerhead motif has a skewed organismal presence, is phylogenetically stable and recent work on a schistosome version confirms its in vivo biological activity. The under-representation of the valine-binding motif and the Rev-binding element in GenBank hints at a detrimental effect on cell growth or viability. Data on the presence and the location of these motifs may provide critical guidance in the design of experiments directed towards the understanding and the manipulation of RNA complexes and activities in vivo.

  19. Classification of protein motifs based on subcellular localization uncovers evolutionary relationships at both sequence and functional levels

    PubMed Central

    2013-01-01

    Background Most proteins have evolved in specific cellular compartments that limit their functions and potential interactions. On the other hand, motifs define amino acid arrangements conserved between protein family members and represent powerful tools for assigning function to protein sequences. The ideal motif would identify all members of a protein family but in practice many motifs identify both family members and unrelated proteins, referred to as True Positive (TP) and False Positive (FP) sequences, respectively. Results To address the relationship between protein motifs, protein function and cellular localization, we systematically assigned subcellular localization data to motif sequences from the comprehensive PROSITE sequence motif database. Using this data we analyzed relationships between localization and function. We find that TPs and FPs have a strong tendency to localize in different compartments. When multiple localizations are considered, TPs are usually distributed between related cellular compartments. We also identified cases where FPs are concentrated in particular subcellular regions, indicating possible functional or evolutionary relationships with TP sequences of the same motif. Conclusions Our findings suggest that the systematic examination of subcellular localization has the potential to uncover evolutionary and functional relationships between motif-containing sequences. We believe that this type of analysis complements existing motif annotations and could aid in their interpretation. Our results shed light on the evolution of cellular organelles and potentially establish the basis for new subcellular localization and function prediction algorithms. PMID:23865897

  20. Chaotic motif sampler: detecting motifs from biological sequences by using chaotic neurodynamics

    NASA Astrophysics Data System (ADS)

    Matsuura, Takafumi; Ikeguchi, Tohru

    Identification of a region in biological sequences, motif extraction problem (MEP) is solved in bioinformatics. However, the MEP is an NP-hard problem. Therefore, it is almost impossible to obtain an optimal solution within a reasonable time frame. To find near optimal solutions for NP-hard combinatorial optimization problems such as traveling salesman problems, quadratic assignment problems, and vehicle routing problems, chaotic search, which is one of the deterministic approaches, has been proposed and exhibits better performance than stochastic approaches. In this paper, we propose a new alignment method that employs chaotic dynamics to solve the MEPs. It is called the Chaotic Motif Sampler. We show that the performance of the Chaotic Motif Sampler is considerably better than that of the conventional methods such as the Gibbs Site Sampler and the Neighborhood Optimization for Multiple Alignment Discovery.

  1. An evolutionary analysis of flightin reveals a conserved motif unique and widespread in Pancrustacea.

    PubMed

    Soto-Adames, Felipe N; Alvarez-Ortiz, Pedro; Vigoreaux, Jim O

    2014-01-01

    Flightin is a thick filament protein that in Drosophila melanogaster is uniquely expressed in the asynchronous, indirect flight muscles (IFM). Flightin is required for the structure and function of the IFM and is indispensable for flight in Drosophila. Given the importance of flight acquisition in the evolutionary history of insects, here we study the phylogeny and distribution of flightin. Flightin was identified in 69 species of hexapods in classes Collembola (springtails), Protura, Diplura, and insect orders Thysanura (silverfish), Dictyoptera (roaches), Orthoptera (grasshoppers), Pthiraptera (lice), Hemiptera (true bugs), Coleoptera (beetles), Neuroptera (green lacewing), Hymenoptera (bees, ants, and wasps), Lepidoptera (moths), and Diptera (flies and mosquitoes). Flightin was also found in 14 species of crustaceans in orders Anostraca (water flea), Cladocera (brine shrimp), Isopoda (pill bugs), Amphipoda (scuds, sideswimmers), and Decapoda (lobsters, crabs, and shrimps). Flightin was not identified in representatives of chelicerates, myriapods, or any species outside Pancrustacea (Tetraconata, sensu Dohle). Alignment of amino acid sequences revealed a conserved region of 52 amino acids, referred herein as WYR, that is bound by strictly conserved tryptophan (W) and arginine (R) and an intervening sequence with a high content of tyrosines (Y). This motif has no homologs in GenBank or PROSITE and is unique to flightin and paraflightin, a putative flightin paralog identified in decapods. A third motif of unclear affinities to pancrustacean WYR was observed in chelicerates. Phylogenetic analysis of amino acid sequences of the conserved motif suggests that paraflightin originated before the divergence of amphipods, isopods, and decapods. We conclude that flightin originated de novo in the ancestor of Pancrustacea > 500 MYA, well before the divergence of insects (~400 MYA) and the origin of flight (~325 MYA), and that its IFM-specific function in Drosophila is a more

  2. A generalized profile syntax for biomolecular sequence motifs and its function in automatic sequence interpretation

    SciTech Connect

    Bucher, P.; Bairoch, A.

    1994-12-31

    A general syntax for expressing bimolecular sequence motifs is described, which will be used in future releases of the PROSITE data bank and in a similar collection of nucleic acid sequence motifs currently under development. The central part of the syntax is a regular structure which can be viewed as a generalization of the profiles introduced by Gribskov and coworkers. Accessory features implement specific motif search strategies and provide information helpful for the interpretation of predicted matches. Two contrasting examples, representing E. coli promoters and SH3 domains respectively, are shown to demonstrate the versatility of the syntax, and its compatibility with diverse motif search methods. It is argued, that a comprehensive machine-readable motif collection based on the new syntax, in conjunction with a standard search program, can serve as a general-purpose sequence interpretation and function prediction tool.

  3. Computational definition of sequence motifs governing constitutive exon splicing.

    PubMed

    Zhang, Xiang H-F; Chasin, Lawrence A

    2004-06-01

    We have searched for sequence motifs that contribute to the recognition of human pre-mRNA splice sites by comparing the frequency of 8-mers in internal noncoding exons versus unspliced pseudo exons and 5' untranslated regions (5' untranslated regions [UTRs]) of transcripts of intronless genes. This type of comparison avoids the isolation of sequences that are distinguished by their protein-coding information. We classified sequence families comprising 2069 putative exonic enhancers and 974 putative exonic silencers. Representatives of each class functioned as enhancers or silencers when inserted into a test exon and assayed in transfected mammalian cells. As a class, the enhancer sequencers were more prevalent and the silencer elements less prevalent in all exons compared with introns. A survey of 58 reported exonic splicing mutations showed good agreement between the splicing phenotype and the effect of the mutation on the motifs defined here. The large number of effective sequences implied by these results suggests that sequences that influence splicing may be very abundant in pre-mRNA.

  4. Cloning, expression and functional characterization of the putative regeneration and tolerance factor (RTF/TJ6) as a functional vacuolar ATPase proton pump regulatory subunit with a conserved sequence of immunoreceptor tyrosine-based activation motif.

    PubMed

    Babichev, Yael; Tamir, Ami; Park, Meeyoug; Muallem, Shmuel; Isakov, Noah

    2005-10-01

    In an attempt to identify new immunoreceptor tyrosine-based activation motif (ITAM)-containing human molecules that may regulate hitherto unknown immune cell functions, we BLAST searched the National Center for Biotechnology Information database for ITAM-containing sequences. A human expressed sequence tag showing partial homology to the murine TJ6 (mTJ6) gene and encoding a putative ITAM sequence has been identified and used to clone the human TJ6 (hTJ6) gene from an HL-60-derived cDNA library. hTJ6 was found to encode a protein of 856 residues with a calculated mass of 98 155 Da. Immunolocalization and sequence analysis revealed that hTJ6 is a membrane protein with predicted six transmembrane-spanning regions, typical of ion channels, and a single putative ITAM (residues 452-466) in a juxtamembrane or hydrophobic intramembrane region. hTJ6 is highly homologous to Bos taurus 116-kDa subunit of the vacuolar proton-translocating ATPase. Over-expression of hTJ6 in HEK 293 cells increased H+ uptake into intracellular organelles, an effect that was sensitive to inhibition by bafilomycin, a selective inhibitor of vacuolar H+ pump. Northern blot analysis demonstrated three different hybridizing mRNA transcripts corresponding to 3.2, 5.0 and 7.3 kb, indicating the presence of several splice variants. Significant differences in hTJ6 mRNA levels in human tissues of different origins point to possible tissue-specific function. Although hTJ6 was found to be a poor substrate for tyrosine-phosphorylating enzymes, suggesting that its ITAM sequence is non-functional in protein tyrosine kinase-mediated signaling pathways, its role in organellar H+ pumping suggests that hTJ6 function may participate in protein trafficking/processing.

  5. Sequence-motif Detection of NAD(P)-binding Proteins: Discovery of a Unique Antibacterial Drug Target

    NASA Astrophysics Data System (ADS)

    Hua, Yun Hao; Wu, Chih Yuan; Sargsyan, Karen; Lim, Carmay

    2014-09-01

    Many enzymes use nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate (NAD(P)) as essential coenzymes. These enzymes often do not share significant sequence identity and cannot be easily detected by sequence homology. Previously, we determined all distinct locally conserved pyrophosphate-binding structures (3d motifs) from NAD(P)-bound protein structures, from which 1d sequence motifs were derived. Here, we aim to establish the precision of these 3d and 1d motifs to annotate NAD(P)-binding proteins. We show that the pyrophosphate-binding 3d motifs are characteristic of NAD(P)-binding proteins, as they are rarely found in nonNAD(P)-binding proteins. Furthermore, several 1d motifs could distinguish between proteins that bind only NAD and those that bind only NADP. They could also distinguish between NAD(P)-binding proteins from nonNAD(P)-binding ones. Interestingly, one of the pyrophosphate-binding 3d and corresponding 1d motifs was found only in enoyl-acyl carrier protein reductases, which are enzymes essential for bacterial fatty acid biosynthesis. This unique 3d motif serves as an attractive novel drug target, as it is conserved across many bacterial species and is not found in human proteins.

  6. Conserved motifs in prokaryotic and eukaryotic polypeptide release factors: tRNA-protein mimicry hypothesis.

    PubMed Central

    Ito, K; Ebihara, K; Uno, M; Nakamura, Y

    1996-01-01

    Translation termination requires two codon-specific polypeptide release factors in prokaryotes and one omnipotent factor in eukaryotes. Sequences of 17 different polypeptide release factors from prokaryotes and eukaryotes were compared. The prokaryotic release factors share residues split into seven motifs. Conservation of many discrete, perhaps critical, amino acids is observed in eukaryotic release factors, as well as in the C-terminal portion of elongation factor (EF) G. Given that the C-terminal domains of EF-G interacts with ribosomes by mimicry of a tRNA structure, the pattern of conservation of residues in release factors may reflect requirements for a tRNA-mimicry for binding to the A site of the ribosome. This mimicry would explain why release factors recognize stop codons and suggests that all prokaryotic and eukaryotic release factors evolved from the progenitor of EF-G. Images Fig. 2 Fig. 3 PMID:8643594

  7. Nucleotide binding database NBDB – a collection of sequence motifs with specific protein-ligand interactions

    PubMed Central

    Zheng, Zejun; Goncearenco, Alexander; Berezovsky, Igor N.

    2016-01-01

    NBDB database describes protein motifs, elementary functional loops (EFLs) that are involved in binding of nucleotide-containing ligands and other biologically relevant cofactors/coenzymes, including ATP, AMP, ATP, GMP, GDP, GTP, CTP, PAP, PPS, FMN, FAD(H), NAD(H), NADP, cAMP, cGMP, c-di-AMP and c-di-GMP, ThPP, THD, F-420, ACO, CoA, PLP and SAM. The database is freely available online at http://nbdb.bii.a-star.edu.sg. In total, NBDB contains data on 249 motifs that work in interactions with 24 ligands. Sequence profiles of EFL motifs were derived de novo from nonredundant Uniprot proteome sequences. Conserved amino acid residues in the profiles interact specifically with distinct chemical parts of nucleotide-containing ligands, such as nitrogenous bases, phosphate groups, ribose, nicotinamide, and flavin moieties. Each EFL profile in the database is characterized by a pattern of corresponding ligand–protein interactions found in crystallized ligand–protein complexes. NBDB database helps to explore the determinants of nucleotide and cofactor binding in different protein folds and families. NBDB can also detect fragments that match to profiles of particular EFLs in the protein sequence provided by user. Comprehensive information on sequence, structures, and interactions of EFLs with ligands provides a foundation for experimental and computational efforts on design of required protein functions. PMID:26507856

  8. A novel cysteine-rich sequence-specific DNA-binding protein interacts with the conserved X-box motif of the human major histocompatibility complex class II genes via a repeated Cys-His domain and functions as a transcriptional repressor

    PubMed Central

    1994-01-01

    The class II major histocompatibility complex (MHC) molecules function in the presentation of processed peptides to helper T cells. As most mammalian cells can endocytose and process foreign antigen, the critical determinant of an antigen-presenting cell is its ability to express class II MHC molecules. Expression of these molecules is usually restricted to cells of the immune system and dysregulated expression is hypothesized to contribute to the pathogenesis of a severe combined immunodeficiency syndrome and certain autoimmune diseases. Human complementary DNA clones encoding a newly identified, cysteine-rich transcription factor, NF-X1, which binds to the conserved X-box motif of class II MHC genes, were obtained, and the primary amino acid sequence deduced. The major open reading frame encodes a polypeptide of 1,104 amino acids with a symmetrical organization. A central cysteine-rich portion encodes the DNA-binding domain, and is subdivided into seven repeated motifs. This motif is similar to but distinct from the LIM domain and the RING finger family, and is reminiscent of known metal-binding regions. The unique arrangement of cysteines indicates that the consensus sequence CX3CXL-XCGX1- 5HXCX3CHXGXC represents a novel cysteine-rich motif. Two lines of evidence indicate that the polypeptide encodes a potent and biologically relevant repressor of HLA-DRA transcription: (a) overexpression of NF-X1 from a retroviral construct strongly decreases transcription from the HLA-DRA promoter; and (b) the NF-X1 transcript is markedly induced late after induction with interferon gamma (IFN- gamma), coinciding with postinduction attenuation of HLA-DRA transcription. The NF-X1 protein may therefore play an important role in regulating the duration of an inflammatory response by limiting the period in which class II MHC molecules are induced by IFN-gamma. PMID:7964459

  9. Phosphotyrosine Substrate Sequence Motifs for Dual Specificity Phosphatases

    PubMed Central

    Zhao, Bryan M.; Keasey, Sarah L.; Tropea, Joseph E.; Lountos, George T.; Dyas, Beverly K.; Cherry, Scott; Raran-Kurussi, Sreejith; Waugh, David S.; Ulrich, Robert G.

    2015-01-01

    Protein tyrosine phosphatases dephosphorylate tyrosine residues of proteins, whereas, dual specificity phosphatases (DUSPs) are a subgroup of protein tyrosine phosphatases that dephosphorylate not only Tyr(P) residue, but also the Ser(P) and Thr(P) residues of proteins. The DUSPs are linked to the regulation of many cellular functions and signaling pathways. Though many cellular targets of DUSPs are known, the relationship between catalytic activity and substrate specificity is poorly defined. We investigated the interactions of peptide substrates with select DUSPs of four types: MAP kinases (DUSP1 and DUSP7), atypical (DUSP3, DUSP14, DUSP22 and DUSP27), viral (variola VH1), and Cdc25 (A-C). Phosphatase recognition sites were experimentally determined by measuring dephosphorylation of 6,218 microarrayed Tyr(P) peptides representing confirmed and theoretical phosphorylation motifs from the cellular proteome. A broad continuum of dephosphorylation was observed across the microarrayed peptide substrates for all phosphatases, suggesting a complex relationship between substrate sequence recognition and optimal activity. Further analysis of peptide dephosphorylation by hierarchical clustering indicated that DUSPs could be organized by substrate sequence motifs, and peptide-specificities by phylogenetic relationships among the catalytic domains. The most highly dephosphorylated peptides represented proteins from 29 cell-signaling pathways, greatly expanding the list of potential targets of DUSPs. These newly identified DUSP substrates will be important for examining structure-activity relationships with physiologically relevant targets. PMID:26302245

  10. cisExpress: motif detection in DNA sequences

    PubMed Central

    Triska, Martin; Grocutt, David; Southern, James; Murphy, Denis J.; Tatarinova, Tatiana

    2013-01-01

    Motivation: One of the major challenges for contemporary bioinformatics is the analysis and accurate annotation of genomic datasets to enable extraction of useful information about the functional role of DNA sequences. This article describes a novel genome-wide statistical approach to the detection of specific DNA sequence motifs based on similarities between the promoters of similarly expressed genes. This new tool, cisExpress, is especially designed for use with large datasets, such as those generated by publicly accessible whole genome and transcriptome projects. cisExpress uses a task farming algorithm to exploit all available computational cores within a shared memory node. We demonstrate the robust nature and validity of the proposed method. It is applicable for use with a wide range of genomic databases for any species of interest. Availability: cisExpress is available at www.cisexpress.org. Contact: tatiana.tatarinova@usc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23793750

  11. Housekeeping genes tend to show reduced upstream sequence conservation

    PubMed Central

    Farré, Domènec; Bellora, Nicolás; Mularoni, Loris; Messeguer, Xavier; Albà, M Mar

    2007-01-01

    Background Understanding the constraints that operate in mammalian gene promoter sequences is of key importance to understand the evolution of gene regulatory networks. The level of promoter conservation varies greatly across orthologous genes, denoting differences in the strength of the evolutionary constraints. Here we test the hypothesis that the number of tissues in which a gene is expressed is related in a significant manner to the extent of promoter sequence conservation. Results We show that mammalian housekeeping genes, expressed in all or nearly all tissues, show significantly lower promoter sequence conservation, especially upstream of position -500 with respect to the transcription start site, than genes expressed in a subset of tissues. In addition, we evaluate the effect of gene function, CpG island content and protein evolutionary rate on promoter sequence conservation. Finally, we identify a subset of transcription factors that bind to motifs that are specifically over-represented in housekeeping gene promoters. Conclusion This is the first report that shows that the promoters of housekeeping genes show reduced sequence conservation with respect to genes expressed in a more tissue-restricted manner. This is likely to be related to simpler gene expression, requiring a smaller number of functional cis-regulatory motifs. PMID:17626644

  12. QGRS-H Predictor: a web server for predicting homologous quadruplex forming G-rich sequence motifs in nucleotide sequences

    PubMed Central

    Menendez, Camille; Frees, Scott; Bagga, Paramjeet S.

    2012-01-01

    Naturally occurring G-quadruplex structural motifs, formed by guanine-rich nucleic acids, have been reported in telomeric, promoter and transcribed regions of mammalian genomes. G-quadruplex structures have received significant attention because of growing evidence for their role in important biological processes, human disease and as therapeutic targets. Lately, there has been much interest in the potential roles of RNA G-quadruplexes as cis-regulatory elements of post-transcriptional gene expression. Large-scale computational genomics studies on G-quadruplexes have difficulty validating their predictions without laborious testing in ‘wet’ labs. We have developed a bioinformatics tool, QGRS-H Predictor that can map and analyze conserved putative Quadruplex forming 'G'-Rich Sequences (QGRS) in mRNAs, ncRNAs and other nucleotide sequences, e.g. promoter, telomeric and gene flanking regions. Identifying conserved regulatory motifs helps validate computations and enhances accuracy of predictions. The QGRS-H Predictor is particularly useful for mapping homologous G-quadruplex forming sequences as cis-regulatory elements in the context of 5′- and 3′-untranslated regions, and CDS sections of aligned mRNA sequences. QGRS-H Predictor features highly interactive graphic representation of the data. It is a unique and user-friendly application that provides many options for defining and studying G-quadruplexes. The QGRS-H Predictor can be freely accessed at: http://quadruplex.ramapo.edu/qgrs/app/start. PMID:22576365

  13. A conserved motif flags Acyl Carrier Proteins for β-branching in polyketide synthesis

    PubMed Central

    Song, Zhongshu; Farmer, Rohit; Williams, Christopher; Hothersall, Joanne; Płoskoń, Eliza; Wattana-amorn, Pakorn; Stephens, Elton R.; Yamada, Erika; Gurney, Rachel; Takebayashi, Yuiko; Masschelein, Joleen; Cox, Russell J.; Lavigne, Rob; Willis, Christine L.; Simpson, Thomas J.; Crosby, John; Winn, Peter J.; Thomas, Christopher M.; Crump, Matthew P.

    2015-01-01

    Type I PKSs often utilise programmed β-branching, via enzymes of an “HMG-CoA synthase (HCS) cassette”, to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in Acyl Carrier Proteins (ACPs) where β-branching is known. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. While these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modelling and mutagenesis identified ACP Helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality while ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering novel polyketides and lays a basis for determining specificity rules. PMID:24056399

  14. Identification of multiple distinct Snf2 subfamilies with conserved structural motifs

    PubMed Central

    Flaus, Andrew; Martin, David M. A.; Barton, Geoffrey J.; Owen-Hughes, Tom

    2006-01-01

    The Snf2 family of helicase-related proteins includes the catalytic subunits of ATP-dependent chromatin remodelling complexes found in all eukaryotes. These act to regulate the structure and dynamic properties of chromatin and so influence a broad range of nuclear processes. We have exploited progress in genome sequencing to assemble a comprehensive catalogue of over 1300 Snf2 family members. Multiple sequence alignment of the helicase-related regions enables 24 distinct subfamilies to be identified, a considerable expansion over earlier surveys. Where information is known, there is a good correlation between biological or biochemical function and these assignments, suggesting Snf2 family motor domains are tuned for specific tasks. Scanning of complete genomes reveals all eukaryotes contain members of multiple subfamilies, whereas they are less common and not ubiquitous in eubacteria or archaea. The large sample of Snf2 proteins enables additional distinguishing conserved sequence blocks within the helicase-like motor to be identified. The establishment of a phylogeny for Snf2 proteins provides an opportunity to make informed assignments of function, and the identification of conserved motifs provides a framework for understanding the mechanisms by which these proteins function. PMID:16738128

  15. Conserved DNA motifs in the type II-A CRISPR leader region

    PubMed Central

    Babu, Kesavan; Najar, Fares Z.

    2017-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas) systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer) site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3′ end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3′ leader end: ATTTGAG (noted previously in the CRISPR1 locus of Streptococcus thermophilus DGCC7710) and a newly defined CTRCGAG, associated with the CRISPR3 locus of S. thermophilus DGCC7710. A third group with a very short CG DNA conservation at the 3′ leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci. PMID:28392985

  16. Human and mouse introns are linked to the same processes and functions through each genome's most frequent non-conserved motifs.

    PubMed

    Tsirigos, Aristotelis; Rigoutsos, Isidore

    2008-06-01

    We identified the most frequent, variable-length DNA sequence motifs in the human and mouse genomes and sub-selected those with multiple recurrences in the intergenic and intronic regions and at least one additional exonic instance in the corresponding genome. We discovered that these motifs have virtually no overlap with intronic sequences that are conserved between human and mouse, and thus are genome-specific. Moreover, we found that these motifs span a substantial fraction of previously uncharacterized human and mouse intronic space. Surprisingly, we found that these genome-specific motifs are over-represented in the introns of genes belonging to the same biological processes and molecular functions in both the human and mouse genomes even though the underlying sequences are not conserved between the two genomes. In fact, the processes and functions that are linked to these genome-specific sequence-motifs are distinct from the processes and functions which are associated with intronic regions that are conserved between human and mouse. The findings show that intronic regions from different genomes are linked to the same processes and functions in the absence of underlying sequence conservation. We highlight the ramifications of this observation with a concrete example that involves the microsatellite instability gene MLH1.

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

    PubMed

    van Beest, M; Dooijes, D; van De Wetering, M; Kjaerulff, S; Bonvin, A; Nielsen, O; Clevers, H

    2000-09-01

    Sequence-specific high mobility group (HMG) box factors bind and bend DNA via interactions in the minor groove. Three-dimensional NMR analyses have provided the structural basis for this interaction. The cognate HMG domain DNA motif is generally believed to span 6-8 bases. However, alignment of promoter elements controlled by the yeast genes ste11 and Rox1 has indicated strict conservation of a larger DNA motif. By site selection, we identify a highly specific 12-base pair motif for Ste11, AGAACAAAGAAA. Similarly, we show that Tcf1, MatMc, and Sox4 bind unique, highly specific DNA motifs of 12, 12, and 10 base pairs, respectively. Footprinting with a deletion mutant of Ste11 reveals a novel interaction between the 3' base pairs of the extended DNA motif and amino acids C-terminal to the HMG domain. The sequence-specific interaction of Ste11 with these 3' base pairs contributes significantly to binding and bending of the DNA motif.

  18. Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

    PubMed Central

    del Val, Coral; White, Stephen H.

    2014-01-01

    We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide. PMID:22836667

  19. Comprehensive analysis of animal TALE homeobox genes: new conserved motifs and cases of accelerated evolution.

    PubMed

    Mukherjee, Krishanu; Bürglin, Thomas R

    2007-08-01

    TALE homeodomain proteins are an ancient subgroup within the group of homeodomain transcription factors that play important roles in animal, plant, and fungal development. We have extracted the full complement of TALE superclass homeobox genes from the genome projects of seven protostomes, seven deuterostomes, and Nematostella. This was supplemented with TALE homeobox genes from additional species and phylogenetic analyses were carried out with 276 sequences. We found 20 homeobox genes and 4 pseudogenes in humans, 21 genes in mouse, 8 genes in Drosophila, and 5 genes plus one truncated gene in Caenorhabditis elegans. Apart from the previously identified TALE classes MEIS, PBC, IRO, and TGIF, a novel class is identified, termed MOHAWK (MKX). Further, we show that the MEIS class can be divided into two families, PREP and MEIS. Prep genes have previously only been described in vertebrates but are lacking in Drosophila. Here we identify orthologues in other insect taxa as well as in the cnidarian Nematostella. In C. elegans, a divergent Prep protein has lost the homeodomain. Full-length multiple sequence alignment of the protostome and deuterostome sequences allowed us to identify several novel conserved motifs within the MKX, TGIF, and MEIS classes. Phylogenetic analyses revealed fast-evolving PBC class genes; in particular, some X-linked PBC genes in nematodes are subject to rapid evolution. In addition, several instances of gene loss were identified. In conclusion, our comprehensive analysis provides a defining framework for the classification of animal TALE homeobox genes and the understanding of their evolution.

  20. RNA polymerase II senses obstruction in the DNA minor groove via a conserved sensor motif

    PubMed Central

    Xu, Liang; Wang, Wei; Gotte, Deanna; Yang, Fei; Hare, Alissa A.; Welch, Timothy R.; Li, Benjamin C.; Shin, Ji Hyun; Chong, Jenny; Strathern, Jeffrey N.; Dervan, Peter B.; Wang, Dong

    2016-01-01

    RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation. However, the mechanism of pol II inhibition by Py-Im polyamides is unclear. Here we investigate the mechanism of how these minor-groove binders affect pol II transcription elongation. In the presence of site-specifically bound Py-Im polyamides, we find that the pol II elongation complex becomes arrested immediately upstream of the targeted DNA sequence, and is not rescued by transcription factor IIS, which is in contrast to pol II blockage by a nucleosome barrier. Further analysis reveals that two conserved pol II residues in the Switch 1 region contribute to pol II stalling. Our study suggests this motif in pol II can sense the structural changes of the DNA minor groove and can be considered a “minor groove sensor.” Prolonged interference of transcription elongation by sequence-specific minor groove binders may present opportunities to target transcription addiction for cancer therapy. PMID:27791148

  1. Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene

    PubMed Central

    Van den Hoecke, Silvie; Verhelst, Judith; Saelens, Xavier

    2016-01-01

    Green fluorescent protein (GFP) is one of the most used reporter genes. We have used next-generation sequencing (NGS) to analyse the genetic diversity of a recombinant influenza A virus that expresses GFP and found a remarkable coverage dip in the GFP coding sequence. This coverage dip was present when virus-derived RT-PCR product or the parental plasmid DNA was used as starting material for NGS and regardless of whether Nextera XT transposase or Covaris shearing was used for DNA fragmentation. Therefore, the sequence coverage dip in the GFP coding sequence was not the result of emerging GFP mutant viruses or a bias introduced by Nextera XT fragmentation. Instead, we found that the Illumina MiSeq sequencing method disfavours the ‘CCCGCC’ motif in the GFP coding sequence. PMID:27193250

  2. Exhaustive Search for Over-represented DNA Sequence Motifs with CisFinder

    PubMed Central

    Sharov, Alexei A.; Ko, Minoru S.H.

    2009-01-01

    We present CisFinder software, which generates a comprehensive list of motifs enriched in a set of DNA sequences and describes them with position frequency matrices (PFMs). A new algorithm was designed to estimate PFMs directly from counts of n-mer words with and without gaps; then PFMs are extended over gaps and flanking regions and clustered to generate non-redundant sets of motifs. The algorithm successfully identified binding motifs for 12 transcription factors (TFs) in embryonic stem cells based on published chromatin immunoprecipitation sequencing data. Furthermore, CisFinder successfully identified alternative binding motifs of TFs (e.g. POU5F1, ESRRB, and CTCF) and motifs for known and unknown co-factors of genes associated with the pluripotent state of ES cells. CisFinder also showed robust performance in the identification of motifs that were only slightly enriched in a set of DNA sequences. PMID:19740934

  3. Evolutionarily divergent spliceosomal snRNAs and a conserved non-coding RNA processing motif in Giardia lamblia.

    PubMed

    Hudson, Andrew J; Moore, Ashley N; Elniski, David; Joseph, Joella; Yee, Janet; Russell, Anthony G

    2012-11-01

    Non-coding RNAs (ncRNAs) have diverse essential biological functions in all organisms, and in eukaryotes, two such classes of ncRNAs are the small nucleolar (sno) and small nuclear (sn) RNAs. In this study, we have identified and characterized a collection of sno and snRNAs in Giardia lamblia, by exploiting our discovery of a conserved 12 nt RNA processing sequence motif found in the 3' end regions of a large number of G. lamblia ncRNA genes. RNA end mapping and other experiments indicate the motif serves to mediate ncRNA 3' end formation from mono- and di-cistronic RNA precursor transcripts. Remarkably, we find the motif is also utilized in the processing pathway of all four previously identified trans-spliced G. lamblia introns, revealing a common RNA processing pathway for ncRNAs and trans-spliced introns in this organism. Motif sequence conservation then allowed for the bioinformatic and experimental identification of additional G. lamblia ncRNAs, including new U1 and U6 spliceosomal snRNA candidates. The U6 snRNA candidate was then used as a tool to identity novel U2 and U4 snRNAs, based on predicted phylogenetically conserved snRNA-snRNA base-pairing interactions, from a set of previously identified G. lamblia ncRNAs without assigned function. The Giardia snRNAs retain the core features of spliceosomal snRNAs but are sufficiently evolutionarily divergent to explain the difficulties in their identification. Most intriguingly, all of these snRNAs show structural features diagnostic of U2-dependent/major and U12-dependent/minor spliceosomal snRNAs.

  4. Sequence motifs and prokaryotic expression of the reptilian paramyxovirus fusion protein

    USGS Publications Warehouse

    Franke, J.; Batts, W.N.; Ahne, W.; Kurath, G.; Winton, J.R.

    2006-01-01

    Fourteen reptilian paramyxovirus isolates were chosen to represent the known extent of genetic diversity among this novel group of viruses. Selected regions of the fusion (F) gene were sequenced, analyzed and compared. The F gene of all isolates contained conserved motifs homologous to those described for other members of the family Paramyxoviridae including: signal peptide, transmembrane domain, furin cleavage site, fusion peptide, N-linked glycosylation sites, and two heptad repeats, the second of which (HRB-LZ) had the characteristics of a leucine zipper. Selected regions of the fusion gene of isolate Gono-GER85 were inserted into a prokaryotic expression system to generate three recombinant protein fragments of various sizes. The longest recombinant protein was cleaved by furin into two fragments of predicted length. Western blot analysis with virus-neutralizing rabbit-antiserum against this isolate demonstrated that only the longest construct reacted with the antiserum. This construct was unique in containing 30 additional C-terminal amino acids that included most of the HRB-LZ. These results indicate that the F genes of reptilian paramyxoviruses contain highly conserved motifs typical of other members of the family and suggest that the HRB-LZ domain of the reptilian paramyxovirus F protein contains a linear antigenic epitope. ?? Springer-Verlag 2005.

  5. A conserved motif mediates both multimer formation and allosteric activation of phosphoglycerate mutase 5.

    PubMed

    Wilkins, Jordan M; McConnell, Cyrus; Tipton, Peter A; Hannink, Mark

    2014-09-05

    Phosphoglycerate mutase 5 (PGAM5) is an atypical mitochondrial Ser/Thr phosphatase that modulates mitochondrial dynamics and participates in both apoptotic and necrotic cell death. The mechanisms that regulate the phosphatase activity of PGAM5 are poorly understood. The C-terminal phosphoglycerate mutase domain of PGAM5 shares homology with the catalytic domains found in other members of the phosphoglycerate mutase family, including a conserved histidine that is absolutely required for catalytic activity. However, this conserved domain is not sufficient for maximal phosphatase activity. We have identified a highly conserved amino acid motif, WDXNWD, located within the unique N-terminal region, which is required for assembly of PGAM5 into large multimeric complexes. Alanine substitutions within the WDXNWD motif abolish the formation of multimeric complexes and markedly reduce phosphatase activity of PGAM5. A peptide containing the WDXNWD motif dissociates the multimeric complex and reduces but does not fully abolish phosphatase activity. Addition of the WDXNWD-containing peptide in trans to a mutant PGAM5 protein lacking the WDXNWD motif markedly increases phosphatase activity of the mutant protein. Our results are consistent with an intermolecular allosteric regulation mechanism for the phosphatase activity of PGAM5, in which the assembly of PGAM5 into multimeric complexes, mediated by the WDXNWD motif, results in maximal activation of phosphatase activity. Our results suggest the possibility of identifying small molecules that function as allosteric regulators of the phosphatase activity of PGAM5.

  6. Conserved amino acid motifs from the novel Piv/MooV family of transposases and site-specific recombinases are required for catalysis of DNA inversion by Piv.

    PubMed

    Tobiason, D M; Buchner, J M; Thiel, W H; Gernert, K M; Karls, A C

    2001-02-01

    Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.

  7. PairMotifChIP: A Fast Algorithm for Discovery of Patterns Conserved in Large ChIP-seq Data Sets

    PubMed Central

    Huo, Hongwei; Feng, Dazheng

    2016-01-01

    Identifying conserved patterns in DNA sequences, namely, motif discovery, is an important and challenging computational task. With hundreds or more sequences contained, the high-throughput sequencing data set is helpful to improve the identification accuracy of motif discovery but requires an even higher computing performance. To efficiently identify motifs in large DNA data sets, a new algorithm called PairMotifChIP is proposed by extracting and combining pairs of l-mers in the input with relatively small Hamming distance. In particular, a method for rapidly extracting pairs of l-mers is designed, which can be used not only for PairMotifChIP, but also for other DNA data mining tasks with the same demand. Experimental results on the simulated data show that the proposed algorithm can find motifs successfully and runs faster than the state-of-the-art motif discovery algorithms. Furthermore, the validity of the proposed algorithm has been verified on real data. PMID:27843946

  8. Computational generation and screening of RNA motifs in large nucleotide sequence pools

    PubMed Central

    Kim, Namhee; Izzo, Joseph A.; Elmetwaly, Shereef; Gan, Hin Hark; Schlick, Tamar

    2010-01-01

    Although identification of active motifs in large random sequence pools is central to RNA in vitro selection, no systematic computational equivalent of this process has yet been developed. We develop a computational approach that combines target pool generation, motif scanning and motif screening using secondary structure analysis for applications to 1012–1014-sequence pools; large pool sizes are made possible using program redesign and supercomputing resources. We use the new protocol to search for aptamer and ribozyme motifs in pools up to experimental pool size (1014 sequences). We show that motif scanning, structure matching and flanking sequence analysis, respectively, reduce the initial sequence pool by 6–8, 1–2 and 1 orders of magnitude, consistent with the rare occurrence of active motifs in random pools. The final yields match the theoretical yields from probability theory for simple motifs and overestimate experimental yields, which constitute lower bounds, for aptamers because screening analyses beyond secondary structure information are not considered systematically. We also show that designed pools using our nucleotide transition probability matrices can produce higher yields for RNA ligase motifs than random pools. Our methods for generating, analyzing and designing large pools can help improve RNA design via simulation of aspects of in vitro selection. PMID:20448026

  9. Mitoxantrone and Analogues Bind and Stabilize i-Motif Forming DNA Sequences

    PubMed Central

    Wright, Elisé P.; Day, Henry A.; Ibrahim, Ali M.; Kumar, Jeethendra; Boswell, Leo J. E.; Huguin, Camille; Stevenson, Clare E. M.; Pors, Klaus; Waller, Zoë A. E.

    2016-01-01

    There are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences. PMID:28004744

  10. Mitoxantrone and Analogues Bind and Stabilize i-Motif Forming DNA Sequences

    NASA Astrophysics Data System (ADS)

    Wright, Elisé P.; Day, Henry A.; Ibrahim, Ali M.; Kumar, Jeethendra; Boswell, Leo J. E.; Huguin, Camille; Stevenson, Clare E. M.; Pors, Klaus; Waller, Zoë A. E.

    2016-12-01

    There are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences.

  11. Physical-chemical property based sequence motifs and methods regarding same

    DOEpatents

    Braun, Werner; Mathura, Venkatarajan S.; Schein, Catherine H.

    2008-09-09

    A data analysis system, program, and/or method, e.g., a data mining/data exploration method, using physical-chemical property motifs. For example, a sequence database may be searched for identifying segments thereof having physical-chemical properties similar to the physical-chemical property motifs.

  12. MISCORE: a new scoring function for characterizing DNA regulatory motifs in promoter sequences

    PubMed Central

    2012-01-01

    Background Computational approaches for finding DNA regulatory motifs in promoter sequences are useful to biologists in terms of reducing the experimental costs and speeding up the discovery process of de novo binding sites. It is important for rule-based or clustering-based motif searching schemes to effectively and efficiently evaluate the similarity between a k-mer (a k-length subsequence) and a motif model, without assuming the independence of nucleotides in motif models or without employing computationally expensive Markov chain models to estimate the background probabilities of k-mers. Also, it is interesting and beneficial to use a priori knowledge in developing advanced searching tools. Results This paper presents a new scoring function, termed as MISCORE, for functional motif characterization and evaluation. Our MISCORE is free from: (i) any assumption on model dependency; and (ii) the use of Markov chain model for background modeling. It integrates the compositional complexity of motif instances into the function. Performance evaluations with comparison to the well-known Maximum a Posteriori (MAP) score and Information Content (IC) have shown that MISCORE has promising capabilities to separate and recognize functional DNA motifs and its instances from non-functional ones. Conclusions MISCORE is a fast computational tool for candidate motif characterization, evaluation and selection. It enables to embed priori known motif models for computing motif-to-motif similarity, which is more advantageous than IC and MAP score. In addition to these merits mentioned above, MISCORE can automatically filter out some repetitive k-mers from a motif model due to the introduction of the compositional complexity in the function. Consequently, the merits of our proposed MISCORE in terms of both motif signal modeling power and computational efficiency will make it more applicable in the development of computational motif discovery tools. PMID:23282090

  13. Nuclear Magnetic Resonance Structure of a Novel Globular Domain in RBM10 Containing OCRE, the Octamer Repeat Sequence Motif.

    PubMed

    Martin, Bryan T; Serrano, Pedro; Geralt, Michael; Wüthrich, Kurt

    2016-01-05

    The OCtamer REpeat (OCRE) has been annotated as a 42-residue sequence motif with 12 tyrosine residues in the spliceosome trans-regulatory elements RBM5 and RBM10 (RBM [RNA-binding motif]), which are known to regulate alternative splicing of Fas and Bcl-x pre-mRNA transcripts. Nuclear magnetic resonance structure determination showed that the RBM10 OCRE sequence motif is part of a 55-residue globular domain containing 16 aromatic amino acids, which consists of an anti-parallel arrangement of six β strands, with the first five strands containing complete or incomplete Tyr triplets. This OCRE globular domain is a distinctive component of RBM10 and is more widely conserved in RBM10s across the animal kingdom than the ubiquitous RNA recognition components. It is also found in the functionally related RBM5. Thus, it appears that the three-dimensional structure of the globular OCRE domain, rather than the 42-residue OCRE sequence motif alone, confers specificity on RBM10 intermolecular interactions in the spliceosome.

  14. Evolutionarily conserved sequences on human chromosome 21

    SciTech Connect

    Frazer, Kelly A.; Sheehan, John B.; Stokowski, Renee P.; Chen, Xiyin; Hosseini, Roya; Cheng, Jan-Fang; Fodor, Stephen P.A.; Cox, David R.; Patil, Nila

    2001-09-01

    Comparison of human sequences with the DNA of other mammals is an excellent means of identifying functional elements in the human genome. Here we describe the utility of high-density oligonucleotide arrays as a rapid approach for comparing human sequences with the DNA of multiple species whose sequences are not presently available. High-density arrays representing approximately 22.5 Mb of nonrepetitive human chromosome 21 sequence were synthesized and then hybridized with mouse and dog DNA to identify sequences conserved between humans and mice (human-mouse elements) and between humans and dogs (human-dog elements). Our data show that sequence comparison of multiple species provides a powerful empiric method for identifying actively conserved elements in the human genome. A large fraction of these evolutionarily conserved elements are present in regions on chromosome 21 that do not encode known genes.

  15. Examination of the transcription factor NtcA-binding motif by in vitro selection of DNA sequences from a random library.

    PubMed

    Jiang, F; Wisén, S; Widersten, M; Bergman, B; Mannervik, B

    2000-08-25

    A recursive in vitro selection among random DNA sequences was used for analysis of the cyanobacterial transcription factor NtcA-binding motifs. An eight-base palindromic sequence, TGTA-(N(8))-TACA, was found to be the optimal NtcA-binding sequence. The more divergent the binding sequences, compared to this consensus sequence, the lower the NtcA affinity. The second and third bases in each four-nucleotide half of the consensus sequence were crucial for NtcA binding, and they were in general highly conserved. The most frequently occurring sequence in the middle weakly conserved region was similar to that of the NtcA-binding motif of the Anabaena sp. strain PCC 7120 glnA gene, previously known to have high affinity for NtcA. This indicates that the middle sequences were selected for high NtcA affinity. Analysis of natural NtcA-binding motifs showed that these could be classified into two groups based on differences in recognition consensus sequences. It is suggested that NtcA naturally recognizes different DNA-binding motifs, or has differential affinities to these sequences under different physiological conditions.

  16. Factoring local sequence composition in motif significance analysis.

    PubMed

    Ng, Patrick; Keich, Uri

    2008-01-01

    We recently introduced a biologically realistic and reliable significance analysis of the output of a popular class of motif finders. In this paper we further improve our significance analysis by incorporating local base composition information. Relying on realistic biological data simulation, as well as on FDR analysis applied to real data, we show that our method is significantly better than the increasingly popular practice of using the normal approximation to estimate the significance of a finder's output. Finally we turn to leveraging our reliable significance analysis to improve the actual motif finding task. Specifically, endowing a variant of the Gibbs Sampler with our improved significance analysis we demonstrate that de novo finders can perform better than has been perceived. Significantly, our new variant outperforms all the finders reviewed in a recently published comprehensive analysis of the Harbison genome-wide binding location data. Interestingly, many of these finders incorporate additional information such as nucleosome positioning and the significance of binding data.

  17. A Conserved Di-Basic Motif of Drosophila Crumbs Contributes to Efficient ER Export.

    PubMed

    Kumichel, Alexandra; Kapp, Katja; Knust, Elisabeth

    2015-06-01

    The Drosophila type I transmembrane protein Crumbs is an apical determinant required for the maintenance of apico-basal epithelial cell polarity. The level of Crumbs at the plasma membrane is crucial, but how it is regulated is poorly understood. In a genetic screen for regulators of Crumbs protein trafficking we identified Sar1, the core component of the coat protein complex II transport vesicles. sar1 mutant embryos show a reduced plasma membrane localization of Crumbs, a defect similar to that observed in haunted and ghost mutant embryos, which lack Sec23 and Sec24CD, respectively. By pulse-chase assays in Drosophila Schneider cells and analysis of protein transport kinetics based on Endoglycosidase H resistance we identified an RNKR motif in Crumbs, which contributes to efficient ER export. The motif identified fits the highly conserved di-basic RxKR motif and mediates interaction with Sar1. The RNKR motif is also required for plasma membrane delivery of transgene-encoded Crumbs in epithelial cells of Drosophila embryos. Our data are the first to show that a di-basic motif acts as a signal for ER exit of a type I plasma membrane protein in a metazoan organism.

  18. Sequence Fingerprints of MicroRNA Conservation

    PubMed Central

    Shi, Bing; Gao, Wei; Wang, Juan

    2012-01-01

    It is known that the conservation of protein-coding genes is associated with their sequences both various species, such as animals and plants. However, the association between microRNA (miRNA) conservation and their sequences in various species remains unexplored. Here we report the association of miRNA conservation with its sequence features, such as base content and cleavage sites, suggesting that miRNA sequences contain the fingerprints for miRNA conservation. More interestingly, different species show different and even opposite patterns between miRNA conservation and sequence features. For example, mammalian miRNAs show a positive/negative correlation between conservation and AU/GC content, whereas plant miRNAs show a negative/positive correlation between conservation and AU/GC content. Further analysis puts forward the hypothesis that the introns of protein-coding genes may be a main driving force for the origin and evolution of mammalian miRNAs. At the 5′ end, conserved miRNAs have a preference for base U, while less-conserved miRNAs have a preference for a non-U base in mammals. This difference does not exist in insects and plants, in which both conserved miRNAs and less-conserved miRNAs have a preference for base U at the 5′ end. We further revealed that the non-U preference at the 5′ end of less-conserved mammalian miRNAs is associated with miRNA function diversity, which may have evolved from the pressure of a highly sophisticated environmental stimulus the mammals encountered during evolution. These results indicated that miRNA sequences contain the fingerprints for conservation, and these fingerprints vary according to species. More importantly, the results suggest that although species share common mechanisms by which miRNAs originate and evolve, mammals may develop a novel mechanism for miRNA origin and evolution. In addition, the fingerprint found in this study can be predictor of miRNA conservation, and the findings are helpful in achieving a

  19. Analysis of Genomic Sequence Motifs for Deciphering Transcription Factor Binding and Transcriptional Regulation in Eukaryotic Cells

    PubMed Central

    Boeva, Valentina

    2016-01-01

    Eukaryotic genomes contain a variety of structured patterns: repetitive elements, binding sites of DNA and RNA associated proteins, splice sites, and so on. Often, these structured patterns can be formalized as motifs and described using a proper mathematical model such as position weight matrix and IUPAC consensus. Two key tasks are typically carried out for motifs in the context of the analysis of genomic sequences. These are: identification in a set of DNA regions of over-represented motifs from a particular motif database, and de novo discovery of over-represented motifs. Here we describe existing methodology to perform these two tasks for motifs characterizing transcription factor binding. When applied to the output of ChIP-seq and ChIP-exo experiments, or to promoter regions of co-modulated genes, motif analysis techniques allow for the prediction of transcription factor binding events and enable identification of transcriptional regulators and co-regulators. The usefulness of motif analysis is further exemplified in this review by how motif discovery improves peak calling in ChIP-seq and ChIP-exo experiments and, when coupled with information on gene expression, allows insights into physical mechanisms of transcriptional modulation. PMID:26941778

  20. Conserved Hydration Sites in Pin1 Reveal a Distinctive Water Recognition Motif in Proteins.

    PubMed

    Barman, Arghya; Smitherman, Crystal; Souffrant, Michael; Gadda, Giovanni; Hamelberg, Donald

    2016-01-25

    Structurally conserved water molecules are important for biomolecular stability, flexibility, and function. X-ray crystallographic studies of Pin1 have resolved a number of water molecules around the enzyme, including two highly conserved water molecules within the protein. The functional role of these localized water molecules remains unknown and unexplored. Pin1 catalyzes cis/trans isomerizations of peptidyl prolyl bonds that are preceded by a phosphorylated serine or threonine residue. Pin1 is involved in many subcellular signaling processes and is a potential therapeutic target for the treatment of several life threatening diseases. Here, we investigate the significance of these structurally conserved water molecules in the catalytic domain of Pin1 using molecular dynamics (MD) simulations, free energy calculations, analysis of X-ray crystal structures, and circular dichroism (CD) experiments. MD simulations and free energy calculations suggest the tighter binding water molecule plays a crucial role in maintaining the integrity and stability of a critical hydrogen-bonding network in the active site. The second water molecule is exchangeable with bulk solvent and is found in a distinctive helix-turn-coil motif. Structural bioinformatics analysis of nonredundant X-ray crystallographic protein structures in the Protein Data Bank (PDB) suggest this motif is present in several other proteins and can act as a water site, akin to the calcium EF hand. CD experiments suggest the isolated motif is in a distorted PII conformation and requires the protein environment to fully form the α-helix-turn-coil motif. This study provides valuable insights into the role of hydration in the structural integrity of Pin1 that can be exploited in protein engineering and drug design.

  1. Identification of disease-specific motifs in the antibody specificity repertoire via next-generation sequencing

    PubMed Central

    Pantazes, Robert J.; Reifert, Jack; Bozekowski, Joel; Ibsen, Kelly N.; Murray, Joseph A.; Daugherty, Patrick S.

    2016-01-01

    Disease-specific antibodies can serve as highly effective biomarkers but have been identified for only a relatively small number of autoimmune diseases. A method was developed to identify disease-specific binding motifs through integration of bacterial display peptide library screening, next-generation sequencing (NGS) and computational analysis. Antibody specificity repertoires were determined by identifying bound peptide library members for each specimen using cell sorting and performing NGS. A computational algorithm, termed Identifying Motifs Using Next- generation sequencing Experiments (IMUNE), was developed and applied to discover disease- and healthy control-specific motifs. IMUNE performs comprehensive pattern searches, identifies patterns statistically enriched in the disease or control groups and clusters the patterns to generate motifs. Using celiac disease sera as a discovery set, IMUNE identified a consensus motif (QPEQPF[PS]E) with high diagnostic sensitivity and specificity in a validation sera set, in addition to novel motifs. Peptide display and sequencing (Display-Seq) coupled with IMUNE analysis may thus be useful to characterize antibody repertoires and identify disease-specific antibody epitopes and biomarkers. PMID:27481573

  2. Sequence conservation on the Y chromosome

    SciTech Connect

    Gibson, L.H.; Yang-Feng, L.; Lau, C.

    1994-09-01

    The Y chromosome is present in all mammals and is considered to be essential to sex determination. Despite intense genomic research, only a few genes have been identified and mapped to this chromosome in humans. Several of them, such as SRY and ZFY, have been demonstrated to be conserved and Y-located in other mammals. In order to address the issue of sequence conservation on the Y chromosome, we performed fluorescence in situ hybridization (FISH) with DNA from a human Y cosmid library as a probe to study the Y chromosomes from other mammalian species. Total DNA from 3,000-4,500 cosmid pools were labeled with biotinylated-dUTP and hybridized to metaphase chromosomes. For human and primate preparations, human cot1 DNA was included in the hybridization mixture to suppress the hybridization from repeat sequences. FISH signals were detected on the Y chromosomes of human, gorilla, orangutan and baboon (Old World monkey) and were absent on those of squirrel monkey (New World monkey), Indian munjac, wood lemming, Chinese hamster, rat and mouse. Since sequence analysis suggested that specific genes, e.g. SRY and ZFY, are conserved between these two groups, the lack of detectable hybridization in the latter group implies either that conservation of the human Y sequences is limited to the Y chromosomes of the great apes and Old World monkeys, or that the size of the syntenic segment is too small to be detected under the resolution of FISH, or that homologeous sequences have undergone considerable divergence. Further studies with reduced hybridization stringency are currently being conducted. Our results provide some clues as to Y-sequence conservation across species and demonstrate the limitations of FISH across species with total DNA sequences from a particular chromosome.

  3. Using a color-coded ambigraphic nucleic acid notation to visualize conserved palindromic motifs within and across genomes

    PubMed Central

    2014-01-01

    Background Ambiscript is a graphically-designed nucleic acid notation that uses symbol symmetries to support sequence complementation, highlight biologically-relevant palindromes, and facilitate the analysis of consensus sequences. Although the original Ambiscript notation was designed to easily represent consensus sequences for multiple sequence alignments, the notation’s black-on-white ambiguity characters are unable to reflect the statistical distribution of nucleotides found at each position. We now propose a color-augmented ambigraphic notation to encode the frequency of positional polymorphisms in these consensus sequences. Results We have implemented this color-coding approach by creating an Adobe Flash® application ( http://www.ambiscript.org) that shades and colors modified Ambiscript characters according to the prevalence of the encoded nucleotide at each position in the alignment. The resulting graphic helps viewers perceive biologically-relevant patterns in multiple sequence alignments by uniquely combining color, shading, and character symmetries to highlight palindromes and inverted repeats in conserved DNA motifs. Conclusion Juxtaposing an intuitive color scheme over the deliberate character symmetries of an ambigraphic nucleic acid notation yields a highly-functional nucleic acid notation that maximizes information content and successfully embodies key principles of graphic excellence put forth by the statistician and graphic design theorist, Edward Tufte. PMID:24447494

  4. Recurring sequence-structure motifs in (βα)8-barrel proteins and experimental optimization of a chimeric protein designed based on such motifs.

    PubMed

    Wang, Jichao; Zhang, Tongchuan; Liu, Ruicun; Song, Meilin; Wang, Juncheng; Hong, Jiong; Chen, Quan; Liu, Haiyan

    2017-02-01

    An interesting way of generating novel artificial proteins is to combine sequence motifs from natural proteins, mimicking the evolutionary path suggested by natural proteins comprising recurring motifs. We analyzed the βα and αβ modules of TIM barrel proteins by structure alignment-based sequence clustering. A number of preferred motifs were identified. A chimeric TIM was designed by using recurring elements as mutually compatible interfaces. The foldability of the designed TIM protein was then significantly improved by six rounds of directed evolution. The melting temperature has been improved by more than 20°C. A variety of characteristics suggested that the resulting protein is well-folded. Our analysis provided a library of peptide motifs that is potentially useful for different protein engineering studies. The protein engineering strategy of using recurring motifs as interfaces to connect partial natural proteins may be applied to other protein folds.

  5. Targeting of Arabidopsis KNL2 to Centromeres Depends on the Conserved CENPC-k Motif in Its C Terminus[OPEN

    PubMed Central

    Talbert, Paul; Demidov, Dmitri

    2017-01-01

    KINETOCHORE NULL2 (KNL2) is involved in recognition of centromeres and in centromeric localization of the centromere-specific histone cenH3. Our study revealed a cenH3 nucleosome binding CENPC-k motif at the C terminus of Arabidopsis thaliana KNL2, which is conserved among a wide spectrum of eukaryotes. Centromeric localization of KNL2 is abolished by deletion of the CENPC-k motif and by mutating single conserved amino acids, but can be restored by insertion of the corresponding motif of Arabidopsis CENP-C. We showed by electrophoretic mobility shift assay that the C terminus of KNL2 binds DNA sequence-independently and interacts with the centromeric transcripts in vitro. Chromatin immunoprecipitation with anti-KNL2 antibodies indicated that in vivo KNL2 is preferentially associated with the centromeric repeat pAL1. Complete deletion of the CENPC-k motif did not influence its ability to interact with DNA in vitro. Therefore, we suggest that KNL2 recognizes centromeric nucleosomes, similar to CENP-C, via the CENPC-k motif and binds adjoining DNA. PMID:28062749

  6. A conserved motif in Tetrahymena thermophila telomerase reverse transcriptase is proximal to the RNA template and is essential for boundary definition.

    PubMed

    Akiyama, Benjamin M; Gomez, Anastassia; Stone, Michael D

    2013-07-26

    The ends of linear chromosomes are extended by telomerase, a ribonucleoprotein complex minimally consisting of a protein subunit called telomerase reverse transcriptase (TERT) and the telomerase RNA (TER). TERT functions by reverse transcribing a short template region of TER into telomeric DNA. Proper assembly of TERT and TER is essential for telomerase activity; however, a detailed understanding of how TERT interacts with TER is lacking. Previous studies have identified an RNA binding domain (RBD) within TERT, which includes three evolutionarily conserved sequence motifs: CP2, CP, and T. Here, we used site-directed hydroxyl radical probing to directly identify sites of interaction between the TERT RBD and TER, revealing that the CP2 motif is in close proximity to a conserved region of TER known as the template boundary element (TBE). Gel shift assays on CP2 mutants confirmed that the CP2 motif is an RNA binding determinant. Our results explain previous work that established that mutations to the CP2 motif of TERT and to the TBE of TER both permit misincorporation of nucleotides into the growing DNA strand beyond the canonical template. Taken together, these results suggest a model in which the CP2 motif binds the TBE to strictly define which TER nucleotides can be reverse transcribed.

  7. A Conserved Phenylalanine of Motif IV in Superfamily 2 Helicases Is Required for Cooperative, ATP-Dependent Binding of RNA Substrates in DEAD-Box Proteins▿ †

    PubMed Central

    Banroques, Josette; Cordin, Olivier; Doère, Monique; Linder, Patrick; Tanner, N. Kyle

    2008-01-01

    We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-π interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding. PMID:18332124

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

    PubMed

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

    2015-05-01

    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.

  9. 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

  10. Novel hexamerization motif is discovered in a conserved cytoplasmic protein from Salmonella typhimurium.

    SciTech Connect

    Petrova, T.; Cuff, M.; Wu, R.; Kim, Y.; Holzle, D.; Joachimiak, A.; Biosciences Division; Inst. of Mathematical Problems of Biology

    2007-01-01

    The cytoplasmic protein Stm3548 of unknown function obtained from a strain of Salmonella typhimurium was determined by X-ray crystallography at a resolution of 2.25 A. The asymmetric unit contains a hexamer of structurally identical monomers. The monomer is a globular domain with a long beta-hairpin protrusion that distinguishes this structure. This beta-hairpin occupies a central position in the hexamer, and its residues participate in the majority of interactions between subunits of the hexamer. We suggest that the structure of Stm3548 presents a new hexamerization motif. Because the residues participating in interdomain interactions are highly conserved among close members of protein family DUF1355 and buried solvent accessible area for the hexamer is significant, the hexamer is most likely conserved as well. A light scattering experiment confirmed the presence of hexamer in solution.

  11. The transcription factor Spn1 regulates gene expression via a highly conserved novel structural motif

    PubMed Central

    Pujari, Venugopal; Radebaugh, Catherine A.; Chodaparambil, Jayanth V.; Muthurajan, Uma M.; Almeida, Adam R.; Fischbeck, Julie A.; Luger, Karolin; Stargell, Laurie A.

    2010-01-01

    Spn1 plays essential roles in the regulation of gene expression by RNA Polymerase II (RNAPII), and it is highly conserved in organisms ranging from yeast to humans. Spn1 physically and/or genetically interacts with RNAPII, TBP, TFIIS and a number of chromatin remodeling factors (Swi/Snf and Spt6). The central domain of Spn1 (residues 141-305 out of 410) is necessary and sufficient for performing the essential functions of SPN1 in yeast cells. Here we report the high-resolution (1.85Å) crystal structure of the conserved central domain of Saccharomyces cerevisiae Spn1. The central domain is comprised of eight alpha-helices in a right handed super helical arrangement, and exhibits structural similarity to domain I of TFIIS. A unique structural feature of Spn1 is a highly conserved loop, which defines one side of a pronounced cavity. The loop and the other residues forming the cavity are highly conserved at the amino acid level among all Spn1 family members, suggesting that this is a signature motif for Spn1 orthologs. The locations and the molecular characterization of temperature-sensitive mutations in Spn1 indicate that the cavity is a key attribute of Spn1 that is critical for its regulatory functions during RNAPII-mediated transcriptional activity. PMID:20875428

  12. The transcription factor Spn1 regulates gene expression via a highly conserved novel structural motif.

    PubMed

    Pujari, Venugopal; Radebaugh, Catherine A; Chodaparambil, Jayanth V; Muthurajan, Uma M; Almeida, Adam R; Fischbeck, Julie A; Luger, Karolin; Stargell, Laurie A

    2010-11-19

    Spn1/Iws1 plays essential roles in the regulation of gene expression by RNA polymerase II (RNAPII), and it is highly conserved in organisms ranging from yeast to humans. Spn1 physically and/or genetically interacts with RNAPII, TBP (TATA-binding protein), TFIIS (transcription factor IIS), and a number of chromatin remodeling factors (Swi/Snf and Spt6). The central domain of Spn1 (residues 141-305 out of 410) is necessary and sufficient for performing the essential functions of SPN1 in yeast cells. Here, we report the high-resolution (1.85 Å) crystal structure of the conserved central domain of Saccharomyces cerevisiae Spn1. The central domain is composed of eight α-helices in a right-handed superhelical arrangement and exhibits structural similarity to domain I of TFIIS. A unique structural feature of Spn1 is a highly conserved loop, which defines one side of a pronounced cavity. The loop and the other residues forming the cavity are highly conserved at the amino acid level among all Spn1 family members, suggesting that this is a signature motif for Spn1 orthologs. The locations and the molecular characterization of temperature-sensitive mutations in Spn1 indicate that the cavity is a key attribute of Spn1 that is critical for its regulatory functions during RNAPII-mediated transcriptional activity.

  13. Identification of an oligodeoxynucleotide sequence motif that specifically inhibits phosphorylation by protein tyrosine kinases.

    PubMed

    Krieg, A M; Matson, S; Cheng, K; Fisher, E; Koretzky, G A; Koland, J G

    1997-04-01

    Protein tyrosine kinases (PTKs) have central roles in cellular signal transduction. We have identified a sequence motif (CGT[C]GA) in phosphorothioate-modified oligodeoxynucleotides (ODNs) that specifically inhibits the enzymatic activity of recombinant or immunoprecipitated PTK in vitro. Hexamer ODNs containing this motif block both substrate and autophosphorylation of at least four different PTKs but have no apparent effect on the enzymatic activity of a serine/threonine protein kinase. These data suggest possible new applications for ODNs and have implications for the design and interpretation of experiments using antisense or triplex ODNs.

  14. A Conserved GPG-Motif in the HIV-1 Nef Core Is Required for Principal Nef-Activities.

    PubMed

    Martínez-Bonet, Marta; Palladino, Claudia; Briz, Veronica; Rudolph, Jochen M; Fackler, Oliver T; Relloso, Miguel; Muñoz-Fernandez, Maria Angeles; Madrid, Ricardo

    2015-01-01

    To find out new determinants required for Nef activity we performed a functional alanine scanning analysis along a discrete but highly conserved region at the core of HIV-1 Nef. We identified the GPG-motif, located at the 121-137 region of HIV-1 NL4.3 Nef, as a novel protein signature strictly required for the p56Lck dependent Nef-induced CD4-downregulation in T-cells. Since the Nef-GPG motif was dispensable for CD4-downregulation in HeLa-CD4 cells, Nef/AP-1 interaction and Nef-dependent effects on Tf-R trafficking, the observed effects on CD4 downregulation cannot be attributed to structure constraints or to alterations on general protein trafficking. Besides, we found that the GPG-motif was also required for Nef-dependent inhibition of ring actin re-organization upon TCR triggering and MHCI downregulation, suggesting that the GPG-motif could actively cooperate with the Nef PxxP motif for these HIV-1 Nef-related effects. Finally, we observed that the Nef-GPG motif was required for optimal infectivity of those viruses produced in T-cells. According to these findings, we propose the conserved GPG-motif in HIV-1 Nef as functional region required for HIV-1 infectivity and therefore with a potential interest for the interference of Nef activity during HIV-1 infection.

  15. A Conserved GPG-Motif in the HIV-1 Nef Core Is Required for Principal Nef-Activities

    PubMed Central

    Martínez-Bonet, Marta; Palladino, Claudia; Briz, Veronica; Rudolph, Jochen M.; Fackler, Oliver T.; Relloso, Miguel; Muñoz-Fernandez, Maria Angeles; Madrid, Ricardo

    2015-01-01

    To find out new determinants required for Nef activity we performed a functional alanine scanning analysis along a discrete but highly conserved region at the core of HIV-1 Nef. We identified the GPG-motif, located at the 121–137 region of HIV-1 NL4.3 Nef, as a novel protein signature strictly required for the p56Lck dependent Nef-induced CD4-downregulation in T-cells. Since the Nef-GPG motif was dispensable for CD4-downregulation in HeLa-CD4 cells, Nef/AP-1 interaction and Nef-dependent effects on Tf-R trafficking, the observed effects on CD4 downregulation cannot be attributed to structure constraints or to alterations on general protein trafficking. Besides, we found that the GPG-motif was also required for Nef-dependent inhibition of ring actin re-organization upon TCR triggering and MHCI downregulation, suggesting that the GPG-motif could actively cooperate with the Nef PxxP motif for these HIV-1 Nef-related effects. Finally, we observed that the Nef-GPG motif was required for optimal infectivity of those viruses produced in T-cells. According to these findings, we propose the conserved GPG-motif in HIV-1 Nef as functional region required for HIV-1 infectivity and therefore with a potential interest for the interference of Nef activity during HIV-1 infection. PMID:26700863

  16. Protospacer recognition motifs

    PubMed Central

    Shah, Shiraz A.; Erdmann, Susanne; Mojica, Francisco J.M.; Garrett, Roger A.

    2013-01-01

    Protospacer adjacent motifs (PAMs) were originally characterized for CRISPR-Cas systems that were classified on the basis of their CRISPR repeat sequences. A few short 2–5 bp sequences were identified adjacent to one end of the protospacers. Experimental and bioinformatical results linked the motif to the excision of protospacers and their insertion into CRISPR loci. Subsequently, evidence accumulated from different virus- and plasmid-targeting assays, suggesting that these motifs were also recognized during DNA interference, at least for the recently classified type I and type II CRISPR-based systems. The two processes, spacer acquisition and protospacer interference, employ different molecular mechanisms, and there is increasing evidence to suggest that the sequence motifs that are recognized, while overlapping, are unlikely to be identical. In this article, we consider the properties of PAM sequences and summarize the evidence for their dual functional roles. It is proposed to use the terms protospacer associated motif (PAM) for the conserved DNA sequence and to employ spacer acqusition motif (SAM) and target interference motif (TIM), respectively, for acquisition and interference recognition sites. PMID:23403393

  17. Conserved Noncoding Sequences in the Grasses4

    PubMed Central

    Inada, Dan Choffnes; Bashir, Ali; Lee, Chunghau; Thomas, Brian C.; Ko, Cynthia; Goff, Stephen A.; Freeling, Michael

    2003-01-01

    As orthologous genes from related species diverge over time, some sequences are conserved in noncoding regions. In mammals, large phylogenetic footprints, or conserved noncoding sequences (CNSs), are known to be common features of genes. Here we present the first large-scale analysis of plant genes for CNSs. We used maize and rice, maximally diverged members of the grass family of monocots. Using a local sequence alignment set to deliver only significant alignments, we found one or more CNSs in the noncoding regions of the majority of genes studied. Grass genes have dramatically fewer and much smaller CNSs than mammalian genes. Twenty-seven percent of grass gene comparisons revealed no CNSs. Genes functioning in upstream regulatory roles, such as transcription factors, are greatly enriched for CNSs relative to genes encoding enzymes or structural proteins. Further, we show that a CNS cluster in an intron of the knotted1 homeobox gene serves as a site of negative regulation. We showthat CNSs in the adh1 gene do not correlate with known cis-acting sites. We discuss the potential meanings of CNSs and their value as analytical tools and evolutionary characters. We advance the idea that many CNSs function to lock-in gene regulatory decisions. PMID:12952874

  18. Interpreting Frequency Responses to Dose-Conserved Pulsatile Input Signals in Simple Cell Signaling Motifs

    PubMed Central

    Fletcher, Patrick A.; Clément, Frédérique; Vidal, Alexandre; Tabak, Joel; Bertram, Richard

    2014-01-01

    Many hormones are released in pulsatile patterns. This pattern can be modified, for instance by changing pulse frequency, to encode relevant physiological information. Often other properties of the pulse pattern will also change with frequency. How do signaling pathways of cells targeted by these hormones respond to different input patterns? In this study, we examine how a given dose of hormone can induce different outputs from the target system, depending on how this dose is distributed in time. We use simple mathematical models of feedforward signaling motifs to understand how the properties of the target system give rise to preferences in input pulse pattern. We frame these problems in terms of frequency responses to pulsatile inputs, where the amplitude or duration of the pulses is varied along with frequency to conserve input dose. We find that the form of the nonlinearity in the steady state input-output function of the system predicts the optimal input pattern. It does so by selecting an optimal input signal amplitude. Our results predict the behavior of common signaling motifs such as receptor binding with dimerization, and protein phosphorylation. The findings have implications for experiments aimed at studying the frequency response to pulsatile inputs, as well as for understanding how pulsatile patterns drive biological responses via feedforward signaling pathways. PMID:24748217

  19. Evolutionary and molecular analysis of Dof transcription factors identified a conserved motif for intercellular protein trafficking.

    PubMed

    Chen, Huan; Ahmad, Munawar; Rim, Yeonggil; Lucas, William J; Kim, Jae-Yean

    2013-06-01

    · Cell-to-cell trafficking of transcription factors (TFs) has been shown to play an important role in the regulation of plant developmental events, but the evolutionary relationship between cell-autonomous and noncell-autonomous (NCA) TFs remains elusive. · AtDof4.1, named INTERCELLULAR TRAFFICKING DOF 1 (ITD1), was chosen as a representative NCA member to explore this evolutionary relationship. Using domain structure-function analyses and swapping studies, we examined the cell-to-cell trafficking of plant-specific Dof TF family members across Arabidopsis and other species. · We identified a conserved intercellular trafficking motif (ITM) that is necessary and sufficient for selective cell-to-cell trafficking and can impart gain-of-function cell-to-cell movement capacity to an otherwise cell-autonomous TF. The functionality of related motifs from Dof members across the plant kingdom extended, surprisingly, to a unicellular alga that lacked plasmodesmata. By contrast, the algal homeodomain related to the NCA KNOX homeodomain was either inefficient or unable to impart such cell-to-cell movement function. · The Dof ITM appears to predate the evolution of selective plasmodesmal trafficking in the plant kingdom, which may well have acted as a molecular template for the evolution of Dof proteins as NCA TFs. However, the ability to efficiently traffic for KNOX homeodomain (HD) proteins may have been acquired during the evolution of early nonvascular plants.

  20. Sequence motifs associated with paternal transmission of mitochondrial DNA in the horse mussel, Modiolus modiolus (Bivalvia: Mytilidae).

    PubMed

    Robicheau, Brent M; Breton, Sophie; Stewart, Donald T

    2017-03-20

    In the majority of metazoans paternal mitochondria represent evolutionary dead-ends. In many bivalves, however, this paradigm does not hold true; both maternal and paternal mitochondria are inherited. Herein, we characterize maternal and paternal mitochondrial control regions of the horse mussel, Modiolus modiolus (Bivalvia: Mytilidae). The maternal control region is 808bp long, while the paternal control region is longer at 2.3kb. We hypothesize that the size difference is due to a combination of repeated duplications within the control region of the paternal mtDNA genome, as well as an evolutionarily ancient recombination event between two sex-associated mtDNA genomes that led to the insertion of a second control region sequence in the genome that is now transmitted via males. In a comparison to other mytilid male control regions, we identified two evolutionarily Conserved Motifs, CMA and CMB, associated with paternal transmission of mitochondrial DNA. CMA is characterized by a conserved purine/pyrimidine pattern, while CMB exhibits a specific 13bp nucleotide string within a stem and loop structure. The identification of motifs CMA and CMB in M. modiolus extends our understanding of Sperm Transmission Elements (STEs) that have recently been identified as being associated with the paternal transmission of mitochondria in marine bivalves.

  1. Species-Specific Minimal Sequence Motif for Oligodeoxyribonucleotides Activating Mouse TLR9.

    PubMed

    Pohar, Jelka; Lainšček, Duško; Fukui, Ryutaro; Yamamoto, Chikako; Miyake, Kensuke; Jerala, Roman; Benčina, Mojca

    2015-11-01

    Synthetic oligodeoxyribonucleotides (ODNs) containing unmethylated CpG recapitulate the activation of TLR9 by microbial DNA. ODNs are potent stimulators of the immune response in cells expressing TLR9. Despite extensive use of mice as experimental animals in basic and applied immunological research, the key sequence determinants that govern the activation of mouse TLR9 by ODNs have not been well defined. We performed a systematic investigation of the sequence motif of B class phosphodiester ODNs to identify the sequence properties that govern mouse TLR9 activation. In contrast to ODNs activating human TLR9, where the minimal sequence motif for the receptor activation comprises a pair of closely positioned CpGs we found that the mouse TLR9 requires a single CpG positioned 4-6 nt from the 5'-end. Activation is augmented by a 5'TCC sequence one to three nucleotides from the CG. The distance of the CG dinucleotide of four to six nucleotides from the 5'-end and the ODN's length fine-tunes activation of mouse macrophages. Length of the ODN <23 and >29 nt decreases activation of dendritic cells. The ODNs with minimal sequence induce Th1-type cytokine synthesis in dendritic cells and confirm the expression of cell surface markers in B cells. Identification of the minimal sequence provides an insight into the sequence selectivity of mouse TLR9 and points to the differences in the receptor selectivity between species probably as a result of differences in the receptor binding sites.

  2. Using machine learning to predict gene expression and discover sequence motifs

    NASA Astrophysics Data System (ADS)

    Li, Xuejing

    Recently, large amounts of experimental data for complex biological systems have become available. We use tools and algorithms from machine learning to build data-driven predictive models. We first present a novel algorithm to discover gene sequence motifs associated with temporal expression patterns of genes. Our algorithm, which is based on partial least squares (PLS) regression, is able to directly model the flow of information, from gene sequence to gene expression, to learn cis regulatory motifs and characterize associated gene expression patterns. Our algorithm outperforms traditional computational methods e.g. clustering in motif discovery. We then present a study of extending a machine learning model for transcriptional regulation predictive of genetic regulatory response to Caenorhabditis elegans. We show meaningful results both in terms of prediction accuracy on the test experiments and biological information extracted from the regulatory program. The model discovers DNA binding sites ab initio. We also present a case study where we detect a signal of lineage-specific regulation. Finally we present a comparative study on learning predictive models for motif discovery, based on different boosting algorithms: Adaptive Boosting (AdaBoost), Linear Programming Boosting (LPBoost) and Totally Corrective Boosting (TotalBoost). We evaluate and compare the performance of the three boosting algorithms via both statistical and biological validation, for hypoxia response in Saccharomyces cerevisiae.

  3. A Convex Atomic-Norm Approach to Multiple Sequence Alignment and Motif Discovery

    PubMed Central

    Yen, Ian E. H.; Lin, Xin; Zhang, Jiong; Ravikumar, Pradeep; Dhillon, Inderjit S.

    2016-01-01

    Multiple Sequence Alignment and Motif Discovery, known as NP-hard problems, are two fundamental tasks in Bioinformatics. Existing approaches to these two problems are based on either local search methods such as Expectation Maximization (EM), Gibbs Sampling or greedy heuristic methods. In this work, we develop a convex relaxation approach to both problems based on the recent concept of atomic norm and develop a new algorithm, termed Greedy Direction Method of Multiplier, for solving the convex relaxation with two convex atomic constraints. Experiments show that our convex relaxation approach produces solutions of higher quality than those standard tools widely-used in Bioinformatics community on the Multiple Sequence Alignment and Motif Discovery problems. PMID:27559428

  4. Phylogenomics-guided discovery of a novel conserved cassette of short linear motifs in BubR1 essential for the spindle checkpoint

    PubMed Central

    Bade, Debora

    2016-01-01

    The spindle assembly checkpoint (SAC) maintains genomic integrity by preventing progression of mitotic cell division until all chromosomes are stably attached to spindle microtubules. The SAC critically relies on the paralogues Bub1 and BubR1/Mad3, which integrate kinetochore–spindle attachment status with generation of the anaphase inhibitory complex MCC. We previously reported on the widespread occurrences of independent gene duplications of an ancestral ‘MadBub’ gene in eukaryotic evolution and the striking parallel subfunctionalization that lead to loss of kinase function in BubR1/Mad3-like paralogues. Here, we present an elaborate subfunctionalization analysis of the Bub1/BubR1 gene family and perform de novo sequence discovery in a comparative phylogenomics framework to trace the distribution of ancestral sequence features to extant paralogues throughout the eukaryotic tree of life. We show that known ancestral sequence features are consistently retained in the same functional paralogue: GLEBS/CMI/CDII/kinase in the Bub1-like and KEN1/KEN2/D-Box in the BubR1/Mad3-like. The recently described ABBA motif can be found in either or both paralogues. We however discovered two additional ABBA motifs that flank KEN2. This cassette of ABBA1-KEN2-ABBA2 forms a strictly conserved module in all ancestral and BubR1/Mad3-like proteins, suggestive of a specific and crucial SAC function. Indeed, deletion of the ABBA motifs in human BUBR1 abrogates the SAC and affects APC/C–Cdc20 interactions. Our detailed comparative genomics analyses thus enabled discovery of a conserved cassette of motifs essential for the SAC and shows how this approach can be used to uncover hitherto unrecognized functional protein features. PMID:28003474

  5. Functional importance of GGXG sequence motifs in putative reentrant loops of 2HCT and ESS transport proteins.

    PubMed

    Dobrowolski, Adam; Lolkema, Juke S

    2009-08-11

    The 2HCT and ESS families are two families of secondary transporters. Members of the two families are unrelated in amino acid sequence but share similar hydropathy profiles, which suggest a similar folding of the proteins in membranes. Structural models show two homologous domains containing five transmembrane segments (TMSs) each, with a reentrant or pore loop between the fourth and fifth TMSs in each domain. Here we show that GGXG sequence motifs present in the putative reentrant loops are important for the activity of the transporters. Mutation of the conserved Gly residues to Cys in the motifs of the Na(+)-citrate transporter CitS in the 2HCT family and the Na(+)-glutamate transporter GltS in the ESS family resulted in strongly reduced transport activity. Similarly, mutation of the variable residue "X" to Cys in the N-terminal half of GltS essentially inactivated the transporter. The corresponding mutations in the N- and C-terminal halves of CitS reduced transport activity to 60 and 25% of that of the wild type, respectively. Residual activity of any of the mutants could be further reduced by treatment with the membrane permeable thiol reagent N-ethylmaleimide (NEM). The X to Cys mutation (S405C) in the cytoplasmic loop in the C-terminal half of CitS rendered the protein sensitive to the bulky, membrane impermeable thiol reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AmdiS) added at the periplasmic side of the membrane, providing further evidence that this part of the loop is positioned between the transmembrane segments. The putative reentrant loop in the C-terminal half of the ESS family does not contain the GGXG motif, but a conserved stretch rich in Gly residues. Cysteine-scanning mutagenesis of a stretch of 18 residues in the GltS protein revealed two residues important for function. Mutant N356C was completely inactivated by treatment with NEM, and mutant P351C appeared to be the counterpart of mutant S405C of CitS; the mutant was

  6. 'Size leap' algorithm: an efficient extraction of the longest common motifs from a molecular sequence set. Application to the DNA sequence reconstruction.

    PubMed

    Danckaert, A; Chappey, C; Hazout, S

    1991-10-01

    We propose a new method, called 'size leap' algorithm, of search for motifs of maximum size and common to two fragments at least. It allows the creation of a reduced database of motifs from a set of sequences whose size obeys the series of Fibonacci numbers. The convenience lies in the efficiency of the motif extraction. It can be applied in the establishment of overlap regions for DNA sequence reconstruction and multiple alignment of biological sequences. The method of complete DNA sequence reconstruction by extraction of the longest motifs ('anchor motifs') is presented as an application of the size leap algorithm. The details of a reconstruction from three sequenced fragments are given as an example.

  7. The tungsten formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum contains sequence motifs characteristic for enzymes containing molybdopterin dinucleotide.

    PubMed

    Hochheimer, A; Schmitz, R A; Thauer, R K; Hedderich, R

    1995-12-15

    Formylmethanofuran dehydrogenases are molybdenum or tungsten iron-sulfur proteins containing a pterin dinucleotide cofactor. We report here on the primary structures of the four subunits FwdABCD of the tungsten enzyme from Methanobacterium thermoautotrophicum which were determined by cloning and sequencing the encoding genes fwdABCD. FwdB was found to contain sequence motifs characteristic for molybdopterin-dinucleotide-containing enzymes indicating that this subunit harbors the active site. FwdA, FwdC and FwdD showed no significant sequence similarity to proteins in the data bases. Northern blot analysis revealed that the four fwd genes form a transcription unit together with three additional genes designated fwdE, fwdF and fwdG. A 17.8-kDa protein and an 8.6-kDa protein, both containing two [4Fe-4S] cluster binding motifs, were deduced from fwdE and fwdG. The open reading frame fwdF encodes a 38.6-kDa protein containing eight binding motifs for [4Fe-4S] clusters suggesting the gene product to be a novel polyferredoxin. All seven fwd genes were expressed in Escherichia coli yielding proteins of the expected size. The fwd operon was found to be located in a region of the M. thermoautotrophicum genome encoding molybdenum enzymes and proteins involved in molybdopterin biosynthesis.

  8. Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis.

    PubMed

    Alamo, Lorenzo; Qi, Dan; Wriggers, Willy; Pinto, Antonio; Zhu, Jingui; Bilbao, Aivett; Gillilan, Richard E; Hu, Songnian; Padrón, Raúl

    2016-03-27

    Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and single-particle image processing revealed that, in a relaxed state, myosin molecules undergo intramolecular head-head interactions, explaining why head activity switches off. The filament model obtained by rigidly docking a chicken smooth muscle myosin structure to the reconstruction was improved by flexibly fitting an atomic model built by mixing structures from different species to a tilt-corrected 2-nm three-dimensional map of frozen-hydrated tarantula thick filament. We used heavy and light chain sequences from tarantula myosin to build a single-species homology model of two heavy meromyosin interacting-heads motifs (IHMs). The flexibly fitted model includes previously missing loops and shows five intramolecular and five intermolecular interactions that keep the IHM in a compact off structure, forming four helical tracks of IHMs around the backbone. The residues involved in these interactions are oppositely charged, and their sequence conservation suggests that IHM is present across animal species. The new model, PDB 3JBH, explains the structural origin of the ATP turnover rates detected in relaxed tarantula muscle by ascribing the very slow rate to docked unphosphorylated heads, the slow rate to phosphorylated docked heads, and the fast rate to phosphorylated undocked heads. The conservation of intramolecular interactions across animal species and the presence of IHM in bilaterians suggest that a super-relaxed state should be maintained, as it plays a role in saving ATP in skeletal, cardiac, and smooth muscles.

  9. Massive microRNA sequence conservation and prevalence in human and chimpanzee introns.

    PubMed

    Hill, Aubrey E; Sorscher, Eric J

    2013-06-01

    Human and chimpanzee introns contain numerous sequences strongly related to known microRNA hairpin structures. The relative frequency is precisely maintained across all chromosomes, suggesting the possible co-evolution of gene networks dependent upon microRNA regulation and with origins corresponding to the advent of primate transposable elements (TEs). While the motifs are known to be derived from transposable elements, the most common are far more numerous than expected from the number of TEs and their paralogous sequences, and exhibit striking conservation in comparison to the surrounding TE sequence context. Several of these motifs also exhibit structural complimentarity to each other, suggesting a pairing function at the level of DNA or RNA. These "pseudomicroRNAs," in semblance to pseudogenes, include hundreds of thousands of vestigial paralogs of primate microRNAs, many of which may have functioned historically or remain active today.

  10. Polar residues in a conserved motif spanning helices 1 and 2 are functionally important in the SulP transporter family.

    PubMed

    Leves, Fiona P; Tierney, M Louise; Howitt, Susan M

    2008-01-01

    The SulP family (including the SLC26 family) is a diverse family of anion transporters found in all domains of life, with different members transporting different anions. We used sequence and bioinformatics analysis of helices 1 and 2 of SulP family members to identify a conserved motif, extending the previously defined 'sulfate transporter motif'. The analysis showed that in addition to being highly conserved in both sequence and spacing, helices 1 and 2 contain a significant number of polar residues and are predicted to be buried within the protein interior, with at least some faces packed closely against other helices. This suggests a significant functional role for this region and we tested this by mutating polar residues in helices 1 and 2 in the sulfate transporter, SHST1. All mutations made, even those removing only a single hydroxyl group, had significant effects on transport. Many mutations abolished transport without affecting plasma membrane expression of the mutant protein, suggesting a functional role for these residues. Different helical faces appear to have different roles, with the most severe effects being localised to two interacting faces of helices 1 and 2. Our results confirm the predicted importance of conserved polar residues in helices 1 and 2 and suggest that transport of sulfate by SHST1 is dependent on a network of polar and aromatic interactions between these two helices.

  11. Conservation of Tubulin-Binding Sequences in TRPV1 throughout Evolution

    PubMed Central

    Sardar, Puspendu; Kumar, Abhishek; Bhandari, Anita; Goswami, Chandan

    2012-01-01

    Background Transient Receptor Potential Vanilloid sub type 1 (TRPV1), commonly known as capsaicin receptor can detect multiple stimuli ranging from noxious compounds, low pH, temperature as well as electromagnetic wave at different ranges. In addition, this receptor is involved in multiple physiological and sensory processes. Therefore, functions of TRPV1 have direct influences on adaptation and further evolution also. Availability of various eukaryotic genomic sequences in public domain facilitates us in studying the molecular evolution of TRPV1 protein and the respective conservation of certain domains, motifs and interacting regions that are functionally important. Methodology and Principal Findings Using statistical and bioinformatics tools, our analysis reveals that TRPV1 has evolved about ∼420 million years ago (MYA). Our analysis reveals that specific regions, domains and motifs of TRPV1 has gone through different selection pressure and thus have different levels of conservation. We found that among all, TRP box is the most conserved and thus have functional significance. Our results also indicate that the tubulin binding sequences (TBS) have evolutionary significance as these stretch sequences are more conserved than many other essential regions of TRPV1. The overall distribution of positively charged residues within the TBS motifs is conserved throughout evolution. In silico analysis reveals that the TBS-1 and TBS-2 of TRPV1 can form helical structures and may play important role in TRPV1 function. Conclusions and Significance Our analysis identifies the regions of TRPV1, which are important for structure – function relationship. This analysis indicates that tubulin binding sequence-1 (TBS-1) near the TRP-box forms a potential helix and the tubulin interactions with TRPV1 via TBS-1 have evolutionary significance. This interaction may be required for the proper channel function and regulation and may also have significance in the context of Taxol

  12. Peptide sequence motif analysis of tandem MS data with the SALSA algorithm.

    PubMed

    Liebler, Daniel C; Hansen, Beau T; Davey, Sean W; Tiscareno, Laura; Mason, Daniel E

    2002-01-01

    We have developed a pattern recognition algorithm called SALSA (scoring algorithm for spectral analysis) for the detection of specific features in tandem MS (MS-MS) spectra. Application of the SALSA algorithm to the detection of peptide MS-MS ion series enables identification of MS-MS spectra displaying characteristics of specific peptide sequences. SALSA analysis scores MS-MS spectra based on correspondence between theoretical ion series for peptide sequence motifs and actual MS-MS product ion series, regardless of their absolute positions on the m/z axis. Analyses of tryptic digests of bovine serum albumin (BSA) by LC-MS-MS followed by SALSA analysis detected MS-MS spectra for both unmodified and multiple modified forms of several BSA tryptic peptides. SALSA analysis of MS-MS data from mixtures of BSA and human serum albumin (HSA) tryptic digests indicated that ion series searches with BSA peptide sequence motifs identified MS-MS spectra for both BSA and closely related HSA peptides. Optimal discrimination between MS-MS spectra of variant peptide forms is achieved when the SALSA search criteria are optimized to the target peptide. Application of SALSA to LC-MS-MS proteome analysis will facilitate the characterization of modified and sequence variant proteins.

  13. CyanoLyase: a database of phycobilin lyase sequences, motifs and functions

    PubMed Central

    Bretaudeau, Anthony; Coste, François; Humily, Florian; Garczarek, Laurence; Le Corguillé, Gildas; Six, Christophe; Ratin, Morgane; Collin, Olivier; Schluchter, Wendy M.; Partensky, Frédéric

    2013-01-01

    CyanoLyase (http://cyanolyase.genouest.org/) is a manually curated sequence and motif database of phycobilin lyases and related proteins. These enzymes catalyze the covalent ligation of chromophores (phycobilins) to specific binding sites of phycobiliproteins (PBPs). The latter constitute the building bricks of phycobilisomes, the major light-harvesting systems of cyanobacteria and red algae. Phycobilin lyases sequences are poorly annotated in public databases. Sequences included in CyanoLyase were retrieved from all available genomes of these organisms and a few others by similarity searches using biochemically characterized enzyme sequences and then classified into 3 clans and 32 families. Amino acid motifs were computed for each family using Protomata learner. CyanoLyase also includes BLAST and a novel pattern matching tool (Protomatch) that allow users to rapidly retrieve and annotate lyases from any new genome. In addition, it provides phylogenetic analyses of all phycobilin lyases families, describes their function, their presence/absence in all genomes of the database (phyletic profiles) and predicts the chromophorylation of PBPs in each strain. The site also includes a thorough bibliography about phycobilin lyases and genomes included in the database. This resource should be useful to scientists and companies interested in natural or artificial PBPs, which have a number of biotechnological applications, notably as fluorescent markers. PMID:23175607

  14. A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes.

    PubMed Central

    Plumpton, M; McGarvey, M; Beggs, J D

    1994-01-01

    To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibited cell growth by causing a defect in pre-mRNA splicing. This defect was partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2-dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild-type level of RNA-stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein. Images PMID:8112301

  15. Conserved Functional Motifs and Homology Modeling to Predict Hidden Moonlighting Functional Sites

    PubMed Central

    Wong, Aloysius; Gehring, Chris; Irving, Helen R.

    2015-01-01

    Moonlighting functional centers within proteins can provide them with hitherto unrecognized functions. Here, we review how hidden moonlighting functional centers, which we define as binding sites that have catalytic activity or regulate protein function in a novel manner, can be identified using targeted bioinformatic searches. Functional motifs used in such searches include amino acid residues that are conserved across species and many of which have been assigned functional roles based on experimental evidence. Molecules that were identified in this manner seeking cyclic mononucleotide cyclases in plants are used as examples. The strength of this computational approach is enhanced when good homology models can be developed to test the functionality of the predicted centers in silico, which, in turn, increases confidence in the ability of the identified candidates to perform the predicted functions. Computational characterization of moonlighting functional centers is not diagnostic for catalysis but serves as a rapid screening method, and highlights testable targets from a potentially large pool of candidates for subsequent in vitro and in vivo experiments required to confirm the functionality of the predicted moonlighting centers. PMID:26106597

  16. Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif.

    PubMed

    Tesina, Petr; Čermáková, Kateřina; Hořejší, Magdalena; Procházková, Kateřina; Fábry, Milan; Sharma, Subhalakshmi; Christ, Frauke; Demeulemeester, Jonas; Debyser, Zeger; De Rijck, Jan; Veverka, Václav; Řezáčová, Pavlína

    2015-08-06

    Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors.

  17. Identification of sequence motifs involved in Dengue virus-host interactions.

    PubMed

    Asnet Mary, J; Paramasivan, R; Shenbagarathai, R

    2016-01-01

    Dengue fever is a rapidly spreading mosquito-borne virus infection, which remains a serious global public health problem. As there is no specific treatment or commercial vaccine available for effective control of the disease, the attempts on developing novel control strategies are underway. Viruses utilize the surface receptor proteins of host to enter into the cells. Though various proteins were said to be receptors of Dengue virus (DENV) using Virus Overlay Protein Binding Assay, the precise interaction between DENV and host is not explored. Understanding the structural features of domain III envelope glycoprotein would help in developing efficient antiviral inhibitors. Therefore, an attempt was made to identify the sequence motifs present in domain III envelope glycoprotein of Dengue virus. Computational analysis revealed that the NGR motif is present in the domain III envelope glycoprotein of DENV-1 and DENV-3. Similarly, DENV-1, DENV-2 and DENV-4 were found to contain Yxxphi motif which is a tyrosine-based sorting signal responsible for the interaction with a mu subunit of adaptor protein complex. High-throughput virtual screening resulted in five compounds as lead molecules based on glide score, which ranges from -4.664 to -6.52 kcal/Mol. This computational prediction provides an additional tool for understanding the virus-host interactions and helps to identify potential targets in the host. Further, experimental evidence is warranted to confirm the virus-host interactions and also inhibitory activity of reported lead compounds.

  18. Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets.

    PubMed

    Nelson, Christopher S; Fuller, Chris K; Fordyce, Polly M; Greninger, Alexander L; Li, Hao; DeRisi, Joseph L

    2013-07-01

    The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein's DNA-binding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2's-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved.

  19. Conserved motifs II to VI of DNA helicase II from Escherichia coli are all required for biological activity.

    PubMed Central

    Zhang, G; Deng, E; Baugh, L R; Hamilton, C M; Maples, V F; Kushner, S R

    1997-01-01

    There are seven conserved motifs (IA, IB, and II to VI) in DNA helicase II of Escherichia coli that have high homology among a large family of proteins involved in DNA metabolism. To address the functional importance of motifs II to VI, we employed site-directed mutagenesis to replace the charged amino acid residues in each motif with alanines. Cells carrying these mutant alleles exhibited higher UV and methyl methanesulfonate sensitivity, increased rates of spontaneous mutagenesis, and elevated levels of homologous recombination, indicating defects in both the excision repair and mismatch repair pathways. In addition, we also changed the highly conserved tyrosine(600) in motif VI to phenylalanine (uvrD309, Y600F). This mutant displayed a moderate increase in UV sensitivity but a decrease in spontaneous mutation rate, suggesting that DNA helicase II may have different functions in the two DNA repair pathways. Furthermore, a mutation in domain IV (uvrD307, R284A) significantly reduced the viability of some E. coli K-12 strains at 30 degrees C but not at 37 degrees C. The implications of these observations are discussed. PMID:9393722

  20. Functionally conserved enhancers with divergent sequences in distant vertebrates

    DOE PAGES

    Yang, Song; Oksenberg, Nir; Takayama, Sachiko; ...

    2015-10-30

    To examine the contributions of sequence and function conservation in the evolution of enhancers, we systematically identified enhancers whose sequences are not conserved among distant groups of vertebrate species, but have homologous function and are likely to be derived from a common ancestral sequence. In conclusion, our approach combined comparative genomics and epigenomics to identify potential enhancer sequences in the genomes of three groups of distantly related vertebrate species.

  1. A Conserved Structural Motif Mediates Retrograde Trafficking of Shiga Toxin Types 1 and 2.

    PubMed

    Selyunin, Andrey S; Mukhopadhyay, Somshuvra

    2015-12-01

    Shiga toxin-producing Escherichia coli (STEC) produce two types of Shiga toxin (STx): STx1 and STx2. The toxin A-subunits block protein synthesis, while the B-subunits mediate retrograde trafficking. STEC infections do not have definitive treatments, and there is growing interest in generating toxin transport inhibitors for therapy. However, a comprehensive understanding of the mechanisms of toxin trafficking is essential for drug development. While STx2 is more toxic in vivo, prior studies focused on STx1 B-subunit (STx1B) trafficking. Here, we show that, compared with STx1B, trafficking of the B-subunit of STx2 (STx2B) to the Golgi occurs with slower kinetics. Despite this difference, similar to STx1B, endosome-to-Golgi transport of STx2B does not involve transit through degradative late endosomes and is dependent on dynamin II, epsinR, retromer and syntaxin5. Importantly, additional experiments show that a surface-exposed loop in STx2B (β4-β5 loop) is required for its endosome-to-Golgi trafficking. We previously demonstrated that residues in the corresponding β4-β5 loop of STx1B are required for interaction with GPP130, the STx1B-specific endosomal receptor, and for endosome-to-Golgi transport. Overall, STx1B and STx2B share a common pathway and use a similar structural motif to traffic to the Golgi, suggesting that the underlying mechanisms of endosomal sorting may be evolutionarily conserved.

  2. Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs.

    PubMed

    Kazakov, Alexei S; Sokolov, Andrei S; Vologzhannikova, Alisa A; Permyakova, Maria E; Khorn, Polina A; Ismailov, Ramis G; Denessiouk, Konstantin A; Denesyuk, Alexander I; Rastrygina, Victoria A; Baksheeva, Viktoriia E; Zernii, Evgeni Yu; Zinchenko, Dmitry V; Glazatov, Vladimir V; Uversky, Vladimir N; Mirzabekov, Tajib A; Permyakov, Eugene A; Permyakov, Sergei E

    2017-01-01

    Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1-19 μM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, 'black' and 'gray' clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.

  3. Functional conservation of PISTILLATA activity in a pea homolog lacking the PI motif.

    PubMed

    Berbel, Ana; Navarro, Cristina; Ferrándiz, Cristina; Cañas, Luis Antonio; Beltrán, José-Pío; Madueño, Francisco

    2005-09-01

    Current understanding of floral development is mainly based on what we know from Arabidopsis (Arabidopsis thaliana) and Antirrhinum majus. However, we can learn more by comparing developmental mechanisms that may explain morphological differences between species. A good example comes from the analysis of genes controlling flower development in pea (Pisum sativum), a plant with more complex leaves and inflorescences than Arabidopsis and Antirrhinum, and a different floral ontogeny. The analysis of UNIFOLIATA (UNI) and STAMINA PISTILLOIDA (STP), the pea orthologs of LEAFY and UNUSUAL FLORAL ORGANS, has revealed a common link in the regulation of flower and leaf development not apparent in Arabidopsis. While the Arabidopsis genes mainly behave as key regulators of flower development, where they control the expression of B-function genes, UNI and STP also contribute to the development of the pea compound leaf. Here, we describe the characterization of P. sativum PISTILLATA (PsPI), a pea MADS-box gene homologous to B-function genes like PI and GLOBOSA (GLO), from Arabidopsis and Antirrhinum, respectively. PsPI encodes for an atypical PI-type polypeptide that lacks the highly conserved C-terminal PI motif. Nevertheless, constitutive expression of PsPI in tobacco (Nicotiana tabacum) and Arabidopsis shows that it can specifically replace the function of PI, being able to complement the strong pi-1 mutant. Accordingly, PsPI expression in pea flowers, which is dependent on STP, is identical to PI and GLO. Interestingly, PsPI is also transiently expressed in young leaves, suggesting a role of PsPI in pea leaf development, a possibility that fits with the established role of UNI and STP in the control of this process.

  4. Quadfinder: server for identification and analysis of quadruplex-forming motifs in nucleotide sequences

    PubMed Central

    Scaria, Vinod; Hariharan, Manoj; Arora, Amit; Maiti, Souvik

    2006-01-01

    G-quadruplex secondary structures, which play a structural role in repetitive DNA such as telomeres, may also play a functional role at other genomic locations as targetable regulatory elements which control gene expression. The recent interest in application of quadruplexes in biological systems prompted us to develop a tool for the identification and analysis of quadruplex-forming nucleotide sequences especially in the RNA. Here we present Quadfinder, an online server for prediction and bioinformatics of uni-molecular quadruplex-forming nucleotide sequences. The server is designed to be user-friendly and needs minimal intervention by the user, while providing flexibility of defining the variants of the motif. The server is freely available at URL . PMID:16845097

  5. A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication.

    PubMed Central

    Koonin, E V

    1993-01-01

    A new superfamily of (putative) DNA-dependent ATPases is described that includes the ATPase domains of prokaryotic NtrC-related transcription regulators, MCM proteins involved in the initiation of eukaryotic DNA replication, and a group of uncharacterized bacterial and chloroplast proteins. MCM proteins are shown to contain a modified form of the ATP-binding motif and are predicted to mediate ATP-dependent opening of double-stranded DNA in the replication origins. In a second line of investigation, it is demonstrated that the products of unidentified open reading frames from Marchantia mitochondria and from yeast, and a domain of a baculovirus protein involved in viral DNA replication are related to the superfamily III of DNA and RNA helicases that previously has been known to include only proteins of small viruses. Comparison of the multiple alignments showed that the proteins of the NtrC superfamily and the helicases of superfamily III share three related sequence motifs tightly packed in the ATPase domain that consists of 100-150 amino acid residues. A similar array of conserved motifs is found in the family of DnaA-related ATPases. It is hypothesized that the three large groups of nucleic acid-dependent ATPases have similar structure of the core ATPase domain and have evolved from a common ancestor. PMID:8332451

  6. Functional characterization of sequence motifs in the transit peptide of Arabidopsis small subunit of rubisco.

    PubMed

    Lee, Dong Wook; Lee, Sookjin; Lee, Gil-Je; Lee, Kwang Hee; Kim, Sanguk; Cheong, Gang-Won; Hwang, Inhwan

    2006-02-01

    The transit peptides of nuclear-encoded chloroplast proteins are necessary and sufficient for targeting and import of proteins into chloroplasts. However, the sequence information encoded by transit peptides is not fully understood. In this study, we investigated sequence motifs in the transit peptide of the small subunit of the Rubisco complex by examining the ability of various mutant transit peptides to target green fluorescent protein reporter proteins to chloroplasts in Arabidopsis (Arabidopsis thaliana) leaf protoplasts. We divided the transit peptide into eight blocks (T1 through T8), each consisting of eight or 10 amino acids, and generated mutants that had alanine (Ala) substitutions or deletions, of one or two T blocks in the transit peptide. In addition, we generated mutants that had the original sequence partially restored in single- or double-T-block Ala (A) substitution mutants. Analysis of chloroplast import of these mutants revealed several interesting observations. Single-T-block mutations did not noticeably affect targeting efficiency, except in T1 and T4 mutations. However, double-T mutants, T2A/T4A, T3A/T6A, T3A/T7A, T4A/T6A, and T4A/T7A, caused a 50% to 100% loss in targeting ability. T3A/T6A and T4A/T6A mutants produced only precursor proteins, whereas T2A/T4A and T4A/T7A mutants produced only a 37-kD protein. Detailed analyses revealed that sequence motifs ML in T1, LKSSA in T3, FP and RK in T4, CMQVW in T6, and KKFET in T7 play important roles in chloroplast targeting. In T1, the hydrophobicity of ML is important for targeting. LKSSA in T3 is functionally equivalent to CMQVW in T6 and KKFET in T7. Furthermore, subcellular fractionation revealed that Ala substitution in T1, T3, and T6 produced soluble precursors, whereas Ala substitution in T4 and T7 produced intermediates that were tightly associated with membranes. These results demonstrate that the transit peptide contains multiple motifs and that some of them act in concert or

  7. Using Weeder, Pscan, and PscanChIP for the Discovery of Enriched Transcription Factor Binding Site Motifs in Nucleotide Sequences.

    PubMed

    Zambelli, Federico; Pesole, Graziano; Pavesi, Giulio

    2014-09-08

    One of the greatest challenges facing modern molecular biology is understanding the complex mechanisms regulating gene expression. A fundamental step in this process requires the characterization of sequence motifs involved in the regulation of gene expression at transcriptional and post-transcriptional levels. In particular, transcription is modulated by the interaction of transcription factors (TFs) with their corresponding binding sites. Weeder, Pscan, and PscanChIP are software tools freely available for noncommercial users as a stand-alone or Web-based applications for the automatic discovery of conserved motifs in a set of DNA sequences likely to be bound by the same TFs. Input for the tools can be promoter sequences from co-expressed or co-regulated genes (for which Weeder and Pscan are suitable), or regions identified through genome wide ChIP-seq or similar experiments (Weeder and PscanChIP). The motifs are either found by a de novo approach (Weeder) or by using descriptors of the binding specificity of TFs (Pscan and PscanChIP).

  8. A family of cyclin D homologs from plants differentially controlled by growth regulators and containing the conserved retinoblastoma protein interaction motif.

    PubMed Central

    Soni, R; Carmichael, J P; Shah, Z H; Murray, J A

    1995-01-01

    A new family of three related cyclins has been identified in Arabidopsis by complementation of a yeast strain deficient in G1 cyclins. Individual members show tissue-specific expression and are conserved in other plant species. They form a distinctive group of plant cyclins, which we named delta-type cyclins to indicate their similarities with mammalian D-type cyclins. The sequence relationships between delta and D cyclins include the N-terminal sequence LXCXE. This motif was originally identified in certain viral oncoproteins and is strongly implicated in binding to the retinoblastoma protein pRb. By analogy to mammalian cyclin D, these plant homologs may mediate growth and phytohormonal signals into the plant cell cycle. In support of this hypothesis, we show that, on restimulation of suspension-cultured cells, cyclin delta 3 is rapidly induced by the plant growth regulator cytokinin and cyclin delta 2 is induced by carbon source. PMID:7696881

  9. A conserved structural motif reveals the essential transcriptional repression function of Spen proteins and their role in developmental signaling.

    PubMed

    Ariyoshi, Mariko; Schwabe, John W R

    2003-08-01

    Spen proteins regulate the expression of key transcriptional effectors in diverse signaling pathways. They are large proteins characterized by N-terminal RNA-binding motifs and a highly conserved C-terminal SPOC domain. The specific biological role of the SPOC domain (Spen paralog and ortholog C-terminal domain), and hence, the common function of Spen proteins, has been unclear to date. The Spen protein, SHARP (SMRT/HDAC1-associated repressor protein), was identified as a component of transcriptional repression complexes in both nuclear receptor and Notch/RBP-Jkappa signaling pathways. We have determined the 1.8 A crystal structure of the SPOC domain from SHARP. This structure shows that essentially all of the conserved surface residues map to a positively charged patch. Structure-based mutational analysis indicates that this conserved region is responsible for the interaction between SHARP and the universal transcriptional corepressor SMRT/NCoR (silencing mediator for retinoid and thyroid receptors/nuclear receptor corepressor. We demonstrate that this interaction involves a highly conserved acidic motif at the C terminus of SMRT/NCoR. These findings suggest that the conserved function of the SPOC domain is to mediate interaction with SMRT/NCoR corepressors, and that Spen proteins play an essential role in the repression complex.

  10. Candidate disease resistance genes in sunflower cloned using conserved nucleotide-binding site motifs: genetic mapping and linkage to the downy mildew resistance gene Pl1.

    PubMed

    Gedil, M A; Slabaugh, M B; Berry, S; Johnson, R; Michelmore, R; Miller, J; Gulya, T; Knapp, S J

    2001-04-01

    Disease resistance gene candidates (RGCs) belonging to the nucleotide-binding site (NBS) superfamily have been cloned from numerous crop plants using highly conserved DNA sequence motifs. The aims of this research were to (i) isolate genomic DNA clones for RGCs in cultivated sunflower (Helianthus annuus L.) and (ii) map RGC markers and Pl1, a gene for resistance to downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni) race 1. Degenerate oligonucleotide primers targeted to conserved NBS DNA sequence motifs were used to amplify RGC fragments from sunflower genomic DNA. PCR products were cloned, sequenced, and assigned to 11 groups. RFLP analyses mapped six RGC loci to three linkage groups. One of the RGCs (Ha-4W2) was linked to Pl1, a downy mildew resistance gene. A cleaved amplified polymorphic sequence (CAPS) marker was developed for Ha-4W2 using gene-specific oligonucleotide primers. Downy mildew susceptible lines (HA89 and HA372) lacked a 276-bp Tsp5091 restriction fragment that was present in downy mildew resistant lines (HA370, 335, 336, 337, 338, and 339). HA370 x HA372 F2 progeny were genotyped for the Ha-4W2 CAPS marker and phenotyped for resistance to downy mildew race 1. The CAPS marker was linked to but did not completely cosegregate with Pl1 on linkage group 8. Ha-4W2 was found to comprise a gene family with at least five members. Although genetic markers for Ha-4W2 have utility for marker-assisted selection, the RGC detected by the CAPS marker has been ruled out as a candidate gene for Pl1. Three of the RGC probes were monomorphic between HA370 and HA372 and still need to be mapped and screened for linkage to disease resistance loci.

  11. Patterns of sequence conservation in the S-Layer proteins and related sequences in Clostridium difficile.

    PubMed

    Calabi, Emanuela; Fairweather, Neil

    2002-07-01

    Clostridium difficile is the etiological agent of antibiotic-associated diarrhea. Among the factors that may play a role in infection are S-layer proteins (SLPs). Previous work has shown these to consist mainly of two components, resulting from the cleavage of a precursor encoded by the slpA gene. The high-molecular-weight (MW) subunit is related both to amidases from B. subtilis and to at least another 28 gene products in C. difficile strain 630. To gain insight into the functions of the SLPs and related proteins, we have further investigated the pattern of variability both at the slpA locus and at six nearby paralogs. Sequencing of the slpA gene from an S-layer group II strain and a variant S-layer group strain confirms a high degree of divergence in the low-MW SLP, which may result from diversifying selection. A highly conserved motif, however, is found at the C terminus in all low-MW subunits and may be essential for SlpA precursor cleavage. In strain 167, a variant cleavage product is present, suggesting a secondary processing site. Southern blotting analysis shows slpA-like open reading frames (ORFs) 2 to 7 to be conserved in all nine strains tested, with one exception: ORF2, which encodes a 66-kDa polypeptide coextracted at low pH with the main SLPs in strain 630, may be partially deleted in strain 167. Polymorphism within the slpA-ORF7 cluster may be more pronounced in the region proximal to the slpA gene. Unexpectedly, a high-MW subunit probe cross hybridizes to sequences outside the slpA locus, which appear to vary in number in different strains.

  12. Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae

    PubMed Central

    Ostrow, A. Zachary; Gan, Yan; Villwock, Sandra K.; Linke, Christian; Barberis, Matteo; Chen, Lin; Aparicio, Oscar M.

    2017-01-01

    Forkhead Box (Fox) proteins share the Forkhead domain, a winged-helix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified Saccharomyces cerevisiae Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as required for the clustering of a subset of replication origins in G1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here, we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homodimer formation. Fkh1- and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance. PMID:28265091

  13. Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae.

    PubMed

    Ostrow, A Zachary; Kalhor, Reza; Gan, Yan; Villwock, Sandra K; Linke, Christian; Barberis, Matteo; Chen, Lin; Aparicio, Oscar M

    2017-03-21

    Forkhead Box (Fox) proteins share the Forkhead domain, a winged-helix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified Saccharomyces cerevisiae Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as required for the clustering of a subset of replication origins in G1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here, we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homodimer formation. Fkh1- and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance.

  14. Conserved XPB Core Structure and Motifs for DNA Unwinding:Implications for Pathway Selection of Transcription or ExcisionRepair

    SciTech Connect

    Fan, Li; Arval, Andrew S.; Cooper, Priscilla K.; Iwai, Shigenori; Hanaoka, Fumio; Tainer, John A.

    2005-04-01

    The human xeroderma pigmentosum group B (XPB) helicase is essential for transcription, nucleotide excision repair, and TFIIH functional assembly. Here, we determined crystal structures of an Archaeoglobus fulgidus XPB homolog (AfXPB) that characterize two RecA-like XPB helicase domains and discover a DNA damage recognition domain (DRD), a unique RED motif, a flexible thumb motif (ThM), and implied conformational changes within a conserved functional core. RED motif mutations dramatically reduce helicase activity, and the DRD and ThM, which flank the RED motif, appear structurally as well as functionally analogous to the MutS mismatch recognition and DNA polymerase thumb domains. Substrate specificity is altered by DNA damage, such that AfXPB unwinds dsDNA with 3' extensions, but not blunt-ended dsDNA, unless it contains a lesion, as shown for CPD or (6-4) photoproducts. Together, these results provide an unexpected mechanism of DNA unwinding with Implications for XPB damage verification in nucleotide excision repair.

  15. The Carboxy Terminus of YCF1 Contains a Motif Conserved throughout >500 Myr of Streptophyte Evolution

    PubMed Central

    Archibald, John M.; Gould, Sven B.

    2017-01-01

    Plastids evolved from cyanobacteria by endosymbiosis. During the course of evolution, the coding capacity of plastid genomes shrinks due to gene loss or transfer to the nucleus. In the green lineage, however, there were apparent gene gains including that of ycf1. Although its function is still debated, YCF1 has proven to be a useful marker for plastid evolution. YCF1 sequence and predicted structural features unite the plastid genomes of land plants with those of their closest algal relatives, the higher streptophyte algae; YCF1 appears to have undergone pronounced changes during the course of streptophyte algal evolution. Using new data, we show that YCF1 underwent divergent evolution in the common ancestor of higher streptophyte algae and Klebsormidiophycae. This divergence resulted in the origin of an extreme, klebsormidiophycean-specific YCF1 and the higher streptophyte Ste-YCF1. Most importantly, our analysis uncovers a conserved carboxy-terminal sequence stretch within YCF1 that is unique to higher streptophytes and hints at an important, yet unexplored function. PMID:28164224

  16. The Motif Tool Assessment Platform (MTAP) for sequence-based transcription factor binding site prediction tools.

    PubMed

    Quest, Daniel; Ali, Hesham

    2010-01-01

    Predicting transcription factor binding sites (TFBS) from sequence is one of the most challenging problems in computational biology. The development of (semi-)automated computer-assisted prediction methods is needed to find TFBS over an entire genome, which is a first step in reconstructing mechanisms that control gene activity. Bioinformatics journals continue to publish diverse methods for predicting TFBS on a monthly basis. To help practitioners in deciding which method to use to predict for a particular TFBS, we provide a platform to assess the quality and applicability of the available methods. Assessment tools allow researchers to determine how methods can be expected to perform on specific organisms or on specific transcription factor families. This chapter introduces the TFBS detection problem and reviews current strategies for evaluating algorithm effectiveness. In this chapter, a novel and robust assessment tool, the Motif Tool Assessment Platform (MTAP), is introduced and discussed.

  17. The structure of an endogenous Drosophila centromere reveals the prevalence of tandemly repeated sequences able to form i-motifs

    PubMed Central

    Garavís, Miguel; Méndez-Lago, María; Gabelica, Valérie; Whitehead, Siobhan L.; González, Carlos; Villasante, Alfredo

    2015-01-01

    Centromeres are the chromosomal loci at which spindle microtubules attach to mediate chromosome segregation during mitosis and meiosis. In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements). Despite the efforts to establish complete genomic sequences of eukaryotic organisms, the so-called ‘finished’ genomes are not actually complete because the centromeres have not been assembled due to the intrinsic difficulties in constructing both physical maps and complete sequence assemblies of long stretches of tandemly repetitive DNA. Here we show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs. The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures. PMID:26289671

  18. Exceptional motifs in different Markov chain models for a statistical analysis of DNA sequences.

    PubMed

    Schbath, S; Prum, B; de Turckheim, E

    1995-01-01

    Identifying exceptional motifs is often used for extracting information from long DNA sequences. The two difficulties of the method are the choice of the model that defines the expected frequencies of words and the approximation of the variance of the difference T(W) between the number of occurrences of a word W and its estimation. We consider here different Markov chain models, either with stationary or periodic transition probabilities. We estimate the variance of the difference T(W) by the conditional variance of the number of occurrences of W given the oligonucleotides counts that define the model. Two applications show how to use asymptotically standard normal statistics associated with the counts to describe a given sequence in terms of its outlying words. Sequences of Escherichia coli and of Bacillus subtilis are compared with respect to their exceptional tri- and tetranucleotides. For both bacteria, exceptional 3-words are mainly found in the coding frame. E. coli palindrome counts are analyzed in different models, showing that many overabundant words are one-letter mutations of avoided palindromes.

  19. Nucleotide sequence conservation of novel and established cis-regulatory sites within the tyrosine hydroxylase gene promoter

    PubMed Central

    Wang, Meng; Banerjee, Kasturi; Baker, Harriet; Cave, John W.

    2015-01-01

    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis and its gene proximal promoter ( < 1 kb upstream from the transcription start site) is essential for regulating transcription in both the developing and adult nervous systems. Several putative regulatory elements within the TH proximal promoter have been reported, but evolutionary conservation of these elements has not been thoroughly investigated. Since many vertebrate species are used to model development, function and disorders of human catecholaminergic neurons, identifying evolutionarily conserved transcription regulatory mechanisms is a high priority. In this study, we align TH proximal promoter nucleotide sequences from several vertebrate species to identify evolutionarily conserved motifs. This analysis identified three elements (a TATA box, cyclic AMP response element (CRE) and a 5′-GGTGG-3′ site) that constitute the core of an ancient vertebrate TH promoter. Focusing on only eutherian mammals, two regions of high conservation within the proximal promoter were identified: a ∼250 bp region adjacent to the transcription start site and a ∼85 bp region located approximately 350 bp further upstream. Within both regions, conservation of previously reported cis-regulatory motifs and human single nucleotide variants was evaluated. Transcription reporter assays in a TH -expressing cell line demonstrated the functionality of highly conserved motifs in the proximal promoter regions and electromobility shift assays showed that brain-region specific complexes assemble on these motifs. These studies also identified a non-canonical CRE binding (CREB) protein recognition element in the proximal promoter. Together, these studies provide a detailed analysis of evolutionary conservation within the TH promoter and identify potential cis-regulatory motifs that underlie a core set of regulatory mechanisms in mammals. PMID:25774193

  20. Synthesis, anti-mycobacterial activity and DNA sequence-selectivity of a library of biaryl-motifs containing polyamides.

    PubMed

    Brucoli, Federico; Guzman, Juan D; Maitra, Arundhati; James, Colin H; Fox, Keith R; Bhakta, Sanjib

    2015-07-01

    The alarming rise of extensively drug-resistant tuberculosis (XDR-TB) strains, compel the development of new molecules with novel modes of action to control this world health emergency. Distamycin analogues containing N-terminal biaryl-motifs 2(1-5)(1-7) were synthesised using a solution-phase approach and evaluated for their anti-mycobacterial activity and DNA-sequence selectivity. Thiophene dimer motif-containing polyamide 2(2,6) exhibited 10-fold higher inhibitory activity against Mycobacterium tuberculosis compared to distamycin and library member 2(5,7) showed high binding affinity for the 5'-ACATAT-3' sequence.

  1. Inference of transcriptional networks in Arabidopsis through conserved noncoding sequence analysis.

    PubMed

    Van de Velde, Jan; Heyndrickx, Ken S; Vandepoele, Klaas

    2014-07-01

    Transcriptional regulation plays an important role in establishing gene expression profiles during development or in response to (a)biotic stimuli. Transcription factor binding sites (TFBSs) are the functional elements that determine transcriptional activity, and the identification of individual TFBS in genome sequences is a major goal to inferring regulatory networks. We have developed a phylogenetic footprinting approach for the identification of conserved noncoding sequences (CNSs) across 12 dicot plants. Whereas both alignment and non-alignment-based techniques were applied to identify functional motifs in a multispecies context, our method accounts for incomplete motif conservation as well as high sequence divergence between related species. We identified 69,361 footprints associated with 17,895 genes. Through the integration of known TFBS obtained from the literature and experimental studies, we used the CNSs to compile a gene regulatory network in Arabidopsis thaliana containing 40,758 interactions, of which two-thirds act through binding events located in DNase I hypersensitive sites. This network shows significant enrichment toward in vivo targets of known regulators, and its overall quality was confirmed using five different biological validation metrics. Finally, through the integration of detailed expression and function information, we demonstrate how static CNSs can be converted into condition-dependent regulatory networks, offering opportunities for regulatory gene annotation.

  2. Functional Role of Histidine in the Conserved His-x-Asp Motif in the Catalytic Core of Protein Kinases.

    PubMed

    Zhang, Lun; Wang, Jian-Chuan; Hou, Li; Cao, Peng-Rong; Wu, Li; Zhang, Qian-Sen; Yang, Huai-Yu; Zang, Yi; Ding, Jian-Ping; Li, Jia

    2015-05-11

    The His-x-Asp (HxD) motif is one of the most conserved structural components of the catalytic core of protein kinases; however, the functional role of the conserved histidine is unclear. Here we report that replacement of the HxD-histidine with Arginine or Phenylalanine in Aurora A abolishes both the catalytic activity and auto-phosphorylation, whereas the Histidine-to-tyrosine impairs the catalytic activity without affecting its auto-phosphorylation. Comparisons of the crystal structures of wild-type (WT) and mutant Aurora A demonstrate that the impairment of the kinase activity is accounted for by (1) disruption of the regulatory spine in the His-to-Arg mutant, and (2) change in the geometry of backbones of the Asp-Phe-Gly (DFG) motif and the DFG-1 residue in the His-to-Tyr mutant. In addition, bioinformatics analyses show that the HxD-histidine is a mutational hotspot in tumor tissues. Moreover, the H174R mutation of the HxD-histidine, in the tumor suppressor LKB1 abrogates the inhibition of anchorage-independent growth of A549 cells by WT LKB1. Based on these data, we propose that the HxD-histidine is involved in a conserved inflexible organization of the catalytic core that is required for the kinase activity. Mutation of the HxD-histidine may also be involved in the pathogenesis of some diseases including cancer.

  3. A Developmental Sequence of Skills Leading to Conservation

    ERIC Educational Resources Information Center

    Walker, Alice A.

    1978-01-01

    Examines the developmental sequence of skills involved in the understanding of relational concepts and in the development of conservation. Fifty kindergarten children participated in the study. (BD/BR)

  4. Engineering Proteins with Enhanced Mechanical Stability by Force Specific Sequence Motifs

    PubMed Central

    Lu, Wenzhe; Negi, Surendra; Oberhauser, Andres F.; Braun, Werner

    2012-01-01

    Use of atomic force microscopy (AFM) has recently led to a better understanding of the molecular mechanisms of the unfolding process by mechanical forces; however, the rational design of novel proteins with specific mechanical strength remains challenging. We have approached this problem from a new perspective that generates linear physical-chemical properties (PCP) motifs from a limited AFM data set. Guided by our linear sequence analysis we designed and analyzed four new mutants of the titin I1 domain with the goal of increasing the domain's mechanical strength. All four mutants could be cloned and expressed as soluble proteins. AFM data indicate that at least two of the mutants have increased molecular mechanical strength. This observation suggests that the PCP method is useful to graft sequences specific for high mechanical stability to weak proteins to increase their mechanical stability, and represents an additional tool in the design of novel proteins besides steered molecular dynamics calculations, coarse grained simulations and phi-value analysis of the transition state. PMID:22274941

  5. CSTminer: a web tool for the identification of coding and noncoding conserved sequence tags through cross-species genome comparison.

    PubMed

    Castrignanò, Tiziana; Canali, Alessandro; Grillo, Giorgio; Liuni, Sabino; Mignone, Flavio; Pesole, Graziano

    2004-07-01

    The identification and characterization of genome tracts that are highly conserved across species during evolution may contribute significantly to the functional annotation of whole-genome sequences. Indeed, such sequences are likely to correspond to known or unknown coding exons or regulatory motifs. Here, we present a web server implementing a previously developed algorithm that, by comparing user-submitted genome sequences, is able to identify statistically significant conserved blocks and assess their coding or noncoding nature through the measure of a coding potential score. The web tool, available at http://www.caspur.it/CSTminer/, is dynamically interconnected with the Ensembl genome resources and produces a graphical output showing a map of detected conserved sequences and annotated gene features.

  6. Structure of PEP carboxykinase from the succinate-producing Actinobacillus succinogenes: a new conserved active-site motif.

    PubMed

    Leduc, Yvonne A; Prasad, Lata; Laivenieks, Maris; Zeikus, J Gregory; Delbaere, Louis T J

    2005-07-01

    Actinobacillus succinogenes can produce, via fermentation, high concentrations of succinate, an important industrial commodity. A key enzyme in this pathway is phosphoenolpyruvate carboxykinase (PCK), which catalyzes the production of oxaloacetate from phosphoenolpyruvate and carbon dioxide, with the concomitant conversion of adenosine 5'-diphosphate to adenosine 5'-triphosphate. 1.85 and 1.70 A resolution structures of the native and a pyruvate/Mn(2+)/phosphate complex have been solved, respectively. The structure of the complex contains sulfhydryl reducing agents covalently bound to three cysteine residues via disulfide bonds. One of these cysteine residues (Cys285) is located in the active-site cleft and may be analogous to the putative reactive cysteine of PCK from Trypanosoma cruzi. Cys285 is also part of a previously unreported conserved motif comprising residues 280-287 and containing the pattern NXEXGXY(/F)A(/G); this new motif appears to have a structural role in stabilizing and positioning side chains that bind substrates and metal ions. The first few residues of this motif connect the two domains of the enzyme and a fulcrum point appears to be located near Asn280. In addition, an active-site Asp residue forms two coordinate bonds with the Mn(2+) ion present in the structure of the complex in a symmetrical bidentate manner, unlike in other PCK structures that contain a manganese ion.

  7. A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation

    PubMed Central

    Liu, Xin; Zhang, Chen-Song; Lu, Chang; Lin, Sheng-Cai; Wu, Jia-Wei; Wang, Zhi-Xin

    2016-01-01

    Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs). MAPK signalling efficiency and specificity is modulated by protein–protein interactions between individual MAPKs and the docking motifs in cognate binding partners. Two types of docking interactions have been identified: D-motif-mediated interaction and FXF-docking interaction. Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-Å resolution, providing high-resolution structural insight into the FXF-docking interaction. The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. PMID:26988444

  8. Comparative Analysis of P450 Signature Motifs EXXR and CXG in the Large and Diverse Kingdom of Fungi: Identification of Evolutionarily Conserved Amino Acid Patterns Characteristic of P450 Family

    PubMed Central

    Syed, Khajamohiddin; Mashele, Samson Sitheni

    2014-01-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins distributed across the biological kingdoms. P450s are catalytically versatile and play key roles in organisms primary and secondary metabolism. Identification of P450s across the biological kingdoms depends largely on the identification of two P450 signature motifs, EXXR and CXG, in the protein sequence. Once a putative protein has been identified as P450, it will be assigned to a family and subfamily based on the criteria that P450s within a family share more than 40% homology and members of subfamilies share more than 55% homology. However, to date, no evidence has been presented that can distinguish members of a P450 family. Here, for the first time we report the identification of EXXR- and CXG-motifs-based amino acid patterns that are characteristic of the P450 family. Analysis of P450 signature motifs in the under-explored fungal P450s from four different phyla, ascomycota, basidiomycota, zygomycota and chytridiomycota, indicated that the EXXR motif is highly variable and the CXG motif is somewhat variable. The amino acids threonine and leucine are preferred as second and third amino acids in the EXXR motif and proline and glycine are preferred as second and third amino acids in the CXG motif in fungal P450s. Analysis of 67 P450 families from biological kingdoms such as plants, animals, bacteria and fungi showed conservation of a set of amino acid patterns characteristic of a particular P450 family in EXXR and CXG motifs. This suggests that during the divergence of P450 families from a common ancestor these amino acids patterns evolve and are retained in each P450 family as a signature of that family. The role of amino acid patterns characteristic of a P450 family in the structural and/or functional aspects of members of the P450 family is a topic for future research. PMID:24743800

  9. Flow Cytometry-assisted Cloning of Specific Sequence Motifs fromComplex 16S ribosomal RNA Gene Libraries.

    SciTech Connect

    Nielsen, J.L.; Schramm, A.; Bernhard, A.E.; van den Engh, G.J.; Stahl, D.A.

    2004-07-21

    A flow cytometry method was developed for rapid screeningand recovery of cloned DNA containing common sequence motifs. Thisapproach, termed fluorescence-activated cell sorting-assisted cloning,was used to recover sequences affiliated with a unique lineage within theBacteroidetes not abundant in a clone library of environmental 16S rRNAgenes. Retrieval and sequence analysis of phylogenetically informativegenes has become a standard cultivation-independent technique toinvestigate microbial diversity in nature (7, 18). Genes encoding the 16SrRNA, because of the relative ease of their selective amplification, havebeen most frequently employed for general diversity surveys (16).Environmental studies have also focused on specific subpopulationsaffiliated with a phylogenetic group or identified by genes encodingspecific metabolic functions (e.g., ammonia oxidation, sulfaterespiration, and nitrate reduction) (8,15,20). However, specificpopulations may be of low abundance (1,23), or the genes encodingspecific metabolic functions may be insufficiently conserved to providepriming sites for general PCR amplification. Three general approacheshave been used to obtain 16S rRNA sequence information from low-abundancepopulations: screening hundreds to thousands of clones in a general 16SrRNA gene library (21), flow cytometric sorting of a subpopulation ofenvironmentally derived cells labeled by fluorescent in situhybridization (FISH) (27), or selective PCR amplification using primersspecific for the subpopulation (2,23). While the first approach is simplytime-consuming and tedious, the second has been restricted to fairlylarge and strongly fluorescent cells from aquatic samples (5, 27). Thethird approach often generates fragments of only a few hundred bases dueto the limited number of specific priming sites. Partial sequenceinformation often degrades analysis, obscuring or distorting thephylogenetic placement of the new sequences (11, 20). A more robustcharacterization of environ

  10. Structural analysis of the regulatory elements of the type-II procollagen gene. Conservation of promoter and first intron sequences between human and mouse.

    PubMed Central

    Vikkula, M; Metsäranta, M; Syvänen, A C; Ala-Kokko, L; Vuorio, E; Peltonen, L

    1992-01-01

    Transcription of the type-II procollagen gene (COL2A1) is very specifically restricted to a limited number of tissues, particularly cartilages. In order to identify transcription-control motifs we have sequenced the promoter region and the first intron of the human and mouse COL2A1 genes. With the assumption that these motifs should be well conserved during evolution, we have searched for potential elements important for the tissue-specific transcription of the COL2A1 gene by aligning the two sequences with each other and with the available rat type-II procollagen sequence for the promoter. With this approach we could identify specific evolutionarily well-conserved motifs in the promoter area. On the other hand, several suggested regulatory elements in the promoter region did not show evolutionary conservation. In the middle of the first intron we found a cluster of well-conserved transcription-control elements and we conclude that these conserved motifs most probably possess a significant function in the control of the tissue-specific transcription of the COL2A1 gene. We also describe locations of additional, highly conserved nucleotide stretches, which are good candidate regions in the search for binding sites of yet-uncharacterized cartilage-specific transcription regulators of the COL2A1 gene. PMID:1637314

  11. Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction.

    PubMed

    Crowe, Brandon L; Larue, Ross C; Yuan, Chunhua; Hess, Sonja; Kvaratskhelia, Mamuka; Foster, Mark P

    2016-02-23

    The bromodomain and extraterminal domain (BET) protein family are promising therapeutic targets for a range of diseases linked to transcriptional activation, cancer, viral latency, and viral integration. Tandem bromodomains selectively tether BET proteins to chromatin by engaging cognate acetylated histone marks, and the extraterminal (ET) domain is the focal point for recruiting a range of cellular and viral proteins. BET proteins guide γ-retroviral integration to transcription start sites and enhancers through bimodal interaction with chromatin and the γ-retroviral integrase (IN). We report the NMR-derived solution structure of the Brd4 ET domain bound to a conserved peptide sequence from the C terminus of murine leukemia virus (MLV) IN. The complex reveals a protein-protein interaction governed by the binding-coupled folding of disordered regions in both interacting partners to form a well-structured intermolecular three-stranded β sheet. In addition, we show that a peptide comprising the ET binding motif (EBM) of MLV IN can disrupt the cognate interaction of Brd4 with NSD3, and that substitutions of Brd4 ET residues essential for binding MLV IN also impair interaction of Brd4 with a number of cellular partners involved in transcriptional regulation and chromatin remodeling. This suggests that γ-retroviruses have evolved the EBM to mimic a cognate interaction motif to achieve effective integration in host chromatin. Collectively, our findings identify key structural features of the ET domain of Brd4 that allow for interactions with both cellular and viral proteins.

  12. Comparative sequence and structure analysis reveals the conservation and diversity of nucleotide positions and their associated tertiary interactions in the riboswitches.

    PubMed

    Appasamy, Sri D; Ramlan, Effirul Ikhwan; Firdaus-Raih, Mohd

    2013-01-01

    The tertiary motifs in complex RNA molecules play vital roles to either stabilize the formation of RNA 3D structure or to provide important biological functionality to the molecule. In order to better understand the roles of these tertiary motifs in riboswitches, we examined 11 representative riboswitch PDB structures for potential agreement of both motif occurrences and conservations. A total of 61 unique tertiary interactions were found in the reference structures. In addition to the expected common A-minor motifs and base-triples mainly involved in linking distant regions the riboswitch structures three highly conserved variants of A-minor interactions called G-minors were found in the SAM-I and FMN riboswitches where they appear to be involved in the recognition of the respective ligand's functional groups. From our structural survey as well as corresponding structure and sequence alignments, the agreement between motif occurrences and conservations are very prominent across the representative riboswitches. Our analysis provide evidence that some of these tertiary interactions are essential components to form the structure where their sequence positions are conserved despite a high degree of diversity in other parts of the respective riboswitches sequences. This is indicative of a vital role for these tertiary interactions in determining the specific biological function of riboswitch.

  13. Localization of proteins to the 1,2-propanediol utilization microcompartment by non-native signal sequences is mediated by a common hydrophobic motif.

    PubMed

    Jakobson, Christopher M; Kim, Edward Y; Slininger, Marilyn F; Chien, Alex; Tullman-Ercek, Danielle

    2015-10-02

    Various bacteria localize metabolic pathways to proteinaceous organelles known as bacterial microcompartments (MCPs), enabling the metabolism of carbon sources to enhance survival and pathogenicity in the gut. There is considerable interest in exploiting bacterial MCPs for metabolic engineering applications, but little is known about the interactions between MCP signal sequences and the protein shells of different MCP systems. We found that the N-terminal sequences from the ethanolamine utilization (Eut) and glycyl radical-generating protein MCPs are able to target reporter proteins to the 1,2-propanediol utilization (Pdu) MCP, and that this localization is mediated by a conserved hydrophobic residue motif. Recapitulation of this motif by the addition of a single amino acid conferred targeting function on an N-terminal sequence from the ethanol utilization MCP system that previously did not act as a Pdu signal sequence. Moreover, the Pdu-localized signal sequences competed with native Pdu targeting sequences for encapsulation in the Pdu MCP. Salmonella enterica natively possesses both the Pdu and Eut operons, and our results suggest that Eut proteins might be localized to the Pdu MCP in vivo. We further demonstrate that S. enterica LT2 retained the ability to grow on 1,2-propanediol as the sole carbon source when a Pdu enzyme was replaced with its Eut homolog. Although the relevance of this finding to the native system remains to be explored, we show that the Pdu-localized signal sequences described herein allow control over the ratio of heterologous proteins encapsulated within Pdu MCPs.

  14. SarA, a global regulator of virulence determinants in Staphylococcus aureus, binds to a conserved motif essential for sar-dependent gene regulation.

    PubMed

    Chien, Y; Manna, A C; Projan, S J; Cheung, A L

    1999-12-24

    The expression of many virulence determinants in Staphylococcus aureus including alpha-hemolysin-, protein A-, and fibronectin-binding proteins is controlled by global regulatory loci such as sar and agr. In addition to controlling target gene expression via agr (e.g. alpha-hemolysin), the sar locus can also regulate target gene transcription via agr-independent mechanisms. In particular, we have found that SarA, the major regulatory protein encoded within sar, binds to a conserved sequence, homologous to the SarA-binding site on the agr promoter, upstream of the -35 promoter boxes of several target genes including hla (alpha-hemolysin gene), spa (protein A gene), fnb (fibronectin-binding protein genes), and sec (enterotoxin C gene). Deletion of the SarA recognition motif in the promoter regions of agr and hla in shuttle plasmids rendered the transcription of these genes undetectable in agr and hla mutants, respectively. Likewise, the transcription activity of spa (a gene normally repressed by sar), as measured by a XylE reporter fusion assay, became derepressed in a wild type strain containing a shuttle plasmid in which the SarA recognition site had been deleted from the spa promoter region. However, DNase I footprinting assays demonstrated that the SarA-binding region on the spa and hla promoter is more extensive than the predicted consensus sequence, thus raising the possibility that the consensus sequence is an activation site within a larger binding region. Because the sar and agr regulate an assortment of virulence factors in S. aureus, we propose, based on our data, a unifying hypothesis for virulence gene activation in S. aureus whereby SarA is a regulatory protein that binds to its consensus SarA recognition motif to activate (e.g. hla) or repress (e.g. spa) the transcription of sar target genes, thus accounting for both agr-dependent and agr-independent mode of regulation.

  15. Helicobacter pylori CagA: analysis of sequence diversity in relation to phosphorylation motifs and implications for the role of CagA as a virulence factor.

    PubMed

    Evans, D J; Evans, D G

    2001-09-01

    CagA is transported into host target cells and subsequently phosphorylated. Clearly this is a mechanism by which Helicobacter pylori could take control of one or more host cell signal transduction pathways. Presumably the end result of this interaction favors survival of H. pylori, irrespective of eventual damage to the host cell. CagA is noted for its amino acid (AA) sequence diversity, both within and outside the variable region of the molecule. The primary purpose of this review is to examine how variation in the type and number of CagA phosphorylation sites might determine the outcome of infection by different strains of H. pylori. The answer to this question could help to explain the widely disparate results obtained when H. pylori CagA status has been compared to type and severity of disease outcome in different populations, that is in different countries. Analysis of all available CagA sequences revealed that CagA contains both tyrosine phosphorylation motifs (TPMs) and cyclic-AMP-dependent phosphorylation motifs (CPMs). There are two potential CPMs near the N-terminus of CagA and at least two in the repeat region; these are not all equally well conserved. We also defined a 48-residue AA sequence, which includes the N-terminal TPM at tyrosine (Y)-122, which distinguishes between Eastern (Hong Kong-Taiwan-Japan-Thailand) H. pylori isolates and those from the West (Europe-Africa-the Americas-Australia). All 28 of the Eastern type CagA proteins have a functional N-terminal TPM whereas 11 of 47 (23.4%) of the Western type contain an inactive motif, with threonine (T) replacing the critical aspartic acid (D) residue. Only 13 of 24 (54%) known CagA sequences have an active TPM in the repeat region and only one has two TPMs in this region. The potential TPM near the C-terminus of CagA is not likely to be important since only 3 of 24 (12.5%) sequences were found to be intact. Protein database searches revealed that the AA sequence immediately following the TPM at Y

  16. Endocytosis and Trafficking of Natriuretic Peptide Receptor-A: Potential Role of Short Sequence Motifs

    PubMed Central

    Pandey, Kailash N.

    2015-01-01

    The targeted endocytosis and redistribution of transmembrane receptors among membrane-bound subcellular organelles are vital for their correct signaling and physiological functions. Membrane receptors committed for internalization and trafficking pathways are sorted into coated vesicles. Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP) bind to guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) and elicit the generation of intracellular second messenger cyclic guanosine 3',5'-monophosphate (cGMP), which lowers blood pressure and incidence of heart failure. After ligand binding, the receptor is rapidly internalized, sequestrated, and redistributed into intracellular locations. Thus, NPRA is considered a dynamic cellular macromolecule that traverses different subcellular locations through its lifetime. The utilization of pharmacologic and molecular perturbants has helped in delineating the pathways of endocytosis, trafficking, down-regulation, and degradation of membrane receptors in intact cells. This review describes the investigation of the mechanisms of internalization, trafficking, and redistribution of NPRA compared with other cell surface receptors from the plasma membrane into the cell interior. The roles of different short-signal peptide sequence motifs in the internalization and trafficking of other membrane receptors have been briefly reviewed and their potential significance in the internalization and trafficking of NPRA is discussed. PMID:26151885

  17. Finishing and Special Motifs: Lessons Learned from CRISPR Analysis Using Next-Generation Draft Sequences ( 7th Annual SFAF Meeting, 2012)

    SciTech Connect

    Campbell, Catherine

    2012-06-01

    Catherine Campbell on "Finishing and Special Motifs: Lessons learned from CRISPR analysis using next-generation draft sequences" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

  18. Finishing and Special Motifs: Lessons Learned from CRISPR Analysis Using Next-Generation Draft Sequences ( 7th Annual SFAF Meeting, 2012)

    ScienceCinema

    Campbell, Catherine [Noblis

    2016-07-12

    Catherine Campbell on "Finishing and Special Motifs: Lessons learned from CRISPR analysis using next-generation draft sequences" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

  19. Triazine-Based Sequence-Defined Polymers with Side-Chain Diversity and Backbone-Backbone Interaction Motifs.

    PubMed

    Grate, Jay W; Mo, Kai-For; Daily, Michael D

    2016-03-14

    Sequence control in polymers, well-known in nature, encodes structure and functionality. Here we introduce a new architecture, based on the nucleophilic aromatic substitution chemistry of cyanuric chloride, that creates a new class of sequence-defined polymers dubbed TZPs. Proof of concept is demonstrated with two synthesized hexamers, having neutral and ionizable side chains. Molecular dynamics simulations show backbone-backbone interactions, including H-bonding motifs and pi-pi interactions. This architecture is arguably biomimetic while differing from sequence-defined polymers having peptide bonds. The synthetic methodology supports the structural diversity of side chains known in peptides, as well as backbone-backbone hydrogen-bonding motifs, and will thus enable new macromolecules and materials with useful functions.

  20. DNA recognition for virus assembly through multiple sequence-independent interactions with a helix-turn-helix motif

    PubMed Central

    Greive, Sandra J.; Fung, Herman K.H.; Chechik, Maria; Jenkins, Huw T.; Weitzel, Stephen E.; Aguiar, Pedro M.; Brentnall, Andrew S.; Glousieau, Matthieu; Gladyshev, Grigory V.; Potts, Jennifer R.; Antson, Alfred A.

    2016-01-01

    The helix-turn-helix (HTH) motif features frequently in protein DNA-binding assemblies. Viral pac site-targeting small terminase proteins possess an unusual architecture in which the HTH motifs are displayed in a ring, distinct from the classical HTH dimer. Here we investigate how such a circular array of HTH motifs enables specific recognition of the viral genome for initiation of DNA packaging during virus assembly. We found, by surface plasmon resonance and analytical ultracentrifugation, that individual HTH motifs of the Bacillus phage SF6 small terminase bind the packaging regions of SF6 and related SPP1 genome weakly, with little local sequence specificity. Nuclear magnetic resonance chemical shift perturbation studies with an arbitrary single-site substrate suggest that the HTH motif contacts DNA similarly to how certain HTH proteins contact DNA non-specifically. Our observations support a model where specificity is generated through conformational selection of an intrinsically bent DNA segment by a ring of HTHs which bind weakly but cooperatively. Such a system would enable viral gene regulation and control of the viral life cycle, with a minimal genome, conferring a major evolutionary advantage for SPP1-like viruses. PMID:26673721

  1. Defining RNA motif-aminoglycoside interactions via two-dimensional combinatorial screening and structure-activity relationships through sequencing.

    PubMed

    Velagapudi, Sai Pradeep; Disney, Matthew D

    2013-10-15

    RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluable insights into potential bystander targets of these clinically used drugs. We utilized two-dimensional combinatorial screening (2DCS), a library-versus-library screening approach, to select the motifs displayed in a 3×3 nucleotide internal loop library and in a 6-nucleotide hairpin library that bind with high affinity and selectivity to six aminoglycoside derivatives. The selected RNA motifs were then analyzed using structure-activity relationships through sequencing (StARTS), a statistical approach that defines the privileged RNA motif space that binds a small molecule. StARTS allowed for the facile annotation of the selected RNA motif-aminoglycoside interactions in terms of affinity and selectivity. The interactions selected by 2DCS generally have nanomolar affinities, which is higher affinity than the binding of aminoglycosides to a mimic of their therapeutic target, the bacterial rRNA A-site.

  2. DNA consensus sequence motif for binding response regulator PhoP, a virulence regulator of Mycobacterium tuberculosis.

    PubMed

    He, Xiaoyuan; Wang, Shuishu

    2014-12-30

    Tuberculosis has reemerged as a serious threat to human health because of the increasing prevalence of drug-resistant strains and synergetic infection with HIV, prompting an urgent need for new and more efficient treatments. The PhoP-PhoR two-component system of Mycobacterium tuberculosis plays an important role in the virulence of the pathogen and thus represents a potential drug target. To study the mechanism of gene transcription regulation by response regulator PhoP, we identified a high-affinity DNA sequence for PhoP binding using systematic evolution of ligands by exponential enrichment. The sequence contains a direct repeat of two 7 bp motifs separated by a 4 bp spacer, TCACAGC(N4)TCACAGC. The specificity of the direct-repeat sequence for PhoP binding was confirmed by isothermal titration calorimetry and electrophoretic mobility shift assays. PhoP binds to the direct repeat as a dimer in a highly cooperative manner. We found many genes previously identified to be regulated by PhoP that contain the direct-repeat motif in their promoter sequences. Synthetic DNA fragments at the putative promoter-binding sites bind PhoP with variable affinity, which is related to the number of mismatches in the 7 bp motifs, the positions of the mismatches, and the spacer and flanking sequences. Phosphorylation of PhoP increases the affinity but does not change the specificity of DNA binding. Overall, our results confirm the direct-repeat sequence as the consensus motif for PhoP binding and thus pave the way for identification of PhoP directly regulated genes in different mycobacterial genomes.

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

    PubMed Central

    Thomsen, Martin Christen Frølund; Nielsen, Morten

    2012-01-01

    Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino acid composition of binding motifs, active sites, etc. in biological sequences. Accurate generation of sequence logos is often compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation focus on displaying the position-specific enrichment of amino acids, discarding the equally valuable information related to amino acid depletion. Seq2logo aims at resolving these issues allowing the user to include sequence weighting to correct for data redundancy, pseudo counts to correct for low number of observations and different logotype representations each capturing different aspects related to amino acid enrichment and depletion. Besides allowing input in the format of peptides and MSA, Seq2Logo accepts input as Blast sequence profiles, providing easy access for non-expert end-users to characterize and identify functionally conserved/variable amino acids in any given protein of interest. The output from the server is a sequence logo and a PSSM. Seq2Logo is available at http://www.cbs.dtu.dk/biotools/Seq2Logo (14 May 2012, date last accessed). PMID:22638583

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

    PubMed

    Thomsen, Martin Christen Frølund; Nielsen, Morten

    2012-07-01

    Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino acid composition of binding motifs, active sites, etc. in biological sequences. Accurate generation of sequence logos is often compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation focus on displaying the position-specific enrichment of amino acids, discarding the equally valuable information related to amino acid depletion. Seq2logo aims at resolving these issues allowing the user to include sequence weighting to correct for data redundancy, pseudo counts to correct for low number of observations and different logotype representations each capturing different aspects related to amino acid enrichment and depletion. Besides allowing input in the format of peptides and MSA, Seq2Logo accepts input as Blast sequence profiles, providing easy access for non-expert end-users to characterize and identify functionally conserved/variable amino acids in any given protein of interest. The output from the server is a sequence logo and a PSSM. Seq2Logo is available at http://www.cbs.dtu.dk/biotools/Seq2Logo (14 May 2012, date last accessed).

  5. Rust Secreted Protein Ps87 Is Conserved in Diverse Fungal Pathogens and Contains a RXLR-like Motif Sufficient for Translocation into Plant Cells

    PubMed Central

    Gu, Biao; Kale, Shiv D.; Wang, Qinhu; Wang, Dinghe; Pan, Qiaona; Cao, Hua; Meng, Yuling; Kang, Zhensheng; Tyler, Brett M.; Shan, Weixing

    2011-01-01

    Background Effector proteins of biotrophic plant pathogenic fungi and oomycetes are delivered into host cells and play important roles in both disease development and disease resistance response. How obligate fungal pathogen effectors enter host cells is poorly understood. The Ps87 gene of Puccinia striiformis encodes a protein that is conserved in diverse fungal pathogens. Ps87 homologs from a clade containing rust fungi are predicted to be secreted. The aim of this study is to test whether Ps87 may act as an effector during Puccinia striiformis infection. Methodology/Principal Findings Yeast signal sequence trap assay showed that the rust protein Ps87 could be secreted from yeast cells, but a homolog from Magnaporthe oryzae that was not predicted to be secreted, could not. Cell re-entry and protein uptake assays showed that a region of Ps87 containing a conserved RXLR-like motif [K/R]RLTG was confirmed to be capable of delivering oomycete effector Avr1b into soybean leaf cells and carrying GFP into soybean root cells. Mutations in the Ps87 motif (KRLTG) abolished the protein translocation ability. Conclusions/Significance The results suggest that Ps87 and its secreted homologs could utilize similar protein translocation machinery as those of oomycete and other fungal pathogens. Ps87 did not show direct suppression activity on plant defense responses. These results suggest Ps87 may represent an “emerging effector” that has recently acquired the ability to enter plant cells but has not yet acquired the ability to alter host physiology. PMID:22076138

  6. SVM2Motif—Reconstructing Overlapping DNA Sequence Motifs by Mimicking an SVM Predictor

    PubMed Central

    Vidovic, Marina M. -C.; Görnitz, Nico; Müller, Klaus-Robert; Rätsch, Gunnar; Kloft, Marius

    2015-01-01

    Identifying discriminative motifs underlying the functionality and evolution of organisms is a major challenge in computational biology. Machine learning approaches such as support vector machines (SVMs) achieve state-of-the-art performances in genomic discrimination tasks, but—due to its black-box character—motifs underlying its decision function are largely unknown. As a remedy, positional oligomer importance matrices (POIMs) allow us to visualize the significance of position-specific subsequences. Although being a major step towards the explanation of trained SVM models, they suffer from the fact that their size grows exponentially in the length of the motif, which renders their manual inspection feasible only for comparably small motif sizes, typically k ≤ 5. In this work, we extend the work on positional oligomer importance matrices, by presenting a new machine-learning methodology, entitled motifPOIM, to extract the truly relevant motifs—regardless of their length and complexity—underlying the predictions of a trained SVM model. Our framework thereby considers the motifs as free parameters in a probabilistic model, a task which can be phrased as a non-convex optimization problem. The exponential dependence of the POIM size on the oligomer length poses a major numerical challenge, which we address by an efficient optimization framework that allows us to find possibly overlapping motifs consisting of up to hundreds of nucleotides. We demonstrate the efficacy of our approach on a synthetic data set as well as a real-world human splice site data set. PMID:26690911

  7. Detection of conserved segments in proteins: iterative scanning of sequence databases with alignment blocks.

    PubMed Central

    Tatusov, R L; Altschul, S F; Koonin, E V

    1994-01-01

    We describe an approach to analyzing protein sequence databases that, starting from a single uncharacterized sequence or group of related sequences, generates blocks of conserved segments. The procedure involves iterative database scans with an evolving position-dependent weight matrix constructed from a coevolving set of aligned conserved segments. For each iteration, the expected distribution of matrix scores under a random model is used to set a cutoff score for the inclusion of a segment in the next iteration. This cutoff may be calculated to allow the chance inclusion of either a fixed number or a fixed proportion of false positive segments. With sufficiently high cutoff scores, the procedure converged for all alignment blocks studied, with varying numbers of iterations required. Different methods for calculating weight matrices from alignment blocks were compared. The most effective of those tested was a logarithm-of-odds, Bayesian-based approach that used prior residue probabilities calculated from a mixture of Dirichlet distributions. The procedure described was used to detect novel conserved motifs of potential biological importance. Images PMID:7991589

  8. MSDmotif: exploring protein sites and motifs

    PubMed Central

    Golovin, Adel; Henrick, Kim

    2008-01-01

    Background Protein structures have conserved features – motifs, which have a sufficient influence on the protein function. These motifs can be found in sequence as well as in 3D space. Understanding of these fragments is essential for 3D structure prediction, modelling and drug-design. The Protein Data Bank (PDB) is the source of this information however present search tools have limited 3D options to integrate protein sequence with its 3D structure. Results We describe here a web application for querying the PDB for ligands, binding sites, small 3D structural and sequence motifs and the underlying database. Novel algorithms for chemical fragments, 3D motifs, ϕ/ψ sequences, super-secondary structure motifs and for small 3D structural motif associations searches are incorporated. The interface provides functionality for visualization, search criteria creation, sequence and 3D multiple alignment options. MSDmotif is an integrated system where a results page is also a search form. A set of motif statistics is available for analysis. This set includes molecule and motif binding statistics, distribution of motif sequences, occurrence of an amino-acid within a motif, correlation of amino-acids side-chain charges within a motif and Ramachandran plots for each residue. The binding statistics are presented in association with properties that include a ligand fragment library. Access is also provided through the distributed Annotation System (DAS) protocol. An additional entry point facilitates XML requests with XML responses. Conclusion MSDmotif is unique by combining chemical, sequence and 3D data in a single search engine with a range of search and visualisation options. It provides multiple views of data found in the PDB archive for exploring protein structures. PMID:18637174

  9. A combined sequence and structure based method for discovering enriched motifs in RNA from in vivo binding data.

    PubMed

    Polishchuk, Maya; Paz, Inbal; Kohen, Refael; Mesika, Rona; Yakhini, Zohar; Mandel-Gutfreund, Yael

    2017-03-06

    RNA binding proteins (RBPs) play an important role in regulating many processes in the cell. RBPs often recognize their RNA targets in a specific manner. In addition to the RNA primary sequence, the structure of the RNA has been shown to play a central role in RNA recognition by RBPs. In recent years, many experimental approaches, both in vitro and in vivo, were developed and employed to identify and characterize RBP targets and extract their binding specificities. In vivo binding techniques, such as CrossLinking and ImmunoPrecipitation (CLIP)-based methods, enable the characterization of protein binding sites on RNA targets. However, these methods do not provide information regarding the structural preferences of the protein. While methods to obtain the structure of RNA are available, inferring both the sequence and the structure preferences of RBPs remains a challenge. Here we present SMARTIV, a novel computational tool for discovering combined sequence and structure binding motifs from in vivo RNA binding data relying on the sequences of the target sites, the ranking of their binding scores and their predicted secondary structure. The combined motifs are provided in a unified representation that is informative and easy for visual perception. We tested the method on CLIP-seq data from different platforms for a variety of RBPs. Overall, we show that our results are highly consistent with known binding motifs of RBPs, offering additional information on their structural preferences.

  10. Phylogenetic Analysis of Geographically Diverse Radopholus similis via rDNA Sequence Reveals a Monomorphic Motif.

    PubMed

    Kaplan, D T; Thomas, W K; Frisse, L M; Sarah, J L; Stanton, J M; Speijer, P R; Marin, D H; Opperman, C H

    2000-06-01

    The nucleic acid sequences of rDNA ITS1 and the rDNA D2/D3 expansion segment were compared for 57 burrowing nematode isolates collected from Australia, Cameroon, Central America, Cuba, Dominican Republic, Florida, Guadeloupe, Hawaii, Nigeria, Honduras, Indonesia, Ivory Coast, Puerto Rico, South Africa, and Uganda. Of the 57 isolates, 55 were morphologically similar to Radopholus similis and seven were citrus-parasitic. The nucleic acid sequences for PCR-amplified ITS1 and for the D2/D3 expansion segment of the 28S rDNA gene were each identical for all putative R. similis. Sequence divergence for both the ITS1 and the D2/D3 was concordant with morphological differences that distinguish R. similis from other burrowing nematode species. This result substantiates previous observations that the R. similis genome is highly conserved across geographic regions. Autapomorphies that would delimit phylogenetic lineages of non-citrus-parasitic R. similis from those that parasitize citrus were not observed. The data presented herein support the concept that R. similis is comprised of two pathotypes-one that parasitizes citrus and one that does not.

  11. Sequence and domain conservation of the coelacanth Hsp40 and Hsp90 chaperones suggests conservation of function.

    PubMed

    Bishop, Özlem Tastan; Edkins, Adrienne Lesley; Blatch, Gregory Lloyd

    2014-09-01

    Molecular chaperones and their associated co-chaperones play an important role in preserving and regulating the active conformational state of cellular proteins. The chaperone complement of the Indonesian Coelacanth, Latimeria menadoensis, was elucidated using transcriptomic sequences. Heat shock protein 90 (Hsp90) and heat shock protein 40 (Hsp40) chaperones, and associated co-chaperones were focused on, and homologous human sequences were used to search the sequence databases. Coelacanth homologs of the cytosolic, mitochondrial and endoplasmic reticulum (ER) homologs of human Hsp90 were identified, as well as all of the major co-chaperones of the cytosolic isoform. Most of the human Hsp40s were found to have coelacanth homologs, and the data suggested that all of the chaperone machinery for protein folding at the ribosome, protein translocation to cellular compartments such as the ER and protein degradation were conserved. Some interesting similarities and differences were identified when interrogating human, mouse, and zebrafish homologs. For example, DnaJB13 is predicted to be a non-functional Hsp40 in humans, mouse, and zebrafish due to a corrupted histidine-proline-aspartic acid (HPD) motif, while the coelacanth homolog has an intact HPD. These and other comparisons enabled important functional and evolutionary questions to be posed for future experimental studies.

  12. Highly conserved repetitive DNA sequences are present at human centromeres.

    PubMed Central

    Grady, D L; Ratliff, R L; Robinson, D L; McCanlies, E C; Meyne, J; Moyzis, R K

    1992-01-01

    Highly conserved repetitive DNA sequence clones, largely consisting of (GGAAT)n repeats, have been isolated from a human recombinant repetitive DNA library by high-stringency hybridization with rodent repetitive DNA. This sequence, the predominant repetitive sequence in human satellites II and III, is similar to the essential core DNA of the Saccharomyces cerevisiae centromere, centromere DNA element (CDE) III. In situ hybridization to human telophase and Drosophila polytene chromosomes shows localization of the (GGAAT)n sequence to centromeric regions. Hyperchromicity studies indicate that the (GGAAT)n sequence exhibits unusual hydrogen bonding properties. The purine-rich strand alone has the same thermal stability as the duplex. Hyperchromicity studies of synthetic DNA variants indicate that all sequences with the composition (AATGN)n exhibit this unusual thermal stability. DNA-mobility-shift assays indicate that specific HeLa-cell nuclear proteins recognize this sequence with a relative affinity greater than 10(5). The extreme evolutionary conservation of this DNA sequence, its centromeric location, its unusual hydrogen bonding properties, its high affinity for specific nuclear proteins, and its similarity to functional centromeres isolated from yeast suggest that this sequence may be a component of the functional human centromere. Images PMID:1542662

  13. Building dictionaries of 1D and 3D motifs by mining the Unaligned 1D sequences of 17 archaeal and bacterial genomes.

    PubMed

    Rigoutsos, I; Gao, Y; Floratos, A; Parida, L

    1999-01-01

    We have used the Teiresias algorithm to carry out unsupervised pattern discovery in a database containing the unaligned ORFs from the 17 publicly available complete archaeal and bacterial genomes and build a 1D dictionary of motifs. These motifs which we refer to as seqlets account for and cover 97.88% of this genomic input at the level of amino acid positions. Each of the seqlets in this 1D dictionary was located among the sequences in Release 38.0 of the Protein Data Bank and the structural fragments corresponding to each seqlet's instances were identified and aligned in three dimensions: those of the seqlets that resulted in RMSD errors below a pre-selected threshold of 2.5 Angstroms were entered in a 3D dictionary of structurally conserved seqlets. These two dictionaries can be thought of as cross-indices that facilitate the tackling of tasks such as automated functional annotation of genomic sequences, local homology identification, local structure characterization, comparative genomics, etc.

  14. Identification of a Novel Sequence Motif Recognized by the Ankyrin Repeat Domain of zDHHC17/13 S-Acyltransferases*

    PubMed Central

    Lemonidis, Kimon; Sanchez-Perez, Maria C.; Chamberlain, Luke H.

    2015-01-01

    S-Acylation is a major post-translational modification affecting several cellular processes. It is particularly important for neuronal functions. This modification is catalyzed by a family of transmembrane S-acyltransferases that contain a conserved zinc finger DHHC (zDHHC) domain. Typically, eukaryote genomes encode for 7–24 distinct zDHHC enzymes, with two members also harboring an ankyrin repeat (AR) domain at their cytosolic N termini. The AR domain of zDHHC enzymes is predicted to engage in numerous interactions and facilitates both substrate recruitment and S-acylation-independent functions; however, the sequence/structural features recognized by this module remain unknown. The two mammalian AR-containing S-acyltransferases are the Golgi-localized zDHHC17 and zDHHC13, also known as Huntingtin-interacting proteins 14 and 14-like, respectively; they are highly expressed in brain, and their loss in mice leads to neuropathological deficits that are reminiscent of Huntington's disease. Here, we report that zDHHC17 and zDHHC13 recognize, via their AR domain, evolutionary conserved and closely related sequences of a [VIAP][VIT]XXQP consensus in SNAP25, SNAP23, cysteine string protein, Huntingtin, cytoplasmic linker protein 3, and microtubule-associated protein 6. This novel AR-binding sequence motif is found in regions predicted to be unstructured and is present in a number of zDHHC17 substrates and zDHHC17/13-interacting S-acylated proteins. This is the first study to identify a motif recognized by AR-containing zDHHCs. PMID:26198635

  15. Conserved Sequence Preferences Contribute to Substrate Recognition by the Proteasome*

    PubMed Central

    Yu, Houqing; Singh Gautam, Amit K.; Wilmington, Shameika R.; Wylie, Dennis; Martinez-Fonts, Kirby; Kago, Grace; Warburton, Marie; Chavali, Sreenivas; Inobe, Tomonao; Finkelstein, Ilya J.; Babu, M. Madan

    2016-01-01

    The proteasome has pronounced preferences for the amino acid sequence of its substrates at the site where it initiates degradation. Here, we report that modulating these sequences can tune the steady-state abundance of proteins over 2 orders of magnitude in cells. This is the same dynamic range as seen for inducing ubiquitination through a classic N-end rule degron. The stability and abundance of His3 constructs dictated by the initiation site affect survival of yeast cells and show that variation in proteasomal initiation can affect fitness. The proteasome's sequence preferences are linked directly to the affinity of the initiation sites to their receptor on the proteasome and are conserved between Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human cells. These findings establish that the sequence composition of unstructured initiation sites influences protein abundance in vivo in an evolutionarily conserved manner and can affect phenotype and fitness. PMID:27226608

  16. A Conserved Motif within RAP1 Plays Diversified Roles in Telomere Protection and Regulation in Different Organisms

    PubMed Central

    Chen, Yong; Rai, Rekha; Zhou, Zi-Ren; Kanoh, Junko; Ribeyre, Cyril; Yang, Yuting; Zheng, Hong; Damay, Pascal; Wang, Feng; Tsujii, Hisayo; Hiraoka, Yasushi; Shore, David; Hu, Hong-Yu; Chang, Sandy; Lei, Ming

    2013-01-01

    Repressor activator protein 1 (RAP1) is the most highly conserved telomere protein. It is involved in protecting chromosome ends in fission yeast, promoting gene silencing in Saccharomyces cerevisiae while in Kluyveromyces lactis it is required to repress homology directed recombination (HDR) at telomeres. Since mammalian RAP1 requires TRF2 for stable expression, its role in telomere function has remained obscure. To understand how RAP1 plays such diverse functions at telomeres, we solved the crystal or solution structures of the C-terminal RCT domains of RAP1 from multiple organisms in complex with their respective protein-binding partners. Our comparative structural analysis establishes the RCT domain of RAP1 as an evolutionarily conserved protein-protein interaction module. In mammalian and fission yeast cells, this module interacts with TRF2 and Taz1, respectively, targeting RAP1 to chromosome ends for telomere end protection. While RAP1 repress NHEJ at fission yeast telomeres, at mammalian telomeres it is required to repress HDR. In contrast, S. cerevisiae RAP1 utilizes the RCT domain to recruit Sir3 to telomeres to mediate gene silencing. Together, our results reveal that depending on the organism, the evolutionarily conserved RAP1 RCT motif plays diverse functional roles at telomeres. PMID:21217703

  17. Creation of Hybrid Nanorods From Sequences of Natural Trimeric Fibrous Proteins Using the Fibritin Trimerization Motif

    NASA Astrophysics Data System (ADS)

    Papanikolopoulou, Katerina; van Raaij, Mark J.; Mitraki, Anna

    Stable, artificial fibrous proteins that can be functionalized open new avenues in fields such as bionanomaterials design and fiber engineering. An important source of inspiration for the creation of such proteins are natural fibrous proteins such as collagen, elastin, insect silks, and fibers from phages and viruses. The fibrous parts of this last class of proteins usually adopt trimeric, β-stranded structural folds and are appended to globular, receptor-binding domains. It has been recently shown that the globular domains are essential for correct folding and trimerization and can be successfully substituted by a very small (27-amino acid) trimerization motif from phage T4 fibritin. The hybrid proteins are correctly folded nanorods that can withstand extreme conditions. When the fibrous part derives from the adenovirus fiber shaft, different tissue-targeting specificities can be engineered into the hybrid proteins, which therefore can be used as gene therapy vectors. The integration of such stable nanorods in devices is also a big challenge in the field of biomechanical design. The fibritin foldon domain is a versatile trimerization motif and can be combined with a variety of fibrous motifs, such as coiled-coil, collagenous, and triple β-stranded motifs, provided the appropriate linkers are used. The combination of different motifs within the same fibrous molecule to create stable rods with multiple functions can even be envisioned. We provide a comprehensive overview of the experimental procedures used for designing, creating, and characterizing hybrid fibrous nanorods using the fibritin trimerization motif.

  18. Comparison of loline alkaloid gene clusters across fungal endophytes: predicting the co-regulatory sequence motifs and the evolutionary history.

    PubMed

    Kutil, Brandi L; Greenwald, Charles; Liu, Gang; Spiering, Martin J; Schardl, Christopher L; Wilkinson, Heather H

    2007-10-01

    LOL, a fungal secondary metabolite gene cluster found in Epichloë and Neotyphodium species, is responsible for production of insecticidal loline alkaloids. To analyze the genetic architecture and to predict the evolutionary history of LOL, we compared five clusters from four fungal species (single clusters from Epichloë festucae, Neotyphodium sp. PauTG-1, Neotyphodium coenophialum, and two clusters we previously characterized in Neotyphodium uncinatum). Using PhyloCon to compare putative lol gene promoter regions, we have identified four motifs conserved across the lol genes in all five clusters. Each motif has significant similarity to known fungal transcription factor binding sites in the TRANSFAC database. Conservation of these motifs is further support for the hypothesis that the lol genes are co-regulated. Interestingly, the history of asexual Neotyphodium spp. includes multiple interspecific hybridization events. Comparing clusters from three Neotyphodium species and E. festucae allowed us to determine which Epichloë ancestors are the most likely contributors of LOL in these asexual species. For example, while no present day Epichloë typhina isolates are known to produce lolines, our data support the hypothesis that the E. typhina ancestor(s) of three asexual endophyte species contained a LOL gene cluster. Thus, these data support a model of evolution in which the polymorphism in loline alkaloid production phenotypes among endophyte species is likely due to the loss of the trait over time.

  19. Multigenome DNA sequence conservation identifies Hox cis-regulatory elements

    PubMed Central

    Kuntz, Steven G.; Schwarz, Erich M.; DeModena, John A.; De Buysscher, Tristan; Trout, Diane; Shizuya, Hiroaki; Sternberg, Paul W.; Wold, Barbara J.

    2008-01-01

    To learn how well ungapped sequence comparisons of multiple species can predict cis-regulatory elements in Caenorhabditis elegans, we made such predictions across the large, complex ceh-13/lin-39 locus and tested them transgenically. We also examined how prediction quality varied with different genomes and parameters in our comparisons. Specifically, we sequenced ∼0.5% of the C. brenneri and C. sp. 3 PS1010 genomes, and compared five Caenorhabditis genomes (C. elegans, C. briggsae, C. brenneri, C. remanei, and C. sp. 3 PS1010) to find regulatory elements in 22.8 kb of noncoding sequence from the ceh-13/lin-39 Hox subcluster. We developed the MUSSA program to find ungapped DNA sequences with N-way transitive conservation, applied it to the ceh-13/lin-39 locus, and transgenically assayed 21 regions with both high and low degrees of conservation. This identified 10 functional regulatory elements whose activities matched known ceh-13/lin-39 expression, with 100% specificity and a 77% recovery rate. One element was so well conserved that a similar mouse Hox cluster sequence recapitulated the native nematode expression pattern when tested in worms. Our findings suggest that ungapped sequence comparisons can predict regulatory elements genome-wide. PMID:18981268

  20. A Novel Family in Medicago truncatula Consisting of More Than 300 Nodule-Specific Genes Coding for Small, Secreted Polypeptides with Conserved Cysteine Motifs1[w

    PubMed Central

    Mergaert, Peter; Nikovics, Krisztina; Kelemen, Zsolt; Maunoury, Nicolas; Vaubert, Danièle; Kondorosi, Adam; Kondorosi, Eva

    2003-01-01

    Transcriptome analysis of Medicago truncatula nodules has led to the discovery of a gene family named NCR (nodule-specific cysteine rich) with more than 300 members. The encoded polypeptides were short (60–90 amino acids), carried a conserved signal peptide, and, except for a conserved cysteine motif, displayed otherwise extensive sequence divergence. Family members were found in pea (Pisum sativum), broad bean (Vicia faba), white clover (Trifolium repens), and Galega orientalis but not in other plants, including other legumes, suggesting that the family might be specific for galegoid legumes forming indeterminate nodules. Gene expression of all family members was restricted to nodules except for two, also expressed in mycorrhizal roots. NCR genes exhibited distinct temporal and spatial expression patterns in nodules and, thus, were coupled to different stages of development. The signal peptide targeted the polypeptides in the secretory pathway, as shown by green fluorescent protein fusions expressed in onion (Allium cepa) epidermal cells. Coregulation of certain NCR genes with genes coding for a potentially secreted calmodulin-like protein and for a signal peptide peptidase suggests a concerted action in nodule development. Potential functions of the NCR polypeptides in cell-to-cell signaling and creation of a defense system are discussed. PMID:12746522

  1. A conserved 11 nucleotide sequence contains an essential promoter element of the maize mitochondrial atp1 gene.

    PubMed Central

    Rapp, W D; Stern, D B

    1992-01-01

    To determine the structure of a functional plant mitochondrial promoter, we have partially purified an RNA polymerase activity that correctly initiates transcription at the maize mitochondrial atp1 promoter in vitro. Using a series of 5' deletion constructs, we found that essential sequences are located within--19 nucleotides (nt) of the transcription initiation site. The region surrounding the initiation site includes conserved sequence motifs previously proposed to be maize mitochondrial promoter elements. Deletion of a conserved 11 nt sequence showed that it is critical for promoter function, but deletion or alteration of conserved upstream G(A/T)3-4 repeats had no effect. When the atp1 11 nt sequence was inserted into different plasmids lacking mitochondrial promoter activity, transcription was only observed for one of these constructs. We infer from these data that the functional promoter extends beyond this motif, most likely in the 5' direction. The maize mitochondrial cox3 and atp6 promoters also direct transcription initiation in this in vitro system, suggesting that it may be widely applicable for studies of mitochondrial transcription in this species. Images PMID:1372246

  2. A conserved intronic U1 snRNP-binding sequence promotes trans-splicing in Drosophila.

    PubMed

    Gao, Jun-Li; Fan, Yu-Jie; Wang, Xiu-Ye; Zhang, Yu; Pu, Jia; Li, Liang; Shao, Wei; Zhan, Shuai; Hao, Jianjiang; Xu, Yong-Zhen

    2015-04-01

    Unlike typical cis-splicing, trans-splicing joins exons from two separate transcripts to produce chimeric mRNA and has been detected in most eukaryotes. Trans-splicing in trypanosomes and nematodes has been characterized as a spliced leader RNA-facilitated reaction; in contrast, its mechanism in higher eukaryotes remains unclear. Here we investigate mod(mdg4), a classic trans-spliced gene in Drosophila, and report that two critical RNA sequences in the middle of the last 5' intron, TSA and TSB, promote trans-splicing of mod(mdg4). In TSA, a 13-nucleotide (nt) core motif is conserved across Drosophila species and is essential and sufficient for trans-splicing, which binds U1 small nuclear RNP (snRNP) through strong base-pairing with U1 snRNA. In TSB, a conserved secondary structure acts as an enhancer. Deletions of TSA and TSB using the CRISPR/Cas9 system result in developmental defects in flies. Although it is not clear how the 5' intron finds the 3' introns, compensatory changes in U1 snRNA rescue trans-splicing of TSA mutants, demonstrating that U1 recruitment is critical to promote trans-splicing in vivo. Furthermore, TSA core-like motifs are found in many other trans-spliced Drosophila genes, including lola. These findings represent a novel mechanism of trans-splicing, in which RNA motifs in the 5' intron are sufficient to bring separate transcripts into close proximity to promote trans-splicing.

  3. A DNA-binding protein containing two widely separated zinc finger motifs that recognize the same DNA sequence.

    PubMed

    Fan, C M; Maniatis, T

    1990-01-01

    We have isolated a full-length cDNA clone encoding a protein (PRDII-BF1) that binds specifically to a positive regulatory domain (PRDII) of the human IFN-beta gene promoter, and to a similar sequence present in a number of other promoters and enhancers. The sequence of this protein reveals two novel structural features. First, it is the largest sequence-specific DNA-binding protein reported to date (298 kD). Second, it contains two widely separated sets of C2-H2-type zinc fingers. Remarkably, each set of zinc fingers binds to the same DNA sequence motif with similar affinities and methylation interference patterns. Thus, this protein may act by binding simultaneously to reiterated copies of the same recognition sequence. Although the function of PRDII-BF1 is not known, the level of its mRNA is inducible by serum and virus, albeit with different kinetics.

  4. An amino acid sequence motif sufficient for subnuclear localization of an arginine/serine-rich splicing factor.

    PubMed

    Hedley, M L; Amrein, H; Maniatis, T

    1995-12-05

    We have identified an amino acid sequence in the Drosophila Transformer (Tra) protein that is capable of directing a heterologous protein to nuclear speckles, regions of the nucleus previously shown to contain high concentrations of spliceosomal small nuclear RNAs and splicing factors. This sequence contains a nucleoplasmin-like bipartite nuclear localization signal (NLS) and a repeating arginine/serine (RS) dipeptide sequence adjacent to a short stretch of basic amino acids. Sequence comparisons from a number of other splicing factors that colocalize to nuclear speckles reveal the presence of one or more copies of this motif. We propose a two-step subnuclear localization mechanism for splicing factors. The first step is transport across the nuclear envelope via the nucleoplasmin-like NLS, while the second step is association with components in the speckled domain via the RS dipeptide sequence.

  5. Complete mitochondrial genome of the red drum, Sciaenops ocellatus (Perciformes, Sciaenidae): absence of the typical conserved motif in the origin of the light-strand replication.

    PubMed

    Cheng, Yuanzhi; Shi, Ge; Xu, Tianjun; Li, Haiyan; Sun, Yueyan; Wang, Rixin

    2012-04-01

    In this study, the complete mitochondrial genome of the red drum Sciaenops ocellatus was determined first. The genome was 16,500 bp in length and contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 2 main non-coding regions (the control region and the origin of the light-strand replication); the gene composition and order of which were similar to most other vertebrates. The overall base composition of the heavy strand was T 25.5%, C 30.7%, A 27.5%, and G 16.3%, with a slight AT bias of 53%. Within the control region, the discrete and conserved sequence blocks were identified. Motif 5'-ACCGG-3' rather than 5'-GCCGG-3' was detected in the origin of light-strand replication (O(L)) of red drum, which is rare in the mitogenomes of Sciaenidae species. These results would play an important role in elucidating sequence-function relationships of the O(L).

  6. Modeling and analysis of MH1 domain of Smads and their interaction with promoter DNA sequence motif.

    PubMed

    Makkar, Pooja; Metpally, Raghu Prasad R; Sangadala, Sreedhara; Reddy, Boojala Vijay B

    2009-04-01

    The Smads are a group of related intracellular proteins critical for transmitting the signals to the nucleus from the transforming growth factor-beta (TGF-beta) superfamily of proteins at the cell surface. The prototypic members of the Smad family, Mad and Sma, were first described in Drosophila and Caenorhabditis elegans, respectively. Related proteins in Xenopus, Humans, Mice and Rats were subsequently identified, and are now known as Smads. Smad protein family members act downstream in the TGF-beta signaling pathway mediating various biological processes, including cell growth, differentiation, matrix production, apoptosis and development. Smads range from about 400-500 amino acids in length and are grouped into the receptor-regulated Smads (R-Smads), the common Smads (Co-Smads) and the inhibitory Smads (I-Smads). There are eight Smads in mammals, Smad1/5/8 (bone morphogenetic protein regulated) and Smad2/3 (TGF-beta/activin regulated) are termed R-Smads, Smad4 is denoted as Co-Smad and Smad6/7 are inhibitory Smads. A typical Smad consists of a conserved N-terminal Mad Homology 1 (MH1) domain and a C-terminal Mad Homology 2 (MH2) domain connected by a proline rich linker. The MH1 domain plays key role in DNA recognition and also facilitates the binding of Smad4 to the phosphorylated C-terminus of R-Smads to form activated complex. The MH2 domain exhibits transcriptional activation properties. In order to understand the structural basis of interaction of various Smads with their target proteins and the promoter DNA, we modeled MH1 domain of the remaining mammalian Smads based on known crystal structures of Smad3-MH1 domain bound to GTCT Smad box DNA sequence (1OZJ). We generated a B-DNA structure using average base-pair parameters of Twist, Tilt, Roll and base Slide angles. We then modeled interaction pose of the MH1 domain of Smad1/5/8 to their corresponding DNA sequence motif GCCG. These models provide the structural basis towards understanding functional

  7. Evening Expression of Arabidopsis GIGANTEA Is Controlled by Combinatorial Interactions among Evolutionarily Conserved Regulatory Motifs[C][W][OPEN

    PubMed Central

    Nordström, Karl; Cremer, Frédéric; Tóth, Réka; Hartke, Martin; Simon, Samson; Klasen, Jonas R.; Bürstel, Ingmar; Coupland, George

    2014-01-01

    Diurnal patterns of gene transcription are often conferred by complex interactions between circadian clock control and acute responses to environmental cues. Arabidopsis thaliana GIGANTEA (GI) contributes to photoperiodic flowering, circadian clock control, and photoreceptor signaling, and its transcription is regulated by the circadian clock and light. We used phylogenetic shadowing to identify three evolutionarily constrained regions (conserved regulatory modules [CRMs]) within the GI promoter and show that CRM2 is sufficient to confer a similar transcriptional pattern as the full-length promoter. Dissection of CRM2 showed that one subfragment (CRM2-A) contributes light inducibility, while another (CRM2-B) exhibits a diurnal response. Mutational analysis showed that three ABA RESPONSE ELEMENT LIKE (ABREL) motifs in CRM2-A and three EVENING ELEMENTs (EEs) in CRM2-B are essential in combination to confer a high amplitude diurnal pattern of expression. Genome-wide analysis identified characteristic spacing patterns of EEs and 71 A. thaliana promoters containing three EEs. Among these promoters, that of FLAVIN BINDING KELCH REPEAT F-BOX1 was analyzed in detail and shown to harbor a CRM functionally related to GI CRM2. Thus, combinatorial interactions among EEs and ABRELs confer diurnal patterns of transcription via an evolutionarily conserved module present in GI and other evening-expressed genes. PMID:25361953

  8. A sequence upstream of canonical PDZ-binding motif within CFTR COOH-terminus enhances NHERF1 interaction.

    PubMed

    Sharma, Neeraj; LaRusch, Jessica; Sosnay, Patrick R; Gottschalk, Laura B; Lopez, Andrea P; Pellicore, Matthew J; Evans, Taylor; Davis, Emily; Atalar, Melis; Na, Chan-Hyun; Rosson, Gedge D; Belchis, Deborah; Milewski, Michal; Pandey, Akhilesh; Cutting, Garry R

    2016-12-01

    The development of cystic fibrosis transmembrane conductance regulator (CFTR) targeted therapy for cystic fibrosis has generated interest in maximizing membrane residence of mutant forms of CFTR by manipulating interactions with scaffold proteins, such as sodium/hydrogen exchange regulatory factor-1 (NHERF1). In this study, we explored whether COOH-terminal sequences in CFTR beyond the PDZ-binding motif influence its interaction with NHERF1. NHERF1 displayed minimal self-association in blot overlays (NHERF1, Kd = 1,382 ± 61.1 nM) at concentrations well above physiological levels, estimated at 240 nM from RNA-sequencing and 260 nM by liquid chromatography tandem mass spectrometry in sweat gland, a key site of CFTR function in vivo. However, NHERF1 oligomerized at considerably lower concentrations (10 nM) in the presence of the last 111 amino acids of CFTR (20 nM) in blot overlays and cross-linking assays and in coimmunoprecipitations using differently tagged versions of NHERF1. Deletion and alanine mutagenesis revealed that a six-amino acid sequence (1417)EENKVR(1422) and the terminal (1478)TRL(1480) (PDZ-binding motif) in the COOH-terminus were essential for the enhanced oligomerization of NHERF1. Full-length CFTR stably expressed in Madin-Darby canine kidney epithelial cells fostered NHERF1 oligomerization that was substantially reduced (∼5-fold) on alanine substitution of EEN, KVR, or EENKVR residues or deletion of the TRL motif. Confocal fluorescent microscopy revealed that the EENKVR and TRL sequences contribute to preferential localization of CFTR to the apical membrane. Together, these results indicate that COOH-terminal sequences mediate enhanced NHERF1 interaction and facilitate the localization of CFTR, a property that could be manipulated to stabilize mutant forms of CFTR at the apical surface to maximize the effect of CFTR-targeted therapeutics.

  9. Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas.

    PubMed

    Petrov, Anton I; Zirbel, Craig L; Leontis, Neocles B

    2013-10-01

    The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson-Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access.

  10. Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas

    PubMed Central

    Petrov, Anton I.; Zirbel, Craig L.; Leontis, Neocles B.

    2013-01-01

    The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson–Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access. PMID:23970545

  11. Drosophila melanogaster Hox Transcription Factors Access the RNA Polymerase II Machinery through Direct Homeodomain Binding to a Conserved Motif of Mediator Subunit Med19

    PubMed Central

    Boube, Muriel; Hudry, Bruno; Immarigeon, Clément; Carrier, Yannick; Bernat-Fabre, Sandra; Merabet, Samir; Graba, Yacine; Bourbon, Henri-Marc; Cribbs, David L.

    2014-01-01

    Hox genes in species across the metazoa encode transcription factors (TFs) containing highly-conserved homeodomains that bind target DNA sequences to regulate batteries of developmental target genes. DNA-bound Hox proteins, together with other TF partners, induce an appropriate transcriptional response by RNA Polymerase II (PolII) and its associated general transcription factors. How the evolutionarily conserved Hox TFs interface with this general machinery to generate finely regulated transcriptional responses remains obscure. One major component of the PolII machinery, the Mediator (MED) transcription complex, is composed of roughly 30 protein subunits organized in modules that bridge the PolII enzyme to DNA-bound TFs. Here, we investigate the physical and functional interplay between Drosophila melanogaster Hox developmental TFs and MED complex proteins. We find that the Med19 subunit directly binds Hox homeodomains, in vitro and in vivo. Loss-of-function Med19 mutations act as dose-sensitive genetic modifiers that synergistically modulate Hox-directed developmental outcomes. Using clonal analysis, we identify a role for Med19 in Hox-dependent target gene activation. We identify a conserved, animal-specific motif that is required for Med19 homeodomain binding, and for activation of a specific Ultrabithorax target. These results provide the first direct molecular link between Hox homeodomain proteins and the general PolII machinery. They support a role for Med19 as a PolII holoenzyme-embedded “co-factor” that acts together with Hox proteins through their homeodomains in regulated developmental transcription. PMID:24786462

  12. ML2Motif—Reliable extraction of discriminative sequence motifs from learning machines

    PubMed Central

    Kloft, Marius; Müller, Klaus-Robert; Görnitz, Nico

    2017-01-01

    High prediction accuracies are not the only objective to consider when solving problems using machine learning. Instead, particular scientific applications require some explanation of the learned prediction function. For computational biology, positional oligomer importance matrices (POIMs) have been successfully applied to explain the decision of support vector machines (SVMs) using weighted-degree (WD) kernels. To extract relevant biological motifs from POIMs, the motifPOIM method has been devised and showed promising results on real-world data. Our contribution in this paper is twofold: as an extension to POIMs, we propose gPOIM, a general measure of feature importance for arbitrary learning machines and feature sets (including, but not limited to, SVMs and CNNs) and devise a sampling strategy for efficient computation. As a second contribution, we derive a convex formulation of motifPOIMs that leads to more reliable motif extraction from gPOIMs. Empirical evaluations confirm the usefulness of our approach on artificially generated data as well as on real-world datasets. PMID:28346487

  13. Bayesian Markov models consistently outperform PWMs at predicting motifs in nucleotide sequences

    PubMed Central

    Siebert, Matthias; Söding, Johannes

    2016-01-01

    Position weight matrices (PWMs) are the standard model for DNA and RNA regulatory motifs. In PWMs nucleotide probabilities are independent of nucleotides at other positions. Models that account for dependencies need many parameters and are prone to overfitting. We have developed a Bayesian approach for motif discovery using Markov models in which conditional probabilities of order k − 1 act as priors for those of order k. This Bayesian Markov model (BaMM) training automatically adapts model complexity to the amount of available data. We also derive an EM algorithm for de-novo discovery of enriched motifs. For transcription factor binding, BaMMs achieve significantly (P    =  1/16) higher cross-validated partial AUC than PWMs in 97% of 446 ChIP-seq ENCODE datasets and improve performance by 36% on average. BaMMs also learn complex multipartite motifs, improving predictions of transcription start sites, polyadenylation sites, bacterial pause sites, and RNA binding sites by 26–101%. BaMMs never performed worse than PWMs. These robust improvements argue in favour of generally replacing PWMs by BaMMs. PMID:27288444

  14. Conserved structural motifs in the central pair complex of eukaryotic flagella.

    PubMed

    Carbajal-González, Blanca I; Heuser, Thomas; Fu, Xiaofeng; Lin, Jianfeng; Smith, Brandon W; Mitchell, David R; Nicastro, Daniela

    2013-02-01

    Cilia and flagella are conserved hair-like appendages of eukaryotic cells that function as sensing and motility generating organelles. Motility is driven by thousands of axonemal dyneins that require precise regulation. One essential motility regulator is the central pair complex (CPC) and many CPC defects cause paralysis of cilia/flagella. Several human diseases, such as immotile cilia syndrome, show CPC abnormalities, but little is known about the detailed three-dimensional (3D) structure and function of the CPC. The CPC is located in the center of typical [9+2] cilia/flagella and is composed of two singlet microtubules (MTs), each with a set of associated projections that extend toward the surrounding nine doublet MTs. Using cryo-electron tomography coupled with subtomogram averaging, we visualized and compared the 3D structures of the CPC in both the green alga Chlamydomonas and the sea urchin Strongylocentrotus at the highest resolution published to date. Despite the evolutionary distance between these species, their CPCs exhibit remarkable structural conservation. We identified several new projections, including those that form the elusive sheath, and show that the bridge has a more complex architecture than previously thought. Organism-specific differences include the presence of MT inner proteins in Chlamydomonas, but not Strongylocentrotus, and different overall outlines of the highly connected projection network, which forms a round-shaped cylinder in algae, but is more oval in sea urchin. These differences could be adaptations to the mechanical requirements of the rotating CPC in Chlamydomonas, compared to the Strongylocentrotus CPC which has a fixed orientation.

  15. A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.

    PubMed

    Mughal, W; Nguyen, L; Pustylnik, S; da Silva Rosa, S C; Piotrowski, S; Chapman, D; Du, M; Alli, N S; Grigull, J; Halayko, A J; Aliani, M; Topham, M K; Epand, R M; Hatch, G M; Pereira, T J; Kereliuk, S; McDermott, J C; Rampitsch, C; Dolinsky, V W; Gordon, J W

    2015-10-29

    Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

  16. Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

    PubMed Central

    Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

    1995-01-01

    The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

  17. A conserved Glu-Arg salt bridge connects co-evolved motifs that define the eukaryote protein kinase fold

    PubMed Central

    Yang, Jie; Wu, Jian; Steichen, Jon M.; Kornev, Alexandr P.; Deal, Michael S.; Li, Sheng; Sankaran, Banumathi; Woods, Virgil L.; Taylor, Susan S.

    2012-01-01

    Eukaryotic protein kinases (EPK)feature two co-evolved structural segments, the Activation segment which starts with the Asp-Phe-Gly (DFG) and ends with the Ala-Pro-Glu (APE) motifs, and the helical GHI-subdomain that comprises αG-αH-αI helices. Eukaryotic-like kinases have a much shorter Activation segment and lack the GHI-subdomain. They thus lack the conserved salt bridge interaction between the APE Glu and an Arg from the GHI-subdomain, a hallmark signature of EPKs. Although the conservation of this salt bridge in EPKs is well known and its implication in diseases has been illustrated by polymorphism analysis, its function has not been carefully studied. In this work, we use murine cAMP dependent protein kinase (PKA) as the model enzyme (Glu208 and Arg280) to examine the role of these two residues. We showed that Ala replacement of either residue caused a 40–120 fold decrease in catalytic efficiency of the enzyme due to an increase in Km(ATP) and a decrease in kcat. Crystal structures, as well as solution studies, also demonstratethat this ion pair contributes to the hydrophobic network and stability of the enzyme. We show that mutation of either Glu or Arg to Ala renders bothmutant proteins less effective substrates for upstream kinase phosphoinositide dependent kinase 1. We propose that the Glu208-Arg280 pair serves as a center hub of connectivity between these two structurally conserved elements in EPKs. Mutations of either residue disrupt communication not only between the two segments but also within the rest of the molecule leading to altered catalytic activity and enzyme regulation. PMID:22138346

  18. Definition of the tempo of sequence diversity across an alignment and automatic identification of sequence motifs: Application to protein homologous families and superfamilies

    PubMed Central

    May, Alex C.W.

    2002-01-01

    It is often possible to identify sequence motifs that characterize a protein family in terms of its fold and/or function from aligned protein sequences. Such motifs can be used to search for new family members. Partitioning of sequence alignments into regions of similar amino acid variability is usually done by hand. Here, I present a completely automatic method for this purpose: one that is guaranteed to produce globally optimal solutions at all levels of partition granularity. The method is used to compare the tempo of sequence diversity across reliable three-dimensional (3D) structure-based alignments of 209 protein families (HOMSTRAD) and that for 69 superfamilies (CAMPASS). (The mean alignment length for HOMSTRAD and CAMPASS are very similar.) Surprisingly, the optimal segmentation distributions for the closely related proteins and distantly related ones are found to be very similar. Also, optimal segmentation identifies an unusual protein superfamily. Finally, protein 3D structure clues from the tempo of sequence diversity across alignments are examined. The method is general, and could be applied to any area of comparative biological sequence and 3D structure analysis where the constraint of the inherent linear organization of the data imposes an ordering on the set of objects to be clustered. PMID:12441381

  19. Local Function Conservation in Sequence and Structure Space

    PubMed Central

    Weinhold, Nils; Sander, Oliver; Domingues, Francisco S.; Lengauer, Thomas; Sommer, Ingolf

    2008-01-01

    We assess the variability of protein function in protein sequence and structure space. Various regions in this space exhibit considerable difference in the local conservation of molecular function. We analyze and capture local function conservation by means of logistic curves. Based on this analysis, we propose a method for predicting molecular function of a query protein with known structure but unknown function. The prediction method is rigorously assessed and compared with a previously published function predictor. Furthermore, we apply the method to 500 functionally unannotated PDB structures and discuss selected examples. The proposed approach provides a simple yet consistent statistical model for the complex relations between protein sequence, structure, and function. The GOdot method is available online (http://godot.bioinf.mpi-inf.mpg.de). PMID:18604264

  20. Local function conservation in sequence and structure space.

    PubMed

    Weinhold, Nils; Sander, Oliver; Domingues, Francisco S; Lengauer, Thomas; Sommer, Ingolf

    2008-07-04

    We assess the variability of protein function in protein sequence and structure space. Various regions in this space exhibit considerable difference in the local conservation of molecular function. We analyze and capture local function conservation by means of logistic curves. Based on this analysis, we propose a method for predicting molecular function of a query protein with known structure but unknown function. The prediction method is rigorously assessed and compared with a previously published function predictor. Furthermore, we apply the method to 500 functionally unannotated PDB structures and discuss selected examples. The proposed approach provides a simple yet consistent statistical model for the complex relations between protein sequence, structure, and function. The GOdot method is available online (http://godot.bioinf.mpi-inf.mpg.de).

  1. Internal epitope tagging informed by relative lack of sequence conservation

    PubMed Central

    Burg, Leonard; Zhang, Karen; Bonawitz, Tristan; Grajevskaja, Viktorija; Bellipanni, Gianfranco; Waring, Richard; Balciunas, Darius

    2016-01-01

    Many experimental techniques rely on specific recognition and stringent binding of proteins by antibodies. This can readily be achieved by introducing an epitope tag. We employed an approach that uses a relative lack of evolutionary conservation to inform epitope tag site selection, followed by integration of the tag-coding sequence into the endogenous locus in zebrafish. We demonstrate that an internal epitope tag is accessible for antibody binding, and that tagged proteins retain wild type function. PMID:27892520

  2. Composite Conserved Promoter–Terminator Motifs (PeSLs) that Mediate Modular Shuffling in the Diverse T4-Like Myoviruses

    PubMed Central

    Comeau, André M.; Arbiol, Christine; Krisch, Henry M.

    2014-01-01

    The diverse T4-like phages (Tquatrovirinae) infect a wide array of gram-negative bacterial hosts. The genome architecture of these phages is generally well conserved, most of the phylogenetically variable genes being grouped together in a series hyperplastic regions (HPRs) that are interspersed among large blocks of conserved core genes. Recent evidence from a pair of closely related T4-like phages has suggested that small, composite terminator/promoter sequences (promoterearly stem loop [PeSLs]) were implicated in mediating the high levels of genetic plasticity by indels occurring within the HPRs. Here, we present the genome sequence analysis of two T4-like phages, PST (168 kb, 272 open reading frames [ORFs]) and nt-1 (248 kb, 405 ORFs). These two phages were chosen for comparative sequence analysis because, although they are closely related to phages that have been previously sequenced (T4 and KVP40, respectively), they have different host ranges. In each case, one member of the pair infects a bacterial strain that is a human pathogen, whereas the other phage’s host is a nonpathogen. Despite belonging to phylogenetically distant branches of the T4-likes, these pairs of phage have diverged from each other in part by a mechanism apparently involving PeSL-mediated recombination. This analysis confirms a role of PeSL sequences in the generation of genomic diversity by serving as a point of genetic exchange between otherwise unrelated sequences within the HPRs. Finally, the palette of divergent genes swapped by PeSL-mediated homologous recombination is discussed in the context of the PeSLs’ potentially important role in facilitating phage adaption to new hosts and environments. PMID:24951563

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

    NASA Astrophysics Data System (ADS)

    Richardson, Dale N.; Wiehe, Thomas

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

  4. A Glance at Microsatellite Motifs from 454 Sequencing Reads of Watermelon Genomic DNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A single 454 (Life Sciences Sequencing Technology) run of Charleston Gray watermelon (Citrullus lanatus var. lanatus) genomic DNA was performed and sequence data were assembled. A large scale identification of simple sequence repeat (SSR) was performed and SSR sequence data were used for the develo...

  5. Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site

    PubMed Central

    Wongsantichon, Jantana; Robinson, Robert C.; Ketterman, Albert J.

    2015-01-01

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme. PMID:26487708

  6. Conservation patterns in different functional sequence categoriesof divergent Drosophila species

    SciTech Connect

    Papatsenko, Dmitri; Kislyuk, Andrey; Levine, Michael; Dubchak, Inna

    2005-10-01

    We have explored the distributions of fully conservedungapped blocks in genome-wide pairwise alignments of recently completedspecies of Drosophila: D.yakuba, D.ananassae, D.pseudoobscura, D.virilisand D.mojavensis. Based on these distributions we have found that nearlyevery functional sequence category possesses its own distinctiveconservation pattern, sometimes independent of the overall sequenceconservation level. In the coding and regulatory regions, the ungappedblocks were longer than in introns, UTRs and non-functional sequences. Atthe same time, the blocks in the coding regions carried 3N+2 signaturecharacteristic to synonymic substitutions in the 3rd codon positions.Larger block sizes in transcription regulatory regions can be explainedby the presence of conserved arrays of binding sites for transcriptionfactors. We also have shown that the longest ungapped blocks, or'ultraconserved' sequences, are associated with specific gene groups,including those encoding ion channels and components of the cytoskeleton.We discussed how restrained conservation patterns may help in mappingfunctional sequence categories and improving genomeannotation.

  7. One to rule them all: A highly conserved motif in mariner transposase controls multiple steps of transposition.

    PubMed

    Bouuaert, Corentin Claeys; Tellier, Michael; Chalmers, Ronald

    2014-01-01

    The development of transposon-based genome manipulation tools can benefit greatly from understanding transposons' inherent regulatory mechanisms. The Tc1-mariner transposons, which are being widely used in biotechnological applications, are subject to a self-inhibitory mechanism whereby increasing transposase expression beyond a certain point decreases the rate of transposition. In a recent paper, Liu and Chalmers performed saturating mutagenesis on the highly conserved WVPHEL motif in the mariner-family transposase from the Hsmar1 element. Curiously, they found that the majority of all possible single mutations were hyperactive. Biochemical characterizations of the mutants revealed that the hyperactivity is due to a defect in communication between transposase subunits, which normally regulates transposition by reducing the rate of synapsis. This provides important clues for improving transposon-based tools. However, some WVPHEL mutants also showed features that would be undesirable for most biotechnological applications: they showed uncontrolled DNA cleavage activities and defects in the coordination of cleavage between the two transposon ends. The study illustrates how the knowledge of inhibitory mechanisms can help improve transposon tools but also highlights an important challenge, which is to specifically target a regulatory mechanism without affecting other important functions of the transposase.

  8. Evolutionarily Conserved Dual Lysine Motif Determines the Non-Chaperone Function of Secreted Hsp90alpha in Tumor Progression

    PubMed Central

    Sahu, Divya; Hou, Yingping; Tsen, Fred; Tong, Chang; O’Brien, Kathryn; Situ, Alan J.; Schmidt, Thomas; Chen, Mei; Ying, Qilong; Ulmer, Tobias S.; Woodley, David T.; Li, Wei

    2016-01-01

    Both intracellular and extracellular heat shock protein-90 (Hsp90) family proteins (α and β) have been shown to support tumor progression. The tumor-promoting activity of the intracellular Hsp90 proteins is attributed to their N-terminal ATPase-driven chaperone function. What determines the extracellular function of secreted Hsp90 was unclear. Here we show that knocking out Hsp90α nullifies tumor cell abilities to migrate, invade and metastasize without affecting cell survival and growth. Knocking out Hsp90β leads to cell death. Extracellular supplementation with recombinant Hsp90α, but not Hsp90β, protein recovers the tumorigenicity of Hsp90α-knockout cells. Sequential mutagenesis identifies two evolutionarily conserved lysine residues, lys-270 and lys-277, in Hsp90α subfamily that determine the extracellular Hsp90α function. Hsp90β subfamily lacks the dual lysine motif and does not show the same extracellular function. Substitutions of gly-262 and thr-269 in Hsp90β with lysines convert Hsp90β to act as Hsp90α outside the cells. Monoclonal antibody, 1G6-D7, against the dual lysine region of secreted Hsp90α blocks de novo tumor formation and significantly inhibits expansion of already formed tumors. This study suggests an alternative therapeutic approach to selectively target the extracellular Hsp90α to the conventional approach targeting the ATPase of intracellular Hsp90α and Hsp90β in cancer. PMID:27721406

  9. Conservation patterns in angiosperm rDNA ITS2 sequences.

    PubMed Central

    Hershkovitz, M A; Zimmer, E A

    1996-01-01

    The two internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA have become commonly exploited sources of informative variation for interspecific-/intergeneric-level phylogenetic analyses among angiosperms and other eukaryotes. We present an alignment in which one-third to one-half of the ITS2 sequence is alignable above the family level in angiosperms and a phenetic analysis showing that ITS2 contains information sufficient to diagnose lineages at several hierarchical levels. Base compositional analysis shows that angiosperm ITS2 is inherently GC-rich, and that the proportion of T is much more variable than that for other bases. We propose a general model of angiosperm ITS2 secondary structure that shows common pairing relationships for most of the conserved sequence tracts. Variations in our secondary structure predictions for sequences from different taxa indicate that compensatory mutation is not limited to paired positions. PMID:8760866

  10. Conservative Patch Algorithm and Mesh Sequencing for PAB3D

    NASA Technical Reports Server (NTRS)

    Pao, S. P.; Abdol-Hamid, K. S.

    2005-01-01

    A mesh-sequencing algorithm and a conservative patched-grid-interface algorithm (hereafter Patch Algorithm ) have been incorporated into the PAB3D code, which is a computer program that solves the Navier-Stokes equations for the simulation of subsonic, transonic, or supersonic flows surrounding an aircraft or other complex aerodynamic shapes. These algorithms are efficient, flexible, and have added tremendously to the capabilities of PAB3D. The mesh-sequencing algorithm makes it possible to perform preliminary computations using only a fraction of the grid cells (provided the original cell count is divisible by an integer) along any grid coordinate axis, independently of the other axes. The patch algorithm addresses another critical need in multi-block grid situation where the cell faces of adjacent grid blocks may not coincide, leading to errors in calculating fluxes of conserved physical quantities across interfaces between the blocks. The patch algorithm, based on the Stokes integral formulation of the applicable conservation laws, effectively matches each of the interfacial cells on one side of the block interface to the corresponding fractional cell area pieces on the other side. This approach is comprehensive and unified such that all interface topology is automatically processed without user intervention. This algorithm is implemented in a preprocessing code that creates a cell-by-cell database that will maintain flux conservation at any level of full or reduced grid density as the user may choose by way of the mesh-sequencing algorithm. These two algorithms have enhanced the numerical accuracy of the code, reduced the time and effort for grid preprocessing, and provided users with the flexibility of performing computations at any desired full or reduced grid resolution to suit their specific computational requirements.

  11. HIV-1 conserved-element vaccines: relationship between sequence conservation and replicative capacity.

    PubMed

    Rolland, Morgane; Manocheewa, Siriphan; Swain, J Victor; Lanxon-Cookson, Erinn C; Kim, Moon; Westfall, Dylan H; Larsen, Brendan B; Gilbert, Peter B; Mullins, James I

    2013-05-01

    To overcome the problem of HIV-1 variability, candidate vaccine antigens have been designed to be composed of conserved elements of the HIV-1 proteome. Such candidate vaccines could be improved with a better understanding of both HIV-1 evolutionary constraints and the fitness cost of specific mutations. We evaluated the in vitro fitness cost of 23 mutations engineered in the HIV-1 subtype B Gag-p24 Center-of-Tree (COT) protein through fitness competition assays. While some mutations at conserved sites exacted a high fitness cost, as expected under the assumption that the most conserved residue confers the highest fitness, there was no overall strong relationship between sequence conservation and replicative capacity. By comparing sites that have evolved since the beginning of the epidemic to those that have remain unchanged, we found that sites that have evolved over time were more likely to correspond to HLA-associated sites and that their mutation had limited fitness costs. Our data showed no transcendent link between high conservation and high fitness cost, indicating that merely focusing on conserved segments of HIV-1 would not be sufficient for a successful vaccine strategy. Nonetheless, a subset of sites exacted a high fitness cost upon mutation--these sites have been under selective pressure to change since the beginning of the epidemic but have proved virtually nonmutable and could constitute preferred targets for vaccine design.

  12. Inter-specific sequence conservation and intra-individual sequence variation in a spider silk gene.

    PubMed

    Tai, Pei-Ling; Hwang, Guang-Yuh; Tso, I-Min

    2004-10-01

    Currently, studies on major ampullate spidroin 1 (MaSp1) genes of non-orb weaving spiders are few, and it is not clear whether genes of these organisms exhibit the same characteristics as those of orb-weavers. In addition, many studies have proposed that MaSp1 might be a single gene with allelic variants, but supporting evidence is still lacking. In this study, we compared partial DNA and amino acid sequences of MaSp1 cloned from different spider guilds. We also cloned partial MaSp1 sequences from genomic DNA and cDNA of the same individuals of spiders using the same primer combination to see if different molecular forms existed. In the repetitive region of partial MaSp1 sequences obtained, GGX, GA and poly-A motifs were present in all Araneomorphae and Mygalomorpae species examined. An extreme similarity in MaSp1 non-repetitive portions was found in sequences of ecribellate, cribellate and Mygalomorphae web-builders and such a result suggested that this sequence might exhibit an important function. A comparison of sequences amplified from the same individual showed that substitutions in amino acids occurred in both repetitive and non-repetitive regions, with a much higher variation in the former. These results suggest that the MaSp1 of Araneomorphae spiders exhibits several forms in an individual spider and it might be either a multiple gene or a single gene with a multiple exon/intron organization.

  13. In Vivo Enhancer Analysis Chromosome 16 Conserved NoncodingSequences

    SciTech Connect

    Pennacchio, Len A.; Ahituv, Nadav; Moses, Alan M.; Nobrega,Marcelo; Prabhakar, Shyam; Shoukry, Malak; Minovitsky, Simon; Visel,Axel; Dubchak, Inna; Holt, Amy; Lewis, Keith D.; Plajzer-Frick, Ingrid; Akiyama, Jennifer; De Val, Sarah; Afzal, Veena; Black, Brian L.; Couronne, Olivier; Eisen, Michael B.; Rubin, Edward M.

    2006-02-01

    The identification of enhancers with predicted specificitiesin vertebrate genomes remains a significant challenge that is hampered bya lack of experimentally validated training sets. In this study, weleveraged extreme evolutionary sequence conservation as a filter toidentify putative gene regulatory elements and characterized the in vivoenhancer activity of human-fish conserved and ultraconserved1 noncodingelements on human chromosome 16 as well as such elements from elsewherein the genome. We initially tested 165 of these extremely conservedsequences in a transgenic mouse enhancer assay and observed that 48percent (79/165) functioned reproducibly as tissue-specific enhancers ofgene expression at embryonic day 11.5. While driving expression in abroad range of anatomical structures in the embryo, the majority of the79 enhancers drove expression in various regions of the developingnervous system. Studying a set of DNA elements that specifically droveforebrain expression, we identified DNA signatures specifically enrichedin these elements and used these parameters to rank all ~;3,400human-fugu conserved noncoding elements in the human genome. The testingof the top predictions in transgenic mice resulted in a three-foldenrichment for sequences with forebrain enhancer activity. These datadramatically expand the catalogue of in vivo-characterized human geneenhancers and illustrate the future utility of such training sets for avariety of iological applications including decoding the regulatoryvocabulary of the human genome.

  14. Conservation analysis predicts in vivo occupancy of glucocorticoid receptor-binding sequences at glucocorticoid-induced genes.

    PubMed

    So, Alex Yick-Lun; Cooper, Samantha B; Feldman, Brian J; Manuchehri, Mitra; Yamamoto, Keith R

    2008-04-15

    The glucocorticoid receptor (GR) interacts with specific GR-binding sequences (GBSs) at glucocorticoid response elements (GREs) to orchestrate transcriptional networks. Although the sequences of the GBSs are highly variable among different GREs, the precise sequence within an individual GRE is highly conserved. In this study, we examined whether sequence conservation of sites resembling GBSs is sufficient to predict GR occupancy of GREs at genes responsive to glucocorticoids. Indeed, we found that the level of conservation of these sites at genes up-regulated by glucocorticoids in mouse C3H10T1/2 mesenchymal stem-like cells correlated directly with the extent of occupancy by GR. In striking contrast, we failed to observe GR occupancy of GBSs at genes repressed by glucocorticoids, despite the occurrence of these sites at a frequency similar to that of the induced genes. Thus, GR occupancy of the GBS motif correlates with induction but not repression, and GBS conservation alone is sufficient to predict GR occupancy and GRE function at induced genes.

  15. Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

    SciTech Connect

    Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo; Lersch,Robert A.; Gee, Sherry L.; Hou, Victor C.; Lo, Annie J.; Short, Sarah A.; Chasis, Joel A.; Winkelmann, John C.; Conboy, John G.

    2006-03-01

    Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.

  16. New melanocortin 1 receptor binding motif based on the C-terminal sequence of alpha-melanocyte-stimulating hormone.

    PubMed

    Schiöth, Helgi B; Muceniece, Ruta; Mutule, Ilga; Wikberg, Jarl E S

    2006-10-01

    The C-terminal tripeptide of the alpha-melanocyte stimulating hormone (alpha-MSH11-13) possesses strong antiinflammatory activity without known cellular target. In order to better understand the structural requirements for function of such motif, we designed, synthesized and tested out Trp- and Tyr-containing analogues of the alpha-MSH11-13. Seven alpha-MSH11-13 analogues were synthesized and characterized for their binding to the melanocortin receptors recombinantly expressed in insect (Sf9) cells, infected with baculovirus carrying corresponding MC receptor DNA. We also tested these analogues on B16-F1 mouse melanoma cells endogenously expressing the MC1 receptor for binding and for ability to increase cAMP levels as well as on COS-7 cells transfected with the human MC receptors. The data indicate that HS401 (Ac-Tyr-Lys-Pro-Val-NH2) and HS402 (Ac-Lys-Pro-Val-Tyr-NH2) selectively bound to the MC1 receptor and stimulated cAMP generation in a concentration dependent way while the other Tyr- and Trp-containing alpha-MSH11-13 analogues neither bound to MC receptors nor stimulated cAMP. We have thus identified new MC receptor binding motif derived from the C-terminal sequence of alpha-MSH. The tetrapeptides have novel properties as the both act via MC-ergic pathways and also carry the anti-inflammatory alpha-MSH11-13 message sequence.

  17. The Ku-binding motif is a conserved module for recruitment and stimulation of non-homologous end-joining proteins

    PubMed Central

    Grundy, Gabrielle J.; Rulten, Stuart L.; Arribas-Bosacoma, Raquel; Davidson, Kathryn; Kozik, Zuzanna; Oliver, Antony W.; Pearl, Laurence H.; Caldecott, Keith W.

    2016-01-01

    The Ku-binding motif (KBM) is a short peptide module first identified in APLF that we now show is also present in Werner syndrome protein (WRN) and in Modulator of retrovirus infection homologue (MRI). We also identify a related but functionally distinct motif in XLF, WRN, MRI and PAXX, which we denote the XLF-like motif. We show that WRN possesses two KBMs; one at the N terminus next to the exonuclease domain and one at the C terminus next to an XLF-like motif. We reveal that the WRN C-terminal KBM and XLF-like motif function cooperatively to bind Ku complexes and that the N-terminal KBM mediates Ku-dependent stimulation of WRN exonuclease activity. We also show that WRN accelerates DSB repair by a mechanism requiring both KBMs, demonstrating the importance of WRN interaction with Ku. These data define a conserved family of KBMs that function as molecular tethers to recruit and/or stimulate enzymes during NHEJ. PMID:27063109

  18. Co-conservation of rRNA tetraloop sequences and helix length suggests involvement of the tetraloops in higher-order interactions

    NASA Technical Reports Server (NTRS)

    Hedenstierna, K. O.; Siefert, J. L.; Fox, G. E.; Murgola, E. J.

    2000-01-01

    Terminal loops containing four nucleotides (tetraloops) are common in structural RNAs, and they frequently conform to one of three sequence motifs, GNRA, UNCG, or CUUG. Here we compare available sequences and secondary structures for rRNAs from bacteria, and we show that helices capped by phylogenetically conserved GNRA loops display a strong tendency to be of conserved length. The simplest interpretation of this correlation is that the conserved GNRA loops are involved in higher-order interactions, intramolecular or intermolecular, resulting in a selective pressure for maintaining the lengths of these helices. A small number of conserved UNCG loops were also found to be associated with conserved length helices, consistent with the possibility that this type of tetraloop also takes part in higher-order interactions.

  19. Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements.

    PubMed

    Karvelis, Tautvydas; Gasiunas, Giedrius; Young, Joshua; Bigelyte, Greta; Silanskas, Arunas; Cigan, Mark; Siksnys, Virginijus

    2015-11-19

    To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants.

  20. Rice MEL2, the RNA recognition motif (RRM) protein, binds in vitro to meiosis-expressed genes containing U-rich RNA consensus sequences in the 3'-UTR.

    PubMed

    Miyazaki, Saori; Sato, Yutaka; Asano, Tomoya; Nagamura, Yoshiaki; Nonomura, Ken-Ichi

    2015-10-01

    Post-transcriptional gene regulation by RNA recognition motif (RRM) proteins through binding to cis-elements in the 3'-untranslated region (3'-UTR) is widely used in eukaryotes to complete various biological processes. Rice MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2) is the RRM protein that functions in the transition to meiosis in proper timing. The MEL2 RRM preferentially associated with the U-rich RNA consensus, UUAGUU[U/A][U/G][A/U/G]U, dependently on sequences and proportionally to MEL2 protein amounts in vitro. The consensus sequences were located in the putative looped structures of the RNA ligand. A genome-wide survey revealed a tendency of MEL2-binding consensus appearing in 3'-UTR of rice genes. Of 249 genes that conserved the consensus in their 3'-UTR, 13 genes spatiotemporally co-expressed with MEL2 in meiotic flowers, and included several genes whose function was supposed in meiosis; such as Replication protein A and OsMADS3. The proteome analysis revealed that the amounts of small ubiquitin-related modifier-like protein and eukaryotic translation initiation factor3-like protein were dramatically altered in mel2 mutant anthers. Taken together with transcriptome and gene ontology results, we propose that the rice MEL2 is involved in the translational regulation of key meiotic genes on 3'-UTRs to achieve the faithful transition of germ cells to meiosis.

  1. Characteristic motifs for families of allergenic proteins

    PubMed Central

    Ivanciuc, Ovidiu; Garcia, Tzintzuni; Torres, Miguel; Schein, Catherine H.; Braun, Werner

    2008-01-01

    The identification of potential allergenic proteins is usually done by scanning a database of allergenic proteins and locating known allergens with a high sequence similarity. However, there is no universally accepted cut-off value for sequence similarity to indicate potential IgE cross-reactivity. Further, overall sequence similarity may be less important than discrete areas of similarity in proteins with homologous structure. To identify such areas, we first classified all allergens and their subdomains in the Structural Database of Allergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP/) to their closest protein families as defined in Pfam, and identified conserved physicochemical property motifs characteristic of each group of sequences. Allergens populate only a small subset of all known Pfam families, as all allergenic proteins in SDAP could be grouped to only 130 (of 9318 total) Pfams, and 31 families contain more than four allergens. Conserved physicochemical property motifs for the aligned sequences of the most populated Pfam families were identified with the PCPMer program suite and catalogued in the webserver Motif-Mate (http://born.utmb.edu/motifmate/summary.php). We also determined specific motifs for allergenic members of a family that could distinguish them from non-allergenic ones. These allergen specific motifs should be most useful in database searches for potential allergens. We found that sequence motifs unique to the allergens in three families (seed storage proteins, Bet v 1, and tropomyosin) overlap with known IgE epitopes, thus providing evidence that our motif based approach can be used to assess the potential allergenicity of novel proteins. PMID:18951633

  2. Nucleotide sequence conservation in paramyxoviruses; the concept of codon constellation.

    PubMed

    Rima, Bert K

    2015-05-01

    The stability and conservation of the sequences of RNA viruses in the field and the high error rates measured in vitro are paradoxical. The field stability indicates that there are very strong selective constraints on sequence diversity. The nature of these constraints is discussed. Apart from constraints on variation in cis-acting RNA and the amino acid sequences of viral proteins, there are other ones relating to the presence of specific dinucleotides such CpG and UpA as well as the importance of RNA secondary structures and RNA degradation rates. Recent other constraints identified in other RNA viruses, such as effects of secondary RNA structure on protein folding or modification of cellular tRNA complements, are also discussed. Using the family Paramyxoviridae, I show that the codon usage pattern (CUP) is (i) specific for each virus species and (ii) that it is markedly different from the host - it does not vary even in vaccine viruses that have been derived by passage in a number of inappropriate host cells. The CUP might thus be an additional constraint on variation, and I propose the concept of codon constellation to indicate the informational content of the sequences of RNA molecules relating not only to stability and structure but also to the efficiency of translation of a viral mRNA resulting from the CUP and the numbers and position of rare codons.

  3. Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids

    PubMed Central

    Ivanova, Lyudmila; Buch, Anna; Döhner, Katinka; Pohlmann, Anja; Binz, Anne; Prank, Ute; Sandbaumhüter, Malte

    2016-01-01

    ABSTRACT Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs 1766WD1767 and 1862WE1863 are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17+)Lox-pUL36-WD/AA-WE/AA and HSV-1(17+)Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. IMPORTANCE Herpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several

  4. Genetic diversity of the conserved motifs of six bacterial leaf blight resistance genes in a set of rice landraces

    PubMed Central

    2014-01-01

    Background Bacterial leaf blight (BLB) caused by the vascular pathogen Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious diseases leading to crop failure in rice growing countries. A total of 37 resistance genes against Xoo has been identified in rice. Of these, ten BLB resistance genes have been mapped on rice chromosomes, while 6 have been cloned, sequenced and characterized. Diversity analysis at the resistance gene level of this disease is scanty, and the landraces from West Bengal and North Eastern states of India have received little attention so far. The objective of this study was to assess the genetic diversity at conserved domains of 6 BLB resistance genes in a set of 22 rice accessions including landraces and check genotypes collected from the states of Assam, Nagaland, Mizoram and West Bengal. Results In this study 34 pairs of primers were designed from conserved domains of 6 BLB resistance genes; Xa1, xa5, Xa21, Xa21(A1), Xa26 and Xa27. The designed primer pairs were used to generate PCR based polymorphic DNA profiles to detect and elucidate the genetic diversity of the six genes in the 22 diverse rice accessions of known disease phenotype. A total of 140 alleles were identified including 41 rare and 26 null alleles. The average polymorphism information content (PIC) value was 0.56/primer pair. The DNA profiles identified each of the rice landraces unequivocally. The amplified polymorphic DNA bands were used to calculate genetic similarity of the rice landraces in all possible pair combinations. The similarity among the rice accessions ranged from 18% to 89% and the dendrogram produced from the similarity values was divided into 2 major clusters. The conserved domains identified within the sequenced rare alleles include Leucine-Rich Repeat, BED-type zinc finger domain, sugar transferase domain and the domain of the carbohydrate esterase 4 superfamily. Conclusions This study revealed high genetic diversity at conserved domains of six BLB

  5. A Bayesian hidden Markov model for motif discovery through joint modeling of genomic sequence and ChIP-chip data.

    PubMed

    Gelfond, Jonathan A L; Gupta, Mayetri; Ibrahim, Joseph G

    2009-12-01

    We propose a unified framework for the analysis of chromatin (Ch) immunoprecipitation (IP) microarray (ChIP-chip) data for detecting transcription factor binding sites (TFBSs) or motifs. ChIP-chip assays are used to focus the genome-wide search for TFBSs by isolating a sample of DNA fragments with TFBSs and applying this sample to a microarray with probes corresponding to tiled segments across the genome. Present analytical methods use a two-step approach: (i) analyze array data to estimate IP-enrichment peaks then (ii) analyze the corresponding sequences independently of intensity information. The proposed model integrates peak finding and motif discovery through a unified Bayesian hidden Markov model (HMM) framework that accommodates the inherent uncertainty in both measurements. A Markov chain Monte Carlo algorithm is formulated for parameter estimation, adapting recursive techniques used for HMMs. In simulations and applications to a yeast RAP1 dataset, the proposed method has favorable TFBS discovery performance compared to currently available two-stage procedures in terms of both sensitivity and specificity.

  6. A Bayesian hidden Markov model for motif discovery through joint modeling of genomic sequence and ChIP-chip data

    PubMed Central

    Gelfond, Jonathan A. L.; Gupta, Mayetri; Ibrahim, Joseph G.

    2009-01-01

    SUMMARY We propose a unified framework for the analysis of Chromatin (Ch) Immunoprecipitation (IP) microarray (ChIP-chip) data for detecting transcription factor binding sites (TFBSs) or motifs. ChIP-chip assays are used to focus the genome-wide search for TFBSs by isolating a sample of DNA fragments with TFBSs and applying this sample to a microarray with probes corresponding to tiled segments across the genome. Present analytical methods use a two-step approach: (i) analyze array data to estimate IP enrichment peaks then (ii) analyze the corresponding sequences independently of intensity information. The proposed model integrates peak finding and motif discovery through a unified Bayesian hidden Markov model (HMM) framework that accommodates the inherent uncertainty in both measurements. A Markov Chain Monte Carlo algorithm is formulated for parameter estimation, adapting recursive techniques used for HMMs. In simulations and applications to a yeast RAP1 dataset, the proposed method has favorable TFBS discovery performance compared to currently available two-stage procedures in terms of both sensitivity and specificity. PMID:19210737

  7. De novo computational identification of stress-related sequence motifs and microRNA target sites in untranslated regions of a plant translatome

    PubMed Central

    Munusamy, Prabhakaran; Zolotarov, Yevgen; Meteignier, Louis-Valentin; Moffett, Peter; Strömvik, Martina V.

    2017-01-01

    Gene regulation at the transcriptional and translational level leads to diversity in phenotypes and function in organisms. Regulatory DNA or RNA sequence motifs adjacent to the gene coding sequence act as binding sites for proteins that in turn enable or disable expression of the gene. Whereas the known DNA and RNA binding proteins range in the thousands, only a few motifs have been examined. In this study, we have predicted putative regulatory motifs in groups of untranslated regions from genes regulated at the translational level in Arabidopsis thaliana under normal and stressed conditions. The test group of sequences was divided into random subgroups and subjected to three de novo motif finding algorithms (Seeder, Weeder and MEME). In addition to identifying sequence motifs, using an in silico tool we have predicted microRNA target sites in the 3′ UTRs of the translationally regulated genes, as well as identified upstream open reading frames located in the 5′ UTRs. Our bioinformatics strategy and the knowledge generated contribute to understanding gene regulation during stress, and can be applied to disease and stress resistant plant development. PMID:28276452

  8. A common sequence motif determines the Cajal body-specific localization of box H/ACA scaRNAs.

    PubMed

    Richard, Patricia; Darzacq, Xavier; Bertrand, Edouard; Jády, Beáta E; Verheggen, Céline; Kiss, Tamás

    2003-08-15

    Post-transcriptional synthesis of 2'-O-methylated nucleotides and pseudouridines in Sm spliceosomal small nuclear RNAs takes place in the nucleoplasmic Cajal bodies and it is directed by guide RNAs (scaRNAs) that are structurally and functionally indistinguishable from small nucleolar RNAs (snoRNAs) directing rRNA modification in the nucleolus. The scaRNAs are synthesized in the nucleoplasm and specifically targeted to Cajal bodies. Here, mutational analysis of the human U85 box C/D-H/ACA scaRNA, followed by in situ localization, demonstrates that box H/ACA scaRNAs share a common Cajal body-specific localization signal, the CAB box. Two copies of the evolutionarily conserved CAB consensus (UGAG) are located in the terminal loops of the 5' and 3' hairpins of the box H/ACA domains of mammalian, Drosophila and plant scaRNAs. Upon alteration of the CAB boxes, mutant scaRNAs accumulate in the nucleolus. In turn, authentic snoRNAs can be targeted into Cajal bodies by addition of exogenous CAB box motifs. Our results indicate that scaRNAs represent an ancient group of small nuclear RNAs which are localized to Cajal bodies by an evolutionarily conserved mechanism.

  9. A sequence motif enriched in regions bound by the Drosophila dosage compensation complex

    PubMed Central

    2010-01-01

    Background In Drosophila melanogaster, dosage compensation is mediated by the action of the dosage compensation complex (DCC). How the DCC recognizes the fly X chromosome is still poorly understood. Characteristic sequence signatures at all DCC binding sites have not hitherto been found. Results In this study, we compare the known binding sites of the DCC with oligonucleotide profiles that measure the specificity of the sequences of the D. melanogaster X chromosome. We show that the X chromosome regions bound by the DCC are enriched for a particular type of short, repetitive sequences. Their distribution suggests that these sequences contribute to chromosome recognition, the generation of DCC binding sites and/or the local spreading of the complex. Comparative data indicate that the same sequences may be involved in dosage compensation in other Drosophila species. Conclusions These results offer an explanation for the wild-type binding of the DCC along the Drosophila X chromosome, contribute to delineate the forces leading to the establishment of dosage compensation and suggest new experimental approaches to understand the precise biochemical features of the dosage compensation system. PMID:20226017

  10. Uncharacterized conserved motifs outside the HD-Zip domain in HD-Zip subfamily I transcription factors; a potential source of functional diversity

    PubMed Central

    2011-01-01

    Background Plant HD-Zip transcription factors are modular proteins in which a homeodomain is associated to a leucine zipper. Of the four subfamilies in which they are divided, the tested members from subfamily I bind in vitro the same pseudopalindromic sequence CAAT(A/T)ATTG and among them, several exhibit similar expression patterns. However, most experiments in which HD-Zip I proteins were over or ectopically expressed under the control of the constitutive promoter 35S CaMV resulted in transgenic plants with clearly different phenotypes. Aiming to elucidate the structural mechanisms underlying such observation and taking advantage of the increasing information in databases of sequences from diverse plant species, an in silico analysis was performed. In addition, some of the results were also experimentally supported. Results A phylogenetic tree of 178 HD-Zip I proteins together with the sequence conservation presented outside the HD-Zip domains allowed the distinction of six groups of proteins. A motif-discovery approach enabled the recognition of an activation domain in the carboxy-terminal regions (CTRs) and some putative regulatory mechanisms acting in the amino-terminal regions (NTRs) and CTRs involving sumoylation and phosphorylation. A yeast one-hybrid experiment demonstrated that the activation activity of ATHB1, a member of one of the groups, is located in its CTR. Chimerical constructs were performed combining the HD-Zip domain of one member with the CTR of another and transgenic plants were obtained with these constructs. The phenotype of the chimerical transgenic plants was similar to the observed in transgenic plants bearing the CTR of the donor protein, revealing the importance of this module inside the whole protein. Conclusions The bioinformatical results and the experiments conducted in yeast and transgenic plants strongly suggest that the previously poorly analyzed NTRs and CTRs of HD-Zip I proteins play an important role in their function, hence

  11. A comparative analysis of two conserved motifs in bacterial poly(A) polymerase and CCA-adding enzyme.

    PubMed

    Just, Andrea; Butter, Falk; Trenkmann, Michelle; Heitkam, Tony; Mörl, Mario; Betat, Heike

    2008-09-01

    Showing a high sequence similarity, the evolutionary closely related bacterial poly(A) polymerases (PAP) and CCA-adding enzymes catalyze quite different reactions--PAP adds poly(A) tails to RNA 3'-ends, while CCA-adding enzymes synthesize the sequence CCA at the 3'-terminus of tRNAs. Here, two highly conserved structural elements of the corresponding Escherichia coli enzymes were characterized. The first element is a set of amino acids that was identified in CCA-adding enzymes as a template region determining the enzymes' specificity for CTP and ATP. The same element is also present in PAP, where it confers ATP specificity. The second investigated region corresponds to a flexible loop in CCA-adding enzymes and is involved in the incorporation of the terminal A-residue. Although, PAP seems to carry a similar flexible region, the functional relevance of this element in PAP is not known. The presented results show that the template region has an essential function in both enzymes, while the second element is surprisingly dispensable in PAP. The data support the idea that the bacterial PAP descends from CCA-adding enzymes and still carries some of the structural elements required for CCA-addition as an evolutionary relic and is now fixed in a conformation specific for A-addition.

  12. Structural Analysis of a Repetitive Protein Sequence Motif in Strepsirrhine Primate Amelogenin

    PubMed Central

    Bromley, Keith M.; Hacia, Joseph G.; Bromage, Timothy G.; Snead, Malcolm L.; Moradian-Oldak, Janet; Paine, Michael L.

    2011-01-01

    Strepsirrhines are members of a primate suborder that has a distinctive set of features associated with the development of the dentition. Amelogenin (AMEL), the better known of the enamel matrix proteins, forms 90% of the secreted organic matrix during amelogenesis. Although AMEL has been sequenced in numerous mammalian lineages, the only reported strepsirrhine AMEL sequences are those of the ring-tailed lemur and galago, which contain a set of additional proline-rich tandem repeats absent in all other primates species analyzed to date, but present in some non-primate mammals. Here, we first determined that these repeats are present in AMEL from three additional lemur species and thus are likely to be widespread throughout this group. To evaluate the functional relevance of these repeats in strepsirrhines, we engineered a mutated murine amelogenin sequence containing a similar proline-rich sequence to that of Lemur catta. In the monomeric form, the MQP insertions had no influence on the secondary structure or refolding properties, whereas in the assembled form, the insertions increased the hydrodynamic radii. We speculate that increased AMEL nanosphere size may influence enamel formation in strepsirrhine primates. PMID:21437261

  13. A novel human gene (SARM) at chromosome 17q11 encodes a protein with a SAM motif and structural similarity to Armadillo/beta-catenin that is conserved in mouse, Drosophila, and Caenorhabditis elegans.

    PubMed

    Mink, M; Fogelgren, B; Olszewski, K; Maroy, P; Csiszar, K

    2001-06-01

    A novel human gene, SARM, encodes the orthologue of a Drosophila protein (CG7915) and contains a unique combination of the sterile alpha (SAM) and the HEAT/Armadillo motifs. The SARM gene was identified on chromosome 17q11, between markers D17S783 and D17S841 on BAC clone AC002094, which also included a HERV repeat and keratin-18-like, MAC30, TNFAIP1, HSPC017, and vitronectin genes in addition to three unknown genes. The mouse SARM gene was located on a mouse chromosome 11 BAC clone (AC002324). The SARM gene is 1.8 kb centromeric to the vitronectin gene, and the two genes share a promoter region that directs a high level of liver-specific expression of both the SARM and the vitronectin genes. In addition to the liver, the SARM gene was highly expressed in the kidney. A 0.4-kb antisense transcript was coordinately expressed with the SARM gene in the kidney and liver, while in the brain and malignant cell lines, it appeared independent of SARM gene transcription. The SARM gene encodes a protein of 690 amino acids. Based on amino acid sequence homology, we have identified a SAM motif within this derived protein. Structure modeling and protein folding recognition studies confirmed the presence of alpha-alpha right-handed superhelix-like folds consistent with the structure of the Armadillo and HEAT repeats of the beta-catenin and importin protein families. Both motifs are known to be involved in protein-protein interactions promoting the formation of diverse protein complexes. We have identified the same conserved SAM/Armadillo motif combination in the mouse, Drosophila, and Caenorhabditis elegans SARM proteins.

  14. The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats

    PubMed Central

    Bowman, Andrew; Lercher, Lukas; Singh, Hari R.; Zinne, Daria; Timinszky, Gyula; Carlomagno, Teresa; Ladurner, Andreas G.

    2016-01-01

    Eukaryotic chromatin is a complex yet dynamic structure, which is regulated in part by the assembly and disassembly of nucleosomes. Key to this process is a group of proteins termed histone chaperones that guide the thermodynamic assembly of nucleosomes by interacting with soluble histones. Here we investigate the interaction between the histone chaperone sNASP and its histone H3 substrate. We find that sNASP binds with nanomolar affinity to a conserved heptapeptide motif in the globular domain of H3, close to the C-terminus. Through functional analysis of sNASP homologues we identified point mutations in surface residues within the TPR domain of sNASP that disrupt H3 peptide interaction, but do not completely disrupt binding to full length H3 in cells, suggesting that sNASP interacts with H3 through additional contacts. Furthermore, chemical shift perturbations from 1H-15N HSQC experiments show that H3 peptide binding maps to the helical groove formed by the stacked TPR motifs of sNASP. Our findings reveal a new mode of interaction between a TPR repeat domain and an evolutionarily conserved peptide motif found in canonical H3 and in all histone H3 variants, including CenpA and have implications for the mechanism of histone chaperoning within the cell. PMID:26673727

  15. Conserved structural motifs at the C-terminus of baculovirus protein IE0 are important for its functions in transactivation and supporting hr5-mediated DNA replication.

    PubMed

    Luria, Neta; Lu, Liqun; Chejanovsky, Nor

    2012-05-01

    IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.

  16. Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco.

    PubMed

    Felix, Georg; Boller, Thomas

    2003-02-21

    To detect microbial infection multicellular organisms have evolved sensing systems for pathogen-associated molecular patterns (PAMPs). Here, we identify bacterial cold shock protein (CSP) as a new such PAMP that acts as a highly active elicitor of defense responses in tobacco. Tobacco cells perceive a conserved domain of CSP and synthetic peptides representing 15 amino acids of this domain-induced responses at subnanomolar concentrations. Central to the elicitor-active domain is the RNP-1 motif KGFGFITP, a motif conserved also in many RNA- and DNA-binding proteins of eukaryotes. Csp15-Nsyl, a peptide representing the domain with highest homology to csp15 in a protein of Nicotiana sylvestris exhibited only weak activity in tobacco cells. Crystallographic and genetic data from the literature show that the RNP-1 domain of bacterial CSPs resides on a protruding loop and exposes a series of aromatic and basic side chains to the surface that are essential for the nucleotide-binding activity of CSPs. Similarly, these side chains were also essential for elicitor activity and replacement of single residues in csp15 with Ala strongly reduced or abolished activity. Most strikingly, csp15-Ala10, a peptide with the RNP-1 motif modified to KGAGFITP, lacked elicitor activity but acted as a competitive antagonist for CSP-related elicitors. Bacteria commonly have a small family of CSP-like proteins including both cold-inducible and noninducible members, and Csp-related elicitor activity was detected in extracts from all bacteria tested. Thus, the CSP domain containing the RNP-1 motif provides a structure characteristic for bacteria in general, and tobacco plants have evolved a highly sensitive chemoperception system to detect this bacterial PAMP.

  17. Conserved Motifs within Hepatitis C Virus Envelope (E2) RNA and Protein Independently Inhibit T Cell Activation

    PubMed Central

    Bhattarai, Nirjal; McLinden, James H.; Xiang, Jinhua; Kaufman, Thomas M.; Stapleton, Jack T.

    2015-01-01

    T cell receptor (TCR) signaling is required for T-cell activation, proliferation, differentiation, and effector function. Hepatitis C virus (HCV) infection is associated with impaired T-cell function leading to persistent viremia, delayed and inconsistent antibody responses, and mild immune dysfunction. Although multiple factors appear to contribute to T-cell dysfunction, a role for HCV particles in this process has not been identified. Here, we show that incubation of primary human CD4+ and CD8+ T-cells with HCV RNA-containing serum, HCV-RNA containing extracellular vesicles (EVs), cell culture derived HCV particles (HCVcc) and HCV envelope pseudotyped retrovirus particles (HCVpp) inhibited TCR-mediated signaling. Since HCVpp’s contain only E1 and E2, we examined the effect of HCV E2 on TCR signaling pathways. HCV E2 expression recapitulated HCV particle-induced TCR inhibition. A highly conserved, 51 nucleotide (nt) RNA sequence was sufficient to inhibit TCR signaling. Cells expressing the HCV E2 coding RNA contained a short, virus-derived RNA predicted to be a Dicer substrate, which targeted a phosphatase involved in Src-kinase signaling (PTPRE). T-cells and hepatocytes containing HCV E2 RNA had reduced PTPRE protein levels. Mutation of 6 nts abolished the predicted Dicer interactions and restored PTPRE expression and proximal TCR signaling. HCV RNA did not inhibit distal TCR signaling induced by PMA and Ionomycin; however, HCV E2 protein inhibited distal TCR signaling. This inhibition required lymphocyte-specific tyrosine kinase (Lck). Lck phosphorylated HCV E2 at a conserved tyrosine (Y613), and phospho-E2 inhibited nuclear translocation of NFAT. Mutation of Y613 restored distal TCR signaling, even in the context of HCVpps. Thus, HCV particles delivered viral RNA and E2 protein to T-cells, and these inhibited proximal and distal TCR signaling respectively. These effects of HCV particles likely aid in establishing infection and contribute to viral persistence

  18. 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.

  19. Peptide sequences identified by phage display are immunodominant functional motifs of Pet and Pic serine proteases secreted by Escherichia coli and Shigella flexneri.

    PubMed

    Ulises, Hernández-Chiñas; Tatiana, Gazarian; Karlen, Gazarian; Guillermo, Mendoza-Hernández; Juan, Xicohtencatl-Cortes; Carlos, Eslava

    2009-12-01

    Plasmid-encoded toxin (Pet) and protein involved in colonization (Pic), are serine protease autotransporters of Enterobacteriaceae (SPATEs) secreted by enteroaggregative Escherichia coli (EAEC), which display the GDSGSG sequence or the serine motif. Our research was directed to localize functional sites in both proteins using the phage display method. From a 12mer linear and a 7mer cysteine-constrained (C7C) libraries displayed on the M13 phage pIII protein we selected different mimotopes using IgG purified from sera of children naturally infected with EAEC producing Pet and Pic proteins, and anti-Pet and anti-Pic IgG purified from rabbits immunized with each one of these proteins. Children IgG selected a homologous group of sequences forming the consensus sequence, motif, PQPxK, and the motifs PGxI/LN and CxPDDSSxC were selected by the rabbit anti-Pet and anti-Pic IgGs, respectively. Analysis of the amino terminal region of a panel of SPATEs showed the presence in all of them of sequences matching the PGxI/LN or CxPDDSSxC motifs, and in a three-dimensional model (Modeller 9v2) designed for Pet, both these motifs were found in the globular portion of the protein, close to the protease active site GDSGSG. Antibodies induced in mice by mimotopes carrying the three aforementioned motifs were reactive with Pet, Pic, and with synthetic peptides carrying the immunogenic mimotope sequences TYPGYINHSKA and LLPQPPKLLLP, thus confirming that the peptide moiety of the selected phages induced the antibodies specific for the toxins. The antibodies induced in mice to the PGxI/LN and CxPDDSSxC mimotopes inhibited fodrin proteolysis and macrophage chemotaxis biological activities of Pet. Our results showed that we were able to generate, by a phage display procedure, mimotopes with sequence motifs PGxI/LN and CxPDDSSxC, and to identify them as functional motifs of the Pet, Pic and other SPATEs involved in their biological activities.

  20. High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum

    PubMed Central

    Christiansen, Anders; Kringelum, Jens V.; Hansen, Christian S.; Bøgh, Katrine L.; Sullivan, Eric; Patel, Jigar; Rigby, Neil M.; Eiwegger, Thomas; Szépfalusi, Zsolt; Masi, Federico de; Nielsen, Morten; Lund, Ole; Dufva, Martin

    2015-01-01

    Phage display is a prominent screening technique with a multitude of applications including therapeutic antibody development and mapping of antigen epitopes. In this study, phages were selected based on their interaction with patient serum and exhaustively characterised by high-throughput sequencing. A bioinformatics approach was developed in order to identify peptide motifs of interest based on clustering and contrasting to control samples. Comparison of patient and control samples confirmed a major issue in phage display, namely the selection of unspecific peptides. The potential of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage display by (i) enabling the analysis of complex biological samples, (ii) circumventing the traditional laborious picking and functional testing of individual phage clones and (iii) reducing the number of selection rounds. PMID:26246327

  1. CpG island erosion, polycomb occupancy and sequence motif enrichment at bivalent promoters in mammalian embryonic stem cells.

    PubMed

    Mantsoki, Anna; Devailly, Guillaume; Joshi, Anagha

    2015-11-19

    In embryonic stem (ES) cells, developmental regulators have a characteristic bivalent chromatin signature marked by simultaneous presence of both activation (H3K4me3) and repression (H3K27me3) signals and are thought to be in a 'poised' state for subsequent activation or silencing during differentiation. We collected eleven pairs (H3K4me3 and H3K27me3) of ChIP sequencing datasets in human ES cells and eight pairs in murine ES cells, and predicted high-confidence (HC) bivalent promoters. Over 85% of H3K27me3 marked promoters were bivalent in human and mouse ES cells. We found that (i) HC bivalent promoters were enriched for developmental factors and were highly likely to be differentially expressed upon transcription factor perturbation; (ii) murine HC bivalent promoters were occupied by both polycomb repressive component classes (PRC1 and PRC2) and grouped into four distinct clusters with different biological functions; (iii) HC bivalent and active promoters were CpG rich while H3K27me3-only promoters lacked CpG islands. Binding enrichment of distinct sets of regulators distinguished bivalent from active promoters. Moreover, a 'TCCCC' sequence motif was specifically enriched in bivalent promoters. Finally, this analysis will serve as a resource for future studies to further understand transcriptional regulation during embryonic development.

  2. CpG island erosion, polycomb occupancy and sequence motif enrichment at bivalent promoters in mammalian embryonic stem cells

    PubMed Central

    Mantsoki, Anna; Devailly, Guillaume; Joshi, Anagha

    2015-01-01

    In embryonic stem (ES) cells, developmental regulators have a characteristic bivalent chromatin signature marked by simultaneous presence of both activation (H3K4me3) and repression (H3K27me3) signals and are thought to be in a ‘poised’ state for subsequent activation or silencing during differentiation. We collected eleven pairs (H3K4me3 and H3K27me3) of ChIP sequencing datasets in human ES cells and eight pairs in murine ES cells, and predicted high-confidence (HC) bivalent promoters. Over 85% of H3K27me3 marked promoters were bivalent in human and mouse ES cells. We found that (i) HC bivalent promoters were enriched for developmental factors and were highly likely to be differentially expressed upon transcription factor perturbation; (ii) murine HC bivalent promoters were occupied by both polycomb repressive component classes (PRC1 and PRC2) and grouped into four distinct clusters with different biological functions; (iii) HC bivalent and active promoters were CpG rich while H3K27me3-only promoters lacked CpG islands. Binding enrichment of distinct sets of regulators distinguished bivalent from active promoters. Moreover, a ‘TCCCC’ sequence motif was specifically enriched in bivalent promoters. Finally, this analysis will serve as a resource for future studies to further understand transcriptional regulation during embryonic development. PMID:26582124

  3. Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element

    PubMed Central

    Jemt, Elisabeth; Persson, Örjan; Shi, Yonghong; Mehmedovic, Majda; Uhler, Jay P.; Dávila López, Marcela; Freyer, Christoph; Gustafsson, Claes M.; Samuelsson, Tore; Falkenberg, Maria

    2015-01-01

    The majority of mitochondrial DNA replication events are terminated prematurely. The nascent DNA remains stably associated with the template, forming a triple-stranded displacement loop (D-loop) structure. However, the function of the D-loop region of the mitochondrial genome remains poorly understood. Using a comparative genomics approach we here identify two closely related 15 nt sequence motifs of the D-loop, strongly conserved among vertebrates. One motif is at the D-loop 5′-end and is part of the conserved sequence block 1 (CSB1). The other motif, here denoted coreTAS, is at the D-loop 3′-end. Both these sequences may prevent transcription across the D-loop region, since light and heavy strand transcription is terminated at CSB1 and coreTAS, respectively. Interestingly, the replication of the nascent D-loop strand, occurring in a direction opposite to that of heavy strand transcription, is also terminated at coreTAS, suggesting that coreTAS is involved in termination of both transcription and replication. Finally, we demonstrate that the loading of the helicase TWINKLE at coreTAS is reversible, implying that this site is a crucial component of a switch between D-loop formation and full-length mitochondrial DNA replication. PMID:26253742

  4. A type of nucleotide motif that distinguishes tobamovirus species more efficiently than nucleotide signatures.

    PubMed

    Gibbs, A J; Armstrong, J S; Gibbs, M J

    2004-10-01

    The complete genomic sequences of forty-eight tobamoviruses were classified and found to form at least twelve species clusters. Individual species were not conveniently defined by 'nucleotide signatures' (i.e. strings of one or more nucleotides unique to a taxon) as these were scattered sparsely throughout the genomes and were mostly single nucleotides. By contrast all the species were concisely and uniquely distinguished by short nucleotide motifs consisting of conserved genus-specific sites intercalated with variable sites that provided species-specific combinations of nucleotides (nucleotide combination motifs; NC-motifs). We describe the procedure for finding NC-motifs in a convenient and phylogenetically conserved region of the tobamovirus RNA polymerase gene, the '4404-50 motif'. NC-motifs have been found in other sets of homologous sequences, and are convenient for use in published taxonomic descriptions.

  5. Contribution of Sequence Motif, Chromatin State, and DNA Structure Features to Predictive Models of Transcription Factor Binding in Yeast.

    PubMed

    Tsai, Zing Tsung-Yeh; Shiu, Shin-Han; Tsai, Huai-Kuang

    2015-08-01

    Transcription factor (TF) binding is determined by the presence of specific sequence motifs (SM) and chromatin accessibility, where the latter is influenced by both chromatin state (CS) and DNA structure (DS) properties. Although SM, CS, and DS have been used to predict TF binding sites, a predictive model that jointly considers CS and DS has not been developed to predict either TF-specific binding or general binding properties of TFs. Using budding yeast as model, we found that machine learning classifiers trained with either CS or DS features alone perform better in predicting TF-specific binding compared to SM-based classifiers. In addition, simultaneously considering CS and DS further improves the accuracy of the TF binding predictions, indicating the highly complementary nature of these two properties. The contributions of SM, CS, and DS features to binding site predictions differ greatly between TFs, allowing TF-specific predictions and potentially reflecting different TF binding mechanisms. In addition, a "TF-agnostic" predictive model based on three DNA "intrinsic properties" (in silico predicted nucleosome occupancy, major groove geometry, and dinucleotide free energy) that can be calculated from genomic sequences alone has performance that rivals the model incorporating experiment-derived data. This intrinsic property model allows prediction of binding regions not only across TFs, but also across DNA-binding domain families with distinct structural folds. Furthermore, these predicted binding regions can help identify TF binding sites that have a significant impact on target gene expression. Because the intrinsic property model allows prediction of binding regions across DNA-binding domain families, it is TF agnostic and likely describes general binding potential of TFs. Thus, our findings suggest that it is feasible to establish a TF agnostic model for identifying functional regulatory regions in potentially any sequenced genome.

  6. Contribution of Sequence Motif, Chromatin State, and DNA Structure Features to Predictive Models of Transcription Factor Binding in Yeast

    PubMed Central

    Tsai, Zing Tsung-Yeh; Shiu, Shin-Han; Tsai, Huai-Kuang

    2015-01-01

    Transcription factor (TF) binding is determined by the presence of specific sequence motifs (SM) and chromatin accessibility, where the latter is influenced by both chromatin state (CS) and DNA structure (DS) properties. Although SM, CS, and DS have been used to predict TF binding sites, a predictive model that jointly considers CS and DS has not been developed to predict either TF-specific binding or general binding properties of TFs. Using budding yeast as model, we found that machine learning classifiers trained with either CS or DS features alone perform better in predicting TF-specific binding compared to SM-based classifiers. In addition, simultaneously considering CS and DS further improves the accuracy of the TF binding predictions, indicating the highly complementary nature of these two properties. The contributions of SM, CS, and DS features to binding site predictions differ greatly between TFs, allowing TF-specific predictions and potentially reflecting different TF binding mechanisms. In addition, a "TF-agnostic" predictive model based on three DNA “intrinsic properties” (in silico predicted nucleosome occupancy, major groove geometry, and dinucleotide free energy) that can be calculated from genomic sequences alone has performance that rivals the model incorporating experiment-derived data. This intrinsic property model allows prediction of binding regions not only across TFs, but also across DNA-binding domain families with distinct structural folds. Furthermore, these predicted binding regions can help identify TF binding sites that have a significant impact on target gene expression. Because the intrinsic property model allows prediction of binding regions across DNA-binding domain families, it is TF agnostic and likely describes general binding potential of TFs. Thus, our findings suggest that it is feasible to establish a TF agnostic model for identifying functional regulatory regions in potentially any sequenced genome. PMID:26291518

  7. Redundant ERF-VII Transcription Factors Bind to an Evolutionarily Conserved cis-Motif to Regulate Hypoxia-Responsive Gene Expression in Arabidopsis

    PubMed Central

    Gasch, Philipp; Fundinger, Moritz; Müller, Jana T.; Lee, Travis; Mustroph, Angelika

    2016-01-01

    The response of Arabidopsis thaliana to low-oxygen stress (hypoxia), such as during shoot submergence or root waterlogging, includes increasing the levels of ∼50 hypoxia-responsive gene transcripts, many of which encode enzymes associated with anaerobic metabolism. Upregulation of over half of these mRNAs involves stabilization of five group VII ethylene response factor (ERF-VII) transcription factors, which are routinely degraded via the N-end rule pathway of proteolysis in an oxygen- and nitric oxide-dependent manner. Despite their importance, neither the quantitative contribution of individual ERF-VIIs nor the cis-regulatory elements they govern are well understood. Here, using single- and double-null mutants, the constitutively synthesized ERF-VIIs RELATED TO APETALA2.2 (RAP2.2) and RAP2.12 are shown to act redundantly as principle activators of hypoxia-responsive genes; constitutively expressed RAP2.3 contributes to this redundancy, whereas the hypoxia-induced HYPOXIA RESPONSIVE ERF1 (HRE1) and HRE2 play minor roles. An evolutionarily conserved 12-bp cis-regulatory motif that binds to and is sufficient for activation by RAP2.2 and RAP2.12 is identified through a comparative phylogenetic motif search, promoter dissection, yeast one-hybrid assays, and chromatin immunopurification. This motif, designated the hypoxia-responsive promoter element, is enriched in promoters of hypoxia-responsive genes in multiple species. PMID:26668304

  8. Localization and trafficking of an isoform of the AtPRA1 family to the Golgi apparatus depend on both N- and C-terminal sequence motifs.

    PubMed

    Jung, Chan Jin; Lee, Myoung Hui; Min, Myung Ki; Hwang, Inhwan

    2011-02-01

    Prenylated Rab acceptors (PRAs) bind to prenylated Rab proteins and possibly aid in targeting Rabs to their respective compartments. In Arabidopsis, 19 isoforms of PRA1 have been identified and, depending upon the isoforms, they localize to the endoplasmic reticulum (ER), Golgi apparatus and endosomes. Here, we investigated the localization and trafficking of AtPRA1.B6, an isoform of the Arabidopsis PRA1 family. In colocalization experiments with various organellar markers, AtPRA1.B6 tagged with hemagglutinin (HA) at the N-terminus localized to the Golgi apparatus in protoplasts and transgenic plants. The valine residue at the C-terminal end and an EEE motif in the C-terminal cytoplasmic domain were critical for anterograde trafficking from the ER to the Golgi apparatus. The N-terminal region contained a sequence motif for retention of AtPRA1.B6 at the Golgi apparatus. In addition, anterograde trafficking of AtPRA1.B6 from the ER to the Golgi apparatus was highly sensitive to the HA:AtPRA1.B6 level. The region that contains the sequence motif for Golgi retention also conferred the abundance-dependent trafficking inhibition. On the basis of these results, we propose that AtPRA1.B6 localizes to the Golgi apparatus and its ER-to-Golgi trafficking and localization to the Golgi apparatus are regulated by multiple sequence motifs in both the C- and N-terminal cytoplasmic domains.

  9. Discovering conserved insect microRNAs from expressed sequence tags.

    PubMed

    Jia, Qidong; Lin, Kejian; Liang, Jingdong; Yu, Lun; Li, Fei

    2010-12-01

    MicroRNAs (miRNA) participate in regulating diverse biological pathways by translational repression in animals. They have attracted increasing attention recently. However, little work has been done on the miRNA genes in agriculturally important pests. Because the transcripts of most miRNA genes are the products of type-II RNA polymerase, pri-miRNA has a poly(A) tail and appears in expressed sequence tags (EST). We developed a computational pipeline to identify miRNA genes from insect ESTs. First, 980,697 ESTs from 63 insects were collected and used to search the nr database. The ESTs which did not share significant similarities with any known protein-coding genes were treated as non-coding ESTs. Next, known mature miRNAs were used to align with non-coding ESTs. The ESTs which contain the sequence of mature miRNA were treated as candidate ESTs. Finally, putative precursors were extracted flanking the mature miRNA region in candidate ESTs and evaluated by the Triplet-SVM algorithm. As a result, 86 miRNAs from 30 insect species were found based on a strict criterion while 330 miRNAs from 51 species were found based on a loose criterion. Evolution analysis indicated that mir-467, mir-297 and mir-466 were the highest conserved miRNA families in insects. To confirm the reliability of putative insect miRNAs, the expression profile of nine predicted miRNAs in Locusta migratoria was investigated. Eight miRNAs were successfully detected by RT-PCR. Most miRNAs were expressed ubiquitously at all examined tissues and developmental stages whereas Lmi-mir-509 was specifically expressed in the thorax of the 2nd, 4th and 5th instars and adult locust. In all, our work reported an efficient computational strategy for predicting miRNA genes from insect ESTs and presented tens of miRNAs in diverse insect species which are expected to participate in many important physiological processes.

  10. Characterization of the dead ringer gene identifies a novel, highly conserved family of sequence-specific DNA-binding proteins.

    PubMed Central

    Gregory, S L; Kortschak, R D; Kalionis, B; Saint, R

    1996-01-01

    We reported the identification of a new family of DNA-binding proteins from our characterization of the dead ringer (dri) gene of Drosophila melanogaster. We show that dri encodes a nuclear protein that contains a sequence-specific DNA-binding domain that bears no similarity to known DNA-binding domains. A number of proteins were found to contain sequences homologous to this domain. Other proteins containing the conserved motif include yeast SWI1, two human retinoblastoma binding proteins, and other mammalian regulatory proteins. A mouse B-cell-specific regulator exhibits 75% identity with DRI over the 137-amino-acid DNA-binding domains of these proteins, indicating a high degree of conservation of this domain. Gel retardation and optimal binding site screens revealed that the in vitro sequence specificity of DRI is strikingly similar to that of many homeodomain proteins, although the sequence and predicted secondary structure do not resemble a homeodomain. The early general expression of dri and the similarity of DRI and homeodomain in vitro DNA-binding specificity compound the problem of understanding the in vivo specificity of action of these proteins. Maternally derived dri product is found throughout the embryo until germ band extension, when dri is expressed in a developmentally regulated set of tissues, including salivary gland ducts, parts of the gut, and a subset of neural cells. The discovery of this new, conserved DNA-binding domain offers an explanation for the regulatory activity of several important members of this class and predicts significant regulatory roles for the others. PMID:8622680

  11. Identification of a novel calcium binding motif based on the detection of sequence insertions in the animal peroxidase domain of bacterial proteins.

    PubMed

    Santamaría-Hernando, Saray; Krell, Tino; Ramos-González, María-Isabel

    2012-01-01

    Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca(2+) coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+) binding with a K(D) of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life.

  12. The orphan G protein-coupled receptor GPR139 is activated by the peptides: Adrenocorticotropic hormone (ACTH), α-, and β-melanocyte stimulating hormone (α-MSH, and β-MSH), and the conserved core motif HFRW.

    PubMed

    Nøhr, Anne Cathrine; Shehata, Mohamed A; Hauser, Alexander S; Isberg, Vignir; Mokrosinski, Jacek; Andersen, Kirsten B; Farooqi, I Sadaf; Pedersen, Daniel Sejer; Gloriam, David E; Bräuner-Osborne, Hans

    2017-01-01

    GPR139 is an orphan G protein-coupled receptor that is expressed primarily in the brain. Not much is known regarding the function of GPR139. Recently we have shown that GPR139 is activated by the amino acids l-tryptophan and l-phenylalanine (EC50 values of 220 μM and 320 μM, respectively), as well as di-peptides comprised of aromatic amino acids. This led us to hypothesize that GPR139 may be activated by peptides. Sequence alignment of the binding cavities of all class A GPCRs, revealed that the binding pocket of the melanocortin 4 receptor is similar to that of GPR139. Based on the chemogenomics principle "similar targets bind similar ligands", we tested three known endogenous melanocortin 4 receptor agonists; adrenocorticotropic hormone (ACTH) and α- and β-melanocyte stimulating hormone (α-MSH and β-MSH) on CHO-k1 cells stably expressing the human GPR139 in a Fluo-4 Ca(2+)-assay. All three peptides, as well as their conserved core motif HFRW, were found to activate GPR139 in the low micromolar range. Moreover, we found that peptides consisting of nine or ten N-terminal residues of α-MSH activate GPR139 in the submicromolar range. α-MSH1-9 was found to correspond to the product of a predicted cleavage site in the pre-pro-protein pro-opiomelanocortin (POMC). Our results demonstrate that GPR139 is a peptide receptor, activated by ACTH, α-MSH, β-MSH, the conserved core motif HFRW as well as a potential endogenous peptide α-MSH1-9. Further studies are needed to determine the functional relevance of GPR139 mediated signaling by these peptides.

  13. Formation and Dissociation of the Interstrand i-Motif by the Sequences d(XnC4Ym) Monitored with Electrospray Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Cao, Yanwei; Qin, Yujiao; Bruist, Michael; Gao, Shang; Wang, Bing; Wang, Huixin; Guo, Xinhua

    2015-06-01

    Formation and dissociation of the interstrand i-motifs by DNA with the sequence d(XnC4Ym) (X and Y represent thymine, adenine, or guanine, and n, m range from 0 to 2) are studied with electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and UV spectrophotometry. The ion complexes detected in the gas phase and the melting temperatures (Tm) obtained in solution show that a non-C base residue located at 5' end favors formation of the four-stranded structures, with T > A > G for imparting stability. Comparatively, no rule is found when a non-C base is located at the 3' end. Detection of penta- and hexa-stranded ions indicates the formation of i-motifs with more than four strands. In addition, the i-motifs seen in our mass spectra are accompanied by single-, double-, and triple-stranded ions, and the trimeric ions were always less abundant during annealing and heat-induced dissociation process of the DNA strands in solution (pH = 4.5). This provides a direct evidence of a strand-by-strand formation and dissociation pathway of the interstrand i-motif and formation of the triple strands is the rate-limiting step. In contrast, the trimeric ions are abundant when the tetramolecular ions are subjected to collision-induced dissociation (CID) in the gas phase, suggesting different dissociation behaviors of the interstrand i-motif in the gas phase and in solution. Furthermore, hysteretic UV absorption melting and cooling curves reveal an irreversible dissociation and association kinetic process of the interstrand i-motif in solution.

  14. De Novo Regulatory Motif Discovery Identifies Significant Motifs in Promoters of Five Classes of Plant Dehydrin Genes

    PubMed Central

    Zolotarov, Yevgen; Strömvik, Martina

    2015-01-01

    Plants accumulate dehydrins in response to osmotic stresses. Dehydrins are divided into five different classes, which are thought to be regulated in different manners. To better understand differences in transcriptional regulation of the five dehydrin classes, de novo motif discovery was performed on 350 dehydrin promoter sequences from a total of 51 plant genomes. Overrepresented motifs were identified in the promoters of five dehydrin classes. The Kn dehydrin promoters contain motifs linked with meristem specific expression, as well as motifs linked with cold/dehydration and abscisic acid response. KS dehydrin promoters contain a motif with a GATA core. SKn and YnSKn dehydrin promoters contain motifs that match elements connected with cold/dehydration, abscisic acid and light response. YnKn dehydrin promoters contain motifs that match abscisic acid and light response elements, but not cold/dehydration response elements. Conserved promoter motifs are present in the dehydrin classes and across different plant lineages, indicating that dehydrin gene regulation is likely also conserved. PMID:26114291

  15. Mutation of the Conserved Calcium-Binding Motif in Neisseria gonorrhoeae PilC1 Impacts Adhesion but Not Piliation

    PubMed Central

    Cheng, Yuan; Johnson, Michael D. L.; Burillo-Kirch, Christine; Mocny, Jeffrey C.; Anderson, James E.; Garrett, Christopher K.; Redinbo, Matthew R.

    2013-01-01

    Neisseria gonorrhoeae PilC1 is a member of the PilC family of type IV pilus-associated adhesins found in Neisseria species and other type IV pilus-producing genera. Previously, a calcium-binding domain was described in the C-terminal domains of PilY1 of Pseudomonas aeruginosa and in PilC1 and PilC2 of Kingella kingae. Genetic analysis of N. gonorrhoeae revealed a similar calcium-binding motif in PilC1. To evaluate the potential significance of this calcium-binding region in N. gonorrhoeae, we produced recombinant full-length PilC1 and a PilC1 C-terminal domain fragment. We show that, while alterations of the calcium-binding motif disrupted the ability of PilC1 to bind calcium, they did not grossly affect the secondary structure of the protein. Furthermore, we demonstrate that both full-length wild-type PilC1 and full-length calcium-binding-deficient PilC1 inhibited gonococcal adherence to cultured human cervical epithelial cells, unlike the truncated PilC1 C-terminal domain. Similar to PilC1 in K. kingae, but in contrast to the calcium-binding mutant of P. aeruginosa PilY1, an equivalent mutation in N. gonorrhoeae PilC1 produced normal amounts of pili. However, the N. gonorrhoeae PilC1 calcium-binding mutant still had partial defects in gonococcal adhesion to ME180 cells and genetic transformation, which are both essential virulence factors in this human pathogen. Thus, we conclude that calcium binding to PilC1 plays a critical role in pilus function in N. gonorrhoeae. PMID:24002068

  16. Sequence Conservation, Radial Distance and Packing Density in Spherical Viral Capsids

    PubMed Central

    Lee, Chi-Wen; Huang, Tsun-Tsao; Shih, Chung-Shiuan; Hwang, Jenn-Kang

    2015-01-01

    The conservation level of a residue is a useful measure about the importance of that residue in protein structure and function. Much information about sequence conservation comes from aligning homologous sequences. Profiles showing the variation of the conservation level along the sequence are usually interpreted in evolutionary terms and dictated by site similarities of a proper set of homologous sequences. Here, we report that, of the viral icosahedral capsids, the sequence conservation profile can be determined by variations in the distances between residues and the centroid of the capsid – with a direct inverse proportionality between the conservation level and the centroid distance – as well as by the spatial variations in local packing density. Examining both the centroid and the packing density models against a dataset of 51 crystal structures of nonhomologous icosahedral capsids, we found that many global patterns and minor features derived from the viral structures are consistent with those present in the sequence conservation profiles. The quantitative link between the level of conservation and structural features like centroid-distance or packing density allows us to look at residue conservation from a structural viewpoint as well as from an evolutionary viewpoint. PMID:26132081

  17. A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs.

    PubMed

    Gromadzka, Agnieszka M; Steckelberg, Anna-Lena; Singh, Kusum K; Hofmann, Kay; Gehring, Niels H

    2016-03-18

    The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs.

  18. Functional interaction between the Fanconi Anemia D2 protein and proliferating cell nuclear antigen (PCNA) via a conserved putative PCNA interaction motif.

    PubMed

    Howlett, Niall G; Harney, Julie A; Rego, Meghan A; Kolling, Frederick W; Glover, Thomas W

    2009-10-16

    Fanconi Anemia (FA) is a rare recessive disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The FA proteins and the familial breast cancer susceptibility gene products, BRCA1 and FANCD1/BRCA2, function cooperatively in the FA-BRCA pathway to repair damaged DNA and to prevent cellular transformation. Activation of this pathway occurs via the mono-ubiquitination of the FANCD2 protein, targeting it to nuclear foci where it co-localizes with FANCD1/BRCA2, RAD51, and PCNA. The regulation of the mono-ubiquitination of FANCD2, as well as its function in DNA repair remain poorly understood. In this study, we have further characterized the interaction between the FANCD2 and PCNA proteins. We have identified a highly conserved, putative FANCD2 PCNA interaction motif (PIP-box), and demonstrate that mutation of this motif disrupts FANCD2-PCNA binding and precludes the mono-ubiquitination of FANCD2. Consequently, the FANCD2 PIP-box mutant protein fails to correct the mitomycin C hypersensitivity of FA-D2 patient cells. Our results suggest that PCNA may function as a molecular platform to facilitate the mono-ubiquitination of FANCD2 and activation of the FA-BRCA pathway.

  19. A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs

    PubMed Central

    Gromadzka, Agnieszka M.; Steckelberg, Anna-Lena; Singh, Kusum K.; Hofmann, Kay; Gehring, Niels H.

    2016-01-01

    The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs. PMID:26773052

  20. Multi-layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid

    PubMed Central

    Montespan, Charlotte; Marvin, Shauna A.; Burrage, Andrew M.; Roger, Benoit; Rayne, Fabienne; Schneider, Carola; Reimer, Rudolph; Wiethoff, Christopher M.

    2017-01-01

    Cells employ active measures to restrict infection by pathogens, even prior to responses from the innate and humoral immune defenses. In this context selective autophagy is activated upon pathogen induced membrane rupture to sequester and deliver membrane fragments and their pathogen contents for lysosomal degradation. Adenoviruses, which breach the endosome upon entry, escape this fate by penetrating into the cytosol prior to autophagosome sequestration of the ruptured endosome. We show that virus induced membrane damage is recognized through Galectin-8 and sequesters the autophagy receptors NDP52 and p62. We further show that a conserved PPxY motif in the viral membrane lytic protein VI is critical for efficient viral evasion of autophagic sequestration after endosomal lysis. Comparing the wildtype with a PPxY-mutant virus we show that depletion of Galectin-8 or suppression of autophagy in ATG5-/- MEFs rescues infectivity of the PPxY-mutant virus while depletion of the autophagy receptors NDP52, p62 has only minor effects. Furthermore we show that wildtype viruses exploit the autophagic machinery for efficient nuclear genome delivery and control autophagosome formation via the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Our data thus demonstrate that a short PPxY-peptide motif in the adenoviral capsid permits multi-layered viral control of autophagic processes during entry. PMID:28192531

  1. Small yet effective: the ethylene responsive element binding factor-associated amphiphilic repression (EAR) motif.

    PubMed

    Kagale, Sateesh; Rozwadowski, Kevin

    2010-06-01

    The Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) motif is a small yet distinct regulatory motif that is conserved in many plant transcriptional regulator (TR) proteins associated with diverse biological functions. We have previously established a list of high-confidence Arabidopsis EAR repressors, the EAR repressome, comprising 219 TRs belonging to 21 different TR families. This class of proteins and the sequence context of the EAR motif exhibited a high degree of conservation across evolutionarily diverse plant species. Our comprehensive genome-wide analysis enabled refining EAR motifs as comprising either LxLxL or DLNxxP. Comparing the representation of these sequence signatures in TRs to that of other repressor motifs we show that the EAR motif is the one most frequently represented, detected in 10 to 25% of the TRs from diverse plant species. The mechanisms involved in regulation of EAR motif function and the cellular fates of EAR repressors are currently not well understood. Our earlier analysis had implicated amino acid residues flanking the EAR motifs in regulation of their functionality. Here, we present additional evidence supporting possible regulation of EAR motif function by phosphorylation of integral or adjacent Ser and/or Thr residues. Additionally, we discuss potential novel roles of EAR motifs in plant-pathogen interaction and processes other than transcriptional repression.

  2. Ovodefensins, an Oviduct-Specific Antimicrobial Gene Family, Have Evolved in Birds and Reptiles to Protect the Egg by Both Sequence and Intra-Six-Cysteine Sequence Motif Spacing.

    PubMed

    Whenham, Natasha; Lu, Tian Chee; Maidin, Maisarah B M; Wilson, Peter W; Bain, Maureen M; Stevenson, M Lynn; Stevens, Mark P; Bedford, Michael R; Dunn, Ian C

    2015-06-01

    Ovodefensins are a novel beta defensin-related family of antimicrobial peptides containing conserved glycine and six cysteine residues. Originally thought to be restricted to the albumen-producing region of the avian oviduct, expression was found in chicken, turkey, duck, and zebra finch in large quantities in many parts of the oviduct, but this varied between species and between gene forms in the same species. Using new search strategies, the ovodefensin family now has 35 members, including reptiles, but no representatives outside birds and reptiles have been found. Analysis of their evolution shows that ovodefensins divide into six groups based on the intra-cysteine amino acid spacing, representing a unique mechanism alongside traditional evolution of sequence. The groups have been used to base a nomenclature for the family. Antimicrobial activity for three ovodefensins from chicken and duck was confirmed against Escherichia coli and a pathogenic E. coli strain as well as a Gram-positive organism, Staphylococcus aureus, for the first time. However, activity varied greatly between peptides, with Gallus gallus OvoDA1 being the most potent, suggesting a link with the different structures. Expression of Gallus gallus OvoDA1 (gallin) in the oviduct was increased by estrogen and progesterone and in the reproductive state. Overall, the results support the hypothesis that ovodefensins evolved to protect the egg, but they are not necessarily restricted to the egg white. Therefore, divergent motif structure and sequence present an interesting area of research for antimicrobial peptide design and understanding protection of the cleidoic egg.

  3. Retinoic acid-induced down-regulation of the interleukin-2 promoter via cis-regulatory sequences containing an octamer motif.

    PubMed Central

    Felli, M P; Vacca, A; Meco, D; Screpanti, I; Farina, A R; Maroder, M; Martinotti, S; Petrangeli, E; Frati, L; Gulino, A

    1991-01-01

    Retinoic acid (RA) is known to influence the proliferation and differentiation of a wide variety of transformed and developing cells. We found that RA and the specific RA receptor (RAR) ligand Ch55 inhibited the phorbol ester and calcium ionophore-induced expression of the T-cell growth factor interleukin-2 (IL-2) gene. Expression of transiently transfected chloramphenicol acetyltransferase vectors containing the 5'-flanking region of the IL-2 gene was also inhibited by RA. RA-induced down-regulation of the IL-2 enhancer is mediated by RAR, since overexpression of transfected RARs increased RA sensitivity of the IL-2 promoter. Functional analysis of chloramphenicol acetyltransferase vectors containing either internal deletion mutants of the region from -317 to +47 bp of the IL-2 enhancer or multimerized cis-regulatory elements showed that the RA-responsive element in the IL-2 promoter mapped to sequences containing an octamer motif. RAR also inhibited the transcriptional activity of the octamer motif of the immunoglobulin heavy chain enhancer. In spite of the transcriptional inhibition of the IL-2 octamer motif, RA did not decrease the in vitro DNA-binding capability of octamer-1 protein. These results identify a regulatory pathway within the IL-2 promoter which involves the octamer motif and RAR. Images PMID:1652063

  4. Trypanosoma cruzi Binds to Cytokeratin through Conserved Peptide Motifs Found in the Laminin-G-Like Domain of the gp85/Trans-sialidase Proteins

    PubMed Central

    Teixeira, Andre Azevedo Reis; de Vasconcelos, Veronica de Cássia Sardinha; Colli, Walter; Alves, Maria Júlia Manso; Giordano, Ricardo José

    2015-01-01

    Background Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is a disease that affects millions of people most of them living in South and Central Americas. There are few treatment options for individuals with Chagas' disease making it important to understand the molecular details of parasite infection, so novel therapeutic alternatives may be developed for these patients. Here, we investigate the interaction between host cell intermediate filament proteins and the T. cruzi gp85 glycoprotein superfamily with hundreds of members that have long been implicated in parasite cell invasion. Methodology/Principal Findings An in silico analysis was utilized to identify peptide motifs shared by the gp85 T. cruzi proteins and, using phage display, these selected peptide motifs were screened for their ability to bind to cells. One peptide, named TS9, showed significant cell binding capacity and was selected for further studies. Affinity chromatography, phage display and invasion assays revealed that peptide TS9 binds to cytokeratins and vimentin, and prevents T. cruzi cell infection. Interestingly, peptide TS9 and a previously identified binding site for intermediate filament proteins are disposed in an antiparallel β-sheet fold, present in a conserved laminin-G-like domain shared by all members of the family. Moreover, peptide TS9 overlaps with an immunodominant T cell epitope. Conclusions/Significance Taken together, the present study reinforces previous results from our group implicating the gp85 superfamily of glycoproteins and the intermediate filament proteins cytokeratin and vimentin in the parasite infection process. It also suggests an important role in parasite biology for the conserved laminin-G-like domain, present in all members of this large family of cell surface proteins. PMID:26398185

  5. Coupling DNA-binding and ATP hydrolysis in Escherichia coli RecQ: role of a highly conserved aromatic-rich sequence.

    PubMed

    Zittel, Morgan C; Keck, James L

    2005-01-01

    RecQ enzymes are broadly conserved Superfamily-2 (SF-2) DNA helicases that play critical roles in DNA metabolism. RecQ proteins use the energy of ATP hydrolysis to drive DNA unwinding; however, the mechanisms by which RecQ links ATPase activity to DNA-binding/unwinding are unknown. In many Superfamily-1 (SF-1) DNA helicases, helicase sequence motif III links these activities by binding both single-stranded (ss) DNA and ATP. However, the ssDNA-binding aromatic-rich element in motif III present in these enzymes is missing from SF-2 helicases, raising the question of how these enzymes link ATP hydrolysis to DNA-binding/unwinding. We show that Escherichia coli RecQ contains a conserved aromatic-rich loop in its helicase domain between motifs II and III. Although placement of the RecQ aromatic-rich loop is topologically distinct relative to the SF-1 enzymes, both loops map to similar tertiary structural positions. We examined the functions of the E.coli RecQ aromatic-rich loop using RecQ variants with single amino acid substitutions within the segment. Our results indicate that the aromatic-rich loop in RecQ is critical for coupling ATPase and DNA-binding/unwinding activities. Our studies also suggest that RecQ's aromatic-rich loop might couple ATP hydrolysis to DNA-binding in a mechanistically distinct manner from SF-1 helicases.

  6. High sequence conservation among cucumber mosaic virus isolates from lily.

    PubMed

    Chen, Y K; Derks, A F; Langeveld, S; Goldbach, R; Prins, M

    2001-08-01

    For classification of Cucumber mosaic virus (CMV) isolates from ornamental crops of different geographical areas, these were characterized by comparing the nucleotide sequences of RNAs 4 and the encoded coat proteins. Within the ornamental-infecting CMV viruses both subgroups were represented. CMV isolates of Alstroemeria and crocus were classified as subgroup II isolates, whereas 8 other isolates, from lily, gladiolus, amaranthus, larkspur, and lisianthus, were identified as subgroup I members. In general, nucleotide sequence comparisons correlated well with geographic distribution, with one notable exception: the analyzed nucleotide sequences of 5 lily isolates showed remarkably high homology despite different origins.

  7. Motif discovery with data mining in 3D protein structure databases: discovery, validation and prediction of the U-shape zinc binding ("Huf-Zinc") motif.

    PubMed

    Maurer-Stroh, Sebastian; Gao, He; Han, Hao; Baeten, Lies; Schymkowitz, Joost; Rousseau, Frederic; Zhang, Louxin; Eisenhaber, Frank

    2013-02-01

    Data mining in protein databases, derivatives from more fundamental protein 3D structure and sequence databases, has considerable unearthed potential for the discovery of sequence motif--structural motif--function relationships as the finding of the U-shape (Huf-Zinc) motif, originally a small student's project, exemplifies. The metal ion zinc is critically involved in universal biological processes, ranging from protein-DNA complexes and transcription regulation to enzymatic catalysis and metabolic pathways. Proteins have evolved a series of motifs to specifically recognize and bind zinc ions. Many of these, so called zinc fingers, are structurally independent globular domains with discontinuous binding motifs made up of residues mostly far apart in sequence. Through a systematic approach starting from the BRIX structure fragment database, we discovered that there exists another predictable subset of zinc-binding motifs that not only have a conserved continuous sequence pattern but also share a characteristic local conformation, despite being included in totally different overall folds. While this does not allow general prediction of all Zn binding motifs, a HMM-based web server, Huf-Zinc, is available for prediction of these novel, as well as conventional, zinc finger motifs in protein sequences. The Huf-Zinc webserver can be freely accessed through this URL (http://mendel.bii.a-star.edu.sg/METHODS/hufzinc/).

  8. Nuclear Magnetic Resonance Solution Structures of Lacticin Q and Aureocin A53 Reveal a Structural Motif Conserved among Leaderless Bacteriocins with Broad-Spectrum Activity.

    PubMed

    Acedo, Jeella Z; van Belkum, Marco J; Lohans, Christopher T; Towle, Kaitlyn M; Miskolzie, Mark; Vederas, John C

    2016-02-02

    Lacticin Q (LnqQ) and aureocin A53 (AucA) are leaderless bacteriocins from Lactococcus lactis QU5 and Staphylococcus aureus A53, respectively. These bacteriocins are characterized by the absence of an N-terminal leader sequence and are active against a broad range of Gram-positive bacteria. LnqQ and AucA consist of 53 and 51 amino acids, respectively, and have 47% identical sequences. In this study, their three-dimensional structures were elucidated using solution nuclear magnetic resonance and were shown to consist of four α-helices that assume a very similar compact, globular overall fold (root-mean-square deviation of 1.7 Å) with a highly cationic surface and a hydrophobic core. The structures of LnqQ and AucA resemble the shorter two-component leaderless bacteriocins, enterocins 7A and 7B, despite having low levels of sequence identity. Homology modeling revealed that the observed structural motif may be shared among leaderless bacteriocins with broad-spectrum activity against Gram-positive organisms. The elucidated structures of LnqQ and AucA also exhibit some resemblance to circular bacteriocins. Despite their similar overall fold, inhibition studies showed that LnqQ and AucA have different antimicrobial potency against the Gram-positive strains tested, suggesting that sequence disparities play a crucial role in their mechanisms of action.

  9. Nuclear Magnetic Resonance Structural Mapping Reveals Promiscuous Interactions between Clathrin-Box Motif Sequences and the N-Terminal Domain of the Clathrin Heavy Chain

    PubMed Central

    2016-01-01

    The recruitment and organization of clathrin at endocytic sites first to form coated pits and then clathrin-coated vesicles depend on interactions between the clathrin N-terminal domain (TD) and multiple clathrin binding sequences on the cargo adaptor and accessory proteins that are concentrated at such sites. Up to four distinct protein binding sites have been proposed to be present on the clathrin TD, with each site proposed to interact with a distinct clathrin binding motif. However, an understanding of how such interactions contribute to clathrin coat assembly must take into account observations that any three of these four sites on clathrin TD can be mutationally ablated without causing loss of clathrin-mediated endocytosis. To take an unbiased approach to mapping binding sites for clathrin-box motifs on clathrin TD, we used isothermal titration calorimetry (ITC) and nuclear magnetic resonance spectroscopy. Our ITC experiments revealed that a canonical clathrin-box motif peptide from the AP-2 adaptor binds to clathrin TD with a stoichiometry of 3:1. Assignment of 90% of the total visible amide resonances in the TROSY-HSQC spectrum of 13C-, 2H-, and 15N-labeled TD40 allowed us to map these three binding sites by analyzing the chemical shift changes as clathrin-box motif peptides were titrated into clathrin TD. We found that three different clathrin-box motif peptides can each simultaneously bind not only to the previously characterized clathrin-box site but also to the W-box site and the β-arrestin splice loop site on a single TD. The promiscuity of these binding sites can help explain why their mutation does not lead to larger effects on clathrin function and suggests a mechanism by which clathrin may be transferred between different proteins during the course of an endocytic event. PMID:25844500

  10. Sequence and spatiotemporal expression analysis of CLE-motif containing genes from the reniform nematode (Rotylenchulus reniformis Linford & Oliveira)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The reniform nematode, Rotylenchulus reniformis, is a sedentary semi-endoparasitic species with a host range that encompasses more than 77 plant families. Nematode effector proteins containing plant-ligand motifs similar to CLAVATA3/ESR (CLE) peptides have been identified in the Heterodera, Globode...

  11. Bivalent Formation 1, a plant-conserved gene, encodes an OmpH/coiled-coil motif-containing protein required for meiotic recombination in rice.

    PubMed

    Zhou, Lian; Han, Jingluan; Chen, Yuanling; Wang, Yingxiang; Liu, Yao-Guang

    2017-03-24

    Meiosis is essential for eukaryotic sexual reproduction and plant fertility. In comparison with over 80 meiotic genes identified in Arabidopsis, there are only ~30 meiotic genes characterized in rice (Oryza sativa L.). Many genes involved in the regulation of meiotic progression remain to be determined. In this study, we identified a sterile rice mutant and cloned a new meiotic gene, OsBVF1 (Bivalent Formation 1) by map-based cloning. Molecular genetics and cytological approaches were carried out to address the function of OsBVF1 in meiosis. Phylogenetic analyses were used to study the evolution of OsBVF1 and its homologs in plant species. Here we showed that the bvf1 male meiocytes were defective in formation of meiotic double strand break, thereby resulting in a failure of bivalent formation in diakinesis and unequal chromosome segregation in anaphase I. The causal gene, OsBVF1, encodes a unique OmpH/coiled-coil motif-containing protein and its homologs are highly conserved in the plant kingdom and seem to be a single-copy gene in the majority of plant species. Our study demonstrates that OsBVF1 is a novel plant-conserved factor involved in meiotic recombination in rice, providing a new insight into understanding of meiotic progression regulation.

  12. Large Putative PEST-like Sequence Motif at the Carboxyl Tail of Human Calcium Receptor Directs Lysosomal Degradation and Regulates Cell Surface Receptor Level*

    PubMed Central

    Zhuang, Xiaolei; Northup, John K.; Ray, Kausik

    2012-01-01

    A deletion between amino acid residues Ser895 and Val1075 in the carboxyl terminus of the human calcium receptor (hCaR), which causes autosomal dominant hypocalcemia, showed enhanced signaling activity and increased cell surface expression in HEK293 cells (Lienhardt, A., Garabédian, M. G., Bai, M., Sinding, C., Zhang, Z., Lagarde, J. P., Boulesteix, J., Rigaud, M., Brown, E. M., and Kottler, M. L. (2000) J. Clin. Endocrinol. Metab. 85, 1695–1702). To identify the underlying mechanism(s) for these increases, we investigated the effects of carboxyl tail truncation and deletion in hCaR mutants using a combination of biochemical and cell imaging approaches to define motifs that participate in regulating cell surface numbers of this G protein-coupled receptor. Our data indicate a rapid constitutive receptor internalization of the cell surface hCaR, accumulating in early (Rab7 positive) and late endosomal (LAMP1 positive) sorting compartments, before targeting to lysosomes for degradation. Recycling of hCaR back to the cell surface was also evident. Truncation and deletion mapping defined a 51-amino acid sequence between residues 920 and 970 that is required for targeting to lysosomes and degradation but not for internalization or recycling of the receptor. No singular sequence motif was identified, instead the required sequence elements seem to distribute throughout this entire interval. This interval includes a high proportion of acidic and hydroxylated amino acid residues, suggesting a similarity to PEST-like degradation motif (PESTfind score of +10) and several glutamine repeats. The results define a novel large PEST-like sequence that participates in the sorting of internalized hCaR routed to the lysosomal/degradation pathway that regulates cell surface receptor numbers. PMID:22158862

  13. Sharing of four DR-beta sequence motifs between HLA-DRB1*1601 and DRB1*1101 correlates with frequent degenerate T-cell recognition of HA306-320 peptide complexed to these two molecules.

    PubMed

    Zeliszewski, D; Dorval, I; Golvano, J J; Prevost, A; Borras-Cuesta, F; Sterkers, G

    1996-02-01

    This paper shows that the seven HA306-320 specific T-cell clones isolated from one individual recognize the peptide complexed to both autologous HLA-DRB1*1101 and allogeneic HLA-DRB1*1601 (or DRB5*0201) molecules. For each T-cell clone, a single T-cell receptor (TCR) is involved in the recognition of these two different peptide-DR complexes as evidenced by cold target competition experiments. Yet, the seven T-cell clones express several different TCRs as judged by V beta-J beta usage and fine specificities. Furthermore, one representative clone has the same fine specificity for HA306-320 analogues mutated at epitopic residues irrespective of the use of DR1101 or DR1601 APC. These results suggest that structural differences between DRB1*1101 and DRB1*1601 (or DRB5*0201) do not dramatically influence the orientation of HA306-320 in the grooves such that most residues interacting with TCRs are conserved. In another individual, the same pattern of restriction, i.e. DR1101 + DR1601, was found for several HA306-320 specific clones. Two additional patterns, DR1101 + DR0801 and DR1101 + DR0801 + DR1601, were identified. By comparing DR sequences the authors found that DRB1*1101 and DRB1*1601 share four important motifs, i.e. beta 85-86, beta 67-71, beta 57 and beta 28-31 supposed to line three distinct HLA-DR pockets. Three of these motifs are also shared with DRB1*0801. All the results further support that the motif similarities allow the peptide to adopt very similar orientations in the cross-reacting DR molecules.

  14. The valine and lysine residues in the conserved FxVTxK motif are important for the function of phylogenetically distant plant cellulose synthases.

    PubMed

    Slabaugh, Erin; Scavuzzo-Duggan, Tess; Chaves, Arielle; Wilson, Liza; Wilson, Carmen; Davis, Jonathan K; Cosgrove, Daniel J; Anderson, Charles T; Roberts, Alison W; Haigler, Candace H

    2016-05-01

    Cellulose synthases (CESAs) synthesize the β-1,4-glucan chains that coalesce to form cellulose microfibrils in plant cell walls. In addition to a large cytosolic (catalytic) domain, CESAs have eight predicted transmembrane helices (TMHs). However, analogous to the structure of BcsA, a bacterial CESA, predicted TMH5 in CESA may instead be an interfacial helix. This would place the conserved FxVTxK motif in the plant cell cytosol where it could function as a substrate-gating loop as occurs in BcsA. To define the functional importance of the CESA region containing FxVTxK, we tested five parallel mutations in Arabidopsis thaliana CESA1 and Physcomitrella patens CESA5 in complementation assays of the relevant cesa mutants. In both organisms, the substitution of the valine or lysine residues in FxVTxK severely affected CESA function. In Arabidopsis roots, both changes were correlated with lower cellulose anisotropy, as revealed by Pontamine Fast Scarlet. Analysis of hypocotyl inner cell wall layers by atomic force microscopy showed that two altered versions of Atcesa1 could rescue cell wall phenotypes observed in the mutant background line. Overall, the data show that the FxVTxK motif is functionally important in two phylogenetically distant plant CESAs. The results show that Physcomitrella provides an efficient model for assessing the effects of engineered CESA mutations affecting primary cell wall synthesis and that diverse testing systems can lead to nuanced insights into CESA structure-function relationships. Although CESA membrane topology needs to be experimentally determined, the results support the possibility that the FxVTxK region functions similarly in CESA and BcsA.

  15. MINER: software for phylogenetic motif identification.

    PubMed

    La, David; Livesay, Dennis R

    2005-07-01

    MINER is web-based software for phylogenetic motif (PM) identification. PMs are sequence regions (fragments) that conserve the overall familial phylogeny. PMs have been shown to correspond to a wide variety of catalytic regions, substrate-binding sites and protein interfaces, making them ideal functional site predictions. The MINER output provides an intuitive interface for interactive PM sequence analysis and structural visualization. The web implementation of MINER is freely available at http://www.pmap.csupomona.edu/MINER/. Source code is available to the academic community on request.

  16. Multilign: an algorithm to predict secondary structures conserved in multiple RNA sequences

    PubMed Central

    Xu, Zhenjiang; Mathews, David H.

    2011-01-01

    Motivation: With recent advances in sequencing, structural and functional studies of RNA lag behind the discovery of sequences. Computational analysis of RNA is increasingly important to reveal structure–function relationships with low cost and speed. The purpose of this study is to use multiple homologous sequences to infer a conserved RNA structure. Results: A new algorithm, called Multilign, is presented to find the lowest free energy RNA secondary structure common to multiple sequences. Multilign is based on Dynalign, which is a program that simultaneously aligns and folds two sequences to find the lowest free energy conserved structure. For Multilign, Dynalign is used to progressively construct a conserved structure from multiple pairwise calculations, with one sequence used in all pairwise calculations. A base pair is predicted only if it is contained in the set of low free energy structures predicted by all Dynalign calculations. In this way, Multilign improves prediction accuracy by keeping the genuine base pairs and excluding competing false base pairs. Multilign has computational complexity that scales linearly in the number of sequences. Multilign was tested on extensive datasets of sequences with known structure and its prediction accuracy is among the best of available algorithms. Multilign can run on long sequences (> 1500 nt) and an arbitrarily large number of sequences. Availability: The algorithm is implemented in ANSI C++ and can be downloaded as part of the RNAstructure package at: http://rna.urmc.rochester.edu Contact: david_mathews@urmc.rochester.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21193521

  17. The Putative Leishmania Telomerase RNA (LeishTER) Undergoes Trans-Splicing and Contains a Conserved Template Sequence

    PubMed Central

    da Silva, Marcelo S.; Segatto, Marcela; Myler, Peter J.; Cano, Maria Isabel N.

    2014-01-01

    Telomerase RNAs (TERs) are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of Leishmania genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five Leishmania species that shares partial synteny with both Trypanosoma brucei TER locus and a putative TER candidate-containing locus of Crithidia fasciculata. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (LeishTER) that contains a 5′ spliced leader (SL) cap, a putative 3′ polyA tail and a predicted C/D box snoRNA domain. LeishTER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5′SL capped LeishTER co-immunoprecipitated and co-localized with the telomerase protein component (TERT) in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a bona fide single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs) and its role in parasite telomere biology. PMID:25391020

  18. The putative Leishmania telomerase RNA (LeishTER) undergoes trans-splicing and contains a conserved template sequence.

    PubMed

    Vasconcelos, Elton J R; Nunes, Vinícius S; da Silva, Marcelo S; Segatto, Marcela; Myler, Peter J; Cano, Maria Isabel N

    2014-01-01

    Telomerase RNAs (TERs) are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of Leishmania genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five Leishmania species that shares partial synteny with both Trypanosoma brucei TER locus and a putative TER candidate-containing locus of Crithidia fasciculata. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (LeishTER) that contains a 5' spliced leader (SL) cap, a putative 3' polyA tail and a predicted C/D box snoRNA domain. LeishTER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5'SL capped LeishTER co-immunoprecipitated and co-localized with the telomerase protein component (TERT) in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a bona fide single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs) and its role in parasite telomere biology.

  19. Conserved intergenic sequences revealed by CTAG-profiling in Salmonella: thermodynamic modeling for function prediction

    PubMed Central

    Tang, Le; Zhu, Songling; Mastriani, Emilio; Fang, Xin; Zhou, Yu-Jie; Li, Yong-Guo; Johnston, Randal N.; Guo, Zheng; Liu, Gui-Rong; Liu, Shu-Lin

    2017-01-01

    Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarily conserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment. PMID:28262684

  20. Conserved intergenic sequences revealed by CTAG-profiling in Salmonella: thermodynamic modeling for function prediction

    NASA Astrophysics Data System (ADS)

    Tang, Le; Zhu, Songling; Mastriani, Emilio; Fang, Xin; Zhou, Yu-Jie; Li, Yong-Guo; Johnston, Randal N.; Guo, Zheng; Liu, Gui-Rong; Liu, Shu-Lin

    2017-03-01

    Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarily conserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment.

  1. Conserved intergenic sequences revealed by CTAG-profiling in Salmonella: thermodynamic modeling for function prediction.

    PubMed

    Tang, Le; Zhu, Songling; Mastriani, Emilio; Fang, Xin; Zhou, Yu-Jie; Li, Yong-Guo; Johnston, Randal N; Guo, Zheng; Liu, Gui-Rong; Liu, Shu-Lin

    2017-03-06

    Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarily conserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment.

  2. Phylogenetic analysis, based on EPIYA repeats in the cagA gene of Indian Helicobacter pylori, and the implications of sequence variation in tyrosine phosphorylation motifs on determining the clinical outcome.

    PubMed

    Tiwari, Santosh K; Sharma, Vishwas; Sharma, Varun Kumar; Gopi, Manoj; Saikant, R; Nandan, Amrita; Bardia, Avinash; Gunisetty, Sivaram; Katikala, Prasanth; Habeeb, Md Aejaz; Khan, Aleem A; Habibullah, C M

    2011-04-01

    The population of India harbors one of the world's most highly diverse gene pools, owing to the influx of successive waves of immigrants over regular periods in time. Several phylogenetic studies involving mitochondrial DNA and Y chromosomal variation have demonstrated Europeans to have been the first settlers in India. Nevertheless, certain controversy exists, due to the support given to the thesis that colonization was by the Austro-Asiatic group, prior to the Europeans. Thus, the aim was to investigate pre-historic colonization of India by anatomically modern humans, using conserved stretches of five amino acid (EPIYA) sequences in the cagA gene of Helicobacter pylori. Simultaneously, the existence of a pathogenic relationship of tyrosine phosphorylation motifs (TPMs), in 32 H. pylori strains isolated from subjects with several forms of gastric diseases, was also explored. High resolution sequence analysis of the above described genes was performed. The nucleotide sequences obtained were translated into amino acids using MEGA (version 4.0) software for EPIYA. An MJ-Network was constructed for obtaining TPM haplotypes by using NETWORK (version 4.5) software. The findings of the study suggest that Indian H. pylori strains share a common ancestry with Europeans. No specific association of haplotypes with the outcome of disease was revealed through additional network analysis of TPMs.

  3. Protein sequence conservation and stable molecular evolution reveals influenza virus nucleoprotein as a universal druggable target.

    PubMed

    Babar, Mustafeez Mujtaba; Zaidi, Najam-us-Sahar Sadaf

    2015-08-01

    The high mutation rate in influenza virus genome and appearance of drug resistance calls for a constant effort to identify alternate drug targets and develop new antiviral strategies. The internal proteins of the virus can be exploited as a potential target for therapeutic interventions. Among these, the nucleoprotein (NP) is the most abundant protein that provides structural and functional support to the viral replication machinery. The current study aims at analysis of protein sequence polymorphism patterns, degree of molecular evolution and sequence conservation as a function of potential druggability of nucleoprotein. We analyzed a universal set of amino acid sequences, (n=22,000) and, in order to identify and correlate the functionally conserved, druggable regions across different parameters, classified them on the basis of host organism, strain type and continental region of sample isolation. The results indicated that around 95% of the sequence length was conserved, with at least 7 regions conserved across the protein among various classes. Moreover, the highly variable regions, though very limited in number, were found to be positively selected indicating, thereby, the high degree of protein stability against various hosts and spatio-temporal references. Furthermore, on mapping the conserved regions on the protein, 7 drug binding pockets in the functionally important regions of the protein were revealed. The results, therefore, collectively indicate that nucleoprotein is a highly conserved and stable viral protein that can potentially be exploited for development of broadly effective antiviral strategies.

  4. Reversibly Bound Chloride in the Atrial Natriuretic Peptide Receptor Hormone Binding Domain: Possible Allosteric Regulation and a Conserved Structural Motif for the Chloride-binding Site

    SciTech Connect

    Ogawa, H.; Qiu, Y; Philo, J; Arakawa, T; Ogata, C; Misono, K

    2010-01-01

    The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(-)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(-) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(-) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis.

  5. Detection of Weakly Conserved Ancestral Mammalian RegulatorySequences by Primate Comparisons

    SciTech Connect

    Wang, Qian-fei; Prabhakar, Shyam; Chanan, Sumita; Cheng,Jan-Fang; Rubin, Edward M.; Boffelli, Dario

    2006-06-01

    Genomic comparisons between human and distant, non-primatemammals are commonly used to identify cis-regulatory elements based onconstrained sequence evolution. However, these methods fail to detectcryptic functional elements, which are too weakly conserved among mammalsto distinguish from nonfunctional DNA. To address this problem, weexplored the potential of deep intra-primate sequence comparisons. Wesequenced the orthologs of 558 kb of human genomic sequence, coveringmultiple loci involved in cholesterol homeostasis, in 6 nonhumanprimates. Our analysis identified 6 noncoding DNA elements displayingsignificant conservation among primates, but undetectable in more distantcomparisons. In vitro and in vivo tests revealed that at least three ofthese 6 elements have regulatory function. Notably, the mouse orthologsof these three functional human sequences had regulatory activity despitetheir lack of significant sequence conservation, indicating that they arecryptic ancestral cis-regulatory elements. These regulatory elementscould still be detected in a smaller set of three primate speciesincluding human, rhesus and marmoset. Since the human and rhesus genomesequences are already available, and the marmoset genome is activelybeing sequenced, the primate-specific conservation analysis describedhere can be applied in the near future on a whole-genome scale, tocomplement the annotation provided by more distant speciescomparisons.

  6. Accelerated Evolution of Conserved Noncoding Sequences in theHuman Genome

    SciTech Connect

    Prambhakar, Shyam; Noonan, James P.; Paabo, Svante; Rubin, EdwardM.

    2006-07-06

    Genomic comparisons between human and distant, non-primatemammals are commonly used to identify cis-regulatory elements based onconstrained sequence evolution. However, these methods fail to detect"cryptic" functional elements, which are too weakly conserved amongmammals to distinguish from nonfunctional DNA. To address this problem,we explored the potential of deep intra-primate sequence comparisons. Wesequenced the orthologs of 558 kb of human genomic sequence, coveringmultiple loci involved in cholesterol homeostasis, in 6 nonhumanprimates. Our analysis identified 6 noncoding DNA elements displayingsignificant conservation among primates, but undetectable in more distantcomparisons. In vitro and in vivo tests revealed that at least three ofthese 6 elements have regulatory function. Notably, the mouse orthologsof these three functional human sequences had regulatory activity despitetheir lack of significant sequence conservation, indicating that they arecryptic ancestral cis-regulatory elements. These regulatory elementscould still be detected in a smaller set of three primate speciesincluding human, rhesus and marmoset. Since the human and rhesus genomesequences are already available, and the marmoset genome is activelybeing sequenced, the primate-specific conservation analysis describedhere can be applied in the near future on a whole-genome scale, tocomplement the annotation provided by more distant speciescomparisons.

  7. Analysis of sequences involved in IE2 transactivation of a baculovirus immediate-early gene promoter and identification of a new regulatory motif.

    PubMed

    Shippam-Brett, C E; Willis, L G; Theilmann, D A

    2001-05-01

    Opep-2 is a unique baculovirus early gene that has only been identified in the Orgyia pseudotsugata multiple capsid nucleopolyhedrovirus (OpMNPV). Previous analyses have shown this gene is expressed at very early times post-infection (p.i.) but is shut down by 36-48 h p.i. The promoter of opep-2 therefore, represents a class of early genes that is temporally regulated. In this study, a detailed analysis of the opep-2 promoter is performed to analyze the role individual motifs play in early gene expression. A new 13 base pair regulatory element was identified and shown to be essential in controlling high-level expression of this gene. In addition, mutational analysis revealed that GATA and CACGTG motifs, which have been shown to bind cellular factors in Sf9 and Ld652Y cells, played minor roles in influencing opep-2 expression in the absence of other viral factors. The OpMNPV transactivator IE2 causes a significant activation of the opep-2 promoter. Cotransfection of an extensive number of promoter deletions and mutations did not show any sequence specificity for IE2 transactivation. This is the first detailed analysis of the sequence requirements for IE2 transactivation, and these results suggest that IE2 does not bind directly to specific elements in the opep-2 promoter.

  8. Conservation of sequence and function in fertilization of the cortical granule serine protease in echinoderms.

    PubMed

    Oulhen, Nathalie; Xu, Dongdong; Wessel, Gary M

    2014-08-01

    Conservation of the cortical granule serine protease during fertilization in echinoderms was tested both functionally in sea stars, and computationally throughout the echinoderm phylum. We find that the inhibitor of serine protease (soybean trypsin inhibitor) effectively blocks proper transition of the sea star fertilization envelope into a protective sperm repellent, whereas inhibitors of the other main types of proteases had no effect. Scanning the transcriptomes of 15 different echinoderm ovaries revealed sequences of high conservation to the originally identified sea urchin cortical serine protease, CGSP1. These conserved sequences contained the catalytic triad necessary for enzymatic activity, and the tandemly repeated LDLr-like repeats. We conclude that the protease involved in the slow block to polyspermy is an essential and conserved element of fertilization in echinoderms, and may provide an important reagent for identification and testing of the cell surface proteins in eggs necessary for sperm binding.

  9. Conservation of sequence and function in fertilization of the cortical granule serine protease in echinoderms

    PubMed Central

    Oulhen, Nathalie; Xu, Dongdong; Wessel, Gary M.

    2014-01-01

    Conservation of the cortical granule serine protease during fertilization in echinoderms was tested both functionally in sea stars, and computationally throughout the echinoderm phylum. We find that the inhibitor of serine protease (soybean trypsin inhibitor) effectively blocks proper transition of the sea star fertilization envelope into a protective sperm repellent, whereas inhibitors of the other main types of proteases had no effect. Scanning the transcriptomes of 15 different echinoderm ovaries revealed sequences of high conservation to the originally identified sea urchin cortical serine protease, CGSP1. These conserved sequences contained the catalytic triad necessary for enzymatic activity, and the tandemly repeated LDLr-like repeats. We conclude that the protease involved in the slow block to polyspermy is an essential and conserved element of fertilization in echinoderms, and may provide an important reagent for identification and testing of the cell surface proteins in eggs necessary for sperm binding. PMID:24878526

  10. Conservation of the human telomere sequence (TTAGGG)n among vertebrates.

    PubMed Central

    Meyne, J; Ratliff, R L; Moyzis, R K

    1989-01-01

    To determine the evolutionary origin of the human telomere sequence (TTAGGG)n, biotinylated oligodeoxynucleotides of this sequence were hybridized to metaphase spreads from 91 different species, including representative orders of bony fish, reptiles, amphibians, birds, and mammals. Under stringent hybridization conditions, fluorescent signals were detected at the telomeres of all chromosomes, in all 91 species. The conservation of the (TTAGGG)n sequence and its telomeric location, in species thought to share a common ancestor over 400 million years ago, strongly suggest that this sequence is the functional vertebrate telomere. Images PMID:2780561

  11. RNAMotifScanX: a graph alignment approach for RNA structural motif identification.

    PubMed

    Zhong, Cuncong; Zhang, Shaojie

    2015-03-01

    RNA structural motifs are recurrent three-dimensional (3D) components found in the RNA architecture. These RNA structural motifs play important structural or functional roles and usually exhibit highly conserved 3D geometries and base-interaction patterns. Analysis of the RNA 3D structures and elucidation of their molecular functions heavily rely on efficient and accurate identification of these motifs. However, efficient RNA structural motif search tools are lacking due to the high complexity of these motifs. In this work, we present RNAMotifScanX, a motif search tool based on a base-interaction graph alignment algorithm. This novel algorithm enables automatic identification of both partially and fully matched motif instances. RNAMotifScanX considers noncanonical base-pairing interactions, base-stacking interactions, and sequence conservation of the motifs, which leads to significantly improved sensitivity and specificity as compared with other state-of-the-art search tools. RNAMotifScanX also adopts a carefully designed branch-and-bound technique, which enables ultra-fast search of large kink-turn motifs against a 23S rRNA. The software package RNAMotifScanX is implemented using GNU C++, and is freely available from http://genome.ucf.edu/RNAMotifScanX.

  12. A mini-RNA containing the tetraloop, wobble-pair and loop E motifs of the central conserved region of potato spindle tuber viroid is processed into a minicircle.

    PubMed

    Schrader, O; Baumstark, T; Riesner, D

    2003-02-01

    A Mini-RNA from potato spindle tuber viroid (PSTVd) was constructed specifically for cleavage and ligation to circles in vitro. It contains the C-domain with the so-called central conserved region (CCR) of PSTVd with a 17 nt duplication in the upper strand and hairpin structures at the left and rights ends of the secondary structure. The CCR was previously shown to be essential for processing of in vitro transcripts. When folded under conditions which favor formation of a kinetically controlled conformation and incubated in a potato nuclear extract, the Mini-RNA is cleaved correctly at the 5'- and the 3'-end and ligated to a circle. Thus, the CCR obviously contains all structural and functional requirements for correct processing and therefore may be regarded as 'processing domain' of PSTVd. Using the Mini-RNA as a model substrate, the structural and functional relevance of its conserved non-canonical motifs GAAA tetraloop, loop E and G:U wobble base pair were studied by mutational analysis. It was found that (i) the conserved GAAA tetraloop is essential for processing by favoring the kinetically controlled conformation, (ii) a G:U wobble base pair at the 5'-cleavage site contributes to its correct recognition and (iii) an unpaired nucleotide in loop E, which is different from the corresponding nucleotide in the conserved loop E motif, is essential for ligation of the 5'- with the 3'-end. Hence all three structural motifs are functional elements for processing in a potato nuclear extract.

  13. Relation between mRNA expression and sequence information in Desulfovibrio vulgaris: Combinatorial contributions of upstream regulatory motifs and coding sequence features to variations in mRNA abundance

    SciTech Connect

    Wu, Gang; Nie, Lei; Zhang, Weiwen

    2006-05-26

    ABSTRACT-The context-dependent expression of genes is the core for biological activities, and significant attention has been given to identification of various factors contributing to gene expression at genomic scale. However, so far this type of analysis has been focused whether on relation between mRNA expression and non-coding sequence features such as upstream regulatory motifs or on correlation between mRN abundance and non-random features in coding sequences (e.g. codon usage and amino acid usage). In this study multiple regression analyses of the mRNA abundance and all sequence information in Desulfovibrio vulgaris were performed, with the goal to investigate how much coding and non-coding sequence features contribute to the variations in mRNA expression, and in what manner they act together...

  14. The nucleotide sequence of the human int-1 mammary oncogene; evolutionary conservation of coding and non-coding sequences.

    PubMed Central

    van Ooyen, A; Kwee, V; Nusse, R

    1985-01-01

    The mouse mammary tumor virus can induce mammary tumors in mice by proviral activation of an evolutionarily conserved cellular oncogene called int-1. Here we present the nucleotide sequence of the human homologue of int-1, and compare it with the mouse gene. Like the mouse gene, the human homologue contains a reading frame of 370 amino acids, with only four substitutions. The amino acid changes are all in the hydrophobic leader domain of the int-1 encoded protein, and do not significantly alter its hydropathic index. The conservation between the mouse and the human int-1 genes is not restricted to exons; extensive parts of the introns are also homologous. Thus, int-1 ranks among the most conserved genes known, a property shared with other oncogenes. PMID:2998762

  15. Inhibition by Avibactam and Clavulanate of the β-Lactamases KPC-2 and CTX-M-15 Harboring the Substitution N(132)G in the Conserved SDN Motif.

    PubMed

    Ourghanlian, Clément; Soroka, Daria; Arthur, Michel

    2017-03-01

    The substitution N(132)G in the SDN motif of class A β-lactamases from rapidly growing mycobacteria was previously shown to impair their inhibition by avibactam but to improve the stability of acyl-enzymes formed with clavulanate. The same substitution was introduced in KPC-2 and CTX-M-15 to assess its impact on β-lactamases from Enterobacteriaceae and evaluate whether it may lead to resistance to the ceftazidime-avibactam combination. Kinetic parameters for the inhibition of the β-lactamases by avibactam and clavulanate were determined by spectrophotometry using nitrocefin as the substrate. The substitution N(132)G impaired (>1,000-fold) the efficacy of carbamylation of KPC-2 and CTX-M-15 by avibactam. The substitution improved the inhibition of KPC-2 by clavulanate due to reduced deacylation, whereas the presence or absence of N(132)G resulted in the inhibition of CTX-M-15 by clavulanate. The hydrolysis of amoxicillin and nitrocefin by KPC-2 and CTX-M-15 was moderately affected by the substitution N(132)G, but that of ceftazidime, ceftaroline, and aztreonam was drastically reduced. Isogenic strains producing KPC-2 and CTX-M-15 were constructed to assess the impact of the substitution N(132)G on the antibacterial activities of β-lactam-inhibitor combinations. For amoxicillin, the substitution resulted in resistance and susceptibility for avibactam and clavulanate, respectively. For ceftazidime, ceftaroline, and aztreonam, the negative impact of the substitution on β-lactamase activity prevented resistance to the β-lactam-avibactam combinations. In conclusion, the N(132)G substitution has profound effects on the substrate and inhibition profiles of class A β-lactamases, which are largely conserved in distantly related enzymes. Fortunately, the substitution does not lead to resistance to the ceftazidime-avibactam combination.

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

    PubMed Central

    2010-01-01

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

  17. Highly conserved d-loop sequences in woolly mouse opossums Marmosa (Micoureus).

    PubMed

    Rocha, Rita Gomes; Leite, Yuri Luiz Reis; Ferreira, Eduardo; Justino, Juliana; Costa, Leonora Pires

    2012-04-01

    This study reports the occurrence of highly conserved d-loop sequences in the mitochondrial genome of the woolly mouse opossum genus Marmosa subgenus Micoureus (Mammalia, Didelphimorphia, Didelphidae). Sixty-six sequences of Marmosa (Micoureus) demerarae, Marmosa (Micoureus) constantiae, and Marmosa (Micoureus) paraguayanus were amplified using universal d-loop primers and virtually no genetic differences were detected within and among species. These sequences matched the control region of the mitochondrial marsupial genome. Analyses of qualitative aspects of these sequences revealed that their structural composition is very similar to the d-loop region of other didelphid species. However, the total lack of variability has not been reported from other closely related species. The data analyzed here support the occurrence of highly conserved d-loop sequences, and we found no support for the hypothesis that these sequences are d-loop-like nuclear pseudogenes. Furthermore, the control and flanking regions obtained with different primers corroborate the lack of variability of the d-loop sequences in the mitochondrial genome of Marmosa (Micoureus).

  18. Role of repetitive nine-residue sequence motifs in secretion, enzymatic activity, and protein conformation of a family I.3 lipase.

    PubMed

    Kwon, Hyun-Ju; Haruki, Mitsuru; Morikawa, Masaaki; Omori, Kenji; Kanaya, Shigenori

    2002-01-01

    A family I.3 lipase from Pseudomonas sp. MIS38 (PML) contains 12 repeats of a nine-residue sequence motif in the C-terminal region. To elucidate the role of these repetitive sequences, mutant proteins PML5, PML4, PML1, and PML0, in which 7, 8, 11, and all 12 of the repetitive sequences are deleted, and PMLdelta19, in which 19 C-terminal residues are truncated, were constructed. Escherichia coli DH5 cells carrying the Serratia marcescens Lip system permitted the secretion of the wild-type and all of the mutant proteins except for PMLdelta19, although they were partially accumulated in the cells in an insoluble form as well. Both the secretion level and cellular content of the proteins decreased in the order PML > PML5 > PML4 > PML1 > PML0, indicating that repetitive sequences are not required for secretion of PML but are important for its stability in the cells. All the mutant proteins were purified in a refolded form and their biochemical properties were characterized. CD spectra, the Ca2+ contents, and susceptibility to chymotryptic digestion strongly suggested that the five repetitive sequences remaining in PML5 are sufficient to form a beta-roll structure, whereas the four in PML4 are not. PML5 and PMLdelta19 showed both lipase and esterase activities, whereas PML4, PML1, and PML0 were inactive. These results suggest that the enzymatic activity of PML is not seriously affected by a deletion or truncation at the C-terminal region as long as a succession of repetitive sequences can build a beta-roll structure.

  19. Limb body wall complex, amniotic band sequence, or new syndrome caused by mutation in IQ Motif containing K (IQCK)?

    PubMed Central

    Kruszka, Paul; Uwineza, Annette; Mutesa, Leon; Martinez, Ariel F; Abe, Yu; Zackai, Elaine H; Ganetzky, Rebecca; Chung, Brian; Stevenson, Roger E; Adelstein, Robert S; Ma, Xuefei; Mullikin, James C; Hong, Sung-Kook; Muenke, Maximilian

    2015-01-01

    Limb body wall complex (LBWC) and amniotic band sequence (ABS) are multiple congenital anomaly conditions with craniofacial, limb, and ventral wall defects. LBWC and ABS are considered separate entities by some, and a continuum of severity of the same condition by others. The etiology of LBWC/ABS remains unknown and multiple hypotheses have been proposed. One individual with features of LBWC and his unaffected parents were whole exome sequenced and Sanger sequenced as confirmation of the mutation. Functional studies were conducted using morpholino knockdown studies followed by human mRNA rescue experiments. Using whole exome sequencing, a de novo heterozygous mutation was found in the gene IQCK: c.667C>G; p.Q223E and confirmed by Sanger sequencing in an individual with LBWC. Morpholino knockdown of iqck mRNA in the zebrafish showed ventral defects including failure of ventral fin to develop and cardiac edema. Human wild-type IQCK mRNA rescued the zebrafish phenotype, whereas human p.Q223E IQCK mRNA did not, but worsened the phenotype of the morpholino knockdown zebrafish. This study supports a genetic etiology for LBWC/ABS, or potentially a new syndrome. PMID:26436108

  20. Combinatorial motif analysis of regulatory gene expression in Mafb deficient macrophages

    PubMed Central

    2011-01-01

    Background Deficiency of the transcription factor MafB, which is normally expressed in macrophages, can underlie cellular dysfunction associated with a range of autoimmune diseases and arteriosclerosis. MafB has important roles in cell differentiation and regulation of target gene expression; however, the mechanisms of this regulation and the identities of other transcription factors with which MafB interacts remain uncertain. Bioinformatics methods provide a valuable approach for elucidating the nature of these interactions with transcriptional regulatory elements from a large number of DNA sequences. In particular, identification of patterns of co-occurrence of regulatory cis-elements (motifs) offers a robust approach. Results Here, the directional relationships among several functional motifs were evaluated using the Log-linear Graphical Model (LGM) after extraction and search for evolutionarily conserved motifs. This analysis highlighted GATA-1 motifs and 5’AT-rich half Maf recognition elements (MAREs) in promoter regions of 18 genes that were down-regulated in Mafb deficient macrophages. GATA-1 motifs and MafB motifs could regulate expression of these genes in both a negative and positive manner, respectively. The validity of this conclusion was tested with data from a luciferase assay that used a C1qa promoter construct carrying both the GATA-1 motifs and MAREs. GATA-1 was found to inhibit the activity of the C1qa promoter with the GATA-1 motifs and MafB motifs. Conclusions These observations suggest that both the GATA-1 motifs and MafB motifs are important for lineage specific expression of C1qa. In addition, these findings show that analysis of combinations of evolutionarily conserved motifs can be successfully used to identify patterns of gene regulation. PMID:22784578

  1. Spectrometric study of the folding process of i-motif-forming DNA sequences upstream of the c-kit transcription initiation site.

    PubMed

    Bucek, Pavel; Gargallo, Raimundo; Kudrev, Andrei

    2010-12-17

    The c-kit oncogene shows a cytosine-rich DNA region upstream of the transcription initiation site which forms an i-motif structure at slightly acidic pH values (Bucek et al. [5]). In the present study, the pH-induced formation of i-motif - forming sequences 5'-CCC CTC CCT CGC GCC CGC CCG-3' (ckitC1, native), 5'-CCC TTC CCT TGT GCC CGC CCG-3' (ckitC2) and 5'-CCCTT CCC TTTTT CCC T CCC T-3' (ckitC3) was studied by spectroscopic techniques, such as UV molecular absorption and circular dichroism (CD), in tandem with two multivariate data analysis methods, the hard modelling-based matrix method and the soft modelling-based MCR-ALS approach. Use of the hard chemical modelling enabled us to propose the equilibrium model, which describes spectral changes as functions of solution acidity. Additionally, the intrinsic protonation constant, K(in), and the cooperativity parameters, ω(c), and ω(a), were calculated from the fitting procedure of the coupled CD and molecular absorption spectra. In the case of ckitC2 and ckitC3, the hard model correctly reproduced the spectral variations observed experimentally. The results indicated that folding was accompanied by a cooperative process, i.e. the enhancement of protonated structure stability upon protonation. In contrast, unfolding was accompanied by an anticooperative process. Finally, folding of the native sequence, ckitC1, seemed to follow a more complex mechanism.

  2. Significance of satellite DNA revealed by conservation of a widespread repeat DNA sequence among angiosperms.

    PubMed

    Mehrotra, Shweta; Goel, Shailendra; Raina, Soom Nath; Rajpal, Vijay Rani

    2014-08-01

    The analysis of plant genome structure and evolution requires comprehensive characterization of repetitive sequences that make up the majority of plant nuclear DNA. In the present study, we analyzed the nature of pCtKpnI-I and pCtKpnI-II tandem repeated sequences, reported earlier in Carthamus tinctorius. Interestingly, homolog of pCtKpnI-I repeat sequence was also found to be present in widely divergent families of angiosperms. pCtKpnI-I showed high sequence similarity but low copy number among various taxa of different families of angiosperms analyzed. In comparison, pCtKpnI-II was specific to the genus Carthamus and was not present in any other taxa analyzed. The molecular structure of pCtKpnI-I was analyzed in various unrelated taxa of angiosperms to decipher the evolutionary conserved nature of the sequence and its possible functional role.

  3. Rice pseudomolecule-anchored cross-species DNA sequence alignments indicate regional genomic variation in expressed sequence conservation

    PubMed Central

    Armstead, Ian; Huang, Lin; King, Julie; Ougham, Helen; Thomas, Howard; King, Ian

    2007-01-01

    Background Various methods have been developed to explore inter-genomic relationships among plant species. Here, we present a sequence similarity analysis based upon comparison of transcript-assembly and methylation-filtered databases from five plant species and physically anchored rice coding sequences. Results A comparison of the frequency of sequence alignments, determined by MegaBLAST, between rice coding sequences in TIGR pseudomolecules and annotations vs 4.0 and comprehensive transcript-assembly and methylation-filtered databases from Lolium perenne (ryegrass), Zea mays (maize), Hordeum vulgare (barley), Glycine max (soybean) and Arabidopsis thaliana (thale cress) was undertaken. Each rice pseudomolecule was divided into 10 segments, each containing 10% of the functionally annotated, expressed genes. This indicated a correlation between relative segment position in the rice genome and numbers of alignments with all the queried monocot and dicot plant databases. Colour-coded moving windows of 100 functionally annotated, expressed genes along each pseudomolecule were used to generate 'heat-maps'. These revealed consistent intra- and inter-pseudomolecule variation in the relative concentrations of significant alignments with the tested plant databases. Analysis of the annotations and derived putative expression patterns of rice genes from 'hot-spots' and 'cold-spots' within the heat maps indicated possible functional differences. A similar comparison relating to ancestral duplications of the rice genome indicated that duplications were often associated with 'hot-spots'. Conclusion Physical positions of expressed genes in the rice genome are correlated with the degree of conservation of similar sequences in the transcriptomes of other plant species. This relative conservation is associated with the distribution of different sized gene families and segmentally duplicated loci and may have functional and evolutionary implications. PMID:17708759

  4. Sense-antisense gene pairs: sequence, transcription, and structure are not conserved between human and mouse

    PubMed Central

    Wood, Emily J.; Chin-Inmanu, Kwanrutai; Jia, Hui; Lipovich, Leonard

    2013-01-01

    Previous efforts to characterize conservation between the human and mouse genomes focused largely on sequence comparisons. These studies are inherently limited because they don't account for gene structure differences, which may exist despite genomic sequence conservation. Recent high-throughput transcriptome studies have revealed widespread and extensive overlaps between genes, and transcripts, encoded on both strands of the genomic sequence. This overlapping gene organization, which produces sense-antisense (SAS) gene pairs, is capable of effecting regulatory cascades through established mechanisms. We present an evolutionary conservation assessment of SAS pairs, on three levels: genomic, transcriptomic, and structural. From a genome-wide dataset of human SAS pairs, we first identified orthologous loci in the mouse genome, then assessed their transcription in the mouse, and finally compared the genomic structures of SAS pairs expressed in both species. We found that approximately half of human SAS loci have single orthologous locations in the mouse genome; however, only half of those orthologous locations have SAS transcriptional activity in the mouse. This suggests that high human-mouse gene conservation overlooks widespread distinctions in SAS pair incidence and expression. We compared gene structures at orthologous SAS loci, finding frequent differences in gene structure between human and orthologous mouse SAS pair members. Our categorization of human SAS pairs with respect to mouse conservation of expression as well as structure points to limitations of mouse models. Gene structure differences, including at SAS loci, may account for some of the phenotypic distinctions between primates and rodents. Genes in non-conserved SAS pairs may contribute to evolutionary lineage-specific regulatory outcomes. PMID:24133500

  5. Studying RNA homology and conservation with Infernal: from single sequences to RNA families

    PubMed Central

    Barquist, Lars; Burge, Sarah W.; Gardner, Paul P.

    2016-01-01

    Emerging high-throughput technologies have led to a deluge of putative non-coding RNA (ncRNA) sequences identified in a wide variety of organisms. Systematic characterization of these transcripts will be a tremendous challenge. Homology detection is critical to making maximal use of functional information gathered about ncRNAs: identifying homologous sequence allows us to transfer information gathered in one organism to another quickly and with a high degree of confidence. ncRNA presents a challenge for homology detection, as the primary sequence is often poorly conserved and de novo secondary structure prediction and search remains difficult. This protocol introduces methods developed by the Rfam database for identifying “families” of homologous ncRNAs starting from single “seed” sequences using manually curated sequence alignments to build powerful statistical models of sequence and structure conservation known as covariance models (CMs), implemented in the Infernal software package. We provide a step-by-step iterative protocol for identifying ncRNA homologs, then constructing an alignment and corresponding CM. We also work through an example for the bacterial small RNA MicA, discovering a previously unreported family of divergent MicA homologs in genus Xenorhabdus in the process. PMID:27322404

  6. Studying RNA Homology and Conservation with Infernal: From Single Sequences to RNA Families.

    PubMed

    Barquist, Lars; Burge, Sarah W; Gardner, Paul P

    2016-06-20

    Emerging high-throughput technologies have led to a deluge of putative non-coding RNA (ncRNA) sequences identified in a wide variety of organisms. Systematic characterization of these transcripts will be a tremendous challenge. Homology detection is critical to making maximal use of functional information gathered about ncRNAs: identifying homologous sequence allows us to transfer information gathered in one organism to another quickly and with a high degree of confidence. ncRNA presents a challenge for homology detection, as the primary sequence is often poorly conserved and de novo secondary structure prediction and search remain difficult. This unit introduces methods developed by the Rfam database for identifying "families" of homologous ncRNAs starting from single "seed" sequences, using manually curated sequence alignments to build powerful statistical models of sequence and structure conservation known as covariance models (CMs), implemented in the Infernal software package. We provide a step-by-step iterative protocol for identifying ncRNA homologs and then constructing an alignment and corresponding CM. We also work through an example for the bacterial small RNA MicA, discovering a previously unreported family of divergent MicA homologs in genus Xenorhabdus in the process. © 2016 by John Wiley & Sons, Inc.

  7. Integrated genome analysis suggests that most conserved non-coding sequences are regulatory factor binding sites

    PubMed Central

    Hemberg, Martin; Gray, Jesse M.; Cloonan, Nicole; Kuersten, Scott; Grimmond, Sean; Greenberg, Michael E.; Kreiman, Gabriel

    2012-01-01

    More than 98% of a typical vertebrate genome does not code for proteins. Although non-coding regions are sprinkled with short (<200 bp) islands of evolutionarily conserved sequences, the function of most of these unannotated conserved islands remains unknown. One possibility is that unannotated conserved islands could encode non-coding RNAs (ncRNAs); alternatively, unannotated conserved islands could serve as promoter-distal regulatory factor binding sites (RFBSs) like enhancers. Here we assess these possibilities by comparing unannotated conserved islands in the human and mouse genomes to transcribed regions and to RFBSs, relying on a detailed case study of one human and one mouse cell type. We define transcribed regions by applying a novel transcript-calling algorithm to RNA-Seq data obtained from total cellular RNA, and we define RFBSs using ChIP-Seq and DNAse-hypersensitivity assays. We find that unannotated conserved islands are four times more likely to coincide with RFBSs than with unannotated ncRNAs. Thousands of conserved RFBSs can be categorized as insulators based on the presence of CTCF or as enhancers based on the presence of p300/CBP and H3K4me1. While many unannotated conserved RFBSs are transcriptionally active to some extent, the transcripts produced tend to be unspliced, non-polyadenylated and expressed at levels 10 to 100-fold lower than annotated coding or ncRNAs. Extending these findings across multiple cell types and tissues, we propose that most conserved non-coding genomic DNA in vertebrate genomes corresponds to promoter-distal regulatory elements. PMID:22684627

  8. The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences.

    PubMed Central

    Fierro, F; Barredo, J L; Díez, B; Gutierrez, S; Fernández, F J; Martín, J F

    1995-01-01

    The penicillin biosynthetic genes (pcbAB, pcbC, penDE) of Penicillium chrysogenum AS-P-78 were located in a 106.5-kb DNA region that is amplified in tandem repeats (five or six copies) linked by conserved TTTACA sequences. The wild-type strains P. chrysogenum NRRL 1951 and Penicillium notatum ATCC 9478 (Fleming's isolate) contain a single copy of the 106.5-kb region. This region was bordered by the same TTTACA hexanucleotide found between tandem repeats in strain AS-P-78. A penicillin overproducer strain, P. chrysogenum E1, contains a large number of copies in tandem of a 57.9-kb DNA fragment, linked by the same hexanucleotide or its reverse complementary TGTAAA sequence. The deletion mutant P. chrysogenum npe10 showed a deletion of 57.9 kb that corresponds exactly to the DNA fragment that is amplified in E1. The conserved hexanucleotide sequence was reconstituted at the deletion site. The amplification has occurred within a single chromosome (chromosome I). The tandem reiteration and deletion appear to arise by mutation-induced site-specific recombination at the conserved hexanucleotide sequences. Images Fig. 3 PMID:7597101

  9. A survey of motif finding Web tools for detecting binding site motifs in ChIP-Seq data.

    PubMed

    Tran, Ngoc Tam L; Huang, Chun-Hsi

    2014-02-20

    ChIP-Seq (chromatin immunoprecipitation sequencing) has provided the advantage for finding motifs as ChIP-Seq experiments narrow down the motif finding to binding site locations. Recent motif finding tools facilitate the motif detection by providing user-friendly Web interface. In this work, we reviewed nine motif finding Web tools that are capable for detecting binding site motifs in ChIP-Seq data. We showed each motif finding Web tool has its own advantages for detecting motifs that other tools may not discover. We recommended the users to use multiple motif finding Web tools that implement different algorithms for obtaining significant motifs, overlapping resemble motifs, and non-overlapping motifs. Finally, we provided our suggestions for future development of motif finding Web tool that better assists researchers for finding motifs in ChIP-Seq data.

  10. A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation

    PubMed Central

    Leen, Eoin N.; Sorgeloos, Frédéric; Correia, Samantha; Chaudhry, Yasmin; Cannac, Fabien; Pastore, Chiara; Xu, Yingqi; Graham, Stephen C.; Matthews, Stephen J.; Goodfellow, Ian G.; Curry, Stephen

    2016-01-01

    Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652–1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy. PMID:26734730

  11. The Bordetella type III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts.

    PubMed

    French, Christopher T; Panina, Ekaterina M; Yeh, Sylvia H; Griffith, Natasha; Arambula, Diego G; Miller, Jeff F

    2009-12-01

    The Bordetella type III secretion system (T3SS) effector protein BteA is necessary and sufficient for rapid cytotoxicity in a wide range of mammalian cells. We show that BteA is highly conserved and functionally interchangeable between Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis. The identification of BteA sequences required for cytotoxicity allowed the construction of non-cytotoxic mutants for localization studies. BteA derivatives were targeted to lipid rafts and showed clear colocalization with cortical actin, ezrin and the lipid raft marker GM1. We hypothesized that BteA associates with the cytoplasmic face of lipid rafts to locally modulate host cell responses to Bordetella attachment. B. bronchiseptica adhered to host cells almost exclusively to GM1-enriched lipid raft microdomains and BteA colocalized to these same sites following T3SS-mediated translocation. Disruption of lipid rafts with methyl-beta-cyclodextrin protected cells from T3SS-induced cytotoxicity. Localization to lipid rafts was mediated by a 130-amino-acid lipid raft targeting domain at the N-terminus of BteA, and homologous domains were identified in virulence factors from other bacterial species. Lipid raft targeting sequences from a T3SS effector (Plu4750) and an RTX-type toxin (Plu3217) from Photorhabdus luminescens directed fusion proteins to lipid rafts in a manner identical to the N-terminus of BteA.

  12. Annotation of cis-regulatory elements by identification, subclassification, and functional assessment of multispecies conserved sequences

    PubMed Central

    Hughes, Jim R.; Cheng, Jan-Fang; Ventress, Nicki; Prabhakar, Shyam; Clark, Kevin; Anguita, Eduardo; De Gobbi, Marco; de Jong, Pieter; Rubin, Eddy; Higgs, Douglas R.

    2005-01-01

    An important step toward improving the annotation of the human genome is to identify cis-acting regulatory elements from primary DNA sequence. One approach is to compare sequences from multiple, divergent species. This approach distinguishes multispecies conserved sequences (MCS) in noncoding regions from more rapidly evolving neutral DNA. Here, we have analyzed a region of ≈238kb containing the human α globin cluster that was sequenced and/or annotated across the syntenic region in 22 species spanning 500 million years of evolution. Using a variety of bioinformatic approaches and correlating the results with many aspects of chromosome structure and function in this region, we were able to identify and evaluate the importance of 24 individual MCSs. This approach sensitively and accurately identified previously characterized regulatory elements but also discovered unidentified promoters, exons, splicing, and transcriptional regulatory elements. Together, these studies demonstrate an integrated approach by which to identify, subclassify, and predict the potential importance of MCSs. PMID:15998734

  13. Identification of conserved and novel microRNAs in Aquilaria sinensis based on small RNA sequencing and transcriptome sequence data.

    PubMed

    Gao, Zhi-Hui; Wei, Jian-He; Yang, Yun; Zhang, Zheng; Xiong, Huan-Ying; Zhao, Wen-Ting

    2012-08-15

    Agarwood is in great demand for its high value in medicine, incense, and perfume across Asia, Middle East, and Europe. As agarwood is formed only when the Aquilaria trees are wounded or infected by some microbes, overharvesting and habitat loss are threatening some populations of agarwood-producing species. Aquilaria sinensis is such a significant economic tree species. To promote the production efficiency and protect the resource of A. sinensis, it would be critical to reveal the regulation mechanisms of stress-induced agarwood formation. MicroRNAs (miRNAs), a key gene expression regulator involved in various plant stress response and metabolic processes, might function in agarwood formation, but no report concerning miRNAs in Aquilaria is available. In this study, the small RNA high-throughput sequencing and 454 transcriptome data were adopted to identify both conserved and novel miRNAs in A. sinensis. Deep sequencing showed that the small RNA (sRNA) population of A. sinensis was complex and the length of sRNAs varied. By in silico analysis of the small RNA deep sequencing data and transcriptome data, we discovered 27 novel miRNAs in A. sinensis. Based on the mature miRNA sequence conservation, we identified 74 putative conserved miRNAs from A. sinensis and 10 of them were confirmed with hairpin forming precursor. Interestingly, a novel miRNA sequence was determined to be the miRNA of asi-miR408, but with accumulation much higher than asi-miR408. The expression levels of ten stress-responsive miRNAs were examined during the time-course after wound treatment. Eight were shown to be wound-responsive. This not only shows the existence of miRNAs in this Asian economically significant tree species but also indicated its critical role in stress-induced agarwood formation. The highly accumulated miRNA of asi-miR408 implied miRNAs would be functional as well as miRNAs in plants.

  14. Automatic identification of highly conserved family regions and relationships in genome wide datasets including remote protein sequences.

    PubMed

    Doğan, Tunca; Karaçalı, Bilge

    2013-01-01

    Identifying shared sequence segments along amino acid sequences generally requires a collection of closely related proteins, most often curated manually from the sequence datasets to suit the purpose at hand. Currently developed statistical methods are strained, however, when the collection contains remote sequences with poor alignment to the rest, or sequences containing multiple domains. In this paper, we propose a completely unsupervised and automated method to identify the shared sequence segments observed in a diverse collection of protein sequences including those present in a smaller fraction of the sequences in the collection, using a combination of sequence alignment, residue conservation scoring and graph-theoretical approaches. Since shared sequence fragments often imply conserved functional or structural attributes, the method produces a table of associations between the sequences and the identified conserved regions that can reveal previously unknown protein families as well as new members to existing ones. We evaluated the biological relevance of the method by clustering the proteins in gold standard datasets and assessing the clustering performance in comparison with previous methods from the literature. We have then applied the proposed method to a genome wide dataset of 17793 human proteins and generated a global association map to each of the 4753 identified conserved regions. Investigations on the major conserved regions revealed that they corresponded strongly to annotated structural domains. This suggests that the method can be useful in predicting novel domains on protein sequences.

  15. Bayesian multiple-instance motif discovery with BAMBI: inference of recombinase and transcription factor binding sites

    PubMed Central

    Jajamovich, Guido H.; Wang, Xiaodong; Arkin, Adam P.; Samoilov, Michael S.

    2011-01-01

    Finding conserved motifs in genomic sequences represents one of essential bioinformatic problems. However, achieving high discovery performance without imposing substantial auxiliary constraints on possible motif features remains a key algorithmic challenge. This work describes BAMBI—a sequential Monte Carlo motif-identification algorithm, which is based on a position weight matrix model that does not require additional constraints and is able to estimate such motif properties as length, logo, number of instances and their locations solely on the basis of primary nucleotide sequence data. Furthermore, should biologically meaningful information about motif attributes be available, BAMBI takes advantage of this knowledge to further refine the discovery results. In practical applications, we show that the proposed approach can be used to find sites of such diverse DNA-binding molecules as the cAMP receptor protein (CRP) and Din-family site-specific serine recombinases. Results obtained by BAMBI in these and other settings demonstrate better statistical performance than any of the four widely-used profile-based motif discovery methods: MEME, BioProspector with BioOptimizer, SeSiMCMC and Motif Sampler as measured by the nucleotide-level correlation coefficient. Additionally, in the case of Din-family recombinase target site discovery, the BAMBI-inferred motif is found to be the only one functionally accurate from the underlying biochemical mechanism standpoint. C++ and Matlab code is available at http://www.ee.columbia.edu/~guido/BAMBI or http://genomics.lbl.gov/BAMBI/. PMID:21948794

  16. The Large Mitochondrial Genome of Symbiodinium minutum Reveals Conserved Noncoding Sequences between Dinoflagellates and Apicomplexans

    PubMed Central

    Shoguchi, Eiichi; Shinzato, Chuya; Hisata, Kanako; Satoh, Nori; Mungpakdee, Sutada

    2015-01-01

    Even though mitochondrial genomes, which characterize eukaryotic cells, were first discovered more than 50 years ago, mitochondrial genomics remains an important topic in molecular biology and genome sciences. The Phylum Alveolata comprises three major groups (ciliates, apicomplexans, and dinoflagellates), the mitochondrial genomes of which have diverged widely. Even though the gene content of dinoflagellate mitochondrial genomes is reportedly comparable to that of apicomplexans, the highly fragmented and rearranged genome structures of dinoflagellates have frustrated whole genomic analysis. Consequently, noncoding sequences and gene arrangements of dinoflagellate mitochondrial genomes have not been well characterized. Here we report that the continuous assembled genome (∼326 kb) of the dinoflagellate, Symbiodinium minutum, is AT-rich (∼64.3%) and that it contains three protein-coding genes. Based upon in silico analysis, the remaining 99% of the genome comprises transcriptomic noncoding sequences. RNA edited sites and unique, possible start and stop codons clarify conserved regions among dinoflagellates. Our massive transcriptome analysis shows that almost all regions of the genome are transcribed, including 27 possible fragmented ribosomal RNA genes and 12 uncharacterized small RNAs that are similar to mitochondrial RNA genes of the malarial parasite, Plasmodium falciparum. Gene map comparisons show that gene order is only slightly conserved between S. minutum and P. falciparum. However, small RNAs and intergenic sequences share sequence similarities with P. falciparum, suggesting that the function of noncoding sequences has been preserved despite development of very different genome structures. PMID:26199191

  17. Sequence-related human proteins cluster by degree of evolutionary conservation

    NASA Astrophysics Data System (ADS)

    Mrowka, Ralf; Patzak, Andreas; Herzel, Hanspeter; Holste, Dirk

    2004-11-01

    Gene duplication followed by adaptive evolution is thought to be a central mechanism for the emergence of novel genes. To illuminate the contribution of duplicated protein-coding sequences to the complexity of the human genome, we study the connectivity of pairwise sequence-related human proteins and construct a network (N) of linked protein sequences with shared similarities. We find that (i) the connectivity distribution P(k) for k sequence-related proteins decays as a power law P(k)˜k-γ with γ≈1.2 , (ii) the top rank of N consists of a single large cluster of proteins (≈70%) , while bottom ranks consist of multiple isolated clusters, and (iii) structural characteristics of N show both a high degree of clustering and an intermediate connectivity (“small-world” features). We gain further insight into structural properties of N by studying the relationship between the connectivity distribution and the phylogenetic conservation of proteins in bacteria, plants, invertebrates, and vertebrates. We find that (iv) the proportion of sequence-related proteins increases with increasing extent of evolutionary conservation. Our results support that small-world network properties constitute a footprint of an evolutionary mechanism and extend the traditional interpretation of protein families.

  18. A mechanism of immunoreceptor tyrosine-based activation motif (ITAM)-like sequences in the capsid protein VP2 in viral growth and pathogenesis of Coxsackievirus B3.

    PubMed

    Kim, Dae-Sun; Park, Jung-Hyun; Kim, Joo-Young; Kim, Dokeun; Nam, Jae-Hwan

    2012-04-01

    Coxsackievirus B3 (CVB3) is an RNA virus that mainly causes myocarditis. We have reported previously that immunoreceptor tyrosine-based activation motif (ITAM)-like sequences are contained in the capsid protein VP2 of CVB3. The substitution of two tyrosines for phenylalanines in the ITAM-like region causes attenuation of CVB3, possibly via defective viral assembly. In this study, we found that Syk, a downstream molecule of ITAM, interacts with the wild-type (WT) CVB3 VP0 protein, but not with the mutant CVB3 VP0 (called YYFF), and that an inhibitor of Syk reduced the growth of CVB3. The WT CVB3 activated nuclear factor kappa B (NF-κB), a protein activated by ITAM, and eventually induced the production of interleukin-6 (IL-6)-one of the proinflammatory cytokines induced by NF-κB-in macrophages. However, the YYFF form did not. In addition, viral VP2 protein may be dependent on the phosphorylation of an ITAM-like region that affected the activation of NF-κB. Taken together, these results suggest that the ITAM-like sequences in CVB3 VP2 can not only affect viral structure but also act as signals in pathogenesis.

  19. Predicting RNA-binding residues from evolutionary information and sequence conservation

    PubMed Central

    2010-01-01

    Abstract Background RNA-binding proteins (RBPs) play crucial roles in post-transcriptional control of RNA. RBPs are designed to efficiently recognize specific RNA sequences after it is derived from the DNA sequence. To satisfy diverse functional requirements, RNA binding proteins are composed of multiple blocks of RNA-binding domains (RBDs) presented in various structural arrangements to provide versatile functions. The ability to computationally predict RNA-binding residues in a RNA-binding protein can help biologists reveal important site-directed mutagenesis in wet-lab experiments. Results The proposed prediction framework named “ProteRNA” combines a SVM-based classifier with conserved residue discovery by WildSpan to identify the residues that interact with RNA in a RNA-binding protein. Although these conserved residues can be either functionally conserved residues or structurally conserved residues, they provide clues on the important residues in a protein sequence. In the independent testing dataset, ProteRNA has been able to deliver overall accuracy of 89.78%, MCC of 0.2628, F-score of 0.3075, and F0.5-score of 0.3546. Conclusions This article presents the design of a sequence-based predictor aiming to identify the RNA-binding residues in a RNA-binding protein by combining machine learning and pattern mining approaches. RNA-binding proteins have diverse functions while interacting with different categories of RNAs because these proteins are composed of multiple copies of RNA-binding domains presented in various structural arrangements to expand the functional repertoire of RNA-binding proteins. Furthermore, predicting RNA-binding residues in a RNA-binding protein can help biologists reveal important site-directed mutagenesis in wet-lab experiments. PMID:21143803

  20. Zinc finger binding motifs do not explain recombination rate variation within or between species of Drosophila.

    PubMed

    Heil, Caiti S S; Noor, Mohamed A F

    2012-01-01

    In humans and mice, the Cys(2)His(2) zinc finger protein PRDM9 binds to a DNA sequence motif enriched in hotspots of recombination, possibly modifying nucleosomes, and recruiting recombination machinery to initiate Double Strand Breaks (DSBs). However, since its discovery, some researchers have suggested that the recombinational effect of PRDM9 is lineage or species specific. To test for a conserved role of PRDM9-like proteins across taxa, we use the Drosophila pseudoobscura species group in an attempt to identify recombination associated zinc finger proteins and motifs. We leveraged the conserved amino acid motifs in Cys(2)His(2) zinc fingers to predict nucleotide binding motifs for all Cys(2)His(2) zinc finger proteins in Drosophila pseudoobscura and identified associations with empirical measures of recombination rate. Additionally, we utilized recombination maps from D. pseudoobscura and D. miranda to explore whether changes in the binding motifs between species can account for changes in the recombination landscape, analogous to the effect observed in PRDM9 among human populations. We identified a handful of potential recombination-associated sequence motifs, but the associations are generally tenuous and their biological relevance remains uncertain. Furthermore, we found no evidence that changes in zinc finger DNA binding explains variation in recombination rate between species. We therefore conclude that there is no protein with a DNA sequence specific human-PRDM9-like function in Drosophila. We suggest these findings could be explained by the existence of a different recombination initiation system in Drosophila.

  1. Predicting protein ligand binding sites by combining evolutionary sequence conservation and 3D structure.

    PubMed

    Capra, John A; Laskowski, Roman A; Thornton, Janet M; Singh, Mona; Funkhouser, Thomas A

    2009-12-01

    Identifying a protein's functional sites is an important step towards characterizing its molecular function. Numerous structure- and sequence-based methods have been developed for this problem. Here we introduce ConCavity, a small molecule binding site prediction algorithm that integrates evolutionary sequence conservation estimates with structure-based methods for identifying protein surface cavities. In large-scale testing on a diverse set of single- and multi-chain protein structures, we show that ConCavity substantially outperforms existing methods for identifying both 3D ligand binding pockets and individual ligand binding residues. As part of our testing, we perform one of the first direct comparisons of conservation-based and structure-based methods. We find that the two approaches provide largely complementary information, which can be combined to improve upon either approach alone. We also demonstrate that ConCavity has state-of-the-art performance in predicting catalytic sites and drug binding pockets. Overall, the algorithms and analysis presented here significantly improve our ability to identify ligand binding sites and further advance our understanding of the relationship between evolutionary sequence conservation and structural and functional attributes of proteins. Data, source code, and prediction visualizations are available on the ConCavity web site (http://compbio.cs.princeton.edu/concavity/).

  2. A phylogenetically conserved sequence within viral 3' untranslated RNA pseudoknots regulates translation.

    PubMed Central

    Leathers, V; Tanguay, R; Kobayashi, M; Gallie, D R

    1993-01-01

    Both the 68-base 5' leader (omega) and the 205-base 3' untranslated region (UTR) of tobacco mosaic virus (TMV) promote efficient translation. A 35-base region within omega is necessary and sufficient for the regulation. Within the 3' UTR, a 52-base region, composed of two RNA pseudoknots, is required for regulation. These pseudoknots are phylogenetically conserved among seven viruses from two different viral groups and one satellite virus. The pseudoknots contained significant conservation at the secondary and tertiary levels and at several positions at the primary sequence level. Mutational analysis of the sequences determined that the primary sequence in several conserved positions, particularly within the third pseudoknot, was essential for function. The higher-order structure of the pseudoknots was also required. Both the leader and the pseudoknot region were specifically recognized by, and competed for, the same proteins in extracts made from carrot cell suspension cells and wheat germ. Binding of the proteins is much stronger to omega than the pseudoknot region. Synergism was observed between the TMV 3' UTR and the cap and to a lesser extent between omega and the 3' UTR. The functional synergism and the protein binding data suggest that the cap, TMV 5' leader, and 3' UTR interact to establish an efficient level of translation. Images PMID:8355685

  3. Conservation of uORF repressiveness and sequence features in mouse, human and zebrafish

    PubMed Central

    Chew, Guo-Liang; Pauli, Andrea; Schier, Alexander F.

    2016-01-01

    Upstream open reading frames (uORFs) are ubiquitous repressive genetic elements in vertebrate mRNAs. While much is known about the regulation of individual genes by their uORFs, the range of uORF-mediated translational repression in vertebrate genomes is largely unexplored. Moreover, it is unclear whether the repressive effects of uORFs are conserved across species. To address these questions, we analyse transcript sequences and ribosome profiling data from human, mouse and zebrafish. We find that uORFs are depleted near coding sequences (CDSes) and have initiation contexts that diminish their translation. Linear modelling reveals that sequence features at both uORFs and CDSes modulate the translation of CDSes. Moreover, the ratio of translation over 5′ leaders and CDSes is conserved between human and mouse, and correlates with the number of uORFs. These observations suggest that the prevalence of vertebrate uORFs may be explained by their conserved role in repressing CDS translation. PMID:27216465

  4. A comparative analysis of distribution and conservation of microsatellites in the transcripts of sequenced Fusarium species and development of genic-SSR markers for polymorphism analysis.

    PubMed

    Mahfooz, Sahil; Srivastava, Arpita; Srivastava, Alok K; Arora, Dilip K

    2015-09-01

    We used an in silico approach to survey and compare microsatellites in transcript sequences of four sequenced members of genus Fusarium. G + C content of transcripts was found to be positively correlated with the frequency of SSRs. Our analysis revealed that, in all the four transcript sequences studied, the occurrence, relative abundance and density of microsatellites varied and was not influenced by transcript sizes. No correlation between relative abundance and transcript sizes was observed. The relative abundance and density of microsatellites were highest in the transcripts of Fusarium solani when compared with F. graminearum, F. verticillioides and F. oxysporum. The maximum frequency of SSRs among all four sequence sets was of trinucleotide repeats (67.8%), whereas the dinucleotide repeat represents <1%. Among all classes of repeats, 36.5% motifs were found conserved within Fusarium species. In order to study polymorphism within Fusarium isolates, 11 polymorphic genic-SSR markers were developed. Of the 11 markers, 5 were from F. oxysporum and remaining 6 belongs to F. solani. SSR markers from F. oxysporum were found to be more polymorphic (38%) as compared to F. solani (26%). Eleven polymorphic markers obtained in this study clearly demonstrate the utility of newly developed SSR markers in establishing genetic relationships among different isolates of Fusarium.

  5. Evolutionary Analysis and Classification of OATs, OCTs, OCTNs, and Other SLC22 Transporters: Structure-Function Implications and Analysis of Sequence Motifs.

    PubMed

    Zhu, Christopher; Nigam, Kabir B; Date, Rishabh C; Bush, Kevin T; Springer, Stevan A; Saier, Milton H; Wu, Wei; Nigam, Sanjay K

    2015-01-01

    The SLC22 family includes organic anion transporters (OATs), organic cation transporters (OCTs) and organic carnitine and zwitterion transporters (OCTNs). These are often referred to as drug transporters even though they interact with many endogenous metabolites and signaling molecules (Nigam, S.K., Nature Reviews Drug Discovery, 14:29-44, 2015). Phylogenetic analysis of SLC22 supports the view that these transporters may have evolved over 450 million years ago. Many OAT members were found to appear after a major expansion of the SLC22 family in mammals, suggesting a physiological and/or toxicological role during the mammalian radiation. Putative SLC22 orthologs exist in worms, sea urchins, flies, and ciona. At least six groups of SLC22 exist. OATs and OCTs form two Major clades of SLC22, within which (apart from Oat and Oct subclades), there are also clear Oat-like, Octn, and Oct-related subclades, as well as a distantly related group we term "Oat-related" (which may have different functions). Based on available data, it is arguable whether SLC22A18, which is related to bacterial drug-proton antiporters, should be assigned to SLC22. Disease-causing mutations, single nucleotide polymorphisms (SNPs) and other functionally analyzed mutations in OAT1, OAT3, URAT1, OCT1, OCT2, OCTN1, and OCTN2 map to the first extracellular domain, the large central intracellular domain, and transmembrane domains 9 and 10. These regions are highly conserved within subclades, but not between subclades, and may be necessary for SLC22 transporter function and functional diversification. Our results not only link function to evolutionarily conserved motifs but indicate the need for a revised sub-classification of SLC22.

  6. Evolutionary Analysis and Classification of OATs, OCTs, OCTNs, and Other SLC22 Transporters: Structure-Function Implications and Analysis of Sequence Motifs

    PubMed Central

    Date, Rishabh C.; Bush, Kevin T.; Springer, Stevan A.; Saier, Milton H.; Wu, Wei; Nigam, Sanjay K.

    2015-01-01

    The SLC22 family includes organic anion transporters (OATs), organic cation transporters (OCTs) and organic carnitine and zwitterion transporters (OCTNs). These are often referred to as drug transporters even though they interact with many endogenous metabolites and signaling molecules (Nigam, S.K., Nature Reviews Drug Discovery, 14:29–44, 2015). Phylogenetic analysis of SLC22 supports the view that these transporters may have evolved over 450 million years ago. Many OAT members were found to appear after a major expansion of the SLC22 family in mammals, suggesting a physiological and/or toxicological role during the mammalian radiation. Putative SLC22 orthologs exist in worms, sea urchins, flies, and ciona. At least six groups of SLC22 exist. OATs and OCTs form two Major clades of SLC22, within which (apart from Oat and Oct subclades), there are also clear Oat-like, Octn, and Oct-related subclades, as well as a distantly related group we term “Oat-related” (which may have different functions). Based on available data, it is arguable whether SLC22A18, which is related to bacterial drug-proton antiporters, should be assigned to SLC22. Disease-causing mutations, single nucleotide polymorphisms (SNPs) and other functionally analyzed mutations in OAT1, OAT3, URAT1, OCT1, OCT2, OCTN1, and OCTN2 map to the first extracellular domain, the large central intracellular domain, and transmembrane domains 9 and 10. These regions are highly conserved within subclades, but not between subclades, and may be necessary for SLC22 transporter function and functional diversification. Our results not only link function to evolutionarily conserved motifs but indicate the need for a revised sub-classification of SLC22. PMID:26536134

  7. Assembly of transmembrane helices of simple polytopic membrane proteins from sequence conservation patterns.

    PubMed

    Park, Yungki; Helms, Volkhard

    2006-09-01

    The transmembrane (TM) domains of most membrane proteins consist of helix bundles. The seemingly simple task of TM helix bundle assembly has turned out to be extremely difficult. This is true even for simple TM helix bundle proteins, i.e., those that have the simple form of compact TM helix bundles. Herein, we present a computational method that is capable of generating native-like structural models for simple TM helix bundle proteins having modest numbers of TM helices based on sequence conservation patterns. Thus, the only requirement for our method is the presence of more than 30 homologous sequences for an accurate extraction of sequence conservation patterns. The prediction method first computes a number of representative well-packed conformations for each pair of contacting TM helices, and then a library of tertiary folds is generated by overlaying overlapping TM helices of the representative conformations. This library is scored using sequence conservation patterns, and a subsequent clustering analysis yields five final models. Assuming that neighboring TM helices in the sequence contact each other (but not that TM helices A and G contact each other), the method produced structural models of Calpha atom root-mean-square deviation (CA RMSD) of 3-5 A from corresponding crystal structures for bacteriorhodopsin, halorhodopsin, sensory rhodopsin II, and rhodopsin. In blind predictions, this type of contact knowledge is not available. Mimicking this, predictions were made for the rotor of the V-type Na(+)-adenosine triphosphatase without such knowledge. The CA RMSD between the best model and its crystal structure is only 3.4 A, and its contact accuracy reaches 55%. Furthermore, the model correctly identifies the binding pocket for sodium ion. These results demonstrate that the method can be readily applied to ab initio structure prediction of simple TM helix bundle proteins having modest numbers of TM helices.

  8. Conserved Asp327 of Walker B motif in the N-terminal Nucleotide Binding Domain (NBD-1) of Cdr1p of Candida albicans has acquired a new role in ATP hydrolysis

    PubMed Central

    Rai, Versha; Gaur, Manisha; Shukla, Sudhanshu; Shukla, Suneet; Ambudkar, Suresh V.; Komath, Sneha Sudha; Prasad, Rajendra

    2008-01-01

    The Walker A and B motifs of nucleotide binding domains (NBDs) of Cdr1p though almost identical to all ABC transporters, has unique substitutions. We have in the past shown that Trp326 of Walker B and Cys193 of Walker A motifs of N-terminal NBD of Cdr1p have distinct roles in ATP binding and hydrolysis, respectively. In the present study, we have examined the role of a well conserved Asp327 in the Walker B motif of the N-terminal NBD which is preceded (Trp326) and followed (Asn328) by atypical amino acid substitutions and compared it with its equivalent well conserved Asp1026 of the C-terminal NBD of Cdr1p. We observed that the removal of the negative charge by D327N, D327A, D1026N, D1026A and D327N/D1026N substitutions, resulted in Cdr1p mutant variants that were severely impaired in ATPase activity and drug efflux. Importantly, all the mutant variants showed characteristics similar to those of wild type with respect to cell surface expression and photoaffinity drug analogue [125I] IAAP and [3H] azidopine labeling. While Cdr1p D327N mutant variant showed comparable binding with [α-32P] 8-azido ATP, Cdr1p D1026N and Cdr1p D327N/D1026N mutant variants were crippled in nucleotide binding. That the two conserved carboxylate residues Asp327 and Asp1026 are functionally different was further evident from the pH profile of ATPase activity. Cdr1p D327N mutant variant showed ∼40% enhancement of its residual ATPase activity at acidic pH while no such pH effect was seen with Cdr1p D1026N mutant variant. Our experimental data suggest that Asp327 of N-terminal NBD has acquired a new role to act as a catalytic base in ATP hydrolysis, a role normally conserved for Glu present adjacent to the conserved Asp in the Walker B motif of all the non-fungal transporters. PMID:17144665

  9. Variation in conserved non-coding sequences on chromosome 5q andsusceptibility to asthma and atopy

    SciTech Connect

    Donfack, Joseph; Schneider, Daniel H.; Tan, Zheng; Kurz,Thorsten; Dubchak, Inna; Frazer, Kelly A.; Ober, Carole

    2005-09-10

    Background: Evolutionarily conserved sequences likely havebiological function. Methods: To determine whether variation in conservedsequences in non-coding DNA contributes to risk for human disease, westudied six conserved non-coding elements in the Th2 cytokine cluster onhuman chromosome 5q31 in a large Hutterite pedigree and in samples ofoutbred European American and African American asthma cases and controls.Results: Among six conserved non-coding elements (>100 bp,>70percent identity; human-mouse comparison), we identified one singlenucleotide polymorphism (SNP) in each of two conserved elements and sixSNPs in the flanking regions of three conserved elements. We genotypedour samples for four of these SNPs and an additional three SNPs each inthe IL13 and IL4 genes. While there was only modest evidence forassociation with single SNPs in the Hutterite and European Americansamples (P<0.05), there were highly significant associations inEuropean Americans between asthma and haplotypes comprised of SNPs in theIL4 gene (P<0.001), including a SNP in a conserved non-codingelement. Furthermore, variation in the IL13 gene was strongly associatedwith total IgE (P = 0.00022) and allergic sensitization to mold allergens(P = 0.00076) in the Hutterites, and more modestly associated withsensitization to molds in the European Americans and African Americans (P<0.01). Conclusion: These results indicate that there is overalllittle variation in the conserved non-coding elements on 5q31, butvariation in IL4 and IL13, including possibly one SNP in a conservedelement, influence asthma and atopic phenotypes in diversepopulations.

  10. Computational analysis of conserved coil functional residues in the mitochondrial genomic sequences of dermatophytes

    PubMed Central

    Gupta, Bulbul; Kaur, Jaspreet

    2016-01-01

    Dermatophyte is a group of closely related fungi that have the capacity to invade keratinized tissue of humans and other animals. The infection known as dermatophytosis, caused by members of the genera Microsporum, Trichophyton, and Epidermophyton includes infection to the groin (tinea cruris), beard (tinea barbae), scalp (tinea capitis), feet (tinea pedis), glabrous skin (tinea corporis), nail (tinea unguium), and hand (tinea manuum). The identification of evolutionary relationship between these three genera of dermatophyte is epidemiologically important to understand their pathogenicity. Mitochondrial DNA evolves more rapidly than a nuclear DNA due to higher rate of mutation but is very less affected by genetic recombination, making it an important tool for phylogenetic studies. Thus, here we present a novel scheme to identify the conserved coil functional residues of Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum and Microsporum canis. Protein coding sequences of the mitochondrial genome were aligned for their similar sequences and homology modelling was performed for structure and pocket identification. The results obtained from comparative analysis of the protein sequences revealed the presence of functionally active sites in all the species of the genera Trichophyton and Microsporum. However in Epidermophyton floccosum it was observed in three protein sequences of the five studied. The absence of these conserved coil functional residues in E. floccusum may be correlated with lesser infectivity of this organism. The functional residues identified in the present study could be responsible for the disease and thus can act as putative target sites for drug designing. PMID:28149055

  11. A novel conserved phosphotyrosine motif in the Drosophila fibroblast growth factor signaling adaptor Dof with a redundant role in signal transmission.

    PubMed

    Csiszar, Agnes; Vogelsang, Elisabeth; Beug, Hartmut; Leptin, Maria

    2010-04-01

    The fibroblast growth factor receptor (FGFR) signals through adaptors constitutively associated with the receptor. In Drosophila melanogaster, the FGFR-specific adaptor protein Downstream-of-FGFR (Dof) becomes phosphorylated upon receptor activation at several tyrosine residues, one of which recruits Corkscrew (Csw), the Drosophila homolog of SHP2, which provides a molecular link to mitogen-activated protein kinase (MAPK) activation. However, the Csw pathway is not the only link from Dof to MAPK. In this study, we identify a novel phosphotyrosine motif present in four copies in Dof and also found in other insect and vertebrate signaling molecules. We show that these motifs are phosphorylated and contribute to FGF signal transduction. They constitute one of three sets of phosphotyrosines that act redundantly in signal transmission: (i) a Csw binding site, (ii) four consensus Grb2 recognition sites, and (iii) four novel tyrosine motifs. We show that Src64B binds to Dof and that Src kinases contribute to FGFR-dependent MAPK activation. Phosphorylation of the novel tyrosine motifs is required for the interaction of Dof with Src64B. Thus, Src64B recruitment to Dof through the novel phosphosites can provide a new link to MAPK activation and other cellular responses. This may give a molecular explanation for the involvement of Src kinases in FGF-dependent developmental events.

  12. GC Content Heterogeneity Transition of Conserved Noncoding Sequences Occurred at the Emergence of Vertebrates

    PubMed Central

    Hettiarachchi, Nilmini; Saitou, Naruya

    2016-01-01

    Conserved non-coding sequences (CNSs) of Eukaryotes are known to be significantly enriched in regulatory sequences. CNSs of diverse lineages follow different patterns in abundance, sequence composition, and location. Here, we report a thorough analysis of CNSs in diverse groups of Eukaryotes with respect to GC content heterogeneity. We examined 24 fungi, 19 invertebrates, and 12 non-mammalian vertebrates so as to find lineage specific features of CNSs. We found that fungi and invertebrate CNSs are predominantly GC rich as in plants we previously observed, whereas vertebrate CNSs are GC poor. This result suggests that the CNS GC content transition occurred from the ancestral GC rich state of Eukaryotes to GC poor in the vertebrate lineage due to the enrollment of GC poor transcription factor binding sites that are lineage specific. CNS GC content is closely linked with the nucleosome occupancy that determines the location and structural architecture of DNAs. PMID:28040773

  13. Highly conserved D-loop-like nuclear mitochondrial sequences (Numts) in tiger (Panthera tigris).

    PubMed

    Zhang, Wenping; Zhang, Zhihe; Shen, Fujun; Hou, Rong; Lv, Xiaoping; Yue, Bisong

    2006-08-01

    Using oligonucleotide primers designed to match hypervariable segments I (HVS-1) of Panthera tigris mitochondrial DNA (mtDNA), we amplified two different PCR products (500 bp and 287 bp) in the tiger (Panthera tigris), but got only one PCR product (287 bp) in the leopard (Panthera pardus). Sequence analyses indicated that the sequence of 287 bp was a D-loop-like nuclear mitochondrial sequence (Numts), indicating a nuclear transfer that occurred approximately 4.8-17 million years ago in the tiger and 4.6-16 million years ago in the leopard. Although the mtDNA D-loop sequence has a rapid rate of evolution, the 287-bp Numts are highly conserved; they are nearly identical in tiger subspecies and only 1.742% different between tiger and leopard. Thus, such sequences represent molecular 'fossils' that can shed light on evolution of the mitochondrial genome and may be the most appropriate outgroup for phylogenetic analysis. This is also proved by comparing the phylogenetic trees reconstructed using the D-loop sequence of snow leopard and the 287-bp Numts as outgroup.

  14. Low molecular weight serine protease inhibitors from insects are proteins with highly conserved sequences.

    PubMed

    Boigegrain, R A; Pugnière, M; Paroutaud, P; Castro, B; Brehélin, M

    2000-02-01

    A low molecular weight protease inhibitor peptide found in ovaries of the desert locust Schistocerca gregaria (SGPI-2), was purified from plasma of the same locust and sequenced. It was named SGCI. It was found active towards chymotrypsin and human leukocyte elastase. SGCI was synthesized using a solid-phase procedure and the sequence of its reactive site for chymotrypsin was determined. Compared with an inhibitor purified earlier from another locust species, the total sequence of SGCI showed 88% identity. In particular, the sequence of the reactive site of these inhibitors was identical. Our search for a closely related peptide in an insect species far removed from locusts, the lepidopteran Spodoptera littoralis, was unfruitful but a different chymotrypsin inhibitor, belonging to the Kazal family, was found whose mass is greater than that of SGCI (20 vs 3.6 kDa). Its N-terminal sequence shares 80% identity with that of a chymotrypsin inhibitor purified earlier from the haemolymph of another lepidopteran. Conservation of the amino acid sequence in the reactive site seems to be an exception among protease inhibitors.

  15. Sample sequencing of vascular plants demonstrates widespread conservation and divergence of microRNAs.

    PubMed

    Chávez Montes, Ricardo A; de Fátima Rosas-Cárdenas, Flor; De Paoli, Emanuele; Accerbi, Monica; Rymarquis, Linda A; Mahalingam, Gayathri; Marsch-Martínez, Nayelli; Meyers, Blake C; Green, Pamela J; de Folter, Stefan

    2014-04-23

    Small RNAs are pivotal regulators of gene expression that guide transcriptional and post-transcriptional silencing mechanisms in eukaryotes, including plants. Here we report a comprehensive atlas of sRNA and miRNA from 3 species of algae and 31 representative species across vascular plants, including non-model plants. We sequence and quantify sRNAs from 99 different tissues or treatments across species, resulting in a data set of over 132 million distinct sequences. Using miRBase mature sequences as a reference, we identify the miRNA sequences present in these libraries. We apply diverse profiling methods to examine critical sRNA and miRNA features, such as size distribution, tissue-specific regulation and sequence conservation between species, as well as to predict putative new miRNA sequences. We also develop database resources, computational analysis tools and a dedicated website, http://smallrna.udel.edu/. This study provides new insights on plant sRNAs and miRNAs, and a foundation for future studies.

  16. The Crystal Structure of the Extracellular 11-heme Cytochrome UndA Reveals a Conserved 10-heme Motif and Defined Binding Site for Soluble Iron Chelates.

    SciTech Connect

    Edwards, Marcus; Hall, Andrea; Shi, Liang; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David; Clarke, Thomas A.

    2012-07-03

    Members of the genus Shewanella translocate deca- or undeca-heme cytochromes to the external cell surface thus enabling respiration using extracellular minerals and polynuclear Fe(III) chelates. The high resolution structure of the first undeca-heme outer membrane cytochrome, UndA, reveals a crossed heme chain with four potential electron ingress/egress sites arranged within four domains. Sequence and structural alignment of UndA and the deca-heme MtrF reveals the extra heme of UndA is inserted between MtrF hemes 6 and 7. The remaining UndA hemes can be superposed over the heme chain of the decaheme MtrF, suggesting that a ten heme core is conserved between outer membrane cytochromes. The UndA structure is the first outer membrane cytochrome to be crystallographically resolved in complex with substrates, an Fe(III)-nitrilotriacetate dimer or an Fe(III)-citrate trimer. The structural resolution of these UndA-Fe(III)-chelate complexes provides a rationale for previous kinetic measurements on UndA and other outer membrane cytochromes.

  17. Reptiles and mammals have differentially retained long conserved noncoding sequences from the amniote ancestor.

    PubMed

    Janes, D E; Chapus, C; Gondo, Y; Clayton, D F; Sinha, S; Blatti, C A; Organ, C L; Fujita, M K; Balakrishnan, C N; Edwards, S V

    2011-01-01

    Many noncoding regions of genomes appear to be essential to genome function. Conservation of large numbers of noncoding sequences has been reported repeatedly among mammals but not thus far among birds and reptiles. By searching genomes of chicken (Gallus gallus), zebra finch (Taeniopygia guttata), and green anole (Anolis carolinensis), we quantified the conservation among birds and reptiles and across amniotes of long, conserved noncoding sequences (LCNS), which we define as sequences ≥500 bp in length and exhibiting ≥95% similarity between species. We found 4,294 LCNS shared between chicken and zebra finch and 574 LCNS shared by the two birds and Anolis. The percent of genomes comprised by LCNS in the two birds (0.0024%) is notably higher than the percent in mammals (<0.0003% to <0.001%), differences that we show may be explained in part by differences in genome-wide substitution rates. We reconstruct a large number of LCNS for the amniote ancestor (ca. 8,630) and hypothesize differential loss and substantial turnover of these sites in descendent lineages. By contrast, we estimated a small role for recruitment of LCNS via acquisition of novel functions over time. Across amniotes, LCNS are significantly enriched with transcription factor binding sites for many developmental genes, and 2.9% of LCNS shared between the two birds show evidence of expression in brain expressed sequence tag databases. These results show that the rate of retention of LCNS from the amniote ancestor differs between mammals and Reptilia (including birds) and that this may reflect differing roles and constraints in gene regulation.

  18. Reptiles and Mammals Have Differentially Retained Long Conserved Noncoding Sequences from the Amniote Ancestor

    PubMed Central

    Janes, D.E.; Chapus, C.; Gondo, Y.; Clayton, D.F.; Sinha, S.; Blatti, C.A.; Organ, C.L.; Fujita, M.K.; Balakrishnan, C.N.; Edwards, S.V.

    2011-01-01

    Many noncoding regions of genomes appear to be essential to genome function. Conservation of large numbers of noncoding sequences has been reported repeatedly among mammals but not thus far among birds and reptiles. By searching genomes of chicken (Gallus gallus), zebra finch (Taeniopygia guttata), and green anole (Anolis carolinensis), we quantified the conservation among birds and reptiles and across amniotes of long, conserved noncoding sequences (LCNS), which we define as sequences ≥500 bp in length and exhibiting ≥95% similarity between species. We found 4,294 LCNS shared between chicken and zebra finch and 574 LCNS shared by the two birds and Anolis. The percent of genomes comprised by LCNS in the two birds (0.0024%) is notably higher than the percent in mammals (<0.0003% to <0.001%), differences that we show may be explained in part by differences in genome-wide substitution rates. We reconstruct a large number of LCNS for the amniote ancestor (ca. 8,630) and hypothesize differential loss and substantial turnover of these sites in descendent lineages. By contrast, we estimated a small role for recruitment of LCNS via acquisition of novel functions over time. Across amniotes, LCNS are significantly enriched with transcription factor binding sites for many developmental genes, and 2.9% of LCNS shared between the two birds show evidence of expression in brain expressed sequence tag databases. These results show that the rate of retention of LCNS from the amniote ancestor differs between mammals and Reptilia (including birds) and that this may reflect differing roles and constraints in gene regulation. PMID:21183607

  19. Motif types, motif locations and base composition patterns around the RNA polyadenylation site in microorganisms, plants and animals

    PubMed Central

    2014-01-01

    Background The polyadenylation of RNA is critical for gene functioning, but the conserved sequence motifs (often called signal or signature motifs), motif locations and abundances, and base composition patterns around mRNA polyadenylation [poly(A)] sites are still uncharacterized in most species. The evolutionary tendency for poly(A) site selection is still largely unknown. Results We analyzed the poly(A) site regions of 31 species or phyla. Different groups of species showed different poly(A) signal motifs: UUACUU at the poly(A) site in the parasite Trypanosoma cruzi; UGUAAC (approximately 13 bases upstream of the site) in the alga Chlamydomonas reinhardtii; UGUUUG (or UGUUUGUU) at mainly the fourth base downstream of the poly(A) site in the parasite Blastocystis hominis; and AAUAAA at approximately 16 bases and approximately 19 bases upstream of the poly(A) site in animals and plants, respectively. Polyadenylation signal motifs are usually several hundred times more abundant around poly(A) sites than in whole genomes. These predominant motifs usually had very specific locations, whether upstream of, at, or downstream of poly(A) sites, depending on the species or phylum. The poly(A) site was usually an adenosine (A) in all analyzed species except for B. hominis, and there was weak A predominance in C. reinhardtii. Fungi, animals, plants, and the protist Phytophthora infestans shared a general base abundance pattern (or base composition pattern) of “U-rich—A-rich—U-rich—Poly(A) site—U-rich regions”, or U-A-U-A-U for short, with some variation for each kingdom or subkingdom. Conclusion This study identified the poly(A) signal motifs, motif locations, and base composition patterns around mRNA poly(A) sites in protists, fungi, plants, and animals and provided insight into poly(A) site evolution. PMID:25052519

  20. Mitochondrial genome sequences illuminate maternal lineages of conservation concern in a rare carnivore

    PubMed Central

    2011-01-01

    Background Science-based wildlife management relies on genetic information to infer population connectivity and identify conservation units. The most commonly used genetic marker for characterizing animal biodiversity and identifying maternal lineages is the mitochondrial genome. Mitochondrial genotyping figures prominently in conservation and management plans, with much of the attention focused on the non-coding displacement ("D") loop. We used massively parallel multiplexed sequencing to sequence complete mitochondrial genomes from 40 fishers, a threatened carnivore that possesses low mitogenomic diversity. This allowed us to test a key assumption of conservation genetics, specifically, that the D-loop accurately reflects genealogical relationships and variation of the larger mitochondrial genome. Results Overall mitogenomic divergence in fishers is exceedingly low, with 66 segregating sites and an average pairwise distance between genomes of 0.00088 across their aligned length (16,290 bp). Estimates of variation and genealogical relationships from the displacement (D) loop region (299 bp) are contradicted by the complete mitochondrial genome, as well as the protein coding fraction of the mitochondrial genome. The sources of this contradiction trace primarily to the near-absence of mutations marking the D-loop region of one of the most divergent lineages, and secondarily to independent (recurrent) mutations at two nucleotide position in the D-loop amplicon. Conclusions Our study has two important implications. First, inferred genealogical reconstructions based on the fisher D-loop region contradict inferences based on the entire mitogenome to the point that the populations of greatest conservation concern cannot be accurately resolved. Whole-genome analysis identifies Californian haplotypes from the northern-most populations as highly distinctive, with a significant excess of amino acid changes that may be indicative of molecular adaptation; D-loop sequences fail

  1. A short sequence motif in the 5' leader of the HIV-1 genome modulates extended RNA dimer formation and virus replication.

    PubMed

    van Bel, Nikki; Das, Atze T; Cornelissen, Marion; Abbink, Truus E M; Berkhout, Ben

    2014-12-19

    The 5' leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.

  2. Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences

    PubMed Central

    Ivanov, Ivaylo P.; Firth, Andrew E.; Michel, Audrey M.; Atkins, John F.; Baranov, Pavel V.

    2011-01-01

    In eukaryotes, it is generally assumed that translation initiation occurs at the AUG codon closest to the messenger RNA 5′ cap. However, in certain cases, initiation can occur at codons differing from AUG by a single nucleotide, especially the codons CUG, UUG, GUG, ACG, AUA and AUU. While non-AUG initiation has been experimentally verified for a handful of human genes, the full extent to which this phenomenon is utilized—both for increased coding capacity and potentially also for novel regulatory mechanisms—remains unclear. To address this issue, and hence to improve the quality of existing coding sequence annotations, we developed a methodology based on phylogenetic analysis of predicted 5′ untranslated regions from orthologous genes. We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences. Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated extensions in 17 other genes. In several instances, we have been able to obtain independent experimental evidence of the expression of non-AUG-initiated products from the previously published literature and ribosome profiling data. PMID:21266472

  3. Lack of evidence of conserved lentiviral sequences in pigs with post weaning multisystemic wasting syndrome.

    PubMed Central

    Bratanich, A; Lairmore, M; Heneine, W; Konoby, C; Harding, J; West, K; Vasquez, G; Allan, G; Ellis, J

    1999-01-01

    In order to investigate the role of retroviruses in the recently described porcine postweaning multisystemic wasting syndrome (PMWS) serum and leukocytes were screened for reverse transcriptase (RT) activity, and tissues were examined for the presence of conserved lentiviral sequences using degenerate primers in a polymerase chain reaction (PCR). Serum and stimulated leukocytes from the blood and lymph nodes from pigs with PMWS, as well as from control pigs had RT activity that was detected by the sensitive Amp-RT assay. A 257-bp fragment was amplified from DNA from the blood and bone marrow of pigs with PMWS. This fragment was identical in size to conserved lentiviral sequences that were amplified from plasmids containing DNA from several lentiviruses. Cloning and sequencing of the fragment from affected pigs, however, did not reveal homology with the recognized lentiviruses. Together the results of these analyses suggest that the RT activity present in tissues from control and affected pigs is the result of endogenous retrovirus expression, and that a lentivirus is not a primary pathogen in PMWS. Images Figure 1. Figure 2. PMID:10480463

  4. A survey of DNA motif finding algorithms

    PubMed Central

    Das, Modan K; Dai, Ho-Kwok

    2007-01-01

    Background Unraveling the mechanisms that regulate gene expression is a major challenge in biology. An important task in this challenge is to identify regulatory elements, especially the binding sites in deoxyribonucleic acid (DNA) for transcription factors. These binding sites are short DNA segments that are called motifs. Recent advances in genome sequence availability and in high-throughput gene expression analysis technologies have allowed for the development of computational methods for motif finding. As a result, a large number of motif finding algorithms have been implemented and applied to various motif models over the past decade. This survey reviews the latest developments in DNA motif finding algorithms. Results Earlier algorithms use promoter sequences of coregulated genes from single genome and search for statistically overrepresented motifs. Recent algorithms are designed to use phylogenetic footprinting or orthologous sequences and also an integrated approach where promoter sequences of coregulated genes and phylogenetic footprinting are used. All the algorithms studied have been reported to correctly detect the motifs that have been previously detected by laboratory experimental approaches, and some algorithms were able to find novel motifs. However, most of these motif finding algorithms have been shown to work successfully in yeast and other lower organisms, but perform significantly worse in higher organisms. Conclusion Despite considerable efforts to date, DNA motif finding remains a complex challenge for biologists and computer scientists. Researchers have taken many different approaches in developing motif discovery tools and the progress made in this area of research is very encouraging. Performance comparison of different motif finding tools and identification of the best tools have proven to be a difficult task because tools are designed based on algorithms and motif models that are diverse and complex and our incomplete understanding of

  5. A Collection of Conserved Noncoding Sequences to Study Gene Regulation in Flowering Plants1[OPEN

    PubMed Central

    2016-01-01

    Transcription factors (TFs) regulate gene expression by binding cis-regulatory elements, of which the identification remains an ongoing challenge owing to the prevalence of large numbers of nonfunctional TF binding sites. Powerful comparative genomics methods, such as phylogenetic footprinting, can be used for the detection of conserved noncoding sequences (CNSs), which are functionally constrained and can greatly help in reducing the number of false-positive elements. In this study, we applied a phylogenetic footprinting approach for the identification of CNSs in 10 dicot plants, yielding 1,032,291 CNSs associated with 243,187 genes. To annotate CNSs with TF binding sites, we made use of binding site information for 642 TFs originating from 35 TF families in Arabidopsis (Arabidopsis thaliana). In three species, the identified CNSs were evaluated using TF chromatin immunoprecipitation sequencing data, resulting in significant overlap for the majority of data sets. To identify ultraconserved CNSs, we included genomes of additional plant families and identified 715 binding sites for 501 genes conserved in dicots, monocots, mosses, and green algae. Additionally, we found that genes that are part of conserved mini-regulons have a higher coherence in their expression profile than other divergent gene pairs. All identified CNSs were integrated in the PLAZA 3.0 Dicots comparative genomics platform (http://bioinformatics.psb.ugent.be/plaza/versions/plaza_v3_dicots/) together with new functionalities facilitating the exploration of conserved cis-regulatory elements and their associated genes. The availability of this data set in a user-friendly platform enables the exploration of functional noncoding DNA to study gene regulation in a variety of plant species, including crops. PMID:27261064

  6. A highly conserved repeated chromosomal sequence in the radioresistant bacterium Deinococcus radiodurans SARK

    SciTech Connect

    Lennon, E.; Gutman, P.D.; Hanlong Yao; Minton, K.W. )

    1991-03-01

    A DNA fragment containing a portion of a DNA damage-inducible gene from Deinococcus radiodurans SARK hybridized to numerous fragments of SARK genomic DNA because of a highly conserved repetitive chromosomal element. The element is of variable length, ranging from 150 to 192 bp, depending on the absence or presence of one or two 21-bp sequences located internally. A putative translational start site of the damage-inducible gene is within the reiterated element. The element contains dyad symmetries that suggest modes of transcriptional and/or translational control.

  7. Substitution of a conserved cysteine-996 in a cysteine-rich motif of the laminin {alpha}2-chain in congenital muscular dystrophy with partial deficiency of the protein

    SciTech Connect

    Nissinen, M.; Xu Zhang; Tryggvason, K.

    1996-06-01

    Congenital muscular dystrophies (CMDs) are autosomal recessive muscle disorders of early onset. Approximately half of CMD patients present laminin {alpha}2-chain (merosin) deficiency in muscle biopsies, and the disease locus has been mapped to the region of the LAMA2 gene (6q22-23) in several families. Recently, two nonsense mutations in the laminin {alpha}2-chain gene were identified in CMD patients exhibiting complete deficiency of the laminin {alpha}2-chain in muscle biopsies. However, a subset of CMD patients with linkage to LAMA2 show only partial absence of the laminin {alpha}2-chain around muscle fibers, by immunocytochemical analysis. In the present study we have identified a homozygous missense mutation in the {alpha}2-chain gene of a consanguineous Turkish family with partial laminin {alpha}2-chain deficiency. The T{r_arrow}C transition at position 3035 in the cDNA sequence results in a Cys996{r_arrow}Arg substitution. The mutation that affects one of the conserved cysteine-rich repeats in the short arm of the laminin {alpha}2-chain should result in normal synthesis of the chain and in formation and secretion of a heterotrimeric laminin molecule. Muscular dysfunction is possibly caused either by abnormal disulfide cross-links and folding of the laminin repeat, leading to the disturbance of an as yet unknown binding function of the laminin {alpha}2-chain and to shorter half-life of the muscle-specific laminin-2 and laminin-4 isoforms, or by increased proteolytic sensitivity, leading to truncation of the short arm. 42 refs., 7 figs.

  8. Repression domains of class II ERF transcriptional repressors share an essential motif for active repression.

    PubMed

    Ohta, M; Matsui, K; Hiratsu, K; Shinshi, H; Ohme-Takagi, M

    2001-08-01

    We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif (L)/(F)DLN(L)/(F)(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.

  9. Isosteric and nonisosteric base pairs in RNA motifs: molecular dynamics and bioinformatics study of the sarcin-ricin internal loop.

    PubMed

    Havrila, Marek; Réblová, Kamila; Zirbel, Craig L; Leontis, Neocles B; Šponer, Jiří

    2013-11-21

    The sarcin-ricin RNA motif (SR motif) is one of the most prominent recurrent RNA building blocks that occurs in many different RNA contexts and folds autonomously, that is, in a context-independent manner. In this study, we combined bioinformatics analysis with explicit-solvent molecular dynamics (MD) simulations to better understand the relation between the RNA sequence and the evolutionary patterns of the SR motif. A SHAPE probing experiment was also performed to confirm the fidelity of the MD simulations. We identified 57 instances of the SR motif in a nonredundant subset of the RNA X-ray structure database and analyzed their base pairing, base-phosphate, and backbone-backbone interactions. We extracted sequences aligned to these instances from large rRNA alignments to determine the frequency of occurrence for different sequence variants. We then used a simple scoring scheme based on isostericity to suggest 10 sequence variants with a highly variable expected degree of compatibility with the SR motif 3D structure. We carried out MD simulations of SR motifs with these base substitutions. Nonisosteric base substitutions led to unstable structures, but so did isosteric substitutions which were unable to make key base-phosphate interactions. The MD technique explains why some potentially isosteric SR motifs are not realized during evolution. We also found that the inability to form stable cWW geometry is an important factor in the case of the first base pair of the flexible region of the SR motif. A comparison of structural, bioinformatics, SHAPE probing, and MD simulation data reveals that explicit solvent MD simulations neatly reflect the viability of different sequence variants of the SR motif. Thus, MD simulations can efficiently complement bioinformatics tools in studies of conservation patterns of RNA motifs and provide atomistic insight into the role of their different signature interactions.

  10. Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences.

    PubMed

    Kalloush, Rawan M; Vivet-Boudou, Valérie; Ali, Lizna M; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A

    2016-06-01

    MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2'hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5' region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses.

  11. Conservation.

    ERIC Educational Resources Information Center

    National Audubon Society, New York, NY.

    This set of teaching aids consists of seven Audubon Nature Bulletins, providing the teacher and student with informational reading on various topics in conservation. The bulletins have these titles: Plants as Makers of Soil, Water Pollution Control, The Ground Water Table, Conservation--To Keep This Earth Habitable, Our Threatened Air Supply,…

  12. New insights into SRY regulation through identification of 5' conserved sequences

    PubMed Central

    Ross, Diana GF; Bowles, Josephine; Koopman, Peter; Lehnert, Sigrid

    2008-01-01

    Background SRY is the pivotal gene initiating male sex determination in most mammals, but how its expression is regulated is still not understood. In this study we derived novel SRY 5' flanking genomic sequence data from bovine and caprine genomic BAC clones. Results We identified four intervals of high homology upstream of SRY by comparison of human, bovine, pig, goat and mouse genomic sequences. These conserved regions contain putative binding sites for a large number of known transcription factor families, including several that have been implicated previously in sex determination and early gonadal development. Conclusion Our results reveal potentially important SRY regulatory elements, mutations in which might underlie cases of idiopathic human XY sex reversal. PMID:18851760

  13. Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within Hup+Rhizobium leguminosarum Strains

    PubMed Central

    Leyva, Antonio; Palacios, José M.; Ruiz-Argüeso, Tomás

    1987-01-01

    Thirteen Rhizobium leguminosarum strains previously reported as H2-uptake hydrogenase positive (Hup+) or negative (Hup−) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H2 oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H2-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H2-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup+ strains in this symbiotic species. Images PMID:16347471

  14. Model peptide studies of sequence regions in the elastomeric biomineralization protein, Lustrin A. I. The C-domain consensus-PG-, -NVNCT-motif.

    PubMed

    Zhang, Bo; Wustman, Brandon A; Morse, Daniel; Evans, John Spencer

    2002-05-01

    The lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layer) in mollusk shell. Recent atomic force microscopy (AFM) pulling studies have demonstrated that the lustrin-containing organic nacre layer in the abalone, Haliotis rufescens, exhibits a typical sawtooth force-extension curve with hysteretic recovery. This force extension behavior is reminiscent of reversible unfolding and refolding in elastomeric proteins such as titin and tenascin. Since secondary structure plays an important role in force-induced protein unfolding and refolding, the question is, What secondary structure(s) exist within the major domains of Lustrin A? Using a model peptide (FPGKNVNCTSGE) representing the 12-residue consensus sequence found near the N-termini of the first eight cysteine-rich domains (C-domains) within the Lustrin A protein, we employed CD, NMR spectroscopy, and simulated annealing/minimization to determine the secondary structure preferences for this sequence. At pH 7.4, we find that the 12-mer sequence adopts a loop conformation, consisting of a "bend" or "turn" involving residues G3-K4 and N7-C8-T9, with extended conformations arising at F1-G3; K4-V6; T9-S10-G11 in the sequence. Minor pH-dependent conformational effects were noted for this peptide; however, there is no evidence for a salt-bridge interaction between the K4 and E12 side chains. The presence of a loop conformation within the highly conserved -PG-, -NVNCT- sequence of C1-C8 domains may have important structural and mechanistic implications for the Lustrin A protein with regard to elastic behavior.

  15. Yeast general transcription factor GFI: sequence requirements for binding to DNA and evolutionary conservation.

    PubMed Central

    Dorsman, J C; van Heeswijk, W C; Grivell, L A

    1990-01-01

    GFI is an abundant DNA binding protein in the yeast S. cerevisiae. The protein binds to specific sequences in both ARS elements and the upstream regions of a large number of genes and is likely to play an important role in yeast cell growth. To get insight into the relative strength of the various GFI-DNA binding sites within the yeast genome, we have determined dissociation rates for several GFI-DNA complexes and found them to vary over a 70-fold range. Strong binding sites for GFI are present in the upstream activating sequences of the gene encoding the 40 kDa subunit II of the QH2:cytochrome c reductase, the gene encoding ribosomal protein S33 and in the intron of the actin gene. The binding site in the ARS1-TRP1 region is of intermediate strength. All strong binding sites conform to the sequence 5' RTCRYYYNNNACG-3'. Modification interference experiments and studies with mutant binding sites indicate that critical bases for GFI recognition are within the two elements of the consensus DNA recognition sequence. Proteins with the DNA binding specificities of GFI and GFII can also be detected in the yeast K. lactis, suggesting evolutionary conservation of at least the respective DNA-binding domains in both yeasts. Images PMID:2187179

  16. Sequence of Radiotherapy and Chemotherapy in Breast Cancer After Breast-Conserving Surgery

    SciTech Connect

    Jobsen, Jan J.; Palen, Job van der; Brinkhuis, Marieel; Ong, Francisca; Struikmans, Henk

    2012-04-01

    Purpose: The optimal sequence of radiotherapy and chemotherapy in breast-conserving therapy is unknown. Methods and Materials: From 1983 through 2007, a total of 641 patients with 653 instances of breast-conserving therapy (BCT), received both chemotherapy and radiotherapy and are the basis of this analysis. Patients were divided into three groups. Groups A and B comprised patients treated before 2005, Group A radiotherapy first and Group B chemotherapy first. Group C consisted of patients treated from 2005 onward, when we had a fixed sequence of radiotherapy first, followed by chemotherapy. Results: Local control did not show any differences among the three groups. For distant metastasis, no difference was shown between Groups A and B. Group C, when compared with Group A, showed, on univariate and multivariate analyses, a significantly better distant metastasis-free survival. The same was noted for disease-free survival. With respect to disease-specific survival, no differences were shown on multivariate analysis among the three groups. Conclusion: Radiotherapy, as an integral part of the primary treatment of BCT, should be administered first, followed by adjuvant chemotherapy.

  17. The Chinese hamster Alu-equivalent sequence: a conserved highly repetitious, interspersed deoxyribonucleic acid sequence in mammals has a structure suggestive of a transposable element.

    PubMed Central

    Haynes, S R; Toomey, T P; Leinwand, L; Jelinek, W R

    1981-01-01

    A consensus sequence has been determined for a major interspersed deoxyribonucleic acid repeat in the genome of Chinese hamster ovary cells (CHO cells). This sequence is extensively homologous to (i) the human Alu sequence (P. L. Deininger et al., J. Mol. Biol., in press), (ii) the mouse B1 interspersed repetitious sequence (Krayev et al., Nucleic Acids Res. 8:1201-1215, 1980) (iii) an interspersed repetitious sequence from African green monkey deoxyribonucleic acid (Dhruva et al., Proc. Natl. Acad. Sci. U.S.A. 77:4514-4518, 1980) and (iv) the CHO and mouse 4.5S ribonucleic acid (this report; F. Harada and N. Kato, Nucleic Acids Res. 8:1273-1285, 1980). Because the CHO consensus sequence shows significant homology to the human Alu sequence it is termed the CHO Alu-equivalent sequence. A conserved structure surrounding CHO Alu-equivalent family members can be recognized. It is similar to that surrounding the human Alu and the mouse B1 sequences, and is represented as follows: direct repeat-CHO-Alu-A-rich sequence-direct repeat. A composite interspersed repetitious sequence has been identified. Its structure is represented as follows: direct repeat-residue 47 to 107 of CHO-Alu-non-Alu repetitious sequence-A-rich sequence-direct repeat. Because the Alu flanking sequences resemble those that flank known transposable elements, we think it likely that the Alu sequence dispersed throughout the mammalian genome by transposition. Images PMID:9279371

  18. Efficient motif search in ranked lists and applications to variable gap motifs.

    PubMed

    Leibovich, Limor; Yakhini, Zohar

    2012-07-01

    Sequence elements, at all levels-DNA, RNA and protein, play a central role in mediating molecular recognition and thereby molecular regulation and signaling. Studies that focus on -measuring and investigating sequence-based recognition make use of statistical and computational tools, including approaches to searching sequence motifs. State-of-the-art motif searching tools are limited in their coverage and ability to address large motif spaces. We develop and present statistical and algorithmic approaches that take as input ranked lists of sequences and return significant motifs. The efficiency of our approach, based on suffix trees, allows searches over motif spaces that are not covered by existing tools. This includes searching variable gap motifs-two half sites with a flexible length gap in between-and searching long motifs over large alphabets. We used our approach to analyze several high-throughput measurement data sets and report some validation results as well as novel suggested motifs and motif refinements. We suggest a refinement of the known estrogen receptor 1 motif in humans, where we observe gaps other than three nucleotides that also serve as significant recognition sites, as well as a variable length motif related to potential tyrosine phosphorylation.

  19. Prevalence of the EH1 Groucho interaction motif in the metazoan Fox family of transcriptional regulators

    PubMed Central

    Yaklichkin, Sergey; Vekker, Alexander; Stayrook, Steven; Lewis, Mitchell; Kessler, Daniel S

    2007-01-01

    Background The Fox gene family comprises a large and functionally diverse group of forkhead-related transcriptional regulators, many of which are essential for metazoan embryogenesis and physiology. Defining conserved functional domains that mediate the transcriptional activity of Fox proteins will contribute to a comprehensive understanding of the biological function of Fox family genes. Results Systematic analysis of 458 protein sequences of the metazoan Fox family was performed to identify the presence of the engrailed homology-1 motif (eh1), a motif known to mediate physical interaction with transcriptional corepressors of the TLE/Groucho family. Greater than 50% of Fox proteins contain sequences with high similarity to the eh1 motif, including ten of the nineteen Fox subclasses (A, B, C, D, E, G, H, I, L, and Q) and Fox proteins of early divergent species such as marine sponge. The eh1 motif is not detected in Fox proteins of the F, J, K, M, N, O, P, R and S subclasses, or in yeast Fox proteins. The eh1-like motifs are positioned C-terminal to the winged helix DNA-binding domain in all subclasses except for FoxG proteins, which have an N-terminal motif. Two similar eh1-like motifs are found in the zebrafish FoxQ1 and in FoxG proteins of sea urchin and amphioxus. The identification of eh1-like motifs by manual sequence alignment was validated by statistical analyses of the Swiss protein database, confirming a high frequency of occurrence of eh1-like sequences in Fox family proteins. Structural predictions suggest that the majority of identified eh1-like motifs are short α-helices, and wheel modeling revealed an amphipathicity that supports this secondary structure prediction. Conclusion A search for eh1 Groucho interaction motifs in the Fox gene family has identified eh1-like sequences in greater than 50% of Fox proteins. The results predict a physical and functional interaction of TLE/Groucho corepressors with many members of the Fox family of transcriptional

  20. A motif rich in charged residues determines product specificity in isomaltulose synthase.

    PubMed

    Zhang, Daohai; Li, Nan; Swaminathan, Kunchithapadam; Zhang, Lian Hui

    2003-01-16

    Isomaltulose synthase (PalI) catalyzes hydrolysis of sucrose and formation of alpha-1,6 and alpha-1,1 bonds to produce isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofranose) and small amount of trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofranose). A potential isomaltulose synthase-specific motif ((325)RLDRD(329)), that contains a 'DxD' motif conserved in many glycosyltransferases, was identified based on sequence comparison with reference to the secondary structural features of PalI and homologs. Site-directed mutagenesis analysis of the motif showed that the four charged amino acid residues (Arg(325), Arg(328), Asp(327) and Asp(329)) influence the enzyme kinetics and determine the product specificity. Mutation of these four residues increased trehalulose formation by 17-61% and decreased isomaltulose by 26-67%. We conclude that the 'RLDRD' motif controls the product specificity of PalI.

  1. DNA motifs determining the accuracy of repeat duplication during CRISPR adaptation in Haloarcula hispanica

    PubMed Central

    Wang, Rui; Li, Ming; Gong, Luyao; Hu, Songnian; Xiang, Hua

    2016-01-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) acquire new spacers to generate adaptive immunity in prokaryotes. During spacer integration, the leader-preceded repeat is always accurately duplicated, leading to speculations of a repeat-length ruler. Here in Haloarcula hispanica, we demonstrate that the accurate duplication of its 30-bp repeat requires two conserved mid-repeat motifs, AACCC and GTGGG. The AACCC motif was essential and needed to be ∼10 bp downstream from the leader-repeat junction site, where duplication consistently started. Interestingly, repeat duplication terminated sequence-independently and usually with a specific distance from the GTGGG motif, which seemingly served as an anchor site for a molecular ruler. Accordingly, altering the spacing between the two motifs led to an aberrant duplication size (29, 31, 32 or 33 bp). We propose the adaptation complex may recognize these mid-repeat elements to enable measuring the repeat DNA for spacer integration. PMID:27085805

  2. Rapid fixation of a distinctive sequence motif in the 3' noncoding region of the clade of West Nile virus invading North America.

    PubMed

    Hughes, Austin L; Piontkivska, Helen; Foppa, Ivo

    2007-09-15

    Phylogenetic analysis of complete genomes of West Nile virus (WNV) by a variety of methods supported the hypothesis that North American isolates of WNV constitute a monophyletic group, together with an isolate from Israel and one from Hungary. We used ancestral sequence reconstruction in order to obtain evidence for evolutionary changes that might be correlated with increased virulence in this clade (designated the N.A. clade). There was one amino acid change (I-->T at residue 356 of the NS3 protein) that occurred in the ancestor of the N.A. clade and remained conserved in all N.A. clade genomes analyzed. There were four changes in the upstream portion of the 3' noncoding region (the AT-enriched region) that occurred in the ancestor of the N.A. clade and remained conserved in all N.A. clade genomes analyzed, changes predicted to alter RNA secondary structure. The AT-enriched region showed a higher rate of substitution in the branch ancestral to the N.A. clade, relative to polymorphism, than did the remainder of the noncoding regions, synonymous sites in coding regions, or nonsynonymous sites in coding regions. The high rate of occurrence of fixed nucleotide substitutions in this region suggests that positive Darwinian selection may have acted on this portion of the 3'NCR and that these fixed changes, possibly in concert with the amino acid change in NS3, may underlie phenotypic effects associated with increased virulence in North American WNV.

  3. SA-Mot: a web server for the identification of motifs of interest extracted from protein loops.

    PubMed

    Regad, Leslie; Saladin, Adrien; Maupetit, Julien; Geneix, Colette; Camproux, Anne-Claude

    2011-07-01

    The detection of functional motifs is an important step for the determination of protein functions. We present here a new web server SA-Mot (Structural Alphabet Motif) for the extraction and location of structural motifs of interest from protein loops. Contrary to other methods, SA-Mot does not focus only on functional motifs, but it extracts recurrent and conserved structural motifs involved in structural redundancy of loops. SA-Mot uses the structural word notion to extract all structural motifs from uni-dimensional sequences corresponding to loop structures. Then, SA-Mot provides a description of these structural motifs using statistics computed in the loop data set and in SCOP superfamily, sequence and structural parameters. SA-Mot results correspond to an interactive table listing all structural motifs extracted from a target structure and their associated descriptors. Using this information, the users can easily locate loop regions that are important for the protein folding and function. The SA-Mot web server is available at http://sa-mot.mti.univ-paris-diderot.fr.

  4. Comparative genomic analysis of equilibrative nucleoside transporters suggests conserved protein structure despite limited sequence identity.

    PubMed

    Sankar, Narendra; Machado, Jerry; Abdulla, Parween; Hilliker, Arthur J; Coe, Imogen R

    2002-10-15

    Equilibrative nucleoside transporters (ENTs) are a recently characterized and poorly understood group of membrane proteins that are important in the uptake of endogenous nucleosides required for nucleic acid and nucleoside triphosphate synthesis. Despite their central importance in cellular metabolism and nucleoside analog chemotherapy, no human ENT gene has been described and nothing is known about gene structure and function. To gain insight into the ENT gene family, we used experimental and in silico comparative genomic approaches to identify ENT genes in three evolutionarily diverse organisms with completely (or almost completely) sequenced genomes, Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster. We describe the chromosomal location, the predicted ENT gene structure and putative structural topologies of predicted ENT proteins derived from the open reading frames. Despite variations in genomic layout and limited ortholog protein sequence identity (< or =27.45%), predicted topologies of ENT proteins are strikingly similar, suggesting an evolutionary conservation of a prototypic structure. In addition, a similar distribution of protein domains on exons is apparent in all three taxa. These data demonstrate that comparative sequence analyses should be combined with other approaches (such as genomic and proteomic analyses) to fully understand structure, function and evolution of protein families.

  5. Evolutionary conservation of sequence and secondary structures inCRISPR repeats

    SciTech Connect

    Kunin, Victor; Sorek, Rotem; Hugenholtz, Philip

    2006-09-01

    Clustered Regularly Interspaced Palindromic Repeats (CRISPRs) are a novel class of direct repeats, separated by unique spacer sequences of similar length, that are present in {approx}40% of bacterial and all archaeal genomes analyzed to date. More than 40 gene families, called CRISPR-associated sequences (CAS), appear in conjunction with these repeats and are thought to be involved in the propagation and functioning of CRISPRs. It has been proposed that the CRISPR/CAS system samples, maintains a record of, and inactivates invasive DNA that the cell has encountered, and therefore constitutes a prokaryotic analog of an immune system. Here we analyze CRISPR repeats identified in 195 microbial genomes and show that they can be organized into multiple clusters based on sequence similarity. All individual repeats in any given cluster were inferred to form characteristic RNA secondary structure, ranging from non-existent to pronounced. Stable secondary structures included G:U base pairs and exhibited multiple compensatory base changes in the stem region, indicating evolutionary conservation and functional importance. We also show that the repeat-based classification corresponds to, and expands upon, a previously reported CAS gene-based classification including specific relationships between CRISPR and CAS subtypes.

  6. The complete nucleotide sequence of the Crossostoma lacustre mitochondrial genome: conservation and variations among vertebrates.

    PubMed Central

    Tzeng, C S; Hui, C F; Shen, S C; Huang, P C

    1992-01-01

    The complete mitochondrial (mt) genome of Crossostoma lacustre, a freshwater loach from mountain stream of Taiwan, has been cloned and sequenced. This fish mt genome, consisting of 16558 base-pairs, encodes genes for 13 proteins, two rRNAs, and 22 tRNAs, in addition to a regulatory sequence for replication and transcription (D-loop), is similar to those of the other vertebrates in both the order and orientation of these genes. The protein-coding and ribosomal RNA genes are highly homologous both in size and composition, to their counterparts in mammals, birds, amphibians, and invertebrates, and using essentially the same set of codons, including both the initiation and termination signals, and the tRNAs. Differences do exist, however, in the lengths and sequences of the D-loop regions, and in space between genes, which account for the variations in total lengths of the genomes. Our observations provide evidence for the first time for the conservation of genetic information in the fish mitochondrial genome, especially among the vertebrates. PMID:1408800

  7. Remarkable intron and exon sequence conservation in human and mouse homeobox Hox 1. 3 genes

    SciTech Connect

    Tournier-Lasserve, E.; Odenwald, W.F.; Garbern, J.; Trojanowski, J.; Lazzarini, R.A.

    1989-05-01

    A high degree of conservation exists between the Hox 1.3 homeobox genes of mice and humans. The two genes occupy the same relative positions in their respective Hox 1 gene clusters, they show extensive sequence similarities in their coding and noncoding portions, and both are transcribed into multiple transcripts of similar sizes. The predicted human Hox 1.3 protein differs from its murine counterpart in only 7 of 270 amino acids. The sequence similarity in the 250 base pairs upstream of the initiation codon is 98%, the similarity between the two introns, both 960 base pairs long, is 72%, and the similarity in the 3' noncoding region from termination codon to polyadenylation signal is 90%. Both mouse and human Hox 1.3 introns contain a sequence with homology to a mating-type-controlled cis element of the yeast Ty1 transposon. DNA-binding studies with a recombinant mouse Hox 1.3 protein identified two binding sites in the intron, both of which were within the region of shared homology with this Ty1 cis element.

  8. Sequence conservation in avian CR1: an interspersed repetitive DNA family evolving under functional constraints.

    PubMed Central

    Chen, Z Q; Ritzel, R G; Lin, C C; Hodgetts, R B

    1991-01-01

    CR1 is a short interspersed repetitive DNA element originally identified in the domestic chicken (Gallus gallus). However, unlike virtually all other such sequences described to date, CR1 is not confined to one or a few closely related species. It is probably a ubiquitous component of the avian genome, having been detected in representatives of nine orders encompassing a wide spectrum of the class Aves. This identification was made possible by using the polymerase chain reaction (PCR), which revealed interspecific similarities not detected by conventional Southern analysis. DNA sequence comparisons between a CR1 element isolated from a sarus crane (Grus antigone) and those isolated from an emu (Dromaius novaehollandiae) showed that two short highly conserved regions are present. These are included within two regions previously characterized in the CR1 units of domestic fowl. One of these behaves as a transcriptional silencer and the other is a binding site for a nuclear protein. Our observations suggest that CR1 has evolved under functional constraints and that interspersed repetitive sequences as a class may constitute a more significant component of the eukaryotic genome than is generally acknowledged. Images PMID:1829530

  9. Mutation of the aspartic acid residues of the GDD sequence motif of poliovirus RNA-dependent RNA polymerase results in enzymes with altered metal ion requirements for activity.

    PubMed Central

    Jablonski, S A; Morrow, C D

    1995-01-01

    The poliovirus RNA-dependent RNA polymerase, 3Dpol, is known to share a region of sequence homology with all RNA polymerases centered at the GDD amino acid motif. The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme. To test this hypothesis, we have utilized oligonucleotide site-directed mutagenesis to generate defined mutations in the aspartic acids of the GDD motif of the 3Dpol gene. The codon for the first aspartate (3D-D-328 [D refers to the single amino acid change, and the number refers to its position in the polymerase]) was changed to that for glutamic acid, histidine, asparagine, or glutamine; the codons for both aspartic acids were simultaneously changed to those for glutamic acids; and the codon for the second aspartic acid (3D-D-329) was changed to that for glutamic acid or asparagine. The mutant enzymes were expressed in Escherichia coli, and the in vitro poly(U) polymerase activity was characterized. All of the mutant 3Dpol enzymes were enzymatically inactive in vitro when tested over a range of Mg2+ concentrations. However, when Mn2+ was substituted for Mg2+ in the in vitro assays, the mutant that substituted the second aspartic acid for asparagine (3D-N-329) was active. To further substantiate this finding, a series of different transition metal ions were substituted for Mg2+ in the poly(U) polymerase assay. The wild-type enzyme was active with all metals except Ca2+, while the 3D-N-329 mutant was active only when FeC6H7O5 was used in the reaction. To determine the effects of the mutations on poliovirus replication, the mutant 3Dpol genes were subcloned into an infectious cDNA of poliovirus. The cDNAs containing the mutant 3Dpol genes did not produce infectious virus when transfected into tissue culture cells under standard conditions. Because of the activity of the 3D-N-329 mutant in the presence of Fe2+ and Mn2+, transfections were also performed in the presence of the

  10. Genome-wide analysis of ethylene-responsive element binding factor-associated amphiphilic repression motif-containing transcriptional regulators in Arabidopsis.

    PubMed

    Kagale, Sateesh; Links, Matthew G; Rozwadowski, Kevin

    2010-03-01

    The ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif is a transcriptional regulatory motif identified in members of the ethylene-responsive element binding factor, C2H2, and auxin/indole-3-acetic acid families of transcriptional regulators. Sequence comparison of the core EAR motif sites from these proteins revealed two distinct conservation patterns: LxLxL and DLNxxP. Proteins containing these motifs play key roles in diverse biological functions by negatively regulating genes involved in developmental, hormonal, and stress signaling pathways. Through a genome-wide bioinformatics analysis, we have identified the complete repertoire of the EAR repressome in Arabidopsis (Arabidopsis thaliana) comprising 219 proteins belonging to 21 different transcriptional regulator families. Approximately 72% of these proteins contain a LxLxL type of EAR motif, 22% contain a DLNxxP type of EAR motif, and the remaining 6% have a motif where LxLxL and DLNxxP are overlapping. Published in vitro and in planta investigations support approximately 40% of these proteins functioning as negative regulators of gene expression. Comparative sequence analysis of EAR motif sites and adjoining regions has identified additional preferred residues and potential posttranslational modification sites that may influence the functionality of the EAR motif. Homology searches against protein databases of poplar (Populus trichocarpa), grapevine (Vitis vinifera), rice (Oryza sativa), and sorghum (Sorghum bicolor) revealed that the EAR motif is conserved across these diverse plant species. This genome-wide analysis represents the most extensive survey of EAR motif-containing proteins in Arabidopsis to date and provides a resource enabling investigations into their biological roles and the mechanism of EAR motif-mediated transcriptional regulation.

  11. Comparative Analysis of Genome and Epigenome in Closely Related Medaka Species Identifies Conserved Sequence Preferences for DNA Hypomethylated Domains.

    PubMed

    Uno, Ayako; Nakamura, Ryohei; Tsukahara, Tatsuya; Qu, Wei; Sugano, Sumio; Suzuki, Yutaka; Morishita, Shinichi; Takeda, Hiroyuki

    2016-08-01

    The genomes of vertebrates are globally methylated, but a small portion of genomic regions are known to be hypomethylated. Although hypomethylated domains (HMDs) have been implicated in transcriptional regulation in various ways, how a HMD is determined in a particular genomic region remains elusive. To search for DNA motifs essential for the formation of HMDs, we performed the genome-wide comparative analysis of genome and DNA methylation patterns of the two medaka inbred lines, Hd-rRII1 and HNI-II, which are derived from northern and southern subpopulations of Japan and exhibit high levels of genetic variations (SNP, ∼ 3%). We successfully mapped > 70% of HMDs in both genomes and found that the majority of those mapped HMDs are conserved between the two lines (common HMDs). Unexpectedly, the average genetic variations are similar in the common HMD and other genome regions. However, we identified short well-conserved motifs that are specifically enriched in HMDs, suggesting that they may play roles in the establishment of HMDs in the medaka genome.

  12. The Calmodulin-Binding, Short Linear Motif, NSCaTE Is Conserved in L-Type Channel Ancestors of Vertebrate Cav1.2 and Cav1.3 Channels

    PubMed Central

    Taiakina, Valentina; Boone, Adrienne N.; Fux, Julia; Senatore, Adriano; Weber-Adrian, Danielle

    2013-01-01

    NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca2+ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca2+-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels. PMID:23626724

  13. The C-Terminal Sequence and PI motif of the Orchid (Oncidium Gower Ramsey) PISTILLATA (PI) Ortholog Determine its Ability to Bind AP3 Orthologs and Enter the Nucleus to Regulate Downstream Genes Controlling Petal and Stamen Formation.

    PubMed

    Mao, Wan-Ting; Hsu, Hsing-Fun; Hsu, Wei-Han; Li, Jen-Ying; Lee, Yung-I; Yang, Chang-Hsien

    2015-11-01

    This study focused on the investigation of the effects of the PI motif and C-terminus of the Oncidium Gower Ramsey MADS box gene 8 (OMADS8), a PISTILLATA (PI) ortholog, on floral organ formation. 35S::OMADS8 completely rescued and 35S::OMADS8-PI (with the PI motif deleted) partially rescued petal/stamen formation, whereas these deficiencies were not rescued by 35S::OMADS8-C (C-terminal 29 amino acids deleted) in pi-1 mutants. OMADS8 could interact with Arabidopsis APETALA3 (AP3) and enter the nucleus. The nuclear entry efficiency was reduced for OMADS8-PI/AP3 and OMADS8-C/AP3. OMADS8 could also interact with OMADS5/OMADS9 (the Oncidium AP3 ortholog) and enter the nucleus with an efficiency only slightly affected by the deletion of the C-terminal sequence or PI motif. However, the stability of the OMADS8/OMADS5 and OMADS8/OMADS9 complexes was significantly reduced by deletion of the C-terminal sequence or PI motif. Further analysis indicated that the expression of genes downstream of AP3/PI (BNQ1/BNQ2/GNC/At4g30270) was compensated by 35S::OMADS8 and 35S::OMADS8-PI to a level similar to wild-type plants but was not affected by 35S::OMADS8-C in the pi-1 mutants. A similar FRET (fluorescence resonance energy transfer) efficiency was observed for Arabidopsis AGAMOUS (AG) and the Oncidium AG ortholog OMADS4 for OMADS8, OMADS8-PI and OMADS8-C. These results indicated that the OMADS8 PI motif and C-terminus were valuable for the interaction of OMADS8 with the AP3 orthologs to form higher order heterotetrameric complexes that regulated petal/stamen formation in both Oncidium orchids and transgenic Arabidopsis. However, the C-terminal sequence and PI motif were dispensable for the interaction of OMADS8 with the AG orthologs.

  14. Position-specific prediction of methylation sites from sequence conservation based on information theory.

    PubMed

    Shi, Yinan; Guo, Yanzhi; Hu, Yayun; Li, Menglong

    2015-07-23

    Protein methylation plays vital roles in many biological processes and has been implicated in various human diseases. To fully understand the mechanisms underlying methylation for use in drug design and work in methylation-related diseases, an initial but crucial step is to identify methylation sites. The use of high-throughput bioinformatics methods has become imperative to predict methylation sites. In this study, we developed a novel method that is based only on sequence conservation to predict protein methylation sites. Conservation difference profiles between methylated and non-methylated peptides were constructed by the information entropy (IE) in a wider neighbor interval around the methylation sites that fully incorporated all of the environmental information. Then, the distinctive neighbor residues were identified by the importance scores of information gain (IG). The most representative model was constructed by support vector machine (SVM) for Arginine and Lysine methylation, respectively. This model yielded a promising result on both the benchmark dataset and independent test set. The model was used to screen the entire human proteome, and many unknown substrates were identified. These results indicate that our method can serve as a useful supplement to elucidate the mechanism of protein methylation and facilitate hypothesis-driven experimental design and validation.

  15. Efficient motif search in ranked lists and applications to variable gap motifs

    PubMed Central

    Leibovich, Limor; Yakhini, Zohar

    2012-01-01

    Sequence elements, at all levels—DNA, RNA and protein, play a central role in mediating molecular recognition and thereby molecular regulation and signaling. Studies that focus on measuring and investigating sequence-based recognition make use of statistical and computational tools, including approaches to searching sequence motifs. State-of-the-art motif searching tools are limited in their coverage and ability to address large motif spaces. We develop and present statistical and algorithmic approaches that take as input ranked lists of sequences and return significant motifs. The efficiency of our approach, based on suffix trees, allows searches over motif spaces that are not covered by existing tools. This includes searching variable gap motifs—two half sites with a flexible length gap in between—and searching long motifs over large alphabets. We used our approach to analyze several high-throughput measurement data sets and report some validation results as well as novel suggested motifs and motif refinements. We suggest a refinement of the known estrogen receptor 1 motif in humans, where we observe gaps other than three nucleotides that also serve as significant recognition sites, as well as a variable length motif related to potential tyrosine phosphorylation. PMID:22416066

  16. Rapid Fixation of a Distinctive Sequence Motif in the 3′Noncoding Region of the Clade of West Nile Virus Invading North America

    PubMed Central

    Hughes, Austin L.; Piontkivska, Helen; Foppa, Ivo

    2007-01-01

    Phylogenetic analysis of complete genomes of West Nile virus (WNV) by a variety of methods supported the hypothesis that North American isolates of WNV constitute a monophyletic group, together with an isolate from Israel and one from Hungary. We used ancestral sequence reconstruction in order to obtain evidence for evolutionary changes that might be correlated with increased virulence in this clade (designated the N.A. clade). There was one amino acid change (I→T at residue 356 of the NS3 protein) that occurred in the ancestor of the N.A. clade and remained conserved in all N.A. clade genomes analyzed. There were four changes in the upstream portion of the 3′ noncoding region (the AT-enriched region) that occurred in the ancestor of the N.A. clade and remained conserved in all N.A. clade genomes analyzed, changes predicted to alter RNA secondary structure. The AT-enriched region showed a higher rate of substitution in the branch ancestral to the N.A. clade, relative to polymorphism, than did the remainder of the non-coding regions, synonymous sites in coding regions, or nonsynonymous sites in coding regions. The high rate of occurrence of fixed nucleotide substitutions in this region suggests that positive Darwinian selection may have acted on this portion of the 3′NCR and that these fixed changes, possibly in concert with the amino acid change in NS3, may underlie phenotypic effects associated with increased virulence in North American WNV. PMID:17587514

  17. High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire.

    PubMed

    Hou, Xianliang; Lu, Chong; Chen, Sisi; Xie, Qian; Cui, Guangying; Chen, Jianing; Chen, Zhi; Wu, Zhongwen; Ding, Yulong; Ye, Ping; Dai, Yong; Diao, Hongyan

    2016-03-01

    The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals.Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR.Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides.Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination.

  18. High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire

    PubMed Central

    Hou, Xianliang; Lu, Chong; Chen, Sisi; Xie, Qian; Cui, Guangying; Chen, Jianing; Chen, Zhi; Wu, Zhongwen; Ding, Yulong; Ye, Ping; Dai, Yong; Diao, Hongyan

    2016-01-01

    Abstract The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals. Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR. Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides. Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination. PMID:26962778

  19. Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins.

    PubMed Central

    Cygler, M.; Schrag, J. D.; Sussman, J. L.; Harel, M.; Silman, I.; Gentry, M. K.; Doctor, B. P.

    1993-01-01

    Based on the recently determined X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three-dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X-ray structures. PMID:8453375

  20. Signature motifs of GDP polyribonucleotidyltransferase, a non-segmented negative strand RNA viral mRNA capping enzyme, domain in the L protein are required for covalent enzyme-pRNA intermediate formation.

    PubMed

    Neubauer, Julie; Ogino, Minako; Green, Todd J; Ogino, Tomoaki

    2016-01-08

    The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase; block V) domain in RNA polymerase L proteins of non-segmented negative strand (NNS) RNA viruses (e.g. rabies, measles, Ebola) contains five collinear sequence elements, Rx(3)Wx(3-8)ΦxGxζx(P/A) (motif A; Φ, hydrophobic; ζ, hydrophilic), (Y/W)ΦGSxT (motif B), W (motif C), HR (motif D) and ζxxΦx(F/Y)QxxΦ (motif E). We performed site-directed mutagenesis of the L protein of vesicular stomatitis virus (VSV, a prototypic NNS RNA virus) to examine participation of these motifs in mRNA capping. Similar to the catalytic residues in motif D, G1100 in motif A, T1157 in motif B, W1188 in motif C, and F1269 and Q1270 in motif E were found to be essential or important for the PRNTase activity in the step of the covalent L-pRNA intermediate formation, but not for the GTPase activity that generates GDP (pRNA acceptor). Cap defective mutations in these residues induced termination of mRNA synthesis at position +40 followed by aberrant stop-start transcription, and abolished virus gene expression in host cells. These results suggest that the conserved motifs constitute the active site of the PRNTase domain and the L-pRNA intermediate formation followed by the cap formation is essential for successful synthesis of full-length mRNAs.

  1. Signature motifs of GDP polyribonucleotidyltransferase, a non-segmented negative strand RNA viral mRNA capping enzyme, domain in the L protein are required for covalent enzyme–pRNA intermediate formation

    PubMed Central

    Neubauer, Julie; Ogino, Minako; Green, Todd J.; Ogino, Tomoaki

    2016-01-01

    The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase; block V) domain in RNA polymerase L proteins of non-segmented negative strand (NNS) RNA viruses (e.g. rabies, measles, Ebola) contains five collinear sequence elements, Rx(3)Wx(3–8)ΦxGxζx(P/A) (motif A; Φ, hydrophobic; ζ, hydrophilic), (Y/W)ΦGSxT (motif B), W (motif C), HR (motif D) and ζxxΦx(F/Y)QxxΦ (motif E). We performed site-directed mutagenesis of the L protein of vesicular stomatitis virus (VSV, a prototypic NNS RNA virus) to examine participation of these motifs in mRNA capping. Similar to the catalytic residues in motif D, G1100 in motif A, T1157 in motif B, W1188 in motif C, and F1269 and Q1270 in motif E were found to be essential or important for the PRNTase activity in the step of the covalent L-pRNA intermediate formation, but not for the GTPase activity that generates GDP (pRNA acceptor). Cap defective mutations in these residues induced termination of mRNA synthesis at position +40 followed by aberrant stop–start transcription, and abolished virus gene expression in host cells. These results suggest that the conserved motifs constitute the active site of the PRNTase domain and the L-pRNA intermediate formation followed by the cap formation is essential for successful synthesis of full-length mRNAs. PMID:26602696

  2. Origin and spread of photosynthesis based upon conserved sequence features in key bacteriochlorophyll biosynthesis proteins.

    PubMed

    Gupta, Radhey S

    2012-11-01

    The origin of photosynthesis and how this capability has spread to other bacterial phyla remain important unresolved questions. I describe here a number of conserved signature indels (CSIs) in key proteins involved in bacteriochlorophyll (Bchl) biosynthesis that provide important insights in these regards. The proteins BchL and BchX, which are essential for Bchl biosynthesis, are derived by gene duplication in a common ancestor of all phototrophs. More ancient gene duplication gave rise to the BchX-BchL proteins and the NifH protein of the nitrogenase complex. The sequence alignment of NifH-BchX-BchL proteins contain two CSIs that are uniquely shared by all NifH and BchX homologs, but not by any BchL homologs. These CSIs and phylogenetic analysis of NifH-BchX-BchL protein sequences strongly suggest that the BchX homologs are ancestral to BchL and that the Bchl-based anoxygenic photosynthesis originated prior to the chlorophyll (Chl)-based photosynthesis in cyanobacteria. Another CSI in the BchX-BchL sequence alignment that is uniquely shared by all BchX homologs and the BchL sequences from Heliobacteriaceae, but absent in all other BchL homologs, suggests that the BchL homologs from Heliobacteriaceae are primitive in comparison to all other photosynthetic lineages. Several other identified CSIs in the BchN homologs are commonly shared by all proteobacterial homologs and a clade consisting of the marine unicellular Cyanobacteria (Clade C). These CSIs in conjunction with the results of phylogenetic analyses and pair-wise sequence similarity on the BchL, BchN, and BchB proteins, where the homologs from Clade C Cyanobacteria and Proteobacteria exhibited close relationship, provide strong evidence that these two groups have incurred lateral gene transfers. Additionally, phylogenetic analyses and several CSIs in the BchL-N-B proteins that are uniquely shared by all Chlorobi and Chloroflexi homologs provide evidence that the genes for these proteins have also been

  3. Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing.

    PubMed

    Wang, Zheng Jia; Huang, Jian Qin; Huang, You Jun; Li, Zheng; Zheng, Bing Song

    2012-08-01

    Hickory (Carya cathayensis Sarg.) is an economically important woody plant in China, but its long juvenile phase delays yield. MicroRNAs (miRNAs) are critical regulators of genes and important for normal plant development and physiology, including flower development. We used Solexa technology to sequence two small RNA libraries from two floral differentiation stages in hickory to identify miRNAs related to flower development. We identified 39 conserved miRNA sequences from 114 loci belonging to 23 families as well as two novel and ten potential novel miRNAs belonging to nine families. Moreover, 35 conserved miRNA*s and two novel miRNA*s were detected. Twenty miRNA sequences from 49 loci belonging to 11 families were differentially expressed; all were up-regulated at the later stage of flower development in hickory. Quantitative real-time PCR of 12 conserved miRNA sequences, five novel miRNA families, and two novel miRNA*s validated that all were expressed during hickory flower development, and the expression patterns were similar to those detected with Solexa sequencing. Finally, a total of 146 targets of the novel and conserved miRNAs were predicted. This study identified a diverse set of miRNAs that were closely related to hickory flower development and that could help in plant floral induction.

  4. Stochastic motif extraction using hidden Markov model

    SciTech Connect

    Fujiwara, Yukiko; Asogawa, Minoru; Konagaya, Akihiko

    1994-12-31

    In this paper, we study the application of an HMM (hidden Markov model) to the problem of representing protein sequences by a stochastic motif. A stochastic protein motif represents the small segments of protein sequences that have a certain function or structure. The stochastic motif, represented by an HMM, has conditional probabilities to deal with the stochastic nature of the motif. This HMM directive reflects the characteristics of the motif, such as a protein periodical structure or grouping. In order to obtain the optimal HMM, we developed the {open_quotes}iterative duplication method{close_quotes} for HMM topology learning. It starts from a small fully-connected network and iterates the network generation and parameter optimization until it achieves sufficient discrimination accuracy. Using this method, we obtained an HMM for a leucine zipper motif. Compared to the accuracy of a symbolic pattern representation with accuracy of 14.8 percent, an HMM achieved 79.3 percent in prediction. Additionally, the method can obtain an HMM for various types of zinc finger motifs, and it might separate the mixed data. We demonstrated that this approach is applicable to the validation of the protein databases; a constructed HMM b as indicated that one protein sequence annotated as {open_quotes}lencine-zipper like sequence{close_quotes} in the database is quite different from other leucine-zipper sequences in terms of likelihood, and we found this discrimination is plausible.

  5. Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity

    PubMed Central

    Sagnol, Sébastien; Yang, Yinshan; Bessin, Yannick; Allemand, Fréderic; Hapkova, Ilona; Notarnicola, Cécile; Guichou, Jean-François; Faure, Sandrine; Labesse, Gilles; de Santa Barbara, Pascal

    2014-01-01

    In vertebrates, smooth muscle cells (SMCs) can reversibly switch between contractile and proliferative phenotypes. This involves various molecular mechanisms to reactivate developmental signaling pathways and induce cell dedifferentiation. The protein RBPMS2 regulates early development and plasticity of digestive SMCs by inhibiting the bone morphogenetic protein pathway through its interaction with NOGGIN mRNA. RBPMS2 contains only one RNA recognition motif (RRM) while this motif is often repeated in tandem or associated with other functional domains in RRM-containing proteins. Herein, we show using an extensive combination of structure/function analyses that RBPMS2 homodimerizes through a particular sequence motif (D-x-K-x-R-E-L-Y-L-L-F: residues 39–51) located in its RRM domain. We also show that this specific motif is conserved among its homologs and paralogs in vertebrates and in its insect and worm orthologs (CPO and MEC-8, respectively) suggesting a conserved molecular mechanism of action. Inhibition of the dimerization process through targeting a conserved leucine inside of this motif abolishes the capacity of RBPMS2 to interact with the translational elongation eEF2 protein, to upregulate NOGGIN mRNA in vivo and to drive SMC dedifferentiation. Our study demonstrates that RBPMS2 possesses an RRM domain harboring both RNA-binding and protein-binding properties and that the newly identified RRM-homodimerization motif is crucial for the function of RBPMS2 at the cell and tissue levels. PMID:25064856

  6. Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity.

    PubMed

    Sagnol, Sébastien; Yang, Yinshan; Bessin, Yannick; Allemand, Fréderic; Hapkova, Ilona; Notarnicola, Cécile; Guichou, Jean-François; Faure, Sandrine; Labesse, Gilles; de Santa Barbara, Pascal

    2014-09-01

    In vertebrates, smooth muscle cells (SMCs) can reversibly switch between contractile and proliferative phenotypes. This involves various molecular mechanisms to reactivate developmental signaling pathways and induce cell dedifferentiation. The protein RBPMS2 regulates early development and plasticity of digestive SMCs by inhibiting the bone morphogenetic protein pathway through its interaction with NOGGIN mRNA. RBPMS2 contains only one RNA recognition motif (RRM) while this motif is often repeated in tandem or associated with other functional domains in RRM-containing proteins. Herein, we show using an extensive combination of structure/function analyses that RBPMS2 homodimerizes through a particular sequence motif (D-x-K-x-R-E-L-Y-L-L-F: residues 39-51) located in its RRM domain. We also show that this specific motif is conserved among its homologs and paralogs in vertebrates and in its insect and worm orthologs (CPO and MEC-8, respectively) suggesting a conserved molecular mechanism of action. Inhibition of the dimerization process through targeting a conserved leucine inside of this motif abolishes the capacity of RBPMS2 to interact with the translational elongation eEF2 protein, to upregulate NOGGIN mRNA in vivo and to drive SMC dedifferentiation. Our study demonstrates that RBPMS2 possesses an RRM domain harboring both RNA-binding and protein-binding properties and that the newly identified RRM-homodimerization motif is crucial for the function of RBPMS2 at the cell and tissue levels.

  7. A sequence dimorphism in a conserved domain of human 28S rRNA. Uneven distribution of variant genes among individuals. Differential expression in HeLa cells.

    PubMed Central

    Qu, L H; Nicoloso, M; Bachellerie, J P

    1991-01-01

    In humans, cellular 28S rRNA displays a sequence dimorphism within an evolutionarily conserved motif, with the presence, at position +60, of either a A (like the metazoan consensus) or a G. The relative abundance of the two forms of variant genes in the genome exhibit large differences among individuals. The two variant forms are generally represented in cellular 28S rRNA in proportion of their relative abundance in the genome, at least for leucocytes. However, in some cases, one form of variant may be markedly underexpressed as compared to the other. Thus, in HeLa cells, A-form genes contribute to only 1% of the cellular content in mature 28S rRNA although amounting to 15% of the ribosomal genes. The differential expression seems to result from different transcriptional activities rather than from differences in pre-rRNA processing efficiency or in stabilities of mature rRNAs. G-form ribosomal genes were not detected in other mammals, including chimpanzee, which suggests that the fixation of this variant type is a rather recent event in primate evolution. Images PMID:2020541

  8. Temporal motifs in time-dependent networks

    NASA Astrophysics Data System (ADS)

    Kovanen, Lauri; Karsai, Márton; Kaski, Kimmo; Kertész, János; Saramäki, Jari

    2011-11-01

    Temporal networks are commonly used to represent systems where connections between elements are active only for restricted periods of time, such as telecommunication, neural signal processing, biochemical reaction and human social interaction networks. We introduce the framework of temporal motifs to study the mesoscale topological-temporal structure of temporal networks in which the events of nodes do not overlap in time. Temporal motifs are classes of similar event sequences, where the similarity refers not only to topology but also to the temporal order of the events. We provide a mapping from event sequences to coloured directed graphs that enables an efficient algorithm for identifying temporal motifs. We discuss some aspects of temporal motifs, including causality and null models, and present basic statistics of temporal motifs in a large mobile call network.

  9. Detecting DNA regulatory motifs by incorporating positional trendsin information content

    SciTech Connect

    Kechris, Katherina J.; van Zwet, Erik; Bickel, Peter J.; Eisen,Michael B.

    2004-05-04

    On the basis of the observation that conserved positions in transcription factor binding sites are often clustered together, we propose a simple extension to the model-based motif discovery methods. We assign position-specific prior distributions to the frequency parameters of the model, penalizing deviations from a specified conservation profile. Examples with both simulated and real data show that this extension helps discover motifs as the data become noisier or when there is a competing false motif.

  10. Functional implications of local DNA structures in regulatory motifs.

    PubMed

    Xiang, Qian

    2013-01-01

    The three-dimensional structure of DNA has been proposed to be a major determinant for functional transcription factors (TFs) and DNA interaction. Here, we use hydroxyl radical cleavage pattern as a measure of local DNA structure. We compared the conservation between DNA sequence and structure in terms of information content and attempted to assess the functional implications of DNA structures in regulatory motifs. We used statistical methods to evaluate the structural divergence of substituting a single position within a binding site and applied them to a collection of putative regulatory motifs. The following are our major observations: (i) we observed more information in structural alignment than in the corresponding sequence alignment for most of the transcriptional factors; (ii) for each TF, majority of positions have more information in the structural alignment as compared to the sequence alignment; (iii) we further defined a DNA structural divergence score (SD score) for each wild-type and mutant pair that is distinguished by single-base mutation. The SD score for benign mutations is significantly lower than that of switch mutations. This indicates structural conservation is also important for TFBS to be functional and DNA structures will provide previously unappreciated information for TF to realize the binding specificity.

  11. Phylogenetic Inference From Conserved sites Alignments

    SciTech Connect

    grundy, W.N.; Naylor, G.J.P.

    1999-08-15

    Molecular sequences provide a rich source of data for inferring the phylogenetic relationships among species. However, recent work indicates that even an accurate multiple alignment of a large sequence set may yield an incorrect phylogeny and that the quality of the phylogenetic tree improves when the input consists only of the highly conserved, motif regions of the alignment. This work introduces two methods of producing multiple alignments that include only the conserved regions of the initial alignment. The first method retains conserved motifs, whereas the second retains individual conserved sites in the initial alignment. Using parsimony analysis on a mitochondrial data set containing 19 species among which the phylogenetic relationships are widely accepted, both conserved alignment methods produce better phylogenetic trees than the complete alignment. Unlike any of the 19 inference methods used before to analyze this data, both methods produce trees that are completely consistent with the known phylogeny. The motif-based method employs far fewer alignment sites for comparable error rates. For a larger data set containing mitochondrial sequences from 39 species, the site-based method produces a phylogenetic tree that is largely consistent with known phylogenetic relationships and suggests several novel placements.

  12. THE GRK4 SUBFAMILY OF G PROTEIN-COUPLED RECEPTOR KINASES: ALTERNATIVE SPLICING, GENE ORGANIZATION, AND SEQUENCE CONSERVATION

    EPA Science Inventory

    The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.

    Premont RT, Macrae AD, Aparicio SA, Kendall HE, Welch JE, Lefkowitz RJ.

    Department of Medicine, Howard Hughes Medical Institute, Duke Univer...

  13. Global geno-proteomic analysis reveals cross-continental sequence conservation and druggable sites among influenza virus polymerases.

    PubMed

    Babar, Mustafeez Mujtaba; Zaidi, Najam-us-Sahar Sadaf; Tahir, Muhammad

    2014-12-01

    Influenza virus is one of the major causes of mortality and morbidity associated with respiratory diseases. The high rate of mutation in the viral proteome provides it with the ability to survive in a variety of host species. This property helps it in maintaining and developing its pathogenicity, transmission and drug resistance. Alternate drug targets, particularly the internal proteins, can potentially be exploited for addressing the resistance issues. In the current analysis, the degree of conservation of influenza virus polymerases has been studied as one of the essential elements for establishing its candidature as a potential target of antiviral therapy. We analyzed more than 130,000 nucleotide and amino acid sequences by classifying them on the basis of continental presence of host organisms. Computational analyses including genetic polymorphism study, mutation pattern determination, molecular evolution and geophylogenetic analysis were performed to establish the high degree of conservation among the sequences. These studies lead to establishing the polymerases, in particular PB1, as highly conserved proteins. Moreover, we mapped the conservation percentage on the tertiary structures of proteins to identify the conserved, druggable sites. The research study, hence, revealed that the influenza virus polymerases are highly conserved (95-99%) proteins with a very slow mutation rate. Potential drug binding sites on various polymerases have also been reported. A scheme for drug target candidate development that can be employed to rapidly mutating proteins has been presented. Moreover, the research output can help in designing new therapeutic molecules against the identified targets.

  14. Genotyping by sequencing resolves shallow population structure to inform conservation of Chinook salmon (Oncorhynchus tshawytscha)

    PubMed Central

    Larson, Wesley A; Seeb, Lisa W; Everett, Meredith V; Waples, Ryan K; Templin, William D; Seeb, James E

    2014-01-01

    Recent advances in population genomics have made it possible to detect previously unidentified structure, obtain more accurate estimates of demographic parameters, and explore adaptive divergence, potentially revolutionizing the way genetic data are used to manage wild populations. Here, we identified 10 944 single-nucleotide polymorphisms using restriction-site-associated DNA (RAD) sequencing to explore population structure, demography, and adaptive divergence in five populations of Chinook salmon (Oncorhynchus tshawytscha) from western Alaska. Patterns of population structure were similar to those of past studies, but our ability to assign individuals back to their region of origin was greatly improved (>90% accuracy for all populations). We also calculated effective size with and without removing physically linked loci identified from a linkage map, a novel method for nonmodel organisms. Estimates of effective size were generally above 1000 and were biased downward when physically linked loci were not removed. Outlier tests based on genetic differentiation identified 733 loci and three genomic regions under putative selection. These markers and genomic regions are excellent candidates for future research and can be used to create high-resolution panels for genetic monitoring and population assignment. This work demonstrates the utility of genomic data to inform conservation in highly exploited species with shallow population structure. PMID:24665338

  15. Conserved noncoding genomic sequences associated with a flowering-time quantitative trait locus in maize

    PubMed Central

    Salvi, Silvio; Sponza, Giorgio; Morgante, Michele; Tomes, Dwight; Niu, Xiaomu; Fengler, Kevin A.; Meeley, Robert; Ananiev, Evgueni V.; Svitashev, Sergei; Bruggemann, Edward; Li, Bailin; Hainey, Christine F.; Radovic, Slobodanka; Zaina, Giusi; Rafalski, J.-Antoni; Tingey, Scott V.; Miao, Guo-Hua; Phillips, Ronald L.; Tuberosa, Roberto

    2007-01-01

    Flowering time is a fundamental trait of maize adaptation to different agricultural environments. Although a large body of information is available on the map position of quantitative trait loci for flowering time, little is known about the molecular basis of quantitative trait loci. Through positional cloning and association mapping, we resolved the major flowering-time quantitative trait locus, Vegetative to generative transition 1 (Vgt1), to an ≈2-kb noncoding region positioned 70 kb upstream of an Ap2-like transcription factor that we have shown to be involved in flowering-time control. Vgt1 functions as a cis-acting regulatory element as indicated by the correlation of the Vgt1 alleles with the transcript expression levels of the downstream gene. Additionally, within Vgt1, we identified evolutionarily conserved noncoding sequences across the maize–sorghum–rice lineages. Our results support the notion that changes in distant cis-acting regulatory regions are a key component of plant genetic adaptation throughout breeding and evolution. PMID:17595297

  16. Nucleotide sequence of a cluster of early and late genes in a conserved segment of the vaccinia virus genome.

    PubMed Central

    Plucienniczak, A; Schroeder, E; Zettlmeissl, G; Streeck, R E

    1985-01-01

    The nucleotide sequence of a 7.6 kb vaccinia DNA segment from a genomic region conserved among different orthopox virus has been determined. This segment contains a tight cluster of 12 partly overlapping open reading frames most of which can be correlated with previously identified early and late proteins and mRNAs. Regulatory signals used by vaccinia virus have been studied. Presumptive promoter regions are rich in A, T and carry the consensus sequences TATA and AATAA spaced at 20-24 base pairs. Tandem repeats of a CTATTC consensus sequence are proposed to be involved in the termination of early transcription. PMID:2987815

  17. RSAT peak-motifs: motif analysis in full-size ChIP-seq datasets.

    PubMed

    Thomas-Chollier, Morgane; Herrmann, Carl; Defrance, Matthieu; Sand, Olivier; Thieffry, Denis; van Helden, Jacques

    2012-02-01

    ChIP-seq is increasingly used to characterize transcription factor binding and chromatin marks at a genomic scale. Various tools are now available to extract binding motifs from peak data sets. However, most approaches are only available as command-line programs, or via a website but with size restrictions. We present peak-motifs, a computational pipeline that discovers motifs in peak sequences, compares them with databases, exports putative binding sites for visualization in the UCSC genome browser and generates an extensive report suited for both naive and expert users. It relies on time- and memory-efficient algorithms enabling the treatment of several thousand peaks within minutes. Regarding time efficiency, peak-motifs outperforms all comparable tools by several orders of magnitude. We demonstrate its accuracy by analyzing data sets ranging from 4000 to 1,28,000 peaks for 12 embryonic stem cell-specific transcription factors. In all cases, the program finds the expected motifs and returns additional motifs potentially bound by cofactors. We further apply peak-motifs to discover tissue-specific motifs in peak collections for the p300 transcriptional co-activator. To our knowledge, peak-motifs is the only tool that performs a complete motif analysis and offers a user-friendly web interface without any restriction on sequence size or number of peaks.

  18. The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs.

    PubMed

    Pedersen, Kim Brint; Chhabra, Kavaljit H; Nguyen, Van K; Xia, Huijing; Lazartigues, Eric

    2013-11-01

    Pancreatic angiotensin-converting enzyme 2 (ACE2) has previously been shown to be critical for maintaining glycemia and β-cell function. Efforts to maintain or increase ACE2 expression in pancreatic β-cells might therefore have therapeutic potential for treating diabetes. In our study, we investigated the transcriptional role of hepatocyte nuclear factor 1α (HNF1α) and hepatocyte nuclear factor 1β (HNF1β) in induction of ACE2 expression in insulin-secreting cells. A deficient allele of HNF1α or HNF1β causes maturity-onset diabetes of the young (MODY) types 3 and 5, respectively, in humans. We found that ACE2 is primarily transcribed from the proximal part of the ACE2 promoter in the pancreas. In the proximal part of the human ACE2 promoter, we further identified three functional HNF1 binding sites, as they have binding affinity for HNF1α and HNF1β and are required for induction of promoter activity by HNF1β in insulinoma cells. These three sites are well-conserved among mammalian species. Both HNF1α and HNF1β induce expression of ACE2 mRNA and lead to elevated levels of ACE2 protein and ACE2 enzymatic activity in insulinoma cells. Furthermore, HNF1α dose-dependently increases ACE2 expression in primary pancreatic islet cells. We conclude that HNF1α can induce the expression of ACE2 in pancreatic islet cells via evolutionarily conserved HNF1 binding sites in the ACE2 promoter. Potential therapeutics aimed at counteracting functional HNF1α depletion in diabetes and MODY3 will thus have ACE2 induction in pancreatic islets as a likely beneficial effect.

  19. A highly conserved interaction involving the middle residue of the SXN active-site motif is crucial for function of class B penicillin-binding proteins: mutational and computational analysis of PBP 2 from N. gonorrhoeae.

    PubMed

    Tomberg, Joshua; Temple, Brenda; Fedarovich, Alena; Davies, Christopher; Nicholas, Robert A

    2012-04-03

    Insertion of an aspartate residue at position 345a in penicillin-binding protein 2 (PBP 2), which lowers the rate of penicillin acylation by ~6-fold, is commonly observed in penicillin-resistant strains of Neisseria gonorrhoeae. Here, we show that insertions of other amino acids also lower the penicillin acylation rate of PBP 2, but none supported growth of N. gonorrhoeae, indicating loss of essential transpeptidase activity. The Asp345a mutation likely acts by altering the interaction between its adjacent residue, Asp346, in the β2a-β2d hairpin loop and Ser363, the middle residue of the SXN active site motif. Because the adjacent aspartate creates ambiguity in the position of the insertion, we also examined if insertions at position 346a could confer decreased susceptibility to penicillin. However, only aspartate insertions were identified, indicating that only an Asp-Asp couple can confer resistance and retain transpeptidase function. The importance of the Asp346-Ser363 interaction was assessed by mutation of each residue to Ala. Although both mutants lowered the acylation rate of penicillin G by 5-fold, neither could support growth of N. gonorrhoeae, again indicating loss of transpeptidase function. Interaction between a residue in the equivalent of the β2a-β2d hairpin loop and the middle residue of the SXN motif is observed in crystal structures of other Class B PBPs, and its importance is also supported by multisequence alignments. Overall, these results suggest that this conserved interaction can be manipulated (e.g., by insertion) to lower the acylation rate by β-lactam antibiotics and increase resistance, but only if essential transpeptidase activity is preserved.

  20. QColors: an algorithm for conservative viral quasispecies reconstruction from short and non-contiguous next generation sequencing reads.

    PubMed

    Huang, Austin; Kantor, Rami; DeLong, Allison; Schreier, Leeann; Istrail, Sorin

    Next generation sequencing technologies have recently been applied to characterize mutational spectra of the heterogeneous population of viral genotypes (known as a quasispecies) within HIV-infected patients. Such information is clinically relevant because minority genetic subpopulations of HIV within patients enable viral escape from selection pressures such as the immune response and antiretroviral therapy. However, methods for quasispecies sequence reconstruction from next generation sequencing reads are not yet widely used and remains an emerging area of research. Furthermore, the majority of research methodology in HIV has focused on 454 sequencing, while many next-generation sequencing platforms used in practice are limited to shorter read lengths relative to 454 sequencing. Little work has been done in determining how best to address the read length limitations of other platforms. The approach described here incorporates graph representations of both read differences and read overlap to conservatively determine the regions of the sequence with sufficient variability to separate quasispecies sequences. Within these tractable regions of quasispecies inference, we use constraint programming to solve for an optimal quasispecies subsequence determination via vertex coloring of the conflict graph, a representation which also lends itself to data with non-contiguous reads such as paired-end sequencing. We demonstrate the utility of the method by applying it to simulations based on actual intra-patient clonal HIV-1 sequencing data.

  1. Designing synthetic RNAs to determine the relevance of structural motifs in picornavirus IRES elements

    NASA Astrophysics Data System (ADS)

    Fernandez-Chamorro, Javier; Lozano, Gloria; Garcia-Martin, Juan Antonio; Ramajo, Jorge; Dotu, Ivan; Clote, Peter; Martinez-Salas, Encarnacion

    2016-04-01

    The function of Internal Ribosome Entry Site (IRES) elements is intimately linked to their RNA structure. Viral IRES elements are organized in modular domains consisting of one or more stem-loops that harbor conserved RNA motifs critical for internal initiation of translation. A conserved motif is the pyrimidine-tract located upstream of the functional initiation codon in type I and II picornavirus IRES. By computationally designing synthetic RNAs to fold into a structure that sequesters the polypyrimidine tract in a hairpin, we establish a correlation between predicted inaccessibility of the pyrimidine tract and IRES activity, as determined in both in vitro and in vivo systems. Our data supports the hypothesis that structural sequestration of the pyrimidine-tract within a stable hairpin inactivates IRES activity, since the stronger the stability of the hairpin the higher the inhibition of protein synthesis. Destabilization of the stem-loop immediately upstream of the pyrimidine-tract also decreases IRES activity. Our work introduces a hybrid computational/experimental method to determine the importance of structural motifs for biological function. Specifically, we show the feasibility of using the software RNAiFold to design synthetic RNAs with particular sequence and structural motifs that permit subsequent experimental determination of the importance of such motifs for biological function.

  2. Deletion of conserved sequences in IG-DMR at Dlk1-Gtl2 locus suggests their involvement in expression of paternally expressed genes in mice

    PubMed Central

    SAITO, Takeshi; HARA, Satoshi; TAMANO, Moe; ASAHARA, Hiroshi; TAKADA, Shuji

    2016-01-01

    Expression regulation of the Dlk1-Dio3 imprinted domain by the intergenic differentially methylated region (IG-DMR) is essential for normal embryonic development in mammals. In this study, we investigated conserved IG-DMR genomic sequences in eutherians to elucidate their role in genomic imprinting of the Dlk1-Dio3 domain. Using a comparative genomics approach, we identified three highly conserved sequences in IG-DMR. To elucidate the functions of these sequences in vivo, we generated mutant mice lacking each of the identified highly conserved sequences using the CRISPR/Cas9 system. Although mutant mice did not exhibit the gross phenotype, deletions of the conserved sequences altered the expression levels of paternally expressed imprinted genes in the mutant embryos without skewing imprinting status. These results suggest that the conserved sequences in IG-DMR are involved in the expression regulation of some of the imprinted genes in the Dlk1-Dio3 domain. PMID:27904015

  3. Deletion of conserved sequences in IG-DMR at Dlk1-Gtl2 locus suggests their involvement in expression of paternally expressed genes in mice.

    PubMed

    Saito, Takeshi; Hara, Satoshi; Tamano, Moe; Asahara, Hiroshi; Takada, Shuji

    2017-02-16

    Expression regulation of the Dlk1-Dio3 imprinted domain by the intergenic differentially methylated region (IG-DMR) is essential for normal embryonic development in mammals. In this study, we investigated conserved IG-DMR genomic sequences in eutherians to elucidate their role in genomic imprinting of the Dlk1-Dio3 domain. Using a comparative genomics approach, we identified three highly conserved sequences in IG-DMR. To elucidate the functions of these sequences in vivo, we generated mutant mice lacking each of the identified highly conserved sequences using the CRISPR/Cas9 system. Although mutant mice did not exhibit the gross phenotype, deletions of the conserved sequences altered the expression levels of paternally expressed imprinted genes in the mutant embryos without skewing imprinting status. These results suggest that the conserved sequences in IG-DMR are involved in the expression regulation of some of the imprinted genes in the Dlk1-Dio3 domain.

  4. The 'helix clamp' in HIV-1 reverse transcriptase: a new nucleic acid binding motif common in nucleic acid polymerases.

    PubMed Central

    Hermann, T; Meier, T; Götte, M; Heumann, H

    1994-01-01

    Amino acid sequences homologous to 259KLVGKL (X)16KLLR284 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) are conserved in several nucleotide polymerizing enzymes. This amino acid motif has been identified in the crystal structure model as an element of the enzyme's nucleic acid binding apparatus. It is part of the helix-turn-helix structure, alpha H-turn-alpha I, within the 'thumb' region of HIV-1 RT. The motif grasps the complexed nucleic acid at one side. Molecular modeling studies on HIV-1 RT in complex with a nucleic acid fragment suggest that the motif has binding function in the p66 subunit as well as in the p51 subunit, acting as a kind of 'helix clamp'. Given its wide distribution within the nucleic acid polymerases, the helix clamp motif is assumed to be a structure of general significance for nucleic acid binding. Images PMID:7527138

  5. Complete nucleotide sequence of the Actinomyces viscosus T14V sialidase gene: presence of a conserved repeating sequence among strains of Actinomyces spp.

    PubMed Central

    Yeung, M K

    1993-01-01

    The nucleotide sequence of the Actinomyces viscosus T14V sialidase gene (nanH) and flanking regions was determined. An open reading frame of 2,703 nucleotides that encodes a predominately hydrophobic protein of 901 amino acids (M(r), 92,871) was identified. The amino acid sequence at the amino terminus of the predicted protein exhibited properties characteristic of a typical leader peptide. Five 12-amino-acid units that shared between 33 and 67% sequence identity were noted within the central domain of the protein. Each unit contained the sequence Ser-X-Asp-X-Gly-X-Thr-Trp, which is conserved among other bacterial and trypanosoma sp. sialidases. Thus, the A. viscosus T14V nanH gene and the other prokaryotic and eukaryotic sialidase genes evolved from a common ancestor. Southern hybridization analyses under conditions of high stringency revealed the existence of DNA sequences homologous to A. viscosus T14V nanH in the genomes of 18 strains of five Actinomyces species that expressed various levels of sialidase activity. The data demonstrate that the sialidase genes from divergent groups of Actinomyces spp. are highly conserved. Images PMID:8418033

  6. Two distinct nuclear factors bind the conserved regulatory sequences of a rabbit major histocompatibility complex class II gene.

    PubMed Central

    Sittisombut, N

    1988-01-01

    The constitutive coexpression of the major histocompatibility complex (MHC) class II genes in B lymphocytes requires positive, trans-acting transcriptional factors. The need for these trans-acting factors has been suggested by the reversion of the MHC class II-negative phenotype of rare B-lymphocyte mutants through somatic cell fusion with B cells or T-cell lines. The mechanism by which the trans-acting factors exert their effect on gene transcription is unknown. The possibility that two highly conserved DNA sequences, located 90 to 100 base pairs (bp) (the A sequence) and 60 to 70 bp (the B sequence) upstream of the transcription start site of the class II genes, are recognized by the trans-acting factors was investigated in this study. By using the gel electrophoresis retardation assay, a minimum of two proteins which specifically bound the conserved A or B sequence of a rabbit DP beta gene were identified in murine nuclear extracts of a B-lymphoma cell line, A20-2J. Fractionation of nuclear extract through a heparin-agarose column allowed the identification of one protein, designated NF-MHCIIB, which bound an oligonucleotide containing the B sequence and protected the entire B sequence in the DNase I protection analysis. Another protein, designated NF-MHCIIA, which bound an oligonucleotide containing the A sequence and partially protected the 3' half of this sequence, was also identified. NF-MHCIIB did not protect a CCAAT sequence located 17 bp downstream of the B sequence. The possible relationship between these DNA-binding factors and the trans-acting factors identified in the cell fusion experiments is discussed. Images PMID:3133552

  7. Conserved Patterns of Microbial Immune Escape: Pathogenic Microbes of Diverse Origin Target the Human Terminal Complement Inhibitor Vitronectin via a Single Common Motif

    PubMed Central

    Kraiczy, Peter; Hammerschmidt, Sven; Skerka, Christine; Zipfel, Peter F.; Riesbeck, Kristian

    2016-01-01

    Pathogenicity of many microbes relies on their capacity to resist innate immunity, and to survive and persist in an immunocompetent human host microbes have developed highly efficient and sophisticated complement evasion strategies. Here we show that different human pathogens including Gram-negative and Gram-positive bacteria, as well as the fungal pathogen Candida albicans, acquire the human terminal complement regulator vitronectin to their surface. By using truncated vitronectin fragments we found that all analyzed microbial pathogens (n = 13) bound human vitronectin via the same C-terminal heparin-binding domain (amino acids 352–374). This specific interaction leaves the terminal complement complex (TCC) regulatory region of vitronectin accessible, allowing inhibition of C5b-7 membrane insertion and C9 polymerization. Vitronectin complexed with the various microbes and corresponding proteins was thus functionally active and inhibited complement-mediated C5b-9 deposition. Taken together, diverse microbial pathogens expressing different structurally unrelated vitronectin-binding molecules interact with host vitronectin via the same conserved region to allow versatile control of the host innate immune response. PMID:26808444

  8. Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase

    PubMed Central

    Ranjani, Velayudhan; Janeček, Štefan; Chai, Kian Piaw; Shahir, Shafinaz; Rahman, Raja Noor Zaliha Raja Abdul; Chan, Kok-Gan; Goh, Kian Mau

    2014-01-01

    The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 of 51 h and 53 h, respectively. Among the mutants, only the A161D had a specific activity, kcat and kcat/Km higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA. PMID:25069018

  9. Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles.

    PubMed

    Wang, Jianbin; Czech, Benjamin; Crunk, Amanda; Wallace, Adam; Mitreva, Makedonka; Hannon, Gregory J; Davis, Richard E

    2011-09-01

    Eukaryotic cells express several classes of small RNAs that regulate gene expression and ensure genome maintenance. Endogenous siRNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) mainly control gene and transposon expression in the germline, while microRNAs (miRNAs) generally function in post-transcriptional gene silencing in both somatic and germline cells. To provide an evolutionary and developmental perspective on small RNA pathways in nematodes, we identified and characterized known and novel small RNA classes through gametogenesis and embryo development in the parasitic nematode Ascaris suum and compared them with known small RNAs of Caenorhabditis elegans. piRNAs, Piwi-clade Argonautes, and other proteins associated with the piRNA pathway have been lost in Ascaris. miRNAs are synthesized immediately after fertilization in utero, before pronuclear fusion, and before the first cleavage of the zygote. This is the earliest expression of small RNAs ever described at a developmental stage long thought to be transcriptionally quiescent. A comparison of the two classes of Ascaris endo-siRNAs, 22G-RNAs and 26G-RNAs, to those in C. elegans, suggests great diversification and plasticity in the use of small RNA pathways during spermatogenesis in different nematodes. Our data reveal conserved characteristics of nematode small RNAs as well as features unique to Ascaris that illustrate significant flexibility in the use of small RNAs pathways, some of which are likely an adaptation to Ascaris' life cycle and parasitism. The transcriptome assembly has been submitted to NCBI Transcriptome Shotgun Assembly Sequence Database(http://www.ncbi.nlm.nih.gov/genbank/TSA.html) under accession numbers JI163767–JI182837 and JI210738–JI257410.

  10. tRNase Z Catalysis and Conserved Residues on the Carboxy Side of the His Cluster

    PubMed Central

    Karkashon, Shay; Hopkinson, Angela; Levinger, Louis

    2008-01-01

    tRNAs are transcribed as precursors and processed in a series of required reactions leading to aminoacylation and translation. The 3′ end trailer can be removed by the pre-tRNA processing endonuclease tRNase Z, an ancient, conserved member of the β-lactamase superfamily of metal-dependent hydrolases. The signature sequence of this family, the His domain (HxHxDH, Motif II), and histidines in Motifs III and V and aspartate in Motif IV contribute seven side chains for coordination of two divalent metal ions. We previously investigated the effects on catalysis of substitutions in Motif II, and in the PxKxRN loop and Motif I on the amino side of Motif II. Herein we present the effects of substitutions on the carboxy side of Motif II within Motifs III, IV, the HEAT and HST loops and Motif V. Substitution of the Motif IV aspartate reduces catalytic efficiency more than 10,000-fold. Histidines in Motif III, V and the HST loop are also functionally important. Strikingly, replacement of Glu in the HEAT loop with Ala reducesefficiency by ~1000-fold. Proximity and orientation of this Glu side chain relative to His in the HST loop and the importance of both residues for catalysis suggest that they function as a duo in a proton transfer at the final stage of reaction, characteristic of the tRNase Z class of RNA endonucleases. PMID:17655328

  11. Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure.

    PubMed Central

    Thayer, M M; Ahern, H; Xing, D; Cunningham, R P; Tainer, J A

    1995-01-01

    The 1.85 A crystal structure of endonuclease III, combined with mutational analysis, suggests the structural basis for the DNA binding and catalytic activity of the enzyme. Helix-hairpin-helix (HhH) and [4Fe-4S] cluster loop (FCL) motifs, which we have named for their secondary structure, bracket the cleft separating the two alpha-helical domains of the enzyme. These two novel DNA binding motifs and the solvent-filled pocket in the cleft between them all lie within a positively charged and sequence-conserved surface region. Lys120 and Asp138, both shown by mutagenesis to be catalytically important, lie at the mouth of this pocket, suggesting that this pocket is part of the active site. The positions of the HhH motif and protruding FCL motif, which contains the DNA binding residue Lys191, can accommodate B-form DNA, with a flipped-out base bound within the active site pocket. The identification of HhH and FCL sequence patterns in other DNA binding proteins suggests that these motifs may be a recurrent structural theme for DNA binding proteins. Images PMID:7664751

  12. The VQ Motif-Containing Protein Family of Plant-Specific Transcriptional Regulators1

    PubMed Central

    Jing, Yanjun; Lin, Rongcheng

    2015-01-01

    The VQ motif-containing proteins (designated as VQ proteins) are a class of plant-specific proteins with a conserved and single short FxxhVQxhTG amino acid sequence motif. VQ proteins regulate diverse developmental processes, including responses to biotic and abiotic stresses, seed development, and photomorphogenesis. In this Update, we summarize and discuss recent advances in our understanding of the regulation and function of VQ proteins and the role of the VQ motif in mediating transcriptional regulation and protein-protein interactions in signaling pathways. Based on the accumulated evidence, we propose a general mechanism of action for the VQ protein family, which likely defines a novel class of transcriptional regulators specific to plants. PMID:26220951

  13. Experimental Support for the Evolution of Symmetric Protein Architecture from a Simple Peptide Motif

    SciTech Connect

    J Lee; M Blaber

    2011-12-31

    The majority of protein architectures exhibit elements of structural symmetry, and 'gene duplication and fusion' is the evolutionary mechanism generally hypothesized to be responsible for their emergence from simple peptide motifs. Despite the central importance of the gene duplication and fusion hypothesis, experimental support for a plausible evolutionary pathway for a specific protein architecture has yet to be effectively demonstrated. To address this question, a unique 'top-down symmetric deconstruction' strategy was utilized to successfully identify a simple peptide motif capable of recapitulating, via gene duplication and fusion processes, a symmetric protein architecture (the threefold symmetric {beta}-trefoil fold). The folding properties of intermediary forms in this deconstruction agree precisely with a previously proposed 'conserved architecture' model for symmetric protein evolution. Furthermore, a route through foldable sequence-space between the simple peptide motif and extant protein fold is demonstrated. These results provide compelling experimental support for a plausible evolutionary pathway of symmetric protein architecture via gene duplication and fusion processes.

  14. 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

  15. Two overlapping sequence motifs within the polyomavirus enhancer are independently the targets of stimulation by both the tumor promoter 12-O-tetradecanoylphorbol-3-acetate and the Ha-ras oncogene

    SciTech Connect

    Yamaguchi, Yyko; Satake, Masanobu; Ito, Yoshiaki

    1989-03-01

    A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), strongly stimulates the activity of polyomavirus enhancer in a human erythroleukemia cell line, K562. The target of stimulation was the previously defined A element (from nucleotides 5107 to 5130) of the enhancer. The authors found that within the A element, two partly overlapping sequence motifs (one from nucleotides 5107 to 5117, the other from nucleotides 5113 to 5121) were independently the targets of TPA stimulation. The former is homologous to the enhancer core sequence of the adenovirus type 5 E1A gene, and the latter shares the consensus AP-1-binding site. In addition, transiently expressed Ha-ras oncogene also stimulated these two subelements in K562 cells, as they reported for NIH 3T3 cells previously.

  16. Sampling Motif-Constrained Ensembles of Networks

    NASA Astrophysics Data System (ADS)

    Fischer, Rico; Leitão, Jorge C.; Peixoto, Tiago P.; Altmann, Eduardo G.

    2015-10-01

    The statistical significance of network properties is conditioned on null models which satisfy specified properties but that are otherwise random. Exponential random graph models are a principled theoretical framework to generate such constrained ensembles, but which often fail in practice, either due to model inconsistency or due to the impossibility to sample networks from them. These problems affect the important case of networks with prescribed clustering coefficient or number of small connected subgraphs (motifs). In this Letter we use the Wang-Landau method to obtain a multicanonical sampling that overcomes both these problems. We sample, in polynomial time, networks with arbitrary degree sequences from ensembles with imposed motifs counts. Applying this method to social networks, we investigate the relation between transitivity and homophily, and we quantify the correlation between different types of motifs, finding that single motifs can explain up to 60% of the variation of motif profiles.

  17. A Novel Kleefstra Syndrome-associated Variant That Affects the Conserved TPLX Motif within the Ankyrin Repeat of EHMT1 Leads to Abnormal Protein Folding*

    PubMed Central

    Blackburn, Patrick R.; Tischer, Alexander; Zimmermann, Michael T.; Kemppainen, Jennifer L.; Sastry, Sujatha; Knight Johnson, Amy E.; Cousin, Margot A.; Boczek, Nicole J.; Oliver, Gavin; Misra, Vinod K.; Gavrilova, Ralitza H.; Lomberk, Gwen; Auton, Matthew; Urrutia, Raul; Klee, Eric W.

    2017-01-01

    Kleefstra syndrome (KS) (Mendelian Inheritance in Man (MIM) no. 610253), also known as 9q34 deletion syndrome, is an autosomal dominant disorder caused by haploinsufficiency of euchromatic histone methyltransferase-1 (EHMT1). The clinical phenotype of KS includes moderate to severe intellectual disability with absent speech, hypotonia, brachycephaly, congenital heart defects, and dysmorphic facial features with hypertelorism, synophrys, macroglossia, protruding tongue, and prognathism. Only a few cases of de novo missense mutations in EHMT1 giving rise to KS have been described. However, some EHMT1 variants have been described in individuals presenting with autism spectrum disorder or mild intellectual disability, suggesting that the phenotypic spectrum resulting from EHMT1 alterations may be quite broad. In this report, we describe two unrelated patients with complex medical histories consistent with KS in whom next generation sequencing identified the same novel c.2426C>T (p.P809L) missense variant in EHMT1. To examine the functional significance of this novel variant, we performed molecular dynamics simulations of the wild type and p.P809L variant, which predicted that the latter would have a propensity to misfold, leading to abnormal histone mark binding. Recombinant EHMT1 p.P809L was also studied using far UV circular dichroism spectroscopy and intrinsic protein fluorescence. These functional studies confirmed the model-based hypotheses and provided evidence for protein misfolding and aberrant target recognition as the underlying pathogenic mechanism for this novel KS-associated variant. This is the first report to suggest that missense variants in EHMT1 that lead to protein misfolding and disrupted histone mark binding can lead to KS. PMID:28057753

  18. The Drosophila Juvenile Hormone Receptor Candidates Methoprene-tolerant (MET) and Germ Cell-expressed (GCE) Utilize a Conserved LIXXL Motif to Bind the FTZ-F1 Nuclear Receptor*

    PubMed Central

    Bernardo, Travis J.; Dubrovsky, Edward B.

    2012-01-01

    Juvenile hormone (JH) has been implicated in many developmental processes in holometabolous insects, but its mechanism of signaling remains controversial. We previously found that in Drosophila Schneider 2 cells, the nuclear receptor FTZ-F1 is required for activation of the E75A gene by JH. Here, we utilized insect two-hybrid assays to show that FTZ-F1 interacts with two JH receptor candidates, the bHLH-PAS paralogs MET and GCE, in a JH-dependent manner. These interactions are severely reduced when helix 12 of the FTZ-F1 activation function 2 (AF2) is removed, implicating AF2 as an interacting site. Through homology modeling, we found that MET and GCE possess a C-terminal α-helix featuring a conserved motif LIXXL that represents a novel nuclear receptor (NR) box. Docking simulations supported by two-hybrid experiments revealed that FTZ-F1·MET and FTZ-F1·GCE heterodimer formation involves a typical NR box-AF2 interaction but does not require the canonical charge clamp residues of FTZ-F1 and relies primarily on hydrophobic contacts, including a unique interaction with helix 4. Moreover, we identified paralog-specific features, including a secondary interaction site found only in MET. Our findings suggest that a novel NR box enables MET and GCE to interact JH-dependently with the AF2 of FTZ-F1. PMID:22249180

  19. Fitting a mixture model by expectation maximization to discover motifs in biopolymers

    SciTech Connect

    Bailey, T.L.; Elkan, C.

    1994-12-31

    The algorithm described in this paper discovers one or more motifs in a collection of DNA or protein sequences by using the technique of expectation maximization to fit a two-component finite mixture model to the set of sequences. Multiple motifs are found by fitting a mixture model to the data, probabilistically erasing the occurrences of the motif thus found, and repeating the process to find successive motifs. The algorithm requires only a set of unaligned sequences and a number specifying the width of the motifs as input. It returns a model of each motif and a threshold which together can be used as a Bayes-optimal classifier for searching for occurrences of the motif in other databases. The algorithm estimates how many times each motif occurs in each sequence in the dataset and outputs an alignment of the occurrences of the motif. The algorithm is capable of discovering several different motifs with differing numbers of occurrences in a single dataset.

  20. Dominant sequences of human major histocompatibility complex conserved extended haplotypes from HLA-DQA2 to DAXX.

    PubMed

    Larsen, Charles E; Alford, Dennis R; Trautwein, Michael R; Jalloh, Yanoh K; Tarnacki, Jennifer L; Kunnenkeri, Sushruta K; Fici, Dolores A; Yunis, Edmond J; Awdeh, Zuheir L; Alper, Chester A

    2014-10-01

    We resequenced and phased 27 kb of DNA within 580 kb of the MHC class II region in 158 population chromosomes, most of which were conserved extended haplotypes (CEHs) of European descent or contained their centromeric fragments. We determined the single nucleotide polymorphism and deletion-insertion polymorphism alleles of the dominant sequences from HLA-DQA2 to DAXX for these CEHs. Nine of 13 CEHs remained sufficiently intact to possess a dominant sequence extending at least to DAXX, 230 kb centromeric to HLA-DPB1. We identified the regions centromeric to HLA-DQB1 within which single instances of eight "common" European MHC haplotypes previously sequenced by the MHC Haplotype Project (MHP) were representative of those dominant CEH sequences. Only two MHP haplotypes had a dominant CEH sequence throughout the centromeric and extended class II region and one MHP haplotype did not represent a known European CEH anywhere in the region. We identified the centromeric recombination transition points of other MHP sequences from CEH representation to non-representation. Several CEH pairs or groups shared sequence identity in small blocks but had significantly different (although still conserved for each separate CEH) sequences in surrounding regions. These patterns partly explain strong calculated linkage disequilibrium over only short (tens to hundreds of kilobases) distances in the context of a finite number of observed megabase-length CEHs comprising half a population's haplotypes. Our results provide a clearer picture of European CEH class II allelic structure and population haplotype architecture, improved regional CEH markers, and raise questions concerning regional recombination hotspots.

  1. EAR motif-mediated transcriptional repression in plants: an underlying mechanism for epigenetic regulation of gene expression.

    PubMed

    Kagale, Sateesh; Rozwadowski, Kevin

    2011-02-01

    Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) motif-mediated transcriptional repression is emerging as one of the principal mechanisms of plant gene regulation. The EAR motif, defined by the consensus sequence patterns of either LxLxL or DLNxxP, is the most predominant form of transcriptional repression motif so far identified in plants. Additionally, this active repression motif is highly conserved in transcriptional regulators known to function as negative regulators in a broad range of developmental and physiological processes across evolutionarily diverse plant species. Recent discoveries of co-repressors interacting with EAR motifs, such as TOPLESS (TPL) and AtSAP18, have begun to unravel the mechanisms of EAR motif-mediated repression. The demonstration of genetic interaction between mutants of TPL and AtHDA19, co-complex formation between TPL-related 1 (TPR1) and AtHDA19, as well as direct physical interaction between AtSAP18 and AtHDA19 support a model where EAR repressors, via recruitment of chromatin remodeling factors, facilitate epigenetic regulation of gene expression. Here, we discuss the biological significance of EAR-mediated gene regulation in the broader context of plant biology and present literature evidence in support of a model for EAR motif-mediated repression via the recruitment and action of chromatin modifiers. Additionally, we discuss the possible influences of phosphorylation and ubiquitination on the function and turnover of EAR repressors.

  2. High-throughput genomic sequencing of cassava bacterial blight strains identifies conserved effectors to target for durable resistance.

    PubMed

    Bart, Rebecca; Cohn, Megan; Kassen, Andrew; McCallum, Emily J; Shybut, Mikel; Petriello, Annalise; Krasileva, Ksenia; Dahlbeck, Douglas; Medina, Cesar; Alicai, Titus; Kumar, Lava; Moreira, Leandro M; Rodrigues Neto, Júlio; Verdier, Valerie; Santana, María Angélica; Kositcharoenkul, Nuttima; Vanderschuren, Hervé; Gruissem, Wilhelm; Bernal, Adriana; Staskawicz, Brian J

    2012-07-10

    Cassava bacterial blight (CBB), incited by Xanthomonas axonopodis pv. manihotis (Xam), is the most important bacterial disease of cassava, a staple food source for millions of people in developing countries. Here we present a widely applicable strategy for elucidating the virulence components of a pathogen population. We report Illumina-based draft genomes for 65 Xam strains and deduce the phylogenetic relatedness of Xam across the areas where cassava is grown. Using an extensive database of effector proteins from animal and plant pathogens, we identify the effector repertoire for each sequenced strain and use a comparative sequence analysis to deduce the least polymorphic of the conserved effectors. These highly conserved effectors have been maintained over 11 countries, three continents, and 70 y of evolution and as such represent ideal targets for developing resistance strategies.

  3. Identification of conserved genomic regions and variation therein amongst Cetartiodactyla species using next generation sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Next Generation Sequencing has created an opportunity to genetically characterize an individual both inexpensively and comprehensively. In earlier work produced in our collaboration [1], it was demonstrated that, for animals without a reference genome, their Next Generation Sequence data ...

  4. A survey of motif finding Web tools for detecting binding site motifs in ChIP-Seq data

    PubMed Central

    2014-01-01

    Abstract ChIP-Seq (chromatin immunoprecipitation sequencing) has provided the advantage for finding motifs as ChIP-Seq experiments narrow down the motif finding to binding site locations. Recent motif finding tools facilitate the motif detection by providing user-friendly Web interface. In this work, we reviewed nine motif finding Web tools that are capable for detecting binding site motifs in ChIP-Seq data. We showed each motif finding Web tool has its own advantages for detecting motifs that other tools may not discover. We recommended the users to use multiple motif finding Web tools that implement different algorithms for obtaining significant motifs, overlapping resemble motifs, and non-overlapping motifs. Finally, we provided our suggestions for future development of motif finding Web tool that better assists researchers for finding motifs in ChIP-Seq data. Reviewers This article was reviewed by Prof. Sandor Pongor, Dr. Yuriy Gusev, and Dr. Shyam Prabhakar (nominated by Prof. Limsoon Wong). PMID:24555784

  5. Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing.

    PubMed

    Jain, Mukesh; Chevala, V V S Narayana; Garg, Rohini

    2014-11-01

    MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes.

  6. VARUN: discovering extensible motifs under saturation constraints.

    PubMed

    Apostolico, Alberto; Comin, Matteo; Parida, Laxmi

    2010-01-01

    The discovery of motifs in biosequences is frequently torn between the rigidity of the model on one hand and the abundance of candidates on the other hand. In particular, motifs that include wild cards or "don't cares" escalate exponentially with their number, and this gets only worse if a don't care is allowed to stretch up to some prescribed maximum length. In this paper, a notion of extensible motif in a sequence is introduced and studied, which tightly combines the structure of the motif pattern, as described by its syntactic specification, with the statistical measure of its occurrence count. It is shown that a combination of appropriate saturation conditions and the monotonicity of probabilistic scores over regions of constant frequency afford us significant parsimony in the generation and testing of candidate overrepresented motifs. A suite of software programs called Varun is described, implementing the discovery of extensible motifs of the type considered. The merits of the method are then documented by results obtained in a variety of experiments primarily targeting protein sequence families. Of equal importance seems the fact that the sets of all surprising motifs returned in each experiment are extracted faster and come in much more manageable sizes than would be obtained in the absence of saturation constraints.

  7. Primary structure of the merozoite surface antigen 1 of Plasmodium vivax reveals sequences conserved between different Plasmodium species.

    PubMed Central

    del Portillo, H A; Longacre, S; Khouri, E; David, P H

    1991-01-01

    Merozoite surface antigen 1 (MSA1) of several species of plasmodia has been shown to be a promising candidate for a vaccine directed against the asexual blood stages of malaria. We report the cloning and characterization of the MSA1 gene of the human malaria parasite Plasmodium vivax. This gene, which we call Pv200, encodes a polypeptide of 1726 amino acids and displays features described for MSA1 genes of other species, such as signal peptide and anchoring sequences, conserved cysteine residues, number of potential N-glycosylation sites, and repeats consisting here of 23 glutamine residues in a row. When the nucleotide and deduced amino acid sequences of the MSA1 of P. vivax are compared to those of another human malaria parasite, Plasmodium falciparum, and to those of the rodent parasite Plasmodium yoelii, 10 regions of high amino acid similarity are observed despite the very different dG + dC contents of the corresponding genes. All of the interspecies conserved regions reside within the conserved or semiconserved blocks delimited by the sequences of different alleles of the MSA1 gene of P. falciparum. Images PMID:2023952

  8. Conserved sequence-specific lincRNA-steroid receptor interactions drive transcriptional repression and direct cell fate

    SciTech Connect

    Hudson, William H.; Pickard, Mark R.; de Vera, Ian Mitchelle S.; Kuiper, Emily G.; Mourtada-Maarabouni, Mirna; Conn, Graeme L.; Kojetin, Douglas J.; Williams, Gwyn T.; Ortlund, Eric A.

    2014-12-23

    The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5 lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.

  9. Identification and characterization of novel and conserved microRNAs in radish (Raphanus sativus L.) using high-throughput sequencing.

    PubMed

    Xu, Liang; Wang, Yan; Xu, Yuanyuan; Wang, Liangju; Zhai, Lulu; Zhu, Xianwen; Gong, Yiqin; Ye, Shan; Liu, Liwang

    2013-03-01

    MicroRNAs (miRNAs) are endogenous, non-coding, small RNAs that play significant regulatory roles in plant growth, development, and biotic and abiotic stress responses. To date, a great number of conserved and species-specific miRNAs have been identified in many important plant species such as Arabidopsis, rice and poplar. However, little is known about identification of miRNAs and their target genes in radish (Raphanus sativus L.). In the present study, a small RNA library from radish root was constructed and sequenced using the high-throughput Solexa sequencing. Through sequence alignment and secondary structure prediction, a total of 545 conserved miRNA families as well as 15 novel (with their miRNA* strand) and 64 potentially novel miRNAs were identified. Quantitative real-time PCR (qRT-PCR) analysis confirmed that both conserved and novel miRNAs were expressed in radish, and some of them were preferentially expressed in certain tissues. A total of 196 potential target genes were predicted for 42 novel radish miRNAs. Gene ontology (GO) analysis showed that most of the targets were involved in plant growth, development, metabolism and stress responses. This study represents a first large-scale identification and characterization of radish miRNAs and their potential target genes. These results could lead to the further identification of radish miRNAs and enhance our understanding of radish miRNA regulatory mechanisms in diverse biological and metabolic processes.

  10. Regulation of ABCC6 trafficking and stability by a conserved C-terminal PDZ-like sequence.

    PubMed

    Xue, Peng; Crum, Chelsea M; Thibodeau, Patrick H

    2014-01-01

    Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE). The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers. Premature arteriosclerosis, loss of skin and vascular tone, and a progressive loss of vision result from this ectopic mineralization. However, the identity of the circulatory factor and the specific role of ABCC6 in disease pathophysiology are not known. Though recessive loss-of-function alleles are associated with alterations in ABCC6 expression and function, the molecular pathologies associated with the majority of PXE-causing mutations are also not known. Sequence analysis of orthologous ABCC6 proteins indicates the C-terminal sequences are highly conserved and share high similarity to the PDZ sequences found in other ABCC subfamily members. Genetic testing of PXE patients suggests that at least one disease-causing mutation is located in a PDZ-like sequence at the extreme C-terminus of the ABCC6 protein. To evaluate the role of this C-terminal sequence in the biosynthesis and trafficking of ABCC6, a series of mutations were utilized to probe changes in ABCC6 biosynthesis, membrane stability and turnover. Removal of this PDZ-like sequence resulted in decreased steady-state ABCC6 levels, decreased cell surface expression and stability, and mislocalization of the ABCC6 protein in polarized cells. These data suggest that the conserved, PDZ-like sequence promotes the proper biosynthesis and trafficking of the ABCC6 protein.

  11. An update on cell surface proteins containing extensin-motifs.

    PubMed

    Borassi, Cecilia; Sede, Ana R; Mecchia, Martin A; Salgado Salter, Juan D; Marzol, Eliana; Muschietti, Jorge P; Estevez, Jose M

    2016-01-01

    In recent years it has become clear that there are several molecular links that interconnect the plant cell surface continuum, which is highly important in many biological processes such as plant growth, development, and interaction with the environment. The plant cell surface continuum can be defined as the space that contains and interlinks the cell wall, plasma membrane and cytoskeleton compartments. In this review, we provide an updated view of cell surface proteins that include modular domains with an extensin (EXT)-motif followed by a cytoplasmic kinase-like domain, known as PERKs (for proline-rich extensin-like receptor kinases); with an EXT-motif and an actin binding domain, known as formins; and with extracellular hybrid-EXTs. We focus our attention on the EXT-motifs with the short sequence Ser-Pro(3-5), which is found in several different protein contexts within the same extracellular space, highlighting a putative conserved structural and functional role. A closer understanding of the dynamic regulation of plant cell surface continuum and its relationship with the downstream signalling cascade is a crucial forthcoming challenge.

  12. The Thiamin Pyrophosphate-Motif

    NASA Technical Reports Server (NTRS)

    Dominiak, Paulina M.; Ciszak, Ewa M.

    2003-01-01

    Using databases the authors have identified a common thiamin pyrophosphate (TPP)-motif in the family of functionally diverse TPP-dependent enzymes. This common motif consists of multimeric organization of subunits, two catalytic centers, common amino acid sequence, and specific contacts to provide a flip-flop, or alternate site, mechanism of action. Each catalytic center [PP:PYR] is formed at the interface of the PP-domain binding the magnesium ion, pyrophosphate and aminopyrimidine ring of TPP, and the PYR-domain binding the aminopyrimidine ring of that cofactor. A pair of these catalytic centers constitutes the catalytic core [PP:PYR]* within these enzymes. Analysis of the structural elements of this catalytic core reveals novel definition of the common amino acid sequences, which are GX@&(G)@XXGQ, and GDGX25-30 within the PP- domain, and the E&(G)@XXG@ within the PYR-domain, where Q, corresponds to a hydrophobic amino acid. This TPP-motif provides a novel tool for annotation of TPP-dependent enzymes useful in advancing functional proteomics.

  13. Characterization of an Unusually Conserved Alui Highly Reiterated DNA Sequence Family from the Honeybee, Apis Mellifera

    PubMed Central

    Tares, S.; Cornuet, J. M.; Abad, P.

    1993-01-01

    An AluI family of highly reiterated nontranscribed sequences has been found in the genome of the honeybee Apis mellifera. This repeated sequence is shown to be present at approximately 23,000 copies per haploid genome constituting about 2% of the total genomic DNA. The nucleotide sequence of 10 monomers was determined. The consensus sequence is 176 nucleotides long and has an A + T content of 58%. There are clusters of both direct and inverted repeats. Internal subrepeating units ranging from 11 to 17 nucleotides are observed, suggesting that it could have evolved from a shorter sequence. DNA sequence data reveal that this repeat class is unusually homogeneous compared to the other class of invertebrate highly reiterated DNA sequences. The average pairwise sequence divergence between the repeats is 2.5%. In spite of this unusual homogeneity, divergence has been found in the repeated sequence hybridization ladder between four different honeybee subspecies. Therefore, the AluI highly reiterated sequences provide a new probe for fingerprinting in A. m. mellifera. PMID:8104160

  14. Septal localization by membrane targeting sequences and a conserved sequence essential for activity at the COOH-terminus of Bacillus subtilis cardiolipin synthase.

    PubMed

    Kusaka, Jin; Shuto, Satoshi; Imai, Yukiko; Ishikawa, Kazuki; Saito, Tomo; Natori, Kohei; Matsuoka, Satoshi; Hara, Hiroshi; Matsumoto, Kouji

    2016-04-01

    The acidic phospholipid cardiolipin (CL) is localized on polar and septal membranes and plays an important physiological role in Bacillus subtilis cells. ClsA, the enzyme responsible for CL synthesis, is also localized on septal membranes. We found that GFP fusion proteins of the enzyme with NH2-terminal and internal deletions retained septal localization. However, derivatives with deletions starting from the COOH-terminus (Leu482) ceased to localize to the septum once the deletion passed the Ile residue at 448, indicating that the sequence responsible for septal localization is confined within a short distance from the COOH-terminus. Two sequences, Ile436-Leu450 and Leu466-Leu478, are predicted to individually form an amphipathic α-helix. This configuration is known as a membrane targeting sequence (MTS) and we therefore refer to them as MTS2 and MTS1, respectively. Either one has the ability to affect septal localization, and each of these sequences by itself localizes to the septum. Membrane association of the constructs of this enzyme containing the MTSs was verified by subcellular fractionation of the cells. CL synthesis, in contrast, was abolished after deleting just the last residue, Leu482, in the COOH-terminal four amino acid residue sequence, Ser-Pro-Ile-Leu, which is highly conserved among bacterial CL synthases.

  15. The complete mitochondrial genome sequence of the liverwort Pleurozia purpurea reveals extremely conservative mitochondrial genome evolution in liverworts.

    PubMed

    Wang, Bin; Xue, Jiayu; Li, Libo; Liu, Yang; Qiu, Yin-Long

    2009-12-01

    Plant mitochondrial genomes have been known to be highly unusual in their large sizes, frequent intra-genomic rearrangement, and generally conservative sequence evolution. Recent studies show that in early land plants the mitochondrial genomes exhibit a mixed mode of conservative yet dynamic evolution. Here, we report the completely sequenced mitochondrial genome from the liverwort Pleurozia purpurea. The circular genome has a size of 168,526 base pairs, containing 43 protein-coding genes, 3 rRNA genes, 25 tRNA genes, and 31 group I or II introns. It differs from the Marchantia polymorpha mitochondrial genome, the only other liverwort chondriome that has been sequenced, in lacking two genes (trnRucg and trnTggu) and one intron (rrn18i1065gII). The two genomes have identical gene orders and highly similar sequences in exons, introns, and intergenic spacers. Finally, a comparative analysis of duplicated trnRucu and other trnR genes from the two liverworts and several other organisms identified the recent lateral origin of trnRucg in Marchantia mtDNA through modification of a duplicated trnRucu. This study shows that the mitochondrial genomes evolve extremely slowly in liverworts, the earliest-diverging lineage of extant land plants, in stark contrast to what is known of highly dynamic evolution of mitochondrial genomes in seed plants.

  16. Crystallization and Preliminary X-ray Diffraction Analysis of motif N from Saccharomyces cerevisiae Dbf4

    SciTech Connect

    Matthews, L.; Duong, A; Prasad, A; Duncker, B; Guarne, A

    2009-01-01

    The Cdc7-Dbf4 complex plays an instrumental role in the initiation of DNA replication and is a target of replication-checkpoint responses in Saccharomyces cerevisiae. Cdc7 is a conserved serine/threonine kinase whose activity depends on association with its regulatory subunit, Dbf4. A conserved sequence near the N-terminus of Dbf4 (motif N) is necessary for the interaction of Cdc7-Dbf4 with the checkpoint kinase Rad53. To understand the role of the Cdc7-Dbf4 complex in checkpoint responses, a fragment of Saccharomyces cerevisiae Dbf4 encompassing motif N was isolated, overproduced and crystallized. A complete native data set was collected at 100 K from crystals that diffracted X-rays to 2.75 {angstrom} resolution and structure determination is currently under way.

  17. Structural Relationships in the Lysozyme Superfamily: Significant Evidence for Glycoside Hydrolase Signature Motifs

    PubMed Central

    Wohlkönig, Alexandre; Huet, Joëlle; Looze, Yvan; Wintjens, René

    2010-01-01

    Background Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily. Results Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46) share in common two structural elements: the central helix of the all-α domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a β-hairpin pointed towards the substrate binding cleft. The invariant β-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism. Conclusions The seven families and subfamilies of the lysozyme superfamily all have in common a β-hairpin structure which displays a family-specific sequence motif. These GH β-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily. PMID:21085702

  18. Allele drop-out in the MECP2 gene due to G-quadruplex and i-motif sequences when using polymerase chain reaction-based diagnosis for Rett syndrome.

    PubMed

    Saunders, Carol J; Friez, Michael J; Patterson, Melanie; Nzabi, Masha; Zhao, Weiwei; Bi, Chengpeng

    2010-04-01

    Although few examples are formally documented, all polymerase chain reaction-based testing is theoretically vulnerable to allele drop-out (ADO), the failure to amplify one of the two alleles present in a cell. In a clinical setting, this can lead to false positive or negative diagnosis. We investigated the mechanisms leading to ADO in the MECP2 gene in two unrelated female patients undergoing testing for Rett syndrome. Both the patients had two benign DNA variations, c.819G > T and c.1161C > T, that appeared homozygous due to ADO. Bioinformatics analyses indicate that this region of the MECP2 gene is rich in complex tertiary structures called G-quadruplex and i-motifs, the disruption of which by the c.819G > T and c.1161C > T variants leads to preferential amplification of the variant allele. Other examples of ADO likely occur, and consideration of disrupting G-quadruplex and i-motif structures should be given when this phenomenon is unexpected. We identify factors in both the polymerase chain reaction amplification and the sequencing steps that help overcome ADO.

  19. The mouse B cell-specific mb-1 gene encodes an immunoreceptor tyrosine-based activation motif (ITAM) protein that may be evolutionarily conserved in diverse species by purifying selection.

    PubMed

    Sims, Richard; Vandergon, Virginia Oberholzer; Malone, Cindy S

    2012-03-01

    The B-lymphocyte accessory molecule Ig-alpha (Ig-α) is encoded by the mouse B cell-specific gene (mb-1), and along with the Ig-beta (Ig-β) molecule and a membrane bound immunoglobulin (mIg) makes up the B-cell receptor (BCR). Ig-α and Ig-β form a heterodimer structure that upon antigen binding and receptor clusterin