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Sample records for highly conserved sequences

  1. 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. PMID:11676424

  2. The tryptophan repressor sequence is highly conserved among the Enterobacteriaceae.

    PubMed Central

    Arvidson, D N; Arvidson, C G; Lawson, C L; Miner, J; Adams, C; Youderian, P

    1994-01-01

    Tryptophan biosynthesis in Escherichia coli is regulated by the product of the trpR gene, the tryptophan (Trp) repressor. Trp aporepressor binds the corepressor, L-tryptophan, to form a holorepressor complex, which binds trp operator DNA tightly, and inhibits transcription of the tryptophan biosynthetic operon. The conservation of trp operator sequences among enteric Gram-negative bacteria suggests that trpR genes from other bacterial species can be cloned by complementation in E. coli. To clone trpR homologues, a deletion of the E. coli trpR gene, delta trpR504, was made on a plasmid by site-directed mutagenesis, then crossed onto the E. coli genome. Plasmid clones of the trpR genes of Enterobacter aerogenes and Enterobacter cloacae were isolated by complementation of the delta trpR504 allele, scored as the ability to repress beta-galactosidase synthesis from a prophage-borne trpE-lacZ gene fusion. The predicted amino acid sequences of four enteric TrpR proteins show differences, clustered on the backside of the folded repressor, opposite the DNA-binding helix-turn-helix substructures. These differences are predicted to have little effect on the interactions of the aporepressor with tryptophan, holorepressor with operator DNA, or tandemly bound holorepressor dimers with one another. Although there is some variation observed at the dimer interface, interactions predicted to stabilize the interface are conserved. The phylogenetic relationships revealed by the TrpR amino acid sequence alignment agree with the results of others. PMID:8208606

  3. 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. PMID:17072079

  4. Genomes of sequence type 121 Listeria monocytogenes strains harbor highly conserved plasmids and prophages

    PubMed Central

    Schmitz-Esser, Stephan; Müller, Anneliese; Stessl, Beatrix; Wagner, Martin

    2015-01-01

    The food-borne pathogen Listeria (L.) monocytogenes is often found in food production environments. Thus, controlling the occurrence of L. monocytogenes in food production is a great challenge for food safety. Among a great diversity of L. monocytogenes strains from food production, particularly strains belonging to sequence type (ST)121 are prevalent. The molecular reasons for the abundance of ST121 strains are however currently unknown. We therefore determined the genome sequences of three L. monocytogenes ST121 strains: 6179 and 4423, which persisted for up to 8 years in food production plants in Ireland and Austria, and of the strain 3253 and compared them with available L. monocytogenes ST121 genomes. Our results show that the ST121 genomes are highly similar to each other and show a tremendously high degree of conservation among some of their prophages and particularly among their plasmids. This remarkably high level of conservation among prophages and plasmids suggests that strong selective pressure is acting on them. We thus hypothesize that plasmids and prophages are providing important adaptations for survival in food production environments. In addition, the ST121 genomes share common adaptations which might be related to their persistence in food production environments such as the presence of Tn6188, a transposon responsible for increased tolerance against quaternary ammonium compounds, a yet undescribed insertion harboring recombination hotspot (RHS) repeat proteins, which are most likely involved in competition against other bacteria, and presence of homologs of the L. innocua genes lin0464 and lin0465. PMID:25972859

  5. 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.

  6. 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. PMID:22699502

  7. High Sequence Conservation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase under Drug Pressure despite the Continuous Appearance of Mutations

    PubMed Central

    Ceccherini-Silberstein, Francesca; Gago, Federico; Santoro, Maria; Gori, Caterina; Svicher, Valentina; Rodríguez-Barrios, Fátima; d'Arrigo, Roberta; Ciccozzi, Massimo; Bertoli, Ada; Monforte, Antonella d'Arminio; Balzarini, Jan; Antinori, Andrea; Perno, Carlo-Federico

    2005-01-01

    To define the extent of sequence conservation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in vivo, the first 320 amino acids of RT obtained from 2,236 plasma-derived samples from a well-defined cohort of 1,704 HIV-1-infected individuals (457 drug naïve and 1,247 drug treated) were analyzed and examined in structural terms. In naïve patients, 233 out of these 320 residues (73%) were conserved (<1% variability). The majority of invariant amino acids clustered into defined regions comprising between 5 and 29 consecutive residues. Of the nine longest invariant regions identified, some contained residues and domains critical for enzyme stability and function. In patients treated with RT inhibitors, despite profound drug pressure and the appearance of mutations primarily associated with resistance, 202 amino acids (63%) remained highly conserved and appeared mostly distributed in regions of variable length. This finding suggests that participation of consecutive residues in structural domains is strictly required for cooperative functions and sustainability of HIV-1 RT activity. Besides confirming the conservation of amino acids that are already known to be important for catalytic activity, stability of the heterodimer interface, and/or primer/template binding, the other 62 new invariable residues are now identified and mapped onto the three-dimensional structure of the enzyme. This new knowledge could be of help in the structure-based design of novel resistance-evading drugs. PMID:16051864

  8. Highly conserved influenza A virus epitope sequences as candidates of H3N2 flu vaccine targets.

    PubMed

    Wu, Ko-Wen; Chien, Chih-Yi; Li, Shiao-Wen; King, Chwan-Chuen; Chang, Chuan-Hsiung

    2012-08-01

    This study focused on identifying the conserved epitopes in a single subtype A (H3N2)-as candidates for vaccine targets. We identified a total of 32 conserved epitopes in four viral proteins [22 HA, 4PB1, 3 NA, 3 NP]. Evaluation of conserved epitopes in coverage during 1968-2010 revealed that (1) 12 HA conserved epitopes were highly present in the circulating viruses; (2) the remaining 10 HA conserved epitopes appeared with lower percentage but a significantly increasing trend after 1989 [p<0.001]; and (3) the conserved epitopes in NA, NP and PB1 are also highly frequent in wild-type viruses. These conserved epitopes also covered an extremely high percentage of the 16 vaccine strains during the 42 year period. The identification of highly conserved epitopes using our approach can also be applied to develop broad-spectrum vaccines. PMID:22698979

  9. Comparative Mitogenomics of the Genus Odontobutis (Perciformes: Gobioidei: Odontobutidae) Revealed Conserved Gene Rearrangement and High Sequence Variations

    PubMed Central

    Ma, Zhihong; Yang, Xuefen; Bercsenyi, Miklos; Wu, Junjie; Yu, Yongyao; Wei, Kaijian; Fan, Qixue; Yang, Ruibin

    2015-01-01

    To understand the molecular evolution of mitochondrial genomes (mitogenomes) in the genus Odontobutis, the mitogenome of Odontobutis yaluensis was sequenced and compared with those of another four Odontobutis species. Our results displayed similar mitogenome features among species in genome organization, base composition, codon usage, and gene rearrangement. The identical gene rearrangement of trnS-trnL-trnH tRNA cluster observed in mitogenomes of these five closely related freshwater sleepers suggests that this unique gene order is conserved within Odontobutis. Additionally, the present gene order and the positions of associated intergenic spacers of these Odontobutis mitogenomes indicate that this unusual gene rearrangement results from tandem duplication and random loss of large-scale gene regions. Moreover, these mitogenomes exhibit a high level of sequence variation, mainly due to the differences of corresponding intergenic sequences in gene rearrangement regions and the heterogeneity of tandem repeats in the control regions. Phylogenetic analyses support Odontobutis species with shared gene rearrangement forming a monophyletic group, and the interspecific phylogenetic relationships are associated with structural differences among their mitogenomes. The present study contributes to understanding the evolutionary patterns of Odontobutidae species. PMID:26492246

  10. 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

  11. Identification of conserved hepatic transcriptomic responses to 17β-estradiol using high-throughput sequencing in brown trout

    PubMed Central

    Uren Webster, Tamsyn M.; Shears, Janice A.; Moore, Karen

    2015-01-01

    Estrogenic chemicals are major contaminants of surface waters and can threaten the sustainability of natural fish populations. Characterization of the global molecular mechanisms of toxicity of environmental contaminants has been conducted primarily in model species rather than species with limited existing transcriptomic or genomic sequence information. We aimed to investigate the global mechanisms of toxicity of an endocrine disrupting chemical of environmental concern [17β-estradiol (E2)] using high-throughput RNA sequencing (RNA-Seq) in an environmentally relevant species, brown trout (Salmo trutta). We exposed mature males to measured concentrations of 1.94, 18.06, and 34.38 ng E2/l for 4 days and sequenced three individual liver samples per treatment using an Illumina HiSeq 2500 platform. Exposure to 34.4 ng E2/L resulted in 2,113 differentially regulated transcripts (FDR < 0.05). Functional analysis revealed upregulation of processes associated with vitellogenesis, including lipid metabolism, cellular proliferation, and ribosome biogenesis, together with a downregulation of carbohydrate metabolism. Using real-time quantitative PCR, we validated the expression of eight target genes and identified significant differences in the regulation of several known estrogen-responsive transcripts in fish exposed to the lower treatment concentrations (including esr1 and zp2.5). We successfully used RNA-Seq to identify highly conserved responses to estrogen and also identified some estrogen-responsive transcripts that have been less well characterized, including nots and tgm2l. These results demonstrate the potential application of RNA-Seq as a valuable tool for assessing mechanistic effects of pollutants in ecologically relevant species for which little genomic information is available. PMID:26082144

  12. Identification of conserved hepatic transcriptomic responses to 17β-estradiol using high-throughput sequencing in brown trout.

    PubMed

    Uren Webster, Tamsyn M; Shears, Janice A; Moore, Karen; Santos, Eduarda M

    2015-09-01

    Estrogenic chemicals are major contaminants of surface waters and can threaten the sustainability of natural fish populations. Characterization of the global molecular mechanisms of toxicity of environmental contaminants has been conducted primarily in model species rather than species with limited existing transcriptomic or genomic sequence information. We aimed to investigate the global mechanisms of toxicity of an endocrine disrupting chemical of environmental concern [17β-estradiol (E2)] using high-throughput RNA sequencing (RNA-Seq) in an environmentally relevant species, brown trout (Salmo trutta). We exposed mature males to measured concentrations of 1.94, 18.06, and 34.38 ng E2/l for 4 days and sequenced three individual liver samples per treatment using an Illumina HiSeq 2500 platform. Exposure to 34.4 ng E2/L resulted in 2,113 differentially regulated transcripts (FDR < 0.05). Functional analysis revealed upregulation of processes associated with vitellogenesis, including lipid metabolism, cellular proliferation, and ribosome biogenesis, together with a downregulation of carbohydrate metabolism. Using real-time quantitative PCR, we validated the expression of eight target genes and identified significant differences in the regulation of several known estrogen-responsive transcripts in fish exposed to the lower treatment concentrations (including esr1 and zp2.5). We successfully used RNA-Seq to identify highly conserved responses to estrogen and also identified some estrogen-responsive transcripts that have been less well characterized, including nots and tgm2l. These results demonstrate the potential application of RNA-Seq as a valuable tool for assessing mechanistic effects of pollutants in ecologically relevant species for which little genomic information is available. PMID:26082144

  13. The highly conserved codon following the slippery sequence supports -1 frameshift efficiency at the HIV-1 frameshift site.

    PubMed

    Mathew, Suneeth F; Crowe-McAuliffe, Caillan; Graves, Ryan; Cardno, Tony S; McKinney, Cushla; Poole, Elizabeth S; Tate, Warren P

    2015-01-01

    HIV-1 utilises -1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating -1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the 'intercodon') contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules-eRF1 protein or a cognate suppressor tRNA-were able to access and decode the intercodon prior to -1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted) ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1. PMID:25807539

  14. Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants.

    PubMed Central

    Shcherban, T Y; Shi, J; Durachko, D M; Guiltinan, M J; McQueen-Mason, S J; Shieh, M; Cosgrove, D J

    1995-01-01

    Expansins are unusual proteins discovered by virtue of their ability to mediate cell wall extension in plants. We identified cDNA clones for two cucumber expansins on the basis of peptide sequences of proteins purified from cucumber hypocotyls. The expansin cDNAs encode related proteins with signal peptides predicted to direct protein secretion to the cell wall. Northern blot analysis showed moderate transcript abundance in the growing region of the hypocotyl and no detectable transcripts in the nongrowing region. Rice and Arabidopsis expansin cDNAs were identified from collections of anonymous cDNAs (expressed sequence tags). Sequence comparisons indicate at least four distinct expansin cDNAs in rice and at least six in Arabidopsis. Expansins are highly conserved in size and sequence (60-87% amino acid sequence identity and 75-95% similarity between any pairwise comparison), and phylogenetic trees indicate that this multigene family formed before the evolutionary divergence of monocotyledons and dicotyledons. Sequence and motif analyses show no similarities to known functional domains that might account for expansin action on wall extension. A series of highly conserved tryptophans may function in expansin binding to cellulose or other glycans. The high conservation of this multigene family indicates that the mechanism by which expansins promote wall extensin tolerates little variation in protein structure. Images Fig. 2 PMID:7568110

  15. High-Throughput Sequencing Reveals Diverse Sets of Conserved, Nonconserved, and Species-Specific miRNAs in Jute

    PubMed Central

    Islam, Md. Tariqul; Ferdous, Ahlan Sabah; Najnin, Rifat Ara; Sarker, Suprovath Kumar; Khan, Haseena

    2015-01-01

    MicroRNAs play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. A group of plant microRNAs are evolutionarily conserved; however, others are expressed in a species-specific manner. Jute is an agroeconomically important fibre crop; nonetheless, no practical information is available for microRNAs in jute to date. In this study, Illumina sequencing revealed a total of 227 known microRNAs and 17 potential novel microRNA candidates in jute, of which 164 belong to 23 conserved families and the remaining 63 belong to 58 nonconserved families. Among a total of 81 identified microRNA families, 116 potential target genes were predicted for 39 families and 11 targets were predicted for 4 among the 17 identified novel microRNAs. For understanding better the functions of microRNAs, target genes were analyzed by Gene Ontology and their pathways illustrated by KEGG pathway analyses. The presence of microRNAs identified in jute was validated by stem-loop RT-PCR followed by end point PCR and qPCR for randomly selected 20 known and novel microRNAs. This study exhaustively identifies microRNAs and their target genes in jute which will ultimately pave the way for understanding their role in this crop and other crops. PMID:25861616

  16. High-Throughput Sequencing Reveals Diverse Sets of Conserved, Nonconserved, and Species-Specific miRNAs in Jute.

    PubMed

    Islam, Md Tariqul; Ferdous, Ahlan Sabah; Najnin, Rifat Ara; Sarker, Suprovath Kumar; Khan, Haseena

    2015-01-01

    MicroRNAs play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. A group of plant microRNAs are evolutionarily conserved; however, others are expressed in a species-specific manner. Jute is an agroeconomically important fibre crop; nonetheless, no practical information is available for microRNAs in jute to date. In this study, Illumina sequencing revealed a total of 227 known microRNAs and 17 potential novel microRNA candidates in jute, of which 164 belong to 23 conserved families and the remaining 63 belong to 58 nonconserved families. Among a total of 81 identified microRNA families, 116 potential target genes were predicted for 39 families and 11 targets were predicted for 4 among the 17 identified novel microRNAs. For understanding better the functions of microRNAs, target genes were analyzed by Gene Ontology and their pathways illustrated by KEGG pathway analyses. The presence of microRNAs identified in jute was validated by stem-loop RT-PCR followed by end point PCR and qPCR for randomly selected 20 known and novel microRNAs. This study exhaustively identifies microRNAs and their target genes in jute which will ultimately pave the way for understanding their role in this crop and other crops. PMID:25861616

  17. Amplification of human papillomavirus DNA sequences by using conserved primers.

    PubMed Central

    Gregoire, L; Arella, M; Campione-Piccardo, J; Lancaster, W D

    1989-01-01

    The polymerase chain reaction has potential for use in the detection of small amounts of human papillomavirus (HPV) viral nucleic acids present in clinical specimens. However, new HPV types for which no probes exist would remain undetected by using type-specific primers for the polymerase chain reaction before hybridization. Primers corresponding to highly conserved HPV sequences may be useful for detecting low amounts of known HPV DNA as well as new HPV types. Here we analyze a pair of primers derived from conserved sequences within the E1 open reading frame for HPV sequence amplification by using the polymerase chain reaction. The longest perfect homology among HPV sequences is a 12-mer within the first exon of E1M. A region of conserved amino acids coded by the E1 open reading frame allowed the detection of another highly conserved region about 850 base pairs downstream. Two 21-mers derived from these conserved regions were used to amplify sequences from all HPV DNAs used as templates. The amplified DNA was shown to be specific for HPV sequences within the E1 open reading frame. DNA from HPVs whose sequences were not available were amplified by using these two primers. HPV DNA sequences in clinical specimens could also be amplified with the primers. Images PMID:2556429

  18. Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells.

    PubMed

    Kwon, Y K; Hecht, N B

    1991-05-01

    The expression of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated translationally during male germ-cell development. The 3' untranslated region (UTR) of protamine 1 mRNA has been reported to control its time of translation. To understand the mechanisms controlling translation of the protamine mRNAs, we have sought to identify cis elements of the 3' UTR of protamine 2 mRNA that are recognized by cytoplasmic factors. From gel retardation assays, two sequence elements are shown to form specific RNA-protein complexes. Protein binding sites of the two complexes were determined by RNase T1 mapping, by blocking the putative binding sites with antisense oligonucleotides, and by competition assays. The sequences of these elements, located between nucleotides + 537 and + 572 in protamine 2 mRNA, are highly conserved among postmeiotic translationally regulated nuclear proteins of the mammalian testis. Two closely linked protein binding sites were detected. UV-crosslinking studies revealed that a protein of about 18 kDa binds to one of the conserved sequences. These data demonstrate specific protein binding to a highly conserved 3' UTR of translationally regulated testicular mRNA. PMID:2023906

  19. 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.

  20. Conserved noncoding sequences (CNSs) in higher plants.

    PubMed

    Freeling, Michael; Subramaniam, Shabarinath

    2009-04-01

    Plant conserved noncoding sequences (CNSs)--a specific category of phylogenetic footprint--have been shown experimentally to function. No plant CNS is conserved to the extent that ultraconserved noncoding sequences are conserved in vertebrates. Plant CNSs are enriched in known transcription factor or other cis-acting binding sites, and are usually clustered around genes. Genes that encode transcription factors and/or those that respond to stimuli are particularly CNS-rich. Only rarely could this function involve small RNA binding. Some transcribed CNSs encode short translation products as a form of negative control. Approximately 4% of Arabidopsis gene content is estimated to be both CNS-rich and occupies a relatively long stretch of chromosome: Bigfoot genes (long phylogenetic footprints). We discuss a 'DNA-templated protein assembly' idea that might help explain Bigfoot gene CNSs. PMID:19249238

  1. Characterization of the Role of a Highly Conserved Sequence in ATP Binding Cassette Transporter G (ABCG) Family in ABCG1 Stability, Oligomerization, and Trafficking

    PubMed Central

    2013-01-01

    ATP-binding cassette transporter G1 (ABCG1) mediates cholesterol and oxysterol efflux onto lipidated lipoproteins and plays an important role in macrophage reverse cholesterol transport. Here, we identified a highly conserved sequence present in the five ABCG transporter family members. The conserved sequence is located between the nucleotide binding domain and the transmembrane domain and contains five amino acid residues from Asn at position 316 to Phe at position 320 in ABCG1 (NPADF). We found that cells expressing mutant ABCG1, in which Asn316, Pro317, Asp319, and Phe320 in the conserved sequence were replaced with Ala simultaneously, showed impaired cholesterol efflux activity compared with wild type ABCG1-expressing cells. A more detailed mutagenesis study revealed that mutation of Asn316 or Phe 320 to Ala significantly reduced cellular cholesterol and 7-ketocholesterol efflux conferred by ABCG1, whereas replacement of Pro317 or Asp319 with Ala had no detectable effect. To confirm the important role of Asn316 and Phe320, we mutated Asn316 to Asp (N316D) and Gln (N316Q), and Phe320 to Ile (F320I) and Tyr (F320Y). The mutant F320Y showed the same phenotype as wild type ABCG1. However, the efflux of cholesterol and 7-ketocholesterol was reduced in cells expressing ABCG1 mutant N316D, N316Q, or F320I compared with wild type ABCG1. Further, mutations N316Q and F320I impaired ABCG1 trafficking while having no marked effect on the stability and oligomerization of ABCG1. The mutant N316Q and F320I could not be transported to the cell surface efficiently. Instead, the mutant proteins were mainly localized intracellularly. Thus, these findings indicate that the two highly conserved amino acid residues, Asn and Phe, play an important role in ABCG1-dependent export of cellular cholesterol, mainly through the regulation of ABCG1 trafficking. PMID:24320932

  2. A highly conserved DNA replication module from Streptococcus thermophilus phages is similar in sequence and topology to a module from Lactococcus lactis phages.

    PubMed

    Desiere, F; Lucchini, S; Bruttin, A; Zwahlen, M C; Brüssow, H

    1997-08-01

    A highly conserved DNA region extending over 5 kb was observed in Streptococcus thermophilus bacteriophages. Comparative sequencing of one temperate and 26 virulent phages demonstrated in the most extreme case an 18% aa difference for a predicted protein, while the majority of the phages showed fewer, if any aa changes. The relative degree of aa conservation was not homogeneous over the DNA segment investigated. Sequence analysis of the conserved segment revealed genes possibly involved in DNA transactions. Three predicted proteins (orf 233, 443, and 382 gene product (gp)) showed nucleoside triphosphate binding motifs. Orf 443 gp showed in addition a DEAH box motif, characteristically found in a subgroup of helicases, and a variant zinc finger motif known from a phage T7 helicase/primase. Tree analysis classified orf 443 gp as a distant member of the helicase superfamily. Orf 382 gp showed similarity to putative plasmid DNA primases. Downstream of orf 382 a noncoding repeat region was identified that showed similarity to a putative minus origin from a cryptic S. thermophilus plasmid. Four predicted proteins showed not only high degrees of aa identity (34 to 63%) with proteins from Lactococcus lactis phages, but their genes showed a similar topological organization. We interpret this as evidence for a horizontal gene transfer event between phages of the two bacterial genera in the distant past. PMID:9268169

  3. A highly conserved G-rich consensus sequence in hepatitis C virus core gene represents a new anti–hepatitis C target

    PubMed Central

    Wang, Shao-Ru; Min, Yuan-Qin; Wang, Jia-Qi; Liu, Chao-Xing; Fu, Bo-Shi; Wu, Fan; Wu, Ling-Yu; Qiao, Zhi-Xian; Song, Yan-Yan; Xu, Guo-Hua; Wu, Zhi-Guo; Huang, Gai; Peng, Nan-Fang; Huang, Rong; Mao, Wu-Xiang; Peng, Shuang; Chen, Yu-Qi; Zhu, Ying; Tian, Tian; Zhang, Xiao-Lian; Zhou, Xiang

    2016-01-01

    G-quadruplex (G4) is one of the most important secondary structures in nucleic acids. Until recently, G4 RNAs have not been reported in any ribovirus, such as the hepatitis C virus. Our bioinformatics analysis reveals highly conserved guanine-rich consensus sequences within the core gene of hepatitis C despite the high genetic variability of this ribovirus; we further show using various methods that such consensus sequences can fold into unimolecular G4 RNA structures, both in vitro and under physiological conditions. Furthermore, we provide direct evidences that small molecules specifically targeting G4 can stabilize this structure to reduce RNA replication and inhibit protein translation of intracellular hepatitis C. Ultimately, the stabilization of G4 RNA in the genome of hepatitis C represents a promising new strategy for anti–hepatitis C drug development. PMID:27051880

  4. A highly conserved G-rich consensus sequence in hepatitis C virus core gene represents a new anti-hepatitis C target.

    PubMed

    Wang, Shao-Ru; Min, Yuan-Qin; Wang, Jia-Qi; Liu, Chao-Xing; Fu, Bo-Shi; Wu, Fan; Wu, Ling-Yu; Qiao, Zhi-Xian; Song, Yan-Yan; Xu, Guo-Hua; Wu, Zhi-Guo; Huang, Gai; Peng, Nan-Fang; Huang, Rong; Mao, Wu-Xiang; Peng, Shuang; Chen, Yu-Qi; Zhu, Ying; Tian, Tian; Zhang, Xiao-Lian; Zhou, Xiang

    2016-04-01

    G-quadruplex (G4) is one of the most important secondary structures in nucleic acids. Until recently, G4 RNAs have not been reported in any ribovirus, such as the hepatitis C virus. Our bioinformatics analysis reveals highly conserved guanine-rich consensus sequences within the core gene of hepatitis C despite the high genetic variability of this ribovirus; we further show using various methods that such consensus sequences can fold into unimolecular G4 RNA structures, both in vitro and under physiological conditions. Furthermore, we provide direct evidences that small molecules specifically targeting G4 can stabilize this structure to reduce RNA replication and inhibit protein translation of intracellular hepatitis C. Ultimately, the stabilization of G4 RNA in the genome of hepatitis C represents a promising new strategy for anti-hepatitis C drug development. PMID:27051880

  5. Sequence conservation of an avian centromeric repeated DNA component.

    PubMed

    Madsen, C S; Brooks, J E; de Kloet, E; de Kloet, S R

    1994-06-01

    The approximately 190-bp centromeric repeat monomers of the spur-winged lapwing (Vanellus spinosus, Charadriidae), the Chilean flamingo (Phoenicopterus chilensis, Phoenicopteridae), the sarus crane (Grus antigone, Gruidae), parrots (Psittacidae), waterfowl (Anatidae), and the merlin (Falco columbarius, Falconidae) contain elements that are interspecifically highly variable, as well as elements (trinucleotides and higher order oligonucleotides) that are highly conserved in sequence and relative location within the repeat. Such conservation suggests that the centromeric repeats of these avian species have evolved from a common ancestral sequence that may date from very early stages of avian radiation. PMID:8034177

  6. Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot.

    PubMed

    Moon, Jae-Su; Lee, Seung-Hoon; Kim, Eun-Jung; Cho, Hee; Lee, Wooseong; Kim, Geon-Woo; Park, Hyun-Ji; Cho, Seung-Woo; Lee, Choongho; Oh, Jong-Won

    2016-01-01

    The hepatitis C virus (HCV) internal ribosome entry site (IRES) that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA)-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts) 277-343. Based on their antiviral activity, we mapped a druggable region (nts 313-343) where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5' or 3' direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens. PMID:26751678

  7. Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot

    PubMed Central

    Kim, Eun-Jung; Cho, Hee; Lee, Wooseong; Kim, Geon-Woo; Park, Hyun-Ji; Cho, Seung-Woo; Lee, Choongho; Oh, Jong-Won

    2016-01-01

    The hepatitis C virus (HCV) internal ribosome entry site (IRES) that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA)-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts) 277–343. Based on their antiviral activity, we mapped a druggable region (nts 313–343) where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5′ or 3′ direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens. PMID:26751678

  8. Large-scale nucleotide sequence alignment and sequence variability assessment to identify the evolutionarily highly conserved regions for universal screening PCR assay design: an example of influenza A virus.

    PubMed

    Nagy, Alexander; Jiřinec, Tomáš; Černíková, Lenka; Jiřincová, Helena; Havlíčková, Martina

    2015-01-01

    The development of a diagnostic polymerase chain reaction (PCR) or quantitative PCR (qPCR) assay for universal detection of highly variable viral genomes is always a difficult task. The purpose of this chapter is to provide a guideline on how to align, process, and evaluate a huge set of homologous nucleotide sequences in order to reveal the evolutionarily most conserved positions suitable for universal qPCR primer and hybridization probe design. Attention is paid to the quantification and clear graphical visualization of the sequence variability at each position of the alignment. In addition, specific problems related to the processing of the extremely large sequence pool are highlighted. All of these steps are performed using an ordinary desktop computer without the need for extensive mathematical or computational skills. PMID:25697651

  9. OVINE HERPESVIRUS-2 GLYCOPROTEIN B SEQUENCES FROM TISSUES OF RUMINANT MALIGNANT CATARRHAL FEVER AND HEALTHY SHEEP ARE HIGHLY CONSERVED.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ovine herpesvirus-2 (OHV-2) infection has been associated with malignant catarrhal fever (MCF) in susceptible ruminants. In order to further investigate whether OHV-2 is an aetiological agent for sheep-associated (SA) MCF in cattle and bison, the entire sequences of OHV-2 glycoprotein B (gB) from di...

  10. Genome-Wide Analysis of Stowaway-Like MITEs in Wheat Reveals High Sequence Conservation, Gene Association, and Genomic Diversification1[C][W

    PubMed Central

    Yaakov, Beery; Ben-David, Smadar; Kashkush, Khalil

    2013-01-01

    The diversity and evolution of wheat (Triticum-Aegilops group) genomes is determined, in part, by the activity of transposable elements that constitute a large fraction of the genome (up to 90%). In this study, we retrieved sequences from publicly available wheat databases, including a 454-pyrosequencing database, and analyzed 18,217 insertions of 18 Stowaway-like miniature inverted-repeat transposable element (MITE) families previously characterized in wheat that together account for approximately 1.3 Mb of sequence. All 18 families showed high conservation in length, sequence, and target site preference. Furthermore, approximately 55% of the elements were inserted in transcribed regions, into or near known wheat genes. Notably, we observed significant correlation between the mean length of the MITEs and their copy number. In addition, the genomic composition of nine MITE families was studied by real-time quantitative polymerase chain reaction analysis in 40 accessions of Triticum spp. and Aegilops spp., including diploids, tetraploids, and hexaploids. The quantitative polymerase chain reaction data showed massive and significant intraspecific and interspecific variation as well as genome-specific proliferation and nonadditive quantities in the polyploids. We also observed significant differences in the methylation status of the insertion sites among MITE families. Our data thus suggest a possible role for MITEs in generating genome diversification and in the establishment of nascent polyploid species in wheat. PMID:23104862

  11. Low sequence identity but high structural and functional conservation: The case of Hsp70/Hsp90 organizing protein (Hop/Sti1) of Leishmania braziliensis.

    PubMed

    Batista, Fernanda A H; Seraphim, Thiago V; Santos, Clelton A; Gonzaga, Marisvanda R; Barbosa, Leandro R S; Ramos, Carlos H I; Borges, Júlio C

    2016-06-15

    Parasites belonging to the genus Leishmania are subjected to extensive environmental changes during their life cycle; molecular chaperones/co-chaperones act as protagonists in this scenario to maintain cellular homeostasis. Hop/Sti1 is a co-chaperone that connects the Hsp90 and Hsp70 systems, modulating their ATPase activities and affecting the fate of client proteins because it facilitates their transfer from the Hsp70 to the Hsp90 chaperone. Hop/Sti1 is one of the most prevalent co-chaperones, highlighting its importance despite the relatively low sequence identity among orthologue proteins. This multi-domain protein comprises three tetratricopeptides domains (TPR1, TPR2A and TPR2B) and two Asp/Pro-rich domains. Given the importance of Hop/Sti1 for the chaperone system and for Leishmania protozoa viability, the Leishmania braziliensis Hop (LbHop) and a truncated mutant (LbHop(TPR2AB)) were characterized. Structurally, both proteins are α-helix-rich and highly elongated monomeric proteins. Functionally, they inhibited the ATPase activity of Leishmania braziliensis Hsp90 (LbHsp90) to a similar extent, and the thermodynamic parameters of their interactions with LbHsp90 were similar, indicating that TPR2A-TPR2B forms the functional center for the LbHop interaction with LbHsp90. These results highlight the structural and functional similarity of Hop/Sti1 proteins, despite their low sequence conservation compared to the Hsp70 and Hsp90 systems, which are phylogenetic highly conserved. PMID:27103305

  12. Unusually high conservation of untranslated sequences in cDNAs for Trimeresurus flavoviridis phospholipase A2 isozymes.

    PubMed Central

    Ogawa, T; Oda, N; Nakashima, K; Sasaki, H; Hattori, M; Sakaki, Y; Kihara, H; Ohno, M

    1992-01-01

    As a step toward understanding the structure and function of phospholipases A2 (PLA2s), we isolated and sequenced several cDNAs encoding Trimeresurus flavoviridis venom PLA2 isozymes including two [Lys49]PLA2s called basic proteins I and II, [Thr37]PLA2, and PLX'-PLA2. Comparison of the nucleotide sequences of these cDNAs with the previously isolated [Asp49]PLA2 cDNA revealed some interesting findings from the viewpoint of evolution. First, the homologies of the 5' and 3' untranslated regions (98% and 89%, respectively) were much higher than that of the protein-coding regions (67%). The predicted secondary structure showed the characteristic stem-loop structures for both the untranslated regions of the mRNAs, suggesting that these regions play some functional role(s) in translation or stability of mRNAs. Second, base substitutions appeared to have occurred at similar rates for the three positions of codons among these PLA2s. The results are discussed in terms of evolution of PLA2s. Northern blot analysis showed that these PLA2s are specific to venom gland. Images PMID:1528861

  13. Use of genotyping by sequencing data to develop a high-throughput and multifunctional SNP panel for conservation applications in Pacific lamprey.

    PubMed

    Hess, Jon E; Campbell, Nathan R; Docker, Margaret F; Baker, Cyndi; Jackson, Aaron; Lampman, Ralph; McIlraith, Brian; Moser, Mary L; Statler, David P; Young, William P; Wildbill, Andrew J; Narum, Shawn R

    2015-01-01

    Next-generation sequencing data can be mined for highly informative single nucleotide polymorphisms (SNPs) to develop high-throughput genomic assays for nonmodel organisms. However, choosing a set of SNPs to address a variety of objectives can be difficult because SNPs are often not equally informative. We developed an optimal combination of 96 high-throughput SNP assays from a total of 4439 SNPs identified in a previous study of Pacific lamprey (Entosphenus tridentatus) and used them to address four disparate objectives: parentage analysis, species identification and characterization of neutral and adaptive variation. Nine of these SNPs are FST outliers, and five of these outliers are localized within genes and significantly associated with geography, run-timing and dwarf life history. Two of the 96 SNPs were diagnostic for two other lamprey species that were morphologically indistinguishable at early larval stages and were sympatric in the Pacific Northwest. The majority (85) of SNPs in the panel were highly informative for parentage analysis, that is, putatively neutral with high minor allele frequency across the species' range. Results from three case studies are presented to demonstrate the broad utility of this panel of SNP markers in this species. As Pacific lamprey populations are undergoing rapid decline, these SNPs provide an important resource to address critical uncertainties associated with the conservation and recovery of this imperiled species. PMID:24842551

  14. 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.

  15. A highly conserved N-terminal sequence for teleost vitellogenin with potential value to the biochemistry, molecular biology and pathology of vitellogenesis

    USGS Publications Warehouse

    Folmar, L.D.; Denslow, N.D.; Wallace, R.A.; LaFleur, G.; Gross, T.S.; Bonomelli, S.; Sullivan, C.V.

    1995-01-01

    N-terminal amino acid sequences for vitellogenin (Vtg) from six species of teleost fish (striped bass, mummichog, pinfish, brown bullhead, medaka, yellow perch and the sturgeon) are compared with published N-terminal Vtg sequences for the lamprey, clawed frog and domestic chicken. Striped bass and mummichog had 100% identical amino acids between positions 7 and 21, while pinfish, brown bullhead, sturgeon, lamprey, Xenopus and chicken had 87%, 93%, 60%, 47%, 47-60%) for four transcripts and had 40% identical, respectively, with striped bass for the same positions. Partial sequences obtained for medaka and yellow perch were 100% identical between positions 5 to 10. The potential utility of this conserved sequence for studies on the biochemistry, molecular biology and pathology of vitellogenesis is discussed.

  16. Functionally conserved enhancers with divergent sequences in distant vertebrates

    SciTech Connect

    Yang, Song; Oksenberg, Nir; Takayama, Sachiko; Heo, Seok -Jin; Poliakov, Alexander; Ahituv, Nadav; Dubchak, Inna; Boffelli, Dario

    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.

  17. High-Throughput Sequencing Identifies Novel and Conserved Cucumber (Cucumis sativus L.) microRNAs in Response to Cucumber Green Mottle Mosaic Virus Infection

    PubMed Central

    Liang, C. Q.; Jiang, N.; Liu, P. F.; Li, J. Q.

    2015-01-01

    Seedlings of Cucumis sativus L. (cv. 'Zhongnong 16') were artificially inoculated with Cucumber green mottle mosaic virus (CGMMV) at the three-true-leaf stage. Leaf and flower samples were collected at different time points post-inoculation (10, 30 and 50 d), and processed by high throughput sequencing analysis to identify candidate miRNA sequences. Bioinformatic analysis using screening criteria, and secondary structure prediction, indicated that 8 novel and 23 known miRNAs (including 15 miRNAs described for the first time in vivo) were produced by cucumber plants in response to CGMMV infection. Moreover, gene expression profiles (p-value <0.01) validated the expression of 3 of the novel miRNAs and 3 of the putative candidate miRNAs and identified a further 82 conserved miRNAs in CGMMV-infected cucumbers. Gene ontology (GO) analysis revealed that the predicted target genes of these 88 miRNAs, which were screened using the psRNATarget and miRanda algorithms, were involved in three functional categories: 2265 in molecular function, 1362 as cellular components and 276 in biological process. The subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the predicted target genes were frequently involved in metabolic processes (166 pathways) and genetic information processes (40 pathways) and to a lesser degree the biosynthesis of secondary metabolites (12 pathways). These results could provide useful clues to help elucidate host-pathogen interactions in CGMMV and cucumber, as well as for the screening of resistance genes. PMID:26076360

  18. Inhibition of Dengue Virus Infections in Cell Cultures and in AG129 Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence

    PubMed Central

    Stein, David A.; Perry, Stuart T.; Buck, Michael D.; Oehmen, Christopher S.; Fischer, Matthew A.; Poore, Elizabeth; Smith, Jessica L.; Lancaster, Alissa M.; Hirsch, Alec J.; Slifka, Mark K.; Nelson, Jay A.; Shresta, Sujan; Früh, Klaus

    2011-01-01

    The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small interfering RNA (siRNA) (DC-3) targeting the highly conserved 5′ cyclization sequence (5′CS) region of the DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype in vitro. To determine if DC-3 siRNA could inhibit DENV in vivo, an “in vivo-ready” version of DC-3 was synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups in several tissues at various time points postinfection. These results suggest that exogenously introduced siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses. PMID:21795337

  19. Identification of novel and conserved miRNAs involved in pollen development in Brassica campestris ssp. chinensis by high-throughput sequencing and degradome analysis

    PubMed Central

    2014-01-01

    Background microRNAs (miRNAs) are endogenous, noncoding, small RNAs that have essential regulatory functions in plant growth, development, and stress response processes. However, limited information is available about their functions in sexual reproduction of flowering plants. Pollen development is an important process in the life cycle of a flowering plant and is a major factor that affects the yield and quality of crop seeds. Results This study aims to identify miRNAs involved in pollen development. Two independent small RNA libraries were constructed from the flower buds of the male sterile line (Bcajh97-01A) and male fertile line (Bcajh97-01B) of Brassica campestris ssp. chinensis. The libraries were subjected to high-throughput sequencing by using the Illumina Solexa system. Eight novel miRNAs on the other arm of known pre-miRNAs, 54 new conserved miRNAs, and 8 novel miRNA members were identified. Twenty-five pairs of novel miRNA/miRNA* were found. Among all the identified miRNAs, 18 differentially expressed miRNAs with over two-fold change between flower buds of male sterile line (Bcajh97-01A) and male fertile line (Bcajh97-01B) were identified. qRT-PCR analysis revealed that most of the differentially expressed miRNAs were preferentially expressed in flower buds of the male fertile line (Bcajh97-01B). Degradome analysis showed that a total of 15 genes were predicted to be the targets of seven miRNAs. Conclusions Our findings provide an overview of potential miRNAs involved in pollen development and interactions between miRNAs and their corresponding targets, which may provide important clues on the function of miRNAs in pollen development. PMID:24559317

  20. Bioinformatic Identification of Conserved Cis-Sequences in Coregulated Genes.

    PubMed

    Bülow, Lorenz; Hehl, Reinhard

    2016-01-01

    Bioinformatics tools can be employed to identify conserved cis-sequences in sets of coregulated plant genes because more and more gene expression and genomic sequence data become available. Knowledge on the specific cis-sequences, their enrichment and arrangement within promoters, facilitates the design of functional synthetic plant promoters that are responsive to specific stresses. The present chapter illustrates an example for the bioinformatic identification of conserved Arabidopsis thaliana cis-sequences enriched in drought stress-responsive genes. This workflow can be applied for the identification of cis-sequences in any sets of coregulated genes. The workflow includes detailed protocols to determine sets of coregulated genes, to extract the corresponding promoter sequences, and how to install and run a software package to identify overrepresented motifs. Further bioinformatic analyses that can be performed with the results are discussed. PMID:27557771

  1. Maize peroxidase Px5 has a highly conserved sequence in inbreds resistant to mycotoxin producing fungi which enhances fungal and insect resistance.

    PubMed

    Dowd, Patrick F; Johnson, Eric T

    2016-01-01

    Mycotoxin presence in maize causes health and economic issues for humans and animals. Although many studies have investigated expression differences of genes putatively governing resistance to producing fungi, few have confirmed a resistance role, or examined putative resistance gene structure in more than a couple of inbreds. The pericarp expression of maize Px5 has previously been associated with resistance to Aspergillus flavus growth and insects in a set of inbreds. Genes from 14 different inbreds that included ones with resistance and susceptibility to A. flavus, Fusarium proliferatum, F. verticillioides and F. graminearum and/or mycotoxin production were cloned using high fidelity enzymes, and sequenced. The sequence of Px5 from all resistant inbreds was identical, except for a single base change in two inbreds, only one of which affected the amino acid sequence. Conversely, the Px5 sequence from several susceptible inbreds had several base variations, some of which affected amino acid sequence that would potentially alter secondary structure, and thus enzyme function. The sequence of the maize peroxidase Px5 common to inbreds resistant to mycotoxigenic fungi was overexpressed in maize callus. Callus transformants overexpressing the gene caused significant reductions in growth for fall armyworms, corn earworms, and F. graminearum compared to transformant callus with a β-glucuronidase gene. This study demonstrates rarer transcripts of potential resistance genes overlooked by expression screens can be identified by sequence comparisons. A role in pest resistance can be verified by callus expression of the candidate genes, which can thereby justify larger scale transformation and regeneration of transgenic plants expressing the resistance gene for further evaluation. PMID:26659597

  2. Polymorphism, monomorphism, and sequences in conserved microsatellites in primate species.

    PubMed

    Blanquer-Maumont, A; Crouau-Roy, B

    1995-10-01

    Dimeric short tandem repeats are a source of highly polymorphic markers in the mammalian genome. Genetic variation at these hypervariable loci is extensively used for linkage analysis, for the identification of individuals, and may be useful for interpopulation and interspecies studies. In this paper, we analyze the variability and the sequences of a segment including three microsatellites, first described in man, in several species of primates (chimpanzee, orangutan, gibbon, and macaque) using the heterologous primers (man primers). This region is located on the human chromosome 6p, near the tumor necrosis factor genes, in the major histocompatibility complex. The fact that these primers work in all species studied indicates that they are conserved throughout the different lineages of the two superfamilies, the Hominoidea and the Cercopithecidea, represented by the macaques. However, the intervening sequence displays intraspecific and interspecific variability. The sites of base substitutions and the insertion/deletion events are not evenly distributed within this region. The data suggest that it is necessary to have a minimal number of repeats to increase the rate of mutation sufficiently to allow the development of polymorphism. In some species, the microsatellites present single base variations which reduce the number of contiguous repeats, thus apparently slowing the rate of additional slippage events. Species with such variations or a low number of repeats are monomorphic. These microsatellite sequences are informative in the comparison of closely related species and reflect the phylogeny of the Old World monkeys, apes, and man. PMID:7563137

  3. Conserved sequence pattern in a wide variety of phosphoesterases.

    PubMed Central

    Koonin, E. V.

    1994-01-01

    A unique sequence pattern, designated the GD/GNH signature, was shown to be conserved in a wide variety of phosphoesterases. The enzymes containing this signature cleave phosphoester bonds in such different substrates as (1) phosphoserine and phosphothreonine in polypeptides; (2) bis(5'-nucleosidyl)-tetraphosphates; (3) nucleoside 5' phosphates; (4) 2',3'-cyclic nucleotide phosphates; (5) polynucleotides; (6) 2'-5' phosphodiesters in RNA (intron) lariats; (7) sphingomyelin; and (7) various phosphomonoesters. Two conserved acidic amino acid residues and a conserved histidine residue may be directly involved in phosphoester bond cleavage. PMID:8003970

  4. 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

  5. Molecular cloning of mouse glycolate oxidase. High evolutionary conservation and presence of an iron-responsive element-like sequence in the mRNA.

    PubMed

    Kohler, S A; Menotti, E; Kühn, L C

    1999-01-22

    Iron regulatory proteins (IRPs) control the synthesis of several proteins in iron metabolism by binding to iron-responsive elements (IREs), a hairpin structure in the untranslated region (UTR) of corresponding mRNAs. Binding of IRPs to IREs in the 5' UTR inhibits translation of ferritin heavy and light chain, erythroid aminolevulinic acid synthase, mitochondrial aconitase, and Drosophila succinate dehydrogenase b, whereas IRP binding to IREs in the 3' UTR of transferrin receptor mRNA prolongs mRNA half-life. To identify new targets of IRPs, we devised a method to enrich IRE-containing mRNAs by using recombinant IRP-1 as an affinity matrix. A cDNA library established from enriched mRNA was screened by an RNA-protein band shift assay. This revealed a novel IRE-like sequence in the 3' UTR of a liver-specific mouse mRNA. The newly identified cDNA codes for a protein with high homology to plant glycolate oxidase (GOX). Recombinant protein expressed in bacteria displayed enzymatic GOX activity. Therefore, this cDNA represents the first vertebrate GOX homologue. The IRE-like sequence in mouse GOX exhibited strong binding to IRPs at room temperature. However, it differs from functional IREs by a mismatch in the middle of its upper stem and did not confer iron-dependent regulation in cells. PMID:9891009

  6. 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

  7. 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

  8. HIGHLY CONSERVED N-TERMINAL SEQUENCE FOR TELEOST VITELLOGENIN WITH POTENTIAL VALUE TO THE BIOCHEMISTRY, MOLECULAR BIOLOGY AND PATHOLOGY OF VITELLOGENESIS

    EPA Science Inventory

    N-terminal amino acid sequences for vitellogenin (Vtg) from six species of teleost fish: striped bass, Morone saxatillus; mummichog, Fundulus heteroclitus; pinfish, Lagodon rhomboides; brown bullhead, Ameiurus nebulosus; medaka, Oryzias latipes; yellow perch, Percaflavescens and ...

  9. High-Throughput Sequencing Technologies

    PubMed Central

    Reuter, Jason A.; Spacek, Damek; Snyder, Michael P.

    2015-01-01

    Summary The human genome sequence has profoundly altered our understanding of biology, human diversity and disease. The path from the first draft sequence to our nascent era of personal genomes and genomic medicine has been made possible only because of the extraordinary advancements in DNA sequencing technologies over the past ten years. Here, we discuss commonly used high-throughput sequencing platforms, the growing array of sequencing assays developed around them as well as the challenges facing current sequencing platforms and their clinical application. PMID:26000844

  10. Sturgeon conservation genomics: SNP discovery and validation using RAD sequencing.

    PubMed

    Ogden, R; Gharbi, K; Mugue, N; Martinsohn, J; Senn, H; Davey, J W; Pourkazemi, M; McEwing, R; Eland, C; Vidotto, M; Sergeev, A; Congiu, L

    2013-06-01

    Caviar-producing sturgeons belonging to the genus Acipenser are considered to be one of the most endangered species groups in the world. Continued overfishing in spite of increasing legislation, zero catch quotas and extensive aquaculture production have led to the collapse of wild stocks across Europe and Asia. The evolutionary relationships among Adriatic, Russian, Persian and Siberian sturgeons are complex because of past introgression events and remain poorly understood. Conservation management, traceability and enforcement suffer a lack of appropriate DNA markers for the genetic identification of sturgeon at the species, population and individual level. This study employed RAD sequencing to discover and characterize single nucleotide polymorphism (SNP) DNA markers for use in sturgeon conservation in these four tetraploid species over three biological levels, using a single sequencing lane. Four population meta-samples and eight individual samples from one family were barcoded separately before sequencing. Analysis of 14.4 Gb of paired-end RAD data focused on the identification of SNPs in the paired-end contig, with subsequent in silico and empirical validation of candidate markers. Thousands of putatively informative markers were identified including, for the first time, SNPs that show population-wide differentiation between Russian and Persian sturgeons, representing an important advance in our ability to manage these cryptic species. The results highlight the challenges of genotyping-by-sequencing in polyploid taxa, while establishing the potential genetic resources for developing a new range of caviar traceability and enforcement tools. PMID:23473098

  11. 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.

  12. 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

  13. 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.

  14. Conserved Sequences at the Origin of Adenovirus DNA Replication

    PubMed Central

    Stillman, Bruce W.; Topp, William C.; Engler, Jeffrey A.

    1982-01-01

    The origin of adenovirus DNA replication lies within an inverted sequence repetition at either end of the linear, double-stranded viral DNA. Initiation of DNA replication is primed by a deoxynucleoside that is covalently linked to a protein, which remains bound to the newly synthesized DNA. We demonstrate that virion-derived DNA-protein complexes from five human adenovirus serological subgroups (A to E) can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenovirus type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein complexes were not as active as the homologous Ad2 DNA, most probably due to inefficient initiation by Ad2 replication factors. In an attempt to identify common features which may permit this replication, we have also sequenced the inverted terminal repeated DNA from human adenovirus serotypes Ad4 (group E), Ad9 and Ad10 (group D), and Ad31 (group A), and we have compared these to previously determined sequences from Ad2 and Ad5 (group C), Ad7 (group B), and Ad12 and Ad18 (group A) DNA. In all cases, the sequence around the origin of DNA replication can be divided into two structural domains: a proximal A · T-rich region which is partially conserved among these serotypes, and a distal G · C-rich region which is less well conserved. The G · C-rich region contains sequences similar to sequences present in papovavirus replication origins. The two domains may reflect a dual mechanism for initiation of DNA replication: adenovirus-specific protein priming of replication, and subsequent utilization of this primer by host replication factors for completion of DNA synthesis. Images PMID:7143575

  15. 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.

  16. Genomic Investigation Reveals Highly Conserved, Mosaic, Recombination Events Associated with Capsular Switching among Invasive Neisseria meningitidis Serogroup W Sequence Type (ST)-11 Strains

    PubMed Central

    Mustapha, Mustapha M.; Marsh, Jane W.; Krauland, Mary G.; Fernandez, Jorge O.; de Lemos, Ana Paula S.; Hotopp, Julie C. Dunning; Wang, Xin; Mayer, Leonard W.; Lawrence, Jeffrey G.; Hiller, N. Luisa; Harrison, Lee H.

    2016-01-01

    Neisseria meningitidis is an important cause of meningococcal disease globally. Sequence type (ST)-11 clonal complex (cc11) is a hypervirulent meningococcal lineage historically associated with serogroup C capsule and is believed to have acquired the W capsule through a C to W capsular switching event. We studied the sequence of capsule gene cluster (cps) and adjoining genomic regions of 524 invasive W cc11 strains isolated globally. We identified recombination breakpoints corresponding to two distinct recombination events within W cc11: A 8.4-kb recombinant region likely acquired from W cc22 including the sialic acid/glycosyl-transferase gene, csw resulted in a C→W change in capsular phenotype and a 13.7-kb recombinant segment likely acquired from Y cc23 lineage includes 4.5 kb of cps genes and 8.2 kb downstream of the cps cluster resulting in allelic changes in capsule translocation genes. A vast majority of W cc11 strains (497/524, 94.8%) retain both recombination events as evidenced by sharing identical or very closely related capsular allelic profiles. These data suggest that the W cc11 capsular switch involved two separate recombination events and that current global W cc11 meningococcal disease is caused by strains bearing this mosaic capsular switch. PMID:27289093

  17. Genetic mapping of legume orthologs reveals high conservation of synteny between lentil species and the sequenced genomes of Medicago and chickpea

    PubMed Central

    Gujaria-Verma, Neha; Vail, Sally L.; Carrasquilla-Garcia, Noelia; Penmetsa, R. Varma; Cook, Douglas R.; Farmer, Andrew D.; Vandenberg, Albert; Bett, Kirstin E.

    2014-01-01

    Lentil (Lens culinaris Medik.) is a global food crop with increasing importance for food security in south Asia and other regions. Lens ervoides, a wild relative of cultivated lentil, is an important source of agronomic trait variation. Lens is a member of the galegoid clade of the Papilionoideae family, which includes other important dietary legumes such as chickpea (Cicer arietinum) and pea (Pisum sativum), and the sequenced model legume Medicago truncatula. Understanding the genetic structure of Lens spp. in relation to more fully sequenced legumes would allow leveraging of genomic resources. A set of 1107 TOG-based amplicons were identified in L. ervoides and a subset thereof used to design SNP markers for mapping. A map of L. ervoides consisting of 377 SNP markers spread across seven linkage groups was developed using a GoldenGate genotyping array and single SNP marker assays. Comparison with maps of M. truncatula and L. culinaris documented considerable shared synteny and led to the identification of a few major translocations and a major inversion that distinguish Lens from M. truncatula, as well as a translocation that distinguishes L. culinaris from L. ervoides. The identification of chromosome-level differences among Lens spp. will aid in the understanding of introgression of genes from L. ervoides into cultivated L. culinaris, furthering genetic research and breeding applications in lentil. PMID:25538716

  18. RNA-Sequencing Analysis of TCDD-Induced Responses in Zebrafish Liver Reveals High Relatedness to In Vivo Mammalian Models and Conserved Biological Pathways

    PubMed Central

    Li, Zhi-Hua; Xu, Hongyan; Zheng, Weiling; Lam, Siew Hong; Gong, Zhiyuan

    2013-01-01

    TCDD is one of the most persistent environmental toxicants in biological systems and its effect through aryl hydrocarbon receptor (AhR) has been well characterized. However, the information on TCDD-induced toxicity in other molecular pathways is rather limited. To fully understand molecular toxicity of TCDD in an in vivo animal model, adult zebrafish were exposed to TCDD at 10 nM for 96 h and the livers were sampled for RNA-sequencing based transcriptomic profiling. A total of 1,058 differently expressed genes were identified based on fold-change>2 and TPM (transcripts per million) >10. Among the top 20 up-regulated genes, 10 novel responsive genes were identified and verified by RT-qPCR analysis on independent samples. Transcriptomic analysis indicated several deregulated pathways associated with cell cycle, endocrine disruptors, signal transduction and immune systems. Comparative analyses of TCDD-induced transcriptomic changes between fish and mammalian models revealed that proteomic pathway is consistently up-regulated while calcium signaling pathway and several immune-related pathways are generally down-regulated. Finally, our study also suggested that zebrafish model showed greater similarity to in vivo mammalian models than in vitro models. Our study indicated that the zebrafish is a valuable in vivo model in toxicogenomic analyses for understanding molecular toxicity of environmental toxicants relevant to human health. The expression profiles associated with TCDD could be useful for monitoring environmental dioxin and dioxin-like contamination. PMID:24204792

  19. Proteome-Wide Discovery of Evolutionary Conserved Sequences in Disordered Regions

    PubMed Central

    Nguyen Ba, Alex N.; Yeh, Brian J.; van Dyk, Dewald; Davidson, Alan R.; Andrews, Brenda J.; Weiss, Eric L.; Moses, Alan M.

    2016-01-01

    At least 30% of human proteins are thought to contain intrinsically disordered regions, which lack stable structural conformation. Despite lacking enzymatic functions and having few protein domains, disordered regions are functionally important for protein regulation and contain short linear motifs (short peptide sequences involved in protein-protein interactions), but in most disordered regions, the functional amino acid residues remain unknown. We searched for evolutionarily conserved sequences within disordered regions according to the hypothesis that conservation would indicate functional residues. Using a phylogenetic hidden Markov model (phylo-HMM), we made accurate, specific predictions of functional elements in disordered regions even when these elements are only two or three amino acids long. Among the conserved sequences that we identified were previously known and newly identified short linear motifs, and we experimentally verified key examples, including a motif that may mediate interaction between protein kinase Cbk1 and its substrates. We also observed that hub proteins, which interact with many partners in a protein interaction network, are highly enriched in these conserved sequences. Our analysis enabled the systematic identification of the functional residues in disordered regions and suggested that at least 5% of amino acids in disordered regions are important for function. PMID:22416277

  20. Molecular characterization of a bovine Y-specific DNA sequence conserved in taurine and zebu breeds.

    PubMed

    Alves, Beatriz C A; Mayer, Mário G; Taber, Anna Paula; Egito, Andréa A; Fagundes, Valéria; McElreavey, Ken; Moreira-Filho, Carlos A

    2006-06-01

    The identification of new bovine male-specific DNA sequences is of great interest because the bovine Y chromosome remains poorly characterized in terms of physical and genetic maps. Since taurine and zebu Y chromosomes are structurally different, the identification of Y-specific sequences present in both sub-species is particularly important: these sequences are of evolutionary significance and can be broadly used for embryo sexing. In this work, we initially used the random amplified polymorphic DNA (RAPD) technique to search for male-specific sequences present as monomorphic markers in genomic DNA from zebu and taurine bulls. A male-specific RAPD band was found to be present and highly conserved in both sub-species, as demonstrated by Southern blotting, fluorescent in situ hybridization (FISH) and DNA sequencing. In a previous work, a pair of primers derived from this marker was successfully used in taurine and zebu embryo sexing. PMID:17286047

  1. Studying RNA Homology and Conservation with Infernal: From Single Sequences to RNA Families.

    PubMed

    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 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. PMID:27322404

  2. Identification of antimicrobial peptides from teleosts and anurans in expressed sequence tag databases using conserved signal sequences.

    PubMed

    Tessera, Valentina; Guida, Filomena; Juretić, Davor; Tossi, Alessandro

    2012-03-01

    The problem of multidrug resistance requires the efficient and accurate identification of new classes of antimicrobial agents. Endogenous antimicrobial peptides produced by most organisms are a promising source of such molecules. We have exploited the high conservation of signal sequences in teleost and anuran antimicrobial peptides to search cDNA (expressed sequence tag) databases for likely candidates. Subject sequences were then analysed for the presence of potential antimicrobial peptides based on physicochemical properties (amphipathic helical structure, cationicity) and use of the D-descriptor model to predict the therapeutic index (relation between the minimum inhibitory concentration and the concentration giving 50% haemolysis). This analysis also suggested mutations to probe the role of the primary structure in determining potency and selectivity. Selected sequences were chemically synthesized and the antimicrobial activity of the peptides was confirmed. In particular, a short (21-residue) sequence, likely of sticklefish origin, showed potent activity and it was possible to tune the spectrum of action and/or selectivity by combining three directed mutations. Membrane permeabilization studies on both bacterial and host cells indicate that the mode of action was prevalently membranolytic. This method opens up the possibility for more effective searching of the vast and continuously growing expressed sequence tag databases for novel antimicrobial peptides, which are likely abundant, and the efficient identification of the most promising candidates among them. PMID:22188679

  3. 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.

  4. 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

  5. The Large Mitochondrial Genome of Symbiodinium minutum Reveals Conserved Noncoding Sequences between Dinoflagellates and Apicomplexans.

    PubMed

    Shoguchi, Eiichi; Shinzato, Chuya; Hisata, Kanako; Satoh, Nori; Mungpakdee, Sutada

    2015-08-01

    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

  6. Sequence conservation and functional constraint on intergenic spacers in reduced genomes of the obligate symbiont Buchnera.

    PubMed

    Degnan, Patrick H; Ochman, Howard; Moran, Nancy A

    2011-09-01

    Analyses of genome reduction in obligate bacterial symbionts typically focus on the removal and retention of protein-coding regions, which are subject to ongoing inactivation and deletion. However, these same forces operate on intergenic spacers (IGSs) and affect their contents, maintenance, and rates of evolution. IGSs comprise both non-coding, non-functional regions, including decaying pseudogenes at varying stages of recognizability, as well as functional elements, such as genes for sRNAs and regulatory control elements. The genomes of Buchnera and other small genome symbionts display biased nucleotide compositions and high rates of sequence evolution and contain few recognizable regulatory elements. However, IGS lengths are highly correlated across divergent Buchnera genomes, suggesting the presence of functional elements. To identify functional regions within the IGSs, we sequenced two Buchnera genomes (from aphid species Uroleucon ambrosiae and Acyrthosiphon kondoi) and applied a phylogenetic footprinting approach to alignments of orthologous IGSs from a total of eight Buchnera genomes corresponding to six aphid species. Inclusion of these new genomes allowed comparative analyses at intermediate levels of divergence, enabling the detection of both conserved elements and previously unrecognized pseudogenes. Analyses of these genomes revealed that 232 of 336 IGS alignments over 50 nucleotides in length displayed substantial sequence conservation. Conserved alignment blocks within these IGSs encompassed 88 Shine-Dalgarno sequences, 55 transcriptional terminators, 5 Sigma-32 binding sites, and 12 novel small RNAs. Although pseudogene formation, and thus IGS formation, are ongoing processes in these genomes, a large proportion of intergenic spacers contain functional sequences. PMID:21912528

  7. Monoclonal antibody production to human and bovine 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase): high-specificity recognition in whole brain acetone powders and conservation of sequence between CNP1 and CNP2.

    PubMed

    Sprinkle, T J; Agee, J F; Tippins, R B; Chamberlain, C R; Faguet, G B; De Vries, G H

    1987-11-24

    Monoclonal antibodies against human and bovine 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) were generated by fusing FOX-NY myeloma cells with spleen cells from RBF/Dn mice previously immunized with the purified brain antigens. The enzyme isolated from bovine brain was quite basic, with an isoelectric point of 9.71 and both the bovine and human enzymes consisted of a closely spaced doublet at approximately 44 and 46 kDa on SDS-PAGE. Six monoclonals were were identified as strongly recognizing the enzyme on both ELISA plates and on immunoblots of whole brain protein. Four monoclonals very weakly cross-reacted with guinea pig myelin basic protein. In contrast with two previous reports, some of our monoclonal antibodies did immunostain 2 or 3 protein bands in peripheral nerve, two bands closely corresponding to those immunostained in central nervous system (CNS) myelin, the Wolfgram protein fraction and in acetone powders of whole brain. Each of the 6 monoclonals reacting strongly on immunoblots recognized the enzyme in from 2 to 5 of the species examined (human, bovine, rat, mouse and rabbit). In addition, all 6 monoclonals that immunostained the enzyme in whole brain, myelin and Wolfgram protein immunoblots recognized both CNP1 (44 kDa) and CNP2 (46 kDa). The two closely spaced protein bands observed on SDS-PAGE and previously stained on immunoblots of CNS CNPase using polyvalent rabbit anti-bovine CNPase antisera, and now different monoclonal antibodies, appear to be immunologically related and to contain highly conserved sequences. PMID:2446713

  8. Energy Conservation Featured in Illinois High School

    ERIC Educational Resources Information Center

    Modern Schools, 1976

    1976-01-01

    The William Fremd High School in Palatine, Illinois, scheduled to open in 1977, is being built with energy conservation uppermost in mind. In this system, 70 heat pumps will heat and cool 300,000 square feet of educational facilities. (Author/MLF)

  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. High-bay Lighting Energy Conservation Measures

    SciTech Connect

    Ian Metzger, Jesse Dean

    2010-12-31

    This software requires inputs of simple high-bay lighting system inventory information and calculates the energy and cost benefits of various retrofit opportunities. This tool includes energy conservation measures for: 1000 Watt to 750 Watt High-pressure Sodium lighting retrofit, 400 Watt to 360 Watt High Pressure Sodium lighting retrofit, High Intensity Discharge to T5 lighting retrofit, High Intensity Discharge to T8 lighting retrofit, and Daylighting. This tool calculates energy savings, demand reduction, cost savings, building life cycle costs including: simple payback, discounted payback, net-present value, and savings to investment ratio. In addition this tool also displays the environmental benefits of a project.

  11. High resolution schemes for hyperbolic conservation laws

    NASA Technical Reports Server (NTRS)

    Harten, A.

    1983-01-01

    A class of new explicit second order accurate finite difference schemes for the computation of weak solutions of hyperbolic conservation laws is presented. These highly nonlinear schemes are obtained by applying a nonoscillatory first order accurate scheme to an appropriately modified flux function. The so-derived second order accurate schemes achieve high resolution while preserving the robustness of the original nonoscillatory first order accurate scheme. Numerical experiments are presented to demonstrate the performance of these new schemes.

  12. Quantification of tertiary structural conservation despite primary sequence drift in the globin fold.

    PubMed

    Aronson, H E; Royer, W E; Hendrickson, W A

    1994-10-01

    The globin family of protein structures was the first for which it was recognized that tertiary structure can be highly conserved even when primary sequences have diverged to a virtually undetectable level of similarity. This principle of structural inertia in molecular evolution is now evident for many other protein families. We have performed a systematic comparison of the sequences and structures of 6 representative hemoglobin subunits as diverse in origin as plants, clams, and humans. Our analysis is based on a 97-residue helical core in common to all 6 structures. Amino acid sequence identities range from 12.4% to 42.3% in pairwise comparisons, and, despite these variations, the maximal RMS deviation in alpha-carbon positions is 3.02 A. Overall, sequence similarity and structural deviation are significantly anticorrelated, with a correlation coefficient of -0.71, but for a set of structures having under 20% pairwise identity, this anticorrelation falls to -0.38, which emphasizes the weak connection between a specific sequence and the tertiary fold. There is substantial variability in structure outside the helical core, and functional characteristics of these globins also differ appreciably. Nevertheless, despite variations in detail that the sequence dissimilarities and functional differences imply, the core structures of these globins remain remarkably preserved. PMID:7849587

  13. Dynamic behavior of an intrinsically unstructured linker domain is conserved in the face of negligible amino acid sequence conservation.

    PubMed

    Daughdrill, Gary W; Narayanaswami, Pranesh; Gilmore, Sara H; Belczyk, Agniezka; Brown, Celeste J

    2007-09-01

    Proteins or regions of proteins that do not form compact globular structures are classified as intrinsically unstructured proteins (IUPs). IUPs are common in nature and have essential molecular functions, but even a limited understanding of the evolution of their dynamic behavior is lacking. The primary objective of this work was to test the evolutionary conservation of dynamic behavior for a particular class of IUPs that form intrinsically unstructured linker domains (IULD) that tether flanking folded domains. This objective was accomplished by measuring the backbone flexibility of several IULD homologues using nuclear magnetic resonance (NMR) spectroscopy. The backbone flexibility of five IULDs, representing three kingdoms, was measured and analyzed. Two IULDs from animals, one IULD from fungi, and two IULDs from plants showed similar levels of backbone flexibility that were consistent with the absence of a compact globular structure. In contrast, the amino acid sequences of the IULDs from these three taxa showed no significant similarity. To investigate how the dynamic behavior of the IULDs could be conserved in the absence of detectable sequence conservation, evolutionary rate studies were performed on a set of nine mammalian IULDs. The results of this analysis showed that many sites in the IULD are evolving neutrally, suggesting that dynamic behavior can be maintained in the absence of natural selection. This work represents the first experimental test of the evolutionary conservation of dynamic behavior and demonstrates that amino acid sequence conservation is not required for the conservation of dynamic behavior and presumably molecular function. PMID:17721672

  14. 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

  15. Polyclonal antibody against conserved sequences of mce1A protein blocks MTB infection in macrophages.

    PubMed

    Sivagnanam, Sasikala; Namasivayam, Nalini; Chellam, Rajamanickam

    2012-03-01

    The pathogenesis of Mycobacterium tuberculosis is largely due to its ability to enter and survive within human macrophages. It is suggested that a specific protein namely mammalian cell entry protein is involved in the pathogenesis and the specific gene for this protein mce1A has been identified in several pathogenic organisms such as Rickettsia, Shigella, Escherichia coli, Helicobacter, Streptomyces, Klebsiella, Vibrio, Neisseria, Rhodococcus, Nocardioides, Saccharopolyspora erthyrae, and Pseudomonas. Analysis of mce1 operons in the above mentioned organisms through bioinformatics tools has revealed the presence of unique sequences (conserved regions) suggesting that these sequences may be involved in the process of infection. Presently, the mce1A full-length (1,365 bp) region from Mycobacterium bovis and its conserved regions (303 bp) were cloned in to an expression vector and the purified expressed proteins of molecular weight ~47 and ~11 kDa, respectively, were injected to rabbits to raise the polyclonal antibodies. The purified polyclonal antibodies were checked for their ability to inhibit the Mycobacterium infection in cultured human macrophages. In macrophage invasion assay, when antibody added at high concentration, decrease in viable counts was observed in all cell cultures within the first 5 days after infection, where the intracellular bacterial CFU obtained from the infected MTB increased by the 3rd day at low concentration of antibody. The macrophage invasion assay has indicated that the purified antibodies of mce1A conserved region can inhibit the infection of Mycobacterium. PMID:22159737

  16. Forest conservation delivers highly variable coral reef conservation outcomes.

    PubMed

    Klein, Carissa J; Jupiter, Stacy D; Selig, Elizabeth R; Watts, Matthew E; Halpern, Benjamin S; Kamal, Muhammad; Roelfsema, Chris; Possingham, Hugh P

    2012-06-01

    Coral reefs are threatened by human activities on both the land (e.g., deforestation) and the sea (e.g., overfishing). Most conservation planning for coral reefs focuses on removing threats in the sea, neglecting management actions on the land. A more integrated approach to coral reef conservation, inclusive of land-sea connections, requires an understanding of how and where terrestrial conservation actions influence reefs. We address this by developing a land-sea planning approach to inform fine-scale spatial management decisions and test it in Fiji. Our aim is to determine where the protection of forest can deliver the greatest return on investment for coral reef ecosystems. To assess the benefits of conservation to coral reefs, we estimate their relative condition as influenced by watershed-based pollution and fishing. We calculate the cost-effectiveness of protecting forest and find that investments deliver rapidly diminishing returns for improvements to relative reef condition. For example, protecting 2% of forest in one area is almost 500 times more beneficial than protecting 2% in another area, making prioritization essential. For the scenarios evaluated, relative coral reef condition could be improved by 8-58% if all remnant forest in Fiji were protected rather than deforested. Finally, we determine the priority of each coral reef for implementing a marine protected area when all remnant forest is protected for conservation. The general results will support decisions made by the Fiji Protected Area Committee as they establish a national protected area network that aims to protect 20% of the land and 30% of the inshore waters by 2020. Although challenges remain, we can inform conservation decisions around the globe by tackling the complex issues relevant to integrated land-sea planning. PMID:22827132

  17. Evolutionary sequence comparisons using high-density oligonucleotide arrays.

    PubMed

    Hacia, J G; Makalowski, W; Edgemon, K; Erdos, M R; Robbins, C M; Fodor, S P; Brody, L C; Collins, F S

    1998-02-01

    We explored the utility of high-density oligonucleotide arrays (DNA chips) for obtaining sequence information from homologous genes in closely related species. Orthologues of the human BRCA1 exon 11, all approximately 3.4 kb in length and ranging from 98.2% to 83.5% nucleotide identity, were subjected to hybridization-based and conventional dideoxysequencing analysis. Retrospective guidelines for identifying high-fidelity hybridization-based sequence calls were formulated based upon dideoxysequencing results. Prospective application of these rules yielded base-calling with at least 98.8% accuracy over orthologous sequence tracts shown to have approximately 99% identity. For higher primate sequences with greater than 97% nucleotide identity, base-calling was made with at least 99.91% accuracy covering a minimum of 97% of the sequence. Using a second-tier confirmatory hybridization chip strategy, shown in several cases to confirm the identity of predicted sequence changes, the complete sequence of the chimpanzee, gorilla and orangutan orthologues should be deducible solely through hybridization-based methodologies. Analysis of less highly conserved orthologues can still identify conserved nucleotide tracts of at least 15 nucleotides and can provide useful information for designing primers. DNA-chip based assays can be a valuable new technology for obtaining high-throughput cost-effective sequence information from related genomes. PMID:9462745

  18. A search for conserved sequences in coding regions reveals that the let-7 microRNA targets Dicer within its coding sequence

    PubMed Central

    Forman, Joshua J.; Legesse-Miller, Aster; Coller, Hilary A.

    2008-01-01

    Recognition sites for microRNAs (miRNAs) have been reported to be located in the 3′ untranslated regions of transcripts. In a computational screen for highly conserved motifs within coding regions, we found an excess of sequences conserved at the nucleotide level within coding regions in the human genome, the highest scoring of which are enriched for miRNA target sequences. To validate our results, we experimentally demonstrated that the let-7 miRNA directly targets the miRNA-processing enzyme Dicer within its coding sequence, thus establishing a mechanism for a miRNA/Dicer autoregulatory negative feedback loop. We also found computational evidence to suggest that miRNA target sites in coding regions and 3′ UTRs may differ in mechanism. This work demonstrates that miRNAs can directly target transcripts within their coding region in animals, and it suggests that a complete search for the regulatory targets of miRNAs should be expanded to include genes with recognition sites within their coding regions. As more genomes are sequenced, the methodological approach that we used for identifying motifs with high sequence conservation will be increasingly valuable for detecting functional sequence motifs within coding regions. PMID:18812516

  19. Vertebrate paralogous conserved noncoding sequences may be related to gene expressions in brain.

    PubMed

    Matsunami, Masatoshi; Saitou, Naruya

    2013-01-01

    Vertebrate genomes include gene regulatory elements in protein-noncoding regions. A part of gene regulatory elements are expected to be conserved according to their functional importance, so that evolutionarily conserved noncoding sequences (CNSs) might be good candidates for those elements. In addition, paralogous CNSs, which are highly conserved among both orthologous loci and paralogous loci, have the possibility of controlling overlapping expression patterns of their adjacent paralogous protein-coding genes. The two-round whole-genome duplications (2R WGDs), which most probably occurred in the vertebrate common ancestors, generated large numbers of paralogous protein-coding genes and their regulatory elements. These events could contribute to the emergence of vertebrate features. However, the evolutionary history and influences of the 2R WGDs are still unclear, especially in noncoding regions. To address this issue, we identified paralogous CNSs. Region-focused Basic Local Alignment Search Tool (BLAST) search of each synteny block revealed 7,924 orthologous CNSs and 309 paralogous CNSs conserved among eight high-quality vertebrate genomes. Paralogous CNSs we found contained 115 previously reported ones and newly detected 194 ones. Through comparisons with VISTA Enhancer Browser and available ChIP-seq data, one-third (103) of paralogous CNSs detected in this study showed gene regulatory activity in the brain at several developmental stages. Their genomic locations are highly enriched near the transcription factor-coding regions, which are expressed in brain and neural systems. These results suggest that paralogous CNSs are conserved mainly because of maintaining gene expression in the vertebrate brain. PMID:23267051

  20. BlockLogo: visualization of peptide and sequence motif conservation.

    PubMed

    Olsen, Lars Rønn; Kudahl, Ulrich Johan; Simon, Christian; Sun, Jing; Schönbach, Christian; Reinherz, Ellis L; Zhang, Guang Lan; Brusic, Vladimir

    2013-12-31

    BlockLogo is a web-server application for the visualization of protein and nucleotide fragments, continuous protein sequence motifs, and discontinuous sequence motifs using calculation of block entropy from multiple sequence alignments. The user input consists of a multiple sequence alignment, selection of motif positions, type of sequence, and output format definition. The output has BlockLogo along with the sequence logo, and a table of motif frequencies. We deployed BlockLogo as an online application and have demonstrated its utility through examples that show visualization of T-cell epitopes and B-cell epitopes (both continuous and discontinuous). Our additional example shows a visualization and analysis of structural motifs that determine the specificity of peptide binding to HLA-DR molecules. The BlockLogo server also employs selected experimentally validated prediction algorithms to enable on-the-fly prediction of MHC binding affinity to 15 common HLA class I and class II alleles as well as visual analysis of discontinuous epitopes from multiple sequence alignments. It enables the visualization and analysis of structural and functional motifs that are usually described as regular expressions. It provides a compact view of discontinuous motifs composed of distant positions within biological sequences. BlockLogo is available at: http://research4.dfci.harvard.edu/cvc/blocklogo/ and http://met-hilab.bu.edu/blocklogo/. PMID:24001880

  1. 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

  2. High-bay Lighting Energy Conservation Measures

    Energy Science and Technology Software Center (ESTSC)

    2010-12-31

    This software requires inputs of simple high-bay lighting system inventory information and calculates the energy and cost benefits of various retrofit opportunities. This tool includes energy conservation measures for: 1000 Watt to 750 Watt High-pressure Sodium lighting retrofit, 400 Watt to 360 Watt High Pressure Sodium lighting retrofit, High Intensity Discharge to T5 lighting retrofit, High Intensity Discharge to T8 lighting retrofit, and Daylighting. This tool calculates energy savings, demand reduction, cost savings, building lifemore » cycle costs including: simple payback, discounted payback, net-present value, and savings to investment ratio. In addition this tool also displays the environmental benefits of a project.« less

  3. Readings in Wildlife and Fish Conservation, High School Conservation Curriculum Project.

    ERIC Educational Resources Information Center

    Ensminger, Jack

    This publication is a tentative edition of readings on Wildlife and Fish Conservation in Louisiana, and as such it forms part of one of the four units of study designed for an experimental high school course, the "High School Conservation Curriculum Project." The other three units are concerned with Forest Conervation, Soil and Water Conservation,…

  4. High speed nucleic acid sequencing

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2011-05-17

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid. Each type of labeled nucleotide comprises an acceptor fluorophore attached to a phosphate portion of the nucleotide such that the fluorophore is removed upon incorporation into a growing strand. Fluorescent signal is emitted via fluorescent resonance energy transfer between the donor fluorophore and the acceptor fluorophore as each nucleotide is incorporated into the growing strand. The sequence is deduced by identifying which base is being incorporated into the growing strand.

  5. Distinct Functional Constraints Partition Sequence Conservation in a cis-Regulatory Element

    PubMed Central

    Ruvinsky, Ilya

    2011-01-01

    Different functional constraints contribute to different evolutionary rates across genomes. To understand why some sequences evolve faster than others in a single cis-regulatory locus, we investigated function and evolutionary dynamics of the promoter of the Caenorhabditis elegans unc-47 gene. We found that this promoter consists of two distinct domains. The proximal promoter is conserved and is largely sufficient to direct appropriate spatial expression. The distal promoter displays little if any conservation between several closely related nematodes. Despite this divergence, sequences from all species confer robustness of expression, arguing that this function does not require substantial sequence conservation. We showed that even unrelated sequences have the ability to promote robust expression. A prominent feature shared by all of these robustness-promoting sequences is an AT-enriched nucleotide composition consistent with nucleosome depletion. Because general sequence composition can be maintained despite sequence turnover, our results explain how different functional constraints can lead to vastly disparate rates of sequence divergence within a promoter. PMID:21655084

  6. Comparison of mealybug (Planococcus lilacinus) and fruit fly genomes: isolation and analysis of conserved sequences and their utility in studying synteny in the mealybug.

    PubMed

    Mohan, K N; Rani, B S; Selvam, S; Debarshi, S; Kadandale, J S

    2007-01-01

    By using ligation-mediated PCR products from mealybug DNA as tester and biotinylated fly DNA as driver, we recovered a fraction of the tester that remains hybridized to driver following high-stringency washing conditions. This fraction is expected to contain mealybug sequences conserved in the fly (MCF). Reciprocal experiments enabled the isolation of fly sequences conserved in the mealybug (FCM). Coding sequences among MCF show amino acid identities >40% with fly proteins, allowing a reliable identification of orthologs. Three sequences from the fly cytogenetic positions 98-99 were hybridized onto mealybug chromosomes and the results identified differences in synteny between the two species. Taken together, our results present a method for direct isolation of sequences conserved between an 'orphan' (mealybug) genome and a 'reference' (fly) genome and showed that these sequences can be used to study chromosome synteny in the mealybug. PMID:18253039

  7. 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.

  8. 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.

  9. 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

  10. The BsaHI restriction-modification system: Cloning, sequencing and analysis of conserved motifs

    PubMed Central

    Neely, Robert K; Roberts, Richard J

    2008-01-01

    Background Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC. Results The DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank accession #EU386360), cloned and expressed in E. coli. Both the restriction endonuclease and methyltransferase enzymes share significant similarity with a group of 6 other enzymes comprising the restriction-modification systems HgiDI and HgiGI and the putative HindVP, NlaCORFDP, NpuORFC228P and SplZORFNP restriction-modification systems. A sequence alignment of these homologues shows that their amino acid sequences are largely conserved and highlights several motifs of interest. We target one such conserved motif, reading SPERRFD, at the C-terminal end of the bsaHIR gene. A mutational analysis of these amino acids indicates that the motif is crucial for enzymatic activity. Sequence alignment of the methyltransferase gene reveals a short motif within the target recognition domain that is conserved among enzymes recognising the same sequences. Thus, this motif may be used as a diagnostic tool to define the recognition sequences of the cytosine C5 methyltransferases. Conclusion We have cloned and sequenced the BsaHI restriction and modification enzymes. We have identified a region of the R. BsaHI enzyme that is crucial for its activity. Analysis of the amino acid sequence of the BsaHI methyltransferase enzyme led us to propose two new motifs that can be used in the diagnosis of the recognition sequence of the cytosine C5-methyltransferases. PMID:18479503

  11. 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

  12. Whole genome sequencing of Ethiopian highlanders reveals conserved hypoxia tolerance genes

    PubMed Central

    2014-01-01

    Background Although it has long been proposed that genetic factors contribute to adaptation to high altitude, such factors remain largely unverified. Recent advances in high-throughput sequencing have made it feasible to analyze genome-wide patterns of genetic variation in human populations. Since traditionally such studies surveyed only a small fraction of the genome, interpretation of the results was limited. Results We report here the results of the first whole genome resequencing-based analysis identifying genes that likely modulate high altitude adaptation in native Ethiopians residing at 3,500 m above sea level on Bale Plateau or Chennek field in Ethiopia. Using cross-population tests of selection, we identify regions with a significant loss of diversity, indicative of a selective sweep. We focus on a 208 kbp gene-rich region on chromosome 19, which is significant in both of the Ethiopian subpopulations sampled. This region contains eight protein-coding genes and spans 135 SNPs. To elucidate its potential role in hypoxia tolerance, we experimentally tested whether individual genes from the region affect hypoxia tolerance in Drosophila. Three genes significantly impact survival rates in low oxygen: cic, an ortholog of human CIC, Hsl, an ortholog of human LIPE, and Paf-AHα, an ortholog of human PAFAH1B3. Conclusions Our study reveals evolutionarily conserved genes that modulate hypoxia tolerance. In addition, we show that many of our results would likely be unattainable using data from exome sequencing or microarray studies. This highlights the importance of whole genome sequencing for investigating adaptation by natural selection. PMID:24555826

  13. Conservation of plasmid DNA sequences in coronatine-producing pathovars of Pseudomonas syringae

    SciTech Connect

    Bender, C.L.; Young, S.A. ); Mitchell, R.E. )

    1991-04-01

    In Pseudomonas syringae pv. tomato PT23.2, plasmid pPT23A (101 kb) is involved in synthesis of the phytotoxin coronatine. The physical characterization of mutations that abolished coronatine production indicated that at least 30 kb of pPT23A DNA are required for toxin synthesis. In the present study, {sup 32}P-labeled DNA fragments from the 30-kb region of pPT23A hybridized to plasmid DNAs from several coronatine-producing pathovars of P. syringae under conditions of high stringency. These experiments indicated that this region of pPT23A was strongly conserved in large plasmids (90 to 105 kb) that reside in P. syringae pv. atropurpurea, glycinea, and morsprunorum. The functional significance of the observed homology was demonstrated in marker-exchange experiments in which Tn5-inactivated sequences from the 30-kb region of pPT23A were used to mutate coronatine synthesis genes in the three heterologous pathovars. Physical characterization of the Tn5 insertions generated by marker exchange indicated that genes controlling coronatine synthesis in P. syringae pv. atropurpurea 1304, glycinea 4180, and morsprunorum 567 and 3714 were located on the large indigenous plasmids where homology was originally detected. Therefore, coronatine biosynthesis genes are strongly conserved in the plasmid DNAs of four producing pathovars, despite their disparate origins (California, Japan, New Zealand, Great Britain, and Italy).

  14. FRESCO: Referential compression of highly similar sequences.

    PubMed

    Wandelt, Sebastian; Leser, Ulf

    2013-01-01

    In many applications, sets of similar texts or sequences are of high importance. Prominent examples are revision histories of documents or genomic sequences. Modern high-throughput sequencing technologies are able to generate DNA sequences at an ever-increasing rate. In parallel to the decreasing experimental time and cost necessary to produce DNA sequences, computational requirements for analysis and storage of the sequences are steeply increasing. Compression is a key technology to deal with this challenge. Recently, referential compression schemes, storing only the differences between a to-be-compressed input and a known reference sequence, gained a lot of interest in this field. In this paper, we propose a general open-source framework to compress large amounts of biological sequence data called Framework for REferential Sequence COmpression (FRESCO). Our basic compression algorithm is shown to be one to two orders of magnitudes faster than comparable related work, while achieving similar compression ratios. We also propose several techniques to further increase compression ratios, while still retaining the advantage in speed: 1) selecting a good reference sequence; and 2) rewriting a reference sequence to allow for better compression. In addition,we propose a new way of further boosting the compression ratios by applying referential compression to already referentially compressed files (second-order compression). This technique allows for compression ratios way beyond state of the art, for instance,4,000:1 and higher for human genomes. We evaluate our algorithms on a large data set from three different species (more than 1,000 genomes, more than 3 TB) and on a collection of versions of Wikipedia pages. Our results show that real-time compression of highly similar sequences at high compression ratios is possible on modern hardware. PMID:24524158

  15. Novel sequences encoding venom C-type lectins are conserved in phylogenetically and geographically distinct Echis and Bitis viper species.

    PubMed

    Harrison, R A; Oliver, J; Hasson, S S; Bharati, K; Theakston, R D G

    2003-10-01

    Envenoming by Echis saw scaled vipers and Bitis arietans puff adders is the leading cause of death and morbidity in Africa due to snake bite. Despite their medical importance, the composition and constituent functionality of venoms from these vipers remains poorly understood. Here, we report the cloning of cDNA sequences encoding seven clusters or isoforms of the haemostasis-disruptive C-type lectin (CTL) proteins from the venom glands of Echis ocellatus, E. pyramidum leakeyi, E. carinatus sochureki and B. arietans. All these CTL sequences encoded the cysteine scaffold that defines the carbohydrate-recognition domain of mammalian CTLs. All but one of the Echis and Bitis CTL sequences showed greater sequence similarity to the beta than alpha CTL subunits in venoms of related Asian and American vipers. Four of the new CTL clusters showed marked inter-cluster sequence conservation across all four viper species which were significantly different from that of previously published viper CTLs. The other three Echis and Bitis CTL clusters showed varying degrees of sequence similarity to published viper venom CTLs. Because viper venom CTLs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis and Bitis CTLs on the basis of sequence alone. The extraordinary level of inter-specific and inter-generic sequence conservation exhibited by the Echis and Bitis CTLs leads us to speculate that antibodies to representative molecules should neutralise the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East and the Indian subcontinent. PMID:14557069

  16. 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

  17. The Most Deeply Conserved Noncoding Sequences in Plants Serve Similar Functions to Those in Vertebrates Despite Large Differences in Evolutionary Rates[W

    PubMed Central

    Burgess, Diane; Freeling, Michael

    2014-01-01

    In vertebrates, conserved noncoding elements (CNEs) are functionally constrained sequences that can show striking conservation over >400 million years of evolutionary distance and frequently are located megabases away from target developmental genes. Conserved noncoding sequences (CNSs) in plants are much shorter, and it has been difficult to detect conservation among distantly related genomes. In this article, we show not only that CNS sequences can be detected throughout the eudicot clade of flowering plants, but also that a subset of 37 CNSs can be found in all flowering plants (diverging ∼170 million years ago). These CNSs are functionally similar to vertebrate CNEs, being highly associated with transcription factor and development genes and enriched in transcription factor binding sites. Some of the most highly conserved sequences occur in genes encoding RNA binding proteins, particularly the RNA splicing–associated SR genes. Differences in sequence conservation between plants and animals are likely to reflect differences in the biology of the organisms, with plants being much more able to tolerate genomic deletions and whole-genome duplication events due, in part, to their far greater fecundity compared with vertebrates. PMID:24681619

  18. 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

  19. Genotyping by sequencing resolves shallow population structure to inform conservation of Chinook salmon (Oncorhynchus tshawytscha).

    PubMed

    Larson, Wesley A; Seeb, Lisa W; Everett, Meredith V; Waples, Ryan K; Templin, William D; Seeb, James E

    2014-03-01

    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

  20. PCR-based study of conserved and variable DNA sequences of Tritrichomonas foetus isolates from Saskatchewan, Canada.

    PubMed Central

    Riley, D E; Wagner, B; Polley, L; Krieger, J N

    1995-01-01

    The protozoan parasite Tritrichomonas foetus causes infertility and spontaneous abortion in cattle. In Saskatchewan, Canada, the culture prevalence of trichomonads was 65 of 1,048 (6%) among 1,048 bulls tested within a 1-year period ending in April 1994. Saskatchewan was previously thought to be free of the parasite. To confirm the culture results, possible T. foetus DNA presence was determined by the PCR. All of the 16 culture-positive isolates tested were PCR positive by a single-band test, but one PCR product was weak. DNA fingerprinting by both T17 PCR and randomly amplified polymorphic DNA PCR revealed genetic variation or polymorphism among the T. foetus isolates. T17 PCR also revealed conserved loci that distinguished these T. foetus isolates from Trichomonas vaginalis, from a variety of other protozoa, and from prokaryotes. TCO-1 PCR, a PCR test designed to sample DNA sequence homologous to the 5' flank of a highly conserved cell division control gene, detected genetic polymorphism at low stringency and a conserved, single locus at higher stringency. These findings suggested that T. foetus isolates exhibit both conserved genetic loci and polymorphic loci detectable by independent PCR methods. Both conserved and polymorphic genetic loci may prove useful for improved clinical diagnosis of T. foetus. The polymorphic loci detected by PCR suggested either a long history of infection or multiple lines of T. foetus infection in Saskatchewan. Polymorphic loci detected by PCR may provide data for epidemiologic studies of T. foetus. PMID:7615746

  1. Auditory sequence processing reveals evolutionarily conserved regions of frontal cortex in macaques and humans.

    PubMed

    Wilson, Benjamin; Kikuchi, Yukiko; Sun, Li; Hunter, David; Dick, Frederic; Smith, Kenny; Thiele, Alexander; Griffiths, Timothy D; Marslen-Wilson, William D; Petkov, Christopher I

    2015-01-01

    An evolutionary account of human language as a neurobiological system must distinguish between human-unique neurocognitive processes supporting language and evolutionarily conserved, domain-general processes that can be traced back to our primate ancestors. Neuroimaging studies across species may determine whether candidate neural processes are supported by homologous, functionally conserved brain areas or by different neurobiological substrates. Here we use functional magnetic resonance imaging in Rhesus macaques and humans to examine the brain regions involved in processing the ordering relationships between auditory nonsense words in rule-based sequences. We find that key regions in the human ventral frontal and opercular cortex have functional counterparts in the monkey brain. These regions are also known to be associated with initial stages of human syntactic processing. This study raises the possibility that certain ventral frontal neural systems, which play a significant role in language function in modern humans, originally evolved to support domain-general abilities involved in sequence processing. PMID:26573340

  2. Auditory sequence processing reveals evolutionarily conserved regions of frontal cortex in macaques and humans

    PubMed Central

    Wilson, Benjamin; Kikuchi, Yukiko; Sun, Li; Hunter, David; Dick, Frederic; Smith, Kenny; Thiele, Alexander; Griffiths, Timothy D.; Marslen-Wilson, William D.; Petkov, Christopher I.

    2015-01-01

    An evolutionary account of human language as a neurobiological system must distinguish between human-unique neurocognitive processes supporting language and evolutionarily conserved, domain-general processes that can be traced back to our primate ancestors. Neuroimaging studies across species may determine whether candidate neural processes are supported by homologous, functionally conserved brain areas or by different neurobiological substrates. Here we use functional magnetic resonance imaging in Rhesus macaques and humans to examine the brain regions involved in processing the ordering relationships between auditory nonsense words in rule-based sequences. We find that key regions in the human ventral frontal and opercular cortex have functional counterparts in the monkey brain. These regions are also known to be associated with initial stages of human syntactic processing. This study raises the possibility that certain ventral frontal neural systems, which play a significant role in language function in modern humans, originally evolved to support domain-general abilities involved in sequence processing. PMID:26573340

  3. Genome-wide identification of conserved regulatory function in diverged sequences

    PubMed Central

    Taher, Leila; McGaughey, David M.; Maragh, Samantha; Aneas, Ivy; Bessling, Seneca L.; Miller, Webb; Nobrega, Marcelo A.; McCallion, Andrew S.; Ovcharenko, Ivan

    2011-01-01

    Plasticity of gene regulatory encryption can permit DNA sequence divergence without loss of function. Functional information is preserved through conservation of the composition of transcription factor binding sites (TFBS) in a regulatory element. We have developed a method that can accurately identify pairs of functional noncoding orthologs at evolutionarily diverged loci by searching for conserved TFBS arrangements. With an estimated 5% false-positive rate (FPR) in approximately 3000 human and zebrafish syntenic loci, we detected approximately 300 pairs of diverged elements that are likely to share common ancestry and have similar regulatory activity. By analyzing a pool of experimentally validated human enhancers, we demonstrated that 7/8 (88%) of their predicted functional orthologs retained in vivo regulatory control. Moreover, in 5/7 (71%) of assayed enhancer pairs, we observed concordant expression patterns. We argue that TFBS composition is often necessary to retain and sufficient to predict regulatory function in the absence of overt sequence conservation, revealing an entire class of functionally conserved, evolutionarily diverged regulatory elements that we term “covert.” PMID:21628450

  4. Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis

    PubMed Central

    Spangler, Jacob B.; Feltus, Frank Alex

    2013-01-01

    Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression. PMID:23675377

  5. 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

  6. Conservation of uORF repressiveness and sequence features in mouse, human and zebrafish.

    PubMed

    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

  7. The complete mitochondrial genome sequence of the tubeworm Lamellibrachia satsuma and structural conservation in the mitochondrial genome control regions of Order Sabellida.

    PubMed

    Patra, Ajit Kumar; Kwon, Yong Min; Kang, Sung Gyun; Fujiwara, Yoshihiro; Kim, Sang-Jin

    2016-04-01

    The control region of the mitochondrial genomes shows high variation in conserved sequence organizations, which follow distinct evolutionary patterns in different species or taxa. In this study, we sequenced the complete mitochondrial genome of Lamellibrachia satsuma from the cold-seep region of Kagoshima Bay, as a part of whole genome study and extensively studied the structural features and patterns of the control region sequences. We obtained 15,037 bp of mitochondrial genome using Illumina sequencing and identified the non-coding AT-rich region or control region (354 bp, AT=83.9%) located between trnH and trnR. We found 7 conserved sequence blocks (CSB), scattered throughout the control region of L. satsuma and other taxa of Annelida. The poly-TA stretches, which commonly form the stem of multiple stem-loop structures, are most conserved in the CSB-I and CSB-II regions. The mitochondrial genome of L. satsuma encodes a unique repetitive sequence in the control region, which forms a unique secondary structure in comparison to Lamellibrachia luymesi. Phylogenetic analyses of all protein-coding genes indicate that L. satsuma forms a monophyletic clade with L. luymesi along with other tubeworms found in cold-seep regions (genera: Lamellibrachia, Escarpia, and Seepiophila). In general, the control region sequences of Annelida could be aligned with certainty within each genus, and to some extent within the family, but with a higher rate of variation in conserved regions. PMID:26776396

  8. Highly multiplexed DNA sequencing by capillary electrophoresis

    SciTech Connect

    Yeung, E.S.; Ueno, K.; Chang, H.T.

    1994-12-31

    It is obvious that irrespective of whichever basic technology is eventually selected to sequence the entire human genome there are substantial gains to be made if a high degree of multiplexing of parallel runs can be implemented. Such multiplexing should not involve expensive instrumentation and should not require additional personnel, or else the main objective of cost reduction will not be satisfied even though the total time for sequencing is reduced. In the last two years, several research groups have shown that capillary electrophoresis (CE) is an attractive alternative for DNA sequencing. Part of the improvement in sequencing speed in CE is counteracted by the inherent ability of slab gels for accommodating multiple lanes in a single run. Recently, the authors have developed several excitation schemes for highly multiplexed capillary electrophoresis. Detection at the pM level was demonstrated. The authors report here the use of a novel excitation geometry to simultaneously monitor 100 capillary tubes during electrophoresis. This represents a truly parallel multiplexing scheme for high-speed DNA sequencing.

  9. Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences.

    PubMed Central

    Center, R J; Kemp, B E; Poumbourios, P

    1997-01-01

    Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization. PMID:9188654

  10. 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

  11. Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase.

    PubMed

    Ranjani, Velayudhan; Janeček, Stefan; Chai, Kian Piaw; Shahir, Shafinaz; Abdul Rahman, Raja Noor Zaliha Raja; 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, k(cat) and k(cat)/K(m) 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

  12. An approach to delineate primers for a group of poorly conserved sequences incorporating the common motif region.

    PubMed

    Sahu, Mousumi; Sahu, Jagajjit; Sahoo, Smita; Dehury, Budheswar; Sarma, Kishore; Sarmah, Ranjan; Sen, Priyabrata; Modi, Mahendra Kumar; Barooah, Madhumita

    2012-01-01

    Glutathione synthetase (gshB) has previously been reported to confer tolerance to acidic soil condition in Rhizobium species. Cloning the gene coding for this enzyme necessitates the designing of proper primer sets which in turn depends on the identification of high quality sequence similarity in multiple global alignments. In this experiment, a group of homologous gene sequences related to gshB gene (accession no: gi-86355669:327589-328536) of Rhizobium etli CFN 42, were extracted from NCBI nucleotide sequence databases using BLASTN and were analyzed for designing degenerate primers. However, the T-coffee multiple global alignment results did not show any block of conserved region for the above sequence set to design the primers. Therefore, we attempted to identify the location of common motif region based on multiple local alignments employing the MEME algorithm supported with MAST and Primer3. The results revealed some common motif regions that enabled us to design the primer sets for related gshB gene sequences. The result will be validated in wet lab. PMID:22419837

  13. Cytochrome Oxidase I (COI) sequence conservation and variation patterns in the yellowfin and longtail tunas.

    PubMed

    Kunal, Swaraj Priyaranjan; Kumar, Girish

    2013-01-01

    Tunas are commercially important fishery worldwide. There are at least 13 species of tuna belonging to three genera, out of which genus Thunnus has maximum eight species. On the basis of their availability, they can be characterised as oceanic such as Thunnus albacares (yellowfin tuna) or coastal such as Thunnus tonggol (longtail tuna). Although these two are different species, morphological differentiation can only be seen in mature individuals, hence misidentification may result in erroneous data set, which ultimately affect conservation strategies. The mitochondrial DNA cytochrome oxidase c subunit 1 (COI) gene is one of the most popular markers for population genetic and phylogeographic studies across the animal kingdom. The present study aims to study the sequence conservation and variation in mitochondrial Cytochrome Oxidase I (COI) between these two species of tuna. COI sequence analysis of yellowfin and longtail revealed the close relationship between them in Thunnus genera. The present study is the first direct comparison of mitochondrial COI sequences of these two tuna species. PMID:23649742

  14. 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

  15. Comparative sequence analysis suggests a conserved gating mechanism for TRP channels

    PubMed Central

    Palovcak, Eugene; Delemotte, Lucie; Klein, Michael L.

    2015-01-01

    The transient receptor potential (TRP) channel superfamily plays a central role in transducing diverse sensory stimuli in eukaryotes. Although dissimilar in sequence and domain organization, all known TRP channels act as polymodal cellular sensors and form tetrameric assemblies similar to those of their distant relatives, the voltage-gated potassium (Kv) channels. Here, we investigated the related questions of whether the allosteric mechanism underlying polymodal gating is common to all TRP channels, and how this mechanism differs from that underpinning Kv channel voltage sensitivity. To provide insight into these questions, we performed comparative sequence analysis on large, comprehensive ensembles of TRP and Kv channel sequences, contextualizing the patterns of conservation and correlation observed in the TRP channel sequences in light of the well-studied Kv channels. We report sequence features that are specific to TRP channels and, based on insight from recent TRPV1 structures, we suggest a model of TRP channel gating that differs substantially from the one mediating voltage sensitivity in Kv channels. The common mechanism underlying polymodal gating involves the displacement of a defect in the H-bond network of S6 that changes the orientation of the pore-lining residues at the hydrophobic gate. PMID:26078053

  16. Conserved Noncoding Sequences Highlight Shared Components of Regulatory Networks in Dicotyledonous Plants[W

    PubMed Central

    Baxter, Laura; Jironkin, Aleksey; Hickman, Richard; Moore, Jay; Barrington, Christopher; Krusche, Peter; Dyer, Nigel P.; Buchanan-Wollaston, Vicky; Tiskin, Alexander; Beynon, Jim; Denby, Katherine; Ott, Sascha

    2012-01-01

    Conserved noncoding sequences (CNSs) in DNA are reliable pointers to regulatory elements controlling gene expression. Using a comparative genomics approach with four dicotyledonous plant species (Arabidopsis thaliana, papaya [Carica papaya], poplar [Populus trichocarpa], and grape [Vitis vinifera]), we detected hundreds of CNSs upstream of Arabidopsis genes. Distinct positioning, length, and enrichment for transcription factor binding sites suggest these CNSs play a functional role in transcriptional regulation. The enrichment of transcription factors within the set of genes associated with CNS is consistent with the hypothesis that together they form part of a conserved transcriptional network whose function is to regulate other transcription factors and control development. We identified a set of promoters where regulatory mechanisms are likely to be shared between the model organism Arabidopsis and other dicots, providing areas of focus for further research. PMID:23110901

  17. 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

  18. A Collection of Conserved Noncoding Sequences to Study Gene Regulation in Flowering Plants.

    PubMed

    Van de Velde, Jan; Van Bel, Michiel; Vaneechoutte, Dries; Vandepoele, Klaas

    2016-08-01

    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

  19. Comparison of Flavivirus Universal Primer Pairs and Development of a Rapid, Highly Sensitive Heminested Reverse Transcription-PCR Assay for Detection of Flaviviruses Targeted to a Conserved Region of the NS5 Gene Sequences

    PubMed Central

    Scaramozzino, Natale; Crance, Jean-Marc; Jouan, Alain; DeBriel, Dominique A.; Stoll, Françoise; Garin, Daniel

    2001-01-01

    Arthropod-transmitted flaviviruses are responsible for considerable morbidity and mortality, causing severe encephalitic, hemorrhagic, and febrile illnesses in humans. Because there are no specific clinical symptoms for infection by a determined virus and because different arboviruses could be present in the same area, a genus diagnosis by PCR would be a useful first-line diagnostic method. The six published Flavivirus genus primer pairs localized in the NS1, NS3, NS5, and 3′ NC regions were evaluated in terms of specificity and sensitivity with flaviviruses (including the main viruses pathogenic for humans) at a titer of 105 50% tissue culture infectious doses (TCID50s) ml−1 with a common identification step by agarose gel electrophoresis. Only one NS5 primer pair allowed the detection of all tested flaviviruses with the sensitivity limit of 105 TCID50s ml−1. Using a heminested PCR with new primers designed in the same region after an alignment of 30 different flaviviruses, the sensitivity of reverse transcription-PCR was improved and allowed the detection of about 200 infectious doses ml−1 with all of the tick- and mosquito-borne flaviviruses tested. It was confirmed that the sequenced amplified products in the NS5 region allowed predictability of flavivirus species by dendrogram, including the New York 99 West Nile strain. This technique was successfully performed with a cerebrospinal fluid sample from a patient hospitalized with West Nile virus encephalitis. PMID:11326014

  20. Expression of cassini, a murine gamma-satellite sequence conserved in evolution, is regulated in normal and malignant hematopoietic cells

    PubMed Central

    2012-01-01

    Background Acute lymphoblastic leukemia (ALL) cells treated with drugs can become drug-tolerant if co-cultured with protective stromal mouse embryonic fibroblasts (MEFs). Results We performed transcriptional profiling on these stromal fibroblasts to investigate if they were affected by the presence of drug-treated ALL cells. These mitotically inactivated MEFs showed few changes in gene expression, but a family of sequences of which transcription is significantly increased was identified. A sequence related to this family, which we named cassini, was selected for further characterization. We found that cassini was highly upregulated in drug-treated ALL cells. Analysis of RNAs from different normal mouse tissues showed that cassini expression is highest in spleen and thymus, and can be further enhanced in these organs by exposure of mice to bacterial endotoxin. Heat shock, but not other types of stress, significantly induced the transcription of this locus in ALL cells. Transient overexpression of cassini in human 293 embryonic kidney cells did not increase the cytotoxic or cytostatic effects of chemotherapeutic drugs but provided some protection. Database searches revealed that sequences highly homologous to cassini are present in rodents, apicomplexans, flatworms and primates, indicating that they are conserved in evolution. Moreover, CASSINI RNA was induced in human ALL cells treated with vincristine. Surprisingly, cassini belongs to the previously reported murine family of γ-satellite/major satellite DNA sequences, which were not known to be present in other species. Conclusions Our results show that the transcription of at least one member of these sequences is regulated, suggesting that this has a function in normal and transformed immune cells. Expression of these sequences may protect cells when they are exposed to specific stress stimuli. PMID:22916712

  1. 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.

  2. Conserved Noncoding Sequences Regulate lhx5 Expression in the Zebrafish Forebrain

    PubMed Central

    Sun, Liu; Chen, Fengjiao; Peng, Gang

    2015-01-01

    The LIM homeobox family protein Lhx5 plays important roles in forebrain development in the vertebrates. The lhx5 gene exhibits complex temporal and spatial expression patterns during early development but its transcriptional regulation mechanisms are not well understood. Here, we have used transgenesis in zebrafish in order to define regulatory elements that drive lhx5 expression in the forebrain. Through comparative genomic analysis we identified 10 non-coding sequences conserved in five teleost species. We next examined the enhancer activities of these conserved non-coding sequences with Tol2 transposon mediated transgenesis. We found a proximately located enhancer gave rise to robust reporter EGFP expression in the forebrain regions. In addition, we identified an enhancer located at approximately 50 kb upstream of lhx5 coding region that is responsible for reporter gene expression in the hypothalamus. We also identify an enhancer located approximately 40 kb upstream of the lhx5 coding region that is required for expression in the prethalamus (ventral thalamus). Together our results suggest discrete enhancer elements control lhx5 expression in different regions of the forebrain. PMID:26147098

  3. 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,…

  4. A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation

    PubMed Central

    Geissmann, Thomas; Chevalier, Clément; Cros, Marie-Josée; Boisset, Sandrine; Fechter, Pierre; Noirot, Céline; Schrenzel, Jacques; François, Patrice; Vandenesch, François; Gaspin, Christine; Romby, Pascale

    2009-01-01

    Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA‐K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other Staphylococci, and RsaE is also present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE‐mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C−rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanism. PMID:19786493

  5. HMMerThread: Detecting Remote, Functional Conserved Domains in Entire Genomes by Combining Relaxed Sequence-Database Searches with Fold Recognition

    PubMed Central

    Bradshaw, Charles Richard; Surendranath, Vineeth; Henschel, Robert; Mueller, Matthias Stefan; Habermann, Bianca Hermine

    2011-01-01

    Conserved domains in proteins are one of the major sources of functional information for experimental design and genome-level annotation. Though search tools for conserved domain databases such as Hidden Markov Models (HMMs) are sensitive in detecting conserved domains in proteins when they share sufficient sequence similarity, they tend to miss more divergent family members, as they lack a reliable statistical framework for the detection of low sequence similarity. We have developed a greatly improved HMMerThread algorithm that can detect remotely conserved domains in highly divergent sequences. HMMerThread combines relaxed conserved domain searches with fold recognition to eliminate false positive, sequence-based identifications. With an accuracy of 90%, our software is able to automatically predict highly divergent members of conserved domain families with an associated 3-dimensional structure. We give additional confidence to our predictions by validation across species. We have run HMMerThread searches on eight proteomes including human and present a rich resource of remotely conserved domains, which adds significantly to the functional annotation of entire proteomes. We find ∼4500 cross-species validated, remotely conserved domain predictions in the human proteome alone. As an example, we find a DNA-binding domain in the C-terminal part of the A-kinase anchor protein 10 (AKAP10), a PKA adaptor that has been implicated in cardiac arrhythmias and premature cardiac death, which upon stress likely translocates from mitochondria to the nucleus/nucleolus. Based on our prediction, we propose that with this HLH-domain, AKAP10 is involved in the transcriptional control of stress response. Further remotely conserved domains we discuss are examples from areas such as sporulation, chromosome segregation and signalling during immune response. The HMMerThread algorithm is able to automatically detect the presence of remotely conserved domains in proteins based on weak

  6. CONSERVED SEQUENCE IN THE AGGRECAN INTERGLOBULAR DOMAIN MODULATES CLEAVAGE BY ADAMTS-4 AND ADAMTS-5

    PubMed Central

    Miwa, Hazuki E; Gerken, Thomas A; Huynh, Tru D; Duesler, Lori R; Cotter, Meghan; Hering, Thomas M.

    2008-01-01

    Background Cleavage of aggrecan by ADAMTS proteinases at specific sites within highly conserved regions may be important to normal physiological enzyme functions, as well as pathological degradation. Methods To examine ADAMTS selectivity, we assayed ADAMTS-4 and -5 cleavage of recombinant bovine aggrecan mutated at amino acids N-terminal or C-terminal to the interglobular domain cleavage site. Results Mutations of conserved amino acids from P18 to P12 to increase hydrophilicity resulted in ADAMTS-4 cleavage inhibition. Mutation of Thr, but not Asn within the conserved N-glycosylation motif Asn-Ile-Thr from P6 to P4 enhanced cleavage. Mutation of conserved Thr residues from P22 to P17 to increase hydrophobicity enhanced ADAMTS-4 cleavage. A P4′ Ser377Gln mutant inhibited cleavage by ADAMTS-4 and -5, while a neutral Ser377Ala mutant and species mimicking mutants Ser377Thr, Ser377Asn, and Arg375Leu were cleaved normally by ADAMTS-4. The Ser377Thr mutant, however, was resistant to cleavage by ADAMTS-5. Conclusion We have identified multiple conserved amino acids within regions N- and C-terminal to the site of scission that may influence enzyme-substrate recognition, and may interact with exosites on ADAMTS-4 and ADAMTS-5. General Significance Inhibition of the binding of ADAMTS-4 and ADAMTS-5 exosites to aggrecan should be explored as a therapeutic intervention for osteoarthritis. PMID:19101611

  7. Comparison of C. elegans and C. briggsae Genome Sequences Reveals Extensive Conservation of Chromosome Organization and Synteny

    PubMed Central

    Hillier, LaDeana W; Miller, Raymond D; Baird, Scott E; Chinwalla, Asif; Fulton, Lucinda A; Koboldt, Daniel C; Waterston, Robert H

    2007-01-01

    To determine whether the distinctive features of Caenorhabditis elegans chromosomal organization are shared with the C. briggsae genome, we constructed a single nucleotide polymorphism–based genetic map to order and orient the whole genome shotgun assembly along the six C. briggsae chromosomes. Although these species are of the same genus, their most recent common ancestor existed 80–110 million years ago, and thus they are more evolutionarily distant than, for example, human and mouse. We found that, like C. elegans chromosomes, C. briggsae chromosomes exhibit high levels of recombination on the arms along with higher repeat density, a higher fraction of intronic sequence, and a lower fraction of exonic sequence compared with chromosome centers. Despite extensive intrachromosomal rearrangements, 1:1 orthologs tend to remain in the same region of the chromosome, and colinear blocks of orthologs tend to be longer in chromosome centers compared with arms. More strikingly, the two species show an almost complete conservation of synteny, with 1:1 orthologs present on a single chromosome in one species also found on a single chromosome in the other. The conservation of both chromosomal organization and synteny between these two distantly related species suggests roles for chromosome organization in the fitness of an organism that are only poorly understood presently. PMID:17608563

  8. Strong conservation of non-coding sequences during vertebrates evolution: potential involvement in post-transcriptional regulation of gene expression.

    PubMed Central

    Duret, L; Dorkeld, F; Gautier, C

    1993-01-01

    Comparison of nucleotide sequences from different classes of vertebrates that diverged more than 300 million years ago, revealed the existence of highly conserved regions (HCRs) with more than 70% similarity over 100 to 1450 nt in non-coding parts of genes. Such a conservation is unexpected because it is much longer and stronger than what is necessary for specifying the binding of a regulatory protein. HCRs are relatively frequent, particularly in genes that are essential to cell life. In multigene families, conserved regions are specific of each isotype and are probably involved in the control of their specific pattern of expression. Studying HCRs distribution within genes showed that functional constraints are generally much stronger in 3'-non-coding regions than in promoters or introns. The 3'-HCRs are particularly A + T-rich and are always located in the transcribed untranslated regions of genes, which suggests that they are involved in post-transcriptional processes. However, current knowledge of mechanisms that regulate mRNA export, localisation, translation, or degradation is not sufficient to explain the strong functional constraints that we have characterised. PMID:8506129

  9. High compression image and image sequence coding

    NASA Technical Reports Server (NTRS)

    Kunt, Murat

    1989-01-01

    The digital representation of an image requires a very large number of bits. This number is even larger for an image sequence. The goal of image coding is to reduce this number, as much as possible, and reconstruct a faithful duplicate of the original picture or image sequence. Early efforts in image coding, solely guided by information theory, led to a plethora of methods. The compression ratio reached a plateau around 10:1 a couple of years ago. Recent progress in the study of the brain mechanism of vision and scene analysis has opened new vistas in picture coding. Directional sensitivity of the neurones in the visual pathway combined with the separate processing of contours and textures has led to a new class of coding methods capable of achieving compression ratios as high as 100:1 for images and around 300:1 for image sequences. Recent progress on some of the main avenues of object-based methods is presented. These second generation techniques make use of contour-texture modeling, new results in neurophysiology and psychophysics and scene analysis.

  10. 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.

  11. Conservation of Shannon's redundancy for proteins. [information theory applied to amino acid sequences

    NASA Technical Reports Server (NTRS)

    Gatlin, L. L.

    1974-01-01

    Concepts of information theory are applied to examine various proteins in terms of their redundancy in natural originators such as animals and plants. The Monte Carlo method is used to derive information parameters for random protein sequences. Real protein sequence parameters are compared with the standard parameters of protein sequences having a specific length. The tendency of a chain to contain some amino acids more frequently than others and the tendency of a chain to contain certain amino acid pairs more frequently than other pairs are used as randomness measures of individual protein sequences. Non-periodic proteins are generally found to have random Shannon redundancies except in cases of constraints due to short chain length and genetic codes. Redundant characteristics of highly periodic proteins are discussed. A degree of periodicity parameter is derived.

  12. Robust high-order space-time conservative schemes for solving conservation laws on hybrid meshes

    NASA Astrophysics Data System (ADS)

    Shen, Hua; Wen, Chih-Yung; Liu, Kaixin; Zhang, Deliang

    2015-01-01

    In this paper, the second-order space-time conservation element and solution element (CE/SE) method proposed by Chang (1995) [3] is implemented on hybrid meshes for solving conservation laws. In addition, the present scheme has been extended to high-order versions including third and fourth order. Most methodologies of proposed schemes are consistent with that of the original CE/SE method, including: (i) a unified treatment of space and time (thereby ensuring good conservation in both space and time); (ii) a highly compact node stencil (the solution node is calculated using only the neighboring mesh nodes) regardless of the order of accuracy at the cost of storing all derivatives. A staggered time marching strategy is adopted and the solutions are updated alternatively between cell centers and vertexes. To construct explicit high-order schemes, second- and third-order derivatives are calculated by a modified finite-difference/weighted-average procedure which is different from that used to calculate the first-order derivatives. The present schemes can be implemented on a wide variety of meshes, including triangular, quadrilateral and hybrid (consisting of both triangular and quadrilateral elements). Beyond that, it can be easily extended to arbitrary-order schemes and arbitrary shape of polygonal elements by using the present methodologies. A series of common benchmark examples are used to confirm the accuracy and robustness of the proposed schemes.

  13. ENERGY CONSERVATION THROUGH POINT SOURCE RECYCLE WITH HIGH TEMPERATURE HYPERFILTRATION

    EPA Science Inventory

    The report gives results of a study of energy conservation effects of point source recycle with high-temperature hyperfiltration (HF) in the textile industry. (HF and ultrafiltration (UF) are pressure-driven membrane processes which have potential for recycle of water, energy, an...

  14. Conserved nucleotide sequences in the open reading frame and 3' untranslated region of selenoprotein P mRNA.

    PubMed Central

    Hill, K E; Lloyd, R S; Burk, R F

    1993-01-01

    Rat liver selenoprotein P contains 10 selenocysteine residues in its primary structure (deduced). It is the only selenoprotein characterized to date that has more than one selenocysteine residue. Selenoprotein P cDNA has been cloned from human liver and heart cDNA libraries and sequenced. The open reading frames are identical and contain a signal peptide, indicating that the protein is secreted by both organs and is therefore not exclusively produced in the liver. Ten selenocysteine residues (deduced) are present. Comparison of the open reading frame of the human cDNA with the rat cDNA reveals a 69% identity of the nucleotide sequence and 72% identity of the deduced amino acid sequence. Two regions in the 3' untranslated portion have high conservation between human and rat. Each of these regions contains a predicted stable stem-loop structure similar to the single stem-loop structures reported in 3' untranslated regions of type I iodothyronine 5'-deiodinase and glutathione peroxidase. The stem-loop structure of type I iodothyronine 5'-deiodinase has been shown to be necessary for incorporation of the selenocysteine residue at the UGA codon. Because only two stem-loop structures are present in the 3' untranslated region of selenoprotein P mRNA, it can be concluded that a separate stem-loop structure is not required for each selenocysteine residue. Images PMID:8421687

  15. In Silico Structure and Sequence Analysis of Bacterial Porins and Specific Diffusion Channels for Hydrophilic Molecules: Conservation, Multimericity and Multifunctionality.

    PubMed

    Vollan, Hilde S; Tannæs, Tone; Vriend, Gert; Bukholm, Geir

    2016-01-01

    Diffusion channels are involved in the selective uptake of nutrients and form the largest outer membrane protein (OMP) family in Gram-negative bacteria. Differences in pore size and amino acid composition contribute to the specificity. Structure-based multiple sequence alignments shed light on the structure-function relations for all eight subclasses. Entropy-variability analysis results are correlated to known structural and functional aspects, such as structural integrity, multimericity, specificity and biological niche adaptation. The high mutation rate in their surface-exposed loops is likely an important mechanism for host immune system evasion. Multiple sequence alignments for each subclass revealed conserved residue positions that are involved in substrate recognition and specificity. An analysis of monomeric protein channels revealed particular sequence patterns of amino acids that were observed in other classes at multimeric interfaces. This adds to the emerging evidence that all members of the family exist in a multimeric state. Our findings are important for understanding the role of members of this family in a wide range of bacterial processes, including bacterial food uptake, survival and adaptation mechanisms. PMID:27110766

  16. In Silico Structure and Sequence Analysis of Bacterial Porins and Specific Diffusion Channels for Hydrophilic Molecules: Conservation, Multimericity and Multifunctionality

    PubMed Central

    Vollan, Hilde S.; Tannæs, Tone; Vriend, Gert; Bukholm, Geir

    2016-01-01

    Diffusion channels are involved in the selective uptake of nutrients and form the largest outer membrane protein (OMP) family in Gram-negative bacteria. Differences in pore size and amino acid composition contribute to the specificity. Structure-based multiple sequence alignments shed light on the structure-function relations for all eight subclasses. Entropy-variability analysis results are correlated to known structural and functional aspects, such as structural integrity, multimericity, specificity and biological niche adaptation. The high mutation rate in their surface-exposed loops is likely an important mechanism for host immune system evasion. Multiple sequence alignments for each subclass revealed conserved residue positions that are involved in substrate recognition and specificity. An analysis of monomeric protein channels revealed particular sequence patterns of amino acids that were observed in other classes at multimeric interfaces. This adds to the emerging evidence that all members of the family exist in a multimeric state. Our findings are important for understanding the role of members of this family in a wide range of bacterial processes, including bacterial food uptake, survival and adaptation mechanisms. PMID:27110766

  17. Regulation of SHOOT MERISTEMLESS genes via an upstream-conserved noncoding sequence coordinates leaf development

    PubMed Central

    Uchida, Naoyuki; Townsley, Brad; Chung, Kook-Hyun; Sinha, Neelima

    2007-01-01

    The indeterminate shoot apical meristem of plants is characterized by the expression of the Class 1 KNOTTED1-LIKE HOMEOBOX (KNOX1) genes. KNOX1 genes have been implicated in the acquisition and/or maintenance of meristematic fate. One of the earliest indicators of a switch in fate from indeterminate meristem to determinate leaf primordium is the down-regulation of KNOX1 genes orthologous to SHOOT MERISTEMLESS (STM) in Arabidopsis (hereafter called STM genes) in the initiating primordia. In simple leafed plants, this down-regulation persists during leaf formation. In compound leafed plants, however, KNOX1 gene expression is reestablished later in the developing primordia, creating an indeterminate environment for leaflet formation. Despite this knowledge, most aspects of how STM gene expression is regulated remain largely unknown. Here, we identify two evolutionarily conserved noncoding sequences within the 5′ upstream region of STM genes in both simple and compound leafed species across monocots and dicots. We show that one of these elements is involved in the regulation of the persistent repression and/or the reestablishment of STM expression in the developing leaves but is not involved in the initial down-regulation in the initiating primordia. We also show evidence that this regulation is developmentally significant for leaf formation in the pathway involving ASYMMETRIC LEAVES1/2 (AS1/2) gene expression; these genes are known to function in leaf development. Together, these findings reveal a regulatory point of leaf development mediated through a conserved, noncoding sequence in STM genes. PMID:17898165

  18. Hemagglutinin Sequence Conservation Guided Stem Immunogen Design from Influenza A H3 Subtype

    PubMed Central

    Mallajosyula, V. Vamsee Aditya; Citron, Michael; Ferrara, Francesca; Temperton, Nigel J.; Liang, Xiaoping; Flynn, Jessica A.; Varadarajan, Raghavan

    2015-01-01

    Seasonal epidemics caused by influenza A (H1 and H3 subtypes) and B viruses are a major global health threat. The traditional, trivalent influenza vaccines have limited efficacy because of rapid antigenic evolution of the circulating viruses. This antigenic variability mediates viral escape from the host immune responses, necessitating annual vaccine updates. Influenza vaccines elicit a protective antibody response, primarily targeting the viral surface glycoprotein hemagglutinin (HA). However, the predominant humoral response is against the hypervariable head domain of HA, thereby restricting the breadth of protection. In contrast, the conserved, subdominant stem domain of HA is a potential “universal” vaccine candidate. We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis. The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, “isoleucine-zipper”, or “foldon”. These immunogens elicited cross-reactive, antiviral antibodies and conferred partial protection against a lethal, homologous HK68 virus challenge in vivo. Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up. PMID:26167164

  19. Comparative Sequence and Structure Analysis Reveals the Conservation and Diversity of Nucleotide Positions and Their Associated Tertiary Interactions in the Riboswitches

    PubMed Central

    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. PMID:24040136

  20. Mammalian mitochondrial D-loop region structural analysis: identification of new conserved sequences and their functional and evolutionary implications.

    PubMed

    Sbisà, E; Tanzariello, F; Reyes, A; Pesole, G; Saccone, C

    1997-12-31

    This paper reports the first comprehensive analysis of Displacement loop (D-loop) region sequences from ten different mammalian orders. It represents a systematic evolutionary study at the molecular level on regulatory homologous regions in organisms belonging to a well defined class, mammalia, which radiated about 150 million years ago (Mya). We have aligned and analyzed 26 complete D-loop region sequences available in the literature and the fat dormouse sequence, recently determined in our laboratory. The novelty of our alignment consists of the extensive manual revision of the preliminary output obtained by computer program to optimize sequence similarity, particularly for the two peripheral domains displaying heterogeneity in length and the presence of repeated sequences. The multialignment is available at the WWW site: http://www.ba.cnr.it/dloop.html. Our comparative study has allowed us to identify new conserved sequence blocks present in all the species under consideration and events of insertion/deletion which have important implications in both functional and evolutionary aspects. In particular we have detected two blocks, about 60 bp long, extended termination associated sequences (ETAS1 and ETAS2) conserved in all the organisms considered. Evaluation against experimental work suggests a possible functional role of ETAS1 and ETAS2 in the regulation of replication and transcription and targeted experimental approaches. The analyses on conserved sequence blocks (CSBs) clearly indicate that CSB1 is the only very essential element, common to all mammalian mt genomes, while CSB2 and CSB3 could be involved in different though related functions, probably species specific, and thus more linked to nuclear mitochondrial coevolutionary processes. Our hypothesis on the different functional implications of the conserved elements, CSBs and TASs, reported so far as main regulatory signals, would explain the different conservation of these elements in evolution. Moreover

  1. The human archain gene, ARCN1, has highly conserved homologs in rice and drosophila

    SciTech Connect

    Radice, P.; Jones, C.; Perry, H.

    1995-03-01

    A novel human gene, ARCN1, has been identified in chromosome band 11q23.3. It maps approximately 50 kb telomeric to MLL, a gene that is disrupted in a number of leukemia-associated translocation chromosomes. cDNA clones representing ARCN1 hybridize to 4-kb mRNA species present in all tissues tested. Sequencing of cDNAs suggests that at least two forms of mRNA with alternative 5 {prime} ends are present within the cell. The mRNA with the longest open reading frame gives rise to a protein of 57 kDa. Although the sequence reported is novel, remarkable similarity is observed with two predicted protein sequences from partial DNA sequences generated by rice (Oryza sativa) and fruit fly (Drosophila melanogaster) genome projects. The degree of sequence conservation is comparable to that observed for highly conserved structural proteins, such as heat shock protein HSP70, and is greater than that of {gamma}-gubulin and heat shock protein HSP60. A more distant relationship to the group of clathrin-associated proteins suggests a possible role in vesicle structure or trafficking. In view of its ancient pedigree and a potential involvement in cellular architecture, the authors propose that the ARCN1 protein be named archain. 20 refs., 5 figs.

  2. High-resolution schemes for hyperbolic conservation laws

    NASA Technical Reports Server (NTRS)

    Harten, A.

    1982-01-01

    A class of new explicit second order accurate finite difference schemes for the computation of weak solutions of hyperbolic conservation laws is presented. These highly nonlinear schemes are obtained by applying a nonoscillatory first order accurae scheme to an appropriately modified flux function. The so derived second order accurate schemes achieve high resolution while preserving the robustness of the original nonoscillatory first order accurate scheme.

  3. Lineage-Specific Conserved Noncoding Sequences of Plant Genomes: Their Possible Role in Nucleosome Positioning

    PubMed Central

    Hettiarachchi, Nilmini; Kryukov, Kirill; Sumiyama, Kenta; Saitou, Naruya

    2014-01-01

    Many studies on conserved noncoding sequences (CNSs) have found that CNSs are enriched significantly in regulatory sequence elements. We conducted whole-genome analysis on plant CNSs to identify lineage-specific CNSs in eudicots, monocots, angiosperms, and vascular plants based on the premise that lineage-specific CNSs define lineage-specific characters and functions in groups of organisms. We identified 27 eudicot, 204 monocot, 6,536 grass, 19 angiosperm, and 2 vascular plant lineage-specific CNSs (lengths range from 16 to 1,517 bp) that presumably originated in their respective common ancestors. A stronger constraint on the CNSs located in the untranslated regions was observed. The CNSs were often flanked by genes involved in transcription regulation. A drop of A+T content near the border of CNSs was observed and CNS regions showed a higher nucleosome occupancy probability. These CNSs are candidate regulatory elements, which are expected to define lineage-specific features of various plant groups. PMID:25364802

  4. G-boxes, bigfoot genes, and environmental response: characterization of intragenomic conserved noncoding sequences in Arabidopsis.

    PubMed

    Freeling, Michael; Rapaka, Lakshmi; Lyons, Eric; Pedersen, Brent; Thomas, Brian C

    2007-05-01

    A tetraploidy left Arabidopsis thaliana with 6358 pairs of homoeologs that, when aligned, generated 14,944 intragenomic conserved noncoding sequences (CNSs). Our previous work assembled these phylogenetic footprints into a database. We show that known transcription factor (TF) binding motifs, including the G-box, are overrepresented in these CNSs. A total of 254 genes spanning long lengths of CNS-rich chromosomes (Bigfoot) dominate this database. Therefore, we made subdatabases: one containing Bigfoot genes and the other containing genes with three to five CNSs (Smallfoot). Bigfoot genes are generally TFs that respond to signals, with their modal CNS positioned 3.1 kb 5' from the ATG. Smallfoot genes encode components of signal transduction machinery, the cytoskeleton, or involve transcription. We queried each subdatabase with each possible 7-nucleotide sequence. Among hundreds of hits, most were purified from CNSs, and almost all of those significantly enriched in CNSs had no experimental history. The 7-mers in CNSs are not 5'- to 3'-oriented in Bigfoot genes but are often oriented in Smallfoot genes. CNSs with one G-box tend to have two G-boxes. CNSs were shared with the homoeolog only and with no other gene, suggesting that binding site turnover impedes detection. Bigfoot genes may function in adaptation to environmental change. PMID:17496117

  5. Evolution of a "conserved" amino acid sequence: a model study of an in silico investigation of the phylogenesis of some immune receptors.

    PubMed

    Panaro, M A; Acquafredda, A; Sisto, M; Lisi, S; Saccia, M; Mitolo, V

    2006-01-01

    In this paper we analyze a 55-amino acid (aa) sequence which is relatively well conserved in several seven-transmembrane receptor families (from Insects to Mammals) and in some Viruses. This sequence, which covers the second transmembrane domain, the first extracellular loop and the third transmembrane domain, appears in its complete configuration in most of the seven-transmembrane receptor families, as well as in the protein products of some viruses. Other seven-transmembrane receptors and viruses exhibit reduced configurations of the conserved sequence, lacking either aa 31 or aa 30-31. 53-aa configurations are typically found in most chemokine receptor (CKR) subfamilies, as well as in some viral protein products. However, the CCR1, CCR3, and CCR6 subfamilies comprise a 54-aa configuration and the CKR-related protein products, ChemR23 and RDC1, include the complete 55-aa sequence. For each CKR subfamily the "modal sequence" of the conserved segment was constructed by selecting the most frequently occurring aa at each position. Then, pairwise alignments were made between: (i) the modal CKR sequences, and (ii) the sequence (53-aa) of the Yaba-like disease virus - 7L protein. From the alignments two consensus matrices were derived: (i) the consensus 1 matrix with reference to the whole conserved segment, and (ii) the consensus 2 matrix with reference to aa 22-29, which appear to be the most variable segment of the sequence. Based on the obtained consensus values and with reference to this specific conserved segment, the following conclusions are proposed: (1) ChemR23 and RDC1 are probably the more primitive CKR forms; (2) CCR1 and CCR3 may be grouped in a single cluster; (3) CCRs 2, 4, and 5 are closely related to each other and may be grouped in a cluster; CCR7 is likely to be evolutionarily related to this cluster; (4) CXCRs 2, 3, and 4 and CCX CKR appear to be evolutionarily related to each other and very likely derived from an CCR6-like gene; (5) CCR2/4/5 and

  6. Discovery of Novel ncRNA Sequences in Multiple Genome Alignments on the Basis of Conserved and Stable Secondary Structures.

    PubMed

    Fu, Yinghan; Xu, Zhenjiang Zech; Lu, Zhi J; Zhao, Shan; Mathews, David H

    2015-01-01

    Recently, non-coding RNAs (ncRNAs) have been discovered with novel functions, and it has been appreciated that there is pervasive transcription of genomes. Moreover, many novel ncRNAs are not conserved on the primary sequence level. Therefore, de novo computational ncRNA detection that is accurate and efficient is desirable. The purpose of this study is to develop a ncRNA detection method based on conservation of structure in more than two genomes. A new method called Multifind, using Multilign, was developed. Multilign predicts the common secondary structure for multiple input sequences. Multifind then uses measures of structure conservation to estimate the probability that the input sequences are a conserved ncRNA using a classification support vector machine. Multilign is based on Dynalign, which folds and aligns two sequences simultaneously using a scoring scheme that does not include sequence identity; its structure prediction quality is therefore not affected by input sequence diversity. Additionally, ensemble defect was introduced to Multifind as an additional discriminating feature that quantifies the compactness of the folding space for a sequence. Benchmarks showed Multifind performs better than RNAz and LocARNATE+RNAz, a method that uses RNAz on structure alignments generated by LocARNATE, on testing sequences extracted from the Rfam database. For de novo ncRNA discovery in three genomes, Multifind and LocARNATE+RNAz had an advantage over RNAz in low similarity regions of genome alignments. Additionally, Multifind and LocARNATE+RNAz found different subsets of known ncRNA sequences, suggesting the two approaches are complementary. PMID:26075601

  7. Precise detection of L. monocytogenes hitting its highly conserved region possessing several specific antibody binding sites.

    PubMed

    Jahangiri, Abolfazl; Rasooli, Iraj; Reza Rahbar, Mohammad; Khalili, Saeed; Amani, Jafar; Ahmadi Zanoos, Kobra

    2012-07-21

    Listeria monocytogenes, a facultative intracellular fast-growing Gram-positive food-borne pathogen, can infect immunocompromised individuals leading to meningitis, meningoencephalitis and septicaemias. From the pool of virulence factors of the organism, ActA, a membrane protein, has a critical role in the life cycle of L. monocytogenes. High mortality rate of listeriosis necessitates a sensitive and rapid diagnostic test for precise identification of L. monocytogenes. We used bioinformatic tools to locate a specific conserved region of ActA for designing and developing an antibody-antigen based diagnostic test for the detection of L. monocytogenes. A number of databases were looked for ActA related sequences. Sequences were analyzed with several online software to find an appropriate region for our purpose. ActA protein was found specific to Listeria species with no homologs in other organisms. We finally introduced a highly conserved region within ActA sequence that possess several antibody binding sites specific to L. monocytogenes. This protein sequence can serve as an antigen for designing a relatively cheap, sensitive, and specific diagnostic test for detection of L. monocytogenes. PMID:22575546

  8. Epidermal surface antigen (MS17S1) is highly conserved between mouse and human.

    PubMed

    Cho, Y J; Chema, D; Moskow, J J; Cho, M; Schroeder, W T; Overbeek, P; Buchberg, A M; Duvic, M

    1995-05-20

    A mouse monoclonal antibody ECS-1 raised to human keratinocytes detects a 35-kDa epidermal surface antigen (ESA) and causes keratinocyte dissociation in vitro. ECS-1 stains skin of 16-day mouse embryo and 8- to 9-week human fetus. Mouse Esa cDNA encodes a 379-amino-acid protein that is 99.2% identical to the human, differing at only 3 amino acids. The gene (M17S1) was mapped to mouse chromosome 11, high-lighting the conserved linkage synteny existing between human chromosome 17 and mouse chromosome 11. Although the nude locus has been mapped to the same region of chromosome 11, no abnormalities in protein, mRNA, or cDNA or genomic sequences were detected in nude mice. However, both nude and control mice were found to have a second Esa mRNA transcript that conserves amino acid sequence and molecular weight. The mouse and human 5' and 3' untranslated sequences are conserved. Similar RNA folding patterns of the 5' untranslated region are predicted despite a 91-bp insertion in the mouse. These data suggest that both the function and the regulation of ESA protein are of importance and that Esa (M17S1) is not the nude locus gene. PMID:7557989

  9. 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. PMID:22481137

  10. The Internally Self-fertilizing Hermaphroditic Teleost Rivulus marmoratus (Cyprinodontiformes, Rivulidae) beta-Actin Gene: Amplification and Sequence Analysis with Conserved Primers.

    PubMed

    Lee

    2000-03-01

    To determine the ease and feasibility of amplifying the beta-actin gene in fish by the polymerase chain reaction (PCR), genomic DNAs of several fish (Rivulus, Southern top mouth minnow, common fat minnow, oily bitterling, carp, Far Eastern catfish, medaka, and European flounder) were extracted and used as a template with conserved primers, designed on the basis of high amino acid homology (approximately 98% or more). Among them, the self-fertilizing hermaphroditic fish Rivulus marmoratus was chosen for further characterization. After amplification of the Rivulus beta-actin PCR product with Taq polymerase, PCR product was subcloned to pCRII vector. After restriction enzyme mapping of Rivulus beta-actin gene, the amplified insert was sequenced using ALF Express automatic DNA sequencer with conserved internal primers. The R. marmoratus beta-actin gene consists of 1763 bp encoding 375 amino acids including 5 exons and 4 introns. The splicing and acceptance sites of the exon and intron boundaries of the Rivulus beta-actin gene were highly conserved with consensus sequences (GT/AG). The amino acid homology of R. marmoratus beta-actin to other species was high: 98.93% to human; 98.93%, Atlantic salmon; 98.93%, common carp; 98.93%, grass carp; 98.93%, zebrafish; 98.67%, medaka; and 98.40%, sea bream. To determine the expression of the R. marmoratus beta-actin gene in liver and ovary, reverse transcriptase-polymerase chain reaction was carried out with internal primers. In conclusion, these universal primers are successful in the rapid cloning of the fish beta-actin gene by PCR, based on a high homology of the beta-actin gene conserved through evolution. This approach will be applicable to the isolation of other beta-actin homologues in the investigation of phylogenetic comparisons of fish species, along with a possible application to cloning strategy in other conserved genes. PMID:10811955

  11. Structural and sequence similarities of hydra xeroderma pigmentosum A protein to human homolog suggest early evolution and conservation.

    PubMed

    Barve, Apurva; Ghaskadbi, Saroj; Ghaskadbi, Surendra

    2013-01-01

    Xeroderma pigmentosum group A (XPA) is a protein that binds to damaged DNA, verifies presence of a lesion, and recruits other proteins of the nucleotide excision repair (NER) pathway to the site. Though its homologs from yeast, Drosophila, humans, and so forth are well studied, XPA has not so far been reported from protozoa and lower animal phyla. Hydra is a fresh-water cnidarian with a remarkable capacity for regeneration and apparent lack of organismal ageing. Cnidarians are among the first metazoa with a defined body axis, tissue grade organisation, and nervous system. We report here for the first time presence of XPA gene in hydra. Putative protein sequence of hydra XPA contains nuclear localization signal and bears the zinc-finger motif. It contains two conserved Pfam domains and various characterized features of XPA proteins like regions for binding to excision repair cross-complementing protein-1 (ERCC1) and replication protein A 70 kDa subunit (RPA70) proteins. Hydra XPA shows a high degree of similarity with vertebrate homologs and clusters with deuterostomes in phylogenetic analysis. Homology modelling corroborates the very close similarity between hydra and human XPA. The protein thus most likely functions in hydra in the same manner as in other animals, indicating that it arose early in evolution and has been conserved across animal phyla. PMID:24083246

  12. Structural and Sequence Similarities of Hydra Xeroderma Pigmentosum A Protein to Human Homolog Suggest Early Evolution and Conservation

    PubMed Central

    Ghaskadbi, Saroj

    2013-01-01

    Xeroderma pigmentosum group A (XPA) is a protein that binds to damaged DNA, verifies presence of a lesion, and recruits other proteins of the nucleotide excision repair (NER) pathway to the site. Though its homologs from yeast, Drosophila, humans, and so forth are well studied, XPA has not so far been reported from protozoa and lower animal phyla. Hydra is a fresh-water cnidarian with a remarkable capacity for regeneration and apparent lack of organismal ageing. Cnidarians are among the first metazoa with a defined body axis, tissue grade organisation, and nervous system. We report here for the first time presence of XPA gene in hydra. Putative protein sequence of hydra XPA contains nuclear localization signal and bears the zinc-finger motif. It contains two conserved Pfam domains and various characterized features of XPA proteins like regions for binding to excision repair cross-complementing protein-1 (ERCC1) and replication protein A 70 kDa subunit (RPA70) proteins. Hydra XPA shows a high degree of similarity with vertebrate homologs and clusters with deuterostomes in phylogenetic analysis. Homology modelling corroborates the very close similarity between hydra and human XPA. The protein thus most likely functions in hydra in the same manner as in other animals, indicating that it arose early in evolution and has been conserved across animal phyla. PMID:24083246

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

    PubMed Central

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

    2015-01-01

    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. PMID:25838544

  14. Comparison of SIV and HIV-1 genomic RNA structures reveals impact of sequence evolution on conserved and non-conserved structural motifs.

    PubMed

    Pollom, Elizabeth; Dang, Kristen K; Potter, E Lake; Gorelick, Robert J; Burch, Christina L; Weeks, Kevin M; Swanstrom, Ronald

    2013-01-01

    RNA secondary structure plays a central role in the replication and metabolism of all RNA viruses, including retroviruses like HIV-1. However, structures with known function represent only a fraction of the secondary structure reported for HIV-1(NL4-3). One tool to assess the importance of RNA structures is to examine their conservation over evolutionary time. To this end, we used SHAPE to model the secondary structure of a second primate lentiviral genome, SIVmac239, which shares only 50% sequence identity at the nucleotide level with HIV-1NL4-3. Only about half of the paired nucleotides are paired in both genomic RNAs and, across the genome, just 71 base pairs form with the same pairing partner in both genomes. On average the RNA secondary structure is thus evolving at a much faster rate than the sequence. Structure at the Gag-Pro-Pol frameshift site is maintained but in a significantly altered form, while the impact of selection for maintaining a protein binding interaction can be seen in the conservation of pairing partners in the small RRE stems where Rev binds. Structures that are conserved between SIVmac239 and HIV-1(NL4-3) also occur at the 5' polyadenylation sequence, in the plus strand primer sites, PPT and cPPT, and in the stem-loop structure that includes the first splice acceptor site. The two genomes are adenosine-rich and cytidine-poor. The structured regions are enriched in guanosines, while unpaired regions are enriched in adenosines, and functionaly important structures have stronger base pairing than nonconserved structures. We conclude that much of the secondary structure is the result of fortuitous pairing in a metastable state that reforms during sequence evolution. However, secondary structure elements with important function are stabilized by higher guanosine content that allows regions of structure to persist as sequence evolution proceeds, and, within the confines of selective pressure, allows structures to evolve. PMID:23593004

  15. Automated conserved non-coding sequence (CNS) discovery reveals differences in gene content and promoter evolution among grasses

    PubMed Central

    Turco, Gina; Schnable, James C.; Pedersen, Brent; Freeling, Michael

    2013-01-01

    Conserved non-coding sequences (CNS) are islands of non-coding sequence that, like protein coding exons, show less divergence in sequence between related species than functionless DNA. Several CNSs have been demonstrated experimentally to function as cis-regulatory regions. However, the specific functions of most CNSs remain unknown. Previous searches for CNS in plants have either anchored on exons and only identified nearby sequences or required years of painstaking manual annotation. Here we present an open source tool that can accurately identify CNSs between any two related species with sequenced genomes, including both those immediately adjacent to exons and distal sequences separated by >12 kb of non-coding sequence. We have used this tool to characterize new motifs, associate CNSs with additional functions, and identify previously undetected genes encoding RNA and protein in the genomes of five grass species. We provide a list of 15,363 orthologous CNSs conserved across all grasses tested. We were also able to identify regulatory sequences present in the common ancestor of grasses that have been lost in one or more extant grass lineages. Lists of orthologous gene pairs and associated CNSs are provided for reference inbred lines of arabidopsis, Japonica rice, foxtail millet, sorghum, brachypodium, and maize. PMID:23874343

  16. A comparative genomics strategy for targeted discovery of single-nucleotide polymorphisms and conserved-noncoding sequences in orphan crops.

    PubMed

    Feltus, F A; Singh, H P; Lohithaswa, H C; Schulze, S R; Silva, T D; Paterson, A H

    2006-04-01

    Completed genome sequences provide templates for the design of genome analysis tools in orphan species lacking sequence information. To demonstrate this principle, we designed 384 PCR primer pairs to conserved exonic regions flanking introns, using Sorghum/Pennisetum expressed sequence tag alignments to the Oryza genome. Conserved-intron scanning primers (CISPs) amplified single-copy loci at 37% to 80% success rates in taxa that sample much of the approximately 50-million years of Poaceae divergence. While the conserved nature of exons fostered cross-taxon amplification, the lesser evolutionary constraints on introns enhanced single-nucleotide polymorphism detection. For example, in eight rice (Oryza sativa) genotypes, polymorphism averaged 12.1 per kb in introns but only 3.6 per kb in exons. Curiously, among 124 CISPs evaluated across Oryza, Sorghum, Pennisetum, Cynodon, Eragrostis, Zea, Triticum, and Hordeum, 23 (18.5%) seemed to be subject to rigid intron size constraints that were independent of per-nucleotide DNA sequence variation. Furthermore, we identified 487 conserved-noncoding sequence motifs in 129 CISP loci. A large CISP set (6,062 primer pairs, amplifying introns from 1,676 genes) designed using an automated pipeline showed generally higher abundance in recombinogenic than in nonrecombinogenic regions of the rice genome, thus providing relatively even distribution along genetic maps. CISPs are an effective means to explore poorly characterized genomes for both DNA polymorphism and noncoding sequence conservation on a genome-wide or candidate gene basis, and also provide anchor points for comparative genomics across a diverse range of species. PMID:16607031

  17. Plastid genome sequences of Gymnochlora stellata, Lotharella vacuolata, and Partenskyella glossopodia reveal remarkable structural conservation among chlorarachniophyte species.

    PubMed

    Suzuki, Shigekatsu; Hirakawa, Yoshihisa; Kofuji, Rumiko; Sugita, Mamoru; Ishida, Ken-Ichiro

    2016-07-01

    Chlorarachniophyte algae have complex plastids acquired by the uptake of a green algal endosymbiont, and this event is called secondary endosymbiosis. Interestingly, the plastids possess a relict endosymbiont nucleus, referred to as the nucleomorph, in the intermembrane space, and the nucleomorphs contain an extremely reduced and compacted genome in comparison with green algal nuclear genomes. Therefore, chlorarachniophyte plastids consist of two endosymbiotically derived genomes, i.e., the plastid and nucleomorph genomes. To date, complete nucleomorph genomes have been sequenced in four different species, whereas plastid genomes have been reported in only two species in chlorarachniophytes. To gain further insight into the evolution of endosymbiotic genomes in chlorarachniophytes, we newly sequenced the plastid genomes of three species, Gymnochlora stellata, Lotharella vacuolata, and Partenskyella glossopodia. Our findings reveal that chlorarachniophyte plastid genomes are highly conserved in size, gene content, and gene order among species, but their nucleomorph genomes are divergent in such features. Accordingly, the current architecture of the plastid genomes of chlorarachniophytes evolved in a common ancestor, and changed very little during their subsequent diversification. Furthermore, our phylogenetic analyses using multiple plastid genes suggest that chlorarachniophyte plastids are derived from a green algal lineage that is closely related to Bryopsidales in the Ulvophyceae group. PMID:26920842

  18. Babesia gibsoni internal transcribed spacer 1 region is highly conserved amongst isolates from dogs across Japan

    PubMed Central

    LIU, Mingming; CAO, Shinuo; VUDRIKO, Patrick; SUZUKI, Hiroshi; SOMA, Takehisa; XUAN, Xuenan

    2016-01-01

    Babesia gibsoni is a tick-borne apicomplexan parasite of dogs that often causes fever and hemolytic anemia with highly variable clinical outcome. In this study, we sequenced the 254bp Internal Transcribed Spacer 1 region (ITS1) of 54 B. gibsoni isolates from 14 different geographical regions of Japan. The 54 isolates shared high sequence identity with each other and with B. gibsoni isolates reported in GenBank database (97.2–100%). Consistent with previous reports, phylogenetic analysis showed that B. gibsoni isolates from Japan formed the same clade with those from U.S.A., Australia, India and Taiwan. Our finding indicates that B. gibsoni ITS1 region is highly conserved among isolates from dogs in Japan, making it a useful genetic marker for molecular epidemiology of the parasite. PMID:26806537

  19. Babesia gibsoni internal transcribed spacer 1 region is highly conserved amongst isolates from dogs across Japan.

    PubMed

    Liu, Mingming; Cao, Shinuo; Vudriko, Patrick; Suzuki, Hiroshi; Soma, Takehisa; Xuan, Xuenan

    2016-06-01

    Babesia gibsoni is a tick-borne apicomplexan parasite of dogs that often causes fever and hemolytic anemia with highly variable clinical outcome. In this study, we sequenced the 254bp Internal Transcribed Spacer 1 region (ITS1) of 54 B. gibsoni isolates from 14 different geographical regions of Japan. The 54 isolates shared high sequence identity with each other and with B. gibsoni isolates reported in GenBank database (97.2-100%). Consistent with previous reports, phylogenetic analysis showed that B. gibsoni isolates from Japan formed the same clade with those from U.S.A., Australia, India and Taiwan. Our finding indicates that B. gibsoni ITS1 region is highly conserved among isolates from dogs in Japan, making it a useful genetic marker for molecular epidemiology of the parasite. PMID:26806537

  20. Integrating bioinformatic resources to predict transcription factors interacting with cis-sequences conserved in co-regulated genes

    PubMed Central

    2014-01-01

    Background Using motif detection programs it is fairly straightforward to identify conserved cis-sequences in promoters of co-regulated genes. In contrast, the identification of the transcription factors (TFs) interacting with these cis-sequences is much more elaborate. To facilitate this, we explore the possibility of using several bioinformatic and experimental approaches for TF identification. This starts with the selection of co-regulated gene sets and leads first to the prediction and then to the experimental validation of TFs interacting with cis-sequences conserved in the promoters of these co-regulated genes. Results Using the PathoPlant database, 32 up-regulated gene groups were identified with microarray data for drought-responsive gene expression from Arabidopsis thaliana. Application of the binding site estimation suite of tools (BEST) discovered 179 conserved sequence motifs within the corresponding promoters. Using the STAMP web-server, 49 sequence motifs were classified into 7 motif families for which similarities with known cis-regulatory sequences were identified. All motifs were subjected to a footprintDB analysis to predict interacting DNA binding domains from plant TF families. Predictions were confirmed by using a yeast-one-hybrid approach to select interacting TFs belonging to the predicted TF families. TF-DNA interactions were further experimentally validated in yeast and with a Physcomitrella patens transient expression system, leading to the discovery of several novel TF-DNA interactions. Conclusions The present work demonstrates the successful integration of several bioinformatic resources with experimental approaches to predict and validate TFs interacting with conserved sequence motifs in co-regulated genes. PMID:24773781

  1. 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...

  2. Optimal assembly for high throughput shotgun sequencing

    PubMed Central

    2013-01-01

    We present a framework for the design of optimal assembly algorithms for shotgun sequencing under the criterion of complete reconstruction. We derive a lower bound on the read length and the coverage depth required for reconstruction in terms of the repeat statistics of the genome. Building on earlier works, we design a de Brujin graph based assembly algorithm which can achieve very close to the lower bound for repeat statistics of a wide range of sequenced genomes, including the GAGE datasets. The results are based on a set of necessary and sufficient conditions on the DNA sequence and the reads for reconstruction. The conditions can be viewed as the shotgun sequencing analogue of Ukkonen-Pevzner's necessary and sufficient conditions for Sequencing by Hybridization. PMID:23902516

  3. 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

  4. Advances in high throughput DNA sequence data compression.

    PubMed

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

    2016-06-01

    Advances in high throughput sequencing technologies and reduction in cost of sequencing have led to exponential growth in high throughput DNA sequence data. This growth has posed challenges such as storage, retrieval, and transmission of sequencing data. Data compression is used to cope with these challenges. Various methods have been developed to compress genomic and sequencing data. In this article, we present a comprehensive review of compression methods for genome and reads compression. Algorithms are categorized as referential or reference free. Experimental results and comparative analysis of various methods for data compression are presented. Finally, key challenges and research directions in DNA sequence data compression are highlighted. PMID:26846812

  5. 77 FR 74167 - Information Collection Request: Highly Erodible Land Conservation and Wetland Conservation

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-13

    ... information is collected in support of the conservation compliance provisions of Title XII of the Food Security Act of 1985 (the 1985 Farm Bill), as amended by the Food, Conservation, and Energy Act of 2008... Security Act of 1985 (16 U.S.C. 3801-3862), as amended by the Food, Conservation, and Energy Act of...

  6. Sequence Evaluation of FGF and FGFR Gene Conserved Non-Coding Elements in Non-Syndromic Cleft Lip and Palate Cases

    PubMed Central

    Riley, Bridget M.; Murray, Jeffrey C.

    2009-01-01

    Non-syndromic cleft lip and palate (NS CLP) is a complex birth defect resulting from multiple genetic and environmental factors. We have previously reported the sequencing of the coding region of genes in the fibroblast growth factor (FGF) signaling pathway, in which missense and non-sense mutations contribute to approximately 5%–6% NS CLP cases. In this article we report the sequencing of conserved non-coding elements (CNEs) in and around 11 of the FGF and FGFR genes, which identified 55 novel variants. Seven of variants are highly conserved among ≥8 species and 31 variants alter transcription factor binding sites, 8 of which are important for craniofacial development. Additionally, 15 NS CLP patients had a combination of coding mutations and CNE variants, suggesting that an accumulation of variants in the FGF signaling pathway may contribute to clefting. PMID:17963255

  7. The sexually dimorphic on the Y-chromosome gene (sdY) is a conserved male-specific Y-chromosome sequence in many salmonids

    PubMed Central

    Yano, Ayaka; Nicol, Barbara; Jouanno, Elodie; Quillet, Edwige; Fostier, Alexis; Guyomard, René; Guiguen, Yann

    2013-01-01

    All salmonid species investigated to date have been characterized with a male heterogametic sex-determination system. However, as these species do not share any Y-chromosome conserved synteny, there remains a debate on whether they share a common master sex-determining gene. In this study, we investigated the extent of conservation and evolution of the rainbow trout (Oncorhynchus mykiss) master sex-determining gene, sdY (sexually dimorphic on the Y-chromosome), in 15 different species of salmonids. We found that the sdY sequence is highly conserved in all salmonids and that sdY is a male-specific Y-chromosome gene in the majority of these species. These findings demonstrate that most salmonids share a conserved sex-determining locus and also strongly suggest that sdY may be this conserved master sex-determining gene. However, in two whitefish species (subfamily Coregoninae), sdY was found both in males and females, suggesting that alternative sex-determination systems may have also evolved in this family. Based on the wide conservation of sdY as a male-specific Y-chromosome gene, efficient and easy molecular sexing techniques can now be developed that will be of great interest for studying these economically and environmentally important species. PMID:23745140

  8. Evolutionary divergence and limits of conserved non-coding sequence detection in plant genomes

    PubMed Central

    Reineke, Anna R.; Bornberg-Bauer, Erich; Gu, Jenny

    2011-01-01

    The discovery of regulatory motifs embedded in upstream regions of plants is a particularly challenging bioinformatics task. Previous studies have shown that motifs in plants are short compared with those found in vertebrates. Furthermore, plant genomes have undergone several diversification mechanisms such as genome duplication events which impact the evolution of regulatory motifs. In this article, a systematic phylogenomic comparison of upstream regions is conducted to further identify features of the plant regulatory genomes, the component of genomes regulating gene expression, to enable future de novo discoveries. The findings highlight differences in upstream region properties between major plant groups and the effects of divergence times and duplication events. First, clear differences in upstream region evolution can be detected between monocots and dicots, thus suggesting that a separation of these groups should be made when searching for novel regulatory motifs, particularly since universal motifs such as the TATA box are rare. Second, investigating the decay rate of significantly aligned regions suggests that a divergence time of ∼100 mya sets a limit for reliable conserved non-coding sequence (CNS) detection. Insights presented here will set a framework to help identify embedded motifs of functional relevance by understanding the limits of bioinformatics detection for CNSs. PMID:21470961

  9. 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. PMID:23202421

  10. Sequence analysis of the L protein of the Ebola 2014 outbreak: Insight into conserved regions and mutations.

    PubMed

    Ayub, Gohar; Waheed, Yasir

    2016-06-01

    The 2014 Ebola outbreak was one of the largest that have occurred; it started in Guinea and spread to Nigeria, Liberia and Sierra Leone. Phylogenetic analysis of the current virus species indicated that this outbreak is the result of a divergent lineage of the Zaire ebolavirus. The L protein of Ebola virus (EBOV) is the catalytic subunit of the RNA‑dependent RNA polymerase complex, which, with VP35, is key for the replication and transcription of viral RNA. Earlier sequence analysis demonstrated that the L protein of all non‑segmented negative‑sense (NNS) RNA viruses consists of six domains containing conserved functional motifs. The aim of the present study was to analyze the presence of these motifs in 2014 EBOV isolates, highlight their function and how they may contribute to the overall pathogenicity of the isolates. For this purpose, 81 2014 EBOV L protein sequences were aligned with 475 other NNS RNA viruses, including Paramyxoviridae and Rhabdoviridae viruses. Phylogenetic analysis of all EBOV outbreak L protein sequences was also performed. Analysis of the amino acid substitutions in the 2014 EBOV outbreak was conducted using sequence analysis. The alignment demonstrated the presence of previously conserved motifs in the 2014 EBOV isolates and novel residues. Notably, all the mutations identified in the 2014 EBOV isolates were tolerant, they were pathogenic with certain examples occurring within previously determined functional conserved motifs, possibly altering viral pathogenicity, replication and virulence. The phylogenetic analysis demonstrated that all sequences with the exception of the 2014 EBOV sequences were clustered together. The 2014 EBOV outbreak has acquired a great number of mutations, which may explain the reasons behind this unprecedented outbreak. Certain residues critical to the function of the polymerase remain conserved and may be targets for the development of antiviral therapeutic agents. PMID:27082438

  11. Advances in DNA sequencing technologies for high resolution HLA typing.

    PubMed

    Cereb, Nezih; Kim, Hwa Ran; Ryu, Jaejun; Yang, Soo Young

    2015-12-01

    This communication describes our experience in large-scale G group-level high resolution HLA typing using three different DNA sequencing platforms - ABI 3730 xl, Illumina MiSeq and PacBio RS II. Recent advances in DNA sequencing technologies, so-called next generation sequencing (NGS), have brought breakthroughs in deciphering the genetic information in all living species at a large scale and at an affordable level. The NGS DNA indexing system allows sequencing multiple genes for large number of individuals in a single run. Our laboratory has adopted and used these technologies for HLA molecular testing services. We found that each sequencing technology has its own strengths and weaknesses, and their sequencing performances complement each other. HLA genes are highly complex and genotyping them is quite challenging. Using these three sequencing platforms, we were able to meet all requirements for G group-level high resolution and high volume HLA typing. PMID:26423536

  12. Phylogenetic comparison of the pre-mRNA adenosine deaminase ADAR2 genes and transcripts: conservation and diversity in editing site sequence and alternative splicing patterns.

    PubMed

    Slavov, D; Gardiner, K

    2002-10-16

    Adenosine deaminase that acts on RNA -2 (ADAR2) is a member of a family of vertebrate genes that encode adenosine (A)-to-inosine (I) RNA deaminases, enzymes that deaminate specific A residues in specific pre-mRNAs to produce I. Known substrates of ADAR2 include sites within the coding regions of pre-mRNAs of the ionotropic glutamate receptors, GluR2-6, and the serotonin receptor, 5HT2C. Mammalian ADAR2 expression is itself regulated by A-to-I editing and by several alternative splicing events. Because the biological consequences of ADAR2 function are significant, we have undertaken a phylogenetic comparison of these features. Here we report a comparison of cDNA sequences, genomic organization, editing site sequences and patterns of alternative splicing of ADAR2 genes from human, mouse, chicken, pufferfish and zebrafish. Coding sequences and intron/exon organization are highly conserved. All ADAR2 genes show evidence of transcript editing with required sequences and predicted secondary structures very highly conserved. Patterns and levels of editing and alternative splicing vary among organisms, and include novel N-terminal exons and splicing events. PMID:12459255

  13. Two evolutionarily conserved sequence elements for Peg3/Usp29 transcription

    PubMed Central

    Kim, Jeong Do; Yu, Sungryul; Choo, Jung Ha; Kim, Joomyeong

    2008-01-01

    Background Two evolutionarily Conserved Sequence Elements, CSE1 and CSE2 (YY1 binding sites), are found within the 3.8-kb CpG island surrounding the bidirectional promoter of two imprinted genes, Peg3 (Paternally expressed gene 3) and Usp29 (Ubiquitin-specific protease 29). This CpG island is a likely ICR (Imprinting Control Region) that controls transcription of the 500-kb genomic region of the Peg3 imprinted domain. Results The current study investigated the functional roles of CSE1 and CSE2 in the transcriptional control of the two genes, Peg3 and Usp29, using cell line-based promoter assays. The mutation of 6 YY1 binding sites (CSE2) reduced the transcriptional activity of the bidirectional promoter in the Peg3 direction in an orientation-dependent manner, suggesting an activator role for CSE2 (YY1 binding sites). However, the activity in the Usp29 direction was not detectable regardless of the presence/absence of YY1 binding sites. In contrast, mutation of CSE1 increased the transcriptional activity of the promoter in both the Peg3 and Usp29 directions, suggesting a potential repressor role for CSE1. The observed repression by CSE1 was also orientation-dependent. Serial mutational analyses further narrowed down two separate 6-bp-long regions within the 42-bp-long CSE1 which are individually responsible for the repression of Peg3 and Usp29. Conclusion CSE2 (YY1 binding sites) functions as an activator for Peg3 transcription, while CSE1 acts as a repressor for the transcription of both Peg3 and Usp29. PMID:19068137

  14. Taking High Conservation Value from Forests to Freshwaters

    NASA Astrophysics Data System (ADS)

    Abell, Robin; Morgan, Siân K.; Morgan, Alexis J.

    2015-07-01

    The high conservation value (HCV) concept, originally developed by the Forest Stewardship Council, has been widely incorporated outside the forestry sector into companies' supply chain assessments and responsible purchasing policies, financial institutions' investment policies, and numerous voluntary commodity standards. Many, if not most, of these newer applications relate to production practices that are likely to affect freshwater systems directly or indirectly, yet there is little guidance as to whether or how HCV can be applied to water bodies. We focus this paper on commodity standards and begin by exploring how prominent standards currently address both HCVs and freshwaters. We then highlight freshwater features of high conservation importance and examine how well those features are captured by the existing HCV framework. We propose a new set of freshwater `elements' for each of the six values and suggest an approach for identifying HCV Areas that takes out-of-fence line impacts into account, thereby spatially extending the scope of existing methods to define HCVs. We argue that virtually any non-marine HCV assessment, regardless of the production sector, should be expanded to include freshwater values, and we suggest how to put those recommendations into practice.

  15. Antibody Recognition of a Highly Conserved Influenza Virus Epitope

    SciTech Connect

    Ekiert, Damian C.; Bhabha, Gira; Elsliger, Marc-André; Friesen, Robert H.E.; Jongeneelen, Mandy; Throsby, Mark; Goudsmit, Jaap; Wilson, Ian A.; Scripps; Crucell

    2009-05-21

    Influenza virus presents an important and persistent threat to public health worldwide, and current vaccines provide immunity to viral isolates similar to the vaccine strain. High-affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Cocrystal structures were determined at 2.2 and 2.7 angstrom resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to other structurally characterized influenza antibodies, CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1 and HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibody-based therapies for the treatment of influenza.

  16. A conserved spiral structure for highly diverged phage tail assembly chaperones.

    PubMed

    Pell, Lisa G; Cumby, Nichole; Clark, Teresa E; Tuite, Ashleigh; Battaile, Kevin P; Edwards, Aled M; Chirgadze, Nickolay Y; Davidson, Alan R; Maxwell, Karen L

    2013-07-24

    Tail assembly chaperones (TACs) are a family of proteins likely required for the morphogenesis of all long-tailed phages. In this study, we determined the crystal structure of gp13, the TAC of phage HK97. This structure is similar to that of the TAC from the Lactococcus phage p2 and two unannotated structures of likely TACs encoded in prophage-derived regions of Bacillus subtilis and Bacillus stearothermophilus. Despite the high sequence divergence of these proteins, gp13 forms a ring structure with similar dimensions to the spirals observed in the crystal lattices of these other proteins. Remarkably, these similar quaternary structures are formed through very different interprotomer interactions. We present functional data supporting the biological relevance of these spiral structures and propose that spiral formation has been the primary requirement for these proteins during evolution. This study presents an unusual example of diverged protein sequences and oligomerization mechanisms in the presence of conserved quaternary structure. PMID:23542344

  17. 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. PMID:25299700

  18. Dominant Sequences of Human Major Histocompatibility Complex Conserved Extended Haplotypes from HLA-DQA2 to DAXX

    PubMed Central

    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.

    2014-01-01

    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. PMID:25299700

  19. Concentration of Specific Amino Acids at the Catalytic/Active Centers of Highly-Conserved ``Housekeeping'' Enzymes of Central Metabolism in Archaea, Bacteria and Eukaryota: Is There a Widely Conserved Chemical Signal of Prebiotic Assembly?

    NASA Astrophysics Data System (ADS)

    Pollack, J. Dennis; Pan, Xueliang; Pearl, Dennis K.

    2010-06-01

    In alignments of 1969 protein sequences the amino acid glycine and others were found concentrated at most-conserved sites within ˜15 Å of catalytic/active centers (C/AC) of highly conserved kinases, dehydrogenases or lyases of Archaea, Bacteria and Eukaryota. Lysine and glutamic acid were concentrated at least-conserved sites furthest from their C/ACs. Logistic-regression analyses corroborated the “movement” of glycine towards and lysine away from their C/ACs: the odds of a glycine occupying a site were decreased by 19%, while the odds for a lysine were increased by 53%, for every 10 Å moving away from the C/AC. Average conservation of MSA consensus sites was highest surrounding the C/AC and directly decreased in transition toward model’s peripheries. Findings held with statistical confidence using sequences restricted to individual Domains or enzyme classes or to both. Our data describe variability in the rate of mutation and likelihoods for phylogenetic trees based on protein sequence data and endorse the extension of substitution models by incorporating data on conservation and distance to C/ACs rather than only using cumulative levels. The data support the view that in the most-conserved environment immediately surrounding the C/AC of taxonomically distant and highly conserved essential enzymes of central metabolism there are amino acids whose identity and degree of occupancy is similar to a proposed amino acid set and frequency associated with prebiotic evolution.

  20. High-throughput sequencing and vaccine design.

    PubMed

    Luciani, F

    2016-04-01

    Next-generation sequencing (NGS) technologies have reshaped genome research. The resulting increase in sequencing depth and resolution has led to an unprecedented level of genomic detail and thus an increasing awareness of the complexity of animal, human and pathogen genomes. This has resulted in new approaches to vaccine research. On the one hand, the increase in genome complexity challenges our ability to study and understand pathogen biology and pathogen-host interactions. On the other hand, the increase in genomic data also provides key information for developing and designing improved vaccines against pathogens that were previously extremely difficult to deal with, such as rapidly mutating RNA viruses or bacteria that have complex interactions with the host immune system. This review describes how the broad application of NGS technologies to genome research is affecting vaccine research. It focuses on implications for the field of viral genomics, and includes recent animal and human studies. PMID:27217168

  1. The putative cell cycle gene, enhancer of rudimentary, encodes a highly conserved protein found in plants and animals.

    PubMed

    Gelsthorpe, M; Pulumati, M; McCallum, C; Dang-Vu, K; Tsubota, S I

    1997-02-28

    The enhancer of rudimentary gene, e(r), in Drosophila melanogaster encodes a protein, ER, whose function has been implicated in pyrimidine biosynthesis and the cell cycle (Wojcik et al. (1994) Genetics 138, 1163-1170). In order to identify conserved regions of the protein and potentially important functional domains, the e(r) gene was cloned and sequenced from two other insects (Drosophila virilis and Aedes aegypti) and three vertebrates (Homo sapiens, Mus musculus, and Brachydanio rerio) and sequenced from a flowering plant (Arabidopsis thaliana). These sequences along with those of a nematode (Caenorhabditis elegans) exhibit a high degree of identity. ER of Drosophila melanogaster is 76% identical to the three vertebrate proteins, 49% identical to the nematode protein, and 40% identical to the plant protein. There is high evolutionary conservation among the vertebrates. The mouse and human proteins are identical and differ from that of the zebrafish by a single conservative amino-acid change (valine for isoleucine). A dramatic sequence conservation is seen in the position of the hydrophobic amino acids. Of the 27 positions occupied by hydrophobic amino acids in ER of Drosophila melanogaster, 25 of the corresponding positions in the human protein, 23 of the positions in Caenorhabditis elegans, and 20 of the positions in Arabidopsis thaliana have hydrophobic amino acids. Most of these residues are present in three conserved amphipathic alpha-helices, which are proposed to function in protein-protein interactions. Two phosphorylation sites for casein kinase II (CKII) have also been conserved within the animal groups. Purified ER from Drosophila melanogaster is phosphorylated in vitro by CKII, arguing that these two sites are functional in vivo. A putative shift in the secondary structure of ER caused by the phosphorylation of these sites suggests that CKII may be regulating the activity of the ER in vivo. PMID:9074495

  2. 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 ...

  3. High-throughput sequencing of cytosine methylation in plant DNA

    PubMed Central

    2013-01-01

    Cytosine methylation is a significant and widespread regulatory factor in plant systems. Methods for the high-throughput sequencing of methylation have allowed a greatly improved characterisation of the methylome. Here we discuss currently available methods for generation and analysis of high-throughput sequencing of methylation data. We also discuss the results previously acquired through sequencing plant methylomes, and highlight remaining challenges in this field. PMID:23758782

  4. cDNA sequence, genomic organization, and evolutionary conservation of a novel gene from the WAGR region

    SciTech Connect

    Schwartz, F.; Eisenman, R.; Knoll, J.; Bruns, G.

    1995-09-20

    A new gene (239FB) with predominant and differential expression in fetal brain has recently been isolated from a chromosome 11p13-p14 boundary area near FSHB. The corresponding mRNA has an open reading frame of 294 amino acids, a 3` untranslated region of 1247 nucleotides, and a highly GC-rich 5` untranslated region. The coding and 3` UT sequence is specified by 6 exons within nearly 87 kb of isolated genomic locus. The 5` end region of the transcript maps adjacent to the only genomically defined CpG island in a chromosomal subregion that may be associated with part of the mental retardation of some WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome patients. In addition to nucleotide and amino acid similarity to an EST from a normalized infant brain cDNA library, the predicted protein has extensive similarity to Caenorhbditis elegans polypeptides of, as yet, unknown function. The 239FB locus is, therefore, likely part of a family of genes with two members expressed in human brain. The extensive conservation of the predicted protein suggests a fundamental function of the gene product and will enable evaluation of the role of the 239FB gene in neurogenesis in model organisms. 48 refs., 4 figs., 1 tab.

  5. Design and analysis of structure-activity relationship of novel antimicrobial peptides derived from the conserved sequence of cecropin.

    PubMed

    Hao, Gang; Shi, Yong-Hui; Han, Jing-Hui; Li, Qi-Hui; Tang, Ya-Li; Le, Guo-Wei

    2008-03-01

    We have de novo designed four antimicrobial peptides AMP-A/B/C/D, the 51-residues peptides, which are based on the conserved sequence of cecropin. In the present study, the four peptides were chemically synthesized and their activities assayed. Their secondary structure, amphipathic property, electric field distribution and transmembrane domain were subsequently predicted by bioinformatics tools. Finally, the structure-activity relationship was analyzed from the results of activity experiments and prediction. The results of activity experiments indicated that AMP-B/C/D clearly possessed excellent broad-spectrum activity against bacteria, whereas AMP-A was almost inactive against most of the bacterial strains tested. AMP-B/C/D showed more potent activity against Gram-positive bacteria than against Gram-negative bacteria. By utilizing bioinformatics analysis tools, we found that the secondary structure of the four cation peptides was mainly alpha-helix, and the result of CD spectrum also displayed that all the peptides had considerable alpha-helix in the presence of either 50% TFE or SDS micelles. AMP-C showed much better activity than other peptides against most of the bacteria tested, owing to its remarkable cation property and the amphipathic character of its N-terminal. The study of structure-activity relationship of the designed peptides confirmed that amphipathic structure and high net positive charge were prerequisites for maintaining their activities. PMID:17929330

  6. Dose-sensitivity, conserved non-coding sequences, and duplicate gene retention through multiple tetraploidies in the grasses.

    PubMed

    Schnable, James C; Pedersen, Brent S; Subramaniam, Sabarinath; Freeling, Michael

    2011-01-01

    Whole genome duplications, or tetraploidies, are an important source of increased gene content. Following whole genome duplication, duplicate copies of many genes are lost from the genome. This loss of genes is biased both in the classes of genes deleted and the subgenome from which they are lost. Many or all classes are genes preferentially retained as duplicate copies are engaged in dose sensitive protein-protein interactions, such that deletion of any one duplicate upsets the status quo of subunit concentrations, and presumably lowers fitness as a result. Transcription factors are also preferentially retained following every whole genome duplications studied. This has been explained as a consequence of protein-protein interactions, just as for other highly retained classes of genes. We show that the quantity of conserved noncoding sequences (CNSs) associated with genes predicts the likelihood of their retention as duplicate pairs following whole genome duplication. As many CNSs likely represent binding sites for transcriptional regulators, we propose that the likelihood of gene retention following tetraploidy may also be influenced by dose-sensitive protein-DNA interactions between the regulatory regions of CNS-rich genes - nicknamed bigfoot genes - and the proteins that bind to them. Using grass genomes, we show that differential loss of CNSs from one member of a pair following the pre-grass tetraploidy reduces its chance of retention in the subsequent maize lineage tetraploidy. PMID:22645525

  7. Dose–Sensitivity, Conserved Non-Coding Sequences, and Duplicate Gene Retention Through Multiple Tetraploidies in the Grasses

    PubMed Central

    Schnable, James C.; Pedersen, Brent S.; Subramaniam, Sabarinath; Freeling, Michael

    2011-01-01

    Whole genome duplications, or tetraploidies, are an important source of increased gene content. Following whole genome duplication, duplicate copies of many genes are lost from the genome. This loss of genes is biased both in the classes of genes deleted and the subgenome from which they are lost. Many or all classes are genes preferentially retained as duplicate copies are engaged in dose sensitive protein–protein interactions, such that deletion of any one duplicate upsets the status quo of subunit concentrations, and presumably lowers fitness as a result. Transcription factors are also preferentially retained following every whole genome duplications studied. This has been explained as a consequence of protein–protein interactions, just as for other highly retained classes of genes. We show that the quantity of conserved noncoding sequences (CNSs) associated with genes predicts the likelihood of their retention as duplicate pairs following whole genome duplication. As many CNSs likely represent binding sites for transcriptional regulators, we propose that the likelihood of gene retention following tetraploidy may also be influenced by dose–sensitive protein–DNA interactions between the regulatory regions of CNS-rich genes – nicknamed bigfoot genes – and the proteins that bind to them. Using grass genomes, we show that differential loss of CNSs from one member of a pair following the pre-grass tetraploidy reduces its chance of retention in the subsequent maize lineage tetraploidy. PMID:22645525

  8. Conservation of sequence in the internal transcribed spacers and 5.8S ribosomal RNA among geographically separated isolates of parasitic scuticociliates (Ciliophora, Orchitophryidae).

    PubMed

    Goggin, C L; Murphy, N E

    2000-02-24

    Nucleotide sequence from the internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene from the ribosomal RNA gene cluster of isolates of the scuticociliate Orchitophrya stellarum from 4 asteroid hosts were compared. Surprisingly, these data (495 bp) were identical for O. stellarum isolated from the testes of Asterias amurensis from Japan; Pisaster ochraceus from British Columbia, Canada; Asterias rubens from The Netherlands; and Asterias vulgaris from Prince Edward Island, Canada. These sequence data were compared to those from 3 scuticociliates which parasitise crustaceans: Mesanophrys pugettensis, M. chesapeakensis and Anophryoides haemophila. No difference was found in this region between the nucleotide sequence of M. pugettensis and M. chesapeakensis. The sequence of Mesanophrys spp. differed by 9.2% in the ITS1 and 4.7% in the ITS2 from that of O. stellarum. The sequence from the ITS1 (135 bp) and ITS2 (233 bp) of A. haemophila differed by 42.6 and 20.5% respectively from those of O. stellarum. Therefore, nucleotide sequence of the ITS regions in these scuticociliates is highly conserved. PMID:10785865

  9. Conserved sequence-specific lincRNA-steroid receptor interactions drive transcriptional repression and direct cell fate

    PubMed Central

    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.

    2014-01-01

    The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic non-coding RNAs (lincRNAs). While 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 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. PMID:25377354

  10. 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.

  11. Control regions for chromosome replication are conserved with respect to sequence and location among Escherichia coli strains

    PubMed Central

    Frimodt-Møller, Jakob; Charbon, Godefroid; Krogfelt, Karen A.; Løbner-Olesen, Anders

    2015-01-01

    In Escherichia coli, chromosome replication is initiated from oriC by the DnaA initiator protein associated with ATP. Three non-coding regions contribute to the activity of DnaA. The datA locus is instrumental in conversion of DnaAATP to DnaAADP (datA dependent DnaAATP hydrolysis) whereas DnaA rejuvenation sequences 1 and 2 (DARS1 and DARS2) reactivate DnaAADP to DnaAATP. The structural organization of oriC, datA, DARS1, and DARS2 were found conserved among 59 fully sequenced E. coli genomes, with differences primarily in the non-functional spacer regions between key protein binding sites. The relative distances from oriC to datA, DARS1, and DARS2, respectively, was also conserved despite of large variations in genome size, suggesting that the gene dosage of either region is important for bacterial growth. Yet all three regions could be deleted alone or in combination without loss of viability. Competition experiments during balanced growth in rich medium and during mouse colonization indicated roles of datA, DARS1, and DARS2 for bacterial fitness although the relative contribution of each region differed between growth conditions. We suggest that this fitness advantage has contributed to conservation of both sequence and chromosomal location for datA, DARS1, and DARS2. PMID:26441936

  12. Genomic Locations of Conserved Noncoding Sequences and Their Proximal Protein-Coding Genes in Mammalian Expression Dynamics.

    PubMed

    Babarinde, Isaac Adeyemi; Saitou, Naruya

    2016-07-01

    Experimental studies have found the involvement of certain conserved noncoding sequences (CNSs) in the regulation of the proximal protein-coding genes in mammals. However, reported cases of long range enhancer activities and inter-chromosomal regulation suggest that proximity of CNSs to protein-coding genes might not be important for regulation. To test the importance of the CNS genomic location, we extracted the CNSs conserved between chicken and four mammalian species (human, mouse, dog, and cattle). These CNSs were confirmed to be under purifying selection. The intergenic CNSs are often found in clusters in gene deserts, where protein-coding genes are in paucity. The distribution pattern, ChIP-Seq, and RNA-Seq data suggested that the CNSs are more likely to be regulatory elements and not corresponding to long intergenic noncoding RNAs. Physical distances between CNS and their nearest protein coding genes were well conserved between human and mouse genomes, and CNS-flanking genes were often found in evolutionarily conserved genomic neighborhoods. ChIP-Seq signal and gene expression patterns also suggested that CNSs regulate nearby genes. Interestingly, genes with more CNSs have more evolutionarily conserved expression than those with fewer CNSs. These computationally obtained results suggest that the genomic locations of CNSs are important for their regulatory functions. In fact, various kinds of evolutionary constraints may be acting to maintain the genomic locations of CNSs and protein-coding genes in mammals to ensure proper regulation. PMID:27017584

  13. Comparative sequence analysis of Solanum and Arabidopsis in a hot spot for pathogen resistance on potato chromosome V reveals a patchwork of conserved and rapidly evolving genome segments

    PubMed Central

    2007-01-01

    Background Quantitative phenotypic variation of agronomic characters in crop plants is controlled by environmental and genetic factors (quantitative trait loci = QTL). To understand the molecular basis of such QTL, the identification of the underlying genes is of primary interest and DNA sequence analysis of the genomic regions harboring QTL is a prerequisite for that. QTL mapping in potato (Solanum tuberosum) has identified a region on chromosome V tagged by DNA markers GP21 and GP179, which contains a number of important QTL, among others QTL for resistance to late blight caused by the oomycete Phytophthora infestans and to root cyst nematodes. Results To obtain genomic sequence for the targeted region on chromosome V, two local BAC (bacterial artificial chromosome) contigs were constructed and sequenced, which corresponded to parts of the homologous chromosomes of the diploid, heterozygous genotype P6/210. Two contiguous sequences of 417,445 and 202,781 base pairs were assembled and annotated. Gene-by-gene co-linearity was disrupted by non-allelic insertions of retrotransposon elements, stretches of diverged intergenic sequences, differences in gene content and gene order. The latter was caused by inversion of a 70 kbp genomic fragment. These features were also found in comparison to orthologous sequence contigs from three homeologous chromosomes of Solanum demissum, a wild tuber bearing species. Functional annotation of the sequence identified 48 putative open reading frames (ORF) in one contig and 22 in the other, with an average of one ORF every 9 kbp. Ten ORFs were classified as resistance-gene-like, 11 as F-box-containing genes, 13 as transposable elements and three as transcription factors. Comparing potato to Arabidopsis thaliana annotated proteins revealed five micro-syntenic blocks of three to seven ORFs with A. thaliana chromosomes 1, 3 and 5. Conclusion Comparative sequence analysis revealed highly conserved collinear regions that flank regions

  14. Large distribution and high sequence identity of a Copia-type retrotransposon in angiosperm families.

    PubMed

    Dias, Elaine Silva; Hatt, Clémence; Hamon, Serge; Hamon, Perla; Rigoreau, Michel; Crouzillat, Dominique; Carareto, Claudia Marcia Aparecida; de Kochko, Alexandre; Guyot, Romain

    2015-09-01

    Retrotransposons are the main component of plant genomes. Recent studies have revealed the complexity of their evolutionary dynamics. Here, we have identified Copia25 in Coffea canephora, a new plant retrotransposon belonging to the Ty1-Copia superfamily. In the Coffea genomes analyzed, Copia25 is present in relatively low copy numbers and transcribed. Similarity sequence searches and PCR analyses show that this retrotransposon with LTRs (Long Terminal Repeats) is widely distributed among the Rubiaceae family and that it is also present in other distantly related species belonging to Asterids, Rosids and monocots. A particular situation is the high sequence identity found between the Copia25 sequences of Musa, a monocot, and Ixora, a dicot species (Rubiaceae). Our results reveal the complexity of the evolutionary dynamics of the ancient element Copia25 in angiosperm, involving several processes including sequence conservation, rapid turnover, stochastic losses and horizontal transfer. PMID:26245353

  15. Sequence conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in human and mouse

    SciTech Connect

    McKay, M.J.; Troelstra, C.; Kanaar, R.

    1996-09-01

    The rad21 gene of Schizosaccharomyces pombe is involved in the repair of ionizing radiation-induced DNA double-strand breaks. The isolation of mouse and human putative homologs of rad21 is reported here. Alignment of the predicted amino acid sequence of Rad21 with the mammalian proteins showed that the similarity was distributed across the length of the proteins, with more highly conserved regions at both termini. The mHR21{sup sp} (mouse homolog of Rad21, S. pombe) and hHR21{sup sp} (human homolog of Rad21, S. pombe) predicted proteins were 96% identical, whereas the human and S. pombe proteins were 25% identical and 47% similar. RNA blot analysis showed that mHR21{sup sp} mRNA was abundant in all adult mouse tissues examined, with highest expression in testis and thymus. In addition to a 3.1-kb constitutive mRNA transcript, a 2.2-kb transcript was present at a high level in postmeiotic spermatids, while expression of the 3.1-kb mRNA in testis was confined to the meiotic compartment. hHR21{sup sp} mRNA was cell-cycle regulated in human cells, increasing in late S phase to a peak in G2 phase. The level of hHR21{sup sp} transcripts was not altered by exposure of normal diploid fibroblasts to 10 Gy ionizing radiation. In situ hybridization showed that mHR21{sup sp} resided on chromosome 15D3, whereas hHR21{sup sp} localized to the syntenic 8q24 region. Elevated expression of mHR21{sup sp} in testis and thymus supports a possible role for the rad21 mammalian homologs in V(D)J and meiotic recombination, respectively. Cell cycle regulation of rad21, retained from S. pombe to human, is consistent with a conservation of function between S. pombe and human rad21 genes. 62 refs., 8 figs., 1 tab.

  16. Denoising DNA deep sequencing data—high-throughput sequencing errors and their correction

    PubMed Central

    Laehnemann, David; Borkhardt, Arndt

    2016-01-01

    Characterizing the errors generated by common high-throughput sequencing platforms and telling true genetic variation from technical artefacts are two interdependent steps, essential to many analyses such as single nucleotide variant calling, haplotype inference, sequence assembly and evolutionary studies. Both random and systematic errors can show a specific occurrence profile for each of the six prominent sequencing platforms surveyed here: 454 pyrosequencing, Complete Genomics DNA nanoball sequencing, Illumina sequencing by synthesis, Ion Torrent semiconductor sequencing, Pacific Biosciences single-molecule real-time sequencing and Oxford Nanopore sequencing. There is a large variety of programs available for error removal in sequencing read data, which differ in the error models and statistical techniques they use, the features of the data they analyse, the parameters they determine from them and the data structures and algorithms they use. We highlight the assumptions they make and for which data types these hold, providing guidance which tools to consider for benchmarking with regard to the data properties. While no benchmarking results are included here, such specific benchmarks would greatly inform tool choices and future software development. The development of stand-alone error correctors, as well as single nucleotide variant and haplotype callers, could also benefit from using more of the knowledge about error profiles and from (re)combining ideas from the existing approaches presented here. PMID:26026159

  17. Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene.

    PubMed Central

    Ullstrom, C A; Siehnel, R; Woodruff, W; Steinbach, S; Hancock, R E

    1991-01-01

    The conservation of the oprF gene for the major outer membrane protein OprF was determined by restriction mapping and Southern blot hybridization with the Pseudomonas aeruginosa oprF gene as a probe. The restriction map was highly conserved among 16 of the 17 serotype strains and 42 clinical isolates of P. aeruginosa. Only the serotype 12 isolate and one clinical isolate showed small differences in restriction pattern. Southern probing of PstI chromosomal digests of 14 species from the family Pseudomonadaceae revealed that only the nine members of rRNA homology group I hybridized with the oprF gene. To reveal the actual extent of homology, the oprF gene and its product were characterized in Pseudomonas syringae. Nine strains of P. syringae from seven different pathovars hybridized with the P. aeruginosa gene to produce five different but related restriction maps. All produced an OprF protein in their outer membranes with the same apparent molecular weight as that of P.aeruginosa OprF. In each case the protein reacted with monoclonal antibody MA4-10 and was similarly heat and 2-mercaptoethanol modifiable. The purified OprF protein of the type strain P. syringae pv. syringae ATCC 19310 reconstituted small channels in lipid bilayer membranes. The oprF gene from this latter strain was cloned and sequenced. Despite the low level of DNA hybridization between P. aeruginosa and P. syringae DNA, the OprF gene was highly conserved between the species with 72% DNA sequence identity and 68% amino acid sequence identity overall. The carboxy terminus-encoding region of P. syringae oprF showed 85 and 33% identity, respectively, with the same regions of the P. aeruginosa oprF and Escherichia coli ompA genes. Images PMID:1898935

  18. Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide.

    PubMed

    Seim, Inge; Jeffery, Penny L; Thomas, Patrick B; Walpole, Carina M; Maugham, Michelle; Fung, Jenny N T; Yap, Pei-Yi; O'Keeffe, Angela J; Lai, John; Whiteside, Eliza J; Herington, Adrian C; Chopin, Lisa K

    2016-06-01

    The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates. PMID:26792793

  19. Chicken interferon consensus sequence-binding protein (ICSBP) and interferon regulatory factor (IRF) 1 genes reveal evolutionary conservation in the IRF gene family.

    PubMed Central

    Jungwirth, C; Rebbert, M; Ozato, K; Degen, H J; Schultz, U; Dawid, I B

    1995-01-01

    Members of the IRF family mediate transcriptional responses to interferons (IFNs) and to virus infection. So far, proteins of this family have been studied only among mammalian species. Here we report the isolation of cDNA clones encoding two members of this family from chicken, interferon consensus sequence-binding protein (ICSBP) and IRF-1. The predicted chicken ICSBP and IRF-1 proteins show high levels of sequence similarity to their corresponding human and mouse counterparts. Sequence identities in the putative DNA-binding domains of chicken and human ICSBP and IRF-1 were 97% and 89%, respectively, whereas the C-terminal regions showed identities of 64% and 51%; sequence relationships with mouse ICSBP and IRF-1 are very similar. Chicken ICSBP was found to be expressed in several embryonic tissues, and both chicken IRF-1 and ICSBP were strongly induced in chicken fibroblasts by IFN treatment, supporting the involvement of these factors in IFN-regulated gene expression. The presence of proteins homologous to mammalian IRF family members, together with earlier observations on the occurrence of functionally homologous IFN-responsive elements in chicken and mammalian genes, highlights the conservation of transcriptional mechanisms in the IFN system, a finding that contrasts with the extensive sequence and functional divergence of the IFNs. Images Fig. 3 Fig. 4 Fig. 5 PMID:7536924

  20. High Throughput Sequence Analysis for Disease Resistance in Maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Preliminary results of a computational analysis of high throughput sequencing data from Zea mays and the fungus Aspergillus are reported. The Illumina Genome Analyzer was used to sequence RNA samples from two strains of Z. mays (Va35 and Mp313) collected over a time course as well as several specie...

  1. Microcollinearity in an ethylene receptor coding gene region of the Coffea canephora genome is extensively conserved with Vitis vinifera and other distant dicotyledonous sequenced genomes

    PubMed Central

    Guyot, Romain; de la Mare, Marion; Viader, Véronique; Hamon, Perla; Coriton, Olivier; Bustamante-Porras, José; Poncet, Valérie; Campa, Claudine; Hamon, Serge; de Kochko, Alexandre

    2009-01-01

    Background Coffea canephora, also called Robusta, belongs to the Rubiaceae, the fourth largest angiosperm family. This diploid species (2x = 2n = 22) has a fairly small genome size of ≈ 690 Mb and despite its extreme economic importance, particularly for developing countries, knowledge on the genome composition, structure and evolution remain very limited. Here, we report the 160 kb of the first C. canephora Bacterial Artificial Chromosome (BAC) clone ever sequenced and its fine analysis. Results This clone contains the CcEIN4 gene, encoding an ethylene receptor, and twenty other predicted genes showing a high gene density of one gene per 7.8 kb. Most of them display perfect matches with C. canephora expressed sequence tags or show transcriptional activities through PCR amplifications on cDNA libraries. Twenty-three transposable elements, mainly Class II transposon derivatives, were identified at this locus. Most of these Class II elements are Miniature Inverted-repeat Transposable Elements (MITE) known to be closely associated with plant genes. This BAC composition gives a pattern similar to those found in gene rich regions of Solanum lycopersicum and Medicago truncatula genomes indicating that the CcEIN4 regions may belong to a gene rich region in the C. canephora genome. Comparative sequence analysis indicated an extensive conservation between C. canephora and most of the reference dicotyledonous genomes studied in this work, such as tomato (S. lycopersicum), grapevine (V. vinifera), barrel medic M. truncatula, black cottonwood (Populus trichocarpa) and Arabidopsis thaliana. The higher degree of microcollinearity was found between C. canephora and V. vinifera, which belong respectively to the Asterids and Rosids, two clades that diverged more than 114 million years ago. Conclusion This study provides a first glimpse of C. canephora genome composition and evolution. Our data revealed a remarkable conservation of the microcollinearity between C. canephora and V

  2. HIVE-Hexagon: High-Performance, Parallelized Sequence Alignment for Next-Generation Sequencing Data Analysis

    PubMed Central

    Santana-Quintero, Luis; Dingerdissen, Hayley; Thierry-Mieg, Jean; Mazumder, Raja; Simonyan, Vahan

    2014-01-01

    Due to the size of Next-Generation Sequencing data, the computational challenge of sequence alignment has been vast. Inexact alignments can take up to 90% of total CPU time in bioinformatics pipelines. High-performance Integrated Virtual Environment (HIVE), a cloud-based environment optimized for storage and analysis of extra-large data, presents an algorithmic solution: the HIVE-hexagon DNA sequence aligner. HIVE-hexagon implements novel approaches to exploit both characteristics of sequence space and CPU, RAM and Input/Output (I/O) architecture to quickly compute accurate alignments. Key components of HIVE-hexagon include non-redundification and sorting of sequences; floating diagonals of linearized dynamic programming matrices; and consideration of cross-similarity to minimize computations. Availability https://hive.biochemistry.gwu.edu/hive/ PMID:24918764

  3. The role of evolutionarily conserved germ-line DH sequence in B-1 cell development and natural antibody production.

    PubMed

    Vale, Andre M; Nobrega, Alberto; Schroeder, Harry W

    2015-12-01

    Because of N addition and variation in the site of VDJ joining, the third complementarity-determining region of the heavy chain (CDR-H3) is the most diverse component of the initial immunoglobulin antigen-binding site repertoire. A large component of the peritoneal cavity B-1 cell component is the product of fetal and perinatal B cell production. The CDR-H3 repertoire is thus depleted of N addition, which increases dependency on germ-line sequence. Cross-species comparisons have shown that DH gene sequence demonstrates conservation of amino acid preferences by reading frame. Preference for reading frame 1, which is enriched for tyrosine and glycine, is created both by rearrangement patterns and by pre-BCR and BCR selection. In previous studies, we have assessed the role of conserved DH sequence by examining peritoneal cavity B-1 cell numbers and antibody production in BALB/c mice with altered DH loci. Here, we review our finding that changes in the constraints normally imposed by germ-line-encoded amino acids within the CDR-H3 repertoire profoundly affect B-1 cell development, especially B-1a cells, and thus natural antibody immunity. Our studies suggest that both natural and somatic selection operate to create a restricted B-1 cell CDR-H3 repertoire. PMID:26104486

  4. Conserved hypothetical protein Rv1977 in Mycobacterium tuberculosis strains contains sequence polymorphisms and might be involved in ongoing immune evasion

    PubMed Central

    Jiang, Yi; Liu, Haican; Wang, Xuezhi; Li, Guilian; Qiu, Yan; Dou, Xiangfeng; Wan, Kanglin

    2015-01-01

    Host immune pressure and associated parasite immune evasion are key features of host-pathogen co-evolution. A previous study showed that human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 151 clinical Mycobacterium tuberculosis isolates from China, amplified gene encoding Rv1977 and compared the sequences. The results showed that Rv1977, a conserved hypothetical protein, is not conserved in M. tuberculosis strains and there are polymorphisms existed in the protein. Some mutations, especially one frameshift mutation, occurred in the antigen Rv1977, which is uncommon in M.tb strains and may lead to the protein function altering. Mutations and deletion in the gene all affect one of three T cell epitopes and the changed T cell epitope contained more than one variable position, which may suggest ongoing immune evasion. PMID:26261576

  5. Characterization of a highly conserved baculovirus structural protein that is specific for occlusion-derived virions.

    PubMed

    Theilmann, D A; Chantler, J K; Stweart, S; Flipsen, H T; Vlak, J M; Crook, N E

    1996-04-01

    A highly conserved baculovirus late gene called odvp-6e was shown to be a structural protein that is specific for occlusion-derived virus (ODV) envelopes. The complete sequence of this gene is presented for both Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) and Cydia pomonella granulosis virus (CpGV). The predicted sizes of the OpMNPV and CpGV ODVP-6E are 40, 241, and 38,655 respectively. The OpMNPV odvp-6e gene was transcriptionally mapped and was shown to initiate from a consensus late gene motif, TTAAG, and is expressed from 18-120 hr postinfection. Polyclonal antiserum was generated against a bacterial fusion protein and used to analyze the cellular steady-state levels of ODVP-6E and to determine if this protein was a component of either budded virus (BV) or ODV. Western blots showed that ODVP-6E is a component of the ODV but not BV. This was confirmed by immunoelectron microscopy of ODV from Autographa californica NPV (AcMNPV) which localized ODVP-6E to the ODV envelope. The sequences of the odvp-6e gene from the baculoviruses Choristoneura fumiferana NPV (CfMNPV), AcMNPV, and Helicoverpa zea NPV (HzSNPV) were obtained from GenBank. Comparisons of the predicted amino acid sequences of OpMNPV, CpGV, AcMNPV, CfMNPV, and HzSNPV show that there are two possible membrane-spanning domains and a cysteine-rich domain that are conserved in all of the proteins. PMID:8615018

  6. Structure-sequence based analysis for identification of conserved regions in proteins

    DOEpatents

    Zemla, Adam T; Zhou, Carol E; Lam, Marisa W; Smith, Jason R; Pardes, Elizabeth

    2013-05-28

    Disclosed are computational methods, and associated hardware and software products for scoring conservation in a protein structure based on a computationally identified family or cluster of protein structures. A method of computationally identifying a family or cluster of protein structures in also disclosed herein.

  7. Species identification using genetic tools: the value of nuclear and mitochondrial gene sequences in whale conservation.

    PubMed

    Palumbi, S R; Cipriano, F

    1998-01-01

    DNA sequence analysis is a powerful tool for identifying the source of samples thought to be derived from threatened or endangered species. Analysis of mitochondrial DNA (mtDNA) from retail whale meat markets has shown consistently that the expected baleen whale in these markets, the minke whale, makes up only about half the products analyzed. The other products are either unregulated small toothed whales like dolphins or are protected baleen whales such as humpback, Bryde's, fin, or blue whales. Independent verification of such mtDNA identifications requires analysis of nuclear genetic loci, but this is technically more difficult than standard mtDNA sequencing. In addition, evolution of species-specific sequences (i.e., fixation of sequence differences to produce reciprocally monophyletic gene trees) is slower in nuclear than in mitochondrial genes primarily because genetic drift is slower at nuclear loci. When will use of nuclear sequences allow forensic DNA identification? Comparison of neutral theories of coalescence of mitochondrial and nuclear loci suggests a simple rule of thumb. The "three-times rule" suggests that phylogenetic sorting at nuclear loci is likely to produce species-specific sequences when mitochondrial alleles are reciprocally monophyletic and the branches leading to the mtDNA sequences of a species are three times longer than the average difference observed within species. A preliminary test of the three-times rule, which depends on many assumptions about the species and genes involved, suggests that blue and fin whales should have species-specific sequences at most neutral nuclear loci, whereas humpback and fin whales should show species-specific sequences at fewer nuclear loci. Partial sequences of actin introns from these species confirm the predictions of the three-times rule and show that blue and fin whales are reciprocally monophyletic at this locus. These intron sequences are thus good tools for the identification of these species

  8. Specific binding of eukaryotic ORC to DNA replication origins depends on highly conserved basic residues.

    PubMed

    Kawakami, Hironori; Ohashi, Eiji; Kanamoto, Shota; Tsurimoto, Toshiki; Katayama, Tsutomu

    2015-01-01

    In eukaryotes, the origin recognition complex (ORC) heterohexamer preferentially binds replication origins to trigger initiation of DNA replication. Crystallographic studies using eubacterial and archaeal ORC orthologs suggested that eukaryotic ORC may bind to origin DNA via putative winged-helix DNA-binding domains and AAA+ ATPase domains. However, the mechanisms how eukaryotic ORC recognizes origin DNA remain elusive. Here, we show in budding yeast that Lys-362 and Arg-367 residues of the largest subunit (Orc1), both outside the aforementioned domains, are crucial for specific binding of ORC to origin DNA. These basic residues, which reside in a putative disordered domain, were dispensable for interaction with ATP and non-specific DNA sequences, suggesting a specific role in recognition. Consistent with this, both residues were required for origin binding of Orc1 in vivo. A truncated Orc1 polypeptide containing these residues solely recognizes ARS sequence with low affinity and Arg-367 residue stimulates sequence specific binding mode of the polypeptide. Lys-362 and Arg-367 residues of Orc1 are highly conserved among eukaryotic ORCs, but not in eubacterial and archaeal orthologs, suggesting a eukaryote-specific mechanism underlying recognition of replication origins by ORC. PMID:26456755

  9. Specific binding of eukaryotic ORC to DNA replication origins depends on highly conserved basic residues

    PubMed Central

    Kawakami, Hironori; Ohashi, Eiji; Kanamoto, Shota; Tsurimoto, Toshiki; Katayama, Tsutomu

    2015-01-01

    In eukaryotes, the origin recognition complex (ORC) heterohexamer preferentially binds replication origins to trigger initiation of DNA replication. Crystallographic studies using eubacterial and archaeal ORC orthologs suggested that eukaryotic ORC may bind to origin DNA via putative winged-helix DNA-binding domains and AAA+ ATPase domains. However, the mechanisms how eukaryotic ORC recognizes origin DNA remain elusive. Here, we show in budding yeast that Lys-362 and Arg-367 residues of the largest subunit (Orc1), both outside the aforementioned domains, are crucial for specific binding of ORC to origin DNA. These basic residues, which reside in a putative disordered domain, were dispensable for interaction with ATP and non-specific DNA sequences, suggesting a specific role in recognition. Consistent with this, both residues were required for origin binding of Orc1 in vivo. A truncated Orc1 polypeptide containing these residues solely recognizes ARS sequence with low affinity and Arg-367 residue stimulates sequence specific binding mode of the polypeptide. Lys-362 and Arg-367 residues of Orc1 are highly conserved among eukaryotic ORCs, but not in eubacterial and archaeal orthologs, suggesting a eukaryote-specific mechanism underlying recognition of replication origins by ORC. PMID:26456755

  10. Mammalian ets-1 and ets-2 genes encode highly conserved proteins

    SciTech Connect

    Watson, D.K.; McWilliams, M.J.; Lapis, P.; Lautenberger, J.A.; Schweinfest, C.W.; Papas, T.S. )

    1988-11-01

    Cellular ets sequences homologous to v-ets of the avian leukemia virus E26 are highly conserved. In mammals the ets sequences are dispersed on two separate chromosomal loci, called ets-1 and ets-2. To determine the structure of these two genes and identify the open reading frames that code for the putative proteins, the authors have sequenced human ets-1 cDNAs and ets-2 cDNA clones obtained from both human and mouse. The human ETS1 gene is capable of encoding a protein of 441 amino acids. This protein is >95% identical to the chicken c-ets-1 gene product. Thus, the human ETS1 gene is homologous to the chicken c-ets-1 gene, the protooncogene that the E26 virus transduced. Human and mouse ets-2 cDNA clones are closely related and contain open reading frames capable of encoding proteins of 469 and 468 residues, respectively. Direct comparison of these data with previously published finding indicates that ets is a family of genes whose members share distinct domains.

  11. Mammalian ets-1 and ets-2 genes encode highly conserved proteins.

    PubMed Central

    Watson, D K; McWilliams, M J; Lapis, P; Lautenberger, J A; Schweinfest, C W; Papas, T S

    1988-01-01

    Cellular ets sequences homologous to v-ets of the avian leukemia virus E26 are highly conserved. In mammals the ets sequences are dispersed on two separate chromosomal loci, called ets-1 and ets-2. To determine the structure of these two genes and identify the open reading frames that code for the putative proteins, we have sequenced human ets-1 cDNAs and ets-2 cDNA clones obtained from both human and mouse. The human ETS1 gene is capable of encoding a protein of 441 amino acids. This protein is greater than 95% identical to the chicken c-ets-1 gene product. Thus, the human ETS1 gene is homologous to the chicken c-ets-1 gene, the protooncogene that the E26 virus transduced. Human and mouse ets-2 cDNA clones are closely related and contain open reading frames capable of encoding proteins of 469 and 468 residues, respectively. Direct comparison of these data with previously published findings indicates that ets is a family of genes whose members share distinct domains. PMID:2847145

  12. A novel satellite DNA isolated in Pecten jacobaeus shows high sequence similarity among molluscs.

    PubMed

    Petraccioli, Agnese; Odierna, Gaetano; Capriglione, Teresa; Barucca, Marco; Forconi, Mariko; Olmo, Ettore; Biscotti, Maria Assunta

    2015-10-01

    The aim of this work is to investigate the sequence conservation and the evolution of repeated DNA in related species. Satellite DNA is a component of eukaryotic genomes and is made up of tandemly repeated sequences. These sequences are affected by high rates of mutation that lead to the occurrence of species-specific satellite DNAs, which are different in terms of both quantity and quality. In this work, a novel repetitive DNA family, named PjHhaI sat, is described in Pecten jacobaeus. The quantitative analyses revealed a different abundance of this element in the molluscan species investigated in agreement with the "library hypothesis" even if, in this case, at a high taxonomic level. In addition, the qualitative analysis demonstrated an astonishing sequence conservation not only among scallops but also in six other molluscan species belonging to three classes. These findings suggest that the PjHhaI sat may be considered as the most ancients of DNA described so far, which remained "frozen" during molluscan evolution. The widespread distribution of this sat DNA in molluscs as well as its long evolutionary preservation open up questions on the functional role of this element. A future challenge might be the identification of proteins or molecules which interact with the PjHhaI sat. PMID:25832354

  13. Use of ancient sedimentary DNA as a novel conservation tool for high-altitude tropical biodiversity.

    PubMed

    Boessenkool, Sanne; McGlynn, Gayle; Epp, Laura S; Taylor, David; Pimentel, Manuel; Gizaw, Abel; Nemomissa, Sileshi; Brochmann, Christian; Popp, Magnus

    2014-04-01

    Conservation of biodiversity may in the future increasingly depend upon the availability of scientific information to set suitable restoration targets. In traditional paleoecology, sediment-based pollen provides a means to define preanthropogenic impact conditions, but problems in establishing the exact provenance and ecologically meaningful levels of taxonomic resolution of the evidence are limiting. We explored the extent to which the use of sedimentary ancient DNA (sedaDNA) may complement pollen data in reconstructing past alpine environments in the tropics. We constructed a record of afro-alpine plants retrieved from DNA preserved in sediment cores from 2 volcanic crater sites in the Albertine Rift, eastern Africa. The record extended well beyond the onset of substantial anthropogenic effects on tropical mountains. To ensure high-quality taxonomic inference from the sedaDNA sequences, we built an extensive DNA reference library covering the majority of the afro-alpine flora, by sequencing DNA from taxonomically verified specimens. Comparisons with pollen records from the same sediment cores showed that plant diversity recovered with sedaDNA improved vegetation reconstructions based on pollen records by revealing both additional taxa and providing increased taxonomic resolution. Furthermore, combining the 2 measures assisted in distinguishing vegetation change at different geographic scales; sedaDNA almost exclusively reflects local vegetation, whereas pollen can potentially originate from a wide area that in highlands in particular can span several ecozones. Our results suggest that sedaDNA may provide information on restoration targets and the nature and magnitude of human-induced environmental changes, including in high conservation priority, biodiversity hotspots, where understanding of preanthropogenic impact (or reference) conditions is highly limited. PMID:24372820

  14. High-Order Space-Time Methods for Conservation Laws

    NASA Technical Reports Server (NTRS)

    Huynh, H. T.

    2013-01-01

    Current high-order methods such as discontinuous Galerkin and/or flux reconstruction can provide effective discretization for the spatial derivatives. Together with a time discretization, such methods result in either too small a time step size in the case of an explicit scheme or a very large system in the case of an implicit one. To tackle these problems, two new high-order space-time schemes for conservation laws are introduced: the first is explicit and the second, implicit. The explicit method here, also called the moment scheme, achieves a Courant-Friedrichs-Lewy (CFL) condition of 1 for the case of one-spatial dimension regardless of the degree of the polynomial approximation. (For standard explicit methods, if the spatial approximation is of degree p, then the time step sizes are typically proportional to 1/p(exp 2)). Fourier analyses for the one and two-dimensional cases are carried out. The property of super accuracy (or super convergence) is discussed. The implicit method is a simplified but optimal version of the discontinuous Galerkin scheme applied to time. It reduces to a collocation implicit Runge-Kutta (RK) method for ordinary differential equations (ODE) called Radau IIA. The explicit and implicit schemes are closely related since they employ the same intermediate time levels, and the former can serve as a key building block in an iterative procedure for the latter. A limiting technique for the piecewise linear scheme is also discussed. The technique can suppress oscillations near a discontinuity while preserving accuracy near extrema. Preliminary numerical results are shown

  15. Structure of the voltage-dependent potassium channel is highly conserved from Drosophila to vertebrate central nervous systems.

    PubMed Central

    Baumann, A; Grupe, A; Ackermann, A; Pongs, O

    1988-01-01

    Voltage-sensitive potassium channels are found in vertebrate and invertebrate central nervous systems. We have isolated a rat brain cDNA by cross-hybridization with a probe of the Drosophila Shaker gene complex. Structural conservation of domains of the deduced protein indicate that the rat brain cDNA encodes a voltage-sensitive potassium channel. Of the deduced amino acid sequence, 82% is homologous to the Drosophila Shaker protein indicating that voltage-sensitive potassium channels have been highly conserved during evolution. Selective pressure was highest on sequences facing the intracellular side and on proposed transmembrane segments S4-S6, suggesting that these domains are crucial for voltage-dependent potassium channel function. The corresponding rat mRNA apparently belongs to a family of mRNA molecules which are preferentially expressed in the central nervous system. Images PMID:3191911

  16. Human immunodeficiency virus type 1 integrase: effect on viral replication of mutations at highly conserved residues.

    PubMed

    Cannon, P M; Wilson, W; Byles, E; Kingsman, S M; Kingsman, A J

    1994-08-01

    Sequence comparisons of the integrase (IN) proteins from different retroviruses have identified several highly conserved residues. We have introduced mutations at 16 of these sites into the integrase gene of human immunodeficiency virus type 1 and analyzed the phenotypes of the resulting viruses. The viruses were all normal for p24 content and reverse transcriptase activity. In addition, all of the mutants could infect T-cell lines and undergo reverse transcription, as assessed by PCR analysis. Most of the mutant viruses also had normal Western blot (immunoblot) profiles, although three of the mutations resulted in reduced signals for IN relative to the wild type on the immunoblots and mutation of residue W235 completely abolished recognition of the protein by pooled sera from human immunodeficiency virus type 1-positive patients. Mutations that have previously been shown to abolish activity in in vitro studies produced noninfectious viruses. The substitution of W235 was notable in producing a noninfectious virus, despite previous reports of this residue being nonessential for IN activity in vitro (A.D. Leavitt, L. Shiue, and H.E. Varmus, J. Biol. Chem. 268:2113-2119, 1993). In addition, we have identified four highly conserved residues that can be mutated without any affect on viral replication in T-cell lines. PMID:8035478

  17. A Highly Conserved Region within H2B Is Important for FACT To Act on Nucleosomes

    PubMed Central

    Zheng, Suting; Crickard, J. Brooks; Srikanth, Abhinaya

    2014-01-01

    Histone N-terminal tails play crucial roles in chromatin-related processes. The tails of histones H3 and H4 are highly conserved and well characterized, but much less is known about the functions of the tails of histones H2A and H2B and their sequences are more divergent among eukaryotes. Here we characterized the function of the only highly conserved region in the H2B tail, the H2B repression (HBR) domain. Once thought to play a role only in repression, it also has an uncharacterized function in gene activation and DNA damage responses. We report that deletion of the HBR domain impairs the eviction of nucleosomes at the promoters and open reading frames of genes. A closer examination of the HBR domain mutants revealed that they displayed phenotypes similar to those of histone chaperone complex FACT mutants, including an increase in intragenic transcription and the accumulation of free histones in cells. Biochemical characterization of recombinant nucleosomes indicates that deletion of the HBR domain impairs FACT-dependent removal of H2A-H2B from nucleosomes, suggesting that the HBR domain plays an important role in allowing FACT to disrupt dimer-DNA interactions. We have uncovered a previously unappreciated role for the HBR domain in regulating chromatin structure and have provided insight into how FACT acts on nucleosomes. PMID:24248595

  18. Biological Processes Discovered by High-Throughput Sequencing.

    PubMed

    Reon, Brian J; Dutta, Anindya

    2016-04-01

    Advances in DNA and RNA sequencing technologies have completely transformed the field of genomics. High-throughput sequencing (HTS) is now a widely used and accessible technology that allows scientists to sequence an entire transcriptome or genome in a timely and cost-effective manner. Application of HTS techniques has led to many key discoveries, including the identification of long noncoding RNAs, microDNAs, a family of small extrachromosomal circular DNA species, and tRNA-derived fragments, which are a group of small non-miRNAs that are derived from tRNAs. Furthermore, public sequencing repositories provide unique opportunities for laboratories to parse large sequencing databases to identify proteins and noncoding RNAs at a scale that was not possible a decade ago. Herein, we review how HTS has led to the discovery of novel nucleic acid species and uncovered new biological processes during the course. PMID:26828742

  19. Conservation of nucleotide sequences for molecular diagnosis of Middle East respiratory syndrome coronavirus, 2015.

    PubMed

    Furuse, Yuki; Okamoto, Michiko; Oshitani, Hitoshi

    2015-11-01

    Infection due to the Middle East respiratory syndrome coronavirus (MERS-CoV) is widespread. The present study was performed to assess the protocols used for the molecular diagnosis of MERS-CoV by analyzing the nucleotide sequences of viruses detected between 2012 and 2015, including sequences from the large outbreak in eastern Asia in 2015. Although the diagnostic protocols were established only 2 years ago, mismatches between the sequences of primers/probes and viruses were found for several of the assays. Such mismatches could lead to a lower sensitivity of the assay, thereby leading to false-negative diagnosis. A slight modification in the primer design is suggested. Protocols for the molecular diagnosis of viral infections should be reviewed regularly after they are established, particularly for viruses that pose a great threat to public health such as MERS-CoV. PMID:26432410

  20. A New DNA Binding Protein Highly Conserved in Diverse Crenarchaeal Viruses

    SciTech Connect

    Larson, E.T.; Eilers, B.J.; Reiter, D.; Ortmann, A.C.; Young, M.J.; Lawrence, C.M.; /Montana State U. /Tubingen U.

    2007-07-09

    Sulfolobus turreted icosahedral virus (STIV) infects Sulfolobus species found in the hot springs of Yellowstone National Park. Its 37 open reading frames (ORFs) generally lack sequence similarity to other genes. One exception, however, is ORF B116. While its function is unknown, orthologs are found in three additional crenarchaeal viral families. Due to the central importance of this protein family to crenarchaeal viruses, we have undertaken structural and biochemical studies of B116. The structure reveals a previously unobserved fold consisting of a five-stranded beta-sheet flanked on one side by three alpha helices. Two subunits come together to form a homodimer with a 10-stranded mixed beta-sheet, where the topology of the central strands resembles an unclosed beta-barrel. Highly conserved loops rise above the surface of the saddle-shaped protein and suggest an interaction with the major groove of DNA. The predicted B116-DNA interaction is confirmed by electrophoretic mobility shift assays.

  1. Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement

    SciTech Connect

    Le Coq, Johanne; Ghosh, Partho

    2012-06-19

    Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein, TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd ({approx}16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 10{sup 20} potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation.

  2. Structure is three to ten times more conserved than sequence--a study of structural response in protein cores.

    PubMed

    Illergård, Kristoffer; Ardell, David H; Elofsson, Arne

    2009-11-15

    Protein structures change during evolution in response to mutations. Here, we analyze the mapping between sequence and structure in a set of structurally aligned protein domains. To avoid artifacts, we restricted our attention only to the core components of these structures. We found that on average, using different measures of structural change, protein cores evolve linearly with evolutionary distance (amino acid substitutions per site). This is true irrespective of which measure of structural change we used, whether RMSD or discrete structural descriptors for secondary structure, accessibility, or contacts. This linear response allows us to quantify the claim that structure is more conserved than sequence. Using structural alphabets of similar cardinality to the sequence alphabet, structural cores evolve three to ten times slower than sequences. Although we observed an average linear response, we found a wide variance. Different domain families varied fivefold in structural response to evolution. An attempt to categorically analyze this variance among subgroups by structural and functional category revealed only one statistically significant trend. This trend can be explained by the fact that beta-sheets change faster than alpha-helices, most likely due to that they are shorter and that change occurs at the ends of the secondary structure elements. PMID:19507241

  3. Storage and retrieval of highly repetitive sequence collections.

    PubMed

    Mäkinen, Veli; Navarro, Gonzalo; Sirén, Jouni; Välimäki, Niko

    2010-03-01

    A repetitive sequence collection is a set of sequences which are small variations of each other. A prominent example are genome sequences of individuals of the same or close species, where the differences can be expressed by short lists of basic edit operations. Flexible and efficient data analysis on such a typically huge collection is plausible using suffix trees. However, the suffix tree occupies much space, which very soon inhibits in-memory analyses. Recent advances in full-text indexing reduce the space of the suffix tree to, essentially, that of the compressed sequences, while retaining its functionality with only a polylogarithmic slowdown. However, the underlying compression model considers only the predictability of the next sequence symbol given the k previous ones, where k is a small integer. This is unable to capture longer-term repetitiveness. For example, r identical copies of an incompressible sequence will be incompressible under this model. We develop new static and dynamic full-text indexes that are able of capturing the fact that a collection is highly repetitive, and require space basically proportional to the length of one typical sequence plus the total number of edit operations. The new indexes can be plugged into a recent dynamic fully-compressed suffix tree, achieving full functionality for sequence analysis, while retaining the reduced space and the polylogarithmic slowdown. Our experimental results confirm the practicality of our proposal. PMID:20377446

  4. Computational identification of riboswitches based on RNA conserved functional sequences and conformations.

    PubMed

    Chang, Tzu-Hao; Huang, Hsien-Da; Wu, Li-Ching; Yeh, Chi-Ta; Liu, Baw-Jhiune; Horng, Jorng-Tzong

    2009-07-01

    Riboswitches are cis-acting genetic regulatory elements within a specific mRNA that can regulate both transcription and translation by interacting with their corresponding metabolites. Recently, an increasing number of riboswitches have been identified in different species and investigated for their roles in regulatory functions. Both the sequence contexts and structural conformations are important characteristics of riboswitches. None of the previously developed tools, such as covariance models (CMs), Riboswitch finder, and RibEx, provide a web server for efficiently searching homologous instances of known riboswitches or considers two crucial characteristics of each riboswitch, such as the structural conformations and sequence contexts of functional regions. Therefore, we developed a systematic method for identifying 12 kinds of riboswitches. The method is implemented and provided as a web server, RiboSW, to efficiently and conveniently identify riboswitches within messenger RNA sequences. The predictive accuracy of the proposed method is comparable with other previous tools. The efficiency of the proposed method for identifying riboswitches was improved in order to achieve a reasonable computational time required for the prediction, which makes it possible to have an accurate and convenient web server for biologists to obtain the results of their analysis of a given mRNA sequence. RiboSW is now available on the web at http://RiboSW.mbc.nctu.edu.tw/. PMID:19460868

  5. Empirical assessment of sequencing errors for high throughput pyrosequencing data

    PubMed Central

    2013-01-01

    Background Sequencing-by-synthesis technologies significantly improve over the Sanger method in terms of speed and cost per base. However, they still usually fail to compete in terms of read length and quality. Current high-throughput implementations of the pyrosequencing technique yield reads whose length approach those of the capillary electrophoresis method. A less obvious question is whether their quality is affected by platform-specific sequencing errors. Results We present an empirical study aimed at assessing the quality and characterising sequencing errors for high throughput pyrosequencing data. We have developed a procedure for extracting sequencing error data from genome assemblies and study their characteristics, in particular the length distribution of indel gaps and their relation to the sequence contexts where they occur. We used this procedure to analyse data from three prokaryotic genomes sequenced with the GS FLX technology. We also compared two models previously employed with success for peptide sequence alignment. Conclusions We observed an overall very low error rate in the analysed data, with indel errors being much more abundant than substitutions. We also observed a dependence between the length of the gaps and that of the homopolymer context where they occur. As with protein alignments, a power-law model seems to approximate the indel errors more accurately, although the results are not so conclusive as to justify a depart from the commonly used affine gap penalty scheme. In whichever case, however, our procedure can be used to estimate more realistic error model parameters. PMID:23339526

  6. Sequence Divergence and Conservation in Genomes of Helicobacter cetorum Strains from a Dolphin and a Whale

    PubMed Central

    Kersulyte, Dangeruta; Rossi, Mirko; Berg, Douglas E.

    2013-01-01

    Background and Objectives Strains of Helicobacter cetorum have been cultured from several marine mammals and have been found to be closely related in 16 S rDNA sequence to the human gastric pathogen H. pylori, but their genomes were not characterized further. Methods The genomes of H. cetorum strains from a dolphin and a whale were sequenced completely using 454 technology and PCR and capillary sequencing. Results These genomes are 1.8 and 1.95 mb in size, some 7–26% larger than H. pylori genomes, and differ markedly from one another in gene content, and sequences and arrangements of shared genes. However, each strain is more related overall to H. pylori and its descendant H. acinonychis than to other known species. These H. cetorum strains lack cag pathogenicity islands, but contain novel alleles of the virulence-associated vacuolating cytotoxin (vacA) gene. Of particular note are (i) an extra triplet of vacA genes with ≤50% protein-level identity to each other in the 5′ two-thirds of the gene needed for host factor interaction; (ii) divergent sets of outer membrane protein genes; (iii) several metabolic genes distinct from those of H. pylori; (iv) genes for an iron-cofactored urease related to those of Helicobacter species from terrestrial carnivores, in addition to genes for a nickel co-factored urease; and (v) members of the slr multigene family, some of which modulate host responses to infection and improve Helicobacter growth with mammalian cells. Conclusions Our genome sequence data provide a glimpse into the novelty and great genetic diversity of marine helicobacters. These data should aid further analyses of microbial genome diversity and evolution and infection and disease mechanisms in vast and often fragile ocean ecosystems. PMID:24358262

  7. 7 CFR 760.821 - Compliance with highly erodible land and wetland conservation.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 7 2010-01-01 2010-01-01 false Compliance with highly erodible land and wetland... Disaster Program § 760.821 Compliance with highly erodible land and wetland conservation. (a) The highly erodible land and wetland conservation provisions of part 12 of this title apply to the receipt of...

  8. 7 CFR 760.821 - Compliance with highly erodible land and wetland conservation.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 7 2013-01-01 2013-01-01 false Compliance with highly erodible land and wetland... Disaster Program § 760.821 Compliance with highly erodible land and wetland conservation. (a) The highly erodible land and wetland conservation provisions of part 12 of this title apply to the receipt of...

  9. 7 CFR 760.821 - Compliance with highly erodible land and wetland conservation.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 7 2012-01-01 2012-01-01 false Compliance with highly erodible land and wetland... Disaster Program § 760.821 Compliance with highly erodible land and wetland conservation. (a) The highly erodible land and wetland conservation provisions of part 12 of this title apply to the receipt of...

  10. 7 CFR 760.821 - Compliance with highly erodible land and wetland conservation.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 7 2014-01-01 2014-01-01 false Compliance with highly erodible land and wetland... Disaster Program § 760.821 Compliance with highly erodible land and wetland conservation. (a) The highly erodible land and wetland conservation provisions of part 12 of this title apply to the receipt of...

  11. 7 CFR 760.821 - Compliance with highly erodible land and wetland conservation.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 7 2011-01-01 2011-01-01 false Compliance with highly erodible land and wetland... Disaster Program § 760.821 Compliance with highly erodible land and wetland conservation. (a) The highly erodible land and wetland conservation provisions of part 12 of this title apply to the receipt of...

  12. Sequence Similarity of Clostridium difficile Strains by Analysis of Conserved Genes and Genome Content Is Reflected by Their Ribotype Affiliation

    PubMed Central

    Kurka, Hedwig; Ehrenreich, Armin; Ludwig, Wolfgang; Monot, Marc; Rupnik, Maja; Barbut, Frederic; Indra, Alexander; Dupuy, Bruno; Liebl, Wolfgang

    2014-01-01

    PCR-ribotyping is a broadly used method for the classification of isolates of Clostridium difficile, an emerging intestinal pathogen, causing infections with increased disease severity and incidence in several European and North American countries. We have now carried out clustering analysis with selected genes of numerous C. difficile strains as well as gene content comparisons of their genomes in order to broaden our view of the relatedness of strains assigned to different ribotypes. We analyzed the genomic content of 48 C. difficile strains representing 21 different ribotypes. The calculation of distance matrix-based dendrograms using the neighbor joining method for 14 conserved genes (standard phylogenetic marker genes) from the genomes of the C. difficile strains demonstrated that the genes from strains with the same ribotype generally clustered together. Further, certain ribotypes always clustered together and formed ribotype groups, i.e. ribotypes 078, 033 and 126, as well as ribotypes 002 and 017, indicating their relatedness. Comparisons of the gene contents of the genomes of ribotypes that clustered according to the conserved gene analysis revealed that the number of common genes of the ribotypes belonging to each of these three ribotype groups were very similar for the 078/033/126 group (at most 69 specific genes between the different strains with the same ribotype) but less similar for the 002/017 group (86 genes difference). It appears that the ribotype is indicative not only of a specific pattern of the amplified 16S–23S rRNA intergenic spacer but also reflects specific differences in the nucleotide sequences of the conserved genes studied here. It can be anticipated that the sequence deviations of more genes of C. difficile strains are correlated with their PCR-ribotype. In conclusion, the results of this study corroborate and extend the concept of clonal C. difficile lineages, which correlate with ribotypes affiliation. PMID:24482682

  13. Sequence similarity of Clostridium difficile strains by analysis of conserved genes and genome content is reflected by their ribotype affiliation.

    PubMed

    Kurka, Hedwig; Ehrenreich, Armin; Ludwig, Wolfgang; Monot, Marc; Rupnik, Maja; Barbut, Frederic; Indra, Alexander; Dupuy, Bruno; Liebl, Wolfgang

    2014-01-01

    PCR-ribotyping is a broadly used method for the classification of isolates of Clostridium difficile, an emerging intestinal pathogen, causing infections with increased disease severity and incidence in several European and North American countries. We have now carried out clustering analysis with selected genes of numerous C. difficile strains as well as gene content comparisons of their genomes in order to broaden our view of the relatedness of strains assigned to different ribotypes. We analyzed the genomic content of 48 C. difficile strains representing 21 different ribotypes. The calculation of distance matrix-based dendrograms using the neighbor joining method for 14 conserved genes (standard phylogenetic marker genes) from the genomes of the C. difficile strains demonstrated that the genes from strains with the same ribotype generally clustered together. Further, certain ribotypes always clustered together and formed ribotype groups, i.e. ribotypes 078, 033 and 126, as well as ribotypes 002 and 017, indicating their relatedness. Comparisons of the gene contents of the genomes of ribotypes that clustered according to the conserved gene analysis revealed that the number of common genes of the ribotypes belonging to each of these three ribotype groups were very similar for the 078/033/126 group (at most 69 specific genes between the different strains with the same ribotype) but less similar for the 002/017 group (86 genes difference). It appears that the ribotype is indicative not only of a specific pattern of the amplified 16S-23S rRNA intergenic spacer but also reflects specific differences in the nucleotide sequences of the conserved genes studied here. It can be anticipated that the sequence deviations of more genes of C. difficile strains are correlated with their PCR-ribotype. In conclusion, the results of this study corroborate and extend the concept of clonal C. difficile lineages, which correlate with ribotypes affiliation. PMID:24482682

  14. 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.

  15. Sorting out relationships among the grouse and ptarmigan using intron, mitochondrial, and ultra-conserved element sequences.

    PubMed

    Persons, Nicholas W; Hosner, Peter A; Meiklejohn, Kelly A; Braun, Edward L; Kimball, Rebecca T

    2016-05-01

    The Holarctic phasianid clade of the grouse and ptarmigan has received substantial attention in areas such as evolution of mating systems, display behavior, and population ecology related to their conservation and management as wild game species. There are multiple molecular phylogenetic studies that focus on grouse and ptarmigan. In spite of this, there is little consensus regarding historical relationships, particularly among genera, which has led to unstable and partial taxonomic revisions. We estimated the phylogeny of all currently recognized species using a combination of novel data from seven nuclear loci (largely intron sequences) and published data from one additional autosomal locus, two W-linked loci, and four mitochondrial regions. To explore relationships among genera and assess paraphyly of one genus more rigorously, we then added over 3000 ultra-conserved element (UCE) loci (over 1.7million bp) gathered using Illumina sequencing. The UCE topology agreed with that of the combined nuclear intron and previously published sequence data with 100% bootstrap support for all relationships. These data strongly support previous studies separating Bonasa from Tetrastes and Dendragapus from Falcipennis. However, the placement of Lagopus differed from previous studies, and we found no support for Falcipennis monophyly. Biogeographic analysis suggests that the ancestors of grouse and ptarmigan were distributed in the New World and subsequently underwent at least four dispersal events between the Old and New Worlds. Divergence time estimates from maternally-inherited and autosomal markers show stark differences across this clade, with divergence time estimates from maternally-inherited markers being nearly half that of the autosomal markers at some nodes, and nearly twice that at other nodes. PMID:26879712

  16. The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.

    PubMed

    Chipman, Ariel D; Ferrier, David E K; Brena, Carlo; Qu, Jiaxin; Hughes, Daniel S T; Schröder, Reinhard; Torres-Oliva, Montserrat; Znassi, Nadia; Jiang, Huaiyang; Almeida, Francisca C; Alonso, Claudio R; Apostolou, Zivkos; Aqrawi, Peshtewani; Arthur, Wallace; Barna, Jennifer C J; Blankenburg, Kerstin P; Brites, Daniela; Capella-Gutiérrez, Salvador; Coyle, Marcus; Dearden, Peter K; Du Pasquier, Louis; Duncan, Elizabeth J; Ebert, Dieter; Eibner, Cornelius; Erikson, Galina; Evans, Peter D; Extavour, Cassandra G; Francisco, Liezl; Gabaldón, Toni; Gillis, William J; Goodwin-Horn, Elizabeth A; Green, Jack E; Griffiths-Jones, Sam; Grimmelikhuijzen, Cornelis J P; Gubbala, Sai; Guigó, Roderic; Han, Yi; Hauser, Frank; Havlak, Paul; Hayden, Luke; Helbing, Sophie; Holder, Michael; Hui, Jerome H L; Hunn, Julia P; Hunnekuhl, Vera S; Jackson, LaRonda; Javaid, Mehwish; Jhangiani, Shalini N; Jiggins, Francis M; Jones, Tamsin E; Kaiser, Tobias S; Kalra, Divya; Kenny, Nathan J; Korchina, Viktoriya; Kovar, Christie L; Kraus, F Bernhard; Lapraz, François; Lee, Sandra L; Lv, Jie; Mandapat, Christigale; Manning, Gerard; Mariotti, Marco; Mata, Robert; Mathew, Tittu; Neumann, Tobias; Newsham, Irene; Ngo, Dinh N; Ninova, Maria; Okwuonu, Geoffrey; Ongeri, Fiona; Palmer, William J; Patil, Shobha; Patraquim, Pedro; Pham, Christopher; Pu, Ling-Ling; Putman, Nicholas H; Rabouille, Catherine; Ramos, Olivia Mendivil; Rhodes, Adelaide C; Robertson, Helen E; Robertson, Hugh M; Ronshaugen, Matthew; Rozas, Julio; Saada, Nehad; Sánchez-Gracia, Alejandro; Scherer, Steven E; Schurko, Andrew M; Siggens, Kenneth W; Simmons, DeNard; Stief, Anna; Stolle, Eckart; Telford, Maximilian J; Tessmar-Raible, Kristin; Thornton, Rebecca; van der Zee, Maurijn; von Haeseler, Arndt; Williams, James M; Willis, Judith H; Wu, Yuanqing; Zou, Xiaoyan; Lawson, Daniel; Muzny, Donna M; Worley, Kim C; Gibbs, Richard A; Akam, Michael; Richards, Stephen

    2014-11-01

    Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific

  17. The First Myriapod Genome Sequence Reveals Conservative Arthropod Gene Content and Genome Organisation in the Centipede Strigamia maritima

    PubMed Central

    Chipman, Ariel D.; Ferrier, David E. K.; Brena, Carlo; Qu, Jiaxin; Hughes, Daniel S. T.; Schröder, Reinhard; Torres-Oliva, Montserrat; Znassi, Nadia; Jiang, Huaiyang; Almeida, Francisca C.; Alonso, Claudio R.; Apostolou, Zivkos; Aqrawi, Peshtewani; Arthur, Wallace; Barna, Jennifer C. J.; Blankenburg, Kerstin P.; Brites, Daniela; Capella-Gutiérrez, Salvador; Coyle, Marcus; Dearden, Peter K.; Du Pasquier, Louis; Duncan, Elizabeth J.; Ebert, Dieter; Eibner, Cornelius; Erikson, Galina; Evans, Peter D.; Extavour, Cassandra G.; Francisco, Liezl; Gabaldón, Toni; Gillis, William J.; Goodwin-Horn, Elizabeth A.; Green, Jack E.; Griffiths-Jones, Sam; Grimmelikhuijzen, Cornelis J. P.; Gubbala, Sai; Guigó, Roderic; Han, Yi; Hauser, Frank; Havlak, Paul; Hayden, Luke; Helbing, Sophie; Holder, Michael; Hui, Jerome H. L.; Hunn, Julia P.; Hunnekuhl, Vera S.; Jackson, LaRonda; Javaid, Mehwish; Jhangiani, Shalini N.; Jiggins, Francis M.; Jones, Tamsin E.; Kaiser, Tobias S.; Kalra, Divya; Kenny, Nathan J.; Korchina, Viktoriya; Kovar, Christie L.; Kraus, F. Bernhard; Lapraz, François; Lee, Sandra L.; Lv, Jie; Mandapat, Christigale; Manning, Gerard; Mariotti, Marco; Mata, Robert; Mathew, Tittu; Neumann, Tobias; Newsham, Irene; Ngo, Dinh N.; Ninova, Maria; Okwuonu, Geoffrey; Ongeri, Fiona; Palmer, William J.; Patil, Shobha; Patraquim, Pedro; Pham, Christopher; Pu, Ling-Ling; Putman, Nicholas H.; Rabouille, Catherine; Ramos, Olivia Mendivil; Rhodes, Adelaide C.; Robertson, Helen E.; Robertson, Hugh M.; Ronshaugen, Matthew; Rozas, Julio; Saada, Nehad; Sánchez-Gracia, Alejandro; Scherer, Steven E.; Schurko, Andrew M.; Siggens, Kenneth W.; Simmons, DeNard; Stief, Anna; Stolle, Eckart; Telford, Maximilian J.; Tessmar-Raible, Kristin; Thornton, Rebecca; van der Zee, Maurijn; von Haeseler, Arndt; Williams, James M.; Willis, Judith H.; Wu, Yuanqing; Zou, Xiaoyan; Lawson, Daniel; Muzny, Donna M.; Worley, Kim C.; Gibbs, Richard A.; Akam, Michael; Richards, Stephen

    2014-01-01

    Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific

  18. Simian immunodeficiency virus (mac 251-32H) transmembrane protein sequence remains conserved throughout the course of infection in macaques.

    PubMed

    Slade, A; Jones, S; Almond, N; Kitchin, P

    1993-02-01

    Two cynomolgus macaques were infected with a genetically complex challenge stock of simian immunodeficiency virus (SIVmac251-32H). The polymerase chain reaction (PCR) was used to amplify the env gp41, rev, and nef overlapping coding sequences from provirus present in the blood of both animals at 1, 6, and 15 months post infection (p.i.). The predominant, env sequences found in both animals at the three time points were very similar to that found in the original 11/88 challenge stock. The functionally important hydrophobic fusion and membrane-spanning domains within gp41 remained conserved throughout the course of infection. Nucleotide variation within the region corresponding to the REV response element (RRE) was limited to four positions, none of which were predicted to cause any significant disruption to the secondary structure of the RRE. Very little genetic variation was observed in and around the cluster of potential glycosylation sites of the external portion of gp41. However, the existence of a previously assigned variable region elsewhere in the cytoplasmic domain of gp41 was confirmed. The three gene loci (env, rev, and nef) examined varied independently. All changes in the predominant protein sequences were brought about by single nucleotide substitutions only. After 15 months of infection with SIV, 1 animal was sick from SIV-induced disease whereas the other remained healthy. In-frame stop codons within the transmembrane protein occurred with a much greater frequency in the healthy animal. PMID:8457380

  19. High-throughput sequencing in veterinary infection biology and diagnostics.

    PubMed

    Belák, S; Karlsson, O E; Leijon, M; Granberg, F

    2013-12-01

    Sequencing methods have improved rapidly since the first versions of the Sanger techniques, facilitating the development of very powerful tools for detecting and identifying various pathogens, such as viruses, bacteria and other microbes. The ongoing development of high-throughput sequencing (HTS; also known as next-generation sequencing) technologies has resulted in a dramatic reduction in DNA sequencing costs, making the technology more accessible to the average laboratory. In this White Paper of the World Organisation for Animal Health (OIE) Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine (Uppsala, Sweden), several approaches and examples of HTS are summarised, and their diagnostic applicability is briefly discussed. Selected future aspects of HTS are outlined, including the need for bioinformatic resources, with a focus on improving the diagnosis and control of infectious diseases in veterinary medicine. PMID:24761741

  20. High-Throughput Sequencing of Complete Mitochondrial Genomes.

    PubMed

    Briscoe, Andrew George; Hopkins, Kevin Peter; Waeschenbach, Andrea

    2016-01-01

    Next-generation sequencing has revolutionized mitogenomics, turning a cottage industry into a high throughput process. This chapter outlines methodologies used to sequence, assemble, and annotate mitogenomes of non-model organisms using Illumina sequencing technology, utilizing either long-range PCR amplicons or gDNA as starting template. Instructions are given on how to extract DNA, conduct long-range PCR amplifications, generate short Sanger barcode tag sequences, prepare equimolar sample pools, construct and assess quality library preparations, assemble Illumina reads using either seeded reference mapping or de novo assembly, and annotate mitogenomes in the absence of an automated pipeline. Notes and recommendations, derived from our own experience, are given throughout this chapter. PMID:27460369

  1. High-Resolution Genuinely Multidimensional Solution of Conservation Laws by the Space-Time Conservation Element and Solution Element Method

    NASA Technical Reports Server (NTRS)

    Himansu, Ananda; Chang, Sin-Chung; Yu, Sheng-Tao; Wang, Xiao-Yen; Loh, Ching-Yuen; Jorgenson, Philip C. E.

    1999-01-01

    In this overview paper, we review the basic principles of the method of space-time conservation element and solution element for solving the conservation laws in one and two spatial dimensions. The present method is developed on the basis of local and global flux conservation in a space-time domain, in which space and time are treated in a unified manner. In contrast to the modern upwind schemes, the approach here does not use the Riemann solver and the reconstruction procedure as the building blocks. The drawbacks of the upwind approach, such as the difficulty of rationally extending the 1D scalar approach to systems of equations and particularly to multiple dimensions is here contrasted with the uniformity and ease of generalization of the Conservation Element and Solution Element (CE/SE) 1D scalar schemes to systems of equations and to multiple spatial dimensions. The assured compatibility with the simplest type of unstructured meshes, and the uniquely simple nonreflecting boundary conditions of the present method are also discussed. The present approach has yielded high-resolution shocks, rarefaction waves, acoustic waves, vortices, ZND detonation waves, and shock/acoustic waves/vortices interactions. Moreover, since no directional splitting is employed, numerical resolution of two-dimensional calculations is comparable to that of the one-dimensional calculations. Some sample applications displaying the strengths and broad applicability of the CE/SE method are reviewed.

  2. High conservation of a 5' element required for RNA editing of a C target in chloroplast psbE transcripts.

    PubMed

    Hayes, Michael L; Hanson, Maureen R

    2008-09-01

    C-to-U editing modifies 30-40 distinct nucleotides within higher-plant chloroplast transcripts. Many C targets are located at the same position in homologous genes from different plants; these either could have emerged independently or could share a common origin. The 5' sequence GCCGUU, required for editing of C214 in tobacco psbE in vitro, is one of the few identified editing cis-elements. We investigated psbE sequences from many plant species to determine in what lineage(s) editing of psbE C214 emerged and whether the cis-element identified in tobacco is conserved in plants with a C214. The GCCGUU sequence is present at a high frequency in plants that carry a C214 in psbE. However, Sciadopitys verticillata (Pinophyta) edits C214 despite the presence of nucleotide differences compared to the conserved cis-element. The C214 site in psbE genes is represented in members of four branches of spermatophytes but not in gnetophytes, resulting in the parsimonious prediction that editing of psbE C214 was present in the ancestor of spermatophytes. Extracts from chloroplasts from a species that has a difference in the motif and lacks the C target are incapable of editing tobacco psbE C214 substrates, implying that the critical trans-acting protein factors were not retained without a C target. Because noncoding sequences are less constrained than coding regions, we analyzed sequences 5' to two C editing targets located within coding regions to search for possible editing-related conserved elements. Putative editing cis-elements were uncovered in the 5' UTRs near editing sites psbL C2 and ndhD C2. PMID:18696032

  3. Canine Polydactyl Mutations With Heterogeneous Origin in the Conserved Intronic Sequence of LMBR1

    PubMed Central

    Park, Kiyun; Kang, Joohyun; Subedi, Krishna Pd.; Ha, Ji-Hong; Park, Chankyu

    2008-01-01

    Canine preaxial polydactyly (PPD) in the hind limb is a developmental trait that restores the first digit lost during canine evolution. Using a linkage analysis, we previously demonstrated that the affected gene in a Korean breed is located on canine chromosome 16. The candidate locus was further limited to a linkage disequilibrium (LD) block of <213 kb composing the single gene, LMBR1, by LD mapping with single nucleotide polymorphisms (SNPs) for affected individuals from both Korean and Western breeds. The ZPA regulatory sequence (ZRS) in intron 5 of LMBR1 was implicated in mammalian polydactyly. An analysis of the LD haplotypes around the ZRS for various dog breeds revealed that only a subset is assigned to Western breeds. Furthermore, two distinct affected haplotypes for Asian and Western breeds were found, each containing different single-base changes in the upstream sequence (pZRS) of the ZRS. Unlike the previously characterized cases of PPD identified in the mouse and human ZRS regions, the canine mutations in pZRS lacked the ectopic expression of sonic hedgehog in the anterior limb bud, distinguishing its role in limb development from that of the ZRS. PMID:18689889

  4. Identification and profiling of novel and conserved microRNAs during the flower opening process in Prunus mume via deep sequencing.

    PubMed

    Wang, Tao; Pan, Huitang; Wang, Jia; Yang, Weiru; Cheng, Tangren; Zhang, Qixiang

    2014-04-01

    MicroRNAs (miRNAs) are small (20-24 nucleotide) RNAs that are critical regulators of genes involved in diverse plant processes, including development, metabolism, abiotic stress and flowering. Prunus mume is a widely cultivated ornamental plant in East Asia that blooms in early spring, even at temperatures below 0 °C. While miRNAs involved in pistil development have been identified in P. mume, few studies have profiled miRNA expression patterns during flower opening. Here, we used high-throughput sequencing and bioinformatic analysis to identify and profile miRNAs that function during flower opening in P. mume. We identified 47 conserved miRNA sequences belonging to 25 miRNA families from 92 loci in P. mume, along with 33 novel miRNA sequences from 43 loci, including their complementary miRNA* strands. The expression levels of most differentially expressed miRNAs decreased during flower opening, while miR156e-f and miR477b were upregulated at the flowering stage. We predicted 88 target genes for conserved and novel miRNAs using computational analysis and annotated their functions. Seven target genes, encoding squamosa promoter binding protein-like (SPL) and auxin response factor (ARF), scarecrow-like transcription factor (SCL) and APETALA2-like transcription factors (AP2), were verified by 5'-RACE to be the targets of miR156, miR167, miR171 and miR172, respectively. Quantitative real-time PCR validated the expression of the miRNAs and seven target genes. The results help lay the foundation for investigating the roles of miRNAs in the blooming of P. mume. PMID:24343764

  5. Conservative forgetful scholars: How people learn causal structure through sequences of interventions.

    PubMed

    Bramley, Neil R; Lagnado, David A; Speekenbrink, Maarten

    2015-05-01

    Interacting with a system is key to uncovering its causal structure. A computational framework for interventional causal learning has been developed over the last decade, but how real causal learners might achieve or approximate the computations entailed by this framework is still poorly understood. Here we describe an interactive computer task in which participants were incentivized to learn the structure of probabilistic causal systems through free selection of multiple interventions. We develop models of participants' intervention choices and online structure judgments, using expected utility gain, probability gain, and information gain and introducing plausible memory and processing constraints. We find that successful participants are best described by a model that acts to maximize information (rather than expected score or probability of being correct); that forgets much of the evidence received in earlier trials; but that mitigates this by being conservative, preferring structures consistent with earlier stated beliefs. We explore 2 heuristics that partly explain how participants might be approximating these models without explicitly representing or updating a hypothesis space. PMID:25329086

  6. Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

    PubMed Central

    2011-01-01

    Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes) and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes) hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease. PMID:21669002

  7. High Throughput Plasmid Sequencing with Illumina and CLC Bio (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

    SciTech Connect

    Athavale, Ajay

    2012-06-01

    Ajay Athavale (Monsanto) presents "High Throughput Plasmid Sequencing with Illumina and CLC Bio" at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

  8. High Throughput Plasmid Sequencing with Illumina and CLC Bio (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

    ScienceCinema

    Athavale, Ajay [Monsanto

    2013-01-25

    Ajay Athavale (Monsanto) presents "High Throughput Plasmid Sequencing with Illumina and CLC Bio" at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

  9. Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication.

    PubMed

    Cannon, P M; Byles, E D; Kingsman, S M; Kingsman, A J

    1996-01-01

    We have previously identified a residue in the carboxyl terminus of human immunodeficiency virus type 1 integrase (HIV-1 IN), W-235, the requirement for which is only revealed in viral assays for integrase function (P. M. Cannon, W. Wilson, E. Byles, S. M. Kingsman, and A. J. Kingsman, J. Virol. 68:4768-4775, 1994). Our further analysis of this region of retroviral IN has now identified several sequence motifs which are conserved in all the retroviruses we examined, apart from human spumaretrovirus. We have made mutations within these motifs in HIV-1 IN and examined their phenotypes when reintroduced into an infectious proviral clone. The deleterious effects of several of these mutations demonstrate the importance of these regions for IN function in vivo. We observed a further discrepancy, at a motif that is only conserved in the lentiviruses, in the ability of mutants to function in in vitro and in vivo assays. Substitutions both in this region and at W-235 abolish HIV-1 infectivity but do not affect particle production, morphology, reverse transcription, or nuclear import in T-cell lines. Taken together with the in vitro data suggesting that neither of these residues is directly involved in the catalytic reactions of IN, it seems likely that we have identified regions of IN that are essential for interactions with other components of the integration machinery. PMID:8523588

  10. Complete mitochondrial DNA sequence of the endangered giant sable antelope (Hippotragus niger variani): insights into conservation and taxonomy.

    PubMed

    Espregueira Themudo, Gonçalo; Rufino, Ana C; Campos, Paula F

    2015-02-01

    The giant sable antelope is one of the most endangered African bovids. Populations of this iconic animal, the national symbol of Angola, were recently rediscovered, after many decades of presumed extinction. Even so, their numbers are scarce and hence conservation plans are essential. However, fundamental information such as its taxonomic position, time of divergence and degree of genetic variation are still lacking. Here, we used a museum preserved horn as a source of DNA to describe, for the first time, the complete mitochondrial genome of the giant sable antelope, and provide insights into its evolutionary history. Reads generated by shotgun sequencing were mapped against the mitochondrial genome of common sable antelope and the nuclear genomes of cow and sheep. Phylogenetic reconstruction and divergence time estimate give support to the monophyly of the giant sable and a maximum divergence time of 170 thousand years to the closest subspecies. About 7% of the nuclear genome was mapped against the reference. The genetic resources reported here are now available for future work in the field of conservation genetics and phylogeny, in this and related species. PMID:25527983

  11. Binary interactions with high accretion rates onto main sequence stars

    NASA Astrophysics Data System (ADS)

    Shiber, Sagiv; Schreier, Ron; Soker, Noam

    2016-07-01

    Energetic outflows from main sequence stars accreting mass at very high rates might account for the powering of some eruptive objects, such as merging main sequence stars, major eruptions of luminous blue variables, e.g., the Great Eruption of Eta Carinae, and other intermediate luminosity optical transients (ILOTs; red novae; red transients). These powerful outflows could potentially also supply the extra energy required in the common envelope process and in the grazing envelope evolution of binary systems. We propose that a massive outflow/jets mediated by magnetic fields might remove energy and angular momentum from the accretion disk to allow such high accretion rate flows. By examining the possible activity of the magnetic fields of accretion disks, we conclude that indeed main sequence stars might accrete mass at very high rates, up to ≈ 10‑2 M ⊙ yr‑1 for solar type stars, and up to ≈ 1 M ⊙ yr‑1 for very massive stars. We speculate that magnetic fields amplified in such extreme conditions might lead to the formation of massive bipolar outflows that can remove most of the disk's energy and angular momentum. It is this energy and angular momentum removal that allows the very high mass accretion rate onto main sequence stars.

  12. 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

  13. 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

    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

  14. High-utility conserved avian microsatellite markers enable parentage and population studies across a wide range of species

    PubMed Central

    2013-01-01

    Background Microsatellites are widely used for many genetic studies. In contrast to single nucleotide polymorphism (SNP) and genotyping-by-sequencing methods, they are readily typed in samples of low DNA quality/concentration (e.g. museum/non-invasive samples), and enable the quick, cheap identification of species, hybrids, clones and ploidy. Microsatellites also have the highest cross-species utility of all types of markers used for genotyping, but, despite this, when isolated from a single species, only a relatively small proportion will be of utility. Marker development of any type requires skill and time. The availability of sufficient “off-the-shelf” markers that are suitable for genotyping a wide range of species would not only save resources but also uniquely enable new comparisons of diversity among taxa at the same set of loci. No other marker types are capable of enabling this. We therefore developed a set of avian microsatellite markers with enhanced cross-species utility. Results We selected highly-conserved sequences with a high number of repeat units in both of two genetically distant species. Twenty-four primer sets were designed from homologous sequences that possessed at least eight repeat units in both the zebra finch (Taeniopygia guttata) and chicken (Gallus gallus). Each primer sequence was a complete match to zebra finch and, after accounting for degenerate bases, at least 86% similar to chicken. We assessed primer-set utility by genotyping individuals belonging to eight passerine and four non-passerine species. The majority of the new Conserved Avian Microsatellite (CAM) markers amplified in all 12 species tested (on average, 94% in passerines and 95% in non-passerines). This new marker set is of especially high utility in passerines, with a mean 68% of loci polymorphic per species, compared with 42% in non-passerine species. Conclusions When combined with previously described conserved loci, this new set of conserved markers will not only

  15. Unique genomic sequences in human chromosome 16p are conserved in the great apes.

    PubMed

    Tarzami, S T; Kringstein, A M; Conte, R A; Verma, R S

    1997-01-27

    In humans, acute myelomonocytic leukemia (AMML) with abnormal bone marrow eosinophilia is diagnosed by the presence of a pericentric inversion in chromosome 16, involving breakpoints p13;q23 [i.e., inv(16)(p13;q23)]. A pericentric inversion involves breaks that have occurred on the p and q arms and the segment in between is rotated 180 degrees and reattaches. The recent development of a "human micro-coatasome" painting probe for 16p contains unique DNA sequences that fluorescently label only the short arm of chromosome 16, which facilitates the identification of such inversions and represents an ideal tool for analyzing the "divergence/convergence" of the equivalent human chromosome 16 (PTR 18, GGO 17 and PPY 19) in the great apes, chimpanzee, gorilla and orangutan. When the probe is used on the type of pericentric inversion characteristic of AMML, signals are observed on the proximal portions (the regions closest to the centromere) of the long and short arms of chromosome 16. The probe hybridized to only the short arm of all three ape chromosomes and signals were not observed on the long arms, suggesting that a pericentric inversion similar to that seen in AMML has not occurred in any of these great apes. PMID:9037113

  16. Assessment of selected conservation measures for high-temperature process industries

    SciTech Connect

    Kusik, C L; Parameswaran, K; Nadkarni, R; O'Neill, J K; Malhotra, S; Hyde, R; Kinneberg, D; Fox, L; Rossetti, M

    1981-01-01

    Energy conservation projects involving high-temperature processes in various stages of development are assessed to quantify their energy conservation potential; to determine their present status of development; to identify their research and development needs and estimate the associated costs; and to determine the most effective role for the Federal government in developing these technologies. The program analyzed 25 energy conserving processes in the iron and steel, aluminium, copper, magnesium, cement, and glassmaking industries. A preliminary list of other potential energy conservation projects in these industries is also presented in the appendix. (MCW)

  17. Conservation of the function counts: homologous neurons express sequence-related neuropeptides that originate from different genes.

    PubMed

    Neupert, Susanne; Huetteroth, Wolf; Schachtner, Joachim; Predel, Reinhard

    2009-11-01

    By means of single-cell matrix assisted laser desorption/ionization time-of-flight mass spectrometry, we analysed neuropeptide expression in all FXPRLamide/pheromone biosynthesis activating neuropeptide synthesizing neurons of the adult tobacco hawk moth, Manduca sexta. Mass spectra clearly suggest a completely identical processing of the pheromone biosynthesis activating neuropeptide-precursor in the mandibular, maxillary and labial neuromeres of the subesophageal ganglion. Only in the pban-neurons of the labial neuromere, products of two neuropeptide genes, namely the pban-gene and the capa-gene, were detected. Both of these genes expressed, amongst others, sequence-related neuropeptides (extended WFGPRLamides). We speculate that the expression of the two neuropeptide genes is a plesiomorph character typical of moths. A detailed examination of the neuroanatomy and the peptidome of the (two) pban-neurons in the labial neuromere of moths with homologous neurons of different insects indicates a strong conservation of the function of this neuroendocrine system. In other insects, however, the labial neurons either express products of the fxprl-gene or products of the capa-gene. The processing of the respective genes is reduced to extended WFGPRLamides in each case and yields a unique peptidome in the labial cells. Thus, sequence-related messenger molecules are always produced in these cells and it seems that the respective neurons recruited different neuropeptide genes for this motif. PMID:19712058

  18. The BEN domain is a novel sequence-specific DNA-binding domain conserved in neural transcriptional repressors

    PubMed Central

    Dai, Qi; Ren, Aiming; Westholm, Jakub O.; Serganov, Artem A.; Patel, Dinshaw J.; Lai, Eric C.

    2013-01-01

    We recently reported that Drosophila Insensitive (Insv) promotes sensory organ development and has activity as a nuclear corepressor for the Notch transcription factor Suppressor of Hairless [Su(H)]. Insv lacks domains of known biochemical function but contains a single BEN domain (i.e., a “BEN-solo” protein). Our chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) analysis confirmed binding of Insensitive to Su(H) target genes in the Enhancer of split gene complex [E(spl)-C]; however, de novo motif analysis revealed a novel site strongly enriched in Insv peaks (TCYAATHRGAA). We validate binding of endogenous Insv to genomic regions bearing such sites, whose associated genes are enriched for neural functions and are functionally repressed by Insv. Unexpectedly, we found that the Insv BEN domain binds specifically to this sequence motif and that Insv directly regulates transcription via this motif. We determined the crystal structure of the BEN–DNA target complex, revealing homodimeric binding of the BEN domain and extensive nucleotide contacts via α helices and a C-terminal loop. Point mutations in key DNA-contacting residues severely impair DNA binding in vitro and capacity for transcriptional regulation in vivo. We further demonstrate DNA-binding and repression activities by the mammalian neural BEN-solo protein BEND5. Altogether, we define novel DNA-binding activity in a conserved family of transcriptional repressors, opening a molecular window on this extensive gene family. PMID:23468431

  19. cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera

    SciTech Connect

    Aris, J.P.; Blobel, G. )

    1991-02-01

    The authors have isolated a 1.1-kilobase cDNA clone that encodes human fibrillarin by screening a hepatoma library in parallel with DNA probes derived from the fibrillarin genes of Saccharomyces cerevisiae (NOP1) and Xenopus laevis. RNA blot analysis indicates that the corresponding mRNA is {approximately}1,300 nucleotides in length. Human fibrillarin expressed in vitro migrates on SDS gels as a 36-kDa protein that is specifically immunoprecipitated by antisera from humans with scleroderma autoimmune disease. Human fibrillarin contains an amino-terminal repetitive domain {approximately}75-80 amino acids in length that is rich in glycine and arginine residues and is similar to amino-terminal domains in the yeast and Xenopus fibrillarins. The occurrence of a putative RNA-binding domain and an RNP consensus sequence within the protein is consistent with the association of fibrillarin with small nucleolar RNAs. Protein sequence alignments show that 67% of amino acids from human fibrillarin are identical to those in yeast fibrillarin and that 81% are identical to those in Xenopus fibrillarin. This identity suggests the evolutionary conservation of an important function early in the pathway for ribosome biosynthesis.

  20. Generating long sequences of high-intensity femtosecond pulses.

    PubMed

    Bitter, M; Milner, V

    2016-02-01

    We present an approach to creating pulse sequences extending beyond 150 ps in duration, comprised of 100 μJ femtosecond pulses. A quarter of the pulse train is produced by a high-resolution pulse shaper, which allows full controllability over the timing of each pulse. Two nested Michelson interferometers follow to quadruple the pulse number and the sequence duration. To boost the pulse energy, the long train is sent through a multipass Ti:sapphire amplifier, followed by an external compressor. A periodic sequence of 84 pulses of 120 fs width and an average pulse energy of 107 μJ, separated by 2 ps, is demonstrated as a proof of principle. PMID:26836087

  1. 7 CFR 1412.68 - Compliance with highly erodible land and wetland conservation provisions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 10 2012-01-01 2012-01-01 false Compliance with highly erodible land and wetland conservation provisions. 1412.68 Section 1412.68 Agriculture Regulations of the Department of Agriculture... and wetland conservation provisions. The provisions of part 12 of this title apply to this part....

  2. 7 CFR 1412.68 - Compliance with highly erodible land and wetland conservation provisions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 10 2014-01-01 2014-01-01 false Compliance with highly erodible land and wetland conservation provisions. 1412.68 Section 1412.68 Agriculture Regulations of the Department of Agriculture... and wetland conservation provisions. The provisions of part 12 of this title apply to this part....

  3. 7 CFR 1412.68 - Compliance with highly erodible land and wetland conservation provisions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Compliance with highly erodible land and wetland conservation provisions. 1412.68 Section 1412.68 Agriculture Regulations of the Department of Agriculture... and wetland conservation provisions. The provisions of part 12 of this title apply to this part....

  4. 7 CFR 1430.225 - Violations of highly erodible land and wetland conservation provisions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 10 2013-01-01 2013-01-01 false Violations of highly erodible land and wetland conservation provisions. 1430.225 Section 1430.225 Agriculture Regulations of the Department of Agriculture... wetland conservation provisions. The provisions of part 12 of this title apply to this part....

  5. 7 CFR 1430.225 - Violations of highly erodible land and wetland conservation provisions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Violations of highly erodible land and wetland conservation provisions. 1430.225 Section 1430.225 Agriculture Regulations of the Department of Agriculture... wetland conservation provisions. The provisions of part 12 of this title apply to this part....

  6. 7 CFR 1430.225 - Violations of highly erodible land and wetland conservation provisions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Violations of highly erodible land and wetland conservation provisions. 1430.225 Section 1430.225 Agriculture Regulations of the Department of Agriculture... wetland conservation provisions. The provisions of part 12 of this title apply to this part....

  7. 7 CFR 1430.225 - Violations of highly erodible land and wetland conservation provisions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 10 2014-01-01 2014-01-01 false Violations of highly erodible land and wetland conservation provisions. 1430.225 Section 1430.225 Agriculture Regulations of the Department of Agriculture... wetland conservation provisions. The provisions of part 12 of this title apply to this part....

  8. 7 CFR 1412.68 - Compliance with highly erodible land and wetland conservation provisions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 10 2013-01-01 2013-01-01 false Compliance with highly erodible land and wetland conservation provisions. 1412.68 Section 1412.68 Agriculture Regulations of the Department of Agriculture... and wetland conservation provisions. The provisions of part 12 of this title apply to this part....

  9. 7 CFR 1430.225 - Violations of highly erodible land and wetland conservation provisions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 10 2012-01-01 2012-01-01 false Violations of highly erodible land and wetland conservation provisions. 1430.225 Section 1430.225 Agriculture Regulations of the Department of Agriculture... wetland conservation provisions. The provisions of part 12 of this title apply to this part....

  10. 7 CFR 1412.68 - Compliance with highly erodible land and wetland conservation provisions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Compliance with highly erodible land and wetland conservation provisions. 1412.68 Section 1412.68 Agriculture Regulations of the Department of Agriculture... and wetland conservation provisions. The provisions of part 12 of this title apply to this part....

  11. Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry

    PubMed Central

    Dorokhov, Yuri L.; Skulachev, Maxim V.; Ivanov, Peter A.; Zvereva, Svetlana D.; Tjulkina, Lydia G.; Merits, Andres; Gleba, Yuri Y.; Hohn, Thomas; Atabekov, Joseph G.

    2002-01-01

    The internal ribosome entry sites (IRES), IRES\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{_{CP,148}^{CR}}}\\end{equation*}\\end{document} and IRES\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{_{MP,75}^{CR}}}\\end{equation*}\\end{document}, precede the coat protein (CP) and movement protein (MP) genes of crucifer-infecting tobamovirus (crTMV), respectively. In the present work, we analyzed the activity of these elements in transgenic plants and other organisms. Comparison of the relative activities of the crTMV IRES elements and the IRES from an animal virus—encephalomyocarditis virus—in plant, yeast, and HeLa cells identified the 148-nt IRES\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{_{CP,148}^{CR}}}\\end{equation*}\\end{document} as the strongest element that also displayed IRES activity across all kingdoms. Deletion analysis suggested that the polypurine (A)-rich sequences (PARSs) contained in IRES\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{_{CP,148}^{CR

  12. Savant: genome browser for high-throughput sequencing data

    PubMed Central

    Fiume, Marc; Williams, Vanessa; Brook, Andrew; Brudno, Michael

    2010-01-01

    Motivation: The advent of high-throughput sequencing (HTS) technologies has made it affordable to sequence many individuals' genomes. Simultaneously the computational analysis of the large volumes of data generated by the new sequencing machines remains a challenge. While a plethora of tools are available to map the resulting reads to a reference genome, and to conduct primary analysis of the mappings, it is often necessary to visually examine the results and underlying data to confirm predictions and understand the functional effects, especially in the context of other datasets. Results: We introduce Savant, the Sequence Annotation, Visualization and ANalysis Tool, a desktop visualization and analysis browser for genomic data. Savant was developed for visualizing and analyzing HTS data, with special care taken to enable dynamic visualization in the presence of gigabases of genomic reads and references the size of the human genome. Savant supports the visualization of genome-based sequence, point, interval and continuous datasets, and multiple visualization modes that enable easy identification of genomic variants (including single nucleotide polymorphisms, structural and copy number variants), and functional genomic information (e.g. peaks in ChIP-seq data) in the context of genomic annotations. Availability: Savant is freely available at http://compbio.cs.toronto.edu/savant Contact: savant@cs.toronto.edu PMID:20562449

  13. Salmonella Serotype Determination Utilizing High-Throughput Genome Sequencing Data

    PubMed Central

    Zhang, Shaokang; Yin, Yanlong; Jones, Marcus B.; Zhang, Zhenzhen; Deatherage Kaiser, Brooke L.; Dinsmore, Blake A.; Fitzgerald, Collette; Fields, Patricia I.

    2015-01-01

    Serotyping forms the basis of national and international surveillance networks for Salmonella, one of the most prevalent foodborne pathogens worldwide (1–3). Public health microbiology is currently being transformed by whole-genome sequencing (WGS), which opens the door to serotype determination using WGS data. SeqSero (www.denglab.info/SeqSero) is a novel Web-based tool for determining Salmonella serotypes using high-throughput genome sequencing data. SeqSero is based on curated databases of Salmonella serotype determinants (rfb gene cluster, fliC and fljB alleles) and is predicted to determine serotype rapidly and accurately for nearly the full spectrum of Salmonella serotypes (more than 2,300 serotypes), from both raw sequencing reads and genome assemblies. The performance of SeqSero was evaluated by testing (i) raw reads from genomes of 308 Salmonella isolates of known serotype; (ii) raw reads from genomes of 3,306 Salmonella isolates sequenced and made publicly available by GenomeTrakr, a U.S. national monitoring network operated by the Food and Drug Administration; and (iii) 354 other publicly available draft or complete Salmonella genomes. We also demonstrated Salmonella serotype determination from raw sequencing reads of fecal metagenomes from mice orally infected with this pathogen. SeqSero can help to maintain the well-established utility of Salmonella serotyping when integrated into a platform of WGS-based pathogen subtyping and characterization. PMID:25762776

  14. Identification of Sequence Specificity of 5-Methylcytosine Oxidation by Tet1 Protein with High-Throughput Sequencing.

    PubMed

    Kizaki, Seiichiro; Chandran, Anandhakumar; Sugiyama, Hiroshi

    2016-03-01

    Tet (ten-eleven translocation) family proteins have the ability to oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic-level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high-throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×10(4) -member mC-containing random DNA-sequence library was constructed. The library was subjected to Tet-reactive pulldown followed by high-throughput sequencing. Analysis of the obtained sequence data identified the Tet1-reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein. PMID:26715454

  15. The Highly Conserved MraZ Protein Is a Transcriptional Regulator in Escherichia coli

    PubMed Central

    Eraso, Jesus M.; Markillie, Lye M.; Mitchell, Hugh D.; Taylor, Ronald C.; Orr, Galya

    2014-01-01

    The mraZ and mraW genes are highly conserved in bacteria, both in sequence and in their position at the head of the division and cell wall (dcw) gene cluster. Located directly upstream of the mraZ gene, the Pmra promoter drives the transcription of mraZ and mraW, as well as many essential cell division and cell wall genes, but no regulator of Pmra has been found to date. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin and MraW is known to methylate the 16S rRNA, mraZ and mraW null mutants have no detectable phenotypes. Here we show that overproduction of Escherichia coli MraZ inhibited cell division and was lethal in rich medium at high induction levels and in minimal medium at low induction levels. Co-overproduction of MraW suppressed MraZ toxicity, and loss of MraW enhanced MraZ toxicity, suggesting that MraZ and MraW have antagonistic functions. MraZ-green fluorescent protein localized to the nucleoid, suggesting that it binds DNA. Consistent with this idea, purified MraZ directly bound a region of DNA containing three direct repeats between Pmra and the mraZ gene. Excess MraZ reduced the expression of an mraZ-lacZ reporter, suggesting that MraZ acts as a repressor of Pmra, whereas a DNA-binding mutant form of MraZ failed to repress expression. Transcriptome sequencing (RNA-seq) analysis suggested that MraZ also regulates the expression of genes outside the dcw cluster. In support of this, purified MraZ could directly bind to a putative operator site upstream of mioC, one of the repressed genes identified by RNA-seq. PMID:24659771

  16. The highly conserved MraZ protein is a transcriptional regulator in Escherichia coli

    SciTech Connect

    Eraso, Jesus M.; Markillie, Lye Meng; Mitchell, Hugh D.; Taylor, Ronald C.; Orr, Galya; Margolin, William

    2014-05-05

    The mraZ and mraW genes are highly conserved in bacteria, both in sequence and location at the head of the division and cell wall (dcw) gene cluster. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin, and MraW is known to methylate ribosomal RNA, mraZ and mraW null mutants have no detectable growth phenotype in any species tested to date, hampering progress in understanding their physiological role. Here we show that overproduction of Escherichia coli MraZ perturbs cell division and the cell envelope, is more lethal at high levels or in minimal growth medium, and that MraW antagonizes these effects. MraZGFP localizes to the nucleoid, suggesting that it binds DNA. Indeed, purified MraZ directly binds a region upstream from its own promoter containing three direct repeats to regulate its own expression and that of downstream cell division and cell wall genes. MraZ-LacZ fusions are repressed by excess MraZ but not when DNA binding by MraZ is inhibited. RNAseq analysis indicates that MraZ is a global transcriptional regulator with numerous targets in addition to dcw genes. One of these targets, mioC, is directly bound by MraZ in a region with three direct repeats.

  17. High-throughput sequencing of small RNAs and anatomical characteristics associated with leaf development in celery.

    PubMed

    Jia, Xiao-Ling; Li, Meng-Yao; Jiang, Qian; Xu, Zhi-Sheng; Wang, Feng; Xiong, Ai-Sheng

    2015-01-01

    MicroRNAs (miRNAs) exhibit diverse and important roles in plant growth, development, and stress responses and regulate gene expression at the post-transcriptional level. Knowledge about the diversity of miRNAs and their roles in leaf development in celery remains unknown. To elucidate the roles of miRNAs in celery leaf development, we identified leaf development-related miRNAs through high-throughput sequencing. Small RNA libraries were constructed using leaves from three stages (10, 20, and 30 cm) of celery cv.'Ventura' and then subjected to high-throughput sequencing and bioinformatics analysis. At Stage 1, Stage 2, and Stage 3 of 'Ventura', a total of 333, 329, and 344 conserved miRNAs (belonging to 35, 35, and 32 families, respectively) were identified. A total of 131 miRNAs were identified as novel in 'Ventura'. Potential miRNA target genes were predicted and annotated using the eggNOG, GO, and KEGG databases to explore gene functions. The abundance of five conserved miRNAs and their corresponding potential target genes were validated. Expression profiles of novel potential miRNAs were also detected. Anatomical characteristics of the leaf blades and petioles at three leaf stages were further analyzed. This study contributes to our understanding on the functions and molecular regulatory mechanisms of miRNAs in celery leaf development. PMID:26057455

  18. Fusion genes and their discovery using high throughput sequencing.

    PubMed

    Annala, M J; Parker, B C; Zhang, W; Nykter, M

    2013-11-01

    Fusion genes are hybrid genes that combine parts of two or more original genes. They can form as a result of chromosomal rearrangements or abnormal transcription, and have been shown to act as drivers of malignant transformation and progression in many human cancers. The biological significance of fusion genes together with their specificity to cancer cells has made them into excellent targets for molecular therapy. Fusion genes are also used as diagnostic and prognostic markers to confirm cancer diagnosis and monitor response to molecular therapies. High-throughput sequencing has enabled the systematic discovery of fusion genes in a wide variety of cancer types. In this review, we describe the history of fusion genes in cancer and the ways in which fusion genes form and affect cellular function. We also describe computational methodologies for detecting fusion genes from high-throughput sequencing experiments, and the most common sources of error that lead to false discovery of fusion genes. PMID:23376639

  19. PTS-Mediated Regulation of the Transcription Activator MtlR from Different Species: Surprising Differences despite Strong Sequence Conservation.

    PubMed

    Joyet, Philippe; Derkaoui, Meriem; Bouraoui, Houda; Deutscher, Josef

    2015-01-01

    The hexitol D-mannitol is transported by many bacteria via a phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). In most Firmicutes, the transcription activator MtlR controls the expression of the genes encoding the D-mannitol-specific PTS components and D-mannitol-1-P dehydrogenase. MtlR contains an N-terminal helix-turn-helix motif followed by an Mga-like domain, two PTS regulation domains (PRDs), an EIIB(Gat)- and an EIIA(Mtl)-like domain. The four regulatory domains are the target of phosphorylation by PTS components. Despite strong sequence conservation, the mechanisms controlling the activity of MtlR from Lactobacillus casei, Bacillus subtilis and Geobacillus stearothermophilus are quite different. Owing to the presence of a tyrosine in place of the second conserved histidine (His) in PRD2, L. casei MtlR is not phosphorylated by Enzyme I (EI) and HPr. When the corresponding His in PRD2 of MtlR from B. subtilis and G. stearothermophilus was replaced with alanine, the transcription regulator was no longer phosphorylated and remained inactive. Surprisingly, L. casei MtlR functions without phosphorylation in PRD2 because in a ptsI (EI) mutant MtlR is constitutively active. EI inactivation prevents not only phosphorylation of HPr, but also of the PTS(Mtl) components, which inactivate MtlR by phosphorylating its EIIB(Gat)- or EIIA(Mtl)-like domain. This explains the constitutive phenotype of the ptsI mutant. The absence of EIIB(Mtl)-mediated phosphorylation leads to induction of the L. caseimtl operon. This mechanism resembles mtlARFD induction in G. stearothermophilus, but differs from EIIA(Mtl)-mediated induction in B. subtilis. In contrast to B. subtilis MtlR, L. casei MtlR activation does not require sequestration to the membrane via the unphosphorylated EIIB(Mtl) domain. PMID:26159071

  20. Marker genes that are less conserved in their sequences are useful for predicting genome-wide similarity levels between closely related prokaryotic strains

    DOE PAGESBeta

    Lan, Yemin; Rosen, Gail; Hershberg, Ruth

    2016-05-03

    The 16s rRNA gene is so far the most widely used marker for taxonomical classification and separation of prokaryotes. Since it is universally conserved among prokaryotes, it is possible to use this gene to classify a broad range of prokaryotic organisms. At the same time, it has often been noted that the 16s rRNA gene is too conserved to separate between prokaryotes at finer taxonomic levels. In this paper, we examine how well levels of similarity of 16s rRNA and 73 additional universal or nearly universal marker genes correlate with genome-wide levels of gene sequence similarity. We demonstrate that themore » percent identity of 16s rRNA predicts genome-wide levels of similarity very well for distantly related prokaryotes, but not for closely related ones. In closely related prokaryotes, we find that there are many other marker genes for which levels of similarity are much more predictive of genome-wide levels of gene sequence similarity. Finally, we show that the identities of the markers that are most useful for predicting genome-wide levels of similarity within closely related prokaryotic lineages vary greatly between lineages. However, the most useful markers are always those that are least conserved in their sequences within each lineage. In conclusion, our results show that by choosing markers that are less conserved in their sequences within a lineage of interest, it is possible to better predict genome-wide gene sequence similarity between closely related prokaryotes than is possible using the 16s rRNA gene. We point readers towards a database we have created (POGO-DB) that can be used to easily establish which markers show lowest levels of sequence conservation within different prokaryotic lineages.« less

  1. Identification of MicroRNAs from Eugenia uniflora by High-Throughput Sequencing and Bioinformatics Analysis

    PubMed Central

    Guzman, Frank; Almerão, Mauricio P.; Körbes, Ana P.; Loss-Morais, Guilherme; Margis, Rogerio

    2012-01-01

    Background microRNAs or miRNAs are small non-coding regulatory RNAs that play important functions in the regulation of gene expression at the post-transcriptional level by targeting mRNAs for degradation or inhibiting protein translation. Eugenia uniflora is a plant native to tropical America with pharmacological and ecological importance, and there have been no previous studies concerning its gene expression and regulation. To date, no miRNAs have been reported in Myrtaceae species. Results Small RNA and RNA-seq libraries were constructed to identify miRNAs and pre-miRNAs in Eugenia uniflora. Solexa technology was used to perform high throughput sequencing of the library, and the data obtained were analyzed using bioinformatics tools. From 14,489,131 small RNA clean reads, we obtained 1,852,722 mature miRNA sequences representing 45 conserved families that have been identified in other plant species. Further analysis using contigs assembled from RNA-seq allowed the prediction of secondary structures of 25 known and 17 novel pre-miRNAs. The expression of twenty-seven identified miRNAs was also validated using RT-PCR assays. Potential targets were predicted for the most abundant mature miRNAs in the identified pre-miRNAs based on sequence homology. Conclusions This study is the first large scale identification of miRNAs and their potential targets from a species of the Myrtaceae family without genomic sequence resources. Our study provides more information about the evolutionary conservation of the regulatory network of miRNAs in plants and highlights species-specific miRNAs. PMID:23166775

  2. The chick and human collagen alpha1(XII) gene promoter--activity of highly conserved regions around the first exon and in the first intron.

    PubMed

    Chiquet, M; Mumenthaler, U; Wittwer, M; Jin, W; Koch, M

    1998-10-15

    A single gene encodes collagen XII, an extracellular matrix protein with three large fibronectin-related subunits connected via a short collagen triple helix. Since collagen XII is a component of a specific subset of collagen fibrils in tissues bearing high tensile stress, we are interested to know how its restricted expression is regulated. To this aim, we have isolated the region around the first exon of both the chick and human collagen alpha1(XII) gene. The upstream sequences of the two genes share common features but are not related. Strong similarity starts about 100 bp 5' of the first exon and ends 100 bp into the first intron. In addition, two large conserved regions (56-63% similarity) were found in the first intron. A single major and two clusters of minor transcription start sites were identified in both the chick and human gene. To test for promoter activity, conserved fragments from the chick gene were cloned into reporter plasmids for transient transfection of fibroblasts. A 70-bp stretch containing a conserved nuclear factor-1 binding sequence just upstream of the first transcription start site was found to work as a basal promoter. An adjacent, but nonoverlapping short segment including the more downstream start sites and a conserved TATTAA sequence exhibited independent promoter activity. GC-rich sequences just 5' and 3' of the minimal promoter fragments were required for full activity. In contrast, inclusion of more upstream sequences (up to 2.4 kb) had no effect. The two conserved regions in the first intron showed no promoter activity on their own but modulated activity when linked to autologous or heterologous promoters. Specifically, one of these intronic regions might contain enhancer element(s) that respond to mechanical stress acting on the fibroblasts. We conclude that the collagen XII gene is driven by a basal promoter with two halves that can act independently; conserved control regions are located around the first exon and in the first

  3. The use of museum specimens with high-throughput DNA sequencers

    PubMed Central

    Burrell, Andrew S.; Disotell, Todd R.; Bergey, Christina M.

    2015-01-01

    Natural history collections have long been used by morphologists, anatomists, and taxonomists to probe the evolutionary process and describe biological diversity. These biological archives also offer great opportunities for genetic research in taxonomy, conservation, systematics, and population biology. They allow assays of past populations, including those of extinct species, giving context to present patterns of genetic variation and direct measures of evolutionary processes. Despite this potential, museum specimens are difficult to work with because natural postmortem processes and preservation methods fragment and damage DNA. These problems have restricted geneticists’ ability to use natural history collections primarily by limiting how much of the genome can be surveyed. Recent advances in DNA sequencing technology, however, have radically changed this, making truly genomic studies from museum specimens possible. We review the opportunities and drawbacks of the use of museum specimens, and suggest how to best execute projects when incorporating such samples. Several high-throughput (HT) sequencing methodologies, including whole genome shotgun sequencing, sequence capture, and restriction digests (demonstrated here), can be used with archived biomaterials. PMID:25532801

  4. Conserved sequences in the current strains of HIV-1 subtype A in Russia are effectively targeted by artificial RNAi in vitro.

    PubMed

    Tchurikov, Nickolai A; Fedoseeva, Daria M; Gashnikova, Natalya M; Sosin, Dmitri V; Gorbacheva, Maria A; Alembekov, Ildar R; Chechetkin, Vladimir R; Kravatsky, Yuri V; Kretova, Olga V

    2016-05-25

    Highly active antiretroviral therapy has greatly reduced the morbidity and mortality of AIDS. However, many of the antiretroviral drugs are toxic with long-term use, and all currently used anti-HIV agents generate drug-resistant mutants. Therefore, there is a great need for new approaches to AIDS therapy. RNAi is a powerful means of inhibiting HIV-1 production in human cells. We propose to use RNAi for gene therapy of HIV/AIDS. Previously we identified a number of new biologically active siRNAs targeting several moderately conserved regions in HIV-1 transcripts. Here we analyze the heterogeneity of nucleotide sequences in three RNAi targets in sequences encoding the reverse transcriptase and integrase domains of current isolates of HIV-1 subtype A in Russia. These data were used to generate genetic constructs expressing short hairpin RNAs 28-30-bp in length that could be processed in cells into siRNAs. After transfection of the constructs we observed siRNAs that efficiently attacked the selected targets. We expect that targeting several viral genes important for HIV-1 reproduction will help overcome the problem of viral adaptation and will prevent the appearance of RNAi escape mutants in current virus strains, an important feature of gene therapy of HIV/AIDS. PMID:26947394

  5. Discovery of highly conserved unique peanut and tree nut peptides by LC-MS/MS for multi-allergen detection.

    PubMed

    Sealey-Voyksner, Jennifer; Zweigenbaum, Jerry; Voyksner, Robert

    2016-03-01

    Proteins unique to peanuts and various tree nuts have been extracted, subjected to trypsin digestion and analysis by liquid chromatography/quadrupole time-of-flight mass spectrometry, in order to find highly conserved peptides that can be used as markers to detect peanuts and tree nuts in food. The marker peptide sequences chosen were those found to be present in both native (unroasted) and thermally processed (roasted) forms of peanuts and tree nuts. Each peptide was selected by assuring its presence in food that was processed or unprocessed, its abundance for sensitivity, sequence size, and uniqueness for peanut and each specific variety of tree nut. At least two peptides were selected to represent peanut, almond, pecan, cashew, walnut, hazelnut, pine nut, Brazil nut, macadamia nut, pistachio nut, chestnut and coconut; to determine the presence of trace levels of peanut and tree nuts in food by a novel multiplexed LC-MS method. PMID:26471545

  6. A conserved predicted pseudoknot in the NS2A-encoding sequence of West Nile and Japanese encephalitis flaviviruses suggests NS1' may derive from ribosomal frameshifting

    PubMed Central

    Firth, Andrew E; Atkins, John F

    2009-01-01

    Japanese encephalitis, West Nile, Usutu and Murray Valley encephalitis viruses form a tight subgroup within the larger Flavivirus genus. These viruses utilize a single-polyprotein expression strategy, resulting in ~10 mature proteins. Plotting the conservation at synonymous sites along the polyprotein coding sequence reveals strong conservation peaks at the very 5' end of the coding sequence, and also at the 5' end of the sequence encoding the NS2A protein. Such peaks are generally indicative of functionally important non-coding sequence elements. The second peak corresponds to a predicted stable pseudoknot structure whose biological importance is supported by compensatory mutations that preserve the structure. The pseudoknot is preceded by a conserved slippery heptanucleotide (Y CCU UUU), thus forming a classical stimulatory motif for -1 ribosomal frameshifting. We hypothesize, therefore, that the functional importance of the pseudoknot is to stimulate a portion of ribosomes to shift -1 nt into a short (45 codon), conserved, overlapping open reading frame, termed foo. Since cleavage at the NS1-NS2A boundary is known to require synthesis of NS2A in cis, the resulting transframe fusion protein is predicted to be NS1-NS2AN-term-FOO. We hypothesize that this may explain the origin of the previously identified NS1 'extension' protein in JEV-group flaviviruses, known as NS1'. PMID:19196463

  7. High-Throughput Sequencing of a South American Amerindian

    PubMed Central

    Almeida, Renan; Alencar, Dayse O.; Barbosa, Maria Silvanira; Gusmão, Leonor; Silva, Wilson A.; de Souza, Sandro J.; Silva, Artur; Ribeiro-dos-Santos, Ândrea; Darnet, Sylvain; Santos, Sidney

    2013-01-01

    The emergence of next-generation sequencing technologies allowed access to the vast amounts of information that are contained in the human genome. This information has contributed to the understanding of individual and population-based variability and improved the understanding of the evolutionary history of different human groups. However, the genome of a representative of the Amerindian populations had not been previously sequenced. Thus, the genome of an individual from a South American tribe was completely sequenced to further the understanding of the genetic variability of Amerindians. A total of 36.8 giga base pairs (Gbp) were sequenced and aligned with the human genome. These Gbp corresponded to 95.92% of the human genome with an estimated miscall rate of 0.0035 per sequenced bp. The data obtained from the alignment were used for SNP (single-nucleotide) and INDEL (insertion-deletion) calling, which resulted in the identification of 502,017 polymorphisms, of which 32,275 were potentially new high-confidence SNPs and 33,795 new INDELs, specific of South Native American populations. The authenticity of the sample as a member of the South Native American populations was confirmed through the analysis of the uniparental (maternal and paternal) lineages. The autosomal comparison distinguished the investigated sample from others continental populations and revealed a close relation to the Eastern Asian populations and Aboriginal Australian. Although, the findings did not discard the classical model of America settlement; it brought new insides to the understanding of the human population history. The present study indicates a remarkable genetic variability in human populations that must still be identified and contributes to the understanding of the genetic variability of South Native American populations and of the human populations history. PMID:24386182

  8. Localization of a highly conserved human potassium channel gene (NGK2-KV4-KCNC1) to chromosome 11p15

    SciTech Connect

    Ried, T.; Ward, D.C. ); Rudy, B.; Miera, V.S. de; Lau, D.; Sen, K. )

    1993-02-01

    Several genes (the Shaker or Sh gene family) encoding components of voltage-gated K[sub +] channels have been identified in various species. Based on sequence similarities Sh genes are classified into four groups or subfamilies. Mammalian genes of each one of these subfamilies also show high levels of sequence similarity to one of four related Drosophila genes: Shaker, Shab, Shaw, and Shal. Here we report the isolation of human cDNAs for a Shaw-related product (NGK2,KV2.1a) previously identified in rat and mice. A comparison of the nucleotide and deduced amino acid sequence of NGK2 in rodents and humans shows that this product is highly conserved in mammals; the human NGK2 protein shows over 99% amino acid sequence identity to its rodent homologue. The gene (NGK2-KV4; KCNC1) encoding NGK2 was mapped to human chromosome 11p15 by fluorescence in situ hybridization with the human NGK2 cDNAs. 65 refs., 2 figs., 1 tab.

  9. A HIGH COVERAGE GENOME SEQUENCE FROM AN ARCHAIC DENISOVAN INDIVIDUAL

    PubMed Central

    Meyer, Matthias; Kircher, Martin; Gansauge, Marie-Theres; Li, Heng; Racimo, Fernando; Mallick, Swapan; Schraiber, Joshua G.; Jay, Flora; Prüfer, Kay; de Filippo, Cesare; Sudmant, Peter H.; Alkan, Can; Fu, Qiaomei; Do, Ron; Rohland, Nadin; Tandon, Arti; Siebauer, Michael; Green, Richard E.; Bryc, Katarzyna; Briggs, Adrian W.; Stenzel, Udo; Dabney, Jesse; Shendure, Jay; Kitzman, Jacob; Hammer, Michael F.; Shunkov, Michael V.; Derevianko, Anatoli P.; Patterson, Nick; Andrés, Aida M.; Eichler, Evan E.; Slatkin, Montgomery; Reich, David; Kelso, Janet; Pääbo, Svante

    2013-01-01

    We present a DNA library preparation method that has allowed us to reconstruct a high coverage (30X) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of “missing evolution” in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans. PMID:22936568

  10. A novel mosaic protein containing LDL receptor elements is highly conserved in humans and chickens.

    PubMed

    Mörwald, S; Yamazaki, H; Bujo, H; Kusunoki, J; Kanaki, T; Seimiya, K; Morisaki, N; Nimpf, J; Schneider, W J; Saito, Y

    1997-05-01

    Certain receptors belonging to the LDL receptor (LDLR) gene family appear to constitute a newly identified branch whose members are expressed in brain, in addition to other tissues. In support of this concept, we have now discovered the expression and delineated the molecular structures of a representative of this emerging branch from two such diverse species as human and chicken. This membrane receptor, called LR11 and thus far only known to exist in the rabbit, is a complex seven-domain mosaic protein containing, among other structural elements, a cluster of 11 LDLR ligand-binding repeats and a domain with homology to VPS10, a yeast receptor for vacuolar protein sorting. Cytoplasmic signature sequences define the receptor as competent for endocytosis. The most striking properties of LR11s are their (1) high degree of structural conservation (>80% identity among mammals and birds), with 100% identity in the membrane-spanning and cytoplasmic domains of rabbit and human; (2) lack of regulation by cholesterol and estrogen; and (3) expression in brain. The features of LR11 suggest important roles in intercellular and intracellular ligand transport processes, some of which it may share with other brain-specific LDLR family members. PMID:9157966

  11. Highly Conserved Mitochondrial Genomes among Multicellular Red Algae of the Florideophyceae.

    PubMed

    Yang, Eun Chan; Kim, Kyeong Mi; Kim, Su Yeon; Lee, JunMo; Boo, Ga Hun; Lee, Jung-Hyun; Nelson, Wendy A; Yi, Gangman; Schmidt, William E; Fredericq, Suzanne; Boo, Sung Min; Bhattacharya, Debashish; Yoon, Hwan Su

    2015-08-01

    Two red algal classes, the Florideophyceae (approximately 7,100 spp.) and Bangiophyceae (approximately 193 spp.), comprise 98% of red algal diversity in marine and freshwater habitats. These two classes form well-supported monophyletic groups in most phylogenetic analyses. Nonetheless, the interordinal relationships remain largely unresolved, in particular in the largest subclass Rhodymeniophycidae that includes 70% of all species. To elucidate red algal phylogenetic relationships and study organelle evolution, we determined the sequence of 11 mitochondrial genomes (mtDNA) from 5 florideophycean subclasses. These mtDNAs were combined with existing data, resulting in a database of 25 florideophytes and 12 bangiophytes (including cyanidiophycean species). A concatenated alignment of mt proteins was used to resolve ordinal relationships in the Rhodymeniophycidae. Red algal mtDNA genome comparisons showed 47 instances of gene rearrangement including 12 that distinguish Bangiophyceae from Hildenbrandiophycidae, and 5 that distinguish Hildenbrandiophycidae from Nemaliophycidae. These organelle data support a rapid radiation and surprisingly high conservation of mtDNA gene syntheny among the morphologically divergent multicellular lineages of Rhodymeniophycidae. In contrast, we find extensive mitochondrial gene rearrangements when comparing Bangiophyceae and Florideophyceae and multiple examples of gene loss among the different red algal lineages. PMID:26245677

  12. Highly Conserved Mitochondrial Genomes among Multicellular Red Algae of the Florideophyceae

    PubMed Central

    Yang, Eun Chan; Kim, Kyeong Mi; Kim, Su Yeon; Lee, JunMo; Boo, Ga Hun; Lee, Jung-Hyun; Nelson, Wendy A.; Yi, Gangman; Schmidt, William E.; Fredericq, Suzanne; Boo, Sung Min; Bhattacharya, Debashish; Yoon, Hwan Su

    2015-01-01

    Two red algal classes, the Florideophyceae (approximately 7,100 spp.) and Bangiophyceae (approximately 193 spp.), comprise 98% of red algal diversity in marine and freshwater habitats. These two classes form well-supported monophyletic groups in most phylogenetic analyses. Nonetheless, the interordinal relationships remain largely unresolved, in particular in the largest subclass Rhodymeniophycidae that includes 70% of all species. To elucidate red algal phylogenetic relationships and study organelle evolution, we determined the sequence of 11 mitochondrial genomes (mtDNA) from 5 florideophycean subclasses. These mtDNAs were combined with existing data, resulting in a database of 25 florideophytes and 12 bangiophytes (including cyanidiophycean species). A concatenated alignment of mt proteins was used to resolve ordinal relationships in the Rhodymeniophycidae. Red algal mtDNA genome comparisons showed 47 instances of gene rearrangement including 12 that distinguish Bangiophyceae from Hildenbrandiophycidae, and 5 that distinguish Hildenbrandiophycidae from Nemaliophycidae. These organelle data support a rapid radiation and surprisingly high conservation of mtDNA gene syntheny among the morphologically divergent multicellular lineages of Rhodymeniophycidae. In contrast, we find extensive mitochondrial gene rearrangements when comparing Bangiophyceae and Florideophyceae and multiple examples of gene loss among the different red algal lineages. PMID:26245677

  13. Population genomic analysis reveals highly conserved mitochondrial genomes in the yeast species Lachancea thermotolerans.

    PubMed

    Freel, Kelle C; Friedrich, Anne; Hou, Jing; Schacherer, Joseph

    2014-10-01

    The increasing availability of mitochondrial (mt) sequence data from various yeasts provides a tool to study genomic evolution within and between different species. While the genomes from a range of lineages are available, there is a lack of information concerning intraspecific mtDNA diversity. Here, we analyzed the mt genomes of 50 strains from Lachancea thermotolerans, a protoploid yeast species that has been isolated from several locations (Europe, Asia, Australia, South Africa, and North / South America) and ecological sources (fruit, tree exudate, plant material, and grape and agave fermentations). Protein-coding genes from the mtDNA were used to construct a phylogeny, which reflected a similar, yet less resolved topology than the phylogenetic tree of 50 nuclear genes. In comparison to its sister species Lachancea kluyveri, L. thermotolerans has a smaller mt genome. This is due to shorter intergenic regions and fewer introns, of which the latter are only found in COX1. We revealed that L. kluyveri and L. thermotolerans share similar levels of intraspecific divergence concerning the nuclear genomes. However, L. thermotolerans has a more highly conserved mt genome with the coding regions characterized by low rates of nonsynonymous substitution. Thus, in the mt genomes of L. thermotolerans, stronger purifying selection and lower mutation rates potentially shape genome diversity in contract to what was found for L. kluyveri, demonstrating that the factors driving mt genome evolution are different even between closely related species. PMID:25212859

  14. A highly conserved molecular switch binds MSY-3 to regulate myogenin repression in postnatal muscle

    PubMed Central

    Berghella, Libera; De Angelis, Luciana; De Buysscher, Tristan; Mortazavi, Ali; Biressi, Stefano; Forcales, Sonia V.; Sirabella, Dario; Cossu, Giulio; Wold, Barbara J.

    2008-01-01

    Myogenin is the dominant transcriptional regulator of embryonic and fetal muscle differentiation and during maturation is profoundly down-regulated. We show that a highly conserved 17-bp DNA cis-acting sequence element located upstream of the myogenin promoter (myogHCE) is essential for postnatal repression of myogenin in transgenic animals. We present multiple lines of evidence supporting the idea that repression is mediated by the Y-box protein MSY-3. Electroporation in vivo shows that myogHCE and MSY-3 are required for postnatal repression. We further show that, in the C2C12 cell culture system, ectopic MSY-3 can repress differentiation, while reduced MSY-3 promotes premature differentiation. MSY-3 binds myogHCE simultaneously with the homeodomain protein Pbx in postnatal innervated muscle. We therefore propose a model in which the myogHCE motif operates as a switch by specifying opposing functions; one that was shown previously is regulated by MyoD and Pbx and it specifies a chromatin opening, gene-activating function at the time myoblasts begin to differentiate; the other includes MYS-3 and Pbx, and it specifies a repression function that operates during and after postnatal muscle maturation in vivo and in myoblasts before they begin to differentiate. PMID:18676817

  15. A new DNA binding protein highly conserved in diverse crenarchaeal viruses.

    PubMed

    Larson, Eric T; Eilers, Brian J; Reiter, Dirk; Ortmann, Alice C; Young, Mark J; Lawrence, C Martin

    2007-07-01

    Sulfolobus turreted icosahedral virus (STIV) infects Sulfolobus species found in the hot springs of Yellowstone National Park. Its 37 open reading frames (ORFs) generally lack sequence similarity to other genes. One exception, however, is ORF B116. While its function is unknown, orthologs are found in three additional crenarchaeal viral families. Due to the central importance of this protein family to crenarchaeal viruses, we have undertaken structural and biochemical studies of B116. The structure reveals a previously unobserved fold consisting of a five-stranded beta-sheet flanked on one side by three alpha helices. Two subunits come together to form a homodimer with a 10-stranded mixed beta-sheet, where the topology of the central strands resembles an unclosed beta-barrel. Highly conserved loops rise above the surface of the saddle-shaped protein and suggest an interaction with the major groove of DNA. The predicted B116-DNA interaction is confirmed by electrophoretic mobility shift assays. PMID:17336360

  16. Epidermal surface antigen (MS17S1) is highly conserved between mouse and human

    SciTech Connect

    Cho, Y.J.; Chema, D.; Cho, M.

    1995-05-20

    A mouse monoclonal antibody ECS-1 raised to human keratinocytes detects a 35-kDa epidermal surface antigen (ESA) and causes keratinocyte dissociation in vitro. ECS-1 stains skin of 16-day mouse embryo and 8- to 9-week human fetus. Mouse Esa cDNA encodes a 379-amino-acid protein that is 99.2% identical to the human, differing at only 3 amino acids. The gene (M17S1) was mapped to mouse chromosome 11, highlighting the conserved linkage synteny existing between human chromosome 17 and mouse chromosome 11. Although the nude locus has been mapped to the same region of chromosome 11, no abnormalities in protein, mRNA, or cDNA or genomic sequences were detected in nude mice. However, both nude and control mice were found to have a second Esa mRNA transcript that conserves amino acid sequence and molecular weight. The mouse and human 5{prime} and 3{prime} untranslated sequences are conserved. Similar RNA folding patterns of the 5{prime} untranslated region are predicted despite a 91-bp insertion in the mouse. These data suggest that both the function and the regulation of ESA protein are of importance and that Esa (M17S1) is not the nude locus gene. 42 refs., 7 figs., 3 tabs.

  17. Validation of high throughput sequencing and microbial forensics applications

    PubMed Central

    2014-01-01

    High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security. PMID:25101166

  18. Advances, practice, and clinical perspectives in high-throughput sequencing.

    PubMed

    Park, S-J; Saito-Adachi, M; Komiyama, Y; Nakai, K

    2016-07-01

    Remarkable advances in high-throughput sequencing technologies have fundamentally changed our understanding of the genetic and epigenetic molecular bases underlying human health and diseases. As these technologies continue to revolutionize molecular biology leading to fresh perspectives, it is imperative to thoroughly consider the enormous excitement surrounding the technologies by highlighting the characteristics of platforms and their global trends as well as potential benefits and limitations. To date, with a variety of platforms, the technologies provide an impressive range of applications, including sequencing of whole genomes and transcriptomes, identifying of genome modifications, and profiling of protein interactions. Because these applications produce a flood of data, simultaneous development of bioinformatics tools is required to efficiently deal with the big data and to comprehensively analyze them. This review covers the major achievements and performances of the high-throughput sequencing and further summarizes the characteristics of their applications along with introducing applicable bioinformatics tools. Moreover, a step-by-step procedure for a practical transcriptome analysis is described employing an analytical pipeline. Clinical perspectives with special consideration to human oral health and diseases are also covered. PMID:26602181

  19. Validation of high throughput sequencing and microbial forensics applications.

    PubMed

    Budowle, Bruce; Connell, Nancy D; Bielecka-Oder, Anna; Colwell, Rita R; Corbett, Cindi R; Fletcher, Jacqueline; Forsman, Mats; Kadavy, Dana R; Markotic, Alemka; Morse, Stephen A; Murch, Randall S; Sajantila, Antti; Schmedes, Sarah E; Ternus, Krista L; Turner, Stephen D; Minot, Samuel

    2014-01-01

    High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security. PMID:25101166

  20. Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code

    PubMed Central

    Ciribilli, Yari; Monti, Paola; Bisio, Alessandra; Nguyen, H. Thien; Ethayathulla, Abdul S.; Ramos, Ana; Foggetti, Giorgia; Menichini, Paola; Menendez, Daniel; Resnick, Michael A.; Viadiu, Hector; Fronza, Gilberto; Inga, Alberto

    2013-01-01

    Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein–DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis. PMID:23892287

  1. Deep parallel sequencing reveals conserved and novel miRNAs in gill and hepatopancreas of giant freshwater prawn.

    PubMed

    Tan, Tian Tian; Chen, Maoshan; Harikrishna, Jennifer Ann; Khairuddin, Norliana; Mohd Shamsudin, Maizatul Izzah; Zhang, Guojie; Bhassu, Subha

    2013-10-01

    MicroRNAs (miRNAs) are ~20-22 nucleotides, non protein-coding RNA regulatory genes that post-transcriptionally regulate many protein-coding genes, influencing critical biological and metabolic processes. While the number of known microRNA is increasing, there is currently no published data for miRNA from giant freshwater prawns, Macrobrachium rosenbergii (M. rosenbergii), a commercially cultured and economically important food species. In this study, we identified novel miRNAs in the gill and hepatopancreas of M. rosenbergii. Through a deep parallel sequencing analysis and an in silico data analysis approach, 327 miRNA families were identified from small RNA libraries with reference to both the de novo transcriptome of M. rosenbergii obtained from RNA-Seq and to miRBase (Release 18.0, November 2012). Based on the identified mature miRNA and recovered precursor sequences that form appropriate hairpin structures, three conserved miRNA (miR125, miR750, miR993) and 27 novel miRNA candidates encoding messenger-like non-coding RNA were identified. miR-125, miR-750, G-m0002/H-m0009, G-m0005, G-m0008/H-m0016, G-m0011/H-m0027 and G-m0015 were selected for experimental validation with stem-loop quantitative RT-PCR and were found to be coherent with the expression profile of deep sequencing data as evaluated with Pearson's correlation coefficient (r = 0.835178 for miRNA in gill, r = 0.724131 for miRNA in hepatopancreas). Using a combinatorial approach of pathway enrichment analysis and inverse expression relationship of miRNA and mRNA, four co-expressed novel miRNA candidates (G-m0005, G-m0008/H-m0016, G-m0011/H-m0027, and G-m0015) were found to be associated with energy metabolism. In addition, the expression of the three novel miRNA candidates (G-m0005, G-m0008/H-m0016, and G-m0011/H-m0027) were also found to be significantly reduced at 9 and 24 h post infection in M. rosenbergii challenged with infectious hypodermal and hematopoietic necrosis virus, suggesting a functional

  2. 5'-terminal nucleotide sequences of mammalian type C helper viruses are conserved in the genomes of replication-defective mammalian transforming viruses.

    PubMed Central

    Tronick, S R; Cabradilla, C D; Aaronson, S A; Haseltine, W A

    1978-01-01

    The RNAs of replication-defective murine and primate type C transforming viruses were analyzed for the presence of nucleotide sequences homologous to the genomes of their respective helper type C viruses by using DNAs complementary (cDNA) to either the 5'-terminal (cDNA5') or total (cDNAtotal) nucleotide sequences of the helper virus RNA. The defective viruses examined have previously been shown to vary in their ability to express helper viral gag gene proteins. With cDNAtotal as a probe, these transforming viruses were shown to vary in their representation of helper sequences (15 to 60% hybridization of cDNAtotal). In striking contrast, 5'-terminal-specific sequences of the helper virus were conserved in the RNAs of every transforming virus tested (is greater than 80% hybridization of cDNA5'). These findings suggest a critical role for these sequences in the life cycle of the defective transforming virus. PMID:209210

  3. Energy and Energy Conservation Activities for High School Students.

    ERIC Educational Resources Information Center

    Bottinelli, Charles A., Ed.; Dow, John O., Ed.

    This manual contains fifteen energy activities suitable for high school physical and environmental science and mathematics classrooms. The activities are independent, each having its own objectives, introduction, and background information. A special section of each activity is written for the instructor and contains limits, sample data, and…

  4. A root chicory MADS box sequence and the Arabidopsis flowering repressor FLC share common features that suggest conserved function in vernalization and de-vernalization responses.

    PubMed

    Périlleux, Claire; Pieltain, Alexandra; Jacquemin, Guillaume; Bouché, Frédéric; Detry, Nathalie; D'Aloia, Maria; Thiry, Laura; Aljochim, Pierre; Delansnay, Martin; Mathieu, Anne-Sophie; Lutts, Stanley; Tocquin, Pierre

    2013-08-01

    Root chicory (Cichorium intybus var. sativum) is a biennial crop, but is harvested to obtain root inulin at the end of the first growing season before flowering. However, cold temperatures may vernalize seeds or plantlets, leading to incidental early flowering, and hence understanding the molecular basis of vernalization is important. A MADS box sequence was isolated by RT-PCR and named FLC-LIKE1 (CiFL1) because of its phylogenetic positioning within the same clade as the floral repressor Arabidopsis FLOWERING LOCUS C (AtFLC). Moreover, over-expression of CiFL1 in Arabidopsis caused late flowering and prevented up-regulation of the AtFLC target FLOWERING LOCUS T by photoperiod, suggesting functional conservation between root chicory and Arabidopsis. Like AtFLC in Arabidopsis, CiFL1 was repressed during vernalization of seeds or plantlets of chicory, but repression of CiFL1 was unstable when the post-vernalization temperature was favorable to flowering and when it de-vernalized the plants. This instability of CiFL1 repression may be linked to the bienniality of root chicory compared with the annual lifecycle of Arabidopsis. However, re-activation of AtFLC was also observed in Arabidopsis when a high temperature treatment was used straight after seed vernalization, eliminating the promotive effect of cold on flowering. Cold-induced down-regulation of a MADS box floral repressor and its re-activation by high temperature thus appear to be conserved features of the vernalization and de-vernalization responses in distant species. PMID:23581257

  5. Prevalent distribution and conservation of Streptococcus suis Lmb protein and its protective capacity against the Chinese highly virulent strain infection.

    PubMed

    Zhang, Yan-Mei; Shao, Zhu-Qing; Wang, Jing; Wang, Ling; Li, Xianfu; Wang, Changjun; Tang, Jiaqi; Pan, Xiuzhen

    2014-01-01

    Streptococcus suis (S. suis) is an important zoonotic pathogen that causes multiple diseases in both pigs and humans. Many studies suggest that Streptococcus utilizes host extracellular matrix proteins, including laminin, for adhesion and invasion of host cells. Recently, we identified a putative Lmb protein (CDS 0330) of a highly virulent strain of S. suis (serotype 2). In this study, we characterized the ability of CDS 0330 to bind human laminin, and evaluated the protective efficacy of a recombinant protein vaccine. Bioinformatic analysis revealed that both the amino acid sequence and tertiary structure of CDS 0330 were similar to Lmb proteins in other Streptococcus. In addition, the sequence of CDS 0330 was present in the genomes of 26 of the 38 sequenced streptococci species, indicating an early origin and conservation of this gene. Particularly, all 17 sequenced S. suis genomes, regardless of serotype or geographic origin, contained CDS 0330 gene in their genome with a minimum pair-wise amino acid identity of 92%. PCR amplification revealed that CDS 0330 gene is distributed throughout 35 S. suis serotypes in the lmb-htp format. Flow cytometry analysis confirmed that CDS 0330 was expressed on the cell surface of S. suis, and ELISA revealed the recombinant CDS 0330 protein could bind laminin in vitro. Finally, vaccinating mice with recombinant CDS 0330 protein significantly prolonged survival after S. suis infection. Together, these data reveal that CDS 0330 is a laminin binding protein of S. suis 2, and open new avenues for preventing S. suis 2 infection. PMID:24120016

  6. High-throughput sequencing: a roadmap toward community ecology.

    PubMed

    Poisot, Timothée; Péquin, Bérangère; Gravel, Dominique

    2013-04-01

    High-throughput sequencing is becoming increasingly important in microbial ecology, yet it is surprisingly under-used to generate or test biogeographic hypotheses. In this contribution, we highlight how adding these methods to the ecologist toolbox will allow the detection of new patterns, and will help our understanding of the structure and dynamics of diversity. Starting with a review of ecological questions that can be addressed, we move on to the technical and analytical issues that will benefit from an increased collaboration between different disciplines. PMID:23610649

  7. High-Throughput Sequencing: A Roadmap Toward Community Ecology

    PubMed Central

    Poisot, Timothée; Péquin, Bérangère; Gravel, Dominique

    2013-01-01

    High-throughput sequencing is becoming increasingly important in microbial ecology, yet it is surprisingly under-used to generate or test biogeographic hypotheses. In this contribution, we highlight how adding these methods to the ecologist toolbox will allow the detection of new patterns, and will help our understanding of the structure and dynamics of diversity. Starting with a review of ecological questions that can be addressed, we move on to the technical and analytical issues that will benefit from an increased collaboration between different disciplines. PMID:23610649

  8. Identification of Novel and Conserved miRNAs in Leaves of In vitro Grown Citrus reticulata “Lugan” Plantlets by Solexa Sequencing

    PubMed Central

    Guo, Rongfang; Chen, Xiaodong; Lin, Yuling; Xu, Xuhan; Thu, Min Kyaw; Lai, Zhongxiong

    2016-01-01

    MicroRNAs (miRNAs) play essential roles in plant development, but the roles in the in vitro plant development are unknown. Leaves of ponkan plantlets derived from mature embryos at in vitro culture conditions were used to sequence small RNA fraction via Solexa sequencing, and the miRNAs expression was analyzed. The results showed that there were 3,065,625 unique sequences in ponkan, of which 0.79% were miRNAs. The RNA sequences with lengths of 18–25 nt derived from the library were analyzed, leading to the identification of 224 known miRNAs, of which the most abundant were miR157, miR156, and miR166. Three hundred and fifty-eight novel miRNA candidates were also identified, and the number of reads of ponkan novel miRNAs varied from 5 to 168,273. The expression of the most known miRNAs obtained was at low levels, which varied from 5 to 4,946,356. To better understand the role of miRNAs during the preservation of ponkan in vitro plantlet, the expression patterns of cre-miR156a/159b/160a/166a/167a/168a/171/398b were validated by quantitative real-time PCR (qPCR). The results showed that not only the development-associated miRNAs, e.g., cre-miR156/159/166/396, expressed highly at the early preservation period in the in vitro ponkan plantlet leaves but also the stress-related miRNAs, e.g., cre-miR171 and cre-miR398b, expressed highly at the same time. The expression levels of most tested miRNAs were found to decrease after 6 months and the amounts of these miRNAs were kept at low levels at 18 months. After analyzing the expression level of their targets during the reservation of the ponkan in vitro plantlet, development-associated cre-ARF6 and stress-related cre-CSD modules exhibited negative correlation with miR167 and miR398, respectively, indicating an involvement of the miRNAs in the in vitro development of ponkan and function in the conservation of ponkan germplasm. PMID:26779240

  9. Crystal structure of AFV3-109, a highly conserved protein from crenarchaeal viruses

    PubMed Central

    Keller, Jenny; Leulliot, Nicolas; Cambillau, Christian; Campanacci, Valérie; Porciero, Stéphanie; Prangishvili, David; Forterre, Patrick; Cortez, Diego; Quevillon-Cheruel, Sophie; van Tilbeurgh, Herman

    2007-01-01

    The extraordinary morphologies of viruses infecting hyperthermophilic archaea clearly distinguish them from bacterial and eukaryotic viruses. Moreover, their genomes code for proteins that to a large extend have no related sequences in the extent databases. However, a small pool of genes is shared by overlapping subsets of these viruses, and the most conserved gene, exemplified by the ORF109 of the Acidianus Filamentous Virus 3, AFV3, is present on genomes of members of three viral familes, the Lipothrixviridae, Rudiviridae, and "Bicaudaviridae", as well as of the unclassified Sulfolobus Turreted Icosahedral Virus, STIV. We present here the crystal structure of the protein (Mr = 13.1 kD, 109 residues) encoded by the AFV3 ORF 109 in two different crystal forms at 1.5 and 1.3 Å resolution. The structure of AFV3-109 is a five stranded β-sheet with loops on one side and three helices on the other. It forms a dimer adopting the shape of a cradle that encompasses the best conserved regions of the sequence. No protein with a related fold could be identified except for the ortholog from STIV1, whose structure was deposited at the Protein Data Bank. We could clearly identify a well bound glycerol inside the cradle, contacting exclusively totally conserved residues. This interaction was confirmed in solution by fluorescence titration. Although the function of AFV3-109 cannot be deduced directly from its structure, structural homology with the STIV1 protein, and the size and charge distribution of the cavity suggested it could interact with nucleic acids. Fluorescence quenching titrations also showed that AFV3-109 interacts with dsDNA. Genomic sequence analysis revealed bacterial homologs of AFV3-109 as a part of a putative previously unidentified prophage sequences in some Firmicutes. PMID:17241456

  10. Structure Analysis Uncovers a Highly Diverse but Structurally Conserved Effector Family in Phytopathogenic Fungi

    PubMed Central

    Gracy, Jérome; Fournier, Elisabeth; Kroj, Thomas; Padilla, André

    2015-01-01

    Phytopathogenic ascomycete fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The molecular function of these effectors and the evolutionary mechanisms that generate this tremendous number of singleton genes are largely unknown. To get a deeper understanding of fungal effectors, we determined by NMR spectroscopy the 3-dimensional structures of the Magnaporthe oryzae effectors AVR1-CO39 and AVR-Pia. Despite a lack of sequence similarity, both proteins have very similar 6 β-sandwich structures that are stabilized in both cases by a disulfide bridge between 2 conserved cysteins located in similar positions of the proteins. Structural similarity searches revealed that AvrPiz-t, another effector from M. oryzae, and ToxB, an effector of the wheat tan spot pathogen Pyrenophora tritici-repentis have the same structures suggesting the existence of a family of sequence-unrelated but structurally conserved fungal effectors that we named MAX-effectors (Magnaporthe Avrs and ToxB like). Structure-informed pattern searches strengthened this hypothesis by identifying MAX-effector candidates in a broad range of ascomycete phytopathogens. Strong expansion of the MAX-effector family was detected in M. oryzae and M. grisea where they seem to be particularly important since they account for 5–10% of the effector repertoire and 50% of the cloned avirulence effectors. Expression analysis indicated that the majority of M. oryzae MAX-effectors are expressed specifically during early infection suggesting important functions during biotrophic host colonization. We hypothesize that the scenario observed for MAX-effectors can serve as a paradigm for ascomycete effector diversity and that the enormous number of sequence-unrelated ascomycete effectors may in fact belong to a restricted set of structurally conserved effector families. PMID:26506000

  11. A monoclonal antibody targeting a highly conserved epitope in influenza B neuraminidase provides protection against drug resistant strains.

    PubMed

    Doyle, Tracey M; Li, Changgui; Bucher, Doris J; Hashem, Anwar M; Van Domselaar, Gary; Wang, Junzhi; Farnsworth, Aaron; She, Yi-Min; Cyr, Terry; He, Runtao; Brown, Earl G; Hurt, Aeron C; Li, Xuguang

    2013-11-01

    All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222-230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus. PMID:24140051

  12. High-Resolution Satellite Imagery Is an Important yet Underutilized Resource in Conservation Biology

    PubMed Central

    Boyle, Sarah A.; Kennedy, Christina M.; Torres, Julio; Colman, Karen; Pérez-Estigarribia, Pastor E.; de la Sancha, Noé U.

    2014-01-01

    Technological advances and increasing availability of high-resolution satellite imagery offer the potential for more accurate land cover classifications and pattern analyses, which could greatly improve the detection and quantification of land cover change for conservation. Such remotely-sensed products, however, are often expensive and difficult to acquire, which prohibits or reduces their use. We tested whether imagery of high spatial resolution (≤5 m) differs from lower-resolution imagery (≥30 m) in performance and extent of use for conservation applications. To assess performance, we classified land cover in a heterogeneous region of Interior Atlantic Forest in Paraguay, which has undergone recent and dramatic human-induced habitat loss and fragmentation. We used 4 m multispectral IKONOS and 30 m multispectral Landsat imagery and determined the extent to which resolution influenced the delineation of land cover classes and patch-level metrics. Higher-resolution imagery more accurately delineated cover classes, identified smaller patches, retained patch shape, and detected narrower, linear patches. To assess extent of use, we surveyed three conservation journals (Biological Conservation, Biotropica, Conservation Biology) and found limited application of high-resolution imagery in research, with only 26.8% of land cover studies analyzing satellite imagery, and of these studies only 10.4% used imagery ≤5 m resolution. Our results suggest that high-resolution imagery is warranted yet under-utilized in conservation research, but is needed to adequately monitor and evaluate forest loss and conversion, and to delineate potentially important stepping-stone fragments that may serve as corridors in a human-modified landscape. Greater access to low-cost, multiband, high-resolution satellite imagery would therefore greatly facilitate conservation management and decision-making. PMID:24466287

  13. High-resolution satellite imagery is an important yet underutilized resource in conservation biology.

    PubMed

    Boyle, Sarah A; Kennedy, Christina M; Torres, Julio; Colman, Karen; Pérez-Estigarribia, Pastor E; de la Sancha, Noé U

    2014-01-01

    Technological advances and increasing availability of high-resolution satellite imagery offer the potential for more accurate land cover classifications and pattern analyses, which could greatly improve the detection and quantification of land cover change for conservation. Such remotely-sensed products, however, are often expensive and difficult to acquire, which prohibits or reduces their use. We tested whether imagery of high spatial resolution (≤5 m) differs from lower-resolution imagery (≥30 m) in performance and extent of use for conservation applications. To assess performance, we classified land cover in a heterogeneous region of Interior Atlantic Forest in Paraguay, which has undergone recent and dramatic human-induced habitat loss and fragmentation. We used 4 m multispectral IKONOS and 30 m multispectral Landsat imagery and determined the extent to which resolution influenced the delineation of land cover classes and patch-level metrics. Higher-resolution imagery more accurately delineated cover classes, identified smaller patches, retained patch shape, and detected narrower, linear patches. To assess extent of use, we surveyed three conservation journals (Biological Conservation, Biotropica, Conservation Biology) and found limited application of high-resolution imagery in research, with only 26.8% of land cover studies analyzing satellite imagery, and of these studies only 10.4% used imagery ≤5 m resolution. Our results suggest that high-resolution imagery is warranted yet under-utilized in conservation research, but is needed to adequately monitor and evaluate forest loss and conversion, and to delineate potentially important stepping-stone fragments that may serve as corridors in a human-modified landscape. Greater access to low-cost, multiband, high-resolution satellite imagery would therefore greatly facilitate conservation management and decision-making. PMID:24466287

  14. Comparative analyses reveal a highly conserved endoglucanase in the cellulolytic genus Fibrobacter.

    PubMed Central

    Lin, C; Stahl, D A

    1995-01-01

    An RNA probe complementary to the endoglucanase 3 gene (cel-3) of Fibrobacter succinogenes S85 hybridized to chromosomal DNAs from isolates representing the genetic diversity of the genus. The probe was subsequently used to identify putative cel-3-containing clones from genomic libraries of representative Fibrobacter isolates. Comparative sequence analyses of the cloned cel-3 genes confirmed that cel-3 is conserved among Fibrobacter isolates and that the ancestral cel-3 gene appears to have coevolved with the genus, since the same genealogy was inferred from sequence comparisons of 16S rRNAs and cel-3 genes. Hybridization comparisons using a xylanase gene probe suggested similar conservation of this gene. Together the data indicate that the cellulolytic apparatus is conserved among Fibrobacter isolates and that comparative analyses of homologous elements of the apparatus from different members, in relationship to the now established phylogeny of the genus, could serve to better define the enzymatic basis of fiber digestion in this genus. PMID:7730288

  15. High-speed lossless compression for angiography image sequences

    NASA Astrophysics Data System (ADS)

    Kennedy, Jonathon M.; Simms, Michael; Kearney, Emma; Dowling, Anita; Fagan, Andrew; O'Hare, Neil J.

    2001-05-01

    High speed processing of large amounts of data is a requirement for many diagnostic quality medical imaging applications. A demanding example is the acquisition, storage and display of image sequences in angiography. The functional performance requirements for handling angiography data were identified. A new lossless image compression algorithm was developed, implemented in C++ for the Intel Pentium/MS-Windows environment and optimized for speed of operation. Speeds of up to 6M pixels per second for compression and 12M pixels per second for decompression were measured. This represents an improvement of up to 400% over the next best high-performance algorithm (LOCO-I) without significant reduction in compression ratio. Performance tests were carried out at St. James's Hospital using actual angiography data. Results were compared with the lossless JPEG standard and other leading methods such as JPEG-LS (LOCO-I) and the lossless wavelet approach proposed for JPEG 2000. Our new algorithm represents a significant improvement in the performance of lossless image compression technology without using specialized hardware. It has been applied successfully to image sequence decompression at video rate for angiography, one of the most challenging application areas in medical imaging.

  16. Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

    SciTech Connect

    Hashem, Anwar M.; Van Domselaar, Gary; Li, Changgui; Wang, Junzhi; She, Yi-Min; Cyr, Terry D.; Sui, Jianhua; He, Runtao; Marasco, Wayne A.; Li, Xuguang

    2010-12-10

    Research highlights: {yields} The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. {yields} Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. {yields} The universal antibodies cross-neutralize different influenza A subtypes. {yields} The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

  17. High-throughput sequencing of immune repertoires in multiple sclerosis.

    PubMed

    Lossius, Andreas; Johansen, Jorunn N; Vartdal, Frode; Holmøy, Trygve

    2016-04-01

    T cells and B cells are crucial in the initiation and maintenance of multiple sclerosis (MS), and the activation of these cells is believed to be mediated through specific recognition of antigens by the T- and B-cell receptors. The antigen receptors are highly polymorphic due to recombination (T- and B-cell receptors) and mutation (B-cell receptors) of the encoding genes, which can therefore be used as fingerprints to track individual T- and B-cell clones. Such studies can shed light on mechanisms driving the immune responses and provide new insights into the pathogenesis. Here, we summarize studies that have explored the T- and B-cell receptor repertoires using earlier methodological approaches, and we focus on how high-throughput sequencing has provided new knowledge by surveying the immune repertoires in MS in even greater detail and with unprecedented depth. PMID:27081660

  18. Influence of FGD gypsum on the properties of a highly erodible soil under conservation tillage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The performance of conservation tillage practices imposed on highly erodible soils may be improved by the use of amendments with a high solubility rate, and whose dissolution products are translocated at depth in the soil profile faster than normally used agricultural lime and fertilizer products. T...

  19. First High-Quality Draft Genome Sequence of Pasteurella multocida Sequence Type 128 Isolated from Infected Bone.

    PubMed

    Kavousi, Niloofar; Eng, Wilhelm Wei Han; Lee, Yin Peng; Tan, Lian Huat; Thuraisingham, Ravindran; Yule, Catherine M; Gan, Han Ming

    2016-01-01

    We report here the first high-quality draft genome sequence of Pasteurella multocida sequence type 128, which was isolated from the infected finger bone of an adult female who was bitten by a domestic dog. The draft genome will be a valuable addition to the scarce genomic resources available for P. multocida. PMID:26941132

  20. Isolation of a new marker and conserved sequences close to the DiGeorge syndrome marker HP500 (D22S134).

    PubMed Central

    Wadey, R; Daw, S; Wickremasinghe, A; Roberts, C; Wilson, D; Goodship, J; Burn, J; Halford, S; Scambler, P J

    1993-01-01

    End fragment cloning from a YAC at the D22S134 locus allowed the isolation of a new probe HD7k. This marker detects hemizygosity in two patients previously shown to be dizygous for D22S134. This positions the distal deletion breakpoint in these patients to the sequences within the YAC, and confirms that HD7k is proximal to D22S134. In a search for coding sequences within the region commonly deleted in DGS we have identified a conserved sequence at D22S134. Although no cDNAs have yet been isolated, genomic sequencing shows a short open reading frame with weak similarity to collagen proteins. Images PMID:8230156

  1. Potential of high residue conservation tillage to enhance water conservation and water use efficiency in corn production in the Southeast

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Following the adoption of its first Comprehensive State-wide Water Management Plan in early February 2008, Georgia is on course to drafting regionally-based water development and conservation plans. Conservation tillage-based crop production can be one of the management tools available for achieving...

  2. High-Order Entropy Stable Finite Difference Schemes for Nonlinear Conservation Laws: Finite Domains

    NASA Technical Reports Server (NTRS)

    Fisher, Travis C.; Carpenter, Mark H.

    2013-01-01

    Developing stable and robust high-order finite difference schemes requires mathematical formalism and appropriate methods of analysis. In this work, nonlinear entropy stability is used to derive provably stable high-order finite difference methods with formal boundary closures for conservation laws. Particular emphasis is placed on the entropy stability of the compressible Navier-Stokes equations. A newly derived entropy stable weighted essentially non-oscillatory finite difference method is used to simulate problems with shocks and a conservative, entropy stable, narrow-stencil finite difference approach is used to approximate viscous terms.

  3. siVirus: web-based antiviral siRNA design software for highly divergent viral sequences

    PubMed Central

    Naito, Yuki; Ui-Tei, Kumiko; Nishikawa, Toru; Takebe, Yutaka; Saigo, Kaoru

    2006-01-01

    siVirus () is a web-based online software system that provides efficient short interfering RNA (siRNA) design for antiviral RNA interference (RNAi). siVirus searches for functional, off-target minimized siRNAs targeting highly conserved regions of divergent viral sequences. These siRNAs are expected to resist viral mutational escape, since their highly conserved targets likely contain structurally/functionally constrained elements. siVirus will be a useful tool for designing optimal siRNAs targeting highly divergent pathogens, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus and SARS coronavirus, all of which pose enormous threats to global human health. PMID:16845046

  4. The Phylogeographical Pattern and Conservation of the Chinese Cobra (Naja atra) across Its Range Based on Mitochondrial Control Region Sequences

    PubMed Central

    Lin, Long-Hui; Hua, Lei; Qu, Yan-Fu; Gao, Jian-Fang; Ji, Xiang

    2014-01-01

    The vulnerable Chinese cobra (Naja atra) ranges from southeastern China south of the Yangtze River to northern Vietnam and Laos. Large mountain ranges and water bodies may influence the pattern of genetic diversity of this species. We sequenced the mitochondrial DNA control region (1029 bp) using 285 individuals collected from 23 localities across the species' range and obtained 18 sequences unique to Taiwan from GenBank for phylogenetic and population analysis. Two distinct clades were identified, one including haplotypes from the two westernmost localities (Hekou and Miyi) and the other including haplotypes from all sampling sites except Miyi. A strong population structure was found (Φst = 0.76, P<0.0001) with high haplotype diversity (h = 1.00) and low nucleotide diversity (π = 0.0049). The Luoxiao and Nanling Mountains act as historical geographical barriers limiting gene exchange. In the haplotype network there were two “star” clusters. Haplotypes from populations east of the Luoxiao Mountains were represented within one cluster and haplotypes from populations west of the mountain range within the other, with haplotypes from populations south of the Nanling Mountains in between. Lineage sorting between mainland and island populations is incomplete. It remains unknown as to how much adaptive differentiation there is between population groups or within each group. We caution against long-distance transfers within any group, especially when environmental differences are apparent. PMID:25184236

  5. The Sequence of Learning Cycle Activities in High School Chemistry.

    ERIC Educational Resources Information Center

    Abraham, Michael R.; Renner, John W.

    1986-01-01

    Different learning cycle sequences were investigated to determine factors accounting for success of the cycle, compared learning with conventional instruction, and examined relationships between Piaget's theory and learning cycles. Results show that the normal learning cycle sequence is the optimum sequence for achievement of content knowledge in…

  6. Advantages of Single-Molecule Real-Time Sequencing in High-GC Content Genomes

    PubMed Central

    Shin, Seung Chul; Ahn, Do Hwan; Kim, Su Jin; Lee, Hyoungseok; Oh, Tae-Jin; Lee, Jong Eun; Park, Hyun

    2013-01-01

    Next-generation sequencing has become the most widely used sequencing technology in genomics research, but it has inherent drawbacks when dealing with high-GC content genomes. Recently, single-molecule real-time sequencing technology (SMRT) was introduced as a third-generation sequencing strategy to compensate for this drawback. Here, we report that the unbiased and longer read length of SMRT sequencing markedly improved genome assembly with high GC content via gap filling and repeat resolution. PMID:23894349

  7. Geometric conservation law and applications to high-order finite difference schemes with stationary grids

    NASA Astrophysics Data System (ADS)

    Deng, Xiaogang; Mao, Meiliang; Tu, Guohua; Liu, Huayong; Zhang, Hanxin

    2011-02-01

    The geometric conservation law (GCL) includes the volume conservation law (VCL) and the surface conservation law (SCL). Though the VCL is widely discussed for time-depending grids, in the cases of stationary grids the SCL also works as a very important role for high-order accurate numerical simulations. The SCL is usually not satisfied on discretized grid meshes because of discretization errors, and the violation of the SCL can lead to numerical instabilities especially when high-order schemes are applied. In order to fulfill the SCL in high-order finite difference schemes, a conservative metric method (CMM) is presented. This method is achieved by computing grid metric derivatives through a conservative form with the same scheme applied for fluxes. The CMM is proven to be a sufficient condition for the SCL, and can ensure the SCL for interior schemes as well as boundary and near boundary schemes. Though the first-level difference operators δ3 have no effects on the SCL, no extra errors can be introduced as δ3 = δ2. The generally used high-order finite difference schemes are categorized as central schemes (CS) and upwind schemes (UPW) based on the difference operator δ1 which are used to solve the governing equations. The CMM can be applied to CS and is difficult to be satisfied by UPW. Thus, it is critical to select the difference operator δ1 to reduce the SCL-related errors. Numerical tests based on WCNS-E-5 show that the SCL plays a very important role in ensuring free-stream conservation, suppressing numerical oscillations, and enhancing the robustness of the high-order scheme in complex grids.

  8. Sequence conservation in the Ancylostoma secreted protein-2 of Necator americanus (Na-ASP-2) from hookworm infected individuals in Thailand.

    PubMed

    Ungcharoensuk, Charoenchai; Putaporntip, Chaturong; Pattanawong, Urassaya; Jongwutiwes, Somchai

    2012-12-01

    The Ancylostoma secreted protein-2 of Necator americanus (Na-ASP-2) was one of the promising vaccine candidates against the most prevalent human hookworm species as adverse vaccine reaction has compromised further human vaccine trials. To elucidate the gene structure and the extent of sequence diversity, we determined the complete nucleotide sequence of the Na-asp-2 gene of individual larvae from 32 infected subjects living in 3 different endemic areas of Thailand. Sequence analysis revealed that the gene encoding Na-ASP-2 comprised 8 exons. Of 3 nucleotide substitutions in these exons, only one causes an amino acid change from leucine to methionine. A consensus conserved GT and AG at the 5' and the 3' boundaries of each intron was observed akin to those found in other eukaryotic genes. Introns of Na-asp-2 contained 23 nucleotide substitutions and 0-18 indels. The mean number of nucleotide substitutions per site (d) in introns was not significantly different from the mean number of synonymous substitutions per synonymous site (d(S)) in exons whereas d in introns was significantly exceeded d(N) (the mean number of nonsynonymous substitutions per nonsynonymous site) in exons (p<0.05), suggesting that introns and synonymous sites in exons may evolve at a similar rate whereas functional constraints at the amino acid could limit amino acid substitutions in Na-ASP-2. A recombination site was identified in an intron near the 3' portion of the gene. The positions of introns and the intron phases in the Na-asp-2 gene comparing with those in other pathogenesis-related-1 proteins of Loa loa, Onchocerca volvulus, Heterodera glycines, Caenorhabditis elegans and human were relatively conserved, suggesting evolutionary conservation of these genes. Sequence conservation in Na-ASP-2 may not compromise further vaccine design if adverse vaccine effects could be resolved whereas microheterogeneity in introns of this locus may be useful for population genetics analysis of N. americanus

  9. Characterizing immune repertoires by high throughput sequencing: strategies and applications

    PubMed Central

    Calis, Jorg J.A.; Rosenberg, Brad R.

    2014-01-01

    As the key cellular effectors of adaptive immunity, T and B lymphocytes utilize specialized receptors to recognize, respond to, and neutralize a diverse array of extrinsic threats. These receptors (immunoglobulins in B lymphocytes, T cell receptors in T lymphocytes) are incredibly variable, the products of specialized genetic diversification mechanisms that generate complex lymphocyte repertoires with extensive collections of antigen specificities. Recent advances in high throughput sequencing (HTS) technologies have transformed our ability to examine antigen receptor repertoires at single nucleotide, and more recently, single cell, resolution. Here we review current approaches to examining antigen receptor repertoires by HTS, and discuss inherent biological and technical challenges. We further describe emerging applications of this powerful methodology for exploring the adaptive immune system. PMID:25306219

  10. Eomesodermin promotes interferon-γ expression and binds to multiple conserved noncoding sequences across the Ifng locus in mouse thymoma cell lines.

    PubMed

    Fukuoka, Natsuki; Harada, Misuzu; Nishida, Ai; Ito, Yuko; Shiota, Hideki; Kataoka, Takao

    2016-02-01

    The T-box transcription factors T-bet and eomesodermin (Eomes) have been shown to regulate the lineage-specific expression of interferon-γ (IFN-γ). However, in contrast to T-bet, the role of Eomes in the expression of IFN-γ remains unclear. In this study, we investigated the Eomes-dependent expression of IFN-γ in the mouse thymoma BW5147 and EL4 cells, which do not express T-bet or Eomes. The ectopic expression of Eomes induced BW5147 and EL4 cells to produce IFN-γ in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In BW5147 cells, Eomes augmented luciferase activity driven by the Ifng promoter encoding from -2500 to +113 bp; however, it was not increased by a stimulation with PMA and IM. A chromatin immunoprecipitation assay showed that Eomes bound to the Ifng promoter and conserved noncoding sequence (CNS) -22 kb across the Ifng locus with high efficacy in BW5147 cells. Moreover, Eomes increased permissive histone modifications in the Ifng promoter and multiple CNSs. The stimulation with PMA and IM greatly augmented Eomes binding to CNS-54, CNS-34, CNS+19 and CNS+30, which was inhibited by FK506. These results indicated that Eomes bound to the Ifng promoter and multiple CNSs in stimulation-dependent and stimulation-independent manners. PMID:26749212

  11. Characterization of an Egyptian Spodoptera littoralis nucleopolyhedrovirus and a possible use of a highly conserved region from polyhedrin gene for nucleopolyhedrovirus detection

    PubMed Central

    Seufi, AlaaEddeen M

    2008-01-01

    An Egyptian isolate of Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was tested for its potential as biocontrol agent in comparison to Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Comparative assays of SpliNPV and AcMNPV against 2nd instar larvae of Spodoptera littoralis revealed 4-fold greater susceptibility of S. littoralis to AcMNPV than to SpliNPV based on LC50 values for the two viruses. The LT50s determined for SpliNPV and AcMNPV using LC50 of the virus against 2nd instar larvae were 4.2 and 5.8 days, respectively. A DNA segment of 405 bp containing highly conserved region from polyhedrin gene of SpliNPV (Polh-cr) was successfully amplified by PCR. Subsequently, this DNA segment was cloned and sequenced. Nucleotide sequence and its deduced amino acid sequence were compared to all available sequences in GenBank. Sequence alignment results revealed that Polh-cr showed significant similarities with 91 different baculovirus isolates. The percentage of homology ranged from 78% for Plusia orichalcea NPV to 99% for SpliNPV. This highly conserved region provides a candidate that could be used in easy, fast and economic prospective systems for virus detection as well as in biological control strategies. PMID:18215282

  12. The complete mitochondrial genome sequence of the hornwort Phaeoceros laevis: retention of many ancient pseudogenes and conservative evolution of mitochondrial genomes in hornworts.

    PubMed

    Xue, Jia-Yu; Liu, Yang; Li, Libo; Wang, Bin; Qiu, Yin-Long

    2010-02-01

    Plants have large and complex mitochondrial genomes in comparison to other eukaryotes. In bryophytes, the mitochondrial genomes exhibit a mixed mode of conservative and dynamic evolution. Here, we sequenced the complete mitochondrial genome from hornwort Phaeoceros laevis, to investigate the level of conservation in mitochondrial genome evolution within hornworts. The circular molecule consists of 209,482 base pairs and represents the largest known mitochondrial genome of bryophytes. It contains 30 protein genes, 3 rRNA genes, and 21 tRNA genes, with 34 cis-spliced group II introns disrupting 16 protein genes. There are 11 pseudogenes in this genome, and nine of them are shared with the other fully sequenced hornwort chondriome from Megaceros aenigmaticus, a distant relative of P. laevis. These pseudogenes were likely formed during an early stage of hornwort evolution. The two hornwort chondriomes differ by four inversions and translocations, seven genes, and four introns in the genome structure and organization. At the sequence level, they are very similar, with the identity values ranging mostly from 80 to 95% in intergenic spacers, introns, and exons. These data indicate that mitochondrial genome evolution in hornworts is less conservative than in liverworts, but has not reached the dynamic level as seen in seed plants. PMID:19998039

  13. Complete Sequence Construction of the Highly Repetitive Ribosomal RNA Gene Repeats in Eukaryotes Using Whole Genome Sequence Data.

    PubMed

    Agrawal, Saumya; Ganley, Austen R D

    2016-01-01

    The ribosomal RNA genes (rDNA) encode the major rRNA species of the ribosome, and thus are essential across life. These genes are highly repetitive in most eukaryotes, forming blocks of tandem repeats that form the core of nucleoli. The primary role of the rDNA in encoding rRNA has been long understood, but more recently the rDNA has been implicated in a number of other important biological phenomena, including genome stability, cell cycle, and epigenetic silencing. Noncoding elements, primarily located in the intergenic spacer region, appear to mediate many of these phenomena. Although sequence information is available for the genomes of many organisms, in almost all cases rDNA repeat sequences are lacking, primarily due to problems in assembling these intriguing regions during whole genome assemblies. Here, we present a method to obtain complete rDNA repeat unit sequences from whole genome assemblies. Limitations of next generation sequencing (NGS) data make them unsuitable for assembling complete rDNA unit sequences; therefore, the method we present relies on the use of Sanger whole genome sequence data. Our method makes use of the Arachne assembler, which can assemble highly repetitive regions such as the rDNA in a memory-efficient way. We provide a detailed step-by-step protocol for generating rDNA sequences from whole genome Sanger sequence data using Arachne, for refining complete rDNA unit sequences, and for validating the sequences obtained. In principle, our method will work for any species where the rDNA is organized into tandem repeats. This will help researchers working on species without a complete rDNA sequence, those working on evolutionary aspects of the rDNA, and those interested in conducting phylogenetic footprinting studies with the rDNA. PMID:27576718

  14. Spatial overlap between environmental policy instruments and areas of high conservation value in forest.

    PubMed

    Sverdrup-Thygeson, Anne; Søgaard, Gunnhild; Rusch, Graciela M; Barton, David N

    2014-01-01

    In order to safeguard biodiversity in forest we need to know how forest policy instruments work. Here we use a nationwide network of 9400 plots in productive forest to analyze to what extent large-scale policy instruments, individually and together, target forest of high conservation value in Norway. We studied both instruments working through direct regulation; Strict Protection and Landscape Protection, and instruments working through management planning and voluntary schemes of forest certification; Wilderness Area and Mountain Forest. As forest of high conservation value (HCV-forest) we considered the extent of 12 Biodiversity Habitats and the extent of Old-Age Forest. We found that 22% of productive forest area contained Biodiversity Habitats. More than 70% of this area was not covered by any large-scale instruments. Mountain Forest covered 23%, while Strict Protection and Wilderness both covered 5% of the Biodiversity Habitat area. A total of 9% of productive forest area contained Old-Age Forest, and the relative coverage of the four instruments was similar as for Biodiversity Habitats. For all instruments, except Landscape Protection, the targeted areas contained significantly higher proportions of HCV-forest than areas not targeted by these instruments. Areas targeted by Strict Protection had higher proportions of HCV-forest than areas targeted by other instruments, except for areas targeted by Wilderness Area which showed similar proportions of Biodiversity Habitats. There was a substantial amount of spatial overlap between the policy tools, but no incremental conservation effect of overlapping instruments in terms of contributing to higher percentages of targeted HCV-forest. Our results reveal that although the current policy mix has an above average representation of forest of high conservation value, the targeting efficiency in terms of area overlap is limited. There is a need to improve forest conservation and a potential to cover this need by better

  15. Spatial Overlap between Environmental Policy Instruments and Areas of High Conservation Value in Forest

    PubMed Central

    Sverdrup-Thygeson, Anne; Søgaard, Gunnhild; Rusch, Graciela M.; Barton, David N.

    2014-01-01

    In order to safeguard biodiversity in forest we need to know how forest policy instruments work. Here we use a nationwide network of 9400 plots in productive forest to analyze to what extent large-scale policy instruments, individually and together, target forest of high conservation value in Norway. We studied both instruments working through direct regulation; Strict Protection and Landscape Protection, and instruments working through management planning and voluntary schemes of forest certification; Wilderness Area and Mountain Forest. As forest of high conservation value (HCV-forest) we considered the extent of 12 Biodiversity Habitats and the extent of Old-Age Forest. We found that 22% of productive forest area contained Biodiversity Habitats. More than 70% of this area was not covered by any large-scale instruments. Mountain Forest covered 23%, while Strict Protection and Wilderness both covered 5% of the Biodiversity Habitat area. A total of 9% of productive forest area contained Old-Age Forest, and the relative coverage of the four instruments was similar as for Biodiversity Habitats. For all instruments, except Landscape Protection, the targeted areas contained significantly higher proportions of HCV-forest than areas not targeted by these instruments. Areas targeted by Strict Protection had higher proportions of HCV-forest than areas targeted by other instruments, except for areas targeted by Wilderness Area which showed similar proportions of Biodiversity Habitats. There was a substantial amount of spatial overlap between the policy tools, but no incremental conservation effect of overlapping instruments in terms of contributing to higher percentages of targeted HCV-forest. Our results reveal that although the current policy mix has an above average representation of forest of high conservation value, the targeting efficiency in terms of area overlap is limited. There is a need to improve forest conservation and a potential to cover this need by better

  16. Enhancer of Rudimentary(p1), E(r)(p1), a Highly Conserved Enhancer of the Rudimentary Gene

    PubMed Central

    Wojcik, E.; Murphy, A. M.; Fares, H.; Dang-Vu, K.; Tsubota, S. I.

    1994-01-01

    A hybrid dysgenesis-induced mutation, enhancer of rudimentary(p1) (e(r)(p1)), is a recessive enhancer of a weak rudimentary mutant phenotype in Drosophila melanogaster. The e(r) gene was cloned using P element tagging and localized to region 8B on the X chromosome. It encodes a 1.0-kb and a 1.2-kb transcript. The 1.0-kb transcript is present in both adult males and females, while the 1.2-kb transcript is predominantly found in females. The difference in the lengths of the two e(r) transcripts is caused by two different polyadenylation sites spaced 228 bp apart. The amounts of both of these transcripts are drastically reduced in the e(r)(p1) mutant. The P element in e(r)(p1) is inserted in the 5'-untranslated leader region near the start of transcription. It may be producing its effect by suppressing transcription and/or by providing transcription termination and polyadenylation signals. The putative e(r) protein is 104 amino acids in length and bears no striking resemblance to protein sequences in GenBank or PIR. While its biochemical function is unknown at this time, sequence analysis indicates that the e(r) protein is highly conserved and, presumably, functionally very important. The amino acid sequences of the D. melanogaster and the Drosophila virilis proteins are 95% identical. PMID:7896098

  17. An Analysis of Stimuli that Influence Compliance during the High-Probability Instruction Sequence

    ERIC Educational Resources Information Center

    Normand, Matthew P.; Kestner, Kathryn; Jessel, Joshua

    2010-01-01

    When we evaluated variables that influence the effectiveness of the high-probability (high-p) instruction sequence, the sequence was associated with a precipitous decrease in compliance with high-"p" instructions for 1 participant, thereby precluding continued use of the sequence. We investigated the reasons for this decrease. Stimuli associated…

  18. Protein Sequence Annotation Tool (PSAT): A centralized web-based meta-server for high-throughput sequence annotations

    DOE PAGESBeta

    Leung, Elo; Huang, Amy; Cadag, Eithon; Montana, Aldrin; Soliman, Jan Lorenz; Zhou, Carol L. Ecale

    2016-01-20

    In this study, we introduce the Protein Sequence Annotation Tool (PSAT), a web-based, sequence annotation meta-server for performing integrated, high-throughput, genome-wide sequence analyses. Our goals in building PSAT were to (1) create an extensible platform for integration of multiple sequence-based bioinformatics tools, (2) enable functional annotations and enzyme predictions over large input protein fasta data sets, and (3) provide a web interface for convenient execution of the tools. In this paper, we demonstrate the utility of PSAT by annotating the predicted peptide gene products of Herbaspirillum sp. strain RV1423, importing the results of PSAT into EC2KEGG, and using the resultingmore » functional comparisons to identify a putative catabolic pathway, thereby distinguishing RV1423 from a well annotated Herbaspirillum species. This analysis demonstrates that high-throughput enzyme predictions, provided by PSAT processing, can be used to identify metabolic potential in an otherwise poorly annotated genome. Lastly, PSAT is a meta server that combines the results from several sequence-based annotation and function prediction codes, and is available at http://psat.llnl.gov/psat/. PSAT stands apart from other sequencebased genome annotation systems in providing a high-throughput platform for rapid de novo enzyme predictions and sequence annotations over large input protein sequence data sets in FASTA. PSAT is most appropriately applied in annotation of large protein FASTA sets that may or may not be associated with a single genome.« less

  19. Evolution of conserved non-coding sequences within the vertebrate Hox clusters through the two-round whole genome duplications revealed by phylogenetic footprinting analysis.

    PubMed

    Matsunami, Masatoshi; Sumiyama, Kenta; Saitou, Naruya

    2010-12-01

    As a result of two-round whole genome duplications, four or more paralogous Hox clusters exist in vertebrate genomes. The paralogous genes in the Hox clusters show similar expression patterns, implying shared regulatory mechanisms for expression of these genes. Previous studies partly revealed the expression mechanisms of Hox genes. However, cis-regulatory elements that control these paralogous gene expression are still poorly understood. Toward solving this problem, the authors searched conserved non-coding sequences (CNSs), which are candidates of cis-regulatory elements. When comparing orthologous Hox clusters of 19 vertebrate species, 208 intergenic conserved regions were found. The authors then searched for CNSs that were conserved not only between orthologous clusters but also among the four paralogous Hox clusters. The authors found three regions that are conserved among all the four clusters and eight regions that are conserved between intergenic regions of two paralogous Hox clusters. In total, 28 CNSs were identified in the paralogous Hox clusters, and nine of them were newly found in this study. One of these novel regions bears a RARE motif. These CNSs are candidates for gene expression regulatory regions among paralogous Hox clusters. The authors also compared vertebrate CNSs with amphioxus CNSs within the Hox cluster, and found that two CNSs in the HoxA and HoxB clusters retain homology with amphioxus CNSs through the two-round whole genome duplications. PMID:20981416

  20. Conservation Tillage to Effectively Reduce Interrill Erodibility of Highly-Weathered Ultisols

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Highly-weathered, Southeastern soils traditionally cropped under conventional tillage systems, are drought-prone and susceptible to consolidation, runoff, and soil loss. Conservation tillage systems reduce runoff and soil loss. We quantified differences in infiltration, runoff, soil loss, and interr...

  1. MANAGING NITROGEN FOR COTTON IN A HIGH-RESIDUE CONSERVATION TILLAGE SYSTEM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Over 70% of the cotton (Gossypium hirsutum L.) in the Tennessee Valley of northern Alabama is currently raised using conservation tillage techniques. High-residue small grain cover crops are becoming a common tool in these systems, but N immobilization may occur causing previous N recommendations t...

  2. Cotton nitrogen management in a high-residue conservation system: source, rate, method, and timing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Over 70% of the 94,226 ha of cotton (Gossypium hirsutum L.) in the Tennessee Valley of northern Alabama, USA, is produced using conservation tillage systems with cereal cover crops. Decreased N efficiency, as a result of N immobilization and/or ammonia volatilization in high-residue systems, require...

  3. NITROGEN MANAGEMENT FOR COTTON GROWN IN A HIGH-RESIDUE COVER CROP CONSERVATION TILLAGE SYSTEM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Over 70% of the cotton (Gossypium hirsutum L.) in the Tennessee Valley of northern Alabama is currently raised using conservation tillage techniques. High-residue small grain cover crops are becoming a common tool in these systems, but N immobilization may occur causing previous N recommendations t...

  4. Whole Genome Mapping with Feature Sets from High-Throughput Sequencing Data.

    PubMed

    Pan, Yonglong; Wang, Xiaoming; Liu, Lin; Wang, Hao; Luo, Meizhong

    2016-01-01

    A good physical map is essential to guide sequence assembly in de novo whole genome sequencing, especially when sequences are produced by high-throughput sequencing such as next-generation-sequencing (NGS) technology. We here present a novel method, Feature sets-based Genome Mapping (FGM). With FGM, physical map and draft whole genome sequences can be generated, anchored and integrated using the same data set of NGS sequences, independent of restriction digestion. Method model was created and parameters were inspected by simulations using the Arabidopsis genome sequence. In the simulations, when ~4.8X genome BAC library including 4,096 clones was used to sequence the whole genome, ~90% of clones were successfully connected to physical contigs, and 91.58% of genome sequences were mapped and connected to chromosomes. This method was experimentally verified using the existing physical map and genome sequence of rice. Of 4,064 clones covering 115 Mb sequence selected from ~3 tiles of 3 chromosomes of a rice draft physical map, 3,364 clones were reconstructed into physical contigs and 98 Mb sequences were integrated into the 3 chromosomes. The physical map-integrated draft genome sequences can provide permanent frameworks for eventually obtaining high-quality reference sequences by targeted sequencing, gap filling and combining other sequences. PMID:27611682

  5. Phylogenetic and Functional Analysis of Metagenome Sequence from High-Temperature Archaeal Habitats Demonstrate Linkages between Metabolic Potential and Geochemistry

    PubMed Central

    Inskeep, William P.; Jay, Zackary J.; Herrgard, Markus J.; Kozubal, Mark A.; Rusch, Douglas B.; Tringe, Susannah G.; Macur, Richard E.; Jennings, Ryan deM.; Boyd, Eric S.; Spear, John R.; Roberto, Francisco F.

    2013-01-01

    Geothermal habitats in Yellowstone National Park (YNP) provide an unparalleled opportunity to understand the environmental factors that control the distribution of archaea in thermal habitats. Here we describe, analyze, and synthesize metagenomic and geochemical data collected from seven high-temperature sites that contain microbial communities dominated by archaea relative to bacteria. The specific objectives of the study were to use metagenome sequencing to determine the structure and functional capacity of thermophilic archaeal-dominated microbial communities across a pH range from 2.5 to 6.4 and to discuss specific examples where the metabolic potential correlated with measured environmental parameters and geochemical processes occurring in situ. Random shotgun metagenome sequence (∼40–45 Mb Sanger sequencing per site) was obtained from environmental DNA extracted from high-temperature sediments and/or microbial mats and subjected to numerous phylogenetic and functional analyses. Analysis of individual sequences (e.g., MEGAN and G + C content) and assemblies from each habitat type revealed the presence of dominant archaeal populations in all environments, 10 of whose genomes were largely reconstructed from the sequence data. Analysis of protein family occurrence, particularly of those involved in energy conservation, electron transport, and autotrophic metabolism, revealed significant differences in metabolic strategies across sites consistent with differences in major geochemical attributes (e.g., sulfide, oxygen, pH). These observations provide an ecological basis for understanding the distribution of indigenous archaeal lineages across high-temperature systems of YNP. PMID:23720654

  6. Phylogenetic and Functional Analysis of Metagenome Sequence from High-Temperature Archaeal Habitats Demonstrate Linkages between Metabolic Potential and Geochemistry.

    PubMed

    Inskeep, William P; Jay, Zackary J; Herrgard, Markus J; Kozubal, Mark A; Rusch, Douglas B; Tringe, Susannah G; Macur, Richard E; Jennings, Ryan deM; Boyd, Eric S; Spear, John R; Roberto, Francisco F

    2013-01-01

    Geothermal habitats in Yellowstone National Park (YNP) provide an unparalleled opportunity to understand the environmental factors that control the distribution of archaea in thermal habitats. Here we describe, analyze, and synthesize metagenomic and geochemical data collected from seven high-temperature sites that contain microbial communities dominated by archaea relative to bacteria. The specific objectives of the study were to use metagenome sequencing to determine the structure and functional capacity of thermophilic archaeal-dominated microbial communities across a pH range from 2.5 to 6.4 and to discuss specific examples where the metabolic potential correlated with measured environmental parameters and geochemical processes occurring in situ. Random shotgun metagenome sequence (∼40-45 Mb Sanger sequencing per site) was obtained from environmental DNA extracted from high-temperature sediments and/or microbial mats and subjected to numerous phylogenetic and functional analyses. Analysis of individual sequences (e.g., MEGAN and G + C content) and assemblies from each habitat type revealed the presence of dominant archaeal populations in all environments, 10 of whose genomes were largely reconstructed from the sequence data. Analysis of protein family occurrence, particularly of those involved in energy conservation, electron transport, and autotrophic metabolism, revealed significant differences in metabolic strategies across sites consistent with differences in major geochemical attributes (e.g., sulfide, oxygen, pH). These observations provide an ecological basis for understanding the distribution of indigenous archaeal lineages across high-temperature systems of YNP. PMID:23720654

  7. DUC-Curve, a highly compact 2D graphical representation of DNA sequences and its application in sequence alignment

    NASA Astrophysics Data System (ADS)

    Li, Yushuang; Liu, Qian; Zheng, Xiaoqi

    2016-08-01

    A highly compact and simple 2D graphical representation of DNA sequences, named DUC-Curve, is constructed through mapping four nucleotides to a unit circle with a cyclic order. DUC-Curve could directly detect nucleotide, di-nucleotide compositions and microsatellite structure from DNA sequences. Moreover, it also could be used for DNA sequence alignment. Taking geometric center vectors of DUC-Curves as sequence descriptor, we perform similarity analysis on the first exons of β-globin genes of 11 species, oncogene TP53 of 27 species and twenty-four Influenza A viruses, respectively. The obtained reasonable results illustrate that the proposed method is very effective in sequence comparison problems, and will at least play a complementary role in classification and clustering problems.

  8. Creation of a data base for sequences of ribosomal nucleic acids and detection of conserved restriction endonucleases sites through computerized processing.

    PubMed Central

    Patarca, R; Dorta, B; Ramirez, J L

    1982-01-01

    As part of a project pertaining the organization of ribosomal genes in Kinetoplastidae, we have created a data base for published sequences of ribosomal nucleic acids, with information in Spanish. As a first step in their processing, we have written a computer program which introduces the new feature of determining the length of the fragments produced after single or multiple digestion with any of the known restriction enzymes. With this information we have detected conserved SAU 3A sites: (i) at the 5' end of the 5.8S rRNA and at the 3' end of the small subunit rRNA, both included in similar larger sequences; (ii) in the 5.8S rRNA of vertebrates (a second one), which is not present in lower eukaryotes, showing a clear evolutive divergence; and, (iii) at the 5' terminal of the small subunit rRNA, included in a larger conserved sequence. The possible biological importance of these sequences is discussed. PMID:6278402

  9. Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region

    PubMed Central

    Mei, Qin; Turner, Ross E.; Sorial, Vivian; Klivington, Diane; Angus, C. William; Kovacs, Joseph A.

    1998-01-01

    To facilitate studies of Pneumocystis carinii infection in humans, we undertook to better characterize and to express the major surface glycoprotein (MSG) of human P. carinii, an important protein in host-pathogen interactions. Seven MSG genes were cloned from a single isolate by PCR or genomic library screening and were sequenced. The predicted proteins, like rat MSGs, were closely related but unique variants, with a high level of conservation among cysteine residues. A conserved immunodominant region (of approximately 100 amino acids) near the carboxy terminus was expressed at high levels in Escherichia coli and used in Western blot studies. All 49 of the serum samples, which were taken from healthy controls as well as from patients with and without P. carinii pneumonia, were reactive with this peptide by Western blotting, supporting the hypothesis that most adult humans have been infected with P. carinii at some point. This recombinant MSG fragment, which is the first human P. carinii antigen available in large quantities, may be a useful reagent for investigating the epidemiology of P. carinii infection in humans. PMID:9712777

  10. Use of robotics in high-throughput DNA sequencing.

    PubMed

    Keeney, Stephen

    2011-01-01

    Until relatively recently, full sequencing of genes consisting of more than several exons was not considered practicable within a routine diagnostic context. As a result, many approaches to unknown mutation detection in a specific gene involved a mutation pre-screening step to limit the amount of DNA sequencing required. Protocols to pre-screen for mutations and limit the amount of DNA sequencing may not localise every base change present and/or require considerable levels of manual intervention. Advances in technology, allied with careful protocol design, now permit direct DNA sequencing to be applied to larger areas of gene sequence, allowing unequivocal mutation identification in the area of a gene being analysed. The protocol described below utilises robotic systems, allied to custom-designed PCR primers, to facilitate rapid DNA sequencing of multiple gene targets. The general approach is amenable to adaptation for use with multi-channel pipettes. PMID:20938842

  11. Modeling and optimization of defense high level waste removal sequencing

    NASA Astrophysics Data System (ADS)

    Paul, Pran Krishna

    has been successfully implemented with this general scheme to sequence wastes from different waste tanks for precipitate production and provide optimized sequences to ProdMod for simulating the behavior of the SRS waste complex. Parametric studies using this optimization methodology demonstrate that the devised scheme is appropriate for the real life operations of the SRS waste complex. The computational planning tool based on the coupled simulation and optimization methodology developed in this work is in current use to help planners process the SRS's 34 million gallons of high level radioactive waste efficiently and economically all the way to clean up of all the tanks. This methodology can also be directly applicable to the Hanford Site and aid in the final design and operation of its facilities to process 55 million gallons of high level radioactive waste.

  12. Experimental design-based functional mining and characterization of high-throughput sequencing data in the sequence read archive.

    PubMed

    Nakazato, Takeru; Ohta, Tazro; Bono, Hidemasa

    2013-01-01

    High-throughput sequencing technology, also called next-generation sequencing (NGS), has the potential to revolutionize the whole process of genome sequencing, transcriptomics, and epigenetics. Sequencing data is captured in a public primary data archive, the Sequence Read Archive (SRA). As of January 2013, data from more than 14,000 projects have been submitted to SRA, which is double that of the previous year. Researchers can download raw sequence data from SRA website to perform further analyses and to compare with their own data. However, it is extremely difficult to search entries and download raw sequences of interests with SRA because the data structure is complicated, and experimental conditions along with raw sequences are partly described in natural language. Additionally, some sequences are of inconsistent quality because anyone can submit sequencing data to SRA with no quality check. Therefore, as a criterion of data quality, we focused on SRA entries that were cited in journal articles. We extracted SRA IDs and PubMed IDs (PMIDs) from SRA and full-text versions of journal articles and retrieved 2748 SRA ID-PMID pairs. We constructed a publication list referring to SRA entries. Since, one of the main themes of -omics analyses is clarification of disease mechanisms, we also characterized SRA entries by disease keywords, according to the Medical Subject Headings (MeSH) extracted from articles assigned to each SRA entry. We obtained 989 SRA ID-MeSH disease term pairs, and constructed a disease list referring to SRA data. We previously developed feature profiles of diseases in a system called "Gendoo". We generated hyperlinks between diseases extracted from SRA and the feature profiles of it. The developed project, publication and disease lists resulting from this study are available at our web service, called "DBCLS SRA" (http://sra.dbcls.jp/). This service will improve accessibility to high-quality data from SRA. PMID:24167589

  13. Unprecedented High-Resolution View of Bacterial Operon Architecture Revealed by RNA Sequencing

    PubMed Central

    Creecy, James P.; Maddox, Scott M.; Grissom, Joe E.; Conkle, Trevor L.; Shadid, Tyler M.; Teramoto, Jun; San Miguel, Phillip; Shimada, Tomohiro; Ishihama, Akira; Mori, Hirotada

    2014-01-01

    ABSTRACT We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3′ transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5′ ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. PMID:25006232

  14. Data in support of the discovery of alternative splicing variants of quail LEPR and the evolutionary conservation of qLEPRl by nucleotide and amino acid sequences alignment.

    PubMed

    Wang, Dandan; Xu, Chunlin; Wang, Taian; Li, Hong; Li, Yanmin; Ren, Junxiao; Tian, Yadong; Li, Zhuanjian; Jiao, Yuping; Kang, Xiangtao; Liu, Xiaojun

    2016-03-01

    Leptin receptor (LEPR) belongs to the class I cytokine receptor superfamily which share common structural features and signal transduction pathways. Although multiple LEPR isoforms, which are derived from one gene, were identified in mammals, they were rarely found in avian except the long LEPR. Four alternative splicing variants of quail LEPR (qLEPR) had been cloned and sequenced for the first time (Wang et al., 2015 [1]). To define patterns of the four splicing variants (qLEPRl, qLEPR-a, qLEPR-b and qLEPR-c) and locate the conserved regions of qLEPRl, this data article provides nucleotide sequence alignment of qLEPR and amino acid sequence alignment of representative vertebrate LEPR. The detailed analysis was shown in [1]. PMID:26759819

  15. The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin-arginine translocation.

    PubMed

    Ramasamy, Sureshkumar; Abrol, Ravinder; Suloway, Christian J M; Clemons, William M

    2013-05-01

    In bacteria, two signal-sequence-dependent secretion pathways translocate proteins across the cytoplasmic membrane. Although the mechanism of the ubiquitous general secretory pathway is becoming well understood, that of the twin-arginine translocation pathway, responsible for translocation of folded proteins across the bilayer, is more mysterious. TatC, the largest and most conserved of three integral membrane components, provides the initial binding site of the signal sequence prior to pore assembly. Here, we present two crystal structures of TatC from the thermophilic bacteria Aquifex aeolicus at 4.0 Å and 6.8 Å resolution. The membrane architecture of TatC includes a glove-shaped structure with a lipid-exposed pocket predicted by molecular dynamics to distort the membrane. Correlating the biochemical literature to these results suggests that the signal sequence binds in this pocket, leading to structural changes that facilitate higher order assemblies. PMID:23583035

  16. Data in support of the discovery of alternative splicing variants of quail LEPR and the evolutionary conservation of qLEPRl by nucleotide and amino acid sequences alignment

    PubMed Central

    Wang, Dandan; Xu, Chunlin; Wang, Taian; Li, Hong; Li, Yanmin; Ren, Junxiao; Tian, Yadong; Li, Zhuanjian; Jiao, Yuping; Kang, Xiangtao; Liu, Xiaojun

    2015-01-01

    Leptin receptor (LEPR) belongs to the class I cytokine receptor superfamily which share common structural features and signal transduction pathways. Although multiple LEPR isoforms, which are derived from one gene, were identified in mammals, they were rarely found in avian except the long LEPR. Four alternative splicing variants of quail LEPR (qLEPR) had been cloned and sequenced for the first time (Wang et al., 2015 [1]). To define patterns of the four splicing variants (qLEPRl, qLEPR-a, qLEPR-b and qLEPR-c) and locate the conserved regions of qLEPRl, this data article provides nucleotide sequence alignment of qLEPR and amino acid sequence alignment of representative vertebrate LEPR. The detailed analysis was shown in [1]. PMID:26759819

  17. The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin-arginine translocation

    PubMed Central

    Ramasamy, Sureshkumar; Abrol, Ravinder; Suloway, Christian J.M.; Clemons, William M.

    2013-01-01

    SUMMARY In bacteria, two signal sequence dependent secretion pathways translocate proteins across the cytoplasmic membrane. While the mechanism of the ubiquitous general secretory pathway (SEC) is becoming well understood, that of the twin-arginine translocation pathway (TAT), responsible for translocation of folded proteins across the bilayer, is more mysterious. TatC, the largest and most conserved of three integral membrane components, provides the initial binding site of the signal sequence prior to pore assembly. Here, we present two crystal structures of TatC from the thermophilic bacteria Aquifex aeolicus at 4.0Å and 6.8Å resolution. The novel membrane architecture of TatC includes a glove-shaped structure with a lipid-exposed pocket predicted by molecular dynamics to distort the membrane. Correlating the biochemical literature to these results suggests that the signal sequence binds in this pocket leading to structural changes that facilitate higher order assemblies. PMID:23583035

  18. BOOGIE: Predicting Blood Groups from High Throughput Sequencing Data

    PubMed Central

    Giollo, Manuel; Minervini, Giovanni; Scalzotto, Marta; Leonardi, Emanuela; Ferrari, Carlo; Tosatto, Silvio C. E.

    2015-01-01

    Over the last decade, we have witnessed an incredible growth in the amount of available genotype data due to high throughput sequencing (HTS) techniques. This information may be used to predict phenotypes of medical relevance, and pave the way towards personalized medicine. Blood phenotypes (e.g. ABO and Rh) are a purely genetic trait that has been extensively studied for decades, with currently over thirty known blood groups. Given the public availability of blood group data, it is of interest to predict these phenotypes from HTS data which may translate into more accurate blood typing in clinical practice. Here we propose BOOGIE, a fast predictor for the inference of blood groups from single nucleotide variant (SNV) databases. We focus on the prediction of thirty blood groups ranging from the well known ABO and Rh, to the less studied Junior or Diego. BOOGIE correctly predicted the blood group with 94% accuracy for the Personal Genome Project whole genome profiles where good quality SNV annotation was available. Additionally, our tool produces a high quality haplotype phase, which is of interest in the context of ethnicity-specific polymorphisms or traits. The versatility and simplicity of the analysis make it easily interpretable and allow easy extension of the protocol towards other phenotypes. BOOGIE can be downloaded from URL http://protein.bio.unipd.it/download/. PMID:25893845

  19. A high-plex PCR approach for massively parallel sequencing.

    PubMed

    Nguyen-Dumont, Tú; Pope, Bernard J; Hammet, Fleur; Southey, Melissa C; Park, Daniel J

    2013-08-01

    Current methods for targeted massively parallel sequencing (MPS) have several drawbacks, including limited design flexibility, expense, and protocol complexity, which restrict their application to settings involving modest target size and requiring low cost and high throughput. To address this, we have developed Hi-Plex, a PCR-MPS strategy intended for high-throughput screening of multiple genomic target regions that integrates simple, automated primer design software to control product size. Featuring permissive thermocycling conditions and clamp bias reduction, our protocol is simple, cost- and time-effective, uses readily available reagents, does not require expensive instrumentation, and requires minimal optimization. In a 60-plex assay targeting the breast cancer predisposition genes PALB2 and XRCC2, we applied Hi-Plex to 100 ng LCL-derived DNA, and 100 ng and 25 ng FFPE tumor-derived DNA. Altogether, at least 86.94% of the human genome-mapped reads were on target, and 100% of targeted amplicons were represented within 25-fold of the mean. Using 25 ng FFPE-derived DNA, 95.14% of mapped reads were on-target and relative representation ranged from 10.1-fold lower to 5.8-fold higher than the mean. These results were obtained using only the initial automatically-designed primers present in equal concentration. Hi-Plex represents a powerful new approach for screening panels of genomic target regions. PMID:23931594

  20. Balancing forest-regeneration probabilities and maintenance costs in dry grasslands of high conservation priority

    USGS Publications Warehouse

    Bolliger, Janine; Edwards, Thomas C., Jr.; Eggenberg, Stefan; Ismail, Sascha; Seidl, Irmi; Kienast, Felix

    2011-01-01

    Abandonment of agricultural land has resulted in forest regeneration in species-rich dry grasslands across European mountain regions and threatens conservation efforts in this vegetation type. To support national conservation strategies, we used a site-selection algorithm (MARXAN) to find optimum sets of floristic regions (reporting units) that contain grasslands of high conservation priority. We sought optimum sets that would accommodate 136 important dry-grassland species and that would minimize forest regeneration and costs of management needed to forestall predicted forest regeneration. We did not consider other conservation elements of dry grasslands, such as animal species richness, cultural heritage, and changes due to climate change. Optimal sets that included 95–100% of the dry grassland species encompassed an average of 56–59 floristic regions (standard deviation, SD 5). This is about 15% of approximately 400 floristic regions that contain dry-grassland sites and translates to 4800–5300 ha of dry grassland out of a total of approximately 23,000 ha for the entire study area. Projected costs to manage the grasslands in these optimum sets ranged from CHF (Swiss francs) 5.2 to 6.0 million/year. This is only 15–20% of the current total estimated cost of approximately CHF30–45 million/year required if all dry grasslands were to be protected. The grasslands of the optimal sets may be viewed as core sites in a national conservation strategy.

  1. Comparative genomic analysis of a neurotoxigenic Clostridium species using partial genome sequence: Phylogenetic analysis of a few conserved proteins involved in cellular processes and metabolism.

    PubMed

    Alam, Syed Imteyaz; Dixit, Aparna; Tomar, Arvind; Singh, Lokendra

    2010-04-01

    Clostridial organisms produce neurotoxins, which are generally regarded as the most potent toxic substances of biological origin and potential biological warfare agents. Clostridium tetani produces tetanus neurotoxin and is responsible for the fatal tetanus disease. In spite of the extensive immunization regimen, the disease is an important cause of death especially among neonates. Strains of C. tetani have not been genetically characterized except the complete genome sequencing of strain E88. The present study reports the genetic makeup and phylogenetic affiliations of an environmental strain of this bacterium with respect to C. tetani E88 and other clostridia. A shot gun library was constructed from the genomic DNA of C. tetani drde, isolated from decaying fish sample. Unique clones were sequenced and sequences compared with its closest relative C. tetani E88. A total of 275 clones were obtained and 32,457 bases of non-redundant sequence were generated. A total of 150 base changes were observed over the entire length of sequence obtained, including, additions, deletions and base substitutions. Of the total 120 ORFs detected, 48 exhibited closest similarity to E88 proteins of which three are hypothetical proteins. Eight of the ORFs exhibited similarity with hypothetical proteins from other organisms and 10 aligned with other proteins from unrelated organisms. There is an overall conservation of protein sequences among the two strains of C. tetani and. Selected ORFs involved in cellular processes and metabolism were subjected to phylogenetic analysis. PMID:19527791

  2. Highly Conserved Surface Proteins of Oral Spirochetes as Adhesins and Potent Inducers of Proinflammatory and Osteoclastogenic Factors▿

    PubMed Central

    Jun, Hye-Kyoung; Kang, Young-Mi; Lee, Hae-Ri; Lee, Sung-Hoon; Choi, Bong-Kyu

    2008-01-01

    Oral spirochetes include enormously heterogeneous Treponema species, and some have been implicated in the etiology of periodontitis. In this study, we characterized highly conserved surface proteins in four representative oral spirochetes (Treponema denticola, T. lecithinolyticum, T. maltophilum, and T. socranskii subsp. socranskii) that are homologs of T. pallidum Tp92, with opsonophagocytic potential and protective capacity against syphilis. Tp92 homologs of oral spirochetes had predicted signal peptides (20 to 31 amino acids) and molecular masses of 88 to 92 kDa for mature proteins. They showed amino acid sequence identities of 37.9 to 49.3% and similarities of 54.5 to 66.9% to Tp92. The sequence identities and similarities of Tp92 homologs of oral treponemes to one another were 41.6 to 71.6% and 59.9 to 85.6%, respectively. The tp92 gene homologs were successfully expressed in Escherichia coli, and the recombinant proteins were capable of binding to KB cells, an epithelial cell line, and inhibited the binding of the whole bacteria to the cells. Antiserum (the immunoglobulin G fraction) raised against a recombinant form of the T. denticola Tp92 homolog cross-reacted with homologs from three other species of treponemes. The Tp92 homologs stimulated various factors involved in inflammation and osteoclastogenesis, like interleukin-1β (IL-1β), tumor necrosis factor alpha, IL-6, prostaglandin E2, and matrix metalloproteinase 9, in host cells like monocytes and fibroblasts. Our results demonstrate that Tp92 homologs of oral spirochetes are highly conserved and may play an important role in cell attachment, inflammation, and tissue destruction. The coexistence of various Treponema species in a single periodontal pocket and, therefore, the accumulation of multiple Tp92 homologs may amplify the pathological effect in periodontitis. PMID:18390996

  3. A high-density linkage map for Astyanax mexicanus using genotyping-by-sequencing technology.

    PubMed

    Carlson, Brian M; Onusko, Samuel W; Gross, Joshua B

    2015-02-01

    The Mexican tetra, Astyanax mexicanus, is a unique model system consisting of cave-adapted and surface-dwelling morphotypes that diverged >1 million years (My) ago. This remarkable natural experiment has enabled powerful genetic analyses of cave adaptation. Here, we describe the application of next-generation sequencing technology to the creation of a high-density linkage map. Our map comprises more than 2200 markers populating 25 linkage groups constructed from genotypic data generated from a single genotyping-by-sequencing project. We leveraged emergent genomic and transcriptomic resources to anchor hundreds of anonymous Astyanax markers to the genome of the zebrafish (Danio rerio), the most closely related model organism to our study species. This facilitated the identification of 784 distinct connections between our linkage map and the Danio rerio genome, highlighting several regions of conserved genomic architecture between the two species despite ~150 My of divergence. Using a Mendelian cave-associated trait as a proof-of-principle, we successfully recovered the genomic position of the albinism locus near the gene Oca2. Further, our map successfully informed the positions of unplaced Astyanax genomic scaffolds within particular linkage groups. This ability to identify the relative location, orientation, and linear order of unaligned genomic scaffolds will facilitate ongoing efforts to improve on the current early draft and assemble future versions of the Astyanax physical genome. Moreover, this improved linkage map will enable higher-resolution genetic analyses and catalyze the discovery of the genetic basis for cave-associated phenotypes. PMID:25520037

  4. Two Different High Throughput Sequencing Approaches Identify Thousands of De Novo Genomic Markers for the Genetically Depleted Bornean Elephant

    PubMed Central

    Sharma, Reeta; Goossens, Benoit; Kun-Rodrigues, Célia; Teixeira, Tatiana; Othman, Nurzhafarina; Boone, Jason Q.; Jue, Nathaniel K.; Obergfell, Craig; O'Neill, Rachel J.; Chikhi, Lounès

    2012-01-01

    High throughput sequencing technologies are being applied to an increasing number of model species with a high-quality reference genome. The application and analyses of whole-genome sequence data in non-model species with no prior genomic information are currently under way. Recent sequencing technologies provide new opportunities for gathering genomic data in natural populations, laying the empirical foundation for future research in the field of conservation and population genomics. Here we present the case study of the Bornean elephant, which is the most endangered subspecies of Asian elephant and exhibits very low genetic diversity. We used two different sequencing platforms, the Roche 454 FLX (shotgun) and Illumina, GAIIx (Restriction site associated DNA, RAD) to evaluate the feasibility of the two methodologies for the discovery of de novo markers (single nucleotide polymorphism, SNPs and microsatellites) using low coverage data. Approximately, 6,683 (shotgun) and 14,724 (RAD) SNPs were detected within our elephant sequence dataset. Genotyping of a representative sample of 194 SNPs resulted in a SNP validation rate of ∼ 83 to 94% and 17% of the loci were polymorphic with a low diversity (Ho = 0.057). Different numbers of microsatellites were identified through shotgun (27,226) and RAD (868) techniques. Out of all di-, tri-, and tetra-microsatellite loci, 1,706 loci had sufficient flanking regions (shotgun) while only 7 were found with RAD. All microsatellites were monomorphic in the Bornean but polymorphic in another elephant subspecies. Despite using different sample sizes, and the well known differences in the two platforms used regarding sequence length and throughput, the two approaches showed high validation rate. The approaches used here for marker development in a threatened species demonstrate the utility of high throughput sequencing technologies as a starting point for the development of genomic tools in a non-model species and in particular

  5. Evaluation of a Pooled Strategy for High-Throughput Sequencing of Cosmid Clones from Metagenomic Libraries

    PubMed Central

    Lam, Kathy N.; Hall, Michael W.; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D.; Charles, Trevor C.

    2014-01-01

    High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones. PMID:24911009

  6. Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

    PubMed

    Lam, Kathy N; Hall, Michael W; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D; Charles, Trevor C

    2014-01-01

    High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones. PMID:24911009

  7. Nonoverlapping clone pooling for high-throughput sequencing.

    PubMed

    Kuroshu, Reginaldo M

    2013-01-01

    Simultaneously sequencing multiple clones using second-generation sequencers can speed up many essential clone-based sequencing methods. However, in applications such as fosmid clone sequencing and full-length cDNA sequencing, it is important to create pools of clones that do not overlap on the genome for the identification of structural variations and alternatively spliced transcripts, respectively. We define the nonoverlapping clone pooling problem and provide practical solutions based on optimal graph coloring and bin-packing algorithms with constant absolute worst-case ratios, and further extend them to cope with repetitive mappings. Using theoretical analysis and experiments, we also show that the proposed methods are applicable. PMID:24384700

  8. Characterization of Protective Epitopes in a Highly Conserved Plasmodium falciparum Antigenic Protein Containing Repeats of Acidic and Basic Residues

    PubMed Central

    Sharma, Pawan; Kumar, Anil; Singh, Balwan; Bharadwaj, Ashima; Sailaja, V. Naga; Adak, T.; Kushwaha, Ashima; Malhotra, Pawan; Chauhan, V. S.

    1998-01-01

    The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine. By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasite Plasmodium falciparum represents such an antigen. Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences. We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion of P. falciparum malaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria. When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs. These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay. In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria. More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to ∼90%. These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages of P. falciparum. PMID:9596765

  9. The complete sequence of the human CD79b (Ig{beta}/B29) gene: Identification of a conserved exon/intron organization, immunoglobulin-like regulatory regions, and allelic polymorphism

    SciTech Connect

    Hashimoto, S.; Chiorazzi, N.; Gregersen, P.K. |

    1994-12-31

    We determined the complete genomic sequence of the human CD79b (Ig{beta}/B29) gene. The CD79b gene product is associated with the membrane immunoglobulin signaling complex which is composed of immunoglobulin (Ig) itself, associated in a noncovalent fashion with CD79b and a second polypeptide chain, CD79a (Ig{alpha}/mb1). The sequence and exon/intron organization of the human and mouse CD79b genes are highly similar. The gene organization suggests that some variant forms of CD79b may arise by virtue of alternative splicing of mRNA. In addition, a number of conserved regulatory sequences commonly found in Ig genes are present in sequences which flank the human CD79b gene. Some of these sequences are distinct from those found in the CD79a promoter. These differences may explain why transcription of CD79b, but not CD79a, is observed in plasma cells. A new Taq 1 restriction fragment length polymorphism is described that is not associated with any structural polymorphisms of the expressed CD79b polypeptide. 13 refs., 3 figs., 1 tab.

  10. A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing

    PubMed Central

    Green, Richard E.; Malaspinas, Anna-Sapfo; Krause, Johannes; Briggs, Adrian W.; Johnson, Philip L. F.; Uhler, Caroline; Meyer, Matthias; Good, Jeffrey M.; Maricic, Tomislav; Stenzel, Udo; Prüfer, Kay; Siebauer, Michael; Burbano, Hernán A.; Ronan, Michael; Rothberg, Jonathan M.; Egholm, Michael; Rudan, Pavao; Brajković, Dejana; Kućan, Željko; Gušić, Ivan; Wikström, Mårten; Laakkonen, Liisa; Kelso, Janet; Slatkin, Montgomery; Pääbo, Svante

    2008-01-01

    Summary A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000-year-old Neandertal individual using 8,341 mtDNA sequences identified among 4.8 Gb of DNA generated from ~0.3 grams of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs and allows an estimate of the divergence date between the two mtDNA lineages of 660,000±140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared to other primate lineages suggesting that the effective population size of Neandertals was small. PMID:18692465

  11. Conservative surgery for low rectal carcinoma after high-dose radiation. Functional and oncologic results.

    PubMed Central

    Rouanet, P; Fabre, J M; Dubois, J B; Dravet, F; Saint Aubert, B; Pradel, J; Ychou, M; Solassol, C; Pujol, H

    1995-01-01

    OBJECTIVE: Using a prospective, nonrandomized study, the authors evaluated the morbidity and functional and oncologic results of conservative surgery for cancer of the lower third of the rectum after high-dose radiation. SUMMARY BACKGROUND DATA: Colo-anal anastomosis has made sphincter conservation for low rectal carcinoma technically feasible. The limits to conservative surgery currently are oncologic rather than technical. Adjuvant radiotherapy has proven its benefit in terms of regional control, with a dose relationship. METHODS: Since June 1990, 27 patients with distal rectal adenocarcinoma were treated by preoperative radiotherapy (40 + 20 Gy delivered with three fields) and curative surgery. The mean distance from the anal verge was 47 mm (27-57 mm), and none of the tumors were fixed (15 T2, 12 T3). RESULTS: Mortality and morbidity were not increased by high-dose preoperative radiation. Twenty-one patients underwent conservative surgery (78%-17 total proctectomies and colo-anal anastomoses, 4 trans-anal resections). After colo-anal anastomosis, all patients with colonic pouch had good results; two patients had moderate results and one patient had poor results after straight colo-anal anastomosis. With a mean follow-up of 24 months, the authors noted 1 postoperative death, 2 disease-linked deaths, 1 controlled regional recurrence, 2 evolutive patients with pulmonary metastases, and 21 disease-free patients. CONCLUSIONS: These first results confirm the possibility of conservative surgery for low rectal carcinoma after high-dose radiation. A prospective, randomized trial could be induced to determine the real role of the 20 Gy boost on the sphincter-saving decision. PMID:7826163

  12. Highly conserved elements discovered in vertebrates are present in non-syntenic loci of tunicates, act as enhancers and can be transcribed during development

    PubMed Central

    Sanges, Remo; Hadzhiev, Yavor; Gueroult-Bellone, Marion; Roure, Agnes; Ferg, Marco; Meola, Nicola; Amore, Gabriele; Basu, Swaraj; Brown, Euan R.; De Simone, Marco; Petrera, Francesca; Licastro, Danilo; Strähle, Uwe; Banfi, Sandro; Lemaire, Patrick; Birney, Ewan; Müller, Ferenc; Stupka, Elia

    2013-01-01

    Co-option of cis-regulatory modules has been suggested as a mechanism for the evolution of expression sites during development. However, the extent and mechanisms involved in mobilization of cis-regulatory modules remains elusive. To trace the history of non-coding elements, which may represent candidate ancestral cis-regulatory modules affirmed during chordate evolution, we have searched for conserved elements in tunicate and vertebrate (Olfactores) genomes. We identified, for the first time, 183 non-coding sequences that are highly conserved between the two groups. Our results show that all but one element are conserved in non-syntenic regions between vertebrate and tunicate genomes, while being syntenic among vertebrates. Nevertheless, in all the groups, they are significantly associated with transcription factors showing specific functions fundamental to animal development, such as multicellular organism development and sequence-specific DNA binding. The majority of these regions map onto ultraconserved elements and we demonstrate that they can act as functional enhancers within the organism of origin, as well as in cross-transgenesis experiments, and that they are transcribed in extant species of Olfactores. We refer to the elements as ‘Olfactores conserved non-coding elements’. PMID:23393190

  13. Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation

    PubMed Central

    Kondo, Hidemasa; Hanada, Yuichi; Sugimoto, Hiroshi; Hoshino, Tamotsu; Garnham, Christopher P.; Davies, Peter L.; Tsuda, Sakae

    2012-01-01

    Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi. PMID:22645341

  14. Comparative analysis of the full genome sequence of European bat lyssavirus type 1 and type 2 with other lyssaviruses and evidence for a conserved transcription termination and polyadenylation motif in the G-L 3' non-translated region.

    PubMed

    Marston, D A; McElhinney, L M; Johnson, N; Müller, T; Conzelmann, K K; Tordo, N; Fooks, A R

    2007-04-01

    We report the first full-length genomic sequences for European bat lyssavirus type-1 (EBLV-1) and type-2 (EBLV-2). The EBLV-1 genomic sequence was derived from a virus isolated from a serotine bat in Hamburg, Germany, in 1968 and the EBLV-2 sequence was derived from a virus isolate from a human case of rabies that occurred in Scotland in 2002. A long-distance PCR strategy was used to amplify the open reading frames (ORFs), followed by standard and modified RACE (rapid amplification of cDNA ends) techniques to amplify the 3' and 5' ends. The lengths of each complete viral genome for EBLV-1 and EBLV-2 were 11 966 and 11 930 base pairs, respectively, and follow the standard rhabdovirus genome organization of five viral proteins. Comparison with other lyssavirus sequences demonstrates variation in degrees of homology, with the genomic termini showing a high degree of complementarity. The nucleoprotein was the most conserved, both intra- and intergenotypically, followed by the polymerase (L), matrix and glyco- proteins, with the phosphoprotein being the most variable. In addition, we have shown that the two EBLVs utilize a conserved transcription termination and polyadenylation (TTP) motif, approximately 50 nt upstream of the L gene start codon. All available lyssavirus sequences to date, with the exception of Pasteur virus (PV) and PV-derived isolates, use the second TTP site. This observation may explain differences in pathogenicity between lyssavirus strains, dependent on the length of the untranslated region, which might affect transcriptional activity and RNA stability. PMID:17374776

  15. HybPiper: Extracting coding sequence and introns for phylogenetics from high-throughput sequencing reads using target enrichment1

    PubMed Central

    Johnson, Matthew G.; Gardner, Elliot M.; Liu, Yang; Medina, Rafael; Goffinet, Bernard; Shaw, A. Jonathan; Zerega, Nyree J. C.; Wickett, Norman J.

    2016-01-01

    Premise of the study: Using sequence data generated via target enrichment for phylogenetics requires reassembly of high-throughput sequence reads into loci, presenting a number of bioinformatics challenges. We developed HybPiper as a user-friendly platform for assembly of gene regions, extraction of exon and intron sequences, and identification of paralogous gene copies. We test HybPiper using baits designed to target 333 phylogenetic markers and 125 genes of functional significance in Artocarpus (Moraceae). Methods and Results: HybPiper implements parallel execution of sequence assembly in three phases: read mapping, contig assembly, and target sequence extraction. The pipeline was able to recover nearly complete gene sequences for all genes in 22 species of Artocarpus. HybPiper also recovered more than 500 bp of nontargeted intron sequence in over half of the phylogenetic markers and identified paralogous gene copies in Artocarpus. Conclusions: HybPiper was designed for Linux and Mac OS X and is freely available at https://github.com/mossmatters/HybPiper. PMID:27437175

  16. Construction of a high-density genetic map for grape using next generation restriction-site associated DNA sequencing

    PubMed Central

    2012-01-01

    Background Genetic mapping and QTL detection are powerful methodologies in plant improvement and breeding. Construction of a high-density and high-quality genetic map would be of great benefit in the production of superior grapes to meet human demand. High throughput and low cost of the recently developed next generation sequencing (NGS) technology have resulted in its wide application in genome research. Sequencing restriction-site associated DNA (RAD) might be an efficient strategy to simplify genotyping. Combining NGS with RAD has proven to be powerful for single nucleotide polymorphism (SNP) marker development. Results An F1 population of 100 individual plants was developed. In-silico digestion-site prediction was used to select an appropriate restriction enzyme for construction of a RAD sequencing library. Next generation RAD sequencing was applied to genotype the F1 population and its parents. Applying a cluster strategy for SNP modulation, a total of 1,814 high-quality SNP markers were developed: 1,121 of these were mapped to the female genetic map, 759 to the male map, and 1,646 to the integrated map. A comparison of the genetic maps to the published Vitis vinifera genome revealed both conservation and variations. Conclusions The applicability of next generation RAD sequencing for genotyping a grape F1 population was demonstrated, leading to the successful development of a genetic map with high density and quality using our designed SNP markers. Detailed analysis revealed that this newly developed genetic map can be used for a variety of genome investigations, such as QTL detection, sequence assembly and genome comparison. PMID:22908993

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Barley has one of the largest and most complex genomes of all economically important food crops. The rise of new short read sequencing technologies such as Illumina/Solexa permits such large genomes to be effectively sampled at relatively low costs. An MDR (Mathematically Defined Repeat)...

  18. Contamination-controlled high-throughput whole genome sequencing for influenza A viruses using the MiSeq sequencer.

    PubMed

    Lee, Hong Kai; Lee, Chun Kiat; Tang, Julian Wei-Tze; Loh, Tze Ping; Koay, Evelyn Siew-Chuan

    2016-01-01

    Accurate full-length genomic sequences are important for viral phylogenetic studies. We developed a targeted high-throughput whole genome sequencing (HT-WGS) method for influenza A viruses, which utilized an enzymatic cleavage-based approach, the Nextera XT DNA library preparation kit, for library preparation. The entire library preparation workflow was adapted for the Sentosa SX101, a liquid handling platform, to automate this labor-intensive step. As the enzymatic cleavage-based approach generates low coverage reads at both ends of the cleaved products, we corrected this loss of sequencing coverage at the termini by introducing modified primers during the targeted amplification step to generate full-length influenza A sequences with even coverage across the whole genome. Another challenge of targeted HTS is the risk of specimen-to-specimen cross-contamination during the library preparation step that results in the calling of false-positive minority variants. We included an in-run, negative system control to capture contamination reads that may be generated during the liquid handling procedures. The upper limits of 99.99% prediction intervals of the contamination rate were adopted as cut-off values of contamination reads. Here, 148 influenza A/H3N2 samples were sequenced using the HTS protocol and were compared against a Sanger-based sequencing method. Our data showed that the rate of specimen-to-specimen cross-contamination was highly significant in HTS. PMID:27624998

  19. Highly Informative Simple Sequence Repeat (SSR) Markers for Fingerprinting Hazelnut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Simple sequence repeat (SSR) or microsatellite markers have many applications in breeding and genetic studies of plants, including fingerprinting of cultivars and investigations of genetic diversity, and therefore provide information for better management of germplasm collections. They are repeatab...

  20. Camera Trapping: A Contemporary Approach to Monitoring Invasive Rodents in High Conservation Priority Ecosystems

    PubMed Central

    Rendall, Anthony R.; Sutherland, Duncan R.; Cooke, Raylene; White, John

    2014-01-01

    Invasive rodent species have established on 80% of the world's islands causing significant damage to island environments. Insular ecosystems support proportionally more biodiversity than comparative mainland areas, highlighting them as critical for global biodiversity conservation. Few techniques currently exist to adequately detect, with high confidence, species that are trap-adverse such as the black rat, Rattus rattus, in high conservation priority areas where multiple non-target species persist. This study investigates the effectiveness of camera trapping for monitoring invasive rodents in high conservation areas, and the influence of habitat features and density of colonial-nesting seabirds on rodent relative activity levels to provide insights into their potential impacts. A total of 276 camera sites were established and left in situ for 8 days. Identified species were recorded in discrete 15 min intervals, referred to as ‘events’. In total, 19 804 events were recorded. From these, 31 species were identified comprising 25 native species and six introduced. Two introduced rodent species were detected: the black rat (90% of sites), and house mouse Mus musculus (56% of sites). Rodent activity of both black rats and house mice were positively associated with the structural density of habitats. Density of seabird burrows was not strongly associated with relative activity levels of rodents, yet rodents were still present in these areas. Camera trapping enabled a large number of rodents to be detected with confidence in site-specific absences and high resolution to quantify relative activity levels. This method enables detection of multiple species simultaneously with low impact (for both target and non-target individuals); an ideal strategy for monitoring trap-adverse invasive rodents in high conservation areas. PMID:24599307

  1. Assessment of the effects of farming and conservation programs on pesticide deposition in high plains wetlands.

    PubMed

    Belden, Jason B; Hanson, Brittany Rae; McMurry, Scott T; Smith, Loren M; Haukos, David A

    2012-03-20

    We examined pesticide contamination in sediments from depressional playa wetlands embedded in the three dominant land-use types in the western High Plains and Rainwater Basin of the United States including cropland, perennial grassland enrolled in conservation programs (e.g., Conservation Reserve Program [CRP]), and native grassland or reference condition. Two hundred and sixty four playas, selected from the three land-use types, were sampled from Nebraska and Colorado in the north to Texas and New Mexico in the south. Sediments were examined for most of the commonly used agricultural pesticides. Atrazine, acetochlor, metolachlor, and trifluralin were the most commonly detected pesticides in the northern High Plains and Rainwater Basin. Atrazine, metolachlor, trifluralin, and pendimethalin were the most commonly detected pesticides in the southern High Plains. The top 5-10% of playas contained herbicide concentrations that are high enough to pose a hazard for plants. However, insecticides and fungicides were rarely detected. Pesticide occurrence and concentrations were higher in wetlands surrounded by cropland as compared to native grassland and CRP perennial grasses. The CRP, which is the largest conservation program in the U.S., was protective and had lower pesticide concentrations compared to cropland. PMID:22356096

  2. Taxonomic distinctness and conservation of a new high biodiversity subterranean area in Brazil.

    PubMed

    Gallão, Jonas E; Bichuette, Maria Elina

    2015-03-01

    Subterranean environments, even though they do not possess a primary production (photosynthesis), may present high biodiversity, faunistic originality, endemism, phylogenetic isolations and unique ecological and/or evolution events, in addition to rare taxa. Studies investigating the biological diversity in Neotropical caves are relatively rare and recent, and most of them have been conducted in Brazil. We sampled caves from the state of Bahia, northeastern Brazil, and through sampling sufficiency tests and richness estimators, we demonstrate that the normatization for the Brazilian cave laws is not adequate for its conservation and that only α diversity index is not enough to verify faunistic patterns. We suggest that a phylogenetic diversity index be more robust and accurate for conservation purposes, particularly the Taxonomic Distinctness index. Moreover, we propose that the sandstone complex caves from Chapada Diamantina National Park need to be classified as being of high subterranean biodiversity in a global scope. PMID:25673471

  3. Targeted carbon conservation at national scales with high-resolution monitoring.

    PubMed

    Asner, Gregory P; Knapp, David E; Martin, Roberta E; Tupayachi, Raul; Anderson, Christopher B; Mascaro, Joseph; Sinca, Felipe; Chadwick, K Dana; Higgins, Mark; Farfan, William; Llactayo, William; Silman, Miles R

    2014-11-25

    Terrestrial carbon conservation can provide critical environmental, social, and climate benefits. Yet, the geographically complex mosaic of threats to, and opportunities for, conserving carbon in landscapes remain largely unresolved at national scales. Using a new high-resolution carbon mapping approach applied to Perú, a megadiverse country undergoing rapid land use change, we found that at least 0.8 Pg of aboveground carbon stocks are at imminent risk of emission from land use activities. Map-based information on the natural controls over carbon density, as well as current ecosystem threats and protections, revealed three biogeographically explicit strategies that fully offset forthcoming land-use emissions. High-resolution carbon mapping affords targeted interventions to reduce greenhouse gas emissions in rapidly developing tropical nations. PMID:25385593

  4. Targeted carbon conservation at national scales with high-resolution monitoring

    PubMed Central

    Asner, Gregory P.; Knapp, David E.; Martin, Roberta E.; Tupayachi, Raul; Anderson, Christopher B.; Mascaro, Joseph; Sinca, Felipe; Chadwick, K. Dana; Higgins, Mark; Farfan, William; Llactayo, William; Silman, Miles R.

    2014-01-01

    Terrestrial carbon conservation can provide critical environmental, social, and climate benefits. Yet, the geographically complex mosaic of threats to, and opportunities for, conserving carbon in landscapes remain largely unresolved at national scales. Using a new high-resolution carbon mapping approach applied to Perú, a megadiverse country undergoing rapid land use change, we found that at least 0.8 Pg of aboveground carbon stocks are at imminent risk of emission from land use activities. Map-based information on the natural controls over carbon density, as well as current ecosystem threats and protections, revealed three biogeographically explicit strategies that fully offset forthcoming land-use emissions. High-resolution carbon mapping affords targeted interventions to reduce greenhouse gas emissions in rapidly developing tropical nations. PMID:25385593

  5. Bacillus thuringiensis insecticidal Cry1Aa toxin binds to a highly conserved region of aminopeptidase N in the host insect leading to its evolutionary success.

    PubMed

    Nakanishi, K; Yaoi, K; Shimada, N; Kadotani, T; Sato, R

    1999-06-15

    Bacillus thuringiensis insecticidal protein, Cry1Aa toxin, binds to a specific receptor in insect midguts and has insecticidal activity. Therefore, the structure of the receptor molecule is probably a key factor in determining the binding affinity of the toxin and insect susceptibility. The cDNA fragment (PX frg1) encoding the Cry1Aa toxin-binding region of an aminopeptidase N (APN) or an APN family protein from diamondback moth, Plutella xylostella midgut was cloned and sequenced. A comparison between the deduced amino acid sequence of PX frg1 and other insect APN sequences shows that Cry1Aa toxin binds to a highly conserved region of APN family protein. In this paper, we propose a model to explain the mechanism that causes B. thuringiensis evolutionary success and differing insect susceptibility to Cry1Aa toxin. PMID:10366728

  6. PhyPA: Phylogenetic method with pairwise sequence alignment outperforms likelihood methods in phylogenetics involving highly diverged sequences.

    PubMed

    Xia, Xuhua

    2016-09-01

    While pairwise sequence alignment (PSA) by dynamic programming is guaranteed to generate one of the optimal alignments, multiple sequence alignment (MSA) of highly divergent sequences often results in poorly aligned sequences, plaguing all subsequent phylogenetic analysis. One way to avoid this problem is to use only PSA to reconstruct phylogenetic trees, which can only be done with distance-based methods. I compared the accuracy of this new computational approach (named PhyPA for phylogenetics by pairwise alignment) against the maximum likelihood method using MSA (the ML+MSA approach), based on nucleotide, amino acid and codon sequences simulated with different topologies and tree lengths. I present a surprising discovery that the fast PhyPA method consistently outperforms the slow ML+MSA approach for highly diverged sequences even when all optimization options were turned on for the ML+MSA approach. Only when sequences are not highly diverged (i.e., when a reliable MSA can be obtained) does the ML+MSA approach outperforms PhyPA. The true topologies are always recovered by ML with the true alignment from the simulation. However, with MSA derived from alignment programs such as MAFFT or MUSCLE, the recovered topology consistently has higher likelihood than that for the true topology. Thus, the failure to recover the true topology by the ML+MSA is not because of insufficient search of tree space, but by the distortion of phylogenetic signal by MSA methods. I have implemented in DAMBE PhyPA and two approaches making use of multi-gene data sets to derive phylogenetic support for subtrees equivalent to resampling techniques such as bootstrapping and jackknifing. PMID:27377322

  7. Trypanosoma rangeli and Trypanosoma cruzi: molecular characterization of genes encoding putative calcium-binding proteins, highly conserved in trypanosomatids.

    PubMed

    Porcel, B M; Bontempi, E J; Henriksson, J; Rydåker, M; Aslund, L; Segura, E L; Pettersson, U; Ruiz, A M

    1996-12-01

    Genes encoding a 29-kDa flagellar calcium-binding protein (F29) in Trypanosoma cruzi, strongly homologous to EF-hand calcium-binding protein-encoding genes previously reported in this parasite, were isolated by immunoscreening. F29 is encoded by a number of very similar genes, highly conserved among different T. cruzi isolates. The genes are located on a pair of homologous chromosomes, arranged in one or two clusters of tandem repeats. PCR amplification of Trypanosoma rangeli genomic DNA, using primers derived from the T. cruzi F29 sequence made it possible to isolate the homologous gene in T. rangeli, encoding a 23-kDa protein called TrCaBP. Gene sequence comparisons showed homology to EF-hand calcium-binding proteins from T. cruzi (82.8%), Trypanosoma brucei brucei (60.2%), and Entamoeba histolytica (28.4%). Northern blot analysis revealed that the TrCaBP gene is expressed in T. rangeli as a polyadenylated transcript. The TrCaBP-encoding genes are present in at least 20 copies per cell, organized in tandem arrays, on large T. rangeli chromosomes in some isolates and on two smaller ones in others. This gene, however, seems to be absent from Leishmania. PMID:8948328

  8. Genetic Determinants of Sindbis Virus Mosquito Infection Are Associated with a Highly Conserved Alphavirus and Flavivirus Envelope Sequence▿

    PubMed Central

    Pierro, Dennis J.; Powers, Erik L.; Olson, Ken E.

    2008-01-01

    Wild-type Sindbis virus (SINV) strain MRE16 efficiently infects Aedes aegypti midgut epithelial cells (MEC), but laboratory-derived neurovirulent SINV strain TE/5′2J infects MEC poorly. SINV determinants for MEC infection have been localized to the E2 glycoprotein. The E2 amino acid sequences of MRE16 and TE/5′2J differ at 60 residue sites. To identify the genetic determinants of MEC infection of MRE16, the TE/5′2J virus genome was altered to contain either domain chimeras or more focused nucleotide substitutions of MRE16. The growth patterns of derived viruses in cell culture were determined, as were the midgut infection rates (MIR) in A. aegypti mosquitoes. The results showed that substitutions of MRE16 E2 aa 95 to 96 and 116 to 119 into the TE/5′2J virus increased MIR both independently and in combination with each other. In addition, a unique PPF/.GDS amino acid motif was located between these two sites that was found to be a highly conserved sequence among alphaviruses and flaviviruses but not other arboviruses. PMID:18160430

  9. High-order entropy stable finite difference schemes for nonlinear conservation laws: Finite domains

    NASA Astrophysics Data System (ADS)

    Fisher, Travis C.; Carpenter, Mark H.

    2013-11-01

    Nonlinear entropy stability is used to derive provably stable high-order finite difference operators including boundary closure stencils, for the compressible Navier-Stokes equations. A comparison technique is used to derive a new Entropy Stable Weighted Essentially Non-Oscillatory (SSWENO) finite difference method, appropriate for simulations of problems with shocks. Viscous terms are approximated using conservative, entropy stable, narrow-stencil finite difference operators. The efficacy of the new discrete operators is demonstrated using both smooth and discontinuous test cases.

  10. Sequence Conservation and Sexually Dimorphic Expression of the Ftz-F1 Gene in the Crustacean Daphnia magna

    PubMed Central

    Mohamad Ishak, Nur Syafiqah; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2016-01-01

    Identifying the genes required for environmental sex determination is important for understanding the evolution of diverse sex determination mechanisms in animals. Orthologs of Drosophila orphan receptor Fushi tarazu factor-1 (Ftz-F1) are known to function in genetic sex determination. In contrast, their roles in environmental sex determination remain unknown. In this study, we have cloned and characterized the Ftz-F1 ortholog in the branchiopod crustacean Daphnia magna, which produces males in response to environmental stimuli. Similar to that observed in Drosophila, D. magna Ftz-F1 (DapmaFtz-F1) produces two splicing variants, αFtz-F1 and βFtz-F1, which encode 699 and 777 amino acids, respectively. Both isoforms share a DNA-binding domain, a ligand-binding domain, and an AF-2 activation domain and differ only at the A/B domain. The phylogenetic position and genomic structure of DapmaFtz-F1 suggested that this gene has diverged from an ancestral gene common to branchiopod crustacean and insect Ftz-F1 genes. qRT-PCR showed that at the one cell and gastrulation stages, both DapmaFtz-F1 isoforms are two-fold more abundant in males than in females. In addition, in later stages, their sexual dimorphic expressions were maintained in spite of reduced expression. Time-lapse imaging of DapmaFtz-F1 RNAi embryos was performed in H2B-GFP expressing transgenic Daphnia, demonstrating that development of the RNAi embryos slowed down after the gastrulation stage and stopped at 30–48 h after ovulation. DapmaFtz-F1 shows high homology to insect Ftz-F1 orthologs based on its amino acid sequence and exon-intron organization. The sexually dimorphic expression of DapmaFtz-F1 suggests that it plays a role in environmental sex determination of D. magna. PMID:27138373

  11. Sequence Conservation and Sexually Dimorphic Expression of the Ftz-F1 Gene in the Crustacean Daphnia magna.

    PubMed

    Mohamad Ishak, Nur Syafiqah; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2016-01-01

    Identifying the genes required for environmental sex determination is important for understanding the evolution of diverse sex determination mechanisms in animals. Orthologs of Drosophila orphan receptor Fushi tarazu factor-1 (Ftz-F1) are known to function in genetic sex determination. In contrast, their roles in environmental sex determination remain unknown. In this study, we have cloned and characterized the Ftz-F1 ortholog in the branchiopod crustacean Daphnia magna, which produces males in response to environmental stimuli. Similar to that observed in Drosophila, D. magna Ftz-F1 (DapmaFtz-F1) produces two splicing variants, αFtz-F1 and βFtz-F1, which encode 699 and 777 amino acids, respectively. Both isoforms share a DNA-binding domain, a ligand-binding domain, and an AF-2 activation domain and differ only at the A/B domain. The phylogenetic position and genomic structure of DapmaFtz-F1 suggested that this gene has diverged from an ancestral gene common to branchiopod crustacean and insect Ftz-F1 genes. qRT-PCR showed that at the one cell and gastrulation stages, both DapmaFtz-F1 isoforms are two-fold more abundant in males than in females. In addition, in later stages, their sexual dimorphic expressions were maintained in spite of reduced expression. Time-lapse imaging of DapmaFtz-F1 RNAi embryos was performed in H2B-GFP expressing transgenic Daphnia, demonstrating that development of the RNAi embryos slowed down after the gastrulation stage and stopped at 30-48 h after ovulation. DapmaFtz-F1 shows high homology to insect Ftz-F1 orthologs based on its amino acid sequence and exon-intron organization. The sexually dimorphic expression of DapmaFtz-F1 suggests that it plays a role in environmental sex determination of D. magna. PMID:27138373

  12. Nmf9 Encodes a Highly Conserved Protein Important to Neurological Function in Mice and Flies

    PubMed Central

    Zhang, Shuxiao; Ross, Kevin D.; Seidner, Glen A.; Gorman, Michael R.; Poon, Tiffany H.; Wang, Xiaobo; Keithley, Elizabeth M.; Lee, Patricia N.; Martindale, Mark Q.; Joiner, William J.; Hamilton, Bruce A.

    2015-01-01

    Many protein-coding genes identified by genome sequencing remain without functional annotation or biological context. Here we define a novel protein-coding gene, Nmf9, based on a forward genetic screen for neurological function. ENU-induced and genome-edited null mutations in mice produce deficits in vestibular function, fear learning and circadian behavior, which correlated with Nmf9 expression in inner ear, amygdala, and suprachiasmatic nuclei. Homologous genes from unicellular organisms and invertebrate animals predict interactions with small GTPases, but the corresponding domains are absent in mammalian Nmf9. Intriguingly, homozygotes for null mutations in the Drosophila homolog, CG45058, show profound locomotor defects and premature death, while heterozygotes show striking effects on sleep and activity phenotypes. These results link a novel gene orthology group to discrete neurological functions, and show conserved requirement across wide phylogenetic distance and domain level structural changes. PMID:26131556

  13. Identification and analysis of a highly conserved chemotaxis gene cluster in Shewanella species.

    SciTech Connect

    Li, J.; Romine, Margaret F.; Ward, M.

    2007-08-01

    A conserved cluster of chemotaxis genes was identified from the genome sequences of fifteen Shewanella species. An in-frame deletion of the cheA-3 gene, which is located in this cluster, was created in S. oneidensis MR-1 and the gene shown to be essential for chemotactic responses to anaerobic electron acceptors. The CheA-3 protein showed strong similarity to Vibrio cholerae CheA-2 and P. aeruginosa CheA-1, two proteins that are also essential for chemotaxis. The genes encoding these proteins were shown to be located in chemotaxis gene clusters closely related to the cheA-3-containing cluster in Shewanella species. The results of this study suggest that a combination of gene neighborhood and homology analyses may be used to predict which cheA genes are essential for chemotaxis in groups of closely related microorganisms.

  14. Conservative high-order-accurate finite-difference methods for curvilinear grids

    NASA Technical Reports Server (NTRS)

    Rai, Man M.; Chakrvarthy, Sukumar

    1993-01-01

    Two fourth-order-accurate finite-difference methods for numerically solving hyperbolic systems of conservation equations on smooth curvilinear grids are presented. The first method uses the differential form of the conservation equations; the second method uses the integral form of the conservation equations. Modifications to these schemes, which are required near boundaries to maintain overall high-order accuracy, are discussed. An analysis that demonstrates the stability of the modified schemes is also provided. Modifications to one of the schemes to make it total variation diminishing (TVD) are also discussed. Results that demonstrate the high-order accuracy of both schemes are included in the paper. In particular, a Ringleb-flow computation demonstrates the high-order accuracy and the stability of the boundary and near-boundary procedures. A second computation of supersonic flow over a cylinder demonstrates the shock-capturing capability of the TVD methodology. An important contribution of this paper is the dear demonstration that higher order accuracy leads to increased computational efficiency.

  15. High-Quality Draft Genome Sequence of Bacillus subtilis Strain WAUSV36

    PubMed Central

    Town, Jennifer; Audy, Patrice; Boyetchko, Susan M.

    2016-01-01

    Bacillus subtilis strain WAUSV36 inhibits the growth of and decreases disease symptoms caused by the potato pathogen Phytophthora infestans. We determined the sequence of the 4.7-Mbp genome of this strain. WAUSV36 shared very high nucleotide sequence identity with previously sequenced strains of B. subtilis. PMID:27340068

  16. Putting Physics First: Three Case Studies of High School Science Department and Course Sequence Reorganization

    ERIC Educational Resources Information Center

    Larkin, Douglas B.

    2016-01-01

    This article examines the process of shifting to a "Physics First" sequence in science course offerings in three school districts in the United States. This curricular sequence reverses the more common U.S. high school sequence of biology/chemistry/physics, and has gained substantial support in the physics education community over the…

  17. Characterization of chicken octamer-binding proteins demonstrates that POU domain-containing homeobox transcription factors have been highly conserved during vertebrate evolution

    SciTech Connect

    Petryniak, B.; Postema, C.E.; McCormack, W.T.; Thompson, C.B. ); Staudt, L.M. )

    1990-02-01

    The DNA sequence motif ATTTGCAT (octamer) or its inverse complement has been identified as an evolutionarily conserved element in the promoter region of immunoglobulin genes. Two major DNA-binding proteins that bind in a sequence-specific manner to the octamer DNA sequence have been identified in mammalian species--a ubiquitously expressed protein (Oct-1) and a lymphoid-specific protein (Oct-2). During characterization of the promoter region of the chicken immunoglobulin light chain gene, the authors identified two homologous octamer-binding proteins in chicken B cells. when the cloning of the human gene for Oct-2 revealed it to be a member of a distinct family of homeobox genes, they sought to determine if the human Oct-2 cDNA could be used to identify homologous chicken homeobox genes. Using a human Oct-2 homeobox-specific DNA probe, they were able to identify 6-10 homeobox-containing genes in the chicken genome, demonstrating that the Oct-2-related subfamily of homeobox genes exists in avian species. DNA sequence analysis revealed it to be the chicken homologue of the human Oct-1 gene. Together, the data show that the POU-containing subfamily of homeobox genes have been highly conserved during vertebrate evolution, apparently as a result of selection for their DNA-binding and transcriptional regulatory properties.

  18. Proteomic Analysis of Pathogenic Fungi Reveals Highly Expressed Conserved Cell Wall Proteins

    PubMed Central

    Champer, Jackson; Ito, James I.; Clemons, Karl V.; Stevens, David A.; Kalkum, Markus

    2016-01-01

    We are presenting a quantitative proteomics tally of the most commonly expressed conserved fungal proteins of the cytosol, the cell wall, and the secretome. It was our goal to identify fungi-typical proteins that do not share significant homology with human proteins. Such fungal proteins are of interest to the development of vaccines or drug targets. Protein samples were derived from 13 fungal species, cultured in rich or in minimal media; these included clinical isolates of Aspergillus, Candida, Mucor, Cryptococcus, and Coccidioides species. Proteomes were analyzed by quantitative MSE (Mass Spectrometry—Elevated Collision Energy). Several thousand proteins were identified and quantified in total across all fractions and culture conditions. The 42 most abundant proteins identified in fungal cell walls or supernatants shared no to very little homology with human proteins. In contrast, all but five of the 50 most abundant cytosolic proteins had human homologs with sequence identity averaging 59%. Proteomic comparisons of the secreted or surface localized fungal proteins highlighted conserved homologs of the Aspergillus fumigatus proteins 1,3-β-glucanosyltransferases (Bgt1, Gel1-4), Crf1, Ecm33, EglC, and others. The fact that Crf1 and Gel1 were previously shown to be promising vaccine candidates, underlines the value of the proteomics data presented here. PMID:26878023

  19. Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins.

    PubMed

    Kralt, Annemarie; Jagalur, Noorjahan B; van den Boom, Vincent; Lokareddy, Ravi K; Steen, Anton; Cingolani, Gino; Fornerod, Maarten; Veenhoff, Liesbeth M

    2015-09-15

    Endoplasmic reticulum-synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins. PMID:26179916

  20. Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

    PubMed Central

    Kralt, Annemarie; Jagalur, Noorjahan B.; van den Boom, Vincent; Lokareddy, Ravi K.; Steen, Anton; Cingolani, Gino; Fornerod, Maarten; Veenhoff, Liesbeth M.

    2015-01-01

    Endoplasmic reticulum–synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins. PMID:26179916

  1. Conserved sequences of sperm-activating peptide and its receptor throughout evolution, despite speciation in the sea star Asterias amurensis and closely related species.

    PubMed

    Nakachi, Mia; Hoshi, Motonori; Matsumoto, Midori; Moriyama, Hideaki

    2008-08-01

    The asteroidal sperm-activating peptides (asterosaps) from the egg jelly bind to their sperm receptor, a membrane-bound guanylate cyclase, on the tail to activate sperm in sea stars. Asterosaps are produced as single peptides and then cleaved into shorter peptides. Sperm activation is followed by the acrosome reaction, which is subfamily specific. In order to investigate the molecular details of the asterosap-receptor interaction, corresponding cDNAs have been cloned, sequenced and analysed from the Asteriinae subfamily including Asterias amurensis, A. rubens, A. forbesi and Aphelasterias japonica, as well as Distolasterias nipon from the Coscinasteriinae subfamily. Averages of 29% and 86% identity were found from the deduced amino acid sequences in asterosap and its receptor extracellular domains, respectively, across all species examined. The phylogenic tree topology for asterosap and its receptor was similar to that of the mitochondrial cytochrome c oxidase subunit I. In spite of a certain homology, the amino acid sequences exhibited speciation. Conservation was found in the asterosap residues involved in disulphide bonding and proteinase-cleaving sites. Conversely, similarities were detected between potential asterosap-binding sites and the structure of the atrial natriuretic peptide receptor. Although the sperm-activating peptide and its receptor share certain common sequences, they may serve as barriers that ensure speciation in the sea star A. amurensis and closely related species. PMID:18578950

  2. Comparative application of direct sequencing, PCR-RFLP, and cytogenetic markers in the genetic characterization of Pimelodus (Siluriformes: Pimelodidae) species: possible implications for fish conservation.

    PubMed

    Ferreira, M; Bressane, K C O; Moresco, A R C; Moreira-Filho, O; Almeida-Toledo, L F; Garcia, C

    2014-01-01

    Pimelodus (Pimelodidae) is a genus comprising a group of South American species with complex taxonomic relationships. Cytogenetics, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and sequencing data of mitochondrial genes were analyzed to characterize 4 Pimelodus species: P. fur, P. heraldoi, P. maculatus, and Pimelodus sp. All populations presented 2n=56 chromosomes and distinct karyotypic formulae. The heterochromatin distribution pattern and the number and location of 5S and 18S rDNA sites are discussed. The application of PCR-RFLP markers and sequencing of mitochondrial DNA genes provided species-specific haplotypes, which allowed us to differentiate the species studied. The mitochondrial gene sequences presented nucleotide mutations in the restriction sites and throughout the sequences, and they were mostly related to synonymous substitutions in the coded proteins; however, they did not affect the protein and its function. Comparing the data obtained using these 3 methodologies, the existence of a species complex in P. maculatus along the basins studied might be inferred, showing that cytogenetics is an important tool in studies focusing on the conservation or management of both natural and captive populations of these fishes. PMID:25036358

  3. Replication origins and a sequence involved in coordinate induction of the immediate-early gene family are conserved in an intergenic region of herpes simplex virus.

    PubMed Central

    Whitton, J L; Clements, J B

    1984-01-01

    We have determined the structure of the 5' portion of herpes simplex virus type 2 (HSF-2) immediate-early (IE) mRNA-3 and have obtained the DNA sequence specifying the N terminus of its encoded polypeptide, Vmw182, its untranslated leader and the intergenic region between IEmRNAs-3 & 4/5. Comparison of the HSV-2 intergenic sequences with the HSV-1 equivalent region identifies several conserved regions: (1) an AT-rich element with core consensus TAATGARAT which is likely to be the 'activator' sequence through which coordinate induction of the IE gene family is mediated. (2) GC-rich and GA-rich tracts, found in a wide variety of eukaryotic promoters, which vary in position and orientation between HSV-2 and HSV-1 and which represent modulators of transcription. (3) TATA homologies present 15-25 base pairs (bp) upstream of mRNA 5' termini. (4) a 137bp direct repeat in HSV-2 which contains sequence almost identical to the HSV-1 replication origin. Images PMID:6322134

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

    PubMed

    Silva-Sanchez, Aaron; Liu, Cun Ren; Vale, Andre M; Khass, Mohamed; Kapoor, Pratibha; Elgavish, Ada; Ivanov, Ivaylo I; Ippolito, Gregory C; Schelonka, Robert L; Schoeb, Trenton R; Burrows, Peter D; Schroeder, Harry W

    2015-01-01

    Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3), which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies. PMID:25706374

  5. Evaluating areas of high conservation value in Western Oregon with a decision-support model.

    PubMed

    Staus, Nancy L; Strittholt, James R; Dellasala, Dominick A

    2010-06-01

    The Northwest Forest Plan was implemented in 1994 to protect habitat for species associated with old-growth forests, including Northern Spotted Owls (Strix occidentailis caurina) in Washington, Oregon, and northern California (U.S.A.). Nevertheless, 10-year monitoring data indicate mixed success in meeting the ecological goals of the plan. We used the ecosystem management decision-support model to evaluate terrestrial and aquatic habitats across the landscape on the basis of ecological objectives of the Northwest Forest Plan, which included maintenance of late-successional and old-growth forest, recovery, and maintenance of Pacific salmon (Oncorhynchus spp.), and viability of Northern Spotted Owls. Areas of the landscape that contained habitat characteristics that supported these objectives were considered of high conservation value. We used the model to evaluate ecological condition of each of the 36, 180 township and range sections of the study area. Eighteen percent of the study area was identified as habitat of high conservation value. These areas were mostly on public lands. Many of the sections that contained habitat of exceptional conservation value were on Bureau of Land Management land that has been considered for management-plan revisions to increase timber harvests. The results of our model can be used to guide future land management in the Northwest Forest Plan area, and illustrate how decision-support models can help land managers develop strategies to better meet their goals. PMID:20184658

  6. High-speed Viterbi decoding with overlapping code sequences

    NASA Technical Reports Server (NTRS)

    Ross, Michael D.; Osborne, William P.

    1993-01-01

    The Viterbi Algorithm for decoding convolutional codes and Trellis Coded Modulation is suited to VLSI implementation but contains a feedback loop which limits the speed of pipelined architecture. The feedback loop is circumvented by decoding independent sequences simultaneously, resulting in a 5-9 fold speed-up with a two-fold hardware expansion.

  7. Three-Year Sequence for High School Mathematics, Course III.

    ERIC Educational Resources Information Center

    New York State Education Dept., Albany. Bureau of Curriculum Development.

    This publication is designed to aid schools in planning a course of study for students in Course III Mathematics in New York State schools. This guide continues the developmental program begun in Course I. It is intended to serve as the Regents syllabus for the third year of the academic sequence for a New York State Regents diploma. This document…

  8. The Effects of a High-Probability Request Sequencing Technique in Enhancing Transition Behaviors

    ERIC Educational Resources Information Center

    Banda, Devender R.; Kubina, Richard M., Jr.

    2006-01-01

    In this study, an autism support teacher used a high-probability request sequencing technique to help a middle-school student with autism engage in three transition behaviors. High probability request sequencing refers to a procedure in which 2 to 3 preferred questions, highly associated with compliance, are rapidly given before presenting a low…

  9. A High-Order Finite Spectral Volume Method for Conservation Laws on Unstructured Grids

    NASA Technical Reports Server (NTRS)

    Wang, Z. J.; Liu, Yen; Kwak, Dochan (Technical Monitor)

    2001-01-01

    A time accurate, high-order, conservative, yet efficient method named Finite Spectral Volume (FSV) is developed for conservation laws on unstructured grids. The concept of a 'spectral volume' is introduced to achieve high-order accuracy in an efficient manner similar to spectral element and multi-domain spectral methods. In addition, each spectral volume is further sub-divided into control volumes (CVs), and cell-averaged data from these control volumes is used to reconstruct a high-order approximation in the spectral volume. Riemann solvers are used to compute the fluxes at spectral volume boundaries. Then cell-averaged state variables in the control volumes are updated independently. Furthermore, TVD (Total Variation Diminishing) and TVB (Total Variation Bounded) limiters are introduced in the FSV method to remove/reduce spurious oscillations near discontinuities. A very desirable feature of the FSV method is that the reconstruction is carried out only once, and analytically, and is the same for all cells of the same type, and that the reconstruction stencil is always non-singular, in contrast to the memory and CPU-intensive reconstruction in a high-order finite volume (FV) method. Discussions are made concerning why the FSV method is significantly more efficient than high-order finite volume and the Discontinuous Galerkin (DG) methods. Fundamental properties of the FSV method are studied and high-order accuracy is demonstrated for several model problems with and without discontinuities.

  10. High order filtering methods for approximating hyperbolic systems of conservation laws

    NASA Technical Reports Server (NTRS)

    Lafon, F.; Osher, S.

    1991-01-01

    The essentially nonoscillatory (ENO) schemes, while potentially useful in the computation of discontinuous solutions of hyperbolic conservation-law systems, are computationally costly relative to simple central-difference methods. A filtering technique is presented which employs central differencing of arbitrarily high-order accuracy except where a local test detects the presence of spurious oscillations and calls upon the full ENO apparatus to remove them. A factor-of-three speedup is thus obtained over the full-ENO method for a wide range of problems, with high-order accuracy in regions of smooth flow.

  11. CONSERVED REGULATOR ELEMENTS IDENTIFIED FROM A COMPARATIVE PUROINDOLINE GENE SEQUENCE SURVEY OF TRITICUM AND AEGILOPS DIPLOID TAXA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Kernel texture (“hardness”) is an important trait that determines end-use quality of wheat (Triticum aestivum L. and T. turgidum ssp. durum [Desf.] Husn.). Variation in texture is associated with the presence/absence or sequence polymorphism of two proteins, puroindoline a and puroindoline b. This...

  12. Identification of conserved and novel microRNAs in Catharanthus roseus by deep sequencing and computational prediction of their potential targets.

    PubMed

    Prakash, Pravin; Ghosliya, Dolly; Gupta, Vikrant

    2015-01-10

    MicroRNAs are small endogenous non-coding RNAs of ~19-24 nucleotides and perform regulatory roles in many plant processes. To identify miRNAs involved in regulatory networks controlling diverse biological processes including secondary metabolism in Catharanthus roseus, an important medicinal plant, we employed deep sequencing of small RNA from leaf tissue. A total of 88 potential miRNAs comprising of 81 conserved miRNAs belonging to 35 families and seven novel miRNAs were identified. Precursors for 16 conserved and seven novel cro-miRNAs were identified, and their stem-loop hairpin structures were predicted. Selected cro-miRNAs were analyzed by stem-loop qRT-PCR and differential expression patterns were observed in different vegetative tissues of C. roseus. Targets were predicted for conserved and novel cro-miRNAs, which were found to be involved in diverse biological role(s) including secondary metabolism. Our study enriches available resources and information regarding miRNAs and their potential targets for better understanding of miRNA-mediated gene regulation in plants. PMID:25445288

  13. Forecasting Ecological Genomics: High-Tech Animal Instrumentation Meets High-Throughput Sequencing.

    PubMed

    Shafer, Aaron B A; Northrup, Joseph M; Wikelski, Martin; Wittemyer, George; Wolf, Jochen B W

    2016-01-01

    Recent advancements in animal tracking technology and high-throughput sequencing are rapidly changing the questions and scope of research in the biological sciences. The integration of genomic data with high-tech animal instrumentation comes as a natural progression of traditional work in ecological genetics, and we provide a framework for linking the separate data streams from these technologies. Such a merger will elucidate the genetic basis of adaptive behaviors like migration and hibernation and advance our understanding of fundamental ecological and evolutionary processes such as pathogen transmission, population responses to environmental change, and communication in natural populations. PMID:26745372

  14. Forecasting Ecological Genomics: High-Tech Animal Instrumentation Meets High-Throughput Sequencing

    PubMed Central

    Shafer, Aaron B. A.; Northrup, Joseph M.; Wikelski, Martin; Wittemyer, George; Wolf, Jochen B. W.

    2016-01-01

    Recent advancements in animal tracking technology and high-throughput sequencing are rapidly changing the questions and scope of research in the biological sciences. The integration of genomic data with high-tech animal instrumentation comes as a natural progression of traditional work in ecological genetics, and we provide a framework for linking the separate data streams from these technologies. Such a merger will elucidate the genetic basis of adaptive behaviors like migration and hibernation and advance our understanding of fundamental ecological and evolutionary processes such as pathogen transmission, population responses to environmental change, and communication in natural populations. PMID:26745372

  15. Construction and Analysis of High-Density Linkage Map Using High-Throughput Sequencing Data

    PubMed Central

    Liu, Min; Liu, Hui; Zeng, Huaping; Deng, Dejing; Xin, Huaigen; Song, Jun; Xu, Chunhua; Sun, Xiaowen; Hou, Xilin; Wang, Xiaowu; Zheng, Hongkun

    2014-01-01

    Linkage maps enable the study of important biological questions. The construction of high-density linkage maps appears more feasible since the advent of next-generation sequencing (NGS), which eases SNP discovery and high-throughput genotyping of large population. However, the marker number explosion and genotyping errors from NGS data challenge the computational efficiency and linkage map quality of linkage study methods. Here we report the HighMap method for constructing high-density linkage maps from NGS data. HighMap employs an iterative ordering and error correction strategy based on a k-nearest neighbor algorithm and a Monte Carlo multipoint maximum likelihood algorithm. Simulation study shows HighMap can create a linkage map with three times as many markers as ordering-only methods while offering more accurate marker orders and stable genetic distances. Using HighMap, we constructed a common carp linkage map with 10,004 markers. The singleton rate was less than one-ninth of that generated by JoinMap4.1. Its total map distance was 5,908 cM, consistent with reports on low-density maps. HighMap is an efficient method for constructing high-density, high-quality linkage maps from high-throughput population NGS data. It will facilitate genome assembling, comparative genomic analysis, and QTL studies. HighMap is available at http://highmap.biomarker.com.cn/. PMID:24905985

  16. A strategy to recover a high-quality, complete plastid sequence from low-coverage whole-genome sequencing1

    PubMed Central

    Garaycochea, Silvia; Speranza, Pablo; Alvarez-Valin, Fernando

    2015-01-01

    Premise of the study: We developed a bioinformatic strategy to recover and assemble a chloroplast genome using data derived from low-coverage 454 GS FLX/Roche whole-genome sequencing. Methods: A comparative genomics approach was applied to obtain the complete chloroplast genome from a weedy biotype of rice from Uruguay. We also applied appropriate filters to discriminate reads representing novel DNA transfer events between the chloroplast and nuclear genomes. Results: From a set of 295,159 reads (96 Mb data), we assembled the chloroplast genome into two contigs. This weedy rice was classified based on 23 polymorphic regions identified by comparison with reference chloroplast genomes. We detected recent and past events of genetic material transfer between the chloroplast and nuclear genomes and estimated their occurrence frequency. Discussion: We obtained a high-quality complete chloroplast genome sequence from low-coverage sequencing data. Intergenome DNA transfer appears to be more frequent than previously thought. PMID:26504677

  17. Whole Genome Sequencing of Enterovirus species C Isolates by High-Throughput Sequencing: Development of Generic Primers

    PubMed Central

    Bessaud, Maël; Sadeuh-Mba, Serge A.; Joffret, Marie-Line; Razafindratsimandresy, Richter; Polston, Patsy; Volle, Romain; Rakoto-Andrianarivelo, Mala; Blondel, Bruno; Njouom, Richard; Delpeyroux, Francis

    2016-01-01

    Enteroviruses are among the most common viruses infecting humans and can cause diverse clinical syndromes ranging from minor febrile illness to severe and potentially fatal diseases. Enterovirus species C (EV-C) consists of more than 20 types, among which the three serotypes of polioviruses, the etiological agents of poliomyelitis, are included. Biodiversity and evolution of EV-C genomes are shaped by frequent recombination events. Therefore, identification and characterization of circulating EV-C strains require the sequencing of different genomic regions. A simple method was developed to quickly sequence the entire genome of EV-C isolates. Four overlapping fragments were produced separately by RT-PCR performed with generic primers. The four amplicons were then pooled and purified prior to being sequenced by a high-throughput technique. The method was assessed on a panel of EV-Cs belonging to a wide-range of types. It can be used to determine full-length genome sequences through de novo assembly of thousands of reads. It was also able to discriminate reads from closely related viruses in mixtures. By decreasing the workload compared to classical Sanger-based techniques, this method will serve as a precious tool for sequencing large panels of EV-Cs isolated in cell cultures during environmental surveillance or from patients, including vaccine-derived polioviruses. PMID:27617004

  18. Whole Genome Sequencing of Enterovirus species C Isolates by High-Throughput Sequencing: Development of Generic Primers.

    PubMed

    Bessaud, Maël; Sadeuh-Mba, Serge A; Joffret, Marie-Line; Razafindratsimandresy, Richter; Polston, Patsy; Volle, Romain; Rakoto-Andrianarivelo, Mala; Blondel, Bruno; Njouom, Richard; Delpeyroux, Francis

    2016-01-01

    Enteroviruses are among the most common viruses infecting humans and can cause diverse clinical syndromes ranging from minor febrile illness to severe and potentially fatal diseases. Enterovirus species C (EV-C) consists of more than 20 types, among which the three serotypes of polioviruses, the etiological agents of poliomyelitis, are included. Biodiversity and evolution of EV-C genomes are shaped by frequent recombination events. Therefore, identification and characterization of circulating EV-C strains require the sequencing of different genomic regions. A simple method was developed to quickly sequence the entire genome of EV-C isolates. Four overlapping fragments were produced separately by RT-PCR performed with generic primers. The four amplicons were then pooled and purified prior to being sequenced by a high-throughput technique. The method was assessed on a panel of EV-Cs belonging to a wide-range of types. It can be used to determine full-length genome sequences through de novo assembly of thousands of reads. It was also able to discriminate reads from closely related viruses in mixtures. By decreasing the workload compared to classical Sanger-based techniques, this method will serve as a precious tool for sequencing large panels of EV-Cs isolated in cell cultures during environmental surveillance or from patients, including vaccine-derived polioviruses. PMID:27617004

  19. High-throughput sequencing of complete human mtDNA genomes from the Philippines

    PubMed Central

    Gunnarsdóttir, Ellen D.; Li, Mingkun; Bauchet, Marc; Finstermeier, Knut; Stoneking, Mark

    2011-01-01

    Because of the time and cost associated with Sanger sequencing of complete human mtDNA genomes, practically all evolutionary studies have screened samples first to define haplogroups and then either selected a few samples from each haplogroup, or many samples from a particular haplogroup of interest, for complete mtDNA genome sequencing. Such biased sampling precludes many analyses of interest. Here, we used high-throughput sequencing platforms to generate, rapidly and inexpensively, 109 complete mtDNA genome sequences from random samples of individuals from three Filipino groups, including one Negrito group, the Mamanwa. We obtained on average ∼55-fold coverage per sequence, with <1% missing data per sequence. Various analyses attest to the accuracy of the sequences, including comparison to sequences of the first hypervariable segment of the control region generated by Sanger sequencing; patterns of nucleotide substitution and the distribution of polymorphic sites across the genome; and the observed haplogroups. Bayesian skyline plots of population size change through time indicate similar patterns for all three Filipino groups, but sharply contrast with such plots previously constructed from biased sampling of complete mtDNA genomes, as well as with an artificially constructed sample of sequences that mimics the biased sampling. Our results clearly demonstrate that the high-throughput sequencing platforms are the methodology of choice for generating complete mtDNA genome sequences. PMID:21147912

  20. Tracking TCRβ Sequence Clonotype Expansions during Antiviral Therapy Using High-Throughput Sequencing of the Hypervariable Region

    PubMed Central

    Robinson, Mark W.; Hughes, Joseph; Wilkie, Gavin S.; Swann, Rachael; Barclay, Stephen T.; Mills, Peter R.; Patel, Arvind H.; Thomson, Emma C.; McLauchlan, John

    2016-01-01

    To maintain a persistent infection viruses such as hepatitis C virus (HCV) employ a range of mechanisms that subvert protective T cell responses. The suppression of antigen-specific T cell responses by HCV hinders efforts to profile T cell responses during chronic infection and antiviral therapy. Conventional methods of detecting antigen-specific T cells utilize either antigen stimulation (e.g., ELISpot, proliferation assays, cytokine production) or antigen-loaded tetramer staining. This limits the ability to profile T cell responses during chronic infection due to suppressed effector function and the requirement for prior knowledge of antigenic viral peptide sequences. Recently, high-throughput sequencing (HTS) technologies have been developed for the analysis of T cell repertoires. In the present study, we have assessed the feasibility of HTS of the TCRβ complementarity determining region (CDR)3 to track T cell expansions in an antigen-independent manner. Using sequential blood samples from HCV-infected individuals undergoing antiviral therapy, we were able to measure the population frequencies of >35,000 TCRβ sequence clonotypes in each individual over the course of 12 weeks. TRBV/TRBJ gene segment usage varied markedly between individuals but remained relatively constant within individuals across the course of therapy. Despite this stable TRBV/TRBJ gene segment usage, a number of TCRβ sequence clonotypes showed dramatic changes in read frequency. These changes could not be linked to therapy outcomes in the present study; however, the TCRβ CDR3 sequences with the largest fold changes did include sequences with identical TRBV/TRBJ gene segment usage and high junction region homology to previously published CDR3 sequences from HCV-specific T cells targeting the HLA-B*0801-restricted 1395HSKKKCDEL1403 and HLA-A*0101-restricted 1435ATDALMTGY1443 epitopes. The pipeline developed in this proof of concept study provides a platform for the design of future

  1. Water Wisdom: 23 Stand-Alone Activities on Water Supply and Water Conservation for High School Students. 2nd Edition.

    ERIC Educational Resources Information Center

    Massachusetts State Water Resources Authority, Boston.

    This water conservation education program for high schools consists of both stand-alone activities and teacher support materials. Lessons are divided into six broad categories: (1) The Water Cycle; (2) Water and Society; (3) Keeping Water Pure; (4) Visualizing Volumes; (5) The Economics of Water Use; and (6) Domestic Water Conservation. The…

  2. How to Go Green: Creating a Conservation Culture in a Public High School through Education, Modeling, and Communication

    ERIC Educational Resources Information Center

    Schelly, Chelsea; Cross, Jennifer E.; Franzen, William; Hall, Pete; Reeve, Stu

    2012-01-01

    This case study examines how energy conservation efforts in one public high school contributed to both sustainability education and the adoption of sustainable behavior within educational and organizational practice. Individual role models, school facilities, school governance and school culture together support both conservation and environmental…

  3. Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications.

    PubMed

    Gu, W; Crawford, E D; O'Donovan, B D; Wilson, M R; Chow, E D; Retallack, H; DeRisi, J L

    2016-01-01

    Next-generation sequencing has generated a need for a broadly applicable method to remove unwanted high-abundance species prior to sequencing. We introduce DASH (Depletion of Abundant Sequences by Hybridization). Sequencing libraries are 'DASHed' with recombinant Cas9 protein complexed with a library of guide RNAs targeting unwanted species for cleavage, thus preventing them from consuming sequencing space. We demonstrate a more than 99 % reduction of mitochondrial rRNA in HeLa cells, and enrichment of pathogen sequences in patient samples. We also demonstrate an application of DASH in cancer. This simple method can be adapted for any sample type and increases sequencing yield without additional cost. PMID:26944702

  4. Identification and Characterization of Wilt and Salt Stress-Responsive MicroRNAs in Chickpea through High-Throughput Sequencing

    PubMed Central

    Deokar, Amit Atmaram; Bhardwaj, Ankur R.; Agarwal, Manu; Katiyar-Agarwal, Surekha; Srinivasan, Ramamurthy; Jain, Pradeep Kumar

    2014-01-01

    Chickpea (Cicer arietinum) is the second most widely grown legume worldwide and is the most important pulse crop in the Indian subcontinent. Chickpea productivity is adversely affected by a large number of biotic and abiotic stresses. MicroRNAs (miRNAs) have been implicated in the regulation of plant responses to several biotic and abiotic stresses. This study is the first attempt to identify chickpea miRNAs that are associated with biotic and abiotic stresses. The wilt infection that is caused by the fungus Fusarium oxysporum f.sp. ciceris is one of the major diseases severely affecting chickpea yields. Of late, increasing soil salinization has become a major problem in realizing these potential yields. Three chickpea libraries using fungal-infected, salt-treated and untreated seedlings were constructed and sequenced using next-generation sequencing technology. A total of 12,135,571 unique reads were obtained. In addition to 122 conserved miRNAs belonging to 25 different families, 59 novel miRNAs along with their star sequences were identified. Four legume-specific miRNAs, including miR5213, miR5232, miR2111 and miR2118, were found in all of the libraries. Poly(A)-based qRT-PCR (Quantitative real-time PCR) was used to validate eleven conserved and five novel miRNAs. miR530 was highly up regulated in response to fungal infection, which targets genes encoding zinc knuckle- and microtubule-associated proteins. Many miRNAs responded in a similar fashion under both biotic and abiotic stresses, indicating the existence of cross talk between the pathways that are involved in regulating these stresses. The potential target genes for the conserved and novel miRNAs were predicted based on sequence homologies. miR166 targets a HD-ZIPIII transcription factor and was validated by 5′ RLM-RACE. This study has identified several conserved and novel miRNAs in the chickpea that are associated with gene regulation following exposure to wilt and salt stress. PMID:25295754

  5. Sequence conservation, phylogenetic relationships, and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta.

    PubMed

    Cao, Xiaolong; He, Yan; Hu, Yingxia; Zhang, Xiufeng; Wang, Yang; Zou, Zhen; Chen, Yunru; Blissard, Gary W; Kanost, Michael R; Jiang, Haobo

    2015-07-01

    Serine protease (SP) and serine protease homolog (SPH) genes in insects encode a large family of proteins involved in digestion, development, immunity, and other processes. While 68 digestive SPs and their close homologs are reported in a companion paper (Kuwar et al., in preparation), we have identified 125 other SPs/SPHs in Manduca sexta and studied their structure, evolution, and expression. Fifty-two of them contain cystine-stabilized structures for molecular recognition, including clip, LDLa, Sushi, Wonton, TSP, CUB, Frizzle, and SR domains. There are nineteen groups of genes evolved from relatively recent gene duplication and sequence divergence. Thirty-five SPs and seven SPHs contain 1, 2 or 5 clip domains. Multiple sequence alignment and molecular modeling of the 54 clip domains have revealed structural diversity of these regulatory modules. Sequence comparison with their homologs in Drosophila melanogaster, Anopheles gambiae and Tribolium castaneum allows us to classify them into five subfamilies: A are SPHs with 1 or 5 group-3 clip domains, B are SPs with 1 or 2 group-2 clip domains, C, D1 and D2 are SPs with a single clip domain in group-1a, 1b and 1c, respectively. We have classified into six categories the 125 expression profiles of SP-related proteins in fat body, brain, midgut, Malpighian tubule, testis, and ovary at different stages, suggesting that they participate in various physiological processes. Through RNA-Seq-based gene annotation and expression profiling, as well as intragenomic sequence comparisons, we have established a framework of information for future biochemical research of nondigestive SPs and SPHs in this model species. PMID:25530503

  6. In silico identification of conserved microRNAs and their target transcripts from expressed sequence tags of three earthworm species.

    PubMed

    Gong, Ping; Xie, Fuliang; Zhang, Baohong; Perkins, Edward J

    2010-12-01

    MicroRNAs are a recently identified class of small regulatory RNAs that target more than 30% protein-coding genes. Elevating evidence shows that miRNAs play a critical role in many biological processes, including developmental timing, tissue differentiation, and response to chemical exposure. In this study, we applied a computational approach to analyze expressed sequence tags, and identified 32 miRNAs belonging to 22 miRNA families, in three earthworm species Eisenia fetida, Eisenia andrei, and Lumbricus rubellus. These newly identified earthworm miRNAs possess a difference of 2-4 nucleotides from their homologous counterparts in Caenorhabditis elegans. They also share similar features with other known animal miRNAs, for instance, the nucleotide U being dominant in both mature and pre-miRNA sequences, particularly in the first position of mature miRNA sequences at the 5' end. The newly identified earthworm miRNAs putatively regulate mRNA genes that are involved in many important biological processes and pathways related to development, growth, locomotion, and reproduction as well as response to stresses, particularly oxidative stress. Future efforts will focus on experimental validation of their presence and target mRNA genes to further elucidate their biological functions in earthworms. PMID:21030313

  7. Fisheries conservation on the high seas: linking conservation physiology and fisheries ecology for the management of large pelagic fishes.

    PubMed

    Horodysky, Andrij Z; Cooke, Steven J; Graves, John E; Brill, Richard W

    2016-01-01

    Populations of tunas, billfishes and pelagic sharks are fished at or over capacity in many regions of the world. They are captured by directed commercial and recreational fisheries (the latter of which often promote catch and release) or as incidental catch or bycatch in commercial fisheries. Population assessments of pelagic fishes typically incorporate catch-per-unit-effort time-series data from commercial and recreational fisheries; however, there have been notable changes in target species, areas fished and depth-specific gear deployments over the years that may have affected catchability. Some regional fisheries management organizations take into account the effects of time- and area-specific changes in the behaviours of fish and fishers, as well as fishing gear, to standardize catch-per-unit-effort indices and refine population estimates. However, estimates of changes in stock size over time may be very sensitive to underlying assumptions of the effects of oceanographic conditions and prey distribution on the horizontal and vertical movement patterns and distribution of pelagic fishes. Effective management and successful conservation of pelagic fishes requires a mechanistic understanding of their physiological and behavioural responses to environmental variability, potential for interaction with commercial and recreational fishing gear, and the capture process. The interdisciplinary field of conservation physiology can provide insights into pelagic fish demography and ecology (including environmental relationships and interspecific interactions) by uniting the complementary expertise and skills of fish physiologists and fisheries scientists. The iterative testing by one discipline of hypotheses generated by the other can span the fundamental-applied science continuum, leading to the development of robust insights supporting informed management. The resulting species-specific understanding of physiological abilities and tolerances can help to improve stock

  8. Fisheries conservation on the high seas: linking conservation physiology and fisheries ecology for the management of large pelagic fishes

    PubMed Central

    Horodysky, Andrij Z.; Cooke, Steven J.; Graves, John E.; Brill, Richard W.

    2016-01-01

    Populations of tunas, billfishes and pelagic sharks are fished at or over capacity in many regions of the world. They are captured by directed commercial and recreational fisheries (the latter of which often promote catch and release) or as incidental catch or bycatch in commercial fisheries. Population assessments of pelagic fishes typically incorporate catch-per-unit-effort time-series data from commercial and recreational fisheries; however, there have been notable changes in target species, areas fished and depth-specific gear deployments over the years that may have affected catchability. Some regional fisheries management organizations take into account the effects of time- and area-specific changes in the behaviours of fish and fishers, as well as fishing gear, to standardize catch-per-unit-effort indices and refine population estimates. However, estimates of changes in stock size over time may be very sensitive to underlying assumptions of the effects of oceanographic conditions and prey distribution on the horizontal and vertical movement patterns and distribution of pelagic fishes. Effective management and successful conservation of pelagic fishes requires a mechanistic understanding of their physiological and behavioural responses to environmental variability, potential for interaction with commercial and recreational fishing gear, and the capture process. The interdisciplinary field of conservation physiology can provide insights into pelagic fish demography and ecology (including environmental relationships and interspecific interactions) by uniting the complementary expertise and skills of fish physiologists and fisheries scientists. The iterative testing by one discipline of hypotheses generated by the other can span the fundamental–applied science continuum, leading to the development of robust insights supporting informed management. The resulting species-specific understanding of physiological abilities and tolerances can help to improve stock

  9. A high-order conservative collocation scheme and its application to global shallow-water equations

    NASA Astrophysics Data System (ADS)

    Chen, C.; Li, X.; Shen, X.; Xiao, F.

    2015-02-01

    In this paper, an efficient and conservative collocation method is proposed and used to develop a global shallow-water model. Being a nodal type high-order scheme, the present method solves the pointwise values of dependent variables as the unknowns within each control volume. The solution points are arranged as Gauss-Legendre points to achieve high-order accuracy. The time evolution equations to update the unknowns are derived under the flux reconstruction (FR) framework (Huynh, 2007). Constraint conditions used to build the spatial reconstruction for the flux function include the pointwise values of flux function at the solution points, which are computed directly from the dependent variables, as well as the numerical fluxes at the boundaries of the computational element, which are obtained as Riemann solutions between the adjacent elements. Given the reconstructed flux function, the time tendencies of the unknowns can be obtained directly from the governing equations of differential form. The resulting schemes have super convergence and rigorous numerical conservativeness. A three-point scheme of fifth-order accuracy is presented and analyzed in this paper. The proposed scheme is adopted to develop the global shallow-water model on the cubed-sphere grid, where the local high-order reconstruction is very beneficial for the data communications between adjacent patches. We have used the standard benchmark tests to verify the numerical model, which reveals its great potential as a candidate formulation for developing high-performance general circulation models.

  10. Exploring the diploid wheat ancestral A genome through sequence comparison at the high-molecular-weight glutenin locus region.

    PubMed

    Dong, Lingli; Huo, Naxin; Wang, Yi; Deal, Karin; Luo, Ming-Cheng; Wang, Daowen; Anderson, Olin D; Gu, Yong Qiang

    2012-12-01

    The polyploid nature of hexaploid wheat (T. aestivum, AABBDD) often represents a great challenge in various aspects of research including genetic mapping, map-based cloning of important genes, and sequencing and accurately assembly of its genome. To explore the utility of ancestral diploid species of polyploid wheat, sequence variation of T. urartu (A(u)A(u)) was analyzed by comparing its 277-kb large genomic region carrying the important Glu-1 locus with the homologous regions from the A genomes of the diploid T. monococcum (A(m)A(m)), tetraploid T. turgidum (AABB), and hexaploid T. aestivum (AABBDD). Our results revealed that in addition to a high degree of the gene collinearity, nested retroelement structures were also considerably conserved among the A(u) genome and the A genomes in polyploid wheats, suggesting that the majority of the repetitive sequences in the A genomes of polyploid wheats originated from the diploid A(u) genome. The difference in the compared region between A(u) and A is mainly caused by four differential TE insertion and two deletion events between these genomes. The estimated divergence time of A genomes calculated on nucleotide substitution rate in both shared TEs and collinear genes further supports the closer evolutionary relationship of A to A(u) than to A(m). The structure conservation in the repetitive regions promoted us to develop repeat junction markers based on the A(u) sequence for mapping the A genome in hexaploid wheat. Eighty percent of these repeat junction markers were successfully mapped to the corresponding region in hexaploid wheat, suggesting that T. urartu could serve as a useful resource for developing molecular markers for genetic and breeding studies in hexaploid wheat. PMID:23052831

  11. Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits

    PubMed Central

    Ravel, Catherine; Fiquet, Samuel; Boudet, Julie; Dardevet, Mireille; Vincent, Jonathan; Merlino, Marielle; Michard, Robin; Martre, Pierre

    2014-01-01

    The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs) in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS) are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules (CCRMs) including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs) and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator). In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression. PMID:25429295

  12. Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing.

    PubMed

    Yamamoto, F; Höglund, B; Fernandez-Vina, M; Tyan, D; Rastrou, M; Williams, T; Moonsamy, P; Goodridge, D; Anderson, M; Erlich, H A; Holcomb, C L

    2015-12-01

    Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing. PMID:26037172

  13. Identification of cDNAs encoding viper venom hyaluronidases: cross-generic sequence conservation of full-length and unusually short variant transcripts.

    PubMed

    Harrison, Robert A; Ibison, Frances; Wilbraham, Davina; Wagstaff, Simon C

    2007-05-01

    The immobilisation of prey by snakes is most efficiently achieved by the rapid dissemination of venom from its site of injection into the blood stream. Hyaluronidase is a common component of snake venoms and has been termed the "venom spreading factor". In the absence of nucleotide or protein sequence data to confirm the functional identity of this venom component, we interrogated a venom gland EST database for the saw-scaled viper, Echis ocellatus (Nigeria), using the gene ontology (GO) term "carbohydrate metabolism". A single hyalurononglucosaminadase-activity matching sequence (EOC00242) was found and used to design PCR primers to acquire the full-length cDNA sequence. Although very different from the bee venom and mammalian hyaluronidase sequences, the E. ocellatus sequence retained all the catalytic, positional and structural residues that characterise this class of carbohydrate metabolising hydrolases. An extraordinarily high level of sequence identity (>95%) was observed in analogous venom gland cDNA sequences isolated (by PCR) from another saw-scaled viper species, E. pyramidum leakeyi (Kenya), and from the sahara horned viper, Cerastes cerastes cerastes (Egypt) and the puff adder, Bitis arietans (Nigeria). Smaller amplicons, lacking hyaluronidase catalytic residues because of 768 bp or 855 bp central deletions, appear to encode either truncated peptides without hyaluronidase activity, or are non-translated transcripts because they lack consensus translation initiating motifs. PMID:17210232

  14. HetMappsS: Heterozygous mapping strategy for high resolution Genotyping-by-Sequencing Markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reduced representation genotyping approaches, such as genotyping-by-sequencing (GBS), provide opportunities to generate high-resolution genetic maps at a low per-sample cost. However, missing data and non-uniform sequence coverage can complicate map creation in highly heterozygous species. To facili...

  15. MicroRNA from Moringa oleifera: Identification by High Throughput Sequencing and Their Potential Contribution to Plant Medicinal Value

    PubMed Central

    Pirrò, Stefano; Zanella, Letizia; Kenzo, Maurice; Montesano, Carla; Minutolo, Antonella; Potestà, Marina; Sobze, Martin Sanou; Canini, Antonella; Cirilli, Marco; Muleo, Rosario; Colizzi, Vittorio; Galgani, Andrea

    2016-01-01

    Moringa oleifera is a widespread plant with substantial nutritional and medicinal value. We postulated that microRNAs (miRNAs), which are endogenous, noncoding small RNAs regulating gene expression at the post-transcriptional level, might contribute to the medicinal properties of plants of this species after ingestion into human body, regulating human gene expression. However, the knowledge is scarce about miRNA in Moringa. Furthermore, in order to test the hypothesis on the pharmacological potential properties of miRNA, we conducted a high-throughput sequencing analysis using the Illumina platform. A total of 31,290,964 raw reads were produced from a library of small RNA isolated from M. oleifera seeds. We identified 94 conserved and two novel miRNAs that were validated by qRT-PCR assays. Results from qRT-PCR trials conducted on the expression of 20 Moringa miRNA showed that are conserved across multiple plant species as determined by their detection in tissue of other common crop plants. In silico analyses predicted target genes for the conserved miRNA that in turn allowed to relate the miRNAs to the regulation of physiological processes. Some of the predicted plant miRNAs have functional homology to their mammalian counterparts and regulated human genes when they were transfected into cell lines. To our knowledge, this is the first report of discovering M. oleifera miRNAs based on high-throughput sequencing and bioinformatics analysis and we provided new insight into a potential cross-species control of human gene expression. The widespread cultivation and consumption of M. oleifera, for nutritional and medicinal purposes, brings humans into close contact with products and extracts of this plant species. The potential for miRNA transfer should be evaluated as one possible mechanism of action to account for beneficial properties of this valuable species. PMID:26930203

  16. MicroRNA from Moringa oleifera: Identification by High Throughput Sequencing and Their Potential Contribution to Plant Medicinal Value.

    PubMed

    Pirrò, Stefano; Zanella, Letizia; Kenzo, Maurice; Montesano, Carla; Minutolo, Antonella; Potestà, Marina; Sobze, Martin Sanou; Canini, Antonella; Cirilli, Marco; Muleo, Rosario; Colizzi, Vittorio; Galgani, Andrea

    2016-01-01

    Moringa oleifera is a widespread plant with substantial nutritional and medicinal value. We postulated that microRNAs (miRNAs), which are endogenous, noncoding small RNAs regulating gene expression at the post-transcriptional level, might contribute to the medicinal properties of plants of this species after ingestion into human body, regulating human gene expression. However, the knowledge is scarce about miRNA in Moringa. Furthermore, in order to test the hypothesis on the pharmacological potential properties of miRNA, we conducted a high-throughput sequencing analysis using the Illumina platform. A total of 31,290,964 raw reads were produced from a library of small RNA isolated from M. oleifera seeds. We identified 94 conserved and two novel miRNAs that were validated by qRT-PCR assays. Results from qRT-PCR trials conducted on the expression of 20 Moringa miRNA showed that are conserved across multiple plant species as determined by their detection in tissue of other common crop plants. In silico analyses predicted target genes for the conserved miRNA that in turn allowed to relate the miRNAs to the regulation of physiological processes. Some of the predicted plant miRNAs have functional homology to their mammalian counterparts and regulated human genes when they were transfected into cell lines. To our knowledge, this is the first report of discovering M. oleifera miRNAs based on high-throughput sequencing and bioinformatics analysis and we provided new insight into a potential cross-species control of human gene expression. The widespread cultivation and consumption of M. oleifera, for nutritional and medicinal purposes, brings humans into close contact with products and extracts of this plant species. The potential for miRNA transfer should be evaluated as one possible mechanism of action to account for beneficial properties of this valuable species. PMID:26930203

  17. Higher-order structure of bovine mitochondrial tRNA(Phe) lacking the 'conserved' GG and T psi CG sequences as inferred by enzymatic and chemical probing.

    PubMed Central

    Wakita, K; Watanabe, Y; Yokogawa, T; Kumazawa, Y; Nakamura, S; Ueda, T; Watanabe, K; Nishikawa, K

    1994-01-01

    Bovine mitochondrial (mt) phenylalanine tRNA (tRNA(Phe)), which lacks the 'conserved' GG and T psi YCG sequences, was efficiently purified by the selective hybridization method using a solid phase DNA probe. The entire nucleotide sequence of the tRNA, including modified nucleotides, was determined and its higher-order structure was investigated using RNaseT2 and chemical reagents as structural probes. The D and T loop regions as well as the anticodon loop region were accessible to RNaseT2, and the N-3 positions of cytidines present in the D and T loops were easily modified under the native conditions in the presence of 10mM Mg2+. On the other hand, the nucleotides present in the extra loop were protected from the chemical modification under the native conditions. From the results of these probing analyses and a comparison of the sequences of mitochondrial tRNA(Phe) genes from various organisms, it was inferred that bovine mt tRNA(Phe) lacks the D loop/T loop tertiary interactions, but does have the canonical extra loop/D stem interactions, which seem to be the main factor for bovine mt tRNA(Phe) to preserve its L-shaped higher-order structure. Images PMID:7510390

  18. High resolution numerical simulation of the linearized Euler equations in conservation law form

    NASA Technical Reports Server (NTRS)

    Sreenivas, Kidambi; Whitfield, David L.; Huff, Dennis L.

    1993-01-01

    A linearized Euler solver based on a high resolution numerical scheme is presented. The approach is to linearize the flux vector as opposed to carrying through the complete linearization analysis with the dependent variable vector written as a sum of the mean and the perturbed flow. This allows the linearized equations to be maintained in conservation law form. The linearized equations are used to compute unsteady flows in turbomachinery blade rows arising due to blade vibrations. Numerical solutions are compared to theoretical results (where available) and to numerical solutions of the nonlinear Euler equations.

  19. Symmetrization of conservation laws with entropy for high-temperature hypersonic computations

    NASA Technical Reports Server (NTRS)

    Chalot, F.; Hughes, T. J. R.; Shakib, F.

    1990-01-01

    Results of Hughes, France, and Mallet are generalized to conservation law systems taking into account high-temperature effects. Symmetric forms of different equation sets are derived in terms of entropy variables. First, the case of a general divariant gas is studied; it can be specialized to the usual Navier-Stokes equations, as well as to situations where the gas is vibrationally excited, and undergoes equilibrium chemical reactions. The case of gas in thermochemical nonequilibrium is considered next. Transport phenomena, and in particular mass diffusion, are examined in the framework of symmetric advective-diffusive systems.

  20. Effects of species combination on comparative analyses of conserved regulatory elements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cross-species DNA sequence comparison is the primary approach to discover regulatory elements by identifying highly conserved sequences due to evolutionary constraints. Previously, we reported that a systematic approach, combining position-specific weight matrixes (JASPAR) and phylogenetic footprint...

  1. Systematic identification of conserved regulatory elements in upstream promoter regions of the cattle genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cross-species DNA sequence comparison is the primary approach to discover regulatory elements by identifying highly conserved sequences due to evolutionary constraints. Previously, we reported that a systematic approach, combining position-specific weight matrixes (JASPAR) and phylogenetic footprint...

  2. Prediction of conserved regulatory elements in promoter regions of the cattle genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cross-species DNA sequence comparison is the primary approach to discover regulatory elements by identifying highly conserved sequences due to evolutionary constraints. Previously, we reported that a systematic approach, combining position-specific weight matrixes (JASPAR) and phylogenetic footprint...

  3. Structure of the Pseudokinase VRK3 Reveals a Degraded Catalytic Site, a Highly Conserved Kinase Fold, and a Putative Regulatory Binding Site

    PubMed Central

    Scheeff, Eric D.; Eswaran, Jeyanthy; Bunkoczi, Gabor; Knapp, Stefan; Manning, Gerard

    2009-01-01

    Summary About 10% of all protein kinases are predicted to be enzymatically inactive pseudokinases, but the structural details of kinase inactivation have remained unclear. We present the first structure of a pseudokinase, VRK3, and that of its closest active relative, VRK2. Profound changes to the active site region underlie the loss of catalytic activity, and VRK3 cannot bind ATP because of residue substitutions in the binding pocket. However, VRK3 still shares striking structural similarity with VRK2, and appears to be locked in a pseudoactive conformation. VRK3 also conserves residue interactions that are surprising in the absence of enzymatic function; these appear to play important architectural roles required for the residual functions of VRK3. Remarkably, VRK3 has an “inverted” pattern of sequence conservation: although the active site is poorly conserved, portions of the molecular surface show very high conservation, suggesting that they form key interactions that explain the evolutionary retention of VRK3. PMID:19141289

  4. Identification and Characterization of miRNAs in Chondrus crispus by High-Throughput Sequencing and Bioinformatics Analysis.

    PubMed

    Gao, Fan; Nan, FangRu; Song, Wei; Feng, Jia; Lv, JunPing; Xie, ShuLian

    2016-01-01

    Chondrus crispus, an economically and medicinally important red alga, is a medicinally active substance and important for anti-tumor research. In this study, 117 C. crispus miRNAs (108 conserved and 9 novel) were identified from 2,416,181 small-RNA reads using high-throughput sequencing and bioinformatics methods. According to the BLAST search against the miRBase database, these miRNAs belonged to 110 miRNA families. Sequence alignment combined with homology searching revealed both the conservation and diversity of predicted potential miRNA families in different plant species. Four and 19 randomly selected miRNAs were validated by northern blotting and stem-loop quantitative real-time reverse transcription polymerase chain reaction detection, respectively. The validation rates (75% and 94.7%) demonstrated that most of the identified miRNAs could be credible. A total of 160 potential target genes were predicted and functionally annotated by Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis. We also analyzed the interrelationship of miRNAs, miRNA-target genes and target genes in C. crispus by constructing a Cytoscape network. The 117 miRNAs identified in our study should supply large quantities of information that will be important for red algae small RNA research. PMID:27193824

  5. Identification and Characterization of miRNAs in Chondrus crispus by High-Throughput Sequencing and Bioinformatics Analysis

    PubMed Central

    Gao, Fan; Nan, FangRu; Song, Wei; Feng, Jia; Lv, JunPing; Xie, ShuLian

    2016-01-01

    Chondrus crispus, an economically and medicinally important red alga, is a medicinally active substance and important for anti-tumor research. In this study, 117 C. crispus miRNAs (108 conserved and 9 novel) were identified from 2,416,181 small-RNA reads using high-throughput sequencing and bioinformatics methods. According to the BLAST search against the miRBase database, these miRNAs belonged to 110 miRNA families. Sequence alignment combined with homology searching revealed both the conservation and diversity of predicted potential miRNA families in different plant species. Four and 19 randomly selected miRNAs were validated by northern blotting and stem-loop quantitative real-time reverse transcription polymerase chain reaction detection, respectively. The validation rates (75% and 94.7%) demonstrated that most of the identified miRNAs could be credible. A total of 160 potential target genes were predicted and functionally annotated by Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis. We also analyzed the interrelationship of miRNAs, miRNA-target genes and target genes in C. crispus by constructing a Cytoscape network. The 117 miRNAs identified in our study should supply large quantities of information that will be important for red algae small RNA research. PMID:27193824

  6. High-throughput sequencing of black pepper root transcriptome

    PubMed Central

    2012-01-01

    Background Black pepper (Piper nigrum L.) is one of the most popular spices in the world. It is used in cooking and the preservation of food and even has medicinal properties. Losses in production from disease are a major limitation in the culture of this crop. The major diseases are root rot and foot rot, which are results of root infection by Fusarium solani and Phytophtora capsici, respectively. Understanding the molecular interaction between the pathogens and the host’s root region is important for obtaining resistant cultivars by biotechnological breeding. Genetic and molecular data for this species, though, are limited. In this paper, RNA-Seq technology has been employed, for the first time, to describe the root transcriptome of black pepper. Results The root transcriptome of black pepper was sequenced by the NGS SOLiD platform and assembled using the multiple-k method. Blast2Go and orthoMCL methods were used to annotate 10338 unigenes. The 4472 predicted proteins showed about 52% homology with the Arabidopsis proteome. Two root proteomes identified 615 proteins, which seem to define the plant’s root pattern. Simple-sequence repeats were identified that may be useful in studies of genetic diversity and may have applications in biotechnology and ecology. Conclusions This dataset of 10338 unigenes is crucially important for the biotechnological breeding of black pepper and the ecogenomics of the Magnoliids, a major group of basal angiosperms. PMID:22984782

  7. Genome reassembly with high-throughput sequencing data

    PubMed Central

    2013-01-01

    Motivation Recent studies in genomics have highlighted the significance of structural variation in determining individual variation. Current methods for identifying structural variation, however, are predominantly focused on either assembling whole genomes from scratch, or identifying the relatively small changes between a genome and a reference sequence. While significant progress has been made in recent years on both de novo assembly and resequencing (read mapping) methods, few attempts have been made to bridge the gap between them. Results In this paper, we present a computational method for incorporating a reference sequence into an assembly algorithm. We propose a novel graph construction that builds upon the well-known de Bruijn graph to incorporate the reference, and describe a simple algorithm, based on iterative message passing, which uses this information to significantly improve assembly results. We validate our method by applying it to a series of 5 Mb simulation genomes derived from both mammalian and bacterial references. The results of applying our method to this simulation data are presented along with a discussion of the benefits and drawbacks of this technique. PMID:23368744

  8. Low-intensity agricultural landscapes in Transylvania support high butterfly diversity: implications for conservation.

    PubMed

    Loos, Jacqueline; Dorresteijn, Ine; Hanspach, Jan; Fust, Pascal; Rakosy, László; Fischer, Joern

    2014-01-01

    European farmland biodiversity is declining due to land use changes towards agricultural intensification or abandonment. Some Eastern European farming systems have sustained traditional forms of use, resulting in high levels of biodiversity. However, global markets and international policies now imply rapid and major changes to these systems. To effectively protect farmland biodiversity, understanding landscape features which underpin species diversity is crucial. Focusing on butterflies, we addressed this question for a cultural-historic landscape in Southern Transylvania, Romania. Following a natural experiment, we randomly selected 120 survey sites in farmland, 60 each in grassland and arable land. We surveyed butterfly species richness and abundance by walking transects with four repeats in summer 2012. We analysed species composition using Detrended Correspondence Analysis. We modelled species richness, richness of functional groups, and abundance of selected species in response to topography, woody vegetation cover and heterogeneity at three spatial scales, using generalised linear mixed effects models. Species composition widely overlapped in grassland and arable land. Composition changed along gradients of heterogeneity at local and context scales, and of woody vegetation cover at context and landscape scales. The effect of local heterogeneity on species richness was positive in arable land, but negative in grassland. Plant species richness, and structural and topographic conditions at multiple scales explained species richness, richness of functional groups and species abundances. Our study revealed high conservation value of both grassland and arable land in low-intensity Eastern European farmland. Besides grassland, also heterogeneous arable land provides important habitat for butterflies. While butterfly diversity in arable land benefits from heterogeneity by small-scale structures, grasslands should be protected from fragmentation to provide

  9. Low-Intensity Agricultural Landscapes in Transylvania Support High Butterfly Diversity: Implications for Conservation

    PubMed Central

    Loos, Jacqueline; Dorresteijn, Ine; Hanspach, Jan; Fust, Pascal; Rakosy, László; Fischer, Joern

    2014-01-01

    European farmland biodiversity is declining due to land use changes towards agricultural intensification or abandonment. Some Eastern European farming systems have sustained traditional forms of use, resulting in high levels of biodiversity. However, global markets and international policies now imply rapid and major changes to these systems. To effectively protect farmland biodiversity, understanding landscape features which underpin species diversity is crucial. Focusing on butterflies, we addressed this question for a cultural-historic landscape in Southern Transylvania, Romania. Following a natural experiment, we randomly selected 120 survey sites in farmland, 60 each in grassland and arable land. We surveyed butterfly species richness and abundance by walking transects with four repeats in summer 2012. We analysed species composition using Detrended Correspondence Analysis. We modelled species richness, richness of functional groups, and abundance of selected species in response to topography, woody vegetation cover and heterogeneity at three spatial scales, using generalised linear mixed effects models. Species composition widely overlapped in grassland and arable land. Composition changed along gradients of heterogeneity at local and context scales, and of woody vegetation cover at context and landscape scales. The effect of local heterogeneity on species richness was positive in arable land, but negative in grassland. Plant species richness, and structural and topographic conditions at multiple scales explained species richness, richness of functional groups and species abundances. Our study revealed high conservation value of both grassland and arable land in low-intensity Eastern European farmland. Besides grassland, also heterogeneous arable land provides important habitat for butterflies. While butterfly diversity in arable land benefits from heterogeneity by small-scale structures, grasslands should be protected from fragmentation to provide

  10. High order filtering methods for approximating hyberbolic systems of conservation laws

    NASA Technical Reports Server (NTRS)

    Lafon, F.; Osher, S.

    1990-01-01

    In the computation of discontinuous solutions of hyperbolic systems of conservation laws, the recently developed essentially non-oscillatory (ENO) schemes appear to be very useful. However, they are computationally costly compared to simple central difference methods. A filtering method which is developed uses simple central differencing of arbitrarily high order accuracy, except when a novel local test indicates the development of spurious oscillations. At these points, the full ENO apparatus is used, maintaining the high order of accuracy, but removing spurious oscillations. Numerical results indicate the success of the method. High order of accuracy was obtained in regions of smooth flow without spurious oscillations for a wide range of problems and a significant speed up of generally a factor of almost three over the full ENO method.

  11. The first complete mitochondrial genome sequences of Amblypygi (Chelicerata: Arachnida) reveal conservation of the ancestral arthropod gene order.

    PubMed

    Fahrein, Kathrin; Masta, Susan E; Podsiadlowski, Lars

    2009-05-01

    Amblypygi (whip spiders) are terrestrial chelicerates inhabiting the subtropics and tropics. In morphological and rRNA-based phylogenetic analyses, Amblypygi cluster with Uropygi (whip scorpions) and Araneae (spiders) to form the taxon Tetrapulmonata, but there is controversy regarding the interrelationship of these three taxa. Mitochondrial genomes provide an additional large data set of phylogenetic information (sequences, gene order, RNA secondary structure), but in arachnids, mitochondrial genome data are missing for some of the major orders. In the course of an ongoing project concerning arachnid mitochondrial genomics, we present the first two complete mitochondrial genomes from Amblypygi. Both genomes were found to be typical circular duplex DNA molecules with all 37 genes usually present in bilaterian mitochondrial genomes. In both species, gene order is identical to that of Limulus polyphemus (Xiphosura), which is assumed to reflect the putative arthropod ground pattern. All tRNA gene sequences have the potential to fold into structures that are typical of metazoan mitochondrial tRNAs, except for tRNA-Ala, which lacks the D arm in both amblypygids, suggesting the loss of this feature early in amblypygid evolution. Phylogenetic analysis resulted in weak support for Uropygi being the sister group of Amblypygi. PMID:19448726

  12. Deep Sequencing of Mixed Total DNA without Barcodes Allows Efficient Assembly of Highly Plastic Ascidian Mitochondrial Genomes

    PubMed Central

    Rubinstein, Nimrod D.; Feldstein, Tamar; Shenkar, Noa; Botero-Castro, Fidel; Griggio, Francesca; Mastrototaro, Francesco; Delsuc, Frédéric; Douzery, Emmanuel J.P.; Gissi, Carmela; Huchon, Dorothée

    2013-01-01

    Ascidians or sea squirts form a diverse group within chordates, which includes a few thousand members of marine sessile filter-feeding animals. Their mitochondrial genomes are characterized by particularly high evolutionary rates and rampant gene rearrangements. This extreme variability complicates standard polymerase chain reaction (PCR) based techniques for molecular characterization studies, and consequently only a few complete Ascidian mitochondrial genome sequences are available. Using the standard PCR and Sanger sequencing approach, we produced the mitochondrial genome of Ascidiella aspersa only after a great effort. In contrast, we produced five additional mitogenomes (Botrylloides aff. leachii, Halocynthia spinosa, Polycarpa mytiligera, Pyura gangelion, and Rhodosoma turcicum) with a novel strategy, consisting in sequencing the pooled total DNA samples of these five species using one Illumina HiSeq 2000 flow cell lane. Each mitogenome was efficiently assembled in a single contig using de novo transcriptome assembly, as de novo genome assembly generally performed poorly for this task. Each of the new six mitogenomes presents a different and novel gene order, showing that no syntenic block has been conserved at the ordinal level (in Stolidobranchia and in Phlebobranchia). Phylogenetic analyses support the paraphyly of both Ascidiacea and Phlebobranchia, with Thaliacea nested inside Phlebobranchia, although the deepest nodes of the Phlebobranchia–Thaliacea clade are not well resolved. The strategy described here thus provides a cost-effective approach to obtain complete mitogenomes characterized by a highly plastic gene order and a fast nucleotide/amino acid substitution rate. PMID:23709623

  13. Genome-wide synteny through highly sensitive sequence alignment: Satsuma

    PubMed Central

    Grabherr, Manfred G.; Russell, Pamela; Meyer, Miriah; Mauceli, Evan; Alföldi, Jessica; Di Palma, Federica; Lindblad-Toh, Kerstin

    2010-01-01

    Motivation: Comparative genomics heavily relies on alignments of large and often complex DNA sequences. From an engineering perspective, the problem here is to provide maximum sensitivity (to find all there is to find), specificity (to only find real homology) and speed (to accommodate the billions of base pairs of vertebrate genomes). Results: Satsuma addresses all three issues through novel strategies: (i) cross-correlation, implemented via fast Fourier transform; (ii) a match scoring scheme that eliminates almost all false hits; and (iii) an asynchronous ‘battleship’-like search that allows for aligning two entire fish genomes (470 and 217 Mb) in 120 CPU hours using 15 processors on a single machine. Availability: Satsuma is part of the Spines software package, implemented in C++ on Linux. The latest version of Spines can be freely downloaded under the LGPL license from http://www.broadinstitute.org/science/programs/genome-biology/spines/ Contact: grabherr@broadinstitute.org PMID:20208069

  14. High-resolution network biology: connecting sequence with function

    PubMed Central

    Ryan, Colm J.; Cimermančič, Peter; Szpiech, Zachary A.; Sali, Andrej; Hernandez, Ryan D.; Krogan, Nevan J.

    2014-01-01

    Proteins are not monolithic entities; rather, they can contain multiple domains that mediate distinct interactions, and their functionality can be regulated through post-translational modifications at multiple distinct sites. Traditionally, network biology has ignored such properties of proteins and has instead examined either the physical interactions of whole proteins or the consequences of removing entire genes. In this Review, we discuss experimental and computational methods to increase the resolution of protein– protein, genetic and drug–gene interaction studies to the domain and residue levels. Such work will be crucial for using interaction networks to connect sequence and structural information, and to understand the biological consequences of disease-associated mutations, which will hopefully lead to more effective therapeutic strategies. PMID:24197012

  15. High-throughput-sequencing-based identification of a grapevine fanleaf virus satellite RNA in Vitis vinifera.

    PubMed

    Chiumenti, Michela; Mohorianu, Irina; Roseti, Vincenzo; Saldarelli, Pasquale; Dalmay, Tamas; Minafra, Angelantonio

    2016-05-01

    A new satellite RNA (satRNA) of grapevine fanleaf virus (GFLV) was identified by high-throughput sequencing of high-definition (HD) adapter libraries from grapevine plants of the cultivar Panse precoce (PPE) affected by enation disease. The complete nucleotide sequence was obtained by automatic sequencing using primers designed based on next-generation sequencing (NGS) data. The full-length sequence, named satGFLV-PPE, consisted of 1119 nucleotides with a single open reading frame from position 15 to 1034. This satRNA showed maximum nucleotide sequence identity of 87 % to satArMV-86 and satGFLV-R6. Symptomatic grapevines were surveyed for the presence of the satRNA, and no correlation was found between detection of the satRNA and enation symptom expression. PMID:26873812

  16. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega

    PubMed Central

    Sievers, Fabian; Wilm, Andreas; Dineen, David; Gibson, Toby J; Karplus, Kevin; Li, Weizhong; Lopez, Rodrigo; McWilliam, Hamish; Remmert, Michael; Söding, Johannes; Thompson, Julie D; Higgins, Desmond G

    2011-01-01

    Multiple sequence alignments are fundamental to many sequence analysis methods. Most alignments are computed using the progressive alignment heuristic. These methods are starting to become a bottleneck in some analysis pipelines when faced with data sets of the size of many thousands of sequences. Some methods allow computation of larger data sets while sacrificing quality, and others produce high-quality alignments, but scale badly with the number of sequences. In this paper, we describe a new program called Clustal Omega, which can align virtually any number of protein sequences quickly and that delivers accurate alignments. The accuracy of the package on smaller test cases is similar to that of the high-quality aligners. On larger data sets, Clustal Omega outperforms other packages in terms of execution time and quality. Clustal Omega also has powerful features for adding sequences to and exploiting information in existing alignments, making use of the vast amount of precomputed information in public databases like Pfam. PMID:21988835

  17. Amino acid binding by the class I aminoacyl-tRNA synthetases: role for a conserved proline in the signature sequence.

    PubMed Central

    Burbaum, J. J.; Schimmel, P.

    1992-01-01

    Although partial or complete three-dimensional structures are known for three Class I aminoacyl-tRNA synthetases, the amino acid-binding sites in these proteins remain poorly characterized. To explore the methionine binding site of Escherichia coli methionyl-tRNA synthetase, we chose to study a specific, randomly generated methionine auxotroph that contains a mutant methionyl-tRNA synthetase whose defect is manifested in an elevated Km for methionine (Barker, D.G., Ebel, J.-P., Jakes, R.C., & Bruton, C.J., 1982, Eur. J. Biochem. 127, 449-457), and employed the polymerase chain reaction to sequence this mutant synthetase directly. We identified a Pro 14 to Ser replacement (P14S), which accounts for a greater than 300-fold elevation in Km for methionine and has little effect on either the Km for ATP or the kcat of the amino acid activation reaction. This mutation destabilizes the protein in vivo, which may partly account for the observed auxotrophy. The altered proline is found in the "signature sequence" of the Class I synthetases and is conserved. This sequence motif is 1 of 2 found in the 10 Class I aminoacyl-tRNA synthetases and, in the known structures, it is in the nucleotide-binding fold as part of a loop between the end of a beta-strand and the start of an alpha-helix. The phenotype of the mutant and the stability and affinity for methionine of the wild-type and mutant enzymes are influenced by the amino acid that is 25 residues beyond the C-terminus of the signature sequence.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1304356

  18. Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing

    PubMed Central

    Chandran, Anandhakumar; Syed, Junetha; Taylor, Rhys D.; Kashiwazaki, Gengo; Sato, Shinsuke; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

    2016-01-01

    Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding. However, the binding mechanism of PIPs at the chromatinized genome level is poorly understood. Herein, we report a method using high-throughput sequencing to identify the DNA-alkylating sites of PIP-indole-seco-CBI conjugates. High-throughput sequencing analysis of conjugate 2 showed highly similar DNA-alkylating sites on synthetic oligos (histone-free DNA) and on human genomes (chromatinized DNA context). To our knowledge, this is the first report identifying alkylation sites across genomic DNA by alkylating PIP conjugates using high-throughput sequencing. PMID:27098039

  19. Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing.

    PubMed

    Chandran, Anandhakumar; Syed, Junetha; Taylor, Rhys D; Kashiwazaki, Gengo; Sato, Shinsuke; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

    2016-05-19

    Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding. However, the binding mechanism of PIPs at the chromatinized genome level is poorly understood. Herein, we report a method using high-throughput sequencing to identify the DNA-alkylating sites of PIP-indole-seco-CBI conjugates. High-throughput sequencing analysis of conjugate 2: showed highly similar DNA-alkylating sites on synthetic oligos (histone-free DNA) and on human genomes (chromatinized DNA context). To our knowledge, this is the first report identifying alkylation sites across genomic DNA by alkylating PIP conjugates using high-throughput sequencing. PMID:27098039

  20. Nosema ceranae alters a highly conserved hormonal stress pathway in honeybees.

    PubMed

    Mayack, C; Natsopoulou, M E; McMahon, D P

    2015-12-01

    Nosema ceranae, an emerging pathogen of the western honeybee (Apis mellifera), is implicated in recent pollinator losses and causes severe energetic stress. However, whether precocious foraging and accelerated behavioural maturation in infected bees are caused by the infection itself or via indirect energetic stress remains unknown. Using a combination of nutritional and infection treatments, we investigated how starvation and infection alters the regulation of adipokinetic hormone (AKH) and octopamine, two highly conserved physiological pathways that respond to energetic stress by mobilizing fat stores and increasing search activity for food. Although there was no response from AKH when bees were experimentally infected with N. ceranae or starved, supporting the notion that honeybees have lost this pathway, there were significant regulatory changes in the octopamine pathway. Significantly, we found no evidence of acute energetic stress being the only cause of symptoms associated with N. ceranae infection. Therefore, the parasite itself appears to alter regulatory components along a highly conserved physiological pathway in an infection-specific manner. This indicates that pathogen-induced behavioural alteration of chronically infected bees should not just be viewed as a coincidental short-term by-product of pathogenesis (acute energetic stress) and may be a result of a generalist manipulation strategy to obtain energy for reproduction. PMID:26335565

  1. High-Force Generation Is a Conserved Property of Type IV Pilus Systems▿

    PubMed Central

    Clausen, Martin; Jakovljevic, Vladimir; Søgaard-Andersen, Lotte; Maier, Berenike

    2009-01-01

    The type IV pilus (T4P) system of Neisseria gonorrhoeae is the strongest linear molecular motor reported to date, but it is unclear whether high-force generation is conserved between bacterial species. Using laser tweezers, we found that the average stalling force of single-pilus retraction in Myxococcus xanthus of 149 ± 14 pN exceeds the force generated by N. gonorrhoeae. Retraction velocities including a bimodal distribution were similar between M. xanthus and N. gonorrhoeae, but force-dependent directional switching was not. Force generation by pilus retraction is energized by the ATPase PilT. Surprisingly, an M. xanthus mutant lacking PilT apparently still retracted T4P, although at a reduced frequency. The retraction velocity was comparable to the high-velocity mode in the wild type at low forces but decreased drastically when the force increased, with an average stalling force of 70 ± 10 pN. Thus, M. xanthus harbors at least two different retraction motors. Our results demonstrate that the major physical properties are conserved between bacteria that are phylogenetically distant and pursue very different lifestyles. PMID:19429611

  2. Effects of the Conservation Reserve Program on Hydrologic Processes in the Southern High Plains

    NASA Astrophysics Data System (ADS)

    Haacker, E. M.; Smidt, S. J.; Kendall, A. D.; Basso, B.; Hyndman, D. W.

    2015-12-01

    The Southern High Plains Aquifer is a rapidly depleting resource that supports agriculture in parts of New Mexico and the Texas Panhandle. The development of the aquifer has changed the landscape and the water cycle of the region. This study illustrates the evolving patterns of land use and the effects of cultivation, from irrigated to dryland farming to the countermanding influence of the Conservation Reserve Program (CRP). Previous research indicates that greater recharge rates occur under cultivated land in the Southern High Plains than under unbroken soil: the transition to cultivation causes increased recharge, under both dryland and irrigated management, though most recharge still occurs through playa lakes. The Conservation Reserve Program takes land out of crop production, replacing the land cover with something more like the natural ecosystem. This may decrease recharge below fields, and reduce runoff that feeds playa lakes; or, CRP may help stabilize playa lakes, increasing recharge. Changes to the water cycle are investigated at the field scale using the System Approach to Land Use Sustainability (SALUS) crop model, and at the regional scale with the Landscape Hydrology Model (LHM), and compared with historical data and water table elevations.

  3. In vitro selection of RNase P RNA reveals optimized catalytic activity in a highly conserved structural domain.

    PubMed

    Frank, D N; Ellington, A E; Pace, N R

    1996-12-01

    In vitro selection techniques are useful means of dissecting the functions of both natural and artificial ribozymes. Using a self-cleaving conjugate containing the Escherichia coli ribonuclease P RNA and its substrate, pre-tRNA (Frank DN, Harris ME, Pace NR, 1994, Biochemistry 33:10800-10808), we have devised a method to select for catalytically active variants of the RNase P ribozyme. A selection experiment was performed to probe the structural and sequence constraints that operate on a highly conserved region of RNase P: the J3/4-P4-J2/4 region, which lies within the core of RNase P and is thought to bind catalytically essential magnesium ions (Harris ME et al., 1994, EMBO J 13:3953-3963; Hardt WD et al., 1995, EMBO J 14:2935-2944; Harris ME, Pace NR, 1995, RNA 1:210-218). We sought to determine which, if any, of the nearly invariant nucleotides within J3/4-P4-J2/4 are required for ribozyme-mediated catalysis. Twenty-two residues in the J3/4-P4-J2/4 component of RNase P RNA were randomized and, surprisingly, after only 10 generations, each of the randomized positions returned to the wild-type sequence. This indicates that every position in J3/4-P4-J2/4 contributes to optimal catalytic activity. These results contrast sharply with selections involving other large ribozymes, which evolve improved catalytic function readily in vitro (Chapman KB, Szostak JW, 1994, Curr Opin Struct Biol 4:618-622; Joyce GF, 1994, Curr Opin Struct Biol 4:331-336; Kumar PKR, Ellington AE, 1995, FASEB J 9:1183-1195). The phylogenetic conservation of J3/4-P4-J2/4, coupled with the results reported here, suggests that the contribution of this structure to RNA-mediated catalysis was optimized very early in evolution, before the last common ancestor of all life. PMID:8972768

  4. Sources of PCR-induced distortions in high-throughput sequencing data sets

    PubMed Central

    Kebschull, Justus M.; Zador, Anthony M.

    2015-01-01

    PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error—bias, stochasticity, template switches and polymerase errors—on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules. PMID:26187991

  5. FASTAptamer: A Bioinformatic Toolkit for High-throughput Sequence Analysis of Combinatorial Selections

    PubMed Central

    Alam, Khalid K; Chang, Jonathan L; Burke, Donald H

    2015-01-01

    High-throughput sequence (HTS) analysis of combinatorial selection populations accelerates lead discovery and optimization and offers dynamic insight into selection processes. An underlying principle is that selection enriches high-fitness sequences as a fraction of the population, whereas low-fitness sequences are depleted. HTS analysis readily provides the requisite numerical information by tracking the evolutionary trajectory of individual sequences in response to selection pressures. Unlike genomic data, for which a number of software solutions exist, user-friendly tools are not readily available for the combinatorial selections field, leading many users to create custom software. FASTAptamer was designed to address the sequence-level analysis needs of the field. The open source FASTAptamer toolkit counts, normalizes and ranks read counts in a FASTQ file, compares populations for sequence distribution, generates clusters of sequence families, calculates fold-enrichment of sequences throughout the course of a selection and searches for degenerate sequence motifs. While originally designed for aptamer selections, FASTAptamer can be applied to any selection strategy that can utilize next-generation DNA sequencing, such as ribozyme or deoxyribozyme selections, in vivo mutagenesis and various surface display technologies (peptide, antibody fragment, mRNA, etc.). FASTAptamer software, sample data and a user's guide are available for download at http://burkelab.missouri.edu/fastaptamer.html. PMID:25734917

  6. Sources of PCR-induced distortions in high-throughput sequencing data sets.

    PubMed

    Kebschull, Justus M; Zador, Anthony M

    2015-12-01

    PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error-bias, stochasticity, template switches and polymerase errors-on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules. PMID:26187991

  7. Whole-genome sequencing of six dog breeds from continuous altitudes reveals adaptation to high-altitude hypoxia

    PubMed Central

    Gou, Xiao; Wang, Zhen; Li, Ning; Qiu, Feng; Xu, Ze; Yan, Dawei; Yang, Shuli; Jia, Jia; Kong, Xiaoyan; Wei, Zehui; Lu, Shaoxiong; Lian, Linsheng; Wu, Changxin; Wang, Xueyan; Li, Guozhi; Ma, Teng; Jiang, Qiang; Zhao, Xue; Yang, Jiaqiang; Liu, Baohong; Wei, Dongkai; Li, Hong; Yang, Jianfa; Yan, Yulin; Zhao, Guiying; Dong, Xinxing; Li, Mingli; Deng, Weidong; Leng, Jing; Wei, Chaochun; Wang, Chuan; Mao, Huaming; Zhang, Hao; Ding, Guohui; Li, Yixue

    2014-01-01

    The hypoxic environment imposes severe selective pressure on species living at high altitude. To understand the genetic bases of adaptation to high altitude in dogs, we performed whole-genome sequencing of 60 dogs including five breeds living at continuous altitudes along the Tibetan Plateau from 800 to 5100 m as well as one European breed. More than 150× sequencing coverage for each breed provides us with a comprehensive assessment of the genetic polymorphisms of the dogs, including Tibetan Mastiffs. Comparison of the breeds from different altitudes reveals strong signals of population differentiation at the locus of hypoxia-related genes including endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) and beta hemoglobin cluster. Notably, four novel nonsynonymous mutations specific to high-altitude dogs are identified at EPAS1, one of which occurred at a quite conserved site in the PAS domain. The association testing between EPAS1 genotypes and blood-related phenotypes on additional high-altitude dogs reveals that the homozygous mutation is associated with decreased blood flow resistance, which may help to improve hemorheologic fitness. Interestingly, EPAS1 was also identified as a selective target in Tibetan highlanders, though no amino acid changes were found. Thus, our results not only indicate parallel evolution of humans and dogs in adaptation to high-altitude hypoxia, but also provide a new opportunity to study the role of EPAS1 in the adaptive processes. PMID:24721644

  8. The Moraxella catarrhalis immunoglobulin D-binding protein MID has conserved sequences and is regulated by a mechanism corresponding to phase variation.

    PubMed

    Möllenkvist, Andrea; Nordström, Therése; Halldén, Christer; Christensen, Jens Jørgen; Forsgren, Arne; Riesbeck, Kristian

    2003-04-01

    The prevalence of the Moraxella catarrhalis immunoglobulin D (IgD)-binding outer membrane protein MID and its gene was determined in 91 clinical isolates and in 7 culture collection strains. Eighty-four percent of the clinical Moraxella strains expressed MID-dependent IgD binding. The mid gene was detected in all strains as revealed by homology of the signal peptide sequence and a conserved area in the 3' end of the gene. When MID proteins from five different strains were compared, an identity of 65.3 to 85.0% and a similarity of 71.2 to 89.1% were detected. Gene analyses showed several amino acid repeat motifs in the open reading frames, and MID could be called a putative autotransport protein. Interestingly, homopolymeric [polyguanine [poly(G)

  9. Transcriptional trans activators of human and simian foamy viruses contain a small, highly conserved activation domain.

    PubMed Central

    Garrett, E D; He, F; Bogerd, H P; Cullen, B R

    1993-01-01

    The Bel-1 protein of human foamy virus is a potent transcriptional trans activator of its homologous long terminal repeat promoter element. Here, we demonstrate that Bel-1 can also efficiently activate gene expression when targeted to a heterologous promoter by fusion to the DNA-binding motif of the yeast GAL4 protein. Analysis of a series of deletion mutants of Bel-1 generated in this hybrid protein context suggests the presence of a single transcription activation domain that is fully contained within a discrete, approximately 30-amino-acid segment located proximal to the Bel-1 carboxy terminus. Although this short motif can be shown to function effectively in eukaryotic cells of mammalian, avian, and fungal origin, it does not bear any evident sequence homology to the known classes of eukaryotic activation domain. However, this Bel-1 activation domain was found to be fully conserved, in terms of both biological activity and location, in the distantly related Taf trans activator of simian foamy virus type 1. Images PMID:8411385

  10. (dC-dA)n.(dG-dT)n sequences have evolutionarily conserved chromosomal locations in Drosophila with implications for roles in chromosome structure and function.

    PubMed Central

    Pardue, M L; Lowenhaupt, K; Rich, A; Nordheim, A

    1987-01-01

    In situ hybridization of (dC-dA)n.(dG-dT)n to the polytene chromosomes of Drosophila melanogaster reveals a clearly non-random distribution of chromosomal sites for this sequence. Sites are distributed over most euchromatic regions but the density of sites along the X chromosome is significantly higher than the density over the autosomes. All autosomes show approximately equal levels of hybridization except chromosome 4 which has no detectable stretches of (dC-dA)n.(dG-dT)n. Another striking feature is the lack of hybridization of the beta-heterochromatin of the chromocenter. The specific sites are conserved between different strains of D. melanogaster. The same overall chromosomal pattern of hybridization is seen for the other Drosophila species studied, including D. simulans, a sibling species with a much lower content of middle repetitive DNA, and D. virilis, a distantly related species. The evolutionary conservation of the distribution of (dC-dA)n.(dG-dT)n suggests that these sequences are of functional importance. The distribution patterns seen for D. pseudoobscura and D. miranda raise interesting speculations about function. In these species a chromosome equivalent to an autosomal arm of D. melanogaster has been translocated onto the X chromosome and acquired dosage compensation. In each species the new arm of the X also has a higher density of (dC-dA)n.(dG-dT)n similar to that seen on other X chromosomes. In addition to correlations with dosage compensation, the depletion of (dC-dA)n.(dG-dT)n in beta-heterochromatin and chromosome 4 may also be related to the fact that these regions do not normally undergo meiotic recombination. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. Fig. 10. PMID:3111846

  11. A high-throughput de novo sequencing approach for shotgun proteomics using high-resolution tandem mass spectrometry

    SciTech Connect

    Pan, Chongle; Park, Byung H; McDonald, W Hayes; Carey, Patricia A; Banfield, Jillian F.; Verberkmoes, Nathan C; Hettich, Robert {Bob} L; Samatova, Nagiza F

    2010-01-01

    Background High-resolution tandem mass spectra can now be readily acquired with hybrid instruments, such as LTQ-Orbitrap and LTQ-FT, in high-throughput shotgun proteomics workflows. The improved spectral quality enables more accurate de novo sequencing for identification of post-translational modifications and amino acid polymorphisms. Results In this study, a new de novo sequencing algorithm, called Vonode, has been developed specifically for analysis of such high-resolution tandem mass spectra. To fully exploit the high mass accuracy of these spectra, a unique scoring system is proposed to evaluate sequence tags based primarily on mass accuracy information of fragment ions. Consensus sequence tags were inferred for 11,422 spectra with an average peptide length of 5.5 residues from a total of 40,297 input spectra acquired in a 24-hour proteomics measurement of Rhodopseudomonas palustris. The accuracy of inferred consensus sequence tags was 84%. According to our comparison, the performance of Vonode was shown to be superior to the PepNovo v2.0 algorithm, in terms of the number of de novo sequenced spectra and the sequencing accuracy. Conclusions Here, we improved de novo sequencing performance by developing a new algorithm specifically for high-resolution tandem mass spectral data. The Vonode algorithm is freely available for download at http://compbio.ornl.gov/Vonode.

  12. The budding yeast U5 snRNP Prp8 is a highly conserved protein which links RNA splicing with cell cycle progression.

    PubMed Central

    Shea, J E; Toyn, J H; Johnston, L H

    1994-01-01

    The dbf3 mutation was originally obtained in a screen for DNA synthesis mutants with a cell cycle phenotype in the budding yeast Saccharomyces cerevisiae. We have now isolated the DBF3 gene and found it to be an essential gene with an ORF of 7239 nucleotides, potentially encoding a large protein of 268 kDa. We also obtained an allele-specific high copy number suppressor of the dbf3-1 allele, encoded by the known SSB1 gene, a member of the Hsp70 family of heat shock proteins. The sequence of the Dbf3 protein is 58% identical over 2300 amino acid residues to a predicted protein from Caenorhabditis elegans. Furthermore, partial sequences with 61% amino acid sequence identity were deduced from two files of human cDNA in the EST nucleotide database so that Dbf3 is a highly conserved protein. The nucleotide sequence of DBF3 turned out to be identical to the yeast gene PRP8, which encodes a U5 snRNP required for pre-mRNA splicing. This surprising result led us to further characterise the phenotype of dbf3 which confirmed its role in the cell cycle and showed it to function early, around the time of S phase. This data suggests a hitherto unexpected link between pre-mRNA splicing and the cell cycle. Images PMID:7838707

  13. Comparative genomics reveals conserved positioning of essential genomic clusters in highly rearranged Thermococcales chromosomes

    PubMed Central

    Cossu, Matteo; Da Cunha, Violette; Toffano-Nioche, Claire; Forterre, Patrick; Oberto, Jacques

    2015-01-01

    The genomes of the 21 completely sequenced Thermococcales display a characteristic high level of rearrangements. As a result, the prediction of their origin and termination of replication on the sole basis of chromosomal DNA composition or skew is inoperative. Using a different approach based on biologically relevant sequences, we were able to determine oriC position in all 21 genomes. The position of dif, the site where chromosome dimers are resolved before DNA segregation could be predicted in 19 genomes. Computation of the core genome uncovered a number of essential gene clusters with a remarkably stable chromosomal position across species, in sharp contrast with the scrambled nature of their genomes. The active chromosomal reorganization of numerous genes acquired by horizontal transfer, mainly from mobile elements, could explain this phenomenon. PMID:26166067

  14. Strong conservation of rhoptry-associated-protein-1 (RAP-1) locus organization and sequence among Babesia isolates infecting sheep from China (Babesia motasi-like phylogenetic group).

    PubMed

    Niu, Qingli; Valentin, Charlotte; Bonsergent, Claire; Malandrin, Laurence

    2014-12-01

    Rhoptry-associated-protein 1 (RAP-1) is considered as a potential vaccine candidate due to its involvement in red blood cell invasion by parasites in the genus Babesia. We examined its value as a vaccine candidate by studying RAP-1 conservation in isolates of Babesia sp. BQ1 Ningxian, Babesia sp. Tianzhu and Babesia sp. Hebei, responsible for ovine babesiosis in different regions of China. The rap-1 locus in these isolates has very similar features to those described for Babesia sp. BQ1 Lintan, another Chinese isolate also in the B. motasi-like phylogenetic group, namely the presence of three types of rap-1 genes (rap-1a, rap-1b and rap-1c), multiple conserved rap-1b copies (5) interspaced with more or less variable rap-1a copies (6), and the 3' localization of one rap-1c. The isolates Babesia sp. Tianzhu, Babesia sp. BQ1 Lintan and Ningxian were almost identical (average nucleotide identity of 99.9%) over a putative locus of about 31 Kb, including the intergenic regions. Babesia sp. Hebei showed a similar locus organization but differed in the rap-1 locus sequence, for each gene and intergenic region, with an average nucleotide identity of 78%. Our results are in agreement with 18S rDNA phylogenetic studies performed on these isolates. However, in extremely closely related isolates the rap-1 locus seems more conserved (99.9%) than the 18S rDNA (98.7%), whereas in still closely related isolates the identities are much lower (78%) compared with the 18S rDNA (97.7%). The particularities of the rap-1 locus in terms of evolution, phylogeny, diagnosis and vaccine development are discussed. PMID:25200723

  15. The Histone Modification H3K27me3 Is Retained after Gene Duplication and Correlates with Conserved Noncoding Sequences in Arabidopsis

    PubMed Central

    Berke, Lidija; Snel, Berend

    2014-01-01

    The histone modification H3K27me3 is involved in repression of transcription and plays a crucial role in developmental transitions in both animals and plants. It is deposited by PRC2 (Polycomb repressive complex 2), a conserved protein complex. In Arabidopsis thaliana, H3K27me3 is found at 15% of all genes. These tend to encode transcription factors and other regulators important for development. However, it is not known how PRC2 is recruited to target loci nor how this set of target genes arose during Arabidopsis evolution. To resolve the latter, we integrated A. thaliana gene families with five independent genome-wide H3K27me3 data sets. Gene families were either significantly enriched or depleted of H3K27me3, showing a strong impact of shared ancestry to H3K27me3 distribution. To quantify this, we performed ancestral state reconstruction of H3K27me3 on phylogenetic trees of gene families. The set of H3K27me3-marked genes changed less than expected by chance, suggesting that H3K27me3 was retained after gene duplication. This retention suggests that the PRC2-recruiting signal could be encoded in the DNA and also conserved among certain duplicated genes. Indeed, H3K27me3-marked genes were overrepresented among paralogs sharing conserved noncoding sequences (CNSs) that are enriched with transcription factor binding sites. The association of upstream CNSs with H3K27me3-marked genes represents the first genome-wide connection between H3K27me3 and potential regulatory elements in plants. Thus, we propose that CNSs likely function as part of the PRC2 recruitment in plants. PMID:24567304

  16. Digital fragment analysis of short tandem repeats by high-throughput amplicon sequencing.

    PubMed

    Darby, Brian J; Erickson, Shay F; Hervey, Samuel D; Ellis-Felege, Susan N

    2016-07-01

    High-throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high-throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high-throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost-effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence-aware microsatellite data. PMID:27386092

  17. New insights on the transcriptional regulation of CD69 gene through a potent enhancer located in the conserved non-coding sequence 2.

    PubMed

    Laguna, Teresa; Notario, Laura; Pippa, Raffaella; Fontela, Miguel G; Vázquez, Berta N; Maicas, Miren; Aguilera-Montilla, Noemí; Corbí, Ángel L; Odero, María D; Lauzurica, Pilar

    2015-08-01

    The CD69 type II C-type lectin is one of the earliest indicators of leukocyte activation acting in lymphocyte migration and cytokine secretion. CD69 expression in hematopoietic lineage undergoes rapid changes depending on the cell-lineage, the activation state or the localization of the cell where it is expressed, suggesting a complex and tightly controlled regulation. Here we provide new insights on the transcriptional regulation of CD69 gene in mammal species. Through in silico studies, we analyzed several regulatory features of the 4 upstream conserved non-coding sequences (CNS 1-4) previously described, confirming a major function of CNS2 in the transcriptional regulation of CD69. In addition, multiple transcription binding sites are identified in the CNS2 region by DNA cross-species conservation analysis. By functional approaches we defined a core region of 226bp located within CNS2 as the main enhancer element of CD69 transcription in the hematopoietic cells analyzed. By chromatin immunoprecipitation, binding of RUNX1 to the core-CNS2 was shown in a T cell line. In addition, we found an activating but not essential role of RUNX1 in CD69 gene transcription by site-directed mutagenesis and RNA silencing, probably through the interaction with this potent enhancer specifically in the hematopoietic lineage. In summary, in this study we contribute with new evidences to the landscape of the transcriptional regulation of the CD69 gene. PMID:25801305

  18. Highly Conserved Histidine Plays a Dual Catalytic Role in Protein Splicing: a pKa Shift Mechanism

    PubMed Central

    Du, Zhenming; Shemella, Philip T.; Liu, Yangzhong; McCallum, Scott A.; Pereira, Brian; Nayak, Saroj K.; Belfort, Georges; Belfort, Marlene; Wang, Chunyu

    2009-01-01

    Protein splicing is a precise auto-catalytic process in which an intein excises itself from a precursor with the concomitant ligation of the flanking sequences. Protein splicing occurs through acid-base catalysis in which the ionization states of active site residues are crucial to the reaction mechanism. In inteins, several conserved histidines have been shown to play important roles in protein splicing, including the most conserved “B-block” histidine. In this study, we have combined NMR pKa determination with quantum mechanics/molecular mechanics (QM/MM) modeling to study engineered inteins from Mycobacterium tuberculosis (Mtu) RecA intein. We demonstrate a dramatic pKa shift for the invariant B-block histidine, the most conserved residue among inteins. The B-block histidine has a pKa of 7.3 ± 0.6 in a precursor and a pKa of < 3.5 in a spliced intein. The pKa values and QM/MM data suggest that the B-block histidine has a dual role in the acid-base catalysis of protein splicing. This histidine likely acts as a general base to initiate splicing with an acyl shift and then as a general acid to cause the breakdown of the scissile bond. The proposed pKa shift mechanism accounts for the biochemical data supporting the essential role for the B-block histidine and for the absolute sequence conservation of this residue. PMID:19630416

  19. High-performance teams in wildlife conservation: A species reintroduction and recovery example

    NASA Astrophysics Data System (ADS)

    Clark, Tim W.; Westrum, Ron

    1989-11-01

    Reintroduction of animals to the wild to establish free-ranging viable populations is a valuable conservation tool, but ecological skills alone are not enough to ensure a successful reintroduction; also needed to do the work are effectively designed and managed programs. This article suggests general guidelines for organizing and managing reintroduction programs, reviews some basic organizational issues, and considers ways to develop high-performance teams The need to integrate reintroduction programs into their larger interorganizational context is discussed. The reintroduction program's structure must be appropriate for its function and should be properly staffed, led, and buffered from its political environment It should process information well, learn rapidly from its own mistakes, and be creative A high-performance team devotes most of its energies to solving external rather than internal problems

  20. Developing High Quality Dec