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Sample records for highly conserved sequences

  1. 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

  2. An alternative oxidase monoclonal antibody recognises a highly conserved sequence among alternative oxidase subunits.

    PubMed

    Finnegan, P M; Wooding, A R; Day, D A

    1999-03-19

    The alternative oxidase is found in the inner mitochondrial membranes of plants and some fungi and protists. A monoclonal antibody raised against the alternative oxidase from the aroid lily Sauromatum guttatum has been used extensively to detect the enzyme in these organisms. Using an immunoblotting strategy, the antibody binding site has been localised to the sequence RADEAHHRDVNH within the soybean alternative oxidase 2 protein. Examination of sequence variants showed that A2 and residues C-terminal to H7 are required for recognition by the monoclonal antibody raised against the alternative oxidase. The recognition sequence is highly conserved among all alternative oxidase proteins and is absolutely conserved in 12 of 14 higher plant sequences, suggesting that this antibody will continue to be extremely useful in studying the expression and synthesis of the alternative oxidase.

  3. The preference of the mitochondrial endonuclease for a conserved sequence block in mitochondrial DNA is highly conserved during mammalian evolution.

    PubMed Central

    Low, R L; Buzan, J M; Couper, C L

    1988-01-01

    Endonuclease activity identified in crude preparations of rat and human heart mitochondria has each been partially purified and characterized. Both the rat and human activities purify as a single enzyme that closely resembles the endonuclease of bovine-heart mitochondria (Cummings, O.W. et. al. (1987) J. Biol. Chem. 262:2005-2015). All three enzymes, for example elute similarly during gel filtration and DNA-cellulose chromatography, and exhibit similar enzymatic properties. Although the nucleotide sequences of the mtDNAs indicate that there has occurred an unusual degree of divergence in the displacement-loop region during mammalian evolution, the nucleotide specificities of the mt endonucleases appear highly conserved and show a striking preference for an evolutionarily-conserved sequence tract that is located upstream from the heavy (H)-strand origin of DNA replication (OriH). Images PMID:3399407

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

    PubMed

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

    2012-04-01

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

  5. 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.

  6. 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

  7. A highly conserved repeated chromosomal sequence in the radioresistant bacterium Deinococcus radiodurans SARK.

    PubMed

    Lennon, E; Gutman, P D; Yao, H L; Minton, K W

    1991-03-01

    A DNA fragment containing a portion of a DNA damage-inducible gene from Deinococcus radiodurans SARK hybridized to numerous fragments of SARK genomic DNA because of a highly conserved repetitive chromosomal element. The element is of variable length, ranging from 150 to 192 bp, depending on the absence or presence of one or two 21-bp sequences located internally. A putative translational start site of the damage-inducible gene is within the reiterated element. The element contains dyad symmetries that suggest modes of transcriptional and/or translational control.

  8. High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire

    PubMed Central

    Hou, Xianliang; Lu, Chong; Chen, Sisi; Xie, Qian; Cui, Guangying; Chen, Jianing; Chen, Zhi; Wu, Zhongwen; Ding, Yulong; Ye, Ping; Dai, Yong; Diao, Hongyan

    2016-01-01

    Abstract The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals. Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR. Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides. Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination. PMID:26962778

  9. A highly conserved sequence is a novel gene involved in de novo vitamin B6 biosynthesis

    PubMed Central

    Ehrenshaft, Marilyn; Bilski, Piotr; Li, Ming Y.; Chignell, Colin F.; Daub, Margaret E.

    1999-01-01

    The Cercospora nicotianae SOR1 (singlet oxygen resistance) gene was identified previously as a gene involved in resistance of this fungus to singlet-oxygen-generating phototoxins. Although homologues to SOR1 occur in organisms in four kingdoms and encode one of the most highly conserved proteins yet identified, the precise function of this protein has, until now, remained unknown. We show that SOR1 is essential in pyridoxine (vitamin B6) synthesis in C. nicotianae and Aspergillus flavus, although it shows no homology to previously identified pyridoxine synthesis genes identified in Escherichia coli. Sequence database analysis demonstrated that organisms encode either SOR1 or E. coli pyridoxine biosynthesis genes, but not both, suggesting that there are two divergent pathways for de novo pyridoxine biosynthesis in nature. Pathway divergence appears to have occurred during the evolution of the eubacteria. We also present data showing that pyridoxine quenches singlet oxygen at a rate comparable to that of vitamins C and E, two of the most highly efficient biological antioxidants, suggesting a previously unknown role for pyridoxine in active oxygen resistance. PMID:10430950

  10. SVC: structured visualization of evolutionary sequence conservation.

    PubMed

    Roepcke, S; Fiziev, P; Seeburg, P H; Vingron, M

    2005-07-01

    We have developed a web application for the detailed analysis and visualization of evolutionary sequence conservation in complex vertebrate genes. Given a pair of orthologous genes, the protein-coding sequences are aligned. When these sequences are mapped back onto their encoding exons in the genomes, a scaffold of the conserved gene structure naturally emerges. Sequence similarity between exons and introns is analysed and embedded into the gene structure scaffold. The visualization on the SVC server provides detailed information about evolutionarily conserved features of these genes. It further allows concise representation of complex splice patterns in the context of evolutionary conservation. A particular application of our tool arises from the fact that around mRNA editing sites both exonic and intronic sequences are highly conserved. This aids in delineation of these sites. SVC is available at http://svc.molgen.mpg.de.

  11. Sequence conserved for subcellular localization

    PubMed Central

    Nair, Rajesh; Rost, Burkhard

    2002-01-01

    The more proteins diverged in sequence, the more difficult it becomes for bioinformatics to infer similarities of protein function and structure from sequence. The precise thresholds used in automated genome annotations depend on the particular aspect of protein function transferred by homology. Here, we presented the first large-scale analysis of the relation between sequence similarity and identity in subcellular localization. Three results stood out: (1) The subcellular compartment is generally more conserved than what might have been expected given that short sequence motifs like nuclear localization signals can alter the native compartment; (2) the sequence conservation of localization is similar between different compartments; and (3) it is similar to the conservation of structure and enzymatic activity. In particular, we found the transition between the regions of conserved and nonconserved localization to be very sharp, although the thresholds for conservation were less well defined than for structure and enzymatic activity. We found that a simple measure for sequence similarity accounting for pairwise sequence identity and alignment length, the HSSP distance, distinguished accurately between protein pairs of identical and different localizations. In fact, BLAST expectation values outperformed the HSSP distance only for alignments in the subtwilight zone. We succeeded in slightly improving the accuracy of inferring localization through homology by fine tuning the thresholds. Finally, we applied our results to the entire SWISS-PROT database and five entirely sequenced eukaryotes. PMID:12441382

  12. 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.

  13. 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

  14. Identification and characterization of novel and conserved microRNAs in radish (Raphanus sativus L.) using high-throughput sequencing.

    PubMed

    Xu, Liang; Wang, Yan; Xu, Yuanyuan; Wang, Liangju; Zhai, Lulu; Zhu, Xianwen; Gong, Yiqin; Ye, Shan; Liu, Liwang

    2013-03-01

    MicroRNAs (miRNAs) are endogenous, non-coding, small RNAs that play significant regulatory roles in plant growth, development, and biotic and abiotic stress responses. To date, a great number of conserved and species-specific miRNAs have been identified in many important plant species such as Arabidopsis, rice and poplar. However, little is known about identification of miRNAs and their target genes in radish (Raphanus sativus L.). In the present study, a small RNA library from radish root was constructed and sequenced using the high-throughput Solexa sequencing. Through sequence alignment and secondary structure prediction, a total of 545 conserved miRNA families as well as 15 novel (with their miRNA* strand) and 64 potentially novel miRNAs were identified. Quantitative real-time PCR (qRT-PCR) analysis confirmed that both conserved and novel miRNAs were expressed in radish, and some of them were preferentially expressed in certain tissues. A total of 196 potential target genes were predicted for 42 novel radish miRNAs. Gene ontology (GO) analysis showed that most of the targets were involved in plant growth, development, metabolism and stress responses. This study represents a first large-scale identification and characterization of radish miRNAs and their potential target genes. These results could lead to the further identification of radish miRNAs and enhance our understanding of radish miRNA regulatory mechanisms in diverse biological and metabolic processes.

  15. Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice.

    PubMed

    Püschel, A W; Gruss, P; Westerfield, M

    1992-03-01

    Despite obvious differences in the patterns of early embryonic development, vertebrates share a number of developmental mechanisms and control genes, suggesting that they use similar genetic programs at some stages of development. To examine this idea, we isolated and characterized one such gene, pax-6, a member of the pax gene family, from the zebrafish Brachydanio rerio and determined the evolutionary conservation in the structure and expression of this gene by comparison to its homolog in mice. We found two alternatively spliced forms of the zebrafish pax-6 message. Sequence and expression pattern of the zebrafish pax-6 gene are remarkably similar to its murine homolog. pax-6 expression begins during early neurulation. A stripe of cells in the neuroectoderm, including the prospective diencephalon and a part of the telencephalon, expresses pax-6 as well as the hindbrain and the ventral spinal cord extending from the level of the first rhombomere to the posterior end of the CNS. During later development more limited regions of the brain including the eye, the olfactory bulb and the pituitary gland express pax-6. Cells at the midbrain-hindbrain junction express eng genes and are separated from the neighboring pax-6 regions by several cells that express neither gene, indicating a complex subdivision of this region. pax-6 expression appears during processes when cell-to-cell signalling is thought to be important, for example during induction of the eye and regionalization of the spinal cord and brain, suggesting that it may be one component mediating the response to inductive interactions.

  16. High-throughput sequencing discovery of conserved and novel microRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

    PubMed

    Wang, Fengde; Li, Libin; Liu, Lifeng; Li, Huayin; Zhang, Yihui; Yao, Yingyin; Ni, Zhongfu; Gao, Jianwei

    2012-07-01

    MicroRNAs (miRNAs) are a class of 21-24 nucleotide non-coding RNAs that down-regulate gene expression by cleaving or inhibiting the translation of target gene transcripts. miRNAs have been extensively analyzed in a few model plant species such as Arabidopsis, rice and Populus, and partially investigated in other non-model plant species. However, only a few conserved miRNAs have been identified in Chinese cabbage, a common and economically important crop in Asia. To identify novel and conserved miRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis) we constructed a small RNA library. Using high-throughput Solexa sequencing to identify microRNAs we found 11,210 unique sequences belonging to 321 conserved miRNA families and 228 novel miRNAs. We ran a Blast search with these sequences against the Chinese cabbage mRNA database and found 2,308 and 736 potential target genes for 221 conserved and 125 novel miRNAs, respectively. The BlastX search against the Arabidopsis genome and GO analysis suggested most of the targets were involved in plant growth, metabolism, development and stress response. This study provides the first large scale-cloning and characterization of Chinese cabbage miRNAs and their potential targets. These miRNAs add to the growing database of new miRNAs, prompt further study on Chinese cabbage miRNA regulation mechanisms, and help toward a greater understanding of the important roles of miRNAs in Chinese cabbage.

  17. Sequence fingerprints of microRNA conservation.

    PubMed

    Shi, Bing; Gao, Wei; Wang, Juan

    2012-01-01

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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. High-throughput sequencing identification of novel and conserved miRNAs in the Brassica oleracea leaves

    PubMed Central

    2013-01-01

    Background Plant microRNAs are short (~21 nt) non-coding molecules that regulate gene expression by targeting the mRNA cleavage or protein translation inhibition. In this manner, they play many important roles in the cells of living organisms. One of the plant species in which the entire set of miRNAs has not been yet completely identified is Brassica oleracea var. capitata (cabbage). For this reason and for the economic and nutritional importance of this food crop, high-throughput small RNAs sequencing has been performed to discover the novel and conserved miRNAs in mature cabbage leaves. Results In this study, raw reads generated from three small RNA libraries were bioinformatically processed and further analyzed to select sequences homologous to known B. oleracea and other plant miRNAs. As a result of this analysis, 261 conserved miRNAs (belonging to 62 families) have been discovered. MIR169, MIR167 and MIR166 were the largest miRNA families, while the highest abundance molecules were miR167, miR166, miR168c and miR157a. Among the generated sequencing reads, miRNAs* were also found, such as the miR162c*, miR160a* and miR157a*. The unannotated tags were used in the prediction and evaluation of novel miRNAs, which resulted in the 26 potential miRNAs proposal. The expressions of 13 selected miRNAs were analyzed by northern blot hybridization. The target prediction and annotation for identified miRNAs were performed, according to which discovered molecules may target mRNAs encoding several potential proteins – e.g., transcription factors, polypeptides that regulate hormone stimuli and abiotic stress response, and molecules participating in transport and cell communication. Additionally, KEGG maps analysis suggested that the miRNAs in cabbage are involved in important processing pathways, including glycolysis, glycerolipid metabolism, flavonoid biosynthesis and oxidative phosphorylation. Conclusions Conclusively, for the first time, the large set of miRNAs was

  5. Conserved sequence elements associated with exon skipping

    PubMed Central

    Miriami, Elana; Margalit, Hanah; Sperling, Ruth

    2003-01-01

    One of the major forms of alternative splicing, which generates multiple mRNA isoforms differing in the precise combinations of their exon sequences, is exon skipping. While in constitutive splicing all exons are included, in the skipped pattern(s) one or more exons are skipped. The regulation of this process is still not well understood; so far, cis- regulatory elements (such as exonic splicing enhancers) were identified in individual cases. We therefore set to investigate the possibility that exon skipping is controlled by sequences in the adjacent introns. We employed a computer analysis on 54 sequences documented as undergoing exon skipping, and identified two motifs both in the upstream and downstream introns of the skipped exons. One motif is highly enriched in pyrimidines (mostly C residues), and the other motif is highly enriched in purines (mostly G residues). The two motifs differ from the known cis-elements present at the 5′ and 3′ splice site. Interestingly, the two motifs are complementary, and their relative positional order is conserved in the flanking introns. These suggest that base pairing interactions can underlie a mechanism that involves secondary structure to regulate exon skipping. Remarkably, the two motifs are conserved in mouse orthologous genes that undergo exon skipping. PMID:12655015

  6. A highly conserved sequence in the 3'-untranslated region of the drosophila Adh gene plays a functional role in Adh expression.

    PubMed Central

    Parsch, J; Stephan, W; Tanda, S

    1999-01-01

    Phylogenetic analysis identified a highly conserved eight-base sequence (AAGGCTGA) within the 3'-untranslated region (UTR) of the Drosophila alcohol dehydrogenase gene, Adh. To examine the functional significance of this conserved motif, we performed in vitro deletion mutagenesis on the D. melanogaster Adh gene followed by P-element-mediated germline transformation. Deletion of all or part of the eight-base sequence leads to a twofold increase in in vivo ADH enzymatic activity. The increase in activity is temporally and spatially general and is the result of an underlying increase in Adh transcript. These results indicate that the conserved 3'-UTR motif plays a functional role in the negative regulation of Adh gene expression. The evolutionary significance of our results may be understood in the context of the amino acid change that produces the ADH-F allele and also leads to a twofold increase in ADH activity. While there is compelling evidence that the amino acid replacement has been a target of positive selection, the conservation of the 3'-UTR sequence suggests that it is under strong purifying selection. The selective difference between these two sequence changes, which have similar effects on ADH activity, may be explained by different metabolic costs associated with the increase in activity. PMID:9927459

  7. 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.

  8. 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

  9. 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.

  10. Comparison of orthologous and paralogous DNA flanking the wheat high molecular weight glutenin genes: sequence conservation and divergence, transposon distribution, and matrix-attachment regions.

    PubMed

    Anderson, O D; Larka, L; Christoffers, M J; McCue, K F; Gustafson, J P

    2002-04-01

    Extended flanking DNA sequences were characterized for five members of the wheat high molecular weight (HMW) glutenin gene family to understand more of the structure, control, and evolution of these genes. Analysis revealed more sequence conservation among orthologous regions than between paralogous regions, with differences mainly owing to transposition events involving putative retrotransposons and several miniature inverted transposable elements (MITEs). Both gyspy-like long terminal repeat (LTR) and non-LTR retrotransposon sequences are represented in the flanking DNAs. One of the MITEs is a novel class, but another MITE is related to the maize Stowaway family and is widely represented in Triticeae express sequence tags (ESTs). Flanking DNA of the longest sequence, a 20 425-bp fragment including and surrounding the HMW-glutenin Bx7 gene, showed additional cereal gene-like sequences both immediately 5' and 3' to the HMW-glutenin coding region. The transcriptional activities of sequences related to these flanking putative genes and the retrotransposon-related regions were indicated by matches to wheat and other Triticeae ESTs. Predictive analysis of matrix-attachment regions (MARs) of the HMW glutenin and several alpha-, gamma-, and omega-gliadin flanking DNAs indicate potential MARs immediately flanking each of the genes. Matrix binding activity in the predicted regions was confirmed for two of the HMW-glutenin genes.

  11. 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

  12. 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

  13. Evolutionary conservation of long noncoding RNAs; sequence, structure, function

    PubMed Central

    Johnsson, Per; Lipovich, Leonard; Grandér, Dan; Morris, Kevin V.

    2014-01-01

    Background Recent advances in genome wide studies have revealed the abundance of long non-coding RNAs (lncRNAs) in mammalian transcriptomes. The ENCODE Consortium has elucidated the prevalence of human lncRNA genes, which are as numerous as protein-coding genes. Surprisingly, many lncRNAs do not show the same pattern of high interspecies conservation as protein-coding genes. The absence of functional studies and the frequent lack of sequence conservation therefore make functional interpretation of these newly discovered transcripts challenging. Many investigators have suggested the presence and importance of secondary structural elements within lncRNAs, but mammalian lncRNA secondary structure remains poorly understood. It is intriguing to speculate that in this group of genes, RNA secondary structures might be preserved throughout evolution and that this might explain the lack of sequence conservation among many lncRNAs. Scope of review Here, we review the extent of interspecies conservation among different lncRNAs, with a focus on a subset of lncRNAs that have been functionally investigated. The function of lncRNAs is widespread and we investigate whether different forms of functionalities may be conserved. Major conclusions Lack of conservation does not imbue a lack of function. We highlight several examples of lncRNAs where RNA structure appears to be the main functional unit and evolutionary constraint. We survey existing genomewide studies of mammalian lncRNA conservation and summarize their limitations. We further review specific human lncRNAs which lack evolutionary conservation beyond primates but have proven to be both functional and therapeutically relevant. General significance Pioneering studies highlight a role in lncRNAs for secondary structures, and possibly the presence of functional “modules”, which are interspersed with longer and less conserved stretches of nucleotide sequences. Taken together, high-throughput analysis of conservation and

  14. Patterns of sequence conservation in presynaptic neural genes

    PubMed Central

    Hadley, Dexter; Murphy, Tara; Valladares, Otto; Hannenhalli, Sridhar; Ungar, Lyle; Kim, Junhyong; Bućan, Maja

    2006-01-01

    Background The neuronal synapse is a fundamental functional unit in the central nervous system of animals. Because synaptic function is evolutionarily conserved, we reasoned that functional sequences of genes and related genomic elements known to play important roles in neurotransmitter release would also be conserved. Results Evolutionary rate analysis revealed that presynaptic proteins evolve slowly, although some members of large gene families exhibit accelerated evolutionary rates relative to other family members. Comparative sequence analysis of 46 megabases spanning 150 presynaptic genes identified more than 26,000 elements that are highly conserved in eight vertebrate species, as well as a small subset of sequences (6%) that are shared among unrelated presynaptic genes. Analysis of large gene families revealed that upstream and intronic regions of closely related family members are extremely divergent. We also identified 504 exceptionally long conserved elements (≥360 base pairs, ≥80% pair-wise identity between human and other mammals) in intergenic and intronic regions of presynaptic genes. Many of these elements form a highly stable stem-loop RNA structure and consequently are candidates for novel regulatory elements, whereas some conserved noncoding elements are shown to correlate with specific gene expression profiles. The SynapseDB online database integrates these findings and other functional genomic resources for synaptic genes. Conclusion Highly conserved elements in nonprotein coding regions of 150 presynaptic genes represent sequences that may be involved in the transcriptional or post-transcriptional regulation of these genes. Furthermore, comparative sequence analysis will facilitate selection of genes and noncoding sequences for future functional studies and analysis of variation studies in neurodevelopmental and psychiatric disorders. PMID:17096848

  15. High throughput sequencing of small RNA component of leaves and inflorescence revealed conserved and novel miRNAs as well as phasiRNA loci in chickpea.

    PubMed

    Srivastava, Sangeeta; Zheng, Yun; Kudapa, Himabindu; Jagadeeswaran, Guru; Hivrale, Vandana; Varshney, Rajeev K; Sunkar, Ramanjulu

    2015-06-01

    Among legumes, chickpea (Cicer arietinum L.) is the second most important crop after soybean. MicroRNAs (miRNAs) play important roles by regulating target gene expression important for plant development and tolerance to stress conditions. Additionally, recently discovered phased siRNAs (phasiRNAs), a new class of small RNAs, are abundantly produced in legumes. Nevertheless, little is known about these regulatory molecules in chickpea. The small RNA population was sequenced from leaves and flowers of chickpea to identify conserved and novel miRNAs as well as phasiRNAs/phasiRNA loci. Bioinformatics analysis revealed 157 miRNA loci for the 96 highly conserved and known miRNA homologs belonging to 38 miRNA families in chickpea. Furthermore, 20 novel miRNAs belonging to 17 miRNA families were identified. Sequence analysis revealed approximately 60 phasiRNA loci. Potential target genes likely to be regulated by these miRNAs were predicted and some were confirmed by modified 5' RACE assay. Predicted targets are mostly transcription factors that might be important for developmental processes, and others include superoxide dismutases, plantacyanin, laccases and F-box proteins that could participate in stress responses and protein degradation. Overall, this study provides an inventory of miRNA-target gene interactions for chickpea, useful for the comparative analysis of small RNAs among legumes.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. Detection of Borna disease virus (BDV) antibodies and BDV RNA in psychiatric patients: evidence for high sequence conservation of human blood-derived BDV RNA.

    PubMed Central

    Sauder, C; Müller, A; Cubitt, B; Mayer, J; Steinmetz, J; Trabert, W; Ziegler, B; Wanke, K; Mueller-Lantzsch, N; de la Torre, J C; Grässer, F A

    1996-01-01

    In several vertebrate species, Borna disease virus (BDV), the prototype of a new group of animal viruses, causes central nervous system disease accompanied by diverse behavioral abnormalities. Seroepidemiological data indicate that BDV may contribute to the pathophysiology of certain human mental disorders. This hypothesis is further supported by the detection of both BDV antigens and BDV RNA in peripheral blood mononuclear cells (PBMCs) of patients with psychiatric disorders and the isolation of BDV from such PBMCs. Here we describe serological and molecular epidemiological studies on psychiatric patients and healthy individuals from the area of Homburg, Germany. Using a novel Western blot (immunoblot) assay, we found a BDV seroprevalence of 9.6% among 416 neuropsychiatric patients, which is significantly higher than the 1.4% found among 203 healthy control individuals. Human sera displayed a prominent immunoreactivity against the virus nucleoprotein, the p40 antigen. Reverse transcriptase-mediated PCR analysis of RNA extracted from PBMCs of a subset of 26 of the neuropsychiatric patients revealed that 50% were BDV RNA positive. Three of the 13 BDV RNA-positive patients also had BDV-positive serology, whereas one patient with serum antibodies to BDV p40 antigen did not harbor detectable BDV RNA in PBMCs. BDV p40 and p24 sequences derived from human PBMCs exhibited both a high degree of inter- and intrapatient conservation and a close genetic relationship to animal-derived BDV sequences. PMID:8892892

  2. Sepsid even-skipped Enhancers Are Functionally Conserved in Drosophila Despite Lack of Sequence Conservation

    PubMed Central

    Iyer, Venky N.; Meier, Rudolf; Eisen, Michael B.

    2008-01-01

    The gene expression pattern specified by an animal regulatory sequence is generally viewed as arising from the particular arrangement of transcription factor binding sites it contains. However, we demonstrate here that regulatory sequences whose binding sites have been almost completely rearranged can still produce identical outputs. We sequenced the even-skipped locus from six species of scavenger flies (Sepsidae) that are highly diverged from the model species Drosophila melanogaster, but share its basic patterns of developmental gene expression. Although there is little sequence similarity between the sepsid eve enhancers and their well-characterized D. melanogaster counterparts, the sepsid and Drosophila enhancers drive nearly identical expression patterns in transgenic D. melanogaster embryos. We conclude that the molecular machinery that connects regulatory sequences to the transcription apparatus is more flexible than previously appreciated. In exploring this diverse collection of sequences to identify the shared features that account for their similar functions, we found a small number of short (20–30 bp) sequences nearly perfectly conserved among the species. These highly conserved sequences are strongly enriched for pairs of overlapping or adjacent binding sites. Together, these observations suggest that the local arrangement of binding sites relative to each other is more important than their overall arrangement into larger units of cis-regulatory function. PMID:18584029

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

    NASA Astrophysics Data System (ADS)

    Richardson, Dale N.; Wiehe, Thomas

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

  4. Prediction of Secondary Structures Conserved in Multiple RNA Sequences.

    PubMed

    Xu, Zhenjiang Zech; Mathews, David H

    2016-01-01

    RNA structure is conserved by evolution to a greater extent than sequence. Predicting the conserved structure for multiple homologous sequences can be much more accurate than predicting the structure for a single sequence. RNAstructure is a software package that includes the programs Dynalign, Multilign, TurboFold, and PARTS for predicting conserved RNA secondary structure. This chapter provides protocols for using these programs. PMID:27665591

  5. 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.

  6. 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

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

    PubMed

    Liu, H W; Luo, L X; 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.

  8. Naturally processed HLA class II peptides reveal highly conserved immunogenic flanking region sequence preferences that reflect antigen processing rather than peptide-MHC interactions.

    PubMed

    Godkin, A J; Smith, K J; Willis, A; Tejada-Simon, M V; Zhang, J; Elliott, T; Hill, A V

    2001-06-01

    MHC class II heterodimers bind peptides 12-20 aa in length. The peptide flanking residues (PFRs) of these ligands extend from a central binding core consisting of nine amino acids. Increasing evidence suggests that the PFRs can alter the immunogenicity of T cell epitopes. We have previously noted that eluted peptide pool sequence data derived from an MHC class II Ag reflect patterns of enrichment not only in the core binding region but also in the PFRS: We sought to distinguish whether these enrichments reflect cellular processes or direct MHC-peptide interactions. Using the multiple sclerosis-associated allele HLA-DR2, pool sequence data from naturally processed ligands were compared with the patterns of enrichment obtained by binding semicombinatorial peptide libraries to empty HLA-DR2 molecules. Naturally processed ligands revealed patterns of enrichment reflecting both the binding motif of HLA-DR2 (position (P)1, aliphatic; P4, bulky hydrophobic; and P6, polar) as well as the nonbound flanking regions, including acidic residues at the N terminus and basic residues at the C terminus. These PFR enrichments were independent of MHC-peptide interactions. Further studies revealed similar patterns in nine other HLA alleles, with the C-terminal basic residues being as highly conserved as the previously described N-terminal prolines of MHC class II ligands. There is evidence that addition of C-terminal basic PFRs to known peptide epitopes is able to enhance both processing as well as T cell activation. Recognition of these allele-transcending patterns in the PFRs may prove useful in epitope identification and vaccine design.

  9. 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.

  10. 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

  11. 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

  12. A Developmental Sequence of Skills Leading to Conservation

    ERIC Educational Resources Information Center

    Walker, Alice A.

    1978-01-01

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

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

    PubMed

    Rima, Bert K

    2015-05-01

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

  14. 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

  15. 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

  16. 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

  17. 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

  18. Characterization, nucleotide sequence, and conserved genomic locations of insertion sequence ISRm5 in Rhizobium meliloti.

    PubMed Central

    Laberge, S; Middleton, A T; Wheatcroft, R

    1995-01-01

    A target for ISRm3 transposition in Rhizobium meliloti IZ450 is another insertion sequence element, named ISRm5. ISRm5 is 1,340 bp in length and possesses terminal inverted repeats of unequal lengths (27 and 28 bp) and contain five mismatches. An open reading frame that spans 89% of the length of one DNA strand encodes a putative transposase with significant similarity to the putative transposases of 11 insertion sequence elements from diverse bacterial species, including ISRm3 from R. meliloti. Multiple copies and variants of ISRm5 occur in the R. meliloti genome, often in close association with ISRm3. Five ISRm5 copies in two strains were studied, and each was found to be located between 8-bp direct repeats. At two of these loci, which were shown to be highly conserved in R. meliloti, the copies of ISRm5 were found to be associated with pairs of short inverted repeats resembling transcription terminators. This structural arrangement not only may provide a conserved niche for ISRm5 but also may be a preferred target for transposition. PMID:7768811

  19. Identification of a conserved sequence in the non-coding regions of many human genes.

    PubMed Central

    Donehower, L A; Slagle, B L; Wilde, M; Darlington, G; Butel, J S

    1989-01-01

    We have analyzed a sequence of approximately 70 base pairs (bp) that shows a high degree of similarity to sequences present in the non-coding regions of a number of human and other mammalian genes. The sequence was discovered in a fragment of human genomic DNA adjacent to an integrated hepatitis B virus genome in cells derived from human hepatocellular carcinoma tissue. When one of the viral flanking sequences was compared to nucleotide sequences in GenBank, more than thirty human genes were identified that contained a similar sequence in their non-coding regions. The sequence element was usually found once or twice in a gene, either in an intron or in the 5' or 3' flanking regions. It did not share any similarities with known short interspersed nucleotide elements (SINEs) or presently known gene regulatory elements. This element was highly conserved at the same position within the corresponding human and mouse genes for myoglobin and N-myc, indicating evolutionary conservation and possible functional importance. Preliminary DNase I footprinting data suggested that the element or its adjacent sequences may bind nuclear factors to generate specific DNase I hypersensitive sites. The size, structure, and evolutionary conservation of this sequence indicates that it is distinct from other types of short interspersed repetitive elements. It is possible that the element may have a cis-acting functional role in the genome. Images PMID:2536922

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

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

    PubMed

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

    2014-08-01

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

  7. 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.

  8. 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

  9. Sequence conservation of the 12D3 gene in Mexican isolates of Babesia bovis.

    PubMed

    Perez, J; Javier Perez, J; Vargas, P; Antonio Alvarez, J; Rojas, C; Figueroa, J V

    2010-04-01

    The 12D3 antigen present in Babesia bovis has been evaluated as a recombinant vaccine candidate and the 12d3 coding sequence has been reported for an Australian and an USA (Texas) isolate of B. bovis. However, no approach has been conducted to perform analysis of 12d3 sequence conservation on a larger number of B. bovis isolates. This could provide important information to determine whether a recombinant vaccine containing this antigen could be widely used. This study reports the cloning and sequencing analysis of the 12d3 coding region in 20 different B. bovis isolates collected from various geographical regions in the tropics and subtropics of Mexico. Comparative analysis of the consensus nucleotide sequences obtained for each isolate revealed a high degree of conservation (94-99% sequence identity) among the 12d3 alleles present in the Mexican isolates when compared with the 12d3 ORF sequences from the Texan (T2Bo) B. bovis isolate. Similarly, BLASTX sequence homology search showed a high percent identity (93-99%) of the deduced amino acid 12D3 sequence as compared with the T2Bo isolate sequence. The high level of sequence conservation in 12d3 among the 20 B. bovis isolates collected from geographically distant locations in Mexico suggests that there exists a minimal bovine-host immunological pressure which could be translated into antigenic diversity or variation, and most probably this is reflected in the non-inmunodominant characteristic of the 12D3 antigen as it has been previously described in the literature. 12D3 antigen can be considered as a viable candidate for inclusion in a recombinant vaccine for cattle babesiosis caused by B. bovis in Mexico.

  10. Genome-Wide Identification and Comparative Analysis of Conserved and Novel MicroRNAs in Grafted Watermelon by High-Throughput Sequencing

    PubMed Central

    Liu, Na; Yang, Jinghua; Guo, Shaogui; Xu, Yong; Zhang, Mingfang

    2013-01-01

    MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs involved in the post-transcriptional gene regulation and play a critical role in plant growth, development and stresses response. However less is known about miRNAs involvement in grafting behaviors, especially with the watermelon (Citrullus lanatus L.) crop, which is one of the most important agricultural crops worldwide. Grafting method is commonly used in watermelon production in attempts to improve its adaptation to abiotic and biotic stresses, in particular to the soil-borne fusarium wilt disease. In this study, Solexa sequencing has been used to discover small RNA populations and compare miRNAs on genome-wide scale in watermelon grafting system. A total of 11,458,476, 11,614,094 and 9,339,089 raw reads representing 2,957,751, 2,880,328 and 2,964,990 unique sequences were obtained from the scions of self-grafted watermelon and watermelon grafted on-to bottle gourd and squash at two true-leaf stage, respectively. 39 known miRNAs belonging to 30 miRNA families and 80 novel miRNAs were identified in our small RNA dataset. Compared with self-grafted watermelon, 20 (5 known miRNA families and 15 novel miRNAs) and 47 (17 known miRNA families and 30 novel miRNAs) miRNAs were expressed significantly different in watermelon grafted on to bottle gourd and squash, respectively. MiRNAs expressed differentially when watermelon was grafted onto different rootstocks, suggesting that miRNAs might play an important role in diverse biological and metabolic processes in watermelon and grafting may possibly by changing miRNAs expressions to regulate plant growth and development as well as adaptation to stresses. The small RNA transcriptomes obtained in this study provided insights into molecular aspects of miRNA-mediated regulation in grafted watermelon. Obviously, this result would provide a basis for further unravelling the mechanism on how miRNAs information is exchanged between scion and rootstock in grafted

  11. 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

  12. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids.

    PubMed

    Ashkenazy, Haim; Erez, Elana; Martz, Eric; Pupko, Tal; Ben-Tal, Nir

    2010-07-01

    It is informative to detect highly conserved positions in proteins and nucleic acid sequence/structure since they are often indicative of structural and/or functional importance. ConSurf (http://consurf.tau.ac.il) and ConSeq (http://conseq.tau.ac.il) are two well-established web servers for calculating the evolutionary conservation of amino acid positions in proteins using an empirical Bayesian inference, starting from protein structure and sequence, respectively. Here, we present the new version of the ConSurf web server that combines the two independent servers, providing an easier and more intuitive step-by-step interface, while offering the user more flexibility during the process. In addition, the new version of ConSurf calculates the evolutionary rates for nucleic acid sequences. The new version is freely available at: http://consurf.tau.ac.il/.

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

    PubMed

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

    2014-08-01

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

  14. Mouse Brca1: localization sequence analysis and identification of evolutionarily conserved domains.

    PubMed

    Abel, K J; Xu, J; Yin, G Y; Lyons, R H; Meisler, M H; Weber, B L

    1995-12-01

    The human genes BRCA1, conferring susceptibility to early-onset breast and ovarian cancer, has recently been isolated. Here we describe isolation of cDNAs, sequence analysis, and genomic localization of the murine homolog, Brac1. The mouse cDNA sequence predicts a protein of 1812 amino acids; a number of small gaps account for the 51 fewer residues in the mouse protein relative to human BRCA1. While the predicted mouse and human proteins display on the whole a high level of homology (58% identity, 73% similarity), the regions of greatest homology are at the respective amino and carboxyl termini. Most reported disease-associated missense mutations in human BCRA1 occurred within these more highly conserved terminal regions. A predicted zinc-building RING finger domain near the amino terminus lies within a 50 amino acid stretch that is perfectly conserved in both species. The strong conservation during mammalian evolution argues for the importance of this domain, perhaps mediating a role for BRCA1 in DNA and/or protein binding. We have also identified a conserved highly acidic domain in the carboxyl terminal half of the BCRA1 protein resembling acidic transactivation domains of certain transcription factors. Using an interspecific backcross panel, Brca1 was mapped to a region of mouse chromosome 11 that exhibits conserved linkage with 17q21. The sequence and isolated cDNAs will provide useful reagents for studying the expression of Brca1 in the mouse, and for testing the importance of the evolutionarily conserved domains.

  15. 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

  16. Metazoan remaining genes for essential amino acid biosynthesis: sequence conservation and evolutionary analyses.

    PubMed

    Costa, Igor R; Thompson, Julie D; Ortega, José Miguel; Prosdocimi, Francisco

    2014-12-24

    Essential amino acids (EAA) consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS) and betaine-homocysteine S-methyltransferase (BHMT) diverged from the expected Tree of Life (ToL) relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals.

  17. Sequence recombination and conservation of Varroa destructor virus-1 and deformed wing virus in field collected honey bees (Apis mellifera).

    PubMed

    Wang, Hui; Xie, Jiazheng; Shreeve, Tim G; Ma, Jinmin; Pallett, Denise W; King, Linda A; Possee, Robert D

    2013-01-01

    We sequenced small (s) RNAs from field collected honeybees (Apis mellifera) and bumblebees (Bombuspascuorum) using the Illumina technology. The sRNA reads were assembled and resulting contigs were used to search for virus homologues in GenBank. Matches with Varroadestructor virus-1 (VDV1) and Deformed wing virus (DWV) genomic sequences were obtained for A. mellifera but not B. pascuorum. Further analyses suggested that the prevalent virus population was composed of VDV-1 and a chimera of 5'-DWV-VDV1-DWV-3'. The recombination junctions in the chimera genomes were confirmed by using RT-PCR, cDNA cloning and Sanger sequencing. We then focused on conserved short fragments (CSF, size > 25 nt) in the virus genomes by using GenBank sequences and the deep sequencing data obtained in this study. The majority of CSF sites confirmed conservation at both between-species (GenBank sequences) and within-population (dataset of this study) levels. However, conserved nucleotide positions in the GenBank sequences might be variable at the within-population level. High mutation rates (Pi>10%) were observed at a number of sites using the deep sequencing data, suggesting that sequence conservation might not always be maintained at the population level. Virus-host interactions and strategies for developing RNAi treatments against VDV1/DWV infections are discussed.

  18. Strong evolutionary conservation of neuropeptide Y: sequences of chicken, goldfish, and Torpedo marmorata DNA clones.

    PubMed Central

    Blomqvist, A G; Söderberg, C; Lundell, I; Milner, R J; Larhammar, D

    1992-01-01

    Neuropeptide Y (NPY) is an abundant and widespread neuropeptide in the nervous system of mammals. NPY belongs to a family of 36-amino acid peptides that also includes pancreatic polypeptide and the endocrine gut peptide YY as well as the fish pancreatic peptide Y. To study the evolution of this peptide family, we have isolated clones encoding NPY from central nervous system cDNA libraries of chicken, goldfish, and the ray Torpedo marmorata, as well as from a chicken genomic library. The predicted chicken NPY amino acid sequence differs from that of rat at only one position. The goldfish sequence differs at five positions and shows that bony fishes have a true NPY peptide in addition to their pancreatic peptide Y. The Torpedo sequence differs from that of rat at three positions. As Torpedo NPY has no unique positions when compared with the other sequences, it seems to be identical to the NPY of the common ancestor of cartilaginous fishes, bony fishes, and tetrapods after 420 million years of evolution. The 30-amino acid carboxyl-terminal extension of the NPY precursor also displays considerable sequence conservation. These results show that NPY is one of the most highly conserved neuroendocrine peptides. Images PMID:1549597

  19. Strong evolutionary conservation of neuropeptide Y: sequences of chicken, goldfish, and Torpedo marmorata DNA clones.

    PubMed

    Blomqvist, A G; Söderberg, C; Lundell, I; Milner, R J; Larhammar, D

    1992-03-15

    Neuropeptide Y (NPY) is an abundant and widespread neuropeptide in the nervous system of mammals. NPY belongs to a family of 36-amino acid peptides that also includes pancreatic polypeptide and the endocrine gut peptide YY as well as the fish pancreatic peptide Y. To study the evolution of this peptide family, we have isolated clones encoding NPY from central nervous system cDNA libraries of chicken, goldfish, and the ray Torpedo marmorata, as well as from a chicken genomic library. The predicted chicken NPY amino acid sequence differs from that of rat at only one position. The goldfish sequence differs at five positions and shows that bony fishes have a true NPY peptide in addition to their pancreatic peptide Y. The Torpedo sequence differs from that of rat at three positions. As Torpedo NPY has no unique positions when compared with the other sequences, it seems to be identical to the NPY of the common ancestor of cartilaginous fishes, bony fishes, and tetrapods after 420 million years of evolution. The 30-amino acid carboxyl-terminal extension of the NPY precursor also displays considerable sequence conservation. These results show that NPY is one of the most highly conserved neuroendocrine peptides.

  20. Sequence-related human proteins cluster by degree of evolutionary conservation.

    PubMed

    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) approximately k(-gamma) with gamma approximately 1.2 , (ii) the top rank of N consists of a single large cluster of proteins ( approximately 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.

  1. 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.

  2. 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.

  3. 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

  4. 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)

  5. Identification of conserved and novel microRNAs in Aquilaria sinensis based on small RNA sequencing and transcriptome sequence data.

    PubMed

    Gao, Zhi-Hui; Wei, Jian-He; Yang, Yun; Zhang, Zheng; Xiong, Huan-Ying; Zhao, Wen-Ting

    2012-08-15

    Agarwood is in great demand for its high value in medicine, incense, and perfume across Asia, Middle East, and Europe. As agarwood is formed only when the Aquilaria trees are wounded or infected by some microbes, overharvesting and habitat loss are threatening some populations of agarwood-producing species. Aquilaria sinensis is such a significant economic tree species. To promote the production efficiency and protect the resource of A. sinensis, it would be critical to reveal the regulation mechanisms of stress-induced agarwood formation. MicroRNAs (miRNAs), a key gene expression regulator involved in various plant stress response and metabolic processes, might function in agarwood formation, but no report concerning miRNAs in Aquilaria is available. In this study, the small RNA high-throughput sequencing and 454 transcriptome data were adopted to identify both conserved and novel miRNAs in A. sinensis. Deep sequencing showed that the small RNA (sRNA) population of A. sinensis was complex and the length of sRNAs varied. By in silico analysis of the small RNA deep sequencing data and transcriptome data, we discovered 27 novel miRNAs in A. sinensis. Based on the mature miRNA sequence conservation, we identified 74 putative conserved miRNAs from A. sinensis and 10 of them were confirmed with hairpin forming precursor. Interestingly, a novel miRNA sequence was determined to be the miRNA of asi-miR408, but with accumulation much higher than asi-miR408. The expression levels of ten stress-responsive miRNAs were examined during the time-course after wound treatment. Eight were shown to be wound-responsive. This not only shows the existence of miRNAs in this Asian economically significant tree species but also indicated its critical role in stress-induced agarwood formation. The highly accumulated miRNA of asi-miR408 implied miRNAs would be functional as well as miRNAs in plants.

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

    PubMed

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

    2004-07-01

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

  7. 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.

  8. 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.

  9. Host species-specific conservation of a family of repeated DNA sequences in the genome of a fungal plant pathogen.

    PubMed Central

    Hamer, J E; Farrall, L; Orbach, M J; Valent, B; Chumley, F G

    1989-01-01

    We have identified a family of dispersed repetitive DNA sequences in the genome of Magnaporthe grisea, the fungus that causes rice blast disease. We have named this family of DNA sequences "MGR" for M. grisea repeat. Analysis of five MGR clones demonstrates that MGR sequences are highly polymorphic. The segregation of MGR sequences in genetic crosses and hybridization of MGR probes to separated, chromosome-size DNA molecules of M. grisea shows that this family of sequences is distributed among the M. grisea chromosomes. MGR sequences also hybridize to discrete poly(A)+ RNAs. Southern blot analysis using a MGR probe can distinguish rice pathogens from various sources. However, MGR sequences are not highly conserved in the genomes of M. grisea field isolates that do not infect rice. These results suggest that host selection for a specific pathogen genotype has occurred during the breeding and cultivation of rice. Images PMID:2602385

  10. 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.

  11. 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

  12. Mitochondrial genome sequences illuminate maternal lineages of conservation concern in a rare carnivore

    PubMed Central

    2011-01-01

    Background Science-based wildlife management relies on genetic information to infer population connectivity and identify conservation units. The most commonly used genetic marker for characterizing animal biodiversity and identifying maternal lineages is the mitochondrial genome. Mitochondrial genotyping figures prominently in conservation and management plans, with much of the attention focused on the non-coding displacement ("D") loop. We used massively parallel multiplexed sequencing to sequence complete mitochondrial genomes from 40 fishers, a threatened carnivore that possesses low mitogenomic diversity. This allowed us to test a key assumption of conservation genetics, specifically, that the D-loop accurately reflects genealogical relationships and variation of the larger mitochondrial genome. Results Overall mitogenomic divergence in fishers is exceedingly low, with 66 segregating sites and an average pairwise distance between genomes of 0.00088 across their aligned length (16,290 bp). Estimates of variation and genealogical relationships from the displacement (D) loop region (299 bp) are contradicted by the complete mitochondrial genome, as well as the protein coding fraction of the mitochondrial genome. The sources of this contradiction trace primarily to the near-absence of mutations marking the D-loop region of one of the most divergent lineages, and secondarily to independent (recurrent) mutations at two nucleotide position in the D-loop amplicon. Conclusions Our study has two important implications. First, inferred genealogical reconstructions based on the fisher D-loop region contradict inferences based on the entire mitogenome to the point that the populations of greatest conservation concern cannot be accurately resolved. Whole-genome analysis identifies Californian haplotypes from the northern-most populations as highly distinctive, with a significant excess of amino acid changes that may be indicative of molecular adaptation; D-loop sequences fail

  13. High-bay Lighting Energy Conservation Measures

    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

  14. 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,…

  15. Identification of conservative microRNAs in Saanen dairy goat testis through deep sequencing.

    PubMed

    Wu, J; Zhu, H; Song, W; Li, M; Liu, C; Li, N; Tang, F; Mu, H; Liao, M; Li, X; Guan, W; Li, X; Hua, J

    2014-02-01

    MicroRNA (miRNA) is a kind of small non-coding RNA molecules that function as important gene expression regulators by targeting messenger RNAs for post-transcriptional endonucleolytic cleavage or translational inhibition. In this study, small RNA libraries were constructed based on adult dairy goat testicular tissues and sequenced using the Illumina high-throughput sequencing technology. Blasted to miRNAs of cow and sheep in miRBase 19.0, 373 conserved miRNAs were identified in dairy goat testis and 91 novel paired-miRNAs were found. Expression of miRNAs in the dairy goat testis (miR-10b, miR-126-3p, miR-126-5p, miR-34c, miR-449b and miR-1468) was confirmed by qRT-PCR. In addition, the 128 conserved miRNAs were found by comparing the miRNA expression profiles in dairy goat testis with those in cow and mouse, which all might be involved in dairy goat testis development and meiosis. This study reveals the first miRNA profile related to the biology of testis in the dairy goat. The characterization of these miRNAs could contribute to a better understanding of the molecular mechanisms of reproductive physiology and development in the dairy goat.

  16. High speed nucleic acid sequencing

    SciTech Connect

    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.

  17. PETcofold: predicting conserved interactions and structures of two multiple alignments of RNA sequences

    PubMed Central

    Seemann, Stefan E.; Richter, Andreas S.; Gesell, Tanja; Backofen, Rolf; Gorodkin, Jan

    2011-01-01

    Motivation: Predicting RNA–RNA interactions is essential for determining the function of putative non-coding RNAs. Existing methods for the prediction of interactions are all based on single sequences. Since comparative methods have already been useful in RNA structure determination, we assume that conserved RNA–RNA interactions also imply conserved function. Of these, we further assume that a non-negligible amount of the existing RNA–RNA interactions have also acquired compensating base changes throughout evolution. We implement a method, PETcofold, that can take covariance information in intra-molecular and inter-molecular base pairs into account to predict interactions and secondary structures of two multiple alignments of RNA sequences. Results: PETcofold's ability to predict RNA–RNA interactions was evaluated on a carefully curated dataset of 32 bacterial small RNAs and their targets, which was manually extracted from the literature. For evaluation of both RNA–RNA interaction and structure prediction, we were able to extract only a few high-quality examples: one vertebrate small nucleolar RNA and four bacterial small RNAs. For these we show that the prediction can be improved by our comparative approach. Furthermore, PETcofold was evaluated on controlled data with phylogenetically simulated sequences enriched for covariance patterns at the interaction sites. We observed increased performance with increased amounts of covariance. Availability: The program PETcofold is available as source code and can be downloaded from http://rth.dk/resources/petcofold. Contact: gorodkin@rth.dk; backofen@informatik.uni-freiburg.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21088024

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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

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

    PubMed

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

    2015-05-01

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

  6. Analysis of DNA Sequence Variants Detected by High Throughput Sequencing

    PubMed Central

    Adams, David R; Sincan, Murat; Fajardo, Karin Fuentes; Mullikin, James C; Pierson, Tyler M; Toro, Camilo; Boerkoel, Cornelius F; Tifft, Cynthia J; Gahl, William A; Markello, Tom C

    2014-01-01

    The Undiagnosed Diseases Program at the National Institutes of Health uses High Throughput Sequencing (HTS) to diagnose rare and novel diseases. HTS techniques generate large numbers of DNA sequence variants, which must be analyzed and filtered to find candidates for disease causation. Despite the publication of an increasing number of successful exome-based projects, there has been little formal discussion of the analytic steps applied to HTS variant lists. We present the results of our experience with over 30 families for whom HTS sequencing was used in an attempt to find clinical diagnoses. For each family, exome sequence was augmented with high-density SNP-array data. We present a discussion of the theory and practical application of each analytic step and provide example data to illustrate our approach. The paper is designed to provide an analytic roadmap for variant analysis, thereby enabling a wide range of researchers and clinical genetics practitioners to perform direct analysis of HTS data for their patients and projects. PMID:22290882

  7. Position-specific prediction of methylation sites from sequence conservation based on information theory.

    PubMed

    Shi, Yinan; Guo, Yanzhi; Hu, Yayun; Li, Menglong

    2015-07-23

    Protein methylation plays vital roles in many biological processes and has been implicated in various human diseases. To fully understand the mechanisms underlying methylation for use in drug design and work in methylation-related diseases, an initial but crucial step is to identify methylation sites. The use of high-throughput bioinformatics methods has become imperative to predict methylation sites. In this study, we developed a novel method that is based only on sequence conservation to predict protein methylation sites. Conservation difference profiles between methylated and non-methylated peptides were constructed by the information entropy (IE) in a wider neighbor interval around the methylation sites that fully incorporated all of the environmental information. Then, the distinctive neighbor residues were identified by the importance scores of information gain (IG). The most representative model was constructed by support vector machine (SVM) for Arginine and Lysine methylation, respectively. This model yielded a promising result on both the benchmark dataset and independent test set. The model was used to screen the entire human proteome, and many unknown substrates were identified. These results indicate that our method can serve as a useful supplement to elucidate the mechanism of protein methylation and facilitate hypothesis-driven experimental design and validation.

  8. Eukaryotic genomes contain a [2Fez.sbnd;2S] ferredoxin isoform with a conserved C-terminal sequence motif.

    PubMed

    Seeber, Frank

    2002-11-01

    Apicomplexan protists contain a single mitochondrial [2Fe-2S] ferredoxin sequence (mtFd) with a highly conserved C-terminal motif, VDGxxpxPH, that distinguishes it from other mtFd, which have heterogeneous C-termini. This isoform of mtFd, called 'type II ferredoxin', is widespread in eukaryotes, some species having two isoforms and others possessing only one. Because of the known modulating role of the C-terminus of type I mtFd during association with itself and other interacting proteins, the presence of a conserved C-terminus in type II mtFd suggests it evolved either as a means for optimized homodimerization or to allow interaction with a highly conserved partner(s) that is yet to be defined.

  9. Sequence conservation of homeologous bacterial artificial chromosomes and transcription of homeologous genes in soybean (Glycine max L. Merr.).

    PubMed

    Schlueter, Jessica A; Scheffler, Brian E; Schlueter, Shannon D; Shoemaker, Randy C

    2006-10-01

    The paleopolyploid soybean genome was investigated by sequencing homeologous BAC clones anchored by duplicate N-hydroxycinnamoyl/benzoyltransferase (HCBT) genes. The homeologous BACs were genetically mapped to linkage groups C1 and C2. Annotation of the 173,747- and 98,760-bp BACs showed that gene conservation in both order and orientation is high between homeologous regions with only a single gene insertion/deletion and local tandem duplications differing between the regions. The nucleotide sequence conservation extends into intergenic regions as well, probably due to conserved regulatory sequences. Most of the homeologs appear to have a role in either transcription/DNA binding or cellular signaling, suggesting a potential preference for retention of duplicate genes with these functions. Reverse transcriptase-PCR analysis of homeologs showed that in the tissues sampled, most homeologs have not diverged greatly in their transcription profiles. However, four cases of changes in transcription were identified, primarily in the HCBT gene cluster. Because a mapped locus corresponds to a soybean cyst nematode (SCN) QTL, the potential role of HCBT genes in response to SCN is discussed. These results are the first sequenced-based analysis of homeologous BACs in soybean, a diploidized paleopolyploid. PMID:16888343

  10. 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

  11. 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

  12. The complete mitochondrial genome sequence of the liverwort Pleurozia purpurea reveals extremely conservative mitochondrial genome evolution in liverworts.

    PubMed

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

    2009-12-01

    Plant mitochondrial genomes have been known to be highly unusual in their large sizes, frequent intra-genomic rearrangement, and generally conservative sequence evolution. Recent studies show that in early land plants the mitochondrial genomes exhibit a mixed mode of conservative yet dynamic evolution. Here, we report the completely sequenced mitochondrial genome from the liverwort Pleurozia purpurea. The circular genome has a size of 168,526 base pairs, containing 43 protein-coding genes, 3 rRNA genes, 25 tRNA genes, and 31 group I or II introns. It differs from the Marchantia polymorpha mitochondrial genome, the only other liverwort chondriome that has been sequenced, in lacking two genes (trnRucg and trnTggu) and one intron (rrn18i1065gII). The two genomes have identical gene orders and highly similar sequences in exons, introns, and intergenic spacers. Finally, a comparative analysis of duplicated trnRucu and other trnR genes from the two liverworts and several other organisms identified the recent lateral origin of trnRucg in Marchantia mtDNA through modification of a duplicated trnRucu. This study shows that the mitochondrial genomes evolve extremely slowly in liverworts, the earliest-diverging lineage of extant land plants, in stark contrast to what is known of highly dynamic evolution of mitochondrial genomes in seed plants.

  13. Conserved sequence elements in the 5' region of the Ultrabithorax transcription unit

    PubMed Central

    Wilde, C. Deborah; Akam, Michael

    1987-01-01

    Clones homologous to the 5' region of the Ultrabithorax gene of Drosophila melanogaster have been isolated from D. pseudoobscura, D. funebris and Musca domestica. Regions that encode most of the Ubx protein have been sequenced in all three of these species, and the 5' upstream region has been sequenced in D. funebris to a point ˜1000 bases upstream of the probable mRNA start site. Here we compare these sequences with those described elsewhere for D. melanogaster. Deduced amino acid sequences of the Ubx protein show 8% (D. pseudoobscura), 15% (D. funebris) and 22% (M. domestica) divergence from D. melanogaster. However, these figures mask very different rates of evolution in different regions of the protein. A glycine-rich (`hinge') region is conserved in each of these species, although its length is variable. Comparison of D. funebris and D. melanogaster sequences in the long 5' untranslated leader region of the mRNA, and in the region immediately upstream of the start point of transcription, reveals tightly conserved elements embedded in an otherwise non-homologous sequence. These conserved elements include a 118-bp region that spans the mRNA start site, an internally repetitive (TAA)n region in the untranslated leader and a short repeated motif immediately upstream of the ATG codon that initiates the major open reading frame of the Ubx protein. Two other conserved elements were identified upstream of the transcription start site; both elements have structural features consistent with a role as recognition sites for regulatory proteins. ImagesFig. 2. PMID:16453766

  14. Viral detection by high-throughput sequencing.

    PubMed

    Motooka, Daisuke; Nakamura, Shota; Hagiwara, Katsuro; Nakaya, Takaaki

    2015-01-01

    We applied a high-throughput sequencing platform, Ion PGM, for viral detection in fecal samples from adult cows collected in Hokkaido, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.25 ml of fecal specimens (N = 8), and more than 5 μg of cDNA was synthesized. Unbiased high-throughput sequencing using the 318 v2 semiconductor chip of these eight samples yielded 57-580 K (average: 270 K, after data analysis) reads in a single run. As a result, viral genome sequences were detected in each specimen. In addition to bacteriophage, mammal- and insect-derived viruses, partial genome sequences of plant, algal, and protozoal viruses were detected. Thus, this metagenomic analysis of fecal specimens could be useful to comprehensively understand viral populations of the intestine and food sources in animals. PMID:25287501

  15. Viral detection by high-throughput sequencing.

    PubMed

    Motooka, Daisuke; Nakamura, Shota; Hagiwara, Katsuro; Nakaya, Takaaki

    2015-01-01

    We applied a high-throughput sequencing platform, Ion PGM, for viral detection in fecal samples from adult cows collected in Hokkaido, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.25 ml of fecal specimens (N = 8), and more than 5 μg of cDNA was synthesized. Unbiased high-throughput sequencing using the 318 v2 semiconductor chip of these eight samples yielded 57-580 K (average: 270 K, after data analysis) reads in a single run. As a result, viral genome sequences were detected in each specimen. In addition to bacteriophage, mammal- and insect-derived viruses, partial genome sequences of plant, algal, and protozoal viruses were detected. Thus, this metagenomic analysis of fecal specimens could be useful to comprehensively understand viral populations of the intestine and food sources in animals.

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

    PubMed

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

    2014-09-01

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

  17. Identification of polymorphic, conserved simple sequence repeats (SSRs) in cultivated Brassica species.

    PubMed

    Szewc-McFadden, A K; Kresovich, S; Bliek, S M; Mitchell, S E; McFerson, J R

    1996-09-01

    The application of simple sequence repeat (SSR) genotyping for the characterization of genetic variation in crop plants has been hindered by ready access to useful primer pairs and potentially limited conservation of the repeat sequences among related species. In this phase of work, we report on the identification and characterization of SSRs that are conserved in Brassica napus L. (rapeseed) and its putative progenitors, B. oleracea L. (cabbage, and related vegetable types) and B. rapa (vegetable and oil types). Approximately 140 clones from a size-fractionated genomic library of B. napus were sequenced, and primer pairs were designed for 21 dinucleotide SSRs. Seventeen primer pairs amplified products in the three species and, among these, 13 detected variation between and within species. Unlike findings on SSR information content in human, no relationship could be established between the number of tandem repeats within the target sequence and heterozygosity. All primer pairs have been designed to work under identical amplification conditions; therefore, single-reaction, multiplex polymerase chain reaction (PCR) with these SSRs is possible. Once moderate numbers of primer pairs are accessible to the user community, SSR genotyping may provide a useful method for the characterization, conservation, and utilization of agricultural crop diversity.

  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. Structural Conservation Predominates Over Sequence Variability in the Crown of HIV Type 1's V3 Loop

    PubMed Central

    Almond, David; Kimura, Tetsuya; Kong, XiangPeng; Swetnam, James; Zolla-Pazner, Susan

    2010-01-01

    Abstract The diversity of HIV-1 is a confounding problem for vaccine design, as the human immune response appears to favor poor or strain-specific responses to any given HIV-1 virus strain. A significant portion of this diversity is manifested as sequence variability in the loops of HIV-1's surface envelope glycoprotein. Here we show that the most variable sequence positions in the third variable (V3) loop crown cluster to a small zone on the surface of one face of the V3 loop ß-hairpin conformation. These results provide a novel visualization of the gp120 V3 loop, specifically demonstrating a surprising preponderance of conserved three-dimensional structure in a highly sequence-variable region. From a structural point of view, there appears to be less diversity in this region of the HIV-1 “principle neutralizing domain” than previously appreciated. PMID:20560796

  20. Ancient conserved regions in new gene sequences and the protein databases

    SciTech Connect

    Green, P.; Hillier, L.; Waterston, R. ); Lipman, D.; States, D.; Claverie, J.M. )

    1993-03-19

    Sets of new gene sequences from human, nematode, and yeast were compared with each other and with a set of Escherichia coli genes in order to detect ancient evolutionarily conserved regions (ACRs) in the encoded proteins. Nearly all of the ACRs so identified were found to be homologous to sequences in the protein databases. This suggests that currently known proteins may already include representatives of most ACRs and that new sequences not similar to any database sequence are unlikely to contain ACRs. Preliminary analyses indicate that moderately expressed genes may be more likely to contain ACRs than rarely expressed genes. It is estimated that there are fewer than 900 ACRs in all. 20 refs., 2 figs., 4 tabs.

  1. 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

  2. Regulatory function of conserved sequences upstream of the long-wave sensitive opsin genes in teleost fishes.

    PubMed

    Tam, Kevin J; Watson, Corey T; Massah, Shabnam; Kolybaba, Addie M; Breden, Felix; Prefontaine, Gratien G; Beischlag, Timothy V

    2011-11-01

    Vertebrate opsin genes often occur in sets of tandem duplicates, and their expression varies developmentally and in response to environmental cues. We previously identified two highly conserved regions upstream of the long-wave sensitive opsin (LWS) gene cluster in teleosts. This region has since been shown in zebrafish to drive expression of LWS genes in vivo. In order to further investigate how elements in this region control opsin gene expression, we tested constructs encompassing the highly conserved regions and the less conserved portions upstream of the coding sequences in a promoter-less luciferase expression system. A ∼4500 bp construct of the upstream region, including the highly-conserved regions Reg I and Reg II, increased expression 100-fold, and successive 5' deletions reduced expression relative to the full 4.5 Kb region. Gene expression was highest when the transcription factor RORα was co-transfected with the proposed regulatory regions. Because these regions were tested in a promoter-less expression system, they include elements able to initiate and drive transcription. Teleosts exhibit complex color-mediated adaptive behavior and their adaptive significance has been well documented in several species. Therefore these upstream regions of LWS represent a model system for understanding the molecular basis of adaptive variation in gene regulation of color vision.

  3. Marker production by PCR amplification with primer pairs from conserved sequences of WRKY genes in chili pepper.

    PubMed

    Kim, Hyoun-Joung; Lee, Heung-Ryul; Han, Jung-Heon; Yeom, Seon-In; Harn, Chee-Hark; Kim, Byung-Dong

    2008-04-30

    Despite increasing awareness of the importance of WRKY genes in plant defense signaling, the locations of these genes in the Capsicum genome have not been established. To develop WRKY-based markers, primer sequences were deduced from the conserved sequences of the DNA binding motif within the WRKY domains of tomato and pepper genes. These primers were derived from upstream and downstream parts of the conserved sequences of the three WRKY groups. Six primer combinations of each WRKY group were tested for polymorphisms between the mapping parents, C. annuum 'CM334' and C. annuum 'Chilsungcho'. DNA fragments amplified by primer pairs deduced from WRKY Group II genes revealed high levels of polymorphism. Using 32 primer pairs to amplify upstream and downstream parts of the WRKY domain of WRKY group II genes, 60 polymorphic bands were detected. Polymorphisms were not detected with primer pairs from downstream parts of WRKY group II genes. Half of these primers were subjected to F2 genotyping to construct a linkage map. Thirty of 41 markers were located evenly spaced on 20 of the 28 linkage groups, without clustering. This linkage map also consisted of 199 AFLP and 26 SSR markers. This WRKY-based marker system is a rapid and simple method for generating sequence-specific markers for plant gene families.

  4. 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

  5. Gene sequence, localization, and evolutionary conservation of DAZLA, a candidate male sterility gene.

    PubMed

    Seboun, E; Barbaux, S; Bourgeron, T; Nishi, S; Agulnik, A; Egashira, M; Nikkawa, N; Bishop, C; Fellous, M; McElreavey, K; Kasahara, M; Algonik, A

    1997-04-15

    We have isolated the human homologue of the mouse germ cell-specific transcript Tpx2, which we had previously mapped to mouse chromosome 17. Sequence analysis shows that the human gene is part of the DAZ (Deleted in Azoospermia) family, represents the human homologue of the mouse Dazla and Drosophila boule genes, and is termed DAZLA. Like Dazla and boule, DAZLA is single copy and maps to 3p25. This defines a new region of synteny between mouse chromosome 17 and human chromosome 3. Unlike DAZ, which has multiple DAZ repeats, DAZLA encodes a putative RNA-binding protein with a single RNA-binding motif and a single DAZ repeat. DAZLA is more closely related to Dazla in the mouse than to the Y-linked homologue DAZ (88% identity overall with mouse Dazla compared to 76% identity with the human DAZ protein sequence). Southern blot analysis showed that DAZLA is autosomal in all mammals tested and that DAZ has been recently translocated to the Y chromosome, sometime after the divergence of Old World and New World primates. To investigate the evolutionary relatedness of DAZLA and DAZ further, their partial genomic structures were obtained and compared. This revealed that the genomic organization of both genes in the 5' region is highly conserved. DAZLA is a new member of the DAZ family of genes, which is associated with spermatogenesis and male sterility. Familial cases of male infertility in humans show an autosomal recessive mode of inheritance. It is possible that some of these families may carry mutations in the DAZLA gene.

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

    PubMed

    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.

  7. A phylogenetically conserved sequence within viral 3' untranslated RNA pseudoknots regulates translation.

    PubMed Central

    Leathers, V; Tanguay, R; Kobayashi, M; Gallie, D R

    1993-01-01

    Both the 68-base 5' leader (omega) and the 205-base 3' untranslated region (UTR) of tobacco mosaic virus (TMV) promote efficient translation. A 35-base region within omega is necessary and sufficient for the regulation. Within the 3' UTR, a 52-base region, composed of two RNA pseudoknots, is required for regulation. These pseudoknots are phylogenetically conserved among seven viruses from two different viral groups and one satellite virus. The pseudoknots contained significant conservation at the secondary and tertiary levels and at several positions at the primary sequence level. Mutational analysis of the sequences determined that the primary sequence in several conserved positions, particularly within the third pseudoknot, was essential for function. The higher-order structure of the pseudoknots was also required. Both the leader and the pseudoknot region were specifically recognized by, and competed for, the same proteins in extracts made from carrot cell suspension cells and wheat germ. Binding of the proteins is much stronger to omega than the pseudoknot region. Synergism was observed between the TMV 3' UTR and the cap and to a lesser extent between omega and the 3' UTR. The functional synergism and the protein binding data suggest that the cap, TMV 5' leader, and 3' UTR interact to establish an efficient level of translation. Images PMID:8355685

  8. 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

  9. Predicting protein ligand binding sites by combining evolutionary sequence conservation and 3D structure.

    PubMed

    Capra, John A; Laskowski, Roman A; Thornton, Janet M; Singh, Mona; Funkhouser, Thomas A

    2009-12-01

    Identifying a protein's functional sites is an important step towards characterizing its molecular function. Numerous structure- and sequence-based methods have been developed for this problem. Here we introduce ConCavity, a small molecule binding site prediction algorithm that integrates evolutionary sequence conservation estimates with structure-based methods for identifying protein surface cavities. In large-scale testing on a diverse set of single- and multi-chain protein structures, we show that ConCavity substantially outperforms existing methods for identifying both 3D ligand binding pockets and individual ligand binding residues. As part of our testing, we perform one of the first direct comparisons of conservation-based and structure-based methods. We find that the two approaches provide largely complementary information, which can be combined to improve upon either approach alone. We also demonstrate that ConCavity has state-of-the-art performance in predicting catalytic sites and drug binding pockets. Overall, the algorithms and analysis presented here significantly improve our ability to identify ligand binding sites and further advance our understanding of the relationship between evolutionary sequence conservation and structural and functional attributes of proteins. Data, source code, and prediction visualizations are available on the ConCavity web site (http://compbio.cs.princeton.edu/concavity/).

  10. Septal localization by membrane targeting sequences and a conserved sequence essential for activity at the COOH-terminus of Bacillus subtilis cardiolipin synthase.

    PubMed

    Kusaka, Jin; Shuto, Satoshi; Imai, Yukiko; Ishikawa, Kazuki; Saito, Tomo; Natori, Kohei; Matsuoka, Satoshi; Hara, Hiroshi; Matsumoto, Kouji

    2016-04-01

    The acidic phospholipid cardiolipin (CL) is localized on polar and septal membranes and plays an important physiological role in Bacillus subtilis cells. ClsA, the enzyme responsible for CL synthesis, is also localized on septal membranes. We found that GFP fusion proteins of the enzyme with NH2-terminal and internal deletions retained septal localization. However, derivatives with deletions starting from the COOH-terminus (Leu482) ceased to localize to the septum once the deletion passed the Ile residue at 448, indicating that the sequence responsible for septal localization is confined within a short distance from the COOH-terminus. Two sequences, Ile436-Leu450 and Leu466-Leu478, are predicted to individually form an amphipathic α-helix. This configuration is known as a membrane targeting sequence (MTS) and we therefore refer to them as MTS2 and MTS1, respectively. Either one has the ability to affect septal localization, and each of these sequences by itself localizes to the septum. Membrane association of the constructs of this enzyme containing the MTSs was verified by subcellular fractionation of the cells. CL synthesis, in contrast, was abolished after deleting just the last residue, Leu482, in the COOH-terminal four amino acid residue sequence, Ser-Pro-Ile-Leu, which is highly conserved among bacterial CL synthases.

  11. Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates.

    PubMed

    Howard, R F; Ardeshir, F; Reese, R T

    1986-01-01

    Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium falciparum, were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichia coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the K1 isolate [Hall et al., Nature 311 (1984) 379-382], codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 317 (1985) 270-273]. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of greater than or equal to 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous K1 isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. falciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15% of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. 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

  13. Conserved elements in the 3' untranslated region of flavivirus RNAs and potential cyclization sequences.

    PubMed

    Hahn, C S; Hahn, Y S; Rice, C M; Lee, E; Dalgarno, L; Strauss, E G; Strauss, J H

    1987-11-01

    We have isolated a cDNA clone after reverse transcription of the genomic RNA of Asibi yellow fever virus whose structure suggests it was formed by self-priming from a 3'-terminal hairpin of 87 nucleotides in the genomic RNA. We have also isolated a clone from cDNA made to Murray Valley encephalitis virus RNA that also appears to have arisen by self-priming from a 3'-terminal structure very similar or identical to that of yellow fever. In addition, 3'-terminal sequencing of the S1 strain of dengue 2 RNA shows that this RNA is also capable of forming a 3'-terminal hairpin of 79 nucleotides. Furthermore, we have identified two 20-nucleotide sequence elements which are present in the 3' untranslated region of all three viruses; one of these sequence elements is repeated in Murray Valley encephalitis and dengue 2 RNA but not in yellow fever RNA. In all three viruses, which represent the three major serological subgroups of the mosquito-borne flaviviruses, the 3'-proximal conserved sequence element, which is found immediately adjacent to the potential 3'-terminal hairpin, is complementary to another conserved domain near the 5' end of the viral RNAs, suggesting that flavivirus RNAs can cyclize (calculated delta G less than -11 kcal; 1 kcal = 4.184 kJ).

  14. Sample sequencing of vascular plants demonstrates widespread conservation and divergence of microRNAs.

    PubMed

    Chávez Montes, Ricardo A; de Fátima Rosas-Cárdenas, Flor; De Paoli, Emanuele; Accerbi, Monica; Rymarquis, Linda A; Mahalingam, Gayathri; Marsch-Martínez, Nayelli; Meyers, Blake C; Green, Pamela J; de Folter, Stefan

    2014-04-23

    Small RNAs are pivotal regulators of gene expression that guide transcriptional and post-transcriptional silencing mechanisms in eukaryotes, including plants. Here we report a comprehensive atlas of sRNA and miRNA from 3 species of algae and 31 representative species across vascular plants, including non-model plants. We sequence and quantify sRNAs from 99 different tissues or treatments across species, resulting in a data set of over 132 million distinct sequences. Using miRBase mature sequences as a reference, we identify the miRNA sequences present in these libraries. We apply diverse profiling methods to examine critical sRNA and miRNA features, such as size distribution, tissue-specific regulation and sequence conservation between species, as well as to predict putative new miRNA sequences. We also develop database resources, computational analysis tools and a dedicated website, http://smallrna.udel.edu/. This study provides new insights on plant sRNAs and miRNAs, and a foundation for future studies.

  15. Sample sequencing of vascular plants demonstrates widespread conservation and divergence of microRNAs.

    PubMed

    Chávez Montes, Ricardo A; de Fátima Rosas-Cárdenas, Flor; De Paoli, Emanuele; Accerbi, Monica; Rymarquis, Linda A; Mahalingam, Gayathri; Marsch-Martínez, Nayelli; Meyers, Blake C; Green, Pamela J; de Folter, Stefan

    2014-01-01

    Small RNAs are pivotal regulators of gene expression that guide transcriptional and post-transcriptional silencing mechanisms in eukaryotes, including plants. Here we report a comprehensive atlas of sRNA and miRNA from 3 species of algae and 31 representative species across vascular plants, including non-model plants. We sequence and quantify sRNAs from 99 different tissues or treatments across species, resulting in a data set of over 132 million distinct sequences. Using miRBase mature sequences as a reference, we identify the miRNA sequences present in these libraries. We apply diverse profiling methods to examine critical sRNA and miRNA features, such as size distribution, tissue-specific regulation and sequence conservation between species, as well as to predict putative new miRNA sequences. We also develop database resources, computational analysis tools and a dedicated website, http://smallrna.udel.edu/. This study provides new insights on plant sRNAs and miRNAs, and a foundation for future studies. PMID:24759728

  16. Recognition of distantly related protein sequences using conserved motifs and neural networks.

    PubMed

    Frishman, D; Argos, P

    1992-12-01

    A sensitive technique for protein sequence motif recognition based on neural networks has been developed. It involves three major steps. (1) At each appropriate alignment position of a set of N matched sequences, a set of N aligned oligopeptides is specified with preselected window length. N neural nets are subsequently and successively trained on N-1 amino acid spans after eliminating each ith oligopeptide. A test for recognition of each of the ith spans is performed. The average neural net recognition over N such trials is used as a measure of conservation for the particular windowed region of the multiple alignment. This process is repeated for all possible spans of given length in the multiple alignment. (2) The M most conserved regions are regarded as motifs and the oligopeptides within each are used to train intensively M individual neural networks. (3) The M networks are then applied in a search for related primary structures in a databank of known protein sequences. The oligopeptide spans in the database sequence with strongest neural net output for each of the M networks are saved and then scored according to the output signals and the proper combination that follows the expected N- to C-terminal sequence order. The motifs from the database with highest similarity scores can then be used to retrain the M neural nets, which can be subsequently utilized for further searches in the databank, thus providing even greater sensitivity to recognize distant familial proteins. This technique was successfully applied to the integrase, DNA-polymerase and immunoglobulin families.

  17. 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

  18. 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

  19. An rRNA variable region has an evolutionarily conserved essential role despite sequence divergence.

    PubMed Central

    Sweeney, R; Chen, L; Yao, M C

    1994-01-01

    Regions extremely variable in size and sequence occur at conserved locations in eukaryotic rRNAs. The functional importance of one such region was determined by gene reconstruction and replacement in Tetrahymena thermophila. Deletion of the D8 region of the large-subunit rRNA inactivates T. thermophila rRNA genes (rDNA): transformants containing only this type of rDNA are unable to grow. Replacement with an unrelated sequence of similar size or a variable region from a different position in the rRNA also inactivated the rDNA. Mutant rRNAs resulting from such constructs were present only in precursor forms, suggesting that these rRNAs are deficient in either processing or stabilization of the mature form. Replacement with D8 regions from three other organisms restored function, even though the sequences are very different. Thus, these D8 regions share an essential functional feature that is not reflected in their primary sequences. Similar tertiary structures may be the quality these sequences share that allows them to function interchangeably. Images PMID:8196658

  20. Conserved primary sequences of the DNA terminal proteins of five different human adenovirus groups.

    PubMed

    Green, M; Brackmann, K; Wold, W S; Cartas, M; Thornton, H; Elder, J H

    1979-09-01

    The 31 human adenoviruses (Ad) from five groups (A-E) whose DNAs are <20% homologous by molecular hybridization. Ad5 (group C) DNA contains a 55,000-dalton protein probably covalently bound to each 5' terminus. This covalently bound protein may be analogous to polypeptides found in other viral and nonviral systems that are covalently bound to genomic DNAs or RNAs and that are thought to function in DNA or RNA replication. Because of the importance of proteins linked to nucleic acids, we have investigated whether DNAs from all five groups of human adenoviruses have terminal proteins, as well as the peptide relationships among the different terminal proteins. We show here that DNAs from Ad12, 7, 2, 19, and 4, representing Ad groups A-E, respectively, all contain covalently bound proteins of about 55,000 daltons. To investigate the peptide relatedness among the terminal proteins, we prepared microgram quantities of covalently bound protein from Ads in groups A-E and compared their chymotryptic and tryptic (125)I-labeled peptide maps. We find that the covalently bound protein maps of the five Ad groups are highly related and possibly identical. On the other hand, the tryptic and chymotryptic peptide maps of the major virion protein II and the core proteins V and VII of groups B, C, and E Ads show considerable heterology. Assuming that the covalently bound protein is virally coded, the conserved primary sequence of these proteins suggests a major functional role for the protein in Ad replication. Because the genetic origin of the Ad covalently bound proteins is not established, our data are also consistent with the possibility that the protein is coded by a cellular gene.

  1. 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

  2. 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

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

    PubMed Central

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

    1992-01-01

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

  4. Characterization of the Genomic Xist Locus in Rodents Reveals Conservation of Overall Gene Structure and Tandem Repeats but Rapid Evolution of Unique Sequence

    PubMed Central

    Nesterova, Tatyana B.; Slobodyanyuk, Sergey Ya.; Elisaphenko, Eugene A.; Shevchenko, Alexander I.; Johnston, Colette; Pavlova, Marina E.; Rogozin, Igor B.; Kolesnikov, Nikolay N.; Brockdorff, Neil; Zakian, Suren M.

    2001-01-01

    The Xist locus plays a central role in the regulation of X chromosome inactivation in mammals, although its exact mode of action remains to be elucidated. Evolutionary studies are important in identifying conserved genomic regions and defining their possible function. Here we report cloning, sequence analysis, and detailed characterization of the Xist gene from four closely related species of common vole (field mouse), Microtus arvalis. Our analysis reveals that there is overall conservation of Xist gene structure both between different vole species and relative to mouse and human Xist/XIST. Within transcribed sequence, there is significant conservation over five short regions of unique sequence and also over Xist-specific tandem repeats. The majority of unique sequences, however, are evolving at an unexpectedly high rate. This is also evident from analysis of flanking sequences, which reveals a very high rate of rearrangement and invasion of dispersed repeats. We discuss these results in the context of Xist gene function and evolution. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AJ310127–AJ310130 and AJ311670.] PMID:11337478

  5. Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

    PubMed

    Klobutcher, L A; Gygax, S E; Podoloff, J D; Vermeesch, J R; Price, C M; Tebeau, C M; Jahn, C L

    1998-09-15

    During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.

  6. Effects of Mutation in the Conserved GTSRH Sequence of the Motor Protein Prestin on Its Characteristics

    NASA Astrophysics Data System (ADS)

    Kumano, Shun; Iida, Koji; Murakoshi, Michio; Naito, Naoyuki; Tsumoto, Kouhei; Ikeda, Katsuhisa; Kumagai, Izumi; Kobayashi, Toshimitsu; Wada, Hiroshi

    Prestin is a motor protein responsible for the outer hair cell (OHC) electromotility which amplifies the vibration of the organ of Corti in the inner ear. Identification of the functional significance of particular amino acids is necessary to characterize prestin. In this study, an attempt was made to clarify the role of the GTSRH sequence at positions 127-131 in prestin conserved in six proteins of the solute carrier (SLC) 26 family of which prestin is a member. To elucidate what role that sequence plays in the characteristics of prestin, mutations were introduced into the sequence and the characteristics of the constructed point mutants were investigated by Western blotting, immunofluorescence experiments and the whole-cell patch-clamp technique. The localization of T128A was altered, the anion transport function of H131A and that of S129T were lost and such functions of G127A, T128A, S129A and R130A declined. These results suggest that the GTSRH sequence plays an important role in the localization of prestin, as well as in its anion transport function.

  7. 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.

  8. Peptide Vocabulary Analysis Reveals Ultra-Conservation and Homonymity in Protein Sequences

    PubMed Central

    Gatherer, Derek

    2007-01-01

    A new algorithm is presented for vocabulary analysis (word detection) in texts of human origin. It performs at 60%–70% overall accuracy and greater than 80% accuracy for longer words, and approximately 85% sensitivity on Alice in Wonderland, a considerable improvement on previous methods. When applied to protein sequences, it detects short sequences analogous to words in human texts, i.e. intolerant to changes in spelling (mutation), and relatively context-independent in their meaning (function). Some of these are homonyms of up to 7 amino acids, which can assume different structures in different proteins. Others are ultra-conserved stretches of up to 18 amino acids within proteins of less than 40% overall identity, reflecting extreme constraint or convergent evolution. Different species are found to have qualitatively different major peptide vocabularies, e.g. some are dominated by large gene families, while others are rich in simple repeats or dominated by internally repetitive proteins. This suggests the possibility of a peptide vocabulary signature, analogous to genome signatures in DNA. Homonyms may be useful in detecting convergent evolution and positive selection in protein evolution. Ultra-conserved words may be useful in identifying structures intolerant to substitution over long periods of evolutionary time. PMID:20066129

  9. 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

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

    PubMed

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

    1998-07-01

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

  11. Conservation.

    ERIC Educational Resources Information Center

    National Audubon Society, New York, NY.

    This set of teaching aids consists of seven Audubon Nature Bulletins, providing the teacher and student with informational reading on various topics in conservation. The bulletins have these titles: Plants as Makers of Soil, Water Pollution Control, The Ground Water Table, Conservation--To Keep This Earth Habitable, Our Threatened Air Supply,…

  12. Phylogeny, sequence conservation, and functional complementation of the SBDS protein family.

    PubMed

    Boocock, G R B; Marit, M R; Rommens, J M

    2006-06-01

    The Shwachman-Bodian-Diamond syndrome (SBDS) protein family occurs widely in nature, although its function has not been determined. Comprehensive database searches revealed SBDS homologues from 159 species, including examples from all sequenced archaeal and eukaryotic genomes and all eukaryotic kingdoms. Sequence alignment with ClustalX and MUSCLE algorithms led to the identification of conserved residues that occurred predominantly in the amino-terminal FYSH domain where they appeared to contribute to protein folding or stability. Only SBDS residue Gly91 was invariant in all species. Four distantly related protists were found to have two divergent SBDS genes in their genomes. In each case, phylogenetic analyses and the identification of shared sequence features suggested that one gene was derived from lateral gene transfer. We also identified a shared C-terminal zinc finger domain fusion in flowering plants and chromalveolates that may shed light on the function of the protein family and the evolutionary histories of these kingdoms. To assess the extent of SBDS functional conservation, we carried out complementation studies of SBDS homologues and interspecies chimeras in Saccharomyces cerevisiae. We determined that the FYSH domain was widely interchangeable among eukaryotes, while domain 2 imparted species specificity to protein function. Domain 3 was largely dispensable for function in our yeast complementation assay. Overall, the phylogeny of SBDS was shared with a group of proteins that were markedly enriched for RNA metabolism and/or ribosome-associated functions. These findings link Shwachman-Diamond syndrome to other bone marrow failure syndromes with defects in nucleolus-associated processes, including Diamond-Blackfan anemia, cartilage-hair hypoplasia, and dyskeratosis congenita.

  13. Genome-wide analyses of Epstein-Barr virus reveal conserved RNA structures and a novel stable intronic sequence RNA

    PubMed Central

    2013-01-01

    Background Epstein-Barr virus (EBV) is a human herpesvirus implicated in cancer and autoimmune disorders. Little is known concerning the roles of RNA structure in this important human pathogen. This study provides the first comprehensive genome-wide survey of RNA and RNA structure in EBV. Results Novel EBV RNAs and RNA structures were identified by computational modeling and RNA-Seq analyses of EBV. Scans of the genomic sequences of four EBV strains (EBV-1, EBV-2, GD1, and GD2) and of the closely related Macacine herpesvirus 4 using the RNAz program discovered 265 regions with high probability of forming conserved RNA structures. Secondary structure models are proposed for these regions based on a combination of free energy minimization and comparative sequence analysis. The analysis of RNA-Seq data uncovered the first observation of a stable intronic sequence RNA (sisRNA) in EBV. The abundance of this sisRNA rivals that of the well-known and highly expressed EBV-encoded non-coding RNAs (EBERs). Conclusion This work identifies regions of the EBV genome likely to generate functional RNAs and RNA structures, provides structural models for these regions, and discusses potential functions suggested by the modeled structures. Enhanced understanding of the EBV transcriptome will guide future experimental analyses of the discovered RNAs and RNA structures. PMID:23937650

  14. 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.

  15. 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.

  16. 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.

  17. 76 FR 82075 - Highly Erodible Land and Wetland Conservation

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-30

    ... effective July 24, 1971 (36 FR 13804), relating to notices of proposed rulemaking and public participation... 3015, subpart V, published in the Federal Register on June 24, 1983 (48 FR 29115). Executive Order... Secretary 7 CFR Part 12 RIN 0560-AH97 Highly Erodible Land and Wetland Conservation AGENCY: Office of...

  18. Canine and feline trefoil factor family peptides: highly conserved molecules with some unique characteristics.

    PubMed

    Campbell, B G; Jabbes, M

    2008-08-01

    Trefoil factor family (TFF) peptides maintain and repair the gastrointestinal mucosa and are aberrantly expressed in human and rodent inflammatory bowel disease and carcinomas, diseases common in dogs and cats. Study objectives were to sequence and translate canine and feline tff cDNAs and define any unique residues that might influence their structure and/or function. After isolation and reverse transcription of canine and feline gastrointestinal mucosal RNA, TFF cDNAs were amplified, sequenced, and cloned. Dogs and cats had unique amino acids in several places that were highly or completely conserved in other mammals, including a hydrophobic area in the TFF1 functional site, loop 2 of each TFF2 trefoil domain, a TFF3 dimerization site, and the TFF2 C-terminus. By identifying conserved and unique characteristics of canine and feline TFFs, this study establishes a foundation for investigation of dog and cat models of TFF-related diseases in both human and veterinary medicine.

  19. 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

  20. 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

  1. 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.

  2. T-cell recognition is shaped by epitope sequence conservation in the host proteome and microbiome.

    PubMed

    Bresciani, Anne; Paul, Sinu; Schommer, Nina; Dillon, Myles B; Bancroft, Tara; Greenbaum, Jason; Sette, Alessandro; Nielsen, Morten; Peters, Bjoern

    2016-05-01

    Several mechanisms exist to avoid or suppress inflammatory T-cell immune responses that could prove harmful to the host due to targeting self-antigens or commensal microbes. We hypothesized that these mechanisms could become evident when comparing the immunogenicity of a peptide from a pathogen or allergen with the conservation of its sequence in the human proteome or the healthy human microbiome. Indeed, performing such comparisons on large sets of validated T-cell epitopes, we found that epitopes that are similar with self-antigens above a certain threshold showed lower immunogenicity, presumably as a result of negative selection of T cells capable of recognizing such peptides. Moreover, we also found a reduced level of immune recognition for epitopes conserved in the commensal microbiome, presumably as a result of peripheral tolerance. These findings indicate that the existence (and potentially the polarization) of T-cell responses to a given epitope is influenced and to some extent predictable based on its similarity to self-antigens and commensal antigens.

  3. 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.

  4. Mouse annexin V chromosomal localization, cDNA sequence conservation, and molecular evolution

    SciTech Connect

    Rodriguez-Garcia, M.I.; Morgan, R.O.; Kozak, C.A.

    1996-01-15

    A full-length cDNA encoding mouse annexin V (ANX5) was cloned, sequenced, and utilized for chromosomal mapping. The gene lies on mouse chromosome 3 in close linkage with the fibroblast growth factor 2 (basic) gene and is syntenic with other genes known to have orthologous counterparts on human chromosome 4q. The open reading frame encoded a protein of 319 amino acids (aa), with 92-96% identity to ANX5 in other species. Internal repeat 3 of mouse ANX5 exhibited the highest level of nonconservative aa replacements with respect to other annexin subfamilies, but the greatest sequence conservation among ANX5 species members. This region may thus contain features that distinguish ANX5 from other annexins in properties or function. Phylogenetic analysis and homology testing of ANX5 members indicated that the 34-kDa annexin from Torpedo marmorata may also belong to this subfamily. Comparison of nine species of ANX5 led to an estimation of the unit evolutionary mutation rate at 1% aa replacements every 8 million years, comparable to other annexins. 46 refs., 4 figs.

  5. Agricultural capacity and conservation in high biodiversity forest ecosystems.

    PubMed

    Gorenflo, L J; Brandon, Katrina

    2005-05-01

    Agricultural development is a leading cause of habitat destruction that increasingly threatens global biodiversity. To help understand the likelihood and implications of agricultural expansion in areas of high conservation importance, this article examines agricultural suitability in forested portions of biodiversity hotspots and tropical wilderness areas, regions with especially rich concentrations of species found nowhere else. The study employs geographic information system technology to examine suitability for six crop categories in selected conservation localities worldwide: those portions of regions containing high biodiversity, protected areas (e.g. national parks) within these regions, and 10-km bands around the protected areas that are dominated by forest. Analyses reveal low suitability for most crop categories under both commercial and subsistence scenarios, with a few exceptions. In most cases, adequate planning can enable the coexistence of agriculture and biodiversity without compromising either.

  6. Qualifying high-throughput immune repertoire sequencing.

    PubMed

    Niklas, Norbert; Pröll, Johannes; Weinberger, Johannes; Zopf, Agnes; Wiesinger, Karin; Krismer, Konstantin; Bettelheim, Peter; Gabriel, Christian

    2014-01-01

    Diversity of B and T cell receptors, achieved by gene recombination and somatic hypermutation, allows the immune system for recognition and targeted reaction against various threats. Next-generation sequencing for assessment of a cell's gene composition and variation makes deep analysis of one individual's immune spectrum feasible. An easy to apply but detailed analysis and visualization strategy is necessary to process all sequences generated. We performed sequencing utilizing the 454 system for CLL and control samples, utilized the IMGT database and applied the presented analysis tools. With the applied protocol, malignant clones are found and characterized, mutational status compared to germline identity is elaborated in detail showing that the CLL mutation status is not as monoclonal as generally thought. On the other hand, this strategy is not solely applicable to the 454 sequencing system but can easily be transferred to any other next-generation sequencing platform. PMID:24607567

  7. Seminal-type ribonuclease genes in ruminants, sequence conservation without protein expression?

    PubMed

    Kleineidam, R G; Jekel, P A; Beintema, J J; Situmorang, P

    1999-04-29

    Bovine seminal ribonuclease (BS-RNase) is an interesting enzyme both for functional and structural reasons. The enzyme is the product of a gene duplication that occurred in an ancestral ruminant. It is possible to demonstrate the presence of seminal-type genes in all other investigated ruminant species, but they are not expressed and show features of pseudogenes. In this paper we report the determination of two pancreatic and one seminal-type ribonuclease gene sequences of swamp-type water buffalo (Bubalus bubalis). The two pancreatic sequences encode proteins with identical amino acid sequences as previously determined for the enzymes isolated from swamp-type and river-type water buffalo, respectively. The seminal-type sequence has no pseudogene features and codes for an enzyme with no unusual features compared with the active bovine enzyme, except for the replacement of one of the cysteines which takes part in the two intersubunit disulfide bridges. However, Western blotting demonstrates the presence of only small amounts of the pancreatic enzymes in water buffalo semen, suggesting that also in this species the seminal-type sequence is not expressed. But it is still possible that the gene is expressed somewhere else in the body or during development. Reconstruction of seminal-type ribonuclease sequences in ancestors of Bovinae and Bovidae indicates no serious abnormalities in the encoded proteins and leads us to the hypothesis that the ruminant seminal-type ribonuclease gene has not come to expression during most of its evolutionary history, but did not exhibit a high evolutionary rate that is generally observed in pseudogenes.

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

    PubMed

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

    2015-04-01

    Unlike typical cis-splicing, trans-splicing joins exons from two separate transcripts to produce chimeric mRNA and has been detected in most eukaryotes. Trans-splicing in trypanosomes and nematodes has been characterized as a spliced leader RNA-facilitated reaction; in contrast, its mechanism in higher eukaryotes remains unclear. Here we investigate mod(mdg4), a classic trans-spliced gene in Drosophila, and report that two critical RNA sequences in the middle of the last 5' intron, TSA and TSB, promote trans-splicing of mod(mdg4). In TSA, a 13-nucleotide (nt) core motif is conserved across Drosophila species and is essential and sufficient for trans-splicing, which binds U1 small nuclear RNP (snRNP) through strong base-pairing with U1 snRNA. In TSB, a conserved secondary structure acts as an enhancer. Deletions of TSA and TSB using the CRISPR/Cas9 system result in developmental defects in flies. Although it is not clear how the 5' intron finds the 3' introns, compensatory changes in U1 snRNA rescue trans-splicing of TSA mutants, demonstrating that U1 recruitment is critical to promote trans-splicing in vivo. Furthermore, TSA core-like motifs are found in many other trans-spliced Drosophila genes, including lola. These findings represent a novel mechanism of trans-splicing, in which RNA motifs in the 5' intron are sufficient to bring separate transcripts into close proximity to promote trans-splicing. PMID:25838544

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

    PubMed

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

    2015-04-01

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

  10. Identifying Conserved and Novel MicroRNAs in Developing Seeds of Brassica napus Using Deep Sequencing

    PubMed Central

    Körbes, Ana Paula; Machado, Ronei Dorneles; Guzman, Frank; Almerão, Mauricio Pereira; de Oliveira, Luiz Felipe Valter; Loss-Morais, Guilherme; Turchetto-Zolet, Andreia Carina; Cagliari, Alexandro; dos Santos Maraschin, Felipe; Margis-Pinheiro, Marcia; Margis, Rogerio

    2012-01-01

    MicroRNAs (miRNAs) are important post-transcriptional regulators of plant development and seed formation. In Brassica napus, an important edible oil crop, valuable lipids are synthesized and stored in specific seed tissues during embryogenesis. The miRNA transcriptome of B. napus is currently poorly characterized, especially at different seed developmental stages. This work aims to describe the miRNAome of developing seeds of B. napus by identifying plant-conserved and novel miRNAs and comparing miRNA abundance in mature versus developing seeds. Members of 59 miRNA families were detected through a computational analysis of a large number of reads obtained from deep sequencing two small RNA and two RNA-seq libraries of (i) pooled immature developing stages and (ii) mature B. napus seeds. Among these miRNA families, 17 families are currently known to exist in B. napus; additionally 29 families not reported in B. napus but conserved in other plant species were identified by alignment with known plant mature miRNAs. Assembled mRNA-seq contigs allowed for a search of putative new precursors and led to the identification of 13 novel miRNA families. Analysis of miRNA population between libraries reveals that several miRNAs and isomiRNAs have different abundance in developing stages compared to mature seeds. The predicted miRNA target genes encode a broad range of proteins related to seed development and energy storage. This work presents a comparative study of the miRNA transcriptome of mature and developing B. napus seeds and provides a basis for future research on individual miRNAs and their functions in embryogenesis, seed maturation and lipid accumulation in B. napus. PMID:23226347

  11. 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

  12. High-Throughput Sequencing in Mitochondrial DNA Research

    PubMed Central

    Ye, Fei; Samuels, David C.; Clark, Travis; Guo, Yan

    2014-01-01

    Next-generation sequencing, also known as high-throughput sequencing, has greatly enhanced researchers’ ability to conduct biomedical research on all levels. Mitochondrial research has also benefitted greatly from high-throughput sequencing; sequencing technology now allows for screening of all 16569 base pairs of the mitochondrial genome simultaneously for SNPs and low level heteroplasmy and, in some cases, the estimation of mitochondrial DNA copy number. It is important to realize the full potential of high-throughput sequencing for the advancement of mitochondrial research. To this end, we review how high-throughput sequencing has impacted mitochondrial research in the categories of SNPs, low level heteroplasmy, copy number, and structural variants. We also discuss the different types of mitochondrial DNA sequencing and their pros and cons. Based on previous studies conducted by various groups, we provide strategies for processing mitochondrial DNA sequencing data, including assembly, variant calling, and quality control. PMID:24859348

  13. High-throughput sequencing in mitochondrial DNA research.

    PubMed

    Ye, Fei; Samuels, David C; Clark, Travis; Guo, Yan

    2014-07-01

    Next-generation sequencing, also known as high-throughput sequencing, has greatly enhanced researchers' ability to conduct biomedical research on all levels. Mitochondrial research has also benefitted greatly from high-throughput sequencing; sequencing technology now allows for screening of all 16,569 base pairs of the mitochondrial genome simultaneously for SNPs and low level heteroplasmy and, in some cases, the estimation of mitochondrial DNA copy number. It is important to realize the full potential of high-throughput sequencing for the advancement of mitochondrial research. To this end, we review how high-throughput sequencing has impacted mitochondrial research in the categories of SNPs, low level heteroplasmy, copy number, and structural variants. We also discuss the different types of mitochondrial DNA sequencing and their pros and cons. Based on previous studies conducted by various groups, we provide strategies for processing mitochondrial DNA sequencing data, including assembly, variant calling, and quality control.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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...

  19. Analysis of conserved microsatellite sequences suggests closer relationship between water buffalo Bubalus bubalis and sheep Ovis aries.

    PubMed

    Mattapallil, M J; Ali, S

    1999-06-01

    The distribution and evolutionary pattern of the conserved microsatellite repeat sequences (CA)n, (TGG)6, and (GGAT)4 were studied to determine the divergence time and phylogenetic position of the water buffalo, Bubalus bubalis. The mean allelic frequencies of these repeat loci showed a high level of heterozygosity among the euartiodactyls (buffalo, cattle, sheep, and goat). Genetic distances calculated from the allelic frequencies of these microsatellites were used to position Bubalus bubalis in the phylogenetic tree. The tree topology revealed a closer proximity of the Bubalus bubalis to the Ovis aries (sheep) genome than to other domestic species. The estimated time of divergence of the water buffalo genome relative to cattle, goat, sheep, pig, rabbit, and horse was found to be 21, 0.5, 0.7, 94, 20.3, and 408 million years (Myr), respectively. Although water buffaloes share morphological and biochemical similarities with cattle, our study using the microsatellite sequences places the bubaline species in an entirely new phylogenetic position. Our results also suggest that with respect to these repeat loci, the water buffalo genome shares a common ancestry with sheep and goat after the divergence of subfamily Bovinae (Bos taurus) from the family Bovidae.

  20. 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...

  1. 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

  2. Worldwide sequence conservation of transmission-blocking vaccine candidate Pvs230 in Plasmodium vivax

    PubMed Central

    Doi, Masanori; Tanabe, Kazuyuki; Tachibana, Shin-Ichiro; Hamai, Meiko; Tachibana, Mayumi; Mita, Toshihiro; Yagi, Masanori; Zeyrek, Fadile Yildiz; Ferreira, Marcelo U.; Ohmae, Hiroshi; Kaneko, Akira; Randrianarivelojosia, Milijaona; Sattabongkot, Jetsumon; Cao, Ya-Ming; Horii, Toshihiro; Torii, Motomi; Tsuboi, Takafumi

    2011-01-01

    Pfs230, surface protein of gametocyte/gamete of the human malaria parasite, Plasmodium falciparum, is a prime candidate of malaria transmission-blocking vaccine. P. vivax has an ortholog of Pfs230 (Pvs230), however, there has been no study in any aspects on Pvs230 to date. To investigate whether Pvs230 can be a vivax malaria transmission-blocking vaccine, we performed evolutionary and population genetic analysis of the Pvs230 gene (pvs230: PVX_003905). Our analysis of Pvs230 and its orthologs in seven Plasmodium species revealed two distinctive parts: an interspecies variable part (IVP) containing species-specific oligopeptide repeats at the N-terminus and a 7.5 kb interspecies conserved part (ICP) containing 14 cysteine-rich domains. Pvs230 was closely related to its orthologs, Pks230 and Pcys230, in monkey malaria parasites. Analysis of 113 pvs230 sequences obtained from worldwide, showed that nucleotide diversity is remarkably low in the non-repeat 8-kb region of pvs230 (θπ = 0.00118) with 77 polymorphic nucleotide sites, 40 of which resulting in amino acid replacements. A signature of purifying selection but not of balancing selection was seen on pvs230. Functional and/or structural constraints may limit the level of polymorphism in pvs230. The observed limited polymorphism in pvs230 should ground for utilization of Pvs230 as an effective transmission-blocking vaccine. PMID:21514344

  3. ABCE1 Is a Highly Conserved RNA Silencing Suppressor

    PubMed Central

    Kärblane, Kairi; Gerassimenko, Jelena; Nigul, Lenne; Piirsoo, Alla; Smialowska, Agata; Vinkel, Kadri; Kylsten, Per; Ekwall, Karl; Swoboda, Peter; Truve, Erkki; Sarmiento, Cecilia

    2015-01-01

    ATP-binding cassette sub-family E member 1 (ABCE1) is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference. PMID:25659154

  4. Retention of viral infectivity after extensive mutation of the highly conserved immunodominant domain of the feline immunodeficiency virus envelope.

    PubMed Central

    Pancino, G; Sonigo, P

    1997-01-01

    In lentiviruses, including human immunodeficiency virus and feline immunodeficiency virus (FIV), the principal immunodominant domain (PID) of the transmembrane glycoprotein elicits a strong humoral response in infected hosts. The PID is marked by the presence of two cysteines that delimit a sequence, composed of five to seven amino acids in different lentiviruses, which is highly conserved among isolates of the same lentiviral species. While the conservation of the sequence suggests the presence of functional constraints, the conservation of the immunodominance among divergent lentiviruses raises the hypothesis of a selective advantage for the infecting virus conferred by the host humoral response against this domain. We and others have previously shown that an appropriate structure of the PID is required for the production of a functional envelope. In the present work, we analyzed virological functions and immune reactivity of the envelope after random mutagenesis of the PID of FIV. We obtained nine mutant envelopes which were correctly processed and retained fusogenic ability. Mutation of the two C-terminal residues of the PID sequence between the cysteines in a molecular clone of FIV abolished infectivity. In contrast, three molecular clones containing extensive mutations in the four N-terminal amino acids were infectious. However, the mutations affected PID reactivity with sera from infected cats. Our results suggest that functional constraints, although existent, are not sufficient to account for PID sequence conservation. Such conservation may also result from positive selection by anti-PID antibodies which enhance infection. PMID:9151822

  5. CDvist: A webserver for identification and visualization of conserved domains in protein sequences

    DOE PAGESBeta

    Adebali, Ogun; Ortega, Davi R.; Zhulin, Igor B.

    2014-12-18

    Identification of domains in protein sequences allows their assigning to biological functions. Several webservers exist for identification of protein domains using similarity searches against various databases of protein domain models. However, none of them provides comprehensive domain coverage while allowing bulk querying and their visualization schemes can be improved. To address these issues, we developed CDvist (a comprehensive domain visualization tool), which combines the best available search algorithms and databases into a user-friendly framework. First, a given protein sequence is matched to domain models using high-specificity tools and only then unmatched segments are subjected to more sensitive algorithms resulting inmore » a best possible comprehensive coverage. In conclusion, bulk querying and rich visualization and download options provide improved functionality to domain architecture analysis.« less

  6. CDvist: A webserver for identification and visualization of conserved domains in protein sequences

    SciTech Connect

    Adebali, Ogun; Ortega, Davi R.; Zhulin, Igor B.

    2014-12-18

    Identification of domains in protein sequences allows their assigning to biological functions. Several webservers exist for identification of protein domains using similarity searches against various databases of protein domain models. However, none of them provides comprehensive domain coverage while allowing bulk querying and their visualization schemes can be improved. To address these issues, we developed CDvist (a comprehensive domain visualization tool), which combines the best available search algorithms and databases into a user-friendly framework. First, a given protein sequence is matched to domain models using high-specificity tools and only then unmatched segments are subjected to more sensitive algorithms resulting in a best possible comprehensive coverage. In conclusion, bulk querying and rich visualization and download options provide improved functionality to domain architecture analysis.

  7. 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.

  8. High throughput system for DNA sequencing

    NASA Astrophysics Data System (ADS)

    El-Difrawy, Sameh A.; Lam, Roger; Aborn, James H.; Novotny, Mark; Gismondi, Elizabeth A.; Matsudaira, Paul; Mckenna, Brian K.; O'Neil, Thomas; Streechon, Philip; Ehrlich, Daniel J.

    2005-07-01

    A 768-lane DNA sequencing system based on micromachined plates has been designed as a near-term successor to 96-lane capillary arrays. Electrophoretic separations are implemented in large-format (25cm by 50cm) microfabricated devices with the objective of proving realistic read length, parallelism, and the scaled sample requirements for long-read de novo sequencing. Two 384-lane plates are alternatively cycled between electrophoresis and regeneration via a robotic pipettor and switching optical system. The instrument minimizes the DNA sample requirement to "1/32×" Sanger chemistry, equal to typical genome center operation, and a 16-fold improvement in scaling relative to previous microfabricated devices. The 40-cm-long channels permit an increase in read length (several hundred base pairs) relative to previous multichannel microfabricated devices. These advances directly address the cost and automation model for adaptation of the technology.

  9. 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.

  10. 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.

  11. Estimating Protistan Diversity Using High-Throughput Sequencing.

    PubMed

    Hu, Sarah K; Liu, Zhenfeng; Lie, Alle A Y; Countway, Peter D; Kim, Diane Y; Jones, Adriane C; Gast, Rebecca J; Cary, S Craig; Sherr, Evelyn B; Sherr, Barry F; Caron, David A

    2015-01-01

    Sequencing hypervariable regions from the 18S rRNA gene is commonly employed to characterize protistan biodiversity, yet there are concerns that short reads do not provide the same taxonomic resolution as full-length sequences. A total of 7,432 full-length sequences were used to perform an in silico analysis of how sequences of various lengths and target regions impact downstream ecological interpretations. Sequences that were longer than 400 nucleotides and included the V4 hypervariable region generated results similar to those derived from full-length 18S rRNA gene sequences. Present high-throughput sequencing capabilities are approaching protistan diversity estimation comparable to whole gene sequences.

  12. The complex multidomain organization of SCO-spondin protein is highly conserved in mammals.

    PubMed

    Meiniel, Olivier; Meiniel, Annie

    2007-02-01

    The multidomain organization of SCO-spondin protein is a special feature of the chordate phylum. This protein is expressed in the central nervous system (CNS) from the time a dorsal neural tube appears in the course of phylogenetical evolution. With the advance of the systematic whole genomes sequencing, we were able to determine the SCO-spondin amino acid sequence in four mammalian species using the Wise2 software. From the ClustalW alignment of bovine (Bos taurus), human (Homo sapiens), murine (Mus musculus) and rat (Rattus norvegicus) proteins, a consensus sequence for mammalian SCO-spondin was determined and further validated with the dog (Canis familiaris) SCO-spondin sequence. The analysis of this consensus sequence is consistent with a very high degree of conservation in the amino acids composition and multidomain organization of SCO-spondin in mammals. In addition, the identification of conserved domains, namely, Emilin (EMI), von Willebrand factor D (vWD), low-density lipoprotein receptor type A (LDLrA) domains, SCO repeats (SCOR), thrombospondin type 1 repeats (TSR), a coagulation factor 5/8 type C (FA5-8C) or discoidin motif and a C-terminal cystine knot (CTCK) domain, provides a greater insight into the putative function of this multidomain protein. SCO-spondin belongs to the TSR superfamily given the presence of a great number of TSR (26). A finer classification of the TSR motifs in groups 1, 2 and 3 is proposed on the basis of different cysteine patterns. Interestingly, group 2 TSR are present in a number of CNS developmental proteins including R-spondins, F-spondins and Mindins.

  13. An Abundant, Highly Conserved Tonoplast Protein in Seeds 1

    PubMed Central

    Johnson, Kenneth D.; Herman, Eliot M.; Chrispeels, Maarten J.

    1989-01-01

    We have isolated the membranes of the protein storage vacuoles (protein bodies) from Phaseolus vulgaris cotyledons and purified an integral membrane protein with Mr 25,000 (TP 25). Antiserum to TP 25 recognizes an abundant polypeptide in the total cell extracts of many different seeds (monocots, dicots, and a gymnosperm), and specifically labels the vacuolar membranes of thin-sectioned soybean embryonic axes and cotyledons. TP 25 was not found in the starchy endosperm of barley and wheat or the seed coats of bean but was present in all seed parts examined that consist of living cells at seed maturity. The abundance of TP 25 was not correlated with the amount of storage protein in seed tissue, and the protein was not found in leaves that accumulate leaf storage protein. On the basis of its abundance, evolutionary conservation, and distribution in the plant, we propose that TP 25 may play a role in maintaining the integrity of the tonoplast during the dehydration/rehydration sequence of seeds. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:16667102

  14. Conserved Amino Acid Sequence Features in the α Subunits of MoFe, VFe, and FeFe Nitrogenases

    PubMed Central

    Glazer, Alexander N.; Kechris, Katerina J.

    2009-01-01

    Background This study examines the structural features and phylogeny of the α subunits of 69 full-length NifD (MoFe subunit), VnfD (VFe subunit), and AnfD (FeFe subunit) sequences. Methodology/Principal Findings The analyses of this set of sequences included BLAST scores, multiple sequence alignment, examination of patterns of covariant residues, phylogenetic analysis and comparison of the sequences flanking the conserved Cys and His residues that attach the FeMo cofactor to NifD and that are also conserved in the alternative nitrogenases. The results show that NifD nitrogenases fall into two distinct groups. Group I includes NifD sequences from many genera within Bacteria, including all nitrogen-fixing aerobes examined, as well as strict anaerobes and some facultative anaerobes, but no archaeal sequences. In contrast, Group II NifD sequences were limited to a small number of archaeal and bacterial sequences from strict anaerobes. The VnfD and AnfD sequences fall into two separate groups, more closely related to Group II NifD than to Group I NifD. The pattern of perfectly conserved residues, distributed along the full length of the Group I and II NifD, VnfD, and AnfD, confirms unambiguously that these polypeptides are derived from a common ancestral sequence. Conclusions/Significance There is no indication of a relationship between the patterns of covariant residues specific to each of the four groups discussed above that would give indications of an evolutionary pathway leading from one type of nitrogenase to another. Rather the totality of the data, along with the phylogenetic analysis, is consistent with a radiation of Group I and II NifDs, VnfD and AnfD from a common ancestral sequence. All the data presented here strongly support the suggestion made by some earlier investigators that the nitrogenase family had already evolved in the last common ancestor of the Archaea and Bacteria. PMID:19578539

  15. Sequence of the human 40-kDa keratin reveals an unusual structure with very high sequence identity to the corresponding bovine keratin.

    PubMed Central

    Eckert, R L

    1988-01-01

    The complete amino acid and DNA sequences of the human 40-kDa keratin are reported. The DNA sequence encodes a protein of 44,098 Da, which is unique in that it lacks the terminal non-alpha-helical tail segment found in all other keratins. When the human 40-kDa keratin amino acid sequence is compared to the corresponding bovine keratin, the overall identity is 89%. The coil-forming regions are 89% identical and the head regions are 88% identical. This similarity is also evident in the DNA sequence of the coding region, the 5' upstream sequences, and the 3' noncoding sequences. The high degree of cross-species identity between bovine and human 40-kDa keratins suggests that there is strong evolutionary pressure to conserve the structure of this keratin. This in turn suggests an important and universal role for this intermediate filament subunit in all species. Images PMID:2448790

  16. High resolution mapping of Twist to DNA in Drosophila embryos: Efficient functional analysis and evolutionary conservation.

    PubMed

    Ozdemir, Anil; Fisher-Aylor, Katherine I; Pepke, Shirley; Samanta, Manoj; Dunipace, Leslie; McCue, Kenneth; Zeng, Lucy; Ogawa, Nobuo; Wold, Barbara J; Stathopoulos, Angelike

    2011-04-01

    Cis-regulatory modules (CRMs) function by binding sequence specific transcription factors, but the relationship between in vivo physical binding and the regulatory capacity of factor-bound DNA elements remains uncertain. We investigate this relationship for the well-studied Twist factor in Drosophila melanogaster embryos by analyzing genome-wide factor occupancy and testing the functional significance of Twist occupied regions and motifs within regions. Twist ChIP-seq data efficiently identified previously studied Twist-dependent CRMs and robustly predicted new CRM activity in transgenesis, with newly identified Twist-occupied regions supporting diverse spatiotemporal patterns (>74% positive, n = 31). Some, but not all, candidate CRMs require Twist for proper expression in the embryo. The Twist motifs most favored in genome ChIP data (in vivo) differed from those most favored by Systematic Evolution of Ligands by EXponential enrichment (SELEX) (in vitro). Furthermore, the majority of ChIP-seq signals could be parsimoniously explained by a CABVTG motif located within 50 bp of the ChIP summit and, of these, CACATG was most prevalent. Mutagenesis experiments demonstrated that different Twist E-box motif types are not fully interchangeable, suggesting that the ChIP-derived consensus (CABVTG) includes sites having distinct regulatory outputs. Further analysis of position, frequency of occurrence, and sequence conservation revealed significant enrichment and conservation of CABVTG E-box motifs near Twist ChIP-seq signal summits, preferential conservation of ±150 bp surrounding Twist occupied summits, and enrichment of GA- and CA-repeat sequences near Twist occupied summits. Our results show that high resolution in vivo occupancy data can be used to drive efficient discovery and dissection of global and local cis-regulatory logic.

  17. 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.

  18. 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?

    PubMed

    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 approximately 15 A 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 A 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.

  19. 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

  20. 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

  1. Molecular dissection of a contiguous gene syndrome: Frequent submicroscopic deletions, evolutionarily conserved sequences, and a hypomethylated island in the Miller-Dieker chromosome region

    SciTech Connect

    Ledbetter, D.H.; Ledbetter, S.A.; vanTuinen, P.; Summers, K.M.; Robinson, T.J.; Nakamura, Yusuke; Wolff, R.; White, R.; Barker, D.F.; Wallace, M.R.; Collins, F.S.; Dobyns, W.B. )

    1989-07-01

    The Miller-Dieker syndrome (MDS), composed of characteristic facial abnormalities and a severe neuronal migration disorder affecting the cerebral cortex, is caused by visible or submicroscopic deletions of chromosome band 17p13. Twelve anonymous DNA markers were tested against a panel of somatic cell hybrids containing 17p deletions from seven MDS patients. All patients, including three with normal karyotypes, are deleted for a variable set of 5-12 markers. Two highly polymorphic VNTR (variable number of tandem repeats) probes, YNZ22 and YNH37, are codeleted in all patients tested and make molecular diagnosis for this disorder feasible. By pulsed-field gel electrophoresis, YNZ22 and YNH37 were shown to be within 30 kilobases (kb) of each other. Cosmid clones containing both VNTR sequences were identified, and restriction mapping showed them to be <15 kb apart. Three overlapping cosmids spanning >100 kb were completely deleted in all patients, providing a minimum estimate of the size of the MDS critical region. A hypomethylated island and evolutionarily conserved sequences were identified within this 100-kb region, indications of the presence of one or more expressed sequences potentially involved in the pathophysiology of this disorder. The conserved sequences were mapped to mouse chromosome 11 by using mouse-rat somatic cell hybrids, extending the remarkable homology between human chromosome 17 and mouse chromosome 11 by 30 centimorgans, into the 17p telomere region.

  2. An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5' UTRs and its implications for eukaryotic gene translation regulation.

    PubMed

    Pánek, Josef; Kolár, Michal; Vohradský, Jirí; Shivaya Valásek, Leos

    2013-09-01

    There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA-rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5' untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5' UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5' UTRs of mRNAs.

  3. Phylogenetic conservation of the 3' cryptic recombination signal sequence (3'cRSS) in the VH genes of jawed vertebrates.

    PubMed

    Sun, Yi; Liu, Zhancai; Li, Zhaoyong; Lian, Zhengxing; Zhao, Yaofeng

    2012-01-01

    The VH replacement process is a RAG-mediated secondary recombination in which the variable region of a rearranged VHDJH is replaced by a different germline VH gene. In almost all human and mouse VH genes, two sequence features appear to be crucial for VH replacement. First, an embedded heptamer, which is located near the 3' end of the rearranged VH gene, serves as a cryptic recombination signal sequence (3'cRSS) for the VH replacement process. Second, a short stretch of nucleotides located downstream of the 3'cRSS serve as a footprint of the original VH region, frequently encoding charged amino acids. In this review, we show that both of these two features are conserved in the VH genes of all jawed vertebrates, which suggests that the VH replacement process may be a conserved mechanism.

  4. 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 ...

  5. Standards for Sequencing Viral Genomes in the Era of High-Throughput Sequencing

    PubMed Central

    Beitzel, Brett; Chain, Patrick S. G.; Davenport, Matthew G.; Donaldson, Eric; Frieman, Matthew; Kugelman, Jeffrey; Kuhn, Jens H.; O’Rear, Jules; Sabeti, Pardis C.; Wentworth, David E.; Wiley, Michael R.; Yu, Guo-Yun; Sozhamannan, Shanmuga; Bradburne, Christopher

    2014-01-01

    ABSTRACT Thanks to high-throughput sequencing technologies, genome sequencing has become a common component in nearly all aspects of viral research; thus, we are experiencing an explosion in both the number of available genome sequences and the number of institutions producing such data. However, there are currently no common standards used to convey the quality, and therefore utility, of these various genome sequences. Here, we propose five “standard” categories that encompass all stages of viral genome finishing, and we define them using simple criteria that are agnostic to the technology used for sequencing. We also provide genome finishing recommendations for various downstream applications, keeping in mind the cost-benefit trade-offs associated with different levels of finishing. Our goal is to define a common vocabulary that will allow comparison of genome quality across different research groups, sequencing platforms, and assembly techniques. PMID:24939889

  6. Standards for sequencing viral genomes in the era of high-throughput sequencing.

    PubMed

    Ladner, Jason T; Beitzel, Brett; Chain, Patrick S G; Davenport, Matthew G; Donaldson, Eric F; Frieman, Matthew; Kugelman, Jeffrey R; Kuhn, Jens H; O'Rear, Jules; Sabeti, Pardis C; Wentworth, David E; Wiley, Michael R; Yu, Guo-Yun; Sozhamannan, Shanmuga; Bradburne, Christopher; Palacios, Gustavo

    2014-01-01

    Thanks to high-throughput sequencing technologies, genome sequencing has become a common component in nearly all aspects of viral research; thus, we are experiencing an explosion in both the number of available genome sequences and the number of institutions producing such data. However, there are currently no common standards used to convey the quality, and therefore utility, of these various genome sequences. Here, we propose five "standard" categories that encompass all stages of viral genome finishing, and we define them using simple criteria that are agnostic to the technology used for sequencing. We also provide genome finishing recommendations for various downstream applications, keeping in mind the cost-benefit trade-offs associated with different levels of finishing. Our goal is to define a common vocabulary that will allow comparison of genome quality across different research groups, sequencing platforms, and assembly techniques.

  7. Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing.

    PubMed

    Jain, Mukesh; Chevala, V V S Narayana; Garg, Rohini

    2014-11-01

    MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes.

  8. Genome sequence conservation of Hendra virus isolates during spillover to horses, Australia.

    PubMed

    Marsh, Glenn A; Todd, Shawn; Foord, Adam; Hansson, Eric; Davies, Kelly; Wright, Lynda; Morrissy, Chris; Halpin, Kim; Middleton, Deborah; Field, Hume E; Daniels, Peter; Wang, Lin-Fa

    2010-11-01

    Bat-to-horse transmission of Hendra virus has occurred at least 14 times. Although clinical signs in horses have differed, genome sequencing has demonstrated little variation among the isolates. Our sequencing of 5 isolates from recent Hendra virus outbreaks in horses found no correlation between sequences and time or geographic location of outbreaks.

  9. Consistent levels of A-to-I RNA editing across individuals in coding sequences and non-conserved Alu repeats

    PubMed Central

    2010-01-01

    Background Adenosine to inosine (A-to-I) RNA-editing is an essential post-transcriptional mechanism that occurs in numerous sites in the human transcriptome, mainly within Alu repeats. It has been shown to have consistent levels of editing across individuals in a few targets in the human brain and altered in several human pathologies. However, the variability across human individuals of editing levels in other tissues has not been studied so far. Results Here, we analyzed 32 skin samples, looking at A-to-I editing level in three genes within coding sequences and in the Alu repeats of six different genes. We observed highly consistent editing levels across different individuals as well as across tissues, not only in coding targets but, surprisingly, also in the non evolutionary conserved Alu repeats. Conclusions Our findings suggest that A-to-I RNA-editing of Alu elements is a tightly regulated process and, as such, might have been recruited in the course of primate evolution for post-transcriptional regulatory mechanisms. PMID:21029430

  10. 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.

  11. 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.

  12. 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

  13. Management of High-Throughput DNA Sequencing Projects: Alpheus.

    PubMed

    Miller, Neil A; Kingsmore, Stephen F; Farmer, Andrew; Langley, Raymond J; Mudge, Joann; Crow, John A; Gonzalez, Alvaro J; Schilkey, Faye D; Kim, Ryan J; van Velkinburgh, Jennifer; May, Gregory D; Black, C Forrest; Myers, M Kathy; Utsey, John P; Frost, Nicholas S; Sugarbaker, David J; Bueno, Raphael; Gullans, Stephen R; Baxter, Susan M; Day, Steve W; Retzel, Ernest F

    2008-12-26

    High-throughput DNA sequencing has enabled systems biology to begin to address areas in health, agricultural and basic biological research. Concomitant with the opportunities is an absolute necessity to manage significant volumes of high-dimensional and inter-related data and analysis. Alpheus is an analysis pipeline, database and visualization software for use with massively parallel DNA sequencing technologies that feature multi-gigabase throughput characterized by relatively short reads, such as Illumina-Solexa (sequencing-by-synthesis), Roche-454 (pyrosequencing) and Applied Biosystem's SOLiD (sequencing-by-ligation). Alpheus enables alignment to reference sequence(s), detection of variants and enumeration of sequence abundance, including expression levels in transcriptome sequence. Alpheus is able to detect several types of variants, including non-synonymous and synonymous single nucleotide polymorphisms (SNPs), insertions/deletions (indels), premature stop codons, and splice isoforms. Variant detection is aided by the ability to filter variant calls based on consistency, expected allele frequency, sequence quality, coverage, and variant type in order to minimize false positives while maximizing the identification of true positives. Alpheus also enables comparisons of genes with variants between cases and controls or bulk segregant pools. Sequence-based differential expression comparisons can be developed, with data export to SAS JMP Genomics for statistical analysis. PMID:20151039

  14. Direct multiplex sequencing (DMPS)--a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA.

    PubMed

    Stiller, Mathias; Knapp, Michael; Stenzel, Udo; Hofreiter, Michael; Meyer, Matthias

    2009-10-01

    Although the emergence of high-throughput sequencing technologies has enabled whole-genome sequencing from extinct organisms, little progress has been made in accelerating targeted sequencing from highly degraded DNA. Here, we present a novel and highly sensitive method for targeted sequencing of ancient and degraded DNA, which couples multiplex PCR directly with sample barcoding and high-throughput sequencing. Using this approach, we obtained a 96% complete mitochondrial genome data set from 31 cave bear (Ursus spelaeus) samples using only two 454 Life Sciences (Roche) GS FLX runs. In contrast to previous studies relying only on short sequence fragments, the overlapping portion of our data comprises almost 10 kb of replicated mitochondrial genome sequence, allowing for the unambiguous differentiation of three major cave bear clades. Our method opens up the opportunity to simultaneously generate many kilobases of overlapping sequence data from large sets of difficult samples, such as museum specimens, medical collections, or forensic samples. Embedded in our approach, we present a new protocol for the construction of barcoded sequencing libraries, which is compatible with all current high-throughput technologies and can be performed entirely in plate setup.

  15. Direct multiplex sequencing (DMPS)--a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA.

    PubMed

    Stiller, Mathias; Knapp, Michael; Stenzel, Udo; Hofreiter, Michael; Meyer, Matthias

    2009-10-01

    Although the emergence of high-throughput sequencing technologies has enabled whole-genome sequencing from extinct organisms, little progress has been made in accelerating targeted sequencing from highly degraded DNA. Here, we present a novel and highly sensitive method for targeted sequencing of ancient and degraded DNA, which couples multiplex PCR directly with sample barcoding and high-throughput sequencing. Using this approach, we obtained a 96% complete mitochondrial genome data set from 31 cave bear (Ursus spelaeus) samples using only two 454 Life Sciences (Roche) GS FLX runs. In contrast to previous studies relying only on short sequence fragments, the overlapping portion of our data comprises almost 10 kb of replicated mitochondrial genome sequence, allowing for the unambiguous differentiation of three major cave bear clades. Our method opens up the opportunity to simultaneously generate many kilobases of overlapping sequence data from large sets of difficult samples, such as museum specimens, medical collections, or forensic samples. Embedded in our approach, we present a new protocol for the construction of barcoded sequencing libraries, which is compatible with all current high-throughput technologies and can be performed entirely in plate setup. PMID:19635845

  16. Conservation and variation of nucleotide sequences within related bacterial genomes: enterobacteria.

    PubMed Central

    Riley, M; Anilionis, A

    1980-01-01

    We have assessed the degree of relatedness of several portions of the Escherichia coli genome to the corresponding portions of the genomes of representative enteric bacteria, using the Southern transfer and hybridization technique (E. Southern, J. Mol. Biol. 98:503-517, 1975). The degree of relatedness varied among the regions examined. Judging both by the relative amounts of deoxyribonucleic acid in the various enteric genomes that are highly homologous and by the conservation of positions of restriction enzyme cleavage sites in these regions, the enteric genomes have diverged to greater extents in some parts of the genomes than in others. Portions of the genomes (including the tnaA and thyA genes, the trp operon, and one other unassigned segment) appear to have evolved in concert with the genome as a whole. By contrast, the lacZ gene and portions of the genome that are homologous to phage lambda vary more widely, perhaps reflecting a separate evolutionary origin for these segments of deoxyribonucleic acid. Images PMID:6447143

  17. The stripe rust resistance gene Yr10 encodes an evolutionary-conserved and unique CC-NBS-LRR sequence in wheat.

    PubMed

    Liu, Wei; Frick, Michele; Huel, Réné; Nykiforuk, Cory L; Wang, Xiaomin; Gaudet, Denis A; Eudes, François; Conner, Robert L; Kuzyk, Alan; Chen, Qin; Kang, Zhensheng; Laroche, André

    2014-12-01

    The first seedling or all-stage resistance (R) R gene against stripe rust isolated from Moro wheat (Triticum aestivum L.) using a map-based cloning approach was identified as Yr10. Clone 4B of this gene encodes a highly evolutionary-conserved and unique CC-NBS-LRR sequence. Clone 4E, a homolog of Yr10, but lacking transcription start site (TSS) and putative TATA-box and CAAT-box, is likely a non-expressed pseudogene. Clones 4B and 4E are 84% identical and divergent in the intron and the LRR domain. Gene silencing and transgenesis were used in conjunction with inoculation with differentially avirulent and virulent stripe rust strains to demonstrate Yr10 functionality. The Yr10 CC-NBS-LRR sequence is unique among known CC-NBS-LRR R genes in wheat but highly conserved homologs (E = 0.0) were identified in Aegilops tauschii and other monocots including Hordeum vulgare and Brachypodium distachyon. Related sequences were also identified in genomic databases of maize, rice, and in sorghum. This is the first report of a CC-NBS-LRR resistance gene in plants with limited homologies in its native host, but with numerous homologous R genes in related monocots that are either host or non-hosts for stripe rust. These results represent a unique example of gene evolution and dispersion across species.

  18. 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

  19. Mapping the transcription start points of the Staphylococcus aureus eap, emp, and vwb promoters reveals a conserved octanucleotide sequence that is essential for expression of these genes.

    PubMed

    Harraghy, Niamh; Homerova, Dagmar; Herrmann, Mathias; Kormanec, Jan

    2008-01-01

    Mapping the transcription start points of the eap, emp, and vwb promoters revealed a conserved octanucleotide sequence (COS). Deleting this sequence abolished the expression of eap, emp, and vwb. However, electrophoretic mobility shift assays gave no evidence that this sequence was a binding site for SarA or SaeR, known regulators of eap and emp.

  20. Three-Year Sequence for High School Mathematics, Course III.

    ERIC Educational Resources Information Center

    Buchman, Paul; Buchman, Aaron

    This curriculum guide covers Course III of a three-year sequence for high school mathematics in New York which was intended to provide an alternative to the regular Regents sequence of ninth-, tenth-, and eleventh-grade mathematics. A listing of scope and content is provided along with suggested time allotments. Topics covered are: complex…

  1. The sequence of learning cycle activities in high school chemistry

    NASA Astrophysics Data System (ADS)

    Abraham, Michael R.; Renner, John W.

    The sequence of the three phases of two high school learning cycles in chemistry was altered in order to: (I ) give insights into the factors which account for the success of the learning cycle, (2) serve as an indirect test of the association between Piaget's theory and the learning cycle, and (3) to compare the learning cycle with traditional instruction. Each of the six sequences (one n o d and five altered) was studied with content and atritudc measures. The outcomes of the study supported the contention that the normal learning cycle sequence is the optimum sequence for achievement of content knowledge.

  2. High-speed multiple sequence alignment on a reconfigurable platform.

    PubMed

    Oliver, Tim; Schmidt, Bertil; Maskell, Douglas; Nathan, Darran; Clemens, Ralf

    2006-01-01

    Progressive alignment is a widely used approach to compute multiple sequence alignments (MSAs). However, aligning several hundred sequences by popular progressive alignment tools requires hours on sequential computers. Due to the rapid growth of sequence databases biologists have to compute MSAs in a far shorter time. In this paper we present a new approach to MSA on reconfigurable hardware platforms to gain high performance at low cost. We have constructed a linear systolic array to perform pairwise sequence distance computations using dynamic programming. This results in an implementation with significant runtime savings on a standard FPGA.

  3. 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

  4. MLST analysis reveals a highly conserved core genome among poultry isolates of Clostridium septicum.

    PubMed

    Neumann, Anthony P; Rehberger, Thomas G

    2009-06-01

    Clostridium septicum is a highly virulent, anaerobic bacterium capable of establishing necrotizing tissue infections and forming heat resistant endospores. Disease is primarily facilitated by secretion of numerous toxic products including a lethal pore-forming cytolysin. Spontaneously occurring clostridial myonecrosis involving C. septicum has recently reemerged as a concern for many poultry producers. However, despite its increasing prevalence, the epidemiology of infection and population structure of C. septicum remains largely unknown. In this study a multilocus sequence typing (MLST) approach was utilized to examine evolutionary relationships within a diverse collection of C. septicum isolates recovered from poultry flocks experiencing episodes of gangrenous dermatitis. The 109 isolates examined represented 42 turkey flocks and 24 different flocks of broiler chickens as well as C. septicum type strain, ATCC 12464. Isolates were recovered predominantly from gangrenous lesions although isolates from livers, gastrointestinal tracts, spleens and blood were included. The loci analyzed were csa, the major lethal toxin produced by C. septicum, and the housekeeping genes gyrA, groEL, dnaK, recA, tpi, ddl, colA and glpK. These loci were included in part because of their previous use in MLST analysis of Clostridium perfringens and Clostridium difficile. Results indicated a high level of conservation present within these housekeeping gene fragments when compared to what has been previously reported for the aforementioned clostridia. Of the 5352 bp of sequence data examined for each isolate, 99.7% (5335/5352) was absolutely conserved among the 109 isolates. Only one of the ten unique sequence types, or allelic profiles, identified among the isolates was recovered from both turkeys and broiler chickens suggesting some host species preference. Phylogenetic analyses identified two unique clusters, or clonal complexes, among these poultry isolates which may have important

  5. 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.

  6. 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

  7. Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs.

    PubMed

    Yang, Jun-Bo; Li, De-Zhu; Li, Hong-Tao

    2014-09-01

    Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle-scale barcodes. Next-generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high-quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long-range PCR and sequenced using next-generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early-diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome-scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms.

  8. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families.

    PubMed Central

    Gilkes, N R; Henrissat, B; Kilburn, D G; Miller, R C; Warren, R A

    1991-01-01

    Several types of domain occur in beta-1, 4-glycanases. The best characterized of these are the catalytic domains and the cellulose-binding domains. The domains may be joined by linker sequences rich in proline or hydroxyamino acids or both. Some of the enzymes contain repeated sequences up to 150 amino acids in length. The enzymes can be grouped into families on the basis of sequence similarities between the catalytic domains. There are sequence similarities between the cellulose-binding domains, of which two types have been identified, and also between some domains of unknown function. The beta-1, 4-glycanases appear to have arisen by the shuffling of a relatively small number of progenitor sequences. PMID:1886523

  9. 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.

  10. 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

  11. 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

  12. Conservation of plastid sequences in the plant nuclear genome for millions of years facilitates endosymbiotic evolution.

    PubMed

    Rousseau-Gueutin, Mathieu; Ayliffe, Michael A; Timmis, Jeremy N

    2011-12-01

    The nuclear genome of eukaryotes contains large amounts of cytoplasmic organelle DNA (nuclear integrants of organelle DNA [norgs]). The recent sequencing of many mitochondrial and chloroplast genomes has enabled investigation of the potential role of norgs in endosymbiotic evolution. In this article, we describe a new polymerase chain reaction-based method that allows the identification and evolutionary study of recent and older norgs in a range of eukaryotes. We tested this method in the genus Nicotiana and obtained sequences from seven nuclear integrants of plastid DNA (nupts) totaling 25 kb in length. These nupts were estimated to have been transferred 0.033 to 5.81 million years ago. The spectrum of mutations present in the potential protein-coding sequences compared with the noncoding sequences of each nupt revealed that nupts evolve in a nuclear-specific manner and are under neutral evolution. Indels were more frequent in noncoding regions than in potential coding sequences of former chloroplastic DNA, most probably due to the presence of a higher number of homopolymeric sequences. Unexpectedly, some potential protein-coding sequences within the nupts still contained intact open reading frames for up to 5.81 million years. These results suggest that chloroplast genes transferred to the nucleus have in some cases several millions of years to acquire nuclear regulatory elements and become functional. The different factors influencing this time frame and the potential role of nupts in endosymbiotic gene transfer are discussed.

  13. Conserved sequences in both coding and 5' flanking regions of mammalian opal suppressor tRNA genes.

    PubMed Central

    Pratt, K; Eden, F C; You, K H; O'Neill, V A; Hatfield, D

    1985-01-01

    The rabbit genome encodes an opal suppressor tRNA gene. The coding region is strictly conserved between the rabbit gene and the corresponding gene in the human genome. The rabbit opal suppressor gene contains the consensus sequence in the 3' internal control region but like the human and chicken genes, the rabbit 5' internal control region contains two additional nucleotides. The 5' flanking sequences of the rabbit and the human opal suppressor genes contain extensive regions of homology. A subset of these homologies is also present 5' to the chicken opal suppressor gene. Both the rabbit and the human genomes also encode a pseudogene. That of the rabbit lacks the 3' half of the coding region. Neither pseudogene has homologous regions to the 5' flanking regions of the genes. The presence of 5' homologies flanking only the transcribed genes and not the pseudogenes suggests that these regions may be regulatory control elements specifically involved in the expression of the eukaryotic opal suppressor gene. Moreover the strict conservation of coding sequences indicates functional importance for the opal suppressor tRNA genes. Images PMID:4022772

  14. 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.

  15. 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.

  16. 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

  17. 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.

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

    PubMed

    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.

  19. 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

  20. Optimal Packaging of FIV Genomic RNA Depends upon a Conserved Long-range Interaction and a Palindromic Sequence within gag

    PubMed Central

    Rizvi, Tahir A.; Kenyon, Julia C.; Ali, Jahabar; Aktar, Suriya J.; Phillip, Pretty S.; Ghazawi, Akela; Mustafa, Farah; Lever, Andrew M.L.

    2010-01-01

    The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5′ 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5′ and 3′ sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem–loop (SL2) and a small palindromic stem–loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8–5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5–3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary

  1. 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

  2. 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.

  3. The Neurospora crassa carotenoid biosynthetic gene (albino 3) reveals highly conserved regions among prenyltransferases.

    PubMed

    Carattoli, A; Romano, N; Ballario, P; Morelli, G; Macino, G

    1991-03-25

    In the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light. Here we report the characterization of the albino-3 (al-3) gene of N. crassa, which encodes the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthetase. This is the first geranylgeranyl-pyrophosphate synthetase gene isolated. Nucleotide sequence comparison of al-3 genomic and cDNA clones revealed that the al-3 gene is not interrupted by introns. Transcription of the al-3 gene has been examined in dark-grown and light-induced mycelia. The analysis revealed that the al-3 gene is not expressed in the dark and that its transcription is induced by blue light (Nelson, M. A., Morelli, G., Carattoli, A., Romano, N., and Macino, G. (1989) Mol. Cell. Biol. 9, 1271-1276). The al-3 gene encodes a polypeptide of 428 amino acids. Comparison of the deduced amino acid sequence of al-3 with the sequences of prenyltransferases of other species, from bacteria to humans, showed three highly conserved homologous regions. These homologous regions may be involved in the formation of the catalytic site of the prenyltransferases.

  4. 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.

  5. DNA sequence analysis of conserved genes reveals hybridization events that increase genetic diversity in Verticillium dahliae.

    PubMed

    Collado-Romero, Melania; Jiménez-Díaz, Rafael M; Mercado-Blanco, Jesús

    2010-01-01

    The hybrid origin of a Verticillium dahliae isolate belonging to the vegetative compatibility group (VCG) 3 is reported in this work. Moreover, new data supporting the hybrid origin of two V. dahliae var. longisporum (VDLSP) isolates are provided as well as information about putative parentals. Thus, isolates of VDLSP and V. dahliae VCG3 were found harboring multiple sequences of actin (Act), β-tubulin (β-tub), calmodulin (Cal) and histone 3 (H3) genes. Phylogenetic analysis of these sequences, the internal transcribed sequences (ITS-1 and ITS-2) of the rRNA genes and of a V. dahliae-specific sequence provided molecular evidences for the interspecific hybrid origin of those isolates. Sequence analysis suggests that some of VDLSP isolates may have resulted from hybridization events between a V. dahliae isolate of VCG1 and/or VCG4A and, probably, a closely related taxon to Verticillium alboatrum but not this one. Similarly, phylogenetic analysis and PCR markers indicated that a V. dahliae VCG3 isolate might have arisen from a hybridization event between a V. dahliae VCG1B isolate and as yet unidentified parent. This second parental probably does not belong to the Verticillium genus according to the gene sequences dissimilarities found between the VCG3 isolate and Verticillium spp. These results suggest an important role of parasexuality in diversity and evolution in the genus Verticillium and show that interspecific hybrids within this genus may not be rare in nature.

  6. Identification of proteins that form specific complexes with the highly conserved protein Translin in Schizosaccharomyces pombe.

    PubMed

    Eliahoo, Elad; Litovco, Phyana; Ben Yosef, Ron; Bendalak, Keren; Ziv, Tamar; Manor, Haim

    2014-04-01

    Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin-TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin-TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹⁵N and ¹⁴N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein-protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.

  7. Highly informative nature of inter simple sequence repeat (ISSR) sequences amplified using tri- and tetra-nucleotide primers from DNA of cauliflower (Brassica oleracea var. botrytis L.).

    PubMed

    Bornet, B; Muller, C; Paulus, F; Branchard, M

    2002-10-01

    Inter simple sequence repeat (ISSR) sequences as molecular markers can lead to the detection of polymorphism and also be a new approach to the study of SSR distribution and frequency. In this study, ISSR amplification with nonanchored primer was performed in closely related cauliflower lines. Fourty-four different amplified fragments were sequenced. Sequences of PCR products are delimited by the expected motifs and number of repeats, which validates the ISSR nonanchored primer amplification technique. DNA and amino acids homology search between internal sequences and databases (i) show that the majority of the internal regions of ISSR had homologies with known sequences, mainly with genes coding for proteins implicated in DNA interaction or gene expression, which reflected the significance of amplified ISSR sequences and (ii) display long and numerous homologies with the Arabidopsis thaliana genome. ISSR amplifications revealed a high conservation of these sequences between Arabidopsis thaliana and Brassica oleracea var. botrytis. Thirty-four of the 44 ISSRs had one or several perfect or imperfect internal microsatellites. Such distribution indicates the presence in genomes of highly concentrated regions of SSR, or "SSR hot spots." Among the four nonanchored primers used in this study, trinucleotide repeats, and especially (CAA)5, were the most powerful primers for ISSR amplifications regarding the number of amplified bands, level of polymorphism, and their nature.

  8. 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.

  9. Library preparation for highly accurate population sequencing of RNA viruses

    PubMed Central

    Acevedo, Ashley; Andino, Raul

    2015-01-01

    Circular resequencing (CirSeq) is a novel technique for efficient and highly accurate next-generation sequencing (NGS) of RNA virus populations. The foundation of this approach is the circularization of fragmented viral RNAs, which are then redundantly encoded into tandem repeats by ‘rolling-circle’ reverse transcription. When sequenced, the redundant copies within each read are aligned to derive a consensus sequence of their initial RNA template. This process yields sequencing data with error rates far below the variant frequencies observed for RNA viruses, facilitating ultra-rare variant detection and accurate measurement of low-frequency variants. Although library preparation takes ~5 d, the high-quality data generated by CirSeq simplifies downstream data analysis, making this approach substantially more tractable for experimentalists. PMID:24967624

  10. High-throughput DNA sequencing: a genomic data manufacturing process.

    PubMed

    Huang, G M

    1999-01-01

    The progress trends in automated DNA sequencing operation are reviewed. Technological development in sequencing instruments, enzymatic chemistry and robotic stations has resulted in ever-increasing capacity of sequence data production. This progress leads to a higher demand on laboratory information management and data quality assessment. High-throughput laboratories face the challenge of organizational management, as well as technology management. Engineering principles of process control should be adopted in this biological data manufacturing procedure. While various systems attempt to provide solutions to automate different parts of, or even the entire process, new technical advances will continue to change the paradigm and provide new challenges.

  11. Core genome conservation of Staphylococcus haemolyticus limits sequence based population structure analysis.

    PubMed

    Cavanagh, Jorunn Pauline; Klingenberg, Claus; Hanssen, Anne-Merethe; Fredheim, Elizabeth Aarag; Francois, Patrice; Schrenzel, Jacques; Flægstad, Trond; Sollid, Johanna Ericson

    2012-06-01

    The notoriously multi-resistant Staphylococcus haemolyticus is an emerging pathogen causing serious infections in immunocompromised patients. Defining the population structure is important to detect outbreaks and spread of antimicrobial resistant clones. Currently, the standard typing technique is pulsed-field gel electrophoresis (PFGE). In this study we describe novel molecular typing schemes for S. haemolyticus using multi locus sequence typing (MLST) and multi locus variable number of tandem repeats (VNTR) analysis. Seven housekeeping genes (MLST) and five VNTR loci (MLVF) were selected for the novel typing schemes. A panel of 45 human and veterinary S. haemolyticus isolates was investigated. The collection had diverse PFGE patterns (38 PFGE types) and was sampled over a 20 year-period from eight countries. MLST resolved 17 sequence types (Simpsons index of diversity [SID]=0.877) and MLVF resolved 14 repeat types (SID=0.831). We found a low sequence diversity. Phylogenetic analysis clustered the isolates in three (MLST) and one (MLVF) clonal complexes, respectively. Taken together, neither the MLST nor the MLVF scheme was suitable to resolve the population structure of this S. haemolyticus collection. Future MLVF and MLST schemes will benefit from addition of more variable core genome sequences identified by comparing different fully sequenced S. haemolyticus genomes. PMID:22484086

  12. Sequence analysis shows that Lifeguard belongs to a new evolutionarily conserved cytoprotective family.

    PubMed

    Reimers, Kerstin; Choi, Claudia Y-U; Mau-Thek, Eddy; Vogt, Peter M

    2006-10-01

    Cellular sensitivity to apoptotic stimuli is determined by several regulatory proteins. The biological and biomedical impact of these regulatory proteins is of fundamental importance for understanding and controlling apoptotic processes. We used a bioinformatic approach to characterise the antiapoptotic protein Lifeguard (LFG). LFG is an evolutionarily well-conserved protein with homologues in many species. Due to its hydrophobic nature it is predicted to reside in cellular membranes, namely the endoplasmatic reticulum and the plasma membrane, with seven transmembrane spanners and a small cytoplasmic domain. The consensus motif of a protein family with unknown function UPF0005 was found in the C-terminus. The structure of Lifeguard resembles the antiapoptotic protein Bax Inhibitor-1 (BI-1). Concordantly, it was shown that Bax co-immunoprecipitates with LFG. Our results indicate that LFG belongs to a new cytoprotective family with evolutionarily conserved functions in the prevention of programmed cell death.

  13. 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.

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

    PubMed

    Le Coq, Johanne; Ghosh, Partho

    2011-08-30

    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 (∼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(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.

  15. 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.

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

    PubMed Central

    Le Coq, Johanne; Ghosh, Partho

    2011-01-01

    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 (∼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 1020 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. PMID:21873231

  17. Length heterogeneity at conserved sequence block 2 in human mitochondrial DNA acts as a rheostat for RNA polymerase POLRMT activity

    PubMed Central

    Tan, Benedict G.; Wellesley, Frederick C.; Savery, Nigel J.; Szczelkun, Mark D.

    2016-01-01

    The guanine (G)-tract of conserved sequence block 2 (CSB 2) in human mitochondrial DNA can result in transcription termination due to formation of a hybrid G-quadruplex between the nascent RNA and the nontemplate DNA strand. This structure can then influence genome replication, stability and localization. Here we surveyed the frequency of variation in sequence identity and length at CSB 2 amongst human mitochondrial genomes and used in vitro transcription to assess the effects of this length heterogeneity on the activity of the mitochondrial RNA polymerase, POLRMT. In general, increased G-tract length correlated with increased termination levels. However, variation in the population favoured CSB 2 sequences which produced efficient termination while particularly weak or strong signals were avoided. For all variants examined, the 3′ end of the transcripts mapped to the same downstream sequences and were prevented from terminating by addition of the transcription factor TEFM. We propose that CSB 2 length heterogeneity allows variation in the efficiency of transcription termination without affecting the position of the products or the capacity for regulation by TEFM. PMID:27436287

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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

  4. 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

  5. A new strategy for identification of highly conserved microRNAs in non-model insect, Spodoptera litura.

    PubMed

    Gao, Lu; Zuo, Hongliang; Liu, Keling; Li, Haiyi; Zhong, Guohua

    2012-01-01

    The indigenous small non-coding RNAs, known as microRNAs (miRNAs), are important regulators of gene expression and many of them are evolutionarily conserved. Whether stem-loop RT-PCR, as a sensitive method, could be utilized to clone conserved miRNAs from non-model insects lacks information. Here, three miRNAs, sli-miR-14, sli-miR-2a and sli-bantam, were cloned from Spodoptera litura by stem-loop RT-PCR. Two groups of primers were designed, and one of them performed especially well and proved stable. The sequences of two highly conserved miRNAs, sli-miR-14 and sli-miR-2a were identical to those in Drosophila melanogaster. To validate the reliability of this strategy, pre-miR-14 and pre-miR-2a in S. litura as representatives were given as well; this shared high homology with those in D. melanogaster and Bombyx mori, and both mature sequences of sli-miR-14 and sli-miR-2a in their precursors shared 100% identity to the results shown by stem-loop RT-PCR. Moreover, expression patterns of these miRNAs were investigated by real-time quantitative PCR. Sli-miR-14 and sli-miR-2a could be detected successfully and their expression patterns showed similar characteristics with those in model insects, further suggesting stem-loop RT-PCR technology can be used for identification of highly conserved miRNAs in non-model insects. These results provide a simplified and efficient strategy for studying the structure and function of highly conserved miRNAs, especially some critical miRNAs in non-model insects.

  6. 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

  7. Roche genome sequencer FLX based high-throughput sequencing of ancient DNA.

    PubMed

    Alquezar-Planas, David E; Fordyce, Sarah L

    2012-01-01

    Since the development of so-called "next generation" high-throughput sequencing in 2005, this technology has been applied to a variety of fields. Such applications include disease studies, evolutionary investigations, and ancient DNA. Each application requires a specialized protocol to ensure that the data produced is optimal. Although much of the procedure can be followed directly from the manufacturer's protocols, the key differences lie in the library preparation steps. This chapter presents an optimized protocol for the sequencing of fossil remains and museum specimens, commonly referred to as "ancient DNA," using the Roche GS FLX 454 platform.

  8. Roche genome sequencer FLX based high-throughput sequencing of ancient DNA.

    PubMed

    Alquezar-Planas, David E; Fordyce, Sarah L

    2012-01-01

    Since the development of so-called "next generation" high-throughput sequencing in 2005, this technology has been applied to a variety of fields. Such applications include disease studies, evolutionary investigations, and ancient DNA. Each application requires a specialized protocol to ensure that the data produced is optimal. Although much of the procedure can be followed directly from the manufacturer's protocols, the key differences lie in the library preparation steps. This chapter presents an optimized protocol for the sequencing of fossil remains and museum specimens, commonly referred to as "ancient DNA," using the Roche GS FLX 454 platform. PMID:22665278

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

    PubMed Central

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

    2001-01-01

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

  10. Identification of two voltage-dependent anion channel-like protein sequences conserved in Kinetoplastida

    PubMed Central

    Flinner, Nadine; Schleiff, Enrico; Mirus, Oliver

    2012-01-01

    The eukaryotic porin superfamily consists of two families, voltage-dependent anion channel (VDAC) and Tom40, which are both located in the mitochondrial outer membrane. In Trypanosoma brucei, only a single member of the VDAC family has been described. We report the detection of two additional eukaryotic porin-like sequences in T. brucei. By bioinformatic means, we classify both as putative VDAC isoforms. PMID:22219392

  11. Infection of capilloviruses requires subgenomic RNAs whose transcription is controlled by promoter-like sequences conserved among flexiviruses.

    PubMed

    Komatsu, Ken; Hirata, Hisae; Fukagawa, Takako; Yamaji, Yasuyuki; Okano, Yukari; Ishikawa, Kazuya; Adachi, Tatsushi; Maejima, Kensaku; Hashimoto, Masayoshi; Namba, Shigetou

    2012-07-01

    The first open-reading frame (ORF) of apple stem grooving virus (ASGV), of the genus Capillovirus, encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP). However, our previous study revealed that ASGV mutants with distinct and discontinuous Rep- and CP-coding regions successfully infect plants, indicating that CP expressed via a subgenomic RNA (sgRNA) is sufficient for viability of the virus. Here we identified a transcription start site of the CP sgRNA and revealed that CP translated from the sgRNA is essential for ASGV infection. We mapped the transcription start sites of both the CP and the movement protein (MP) sgRNAs of ASGV and found a hexanucleotide motif, UUAGGU, conserved upstream from both sgRNA transcription start sites. Mutational analysis of the putative CP initiation codon and of the UUAGGU sequence upstream from the transcription start site of CP sgRNA demonstrated their importance for ASGV accumulation. Our results also demonstrated that potato virus T (PVT), an unassigned species closely related to ASGV, produces two sgRNAs putatively deployed for the CP and MP expression and that the same hexanucleotide motif as found in ASGV is located upstream from the transcription start sites of both sgRNAs. This motif, which constituted putative core elements of the sgRNA promoter, is broadly conserved among viruses in the families Alphaflexiviridae and Betaflexiviridae, suggesting that the gene expression strategy of the viruses in both families has been conserved throughout evolution.

  12. 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

  13. 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

  14. 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

  15. Analysis and prediction of RNA-binding residues using sequence, evolutionary conservation, and predicted secondary structure and solvent accessibility.

    PubMed

    Zhang, Tuo; Zhang, Hua; Chen, Ke; Ruan, Jishou; Shen, Shiyi; Kurgan, Lukasz

    2010-11-01

    Identification and prediction of RNA-binding residues (RBRs) provides valuable insights into the mechanisms of protein-RNA interactions. We analyzed the contributions of a wide range of factors including amino acid sequence, evolutionary conservation, secondary structure and solvent accessibility, to the prediction/characterization of RBRs. Five feature sets were designed and feature selection was performed to find and investigate relevant features. We demonstrate that (1) interactions with positively charged amino acids Arg and Lys are preferred by the egatively charged nucleotides; (2) Gly provides flexibility for the RNA binding sites; (3) Glu with negatively charged side chain and several hydrophobic residues such as Leu, Val, Ala and Phe are disfavored in the RNA-binding sites; (4) coil residues, especially in long segments, are more flexible (than other secondary structures) and more likely to interact with RNA; (5) helical residues are more rigid and consequently they are less likely to bind RNA; and (6) residues partially exposed to the solvent are more likely to form RNA-binding sites. We introduce a novel sequence-based predictor of RBRs, RBRpred, which utilizes the selected features. RBRpred is comprehensively tested on three datasets with varied atom distance cutoffs by performing both five-fold cross validation and jackknife tests and achieves Matthew's correlation coefficient (MCC) of 0.51, 0.48 and 0.42, respectively. The quality is comparable to or better than that for state-of-the-art predictors that apply the distancebased cutoff definition. We show that the most important factor for RBRs prediction is evolutionary conservation, followed by the amino acid sequence, predicted secondary structure and predicted solvent accessibility. We also investigate the impact of using native vs. predicted secondary structure and solvent accessibility. The predictions are sufficient for the RBR prediction and the knowledge of the actual solvent accessibility

  16. 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.

  17. 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.

  18. Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site

    PubMed Central

    Coey, Aaron; Larsen, Kevin; Puglisi, Joseph D.; Viani Puglisi, Elisabetta

    2016-01-01

    Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5′ UTR of the HIV genomic RNA (vRNA) and a host primer tRNALys3. Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activation signal (PAS) and the primer tRNA occurs in one of these conformers. This intermolecular PAS interaction likely induces strain on a vRNA intramolecular helix, which must be broken for reverse transcription to initiate. We propose a mechanism by which this vRNA/tRNA conformer relieves the kinetic block formed by the vRNA intramolecular helix to initiate reverse transcription. PMID:27613581

  19. High-resolution mapping of protein sequence-function relationships.

    PubMed

    Fowler, Douglas M; Araya, Carlos L; Fleishman, Sarel J; Kellogg, Elizabeth H; Stephany, Jason J; Baker, David; Fields, Stanley

    2010-09-01

    We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity and high-throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position had unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.

  20. Horse domestication and conservation genetics of Przewalski's horse inferred from sex chromosomal and autosomal sequences.

    PubMed

    Lau, Allison N; Peng, Lei; Goto, Hiroki; Chemnick, Leona; Ryder, Oliver A; Makova, Kateryna D

    2009-01-01

    Despite their ability to interbreed and produce fertile offspring, there is continued disagreement about the genetic relationship of the domestic horse (Equus caballus) to its endangered wild relative, Przewalski's horse (Equus przewalskii). Analyses have differed as to whether or not Przewalski's horse is placed phylogenetically as a separate sister group to domestic horses. Because Przewalski's horse and domestic horse are so closely related, genetic data can also be used to infer domestication-specific differences between the two. To investigate the genetic relationship of Przewalski's horse to the domestic horse and to address whether evolution of the domestic horse is driven by males or females, five homologous introns (a total of approximately 3 kb) were sequenced on the X and Y chromosomes in two Przewalski's horses and three breeds of domestic horses: Arabian horse, Mongolian domestic horse, and Dartmoor pony. Five autosomal introns (a total of approximately 6 kb) were sequenced for these horses as well. The sequences of sex chromosomal and autosomal introns were used to determine nucleotide diversity and the forces driving evolution in these species. As a result, X chromosomal and autosomal data do not place Przewalski's horses in a separate clade within phylogenetic trees for horses, suggesting a close relationship between domestic and Przewalski's horses. It was also found that there was a lack of nucleotide diversity on the Y chromosome and higher nucleotide diversity than expected on the X chromosome in domestic horses as compared with the Y chromosome and autosomes. This supports the hypothesis that very few male horses along with numerous female horses founded the various domestic horse breeds. Patterns of nucleotide diversity among different types of chromosomes were distinct for Przewalski's in contrast to domestic horses, supporting unique evolutionary histories of the two species.

  1. [Conservative orthodontic primary care of four newborns with the Pierre-Robin sequence triad].

    PubMed

    Ludwig, Björn; Glasl, Bettina; Sader, Robert; Schopf, Peter

    2007-01-01

    Newborns with Pierre-Robin sequence often suffer from serious or even life-threatening obstructions in the respiratory tract resulting from anatomic malformations (micrognathia, glossoptosis and potentially a median cleft palate). Such babies require immediate effective therapeutic measures. Our case descriptions of four babies with the typical triad illustrate the application of a modified upper plate with an individually-adjustable pharyngeal spur. Precise and individually-modifiable adaptation of the plate's pharyngeal parts--depending on the developmental stage--permit the narrow airway to be opened, which then affects the tongue's position and the sagittal position of the mandible.

  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. 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.

  4. Identification and characterization of miRNA transcriptome in potato by high-throughput sequencing.

    PubMed

    Zhang, Runxuan; Marshall, David; Bryan, Glenn J; Hornyik, Csaba

    2013-01-01

    Micro RNAs (miRNAs) represent a class of short, non-coding, endogenous RNAs which play important roles in post-transcriptional regulation of gene expression. While the diverse functions of miRNAs in model plants have been well studied, the impact of miRNAs in crop plant biology is poorly understood. Here we used high-throughput sequencing and bioinformatics analysis to analyze miRNAs in the tuber bearing crop potato (Solanum tuberosum). Small RNAs were analysed from leaf and stolon tissues. 28 conserved miRNA families were found and potato-specific miRNAs were identified and validated by RNA gel blot hybridization. The size, origin and predicted targets of conserved and potato specific miRNAs are described. The large number of miRNAs and complex population of small RNAs in potato suggest important roles for these non-coding RNAs in diverse physiological and metabolic pathways.

  5. Effects of High-Order Rule Instruction on Preschool Children's Understanding of Conservation.

    ERIC Educational Resources Information Center

    Lawton, Joseph T.; Reddy, P.

    Examined were effects of advance organizer (AO) instruction on acquisition of conservation concepts in 3- and 4-year-olds. Participants were 28 children who were equally divided into experimental and control groups, pretested, and found to be nonconservers. A sequence of two AO lessons and related learning activities was presented to the…

  6. 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

  7. Conservative management in a case of uncomplicated trap sequence: a case report and brief literature review

    PubMed Central

    Pepe, Franco; Teodoro, Maria Cristina; Luca, Carlo; Privitera, Francesca

    2015-01-01

    Introduction twin reversed arterial perfusion (TRAP) sequence is a rare anomaly that occurs in monochorionic twins with overall mortality rate ranging from 50% to 70% in the normal fetus, above all for congestive cardiac failure. Case report a 31-year-old Caucasian gravida was referred to our fetomaternal medicine unit in the 25 gestational age. Ultrasound examination revealed a monochorionic, biamniotic twin pregnancy with a donor fetus showing normal morphology and growth corresponding to gestational age. The recipient twin appeared grossly abnormal with no head, upper limbs, heart, or thoracic structures and massive, diffuse, soft tissue edema. Fetal Doppler and fetal echocardiography revealed normal parameters. The patient refused any treatment and was monitored with weekly ultrasonography and Doppler ultrasound examination. She underwent cesarean section due to premature labor/rupture of membranes secondary to a mild polyhydramnios, at 36 weeks gestational age and delivered an apparent normal female live baby weighing 2550 gr, and another female acardius acephalus twin, birth weight 1300 gr. This baby had rudimental edematous lower limbs, pelvic bone, lower sacral vertebrae, and absence of thorax and cephalic structures. Conclusion although the literature suggest that early intrafetal laser treatment of TRAP sequence is advantageous, our case shows that pregnancies referred late would still require a tailored approach after a risk-benefit assessment. PMID:27358695

  8. High-Throughput Sequencing and Rare Genetic Diseases

    PubMed Central

    Makrythanasis, P.; Antonarakis, S.E.

    2012-01-01

    High-throughput sequencing has drastically changed the research of genes responsible for genetic disorders and is now gradually introduced as an additional genetic diagnostic testing in clinical practice. The current debates on the emerging technical, medical and ethical issues as well as the potential optimum use of the available technology are discussed. PMID:23293577

  9. 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.

  10. 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.

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

    ScienceCinema

    Athavale, Ajay [Monsanto

    2016-07-12

    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.

  12. 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.

  13. DNA sequence analysis of conserved and unique regions of swinepox virus: identification of genetic elements supporting phenotypic observations including a novel G protein-coupled receptor homologue.

    PubMed

    Massung, R F; Jayarama, V; Moyer, R W

    1993-12-01

    Swinepox virus (SPV) contains a double-stranded cross-linked linear DNA genome of approximately 175 kilobase pairs with terminal inverted repetitions (TIRs) of 4.3 kb. The nucleotide sequence was determined for fragments from several regions of the genome including a 2.85-kb fragment from the central potentially conserved portion and two fragments within the presumed variable near-terminal regions which tend to be unique to a given poxvirus. The core sequence contains one partial and two complete open reading frames that are highly conserved and colinear with three contiguous ORFs within the HindIII D fragment of vaccinia virus (VV). The two near-terminal fragments, encompassing 14.2 and 3.6 kb, are respectively located 2.1 kb internal to the left and right cross-linked termini of the DNA and span the TIR junctions. The sequences encode 25 open reading frames including numerous proteins predicted to be membrane-bound or secreted in infected cells. Several ORFs unique to SPV were identified that may be involved in cell attachment, immune modulation, and pathogenesis including a novel poxvirus G protein-coupled receptor. In addition, several polypeptides encoded within the near-terminal regions of vaccinia virus DNA that function as host range or virulence factors are lacking within this region of swinepox virus including the VV growth factor, complement-binding protein, and ORFs C7L and K1L, associated with host range. The lack of these functional homologues could explain the characteristic attenuated phenotype and limited host range of SPV.

  14. 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

  15. FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns

    PubMed Central

    Nakaya, Tadashi; Alexiou, Panagiotis; Maragkakis, Manolis; Chang, Alexandra; Mourelatos, Zissimos

    2013-01-01

    Dominant mutations and mislocalization or aggregation of Fused in Sarcoma (FUS), an RNA-binding protein (RBP), cause neuronal degeneration in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), two incurable neurological diseases. However, the function of FUS in neurons is not well understood. To uncover the impact of FUS in the neuronal transcriptome, we used high-throughput sequencing of immunoprecipitated and cross-linked RNA (HITS–CLIP) of FUS in human brains and mouse neurons differentiated from embryonic stem cells, coupled with RNA-seq and FUS knockdowns. We report conserved neuronal RNA targets and networks that are regulated by FUS. We find that FUS regulates splicing of genes coding for RBPs by binding to their highly conserved introns. Our findings have important implications for understanding the impact of FUS in neurodegenerative diseases and suggest that perturbations of FUS can impact the neuronal transcriptome via perturbations of RBP transcripts. PMID:23389473

  16. 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

  17. Conservation of DNA sequences for plasmid-mediated citrate utilization within the enterobacteria.

    PubMed

    Hirato, T; Ishiguro, N; Shinagawa, M; Sato, G

    1986-01-01

    Southern blot DNA-DNA hybridization experiments with a cloned Cit+ DNA fragment as a probe showed that the plasmid-mediated Cit+ determinants from four Cit plasmids (R726, pOH3001, pOH3035, and pOH30221) were all homologous. Sequences homologous to the plasmid-borne Cit+ gene were also found in total bacterial DNA isolated from Salmonella paratyphi B, Salmonella enteritidis, Salmonella typhimurium LT-2, Citrobacter freundii, ATCC 8090, Citrobacter amalonaticus ATCC 25405, Klebsiella pneumoniae I and IID 977, and Enterobacter aerogenes ATCC 13048. The DNA digest from C. amalonaticus ATCC 25405 contained a 1.4-kilobase BamHI-HincII DNA fragment that was strongly homologous with and identical in size to the plasmid Cit+ probe.

  18. The Number, Organization, and Size of Polymorphic Membrane Protein Coding Sequences as well as the Most Conserved Pmp Protein Differ within and across Chlamydia Species.

    PubMed

    Van Lent, Sarah; Creasy, Heather Huot; Myers, Garry S A; Vanrompay, Daisy

    2016-01-01

    Variation is a central trait of the polymorphic membrane protein (Pmp) family. The number of pmp coding sequences differs between Chlamydia species, but it is unknown whether the number of pmp coding sequences is constant within a Chlamydia species. The level of conservation of the Pmp proteins has previously only been determined for Chlamydia trachomatis. As different Pmp proteins might be indispensible for the pathogenesis of different Chlamydia species, this study investigated the conservation of Pmp proteins both within and across C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci. The pmp coding sequences were annotated in 16 C. trachomatis, 6 C. pneumoniae, 2 C. abortus, and 16 C. psittaci genomes. The number and organization of polymorphic membrane coding sequences differed within and across the analyzed Chlamydia species. The length of coding sequences of pmpA,pmpB, and pmpH was conserved among all analyzed genomes, while the length of pmpE/F and pmpG, and remarkably also of the subtype pmpD, differed among the analyzed genomes. PmpD, PmpA, PmpH, and PmpA were the most conserved Pmp in C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci, respectively. PmpB was the most conserved Pmp across the 4 analyzed Chlamydia species.

  19. Structural sequences are conserved in the genes coding for the alpha, alpha' and beta-subunits of the soybean 7S seed storage protein.

    PubMed Central

    Schuler, M A; Ladin, B F; Pollaco, J C; Freyer, G; Beachy, R N

    1982-01-01

    Cloned DNAs encoding four different proteins have been isolated from recombinant cDNA libraries constructed with Glycine max seed mRNAs. Two cloned DNAs code for the alpha and alpha'-subunits of the 7S seed storage protein (conglycinin). The other cloned cDNAs code for proteins which are synthesized in vitro as 68,000 d., 60,000 d. or 53,000 d. polypeptides. Hybrid selection experiments indicate that, under low stringency hybridization conditions, all four cDNAs hybridize with mRNAs for the alpha and alpha'-subunits and the 68,000 d., 60,000 d. and 53,000 d. in vitro translation products. Within three of the mRNA, there is a conserved sequence of 155 nucleotides which is responsible for this hybridization. The conserved nucleotides in the alpha and alpha'-subunit cDNAs and the 68,000 d. polypeptide cDNAs span both coding and noncoding sequences. The differences in the coding nucleotides outside the conserved region are extensive. This suggests that selective pressure to maintain the 155 conserved nucleotides has been influenced by the structure of the seed mRNA. RNA blot hybridizations demonstrate that mRNA encoding the other major subunit (beta) of the 7S seed storage protein also shares sequence homology with the conserved 155 nucleotide sequence of the alpha and alpha'-subunit mRNAs, but not with other coding sequences. Images PMID:6897678

  20. Salmonella serotype determination utilizing high-throughput genome sequencing data.

    PubMed

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

    2015-05-01

    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.

  1. FeatureMap3D--a tool to map protein features and sequence conservation onto homologous structures in the PDB.

    PubMed

    Wernersson, Rasmus; Rapacki, Kristoffer; Staerfeldt, Hans-Henrik; Sackett, Peter Wad; Mølgaard, Anne

    2006-07-01

    FeatureMap3D is a web-based tool that maps protein features onto 3D structures. The user provides sequences annotated with any feature of interest, such as post-translational modifications, protease cleavage sites or exonic structure and FeatureMap3D will then search the Protein Data Bank (PDB) for structures of homologous proteins. The results are displayed both as an annotated sequence alignment, where the user-provided annotations as well as the sequence conservation between the query and the target sequence are displayed, and also as a publication-quality image of the 3D protein structure with the selected features and sequence conservation enhanced. The results are also returned in a readily parsable text format as well as a PyMol (http://pymol.sourceforge.net/) script file, which allows the user to easily modify the protein structure image to suit a specific purpose. FeatureMap3D can also be used without sequence annotation, to evaluate the quality of the alignment of the input sequences to the most homologous structures in the PDB, through the sequence conservation colored 3D structure visualization tool. FeatureMap3D is available at: http://www.cbs.dtu.dk/services/FeatureMap3D/. PMID:16845115

  2. 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

  3. 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.

  4. Pleiotropic effect of disrupting a conserved sequence involved in a long-range compensatory interaction in the Drosophila Adh gene.

    PubMed Central

    Baines, John F; Parsch, John; Stephan, Wolfgang

    2004-01-01

    Recent advances in experimental analyses of the evolution of RNA secondary structures suggest a more complex scenario than that typically considered by Kimura's classical model of compensatory evolution. In this study, we examine one such case in more detail. Previous experimental analysis of long-range compensatory interactions between the two ends of Drosophila Adh mRNA failed to fit the classical model of compensatory evolution. To further investigate and verify long-range pairing in Drosophila Adh with respect to models of compensatory evolution and its potential functional role, we introduced site-directed mutations in the Drosophila melanogaster Adh gene. We explore two alternative hypotheses for why previous analysis of long-range compensatory interactions failed to fit the classical model. Specifically, we investigate whether the disruption of a conserved short-range pairing within Adh exon 2 has an effect on Adh expression or if there is a dual functional role of a conserved sequence in the 3'-UTR in both long-range pairing and the negative regulation of Adh expression. We find that a classical result was not observed due to the pleiotropic effect of changing a nucleotide involved in both long-range base pairing and the negative regulation of gene expression. PMID:15020421

  5. 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)

  6. 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

  7. 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

  8. 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

  9. 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.

  10. The Conserved Sequence Repeats of IQGAP1 mediate binding to Ezrin

    PubMed Central

    Liu, Jing; Guidry, Jesse J.; Worthylake, David K.

    2014-01-01

    Mammalian IQGAP proteins all feature multiple ~50 amino acid sequence repeats near their N-termini and little is known about the function of these “Repeats”. We have expressed and purified the Repeats from human IQGAP1 in order to identify binding partners. We used mass spectrometry to identify 42 mouse kidney proteins that associate with the IQGAP1 Repeats including the ERM proteins ezrin, radixin and moesin. ERM proteins have an N-terminal FERM domain (four point one, ezrin, radixin, moesin) through which they bind to protein targets and phosphatidylinositol 4,5-bisphosphate (PIP2), and a C-terminal actin-binding domain, and function to link the actin cytoskeleton to distinct locations on the cell cortex. Isothermal titration calorimetry (ITC) reveals that the IQGAP1 Repeats directly bind to the ezrin FERM domain while no binding is seen for full-length “autoinhibited” ezrin or a version of full-length ezrin intended to mimic the activated protein. ITC also indicates that the ezrin FERM domain binds to the Repeats from IQGAP2 but not the Repeats from IQGAP3. We conclude that IQGAP1 and IQGAP2 are positioned at the cell cortex by ERM proteins. We propose that the IQGAP3 Repeats may likewise bind to FERM domains signaling purposes. PMID:24294828

  11. Copper–Peptide Complex Structure and Reactivity When Found in Conserved His-Xaa-His Sequences

    PubMed Central

    2015-01-01

    Oxygen-activating copper proteins may possess His-Xaa-His chelating sequences at their active sites and additionally exhibit imidiazole group δN vs εN tautomeric preferences. As shown here, such variations strongly affect copper ion’s coordination geometry, redox behavior, and oxidative reactivity. Copper(I) complexes bound to either δ-HGH or ε-HGH tripeptides were synthesized and characterized. Structural investigations using X-ray absorption spectroscopy, density functional theory calculations, and solution conductivity measurements reveal that δ-HGH forms the CuI dimer complex [{CuI(δ-HGH)}2]2+ (1) while ε-HGH binds CuI to give the monomeric complex [CuI(ε-HGH)]+ (2). Only 2 exhibits any reactivity, forming a strong CO adduct, [CuI(ε-HGH)(CO)]+, with properties closely matching those of the copper monooxygenase PHM. Also, 2 is reactive toward O2 or H2O2, giving a new type of O2-adduct or CuII–OOH complex, respectively. PMID:25171435

  12. 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....

  13. 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....

  14. 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....

  15. 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....

  16. 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....

  17. 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....

  18. 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....

  19. 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....

  20. 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....

  1. 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....

  2. Identification and characterization of small non-coding RNAs from Chinese fir by high throughput sequencing

    PubMed Central

    2012-01-01

    Background Small non-coding RNAs (sRNAs) play key roles in plant development, growth and responses to biotic and abiotic stresses. At least four classes of sRNAs have been well characterized in plants, including repeat-associated siRNAs (rasiRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs) and natural antisense transcript-derived siRNAs. Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous evergreen tree species in China. No sRNA from Chinese fir has been described to date. Results To obtain sRNAs in Chinese fir, we sequenced a sRNA library generated from seeds, seedlings, leaves, stems and calli, using Illumina high throughput sequencing technology. A comprehensive set of sRNAs were acquired, including conserved and novel miRNAs, rasiRNAs and tasiRNAs. With BLASTN and MIREAP we identified a total of 115 conserved miRNAs comprising 40 miRNA families and one novel miRNA with precursor sequence. The expressions of 16 conserved and one novel miRNAs and one tasiRNA were detected by RT-PCR. Utilizing real time RT-PCR, we revealed that four conserved and one novel miRNAs displayed developmental stage-specific expression patterns in Chinese fir. In addition, 209 unigenes were predicted to be targets of 30 Chinese fir miRNA families, of which five target genes were experimentally verified by 5' RACE, including a squamosa promoter-binding protein gene, a pentatricopeptide (PPR) repeat-containing protein gene, a BolA-like family protein gene, AGO1 and a gene of unknown function. We also demonstrated that the DCL3-dependent rasiRNA biogenesis pathway, which had been considered absent in conifers, existed in Chinese fir. Furthermore, the miR390-TAS3-ARF regulatory pathway was elucidated. Conclusions We unveiled a complex population of sRNAs in Chinese fir through high throughput sequencing. This provides an insight into the composition and function of sRNAs in Chinese fir and sheds new light on land plant sRNA evolution. PMID:22894611

  3. 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

  4. Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

    PubMed Central

    Fujii, Yuki; Tanaka, Shiho; Otsuki, Manami; Hoshino, Yasushi; Morimoto, Chinatsu; Kotani, Takuya; Harashima, Yuko; Endo, Haruka; Yoshizawa, Yasutaka; Sato, Ryoichi

    2012-01-01

    Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein. PMID:23145814

  5. 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

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

    PubMed Central

    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

  7. 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

    2015-06-09

    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.

  8. 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.

  9. Detecting Alu insertions from high-throughput sequencing data

    PubMed Central

    David, Matei; Mustafa, Harun; Brudno, Michael

    2013-01-01

    High-throughput sequencing technologies have allowed for the cataloguing of variation in personal human genomes. In this manuscript, we present alu-detect, a tool that combines read-pair and split-read information to detect novel Alus and their precise breakpoints directly from either whole-genome or whole-exome sequencing data while also identifying insertions directly in the vicinity of existing Alus. To set the parameters of our method, we use simulation of a faux reference, which allows us to compute the precision and recall of various parameter settings using real sequencing data. Applying our method to 100 bp paired Illumina data from seven individuals, including two trios, we detected on average 1519 novel Alus per sample. Based on the faux-reference simulation, we estimate that our method has 97% precision and 85% recall. We identify 808 novel Alus not previously described in other studies. We also demonstrate the use of alu-detect to study the local sequence and global location preferences for novel Alu insertions. PMID:23921633

  10. U17/snR30 is a ubiquitous snoRNA with two conserved sequence motifs essential for 18S rRNA production.

    PubMed

    Atzorn, Vera; Fragapane, Paola; Kiss, Tamás

    2004-02-01

    Saccharomyces cerevisiae snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) required for the processing of 18S rRNA. Here, we show that the previously characterized human, reptilian, amphibian, and fish U17 snoRNAs represent the vertebrate homologues of yeast snR30. We also demonstrate that U17/snR30 is present in the fission yeast Schizosaccharomyces pombe and the unicellular ciliated protozoan Tetrahymena thermophila. Evolutionary comparison revealed that the 3'-terminal hairpins of U17/snR30 snoRNAs contain two highly conserved sequence motifs, the m1 (AUAUUCCUA) and m2 (AAACCAU) elements. Mutation analysis of yeast snR30 demonstrated that the m1 and m2 elements are essential for early cleavages of the 35S pre-rRNA and, consequently, for the production of mature 18S rRNA. The m1 and m2 motifs occupy the opposite strands of an internal loop structure, and they are located invariantly 7 nucleotides upstream from the ACA box of U17/snR30 snoRNAs. U17/snR30 is the first identified box H/ACA snoRNA that possesses an evolutionarily conserved role in the nucleolytic processing of eukaryotic pre-rRNA.

  11. The Autism Sequencing Consortium: Large scale, high throughput sequencing in autism spectrum disorders

    PubMed Central

    Buxbaum, J. D.; Daly, M. J.; Devlin, B.; Lehner, T.; Roeder, K.; State, M. W.

    2013-01-01

    Research during the past decade has seen significant progress toward a model for the genetic architecture of autism spectrum disorders (ASD), with gene discovery accelerating as the characterization of genomic variation has become increasingly comprehensive. At the same time this research has highlighted ongoing challenges. Here we address the enormous impact of high throughput sequencing (HTS) on ASD gene discovery, outline a consensus view for leveraging this technology, and describe a large multi-site collaboration developed to accomplish these goals. Similar approaches could prove effective for severe neurodevelopmental disorders more broadly. PMID:23259942

  12. 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

  13. 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

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

    PubMed

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

    2015-02-01

    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.

  15. 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

  16. 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

  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. High resolution sequence stratigraphic concepts applied to geostatistical modeling

    SciTech Connect

    Desaubliaux, G.; De Lestang, A.P.; Eschard, R.

    1995-08-01

    Lithofacies simulations using a high resolution 3D grid allow to enhance the geometries of internal heterogeneities of reservoirs. In this study the series simulated were the Ness formation, part of the Brent reservoir in the Dunbar field located in the Viking graben of the North Sea. Simulations results have been used to build the reservoir layering supporting the 3D grid used for reservoir engineering, and also used as a frame to study the effects of secondary diagenetic processes on petrophysical properties. The method used is based on a geostatistical study and integrates the following data: a geological model using sequence stratigraphic concepts to define lithofacies sequences and associated bounding surfaces, well data (cores and logs) used as database for geostatistical analysis and simulations, seismic data: a 3D seismic survey has been used to define the internal surfaces bounding the units, outcrop data: The Mesa Verde formation (Colorado, USA) has been used as an outcrop analog to calibrate geostatistical parameters for the simulations (horizontal range of the variograms). This study illustrates the capacity to use high resolution sequence stratigraphic concepts to improve the simulations of reservoirs when the lack of subsurface information reduce the accuracy of geostatistical analysis.

  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. Functional substitution of an essential yeast RNA polymerase subunit by a highly conserved mammalian counterpart.

    PubMed Central

    McKune, K; Woychik, N A

    1994-01-01

    We isolated the cDNA encoding the homolog of the Saccharomyces cerevisiae nuclear RNA polymerase common subunit RPB6 from hamster CHO cells. Alignment of yeast RPB6 with its mammalian counterpart revealed that the subunits have nearly identical carboxy-terminal halves and a short acidic region at the amino terminus. Remarkably, the length and amino acid sequence of the hamster RPB6 are identical to those of the human RPB6 subunit. The conservation in sequence from lower to higher eukaryotes also reflects conservation of function in vivo, since hamster RPB6 supports normal wild-type yeast cell growth in the absence of the essential gene encoding RPB6. Images PMID:8196653

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

    2015-08-04

    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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. Mouse telocentric sequences reveal a high rate of homogenization and possible role in Robertsonian translocation

    PubMed Central

    Kalitsis, Paul; Griffiths, Belinda; Choo, K. H. Andy

    2006-01-01

    The telomere and centromere are two specialized structures of eukaryotic chromosomes that are essential for chromosome stability and segregation. These structures are usually characterized by large tracts of tandemly repeated DNA. In mouse, the two structures are often located in close proximity to form telocentric chromosomes. To date, no detailed sequence information is available across the mouse telocentric regions. The antagonistic mechanisms for the stable maintenance of the mouse telocentric karyotype and the occurrence of whole-arm Robertsonian translocations remain enigmatic. We have identified large-insert fosmid clones that span the telomere and centromere of several mouse chromosome ends. Sequence analysis shows that the distance between the telomeric T2AG3 and centromeric minor satellite repeats range from 1.8 to 11 kb. The telocentric regions of different mouse chromosomes comprise a contiguous linear order of T2AG3 repeats, a highly conserved truncated long interspersed nucleotide element 1 repeat, and varying amounts of a recently discovered telocentric tandem repeat that shares considerable identity with, and is inverted relative to, the centromeric minor satellite DNA. The telocentric domain as a whole exhibits the same polarity and a high sequence identity of >99% between nonhomologous chromosomes. This organization reflects a mechanism of frequent recombinational exchange between nonhomologous chromosomes that should promote the stable evolutionary maintenance of a telocentric karyotype. It also provides a possible mechanism for occasional inverted mispairing and recombination between the oppositely oriented TLC and minor satellite repeats to result in Robertsonian translocations. PMID:16731628

  13. [Cloning and analysis of highly repetitive sequence fragments from takin (Budorcas taxicolor)].

    PubMed

    Qian, Min; Dou, Zhen; Gu, Yong Xi; Zhang, Qin; Lu, Run Long; Zhu, Xue Liang

    2002-03-01

    Takin (Budorcas taxicolor) is a large animal living in China and other adjacent countries, which belongs to Bovidae of Artiodactyla. The anatomy, morphology and behavior of takin are between species of the subfamily Bovinae and Caprinae. It is now in a separated genus or in the same genus with muskox (Ovibos moschatus). To gain insight into the evolution of takin, we purified and cloned its highly repetitive BamHI fragments from the genomic DNA. The sequences of three fragments were highly homologous, indicative of units of a large repetitive DNA arrays. Southern hybridization using these fragments as probes showed identical patterns among individuals in both the same and different subspecies, implying conserved sequences and distributions of the BamHI clusters in takin genome. Sequence comparison with 1.714 and 1.715 satellite DNA of other species in Bovidae supports that takin has a closer relationship with the subfamily Caprinae than with Bovinae. It also suggests that these BamHI fragments may represent repeat units of the centromeric satellite DNA of takin. PMID:15344315

  14. 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

  15. Genomics in cereals: from genome-wide conserved orthologous set (COS) sequences to candidate genes for trait dissection.

    PubMed

    Quraishi, Umar Masood; Abrouk, Michael; Bolot, Stéphanie; Pont, Caroline; Throude, Mickael; Guilhot, Nicolas; Confolent, Carole; Bortolini, Fernanda; Praud, Sébastien; Murigneux, Alain; Charmet, Gilles; Salse, Jerome

    2009-11-01

    Recent updates in comparative genomics among cereals have provided the opportunity to identify conserved orthologous set (COS) DNA sequences for cross-genome map-based cloning of candidate genes underpinning quantitative traits. New tools are described that are applicable to any cereal genome of interest, namely, alignment criterion for orthologous couples identification, as well as the Intron Spanning Marker software to automatically select intron-spanning primer pairs. In order to test the software, it was applied to the bread wheat genome, and 695 COS markers were assigned to 1,535 wheat loci (on average one marker/2.6 cM) based on 827 robust rice-wheat orthologs. Furthermore, 31 of the 695 COS markers were selected to fine map a pentosan viscosity quantitative trait loci (QTL) on wheat chromosome 7A. Among the 31 COS markers, 14 (45%) were polymorphic between the parental lines and 12 were mapped within the QTL confidence interval with one marker every 0.6 cM defining candidate genes among the rice orthologous region.

  16. 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

  17. 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

  18. 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

  19. Low-Cost, High-Throughput Sequencing of DNA Assemblies Using a Highly Multiplexed Nextera Process.

    PubMed

    Shapland, Elaine B; Holmes, Victor; Reeves, Christopher D; Sorokin, Elena; Durot, Maxime; Platt, Darren; Allen, Christopher; Dean, Jed; Serber, Zach; Newman, Jack; Chandran, Sunil

    2015-07-17

    In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction.

  20. Computational identification and characterization of conserved miRNAs and their target genes in garlic (Allium sativum L.) expressed sequence tags.

    PubMed

    Panda, Debashis; Dehury, Budheswar; Sahu, Jagajjit; Barooah, Madhumita; Sen, Priyabrata; Modi, Mahendra K

    2014-03-10

    The endogenous small non-coding functional microRNAs (miRNAs) are short in size, range from ~21 to 24 nucleotides in length, play a pivotal role in gene expression in plants and animals by silencing genes either by destructing or blocking of translation of homologous mRNA. Although various high-throughput, time consuming and expensive techniques like forward genetics and direct cloning are employed to detect miRNAs in plants but comparative genomics complemented with novel bioinformatic tools pave the way for efficient and cost-effective identification of miRNAs through homologous sequence search with previously known miRNAs. In this study, an attempt was made to identify and characterize conserved miRNAs in garlic expressed sequence tags (ESTs) through computational means. For identification of novel miRNAs in garlic, a total 3227 known mature miRNAs of plant kingdom Viridiplantae were searched for homology against 21,637 EST sequences resulting in identification of 6 potential miRNA candidates belonging to 6 different miRNA families. The psRNATarget server predicted 33 potential target genes and their probable functions for the six identified miRNA families in garlic. Most of the garlic miRNA target genes seem to encode transcription factors as well as genes involved in stress response, metabolism, plant growth and development. The results from the present study will shed more light on the understanding of molecular mechanisms of miRNA in garlic which may aid in the development of novel and precise techniques to understand some post-transcriptional gene silencing mechanism in response to stress tolerance.

  1. BatchPrimer3: A high throughput web application for PCR and sequencing primer design

    PubMed Central

    You, Frank M; Huo, Naxin; Gu, Yong Qiang; Luo, Ming-cheng; Ma, Yaqin; Hane, Dave; Lazo, Gerard R; Dvorak, Jan; Anderson, Olin D

    2008-01-01

    Background Microsatellite (simple sequence repeat – SSR) and single nucleotide polymorphism (SNP) markers are two types of important genetic markers useful in genetic mapping and genotyping. Often, large-scale genomic research projects require high-throughput computer-assisted primer design. Numerous such web-based or standard-alone programs for PCR primer design are available but vary in quality and functionality. In particular, most programs lack batch primer design capability. Such a high-throughput software tool for designing SSR flanking primers and SNP genotyping primers is increasingly demanded. Results A new web primer design program, BatchPrimer3, is developed based on Primer3. BatchPrimer3 adopted the Primer3 core program as a major primer design engine to choose the best primer pairs. A new score-based primer picking module is incorporated into BatchPrimer3 and used to pick position-restricted primers. BatchPrimer3 v1.0 implements several types of primer designs including generic primers, SSR primers together with SSR detection, and SNP genotyping primers (including single-base extension primers, allele-specific primers, and tetra-primers for tetra-primer ARMS PCR), as well as DNA sequencing primers. DNA sequences in FASTA format can be batch read into the program. The basic information of input sequences, as a reference of parameter setting of primer design, can be obtained by pre-analysis of sequences. The input sequences can be pre-processed and masked to exclude and/or include specific regions, or set targets for different primer design purposes as in Primer3Web and primer3Plus. A tab-delimited or Excel-formatted primer output also greatly facilitates the subsequent primer-ordering process. Thousands of primers, including wheat conserved intron-flanking primers, wheat genome-specific SNP genotyping primers, and Brachypodium SSR flanking primers in several genome projects have been designed using the program and validated in several laboratories

  2. Analysis of mammalian gene batteries reveals both stable ancestral cores and highly dynamic regulatory sequences

    PubMed Central

    Ettwiller, Laurence; Budd, Aidan; Spitz, François; Wittbrodt, Joachim

    2008-01-01

    Background Changes in gene regulation are suspected to comprise one of the driving forces for evolution. To address the extent of cis-regulatory changes and how they impact on gene regulatory networks across eukaryotes, we systematically analyzed the evolutionary dynamics of target gene batteries controlled by 16 different transcription factors. Results We found that gene batteries show variable conservation within vertebrates, with slow and fast evolving modules. Hence, while a key gene battery associated with the cell cycle is conserved throughout metazoans, the POU5F1 (Oct4) and SOX2 batteries in embryonic stem cells show strong conservation within mammals, with the striking exception of rodents. Within the genes composing a given gene battery, we could identify a conserved core that likely reflects the ancestral function of the corresponding transcription factor. Interestingly, we show that the association between a transcription factor and its target genes is conserved even when we exclude conserved sequence similarities of their promoter regions from our analysis. This supports the idea that turnover, either of the transcription factor binding site or its direct neighboring sequence, is a pervasive feature of proximal regulatory sequences. Conclusions Our study reveals the dynamics of evolutionary changes within metazoan gene networks, including both the composition of gene batteries and the architecture of target gene promoters. This variation provides the playground required for evolutionary innovation around conserved ancestral core functions. PMID:19087242

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

    PubMed

    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 A resolution. The structure of AFV3-109 is a five stranded beta-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.

  4. 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

  5. The Cysteine-Rich Interdomain Region from the Highly Variable Plasmodium falciparum Erythrocyte Membrane Protein-1 Exhibits a Conserved Structure

    PubMed Central

    Su, Hua-Poo; Makobongo, Morris O.; Moore, Jaime M.; Singh, Sanjay; Miller, Louis H.; Garboczi, David N.

    2008-01-01

    Plasmodium falciparum malaria parasites, living in red blood cells, express proteins of the erythrocyte membrane protein-1 (PfEMP1) family on the red blood cell surface. The binding of PfEMP1 molecules to human cell surface receptors mediates the adherence of infected red blood cells to human tissues. The sequences of the 60 PfEMP1 genes in each parasite genome vary greatly from parasite to parasite, yet the variant PfEMP1 proteins maintain receptor binding. Almost all parasites isolated directly from patients bind the human CD36 receptor. Of the several kinds of highly polymorphic cysteine-rich interdomain region (CIDR) domains classified by sequence, only the CIDR1α domains bind CD36. Here we describe the CD36-binding portion of a CIDR1α domain, MC179, as a bundle of three α-helices that are connected by a loop and three additional helices. The MC179 structure, containing seven conserved cysteines and 10 conserved hydrophobic residues, predicts similar structures for the hundreds of CIDR sequences from the many genome sequences now known. Comparison of MC179 with the CIDR domains in the genome of the P. falciparum 3D7 strain provides insights into CIDR domain structure. The CIDR1α three-helix bundle exhibits less than 20% sequence identity with the three-helix bundles of Duffy-binding like (DBL) domains, but the two kinds of bundles are almost identical. Despite the enormous diversity of PfEMP1 sequences, the CIDR1α and DBL protein structures, taken together, predict that a PfEMP1 molecule is a polymer of three-helix bundles elaborated by a variety of connecting helices and loops. From the structures also comes the insight that DBL1α domains are approximately 100 residues larger and that CIDR1α domains are approximately 100 residues smaller than sequence alignments predict. This new understanding of PfEMP1 structure will allow the use of better-defined PfEMP1 domains for functional studies, for the design of candidate vaccines, and for understanding the

  6. 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

  7. 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.

  8. [Detection and analysis of Tetrahymena pyriformis 26S ribosomal DNA domain sequences, differing in degree of evolutionary conservation].

    PubMed

    Mukha, D V; Sidorenko, A P

    1995-01-01

    Domains of different evolutionary conservatism were defined in the 26S rDNA sequence of T. pyriformis. The fragment of studied DNA (1212 bp) showing high evolutionary conservatism was cloned. It was shown this fragment of DNA may be used to a probe for blot-hybridization analysis of the structure of rDNA from various taxa, protists to mammals. Superconservative and hypervariable domains were defined. The first are good for the primers for PCR analysis of rDNA from various taxa, the second--for species specific primers.

  9. Lineage-specific sequence evolution and exon edge conservation partially explain the relationship between evolutionary rate and expression level in A. thaliana.

    PubMed

    Bush, Stephen J; Kover, Paula X; Urrutia, Araxi O

    2015-06-01

    Rapidly evolving proteins can aid the identification of genes underlying phenotypic adaptation across taxa, but functional and structural elements of genes can also affect evolutionary rates. In plants, the 'edges' of exons, flanking intron junctions, are known to contain splice enhancers and to have a higher degree of conservation compared to the remainder of the coding region. However, the extent to which these regions may be masking indicators of positive selection or account for the relationship between dN/dS and other genomic parameters is unclear. We investigate the effects of exon edge conservation on the relationship of dN/dS to various sequence characteristics and gene expression parameters in the model plant Arabidopsis thaliana. We also obtain lineage-specific dN/dS estimates, making use of the recently sequenced genome of Thellungiella parvula, the second closest sequenced relative after the sister species Arabidopsis lyrata. Overall, we find that the effect of exon edge conservation, as well as the use of lineage-specific substitution estimates, upon dN/dS ratios partly explains the relationship between the rates of protein evolution and expression level. Furthermore, the removal of exon edges shifts dN/dS estimates upwards, increasing the proportion of genes potentially under adaptive selection. We conclude that lineage-specific substitutions and exon edge conservation have an important effect on dN/dS ratios and should be considered when assessing their relationship with other genomic parameters.

  10. Lineage-specific sequence evolution and exon edge conservation partially explain the relationship between evolutionary rate and expression level in A. thaliana.

    PubMed

    Bush, Stephen J; Kover, Paula X; Urrutia, Araxi O

    2015-06-01

    Rapidly evolving proteins can aid the identification of genes underlying phenotypic adaptation across taxa, but functional and structural elements of genes can also affect evolutionary rates. In plants, the 'edges' of exons, flanking intron junctions, are known to contain splice enhancers and to have a higher degree of conservation compared to the remainder of the coding region. However, the extent to which these regions may be masking indicators of positive selection or account for the relationship between dN/dS and other genomic parameters is unclear. We investigate the effects of exon edge conservation on the relationship of dN/dS to various sequence characteristics and gene expression parameters in the model plant Arabidopsis thaliana. We also obtain lineage-specific dN/dS estimates, making use of the recently sequenced genome of Thellungiella parvula, the second closest sequenced relative after the sister species Arabidopsis lyrata. Overall, we find that the effect of exon edge conservation, as well as the use of lineage-specific substitution estimates, upon dN/dS ratios partly explains the relationship between the rates of protein evolution and expression level. Furthermore, the removal of exon edges shifts dN/dS estimates upwards, increasing the proportion of genes potentially under adaptive selection. We conclude that lineage-specific substitutions and exon edge conservation have an important effect on dN/dS ratios and should be considered when assessing their relationship with other genomic parameters. PMID:25930165

  11. Identification of Novel and Conserved miRNAs in Leaves of In vitro Grown Citrus reticulata "Lugan" Plantlets by Solexa Sequencing.

    PubMed

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

    2015-01-01

    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.

  12. Identification of Novel and Conserved miRNAs in Leaves of In vitro Grown Citrus reticulata "Lugan" Plantlets by Solexa Sequencing.

    PubMed

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

    2015-01-01

    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

  13. 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

  14. 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

  15. The Use of Coded PCR Primers Enables High-Throughput Sequencing of Multiple Homolog Amplification Products by 454 Parallel Sequencing

    PubMed Central

    Bollback, Jonathan P.; Panitz, Frank; Bendixen, Christian; Nielsen, Rasmus; Willerslev, Eske

    2007-01-01

    Background The invention of the Genome Sequence 20™ DNA Sequencing System (454 parallel sequencing platform) has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR) reactions and subsequent sequencing runs have been unable to combine template DNA from multiple individuals, as homologous sequences cannot be subsequently assigned to their original sources. Methodology We use conventional PCR with 5′-nucleotide tagged primers to generate homologous DNA amplification products from multiple specimens, followed by sequencing through the high-throughput Genome Sequence 20™ DNA Sequencing System (GS20, Roche/454 Life Sciences). Each DNA sequence is subsequently traced back to its individual source through 5′tag-analysis. Conclusions We demonstrate that this new approach enables the assignment of virtually all the generated DNA sequences to the correct source once sequencing anomalies are accounted for (miss-assignment rate<0.4%). Therefore, the method enables accurate sequencing and assignment of homologous DNA sequences from multiple sources in single high-throughput GS20 run. We observe a bias in the distribution of the differently tagged primers that is dependent on the 5′ nucleotide of the tag. In particular, primers 5′ labelled with a cytosine are heavily overrepresented among the final sequences, while those 5′ labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution of the sequences as sorted by the second nucleotide of the dinucleotide tags. As the results are based on a single GS20 run, the general applicability of the approach requires confirmation. However, our experiments demonstrate that 5′primer tagging is a useful method in which the sequencing power of the GS20 can be applied to PCR-based assays of multiple homologous PCR products. The new approach will be of value to a broad range of research areas, such as those of

  16. Conserved interactions of a compact highly active enhancer/promoter upstream of the rhodopsin kinase (GRK1) gene.

    PubMed

    Young, Joyce E; Kasperek, Eileen M; Vogt, Todd M; Lis, Agnieszka; Khani, Shahrokh C

    2007-08-01

    Rhodopsin kinase (RK) is a conserved component of the light adaptation and recovery pathways shared among rod and cone photoreceptors of a variety of species. To gain insight into transcriptional mechanisms driving RK and potentially other genes of similar spatial profile, the components and the interactions of the highly compact enhancer/promoter region (E/P) upstream of the human RK gene were examined. Cross-species comparison outlined an active 49-bp widely shared E/P core as the major site of conservation in the entire 5' flanking sequence. The area consisted of a bicoid-type homeodomain recognition cassette and a unique T-rich module interacting with TATA-binding proteins. Homeodomain interactions involved primarily Crx and secondarily Otx2. Both strongly stimulated the E/P. In the absence of Crx, persistent E/P activity shifted from the outer retina to the inner to follow the Otx2 pattern. The spatial patterns were largely unaffected by the absence of rod transcription factors, Nrl and Nr2e3, and the RK transcriptional activity preceded the surge in rod-specific transcription. Conserved bicoid homeodomain factors thus appear to be the key factors governing localization of RK E/P activity in retina and photoreceptors.

  17. 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.

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

    PubMed Central

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

    2013-01-01

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

  19. Molecular characterization of flavanone 3 beta-hydroxylases. Consensus sequence, comparison with related enzymes and the role of conserved histidine residues.

    PubMed

    Britsch, L; Dedio, J; Saedler, H; Forkmann, G

    1993-10-15

    A heterologous cDNA probe from Petunia hybrida was used to isolate flavanone-3 beta-hydroxylase-encoding cDNA clones from carnation (Dianthus caryophyllus), china aster (Callistephus chinensis) and stock (Matthiola incana). The deduced protein sequences together with the known sequences of the enzyme from P. hybrida, barley (Hordeum vulgare) and snapdragon (Antirrhinum majus) enabled the determination of a consensus sequence which revealed an overall 84% similarity (53% identity) of flavanone 3 beta-hydroxylases from the different sources. Alignment with the sequences of other known enzymes of the same class and to related non-heme iron-(II) enzymes demonstrated the strict genetic conservation of 14 amino acids, in particular, of three histidines and an aspartic acid. The conservation of the histidine motifs provides strong support for the possible conservation of structurally similar iron-binding sites in these enzymes. The putative role of histidines as chelators of ferrous ions in the active site of flavanone 3 beta-hydroxylases was corroborated by diethyl-pyrocarbonate modification of the partially purified recombinant Petunia enzyme.

  20. Towards Experimental Annotation of Genes by High Throughput Sequencing

    SciTech Connect

    Bradbury, Andrew

    2010-06-03

    Andrew Bradbury of Los Alamos National Laboratory discusses turning annotation into a sequencing pipeline on June 3, 2010 at the "Sequencing, Finishing, Analysis in the Future" meeting in Santa Fe, NM

  1. High affinity DNA-microtubule interactions: evidence for a conserved DNA-MAP interaction involving unusual high CsCl density repetitious DNA families.

    PubMed

    Marx, K A; Denial, T

    1992-12-01

    We have examined high affinity interactions of chick brain microtubule proteins with 35S labelled tracer DNAs from chick, mouse and D. melanogaster under equilibrium conditions by the nitrocellulose filter binding technique. Ternary reaction mixtures of the above two components and a third component, an excess of unlabelled competitor DNA from either E. coli., mouse, D. melanogaster or chick, were used to measure small fractions of DNA in each case (1-4%) bound to microtubule protein under high stringency- large competitor DNA concentration and 0.5 M NaCl. As seen in part previously (Marx, K.A. and Denial, T. (1985) in The Molecular Basis of Cancer, 172B, 65-75 (Rein, ed), A. Liss, N.Y.) the measured order of competitor DNA strengths was identical for all three tracer DNAs. That is: chick > mouse > D. melanogaster > E. coli competitor DNA. Since the homologous interaction, chick competitor DNA with chick brain microtubule protein, is always the strongest interaction measured, we interpret this as evidence for a conserved protein-DNA sequence interaction. 35S chick DNA tracer sequences, isolated from nitrocellulose filters following the stringent binding in the presence of 0.9 mM-1 E. coli. competitor DNA, was used in driven reassociation reactions with total chick driver DNA. This fraction was found to be significantly enriched in repetitive chick DNA sequences. Since we have observed a similar phenomenon in mouse, we then compared the stringent binding mouse sequences and showed that the bulk of these sequences did not cross-hybridize with total chick DNA. Finally, all three 35S tracer DNAs binding to nitrocellulose were isolated and sedimented to equilibrium on CsCl density gradients. The CsCl density distributions from all three DNAs showed significant (100-fold) enrichment in classical satellite DNAs as well as higher enrichment in two very unusual high CsCl density families of DNA (1.720-1.740 g/cm3; 1.750-1.765 g/cm3). These families are never observed as

  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. 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

  4. Unprecedented high-resolution view of bacterial operon architecture revealed by RNA sequencing.

    PubMed

    Conway, Tyrrell; 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; Wanner, Barry L

    2014-01-01

    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. Importance: We precisely mapped the 5' and 3' ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are

  5. 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

  6. The phylogeographical pattern and conservation of the Chinese cobra (Naja atra) across its range based on mitochondrial control region sequences.

    PubMed

    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

  7. 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

  8. 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…

  9. A stationary-phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved gene family.

    PubMed Central

    Braun, E L; Fuge, E K; Padilla, P A; Werner-Washburne, M

    1996-01-01

    The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms. In unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, growth and proliferation are regulated by nutrient availability. We have described changes in the pattern of protein synthesis during the growth of S. cerevisiae cells to stationary phase (E. K. Fuge, E. L. Braun, and M. Werner-Washburne, J. Bacteriol. 176:5802-5813, 1994) and noted a protein, which we designated Snz1p (p35), that shows increased synthesis after entry into stationary phase. We report here the identification of the SNZ1 gene, which encodes this protein. We detected increased SNZ1 mRNA accumulation almost 2 days after glucose exhaustion, significantly later than that of mRNAs encoded by other postexponential genes. SNZ1-related sequences were detected in phylogenetically diverse organisms by sequence comparisons and low-stringency hybridization. Multiple SNZ1-related sequences were detected in some organisms, including S. cerevisiae. Snz1p was found to be among the most evolutionarily conserved proteins currently identified, indicating that we have identified a novel, highly conserved protein involved in growth arrest in S. cerevisiae. The broad phylogenetic distribution, the regulation of the SNZ1 mRNA and protein in S. cerevisiae, and identification of a Snz protein modified during sporulation in the gram-positive bacterium Bacillus subtilis support the hypothesis that Snz proteins are part of an ancient response that occurs during nutrient limitation and growth arrest. PMID:8955308

  10. 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

  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. 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

  13. 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.

  14. 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

  15. Sequence-Dependent T:G Base Pair Opening in DNA Double Helix Bound by Cren7, a Chromatin Protein Conserved among Crenarchaea

    PubMed Central

    Tian, Lei; Zhang, Zhenfeng; Wang, Hanqian; Zhao, Mohan; Dong, Yuhui; Gong, Yong

    2016-01-01

    T:G base pair arising from spontaneous deamination of 5mC or polymerase errors is a great challenge for DNA repair of hyperthermophilic archaea, especially Crenarchaea. Most strains in this phylum lack the protein homologues responsible for the recognition of the mismatch in the DNA repair pathways. To investigate whether Cren7, a highly conserved chromatin protein in Crenarchaea, serves a role in the repair of T:G mispairs, the crystal structures of Cren7-GTAATTGC and Cren7-GTGATCGC complexes were solved at 2.0 Å and 2.1 Å. In our structures, binding of Cren7 to the AT-rich DNA duplex (GTAATTGC) induces opening of T2:G15 but not T10:G7 base pair. By contrast, both T:G mispairs in the GC-rich DNA duplex (GTGATCGC) retain the classic wobble type. Structural analysis also showed DNA helical changes of GTAATTGC, especially in the steps around the open T:G base pair, as compared to GTGATCGC or the matched DNAs. Surface plasmon resonance assays revealed a 4-fold lower binding affinity of Cren7 for GTAATTGC than that for GTGATCGC, which was dominantly contributed by the decrease of association rate. These results suggested that binding of Cren7 to DNA leads to T:G mispair opening in a sequence dependent manner, and therefore propose the potential roles of Cren7 in DNA repair. PMID:27685992

  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. 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

  18. 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.

  19. TGA cysteine codons and intron sequences in conserved and nonconserved positions are found in macronuclear RNA polymerase genes of Euplotes octocarinatus.

    PubMed Central

    Kaufmann, J; Florian, V; Klein, A

    1992-01-01

    The gene sequences of the second largest subunits of RNA polymerases I and II of Euplotes octocarinatus, RPA2 and RPB2, were determined and compared to the respective known sequences of Saccharomyces cerevisiae. The similarity of the derived polypeptide sequences permitted their assignment to the respective polymerases and allowed the comparison of the zinc binding regions. In frame TGA codons were detected, which are likely to encode conserved cysteinyl residues in the putative zinc-finger region of the RPA2 gene. They were also found in other positions in both the RPA2 and RPB2 genes. The RPB2 gene contains a 30 bp intron close to the 5'-end of its coding region. The 5'-ends of the coding regions of all three genes encoding the largest subunits of the three different polymerases were also analyzed. The zinc finger structures again show the use of TGA codons for conserved cysteinyl residues in two of the genes. An N-terminal intron is located in the RPB1 gene at a conserved position as compared to the respective genes of several other eucarya. Images PMID:1461731

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

    PubMed Central

    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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. The highly conserved U small nuclear RNA 3'-end formation signal is quite tolerant to mutation.

    PubMed Central

    Ach, R A; Weiner, A M

    1987-01-01

    Formation of the 3' end of U1 and U2 small nuclear RNA (snRNA) precursors is directed by a conserved sequence called the 3' box located 9 to 28 nucleotides downstream of all metazoan U1 to U4 snRNA genes sequenced so far. Deletion of part or all of the 3' box from human U1 and U2 genes drastically reduces 3'-end formation. To define the essential nucleotides within this box that direct 3'-end formation, we constructed a set of point mutations in the conserved residues of the human U1 3' box. The ability of the various mutations to direct 3'-end formation was tested by microinjection into Xenopus oocytes and transfection into HeLa cells. We found that the point mutations had diverse effects on 3'-end formation, ranging from no effect at all to severe inhibition; however, no single or double point mutation we tested completely eliminated 3'-end formation. We also showed that a rat U3 3' flank can effectively substitute for the human U1 3' flank, indicating that the 3' boxes of the different U snRNA genes are functionally equivalent. Images PMID:3037343

  8. Human metapneumovirus G protein is highly conserved within but not between genetic lineages

    PubMed Central

    Yang, Chin-Fen; Wang, Chiaoyin K.; Tollefson, Sharon J.; Lintao, Linda D.; Liem, Alexis; Chu, Marla; Williams, John V.

    2013-01-01

    Background Human metapneumovirus (HMPV) is an important cause of acute respiratory illnesses in children. HMPV encodes two major surface glycoproteins, fusion (F) and glycoprotein (G). The function of G has not been fully established, though it is dispensable for in vitro and in vivo replication. Methods We analyzed 87 full-length HMPV G sequences from isolates collected over 20 years. Results The G sequences fell into four subgroups with a mean 63% amino acid identity (minimum 29%). The length of G varied from 217 to 241 residues. Structural features such as proline content and N- and O-glycosylation sites were present in all strains but quite variable between subgroups. There was minimal drift within the subgroups over 20 years. The estimated time to the most recent common ancestor was 215 years. Conclusions HMPV G was conserved within lineages over 20 years, suggesting functional constraints on diversity. However, G was poorly conserved between subgroups, pointing to potentially distinct roles for G among different viral lineages. PMID:23385328

  9. Balancing forest-regeneration probabilities and maintenance costs in dry grasslands of high conservation priority

    USGS Publications Warehouse

    Bolliger, Janine; Edwards, Thomas C.; 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.

  10. 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

  11. 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.

  12. 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

  13. Two different high throughput sequencing approaches identify thousands of de novo genomic markers for the genetically depleted Bornean elephant.

    PubMed

    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 (H(o)=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 for a

  14. 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

  15. 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

  16. Conservation of Microstructure between a Sequenced Region of the Genome of Rice and Multiple Segments of the Genome of Arabidopsis thaliana

    PubMed Central

    Mayer, Klaus; Murphy, George; Tarchini, Renato; Wambutt, Rolf; Volckaert, Guido; Pohl, Thomas; Düsterhöft, Andreas; Stiekema, Willem; Entian, Karl-Dieter; Terryn, Nancy; Lemcke, Kai; Haase, Dirk; Hall, Caroline R.; van Dodeweerd, Anne-Marie; Tingey, Scott V.; Mewes, Hans-Werner; Bevan, Michael W.; Bancroft, Ian

    2001-01-01

    The nucleotide sequence was determined for a 340-kb segment of rice chromosome 2, revealing 56 putative protein-coding genes. This represents a density of one gene per 6.1 kb, which is higher than was reported for a previously sequenced segment of the rice genome. Sixteen of the putative genes were supported by matches to ESTs. The predicted products of 29 of the putative genes showed similarity to known proteins, and a further 17 genes showed similarity only to predicted or hypothetical proteins identified in genome sequence data. The region contains a few transposable elements: one retrotransposon, and one transposon. The segment of the rice genome studied had previously been identified as representing a part of rice chromosome 2 that may be homologous to a segment of Arabidopsis chromosome 4. We confirmed the conservation of gene content and order between the two genome segments. In addition, we identified a further four segments of the Arabidopsis genome that contain conserved gene content and order. In total, 22 of the 56 genes identified in the rice genome segment were represented in this set of Arabidopsis genome segments, with at least five genes present, in conserved order, in each segment. These data are consistent with the hypothesis that the Arabidopsis genome has undergone multiple duplication events. Our results demonstrate that conservation of the genome microstructure can be identified even between monocot and dicot species. However, the frequent occurrence of duplication, and subsequent microstructure divergence, within plant genomes may necessitate the integration of subsets of genes present in multiple redundant segments to deduce evolutionary relationships and identify orthologous genes. PMID:11435398

  17. 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

  18. 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

  19. 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

  20. MicroRNA expression analysis of rosette and folding leaves in Chinese cabbage using high-throughput Solexa sequencing.

    PubMed

    Wang, Fengde; Li, Huayin; Zhang, Yihui; Li, Jingjuan; Li, Libin; Liu, Lifeng; Wang, Lihua; Wang, Cuihua; Gao, Jianwei

    2013-12-15

    In this study, a global analysis of miRNA expression from rosette leaves (RLs) and folding leaves (FLs) of Chinese cabbage (Brassica rapa L. ssp. pekinensis) was conducted using high-throughput Solexa sequencing. In total, over 12 million clean reads were obtained from each library. Sequence analysis identified 64 conserved miRNA families in each leaf type and 104 and 95 novel miRNAs from RLs and FLs, respectively. Among these, 61 conserved miRNAs and 61 novel miRNAs were detected in both types of leaves. Furthermore, six conserved and 21 novel miRNAs were differentially expressed between the two libraries. Target gene annotation suggested that these differentially expressed miRNAs targeted transcription factors, F-box proteins, auxin and Ca(2+) signaling pathway proteins, protein kinases and other proteins that may function in governing leafy head formation. This study advanced our understanding of the important roles of miRNAs in regulating leafy head development in Chinese cabbage.

  1. 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

  2. Development of a protein-ligand-binding site prediction method based on interaction energy and sequence conservation.

    PubMed

    Tsujikawa, Hiroto; Sato, Kenta; Wei, Cao; Saad, Gul; Sumikoshi, Kazuya; Nakamura, Shugo; Terada, Tohru; Shimizu, Kentaro

    2016-09-01

    We present a new method for predicting protein-ligand-binding sites based on protein three-dimensional structure and amino acid conservation. This method involves calculation of the van der Waals interaction energy between a protein and many probes placed on the protein surface and subsequent clustering of the probes with low interaction energies to identify the most energetically favorable locus. In addition, it uses amino acid conservation among homologous proteins. Ligand-binding sites were predicted by combining the interaction energy and the amino acid conservation score. The performance of our prediction method was evaluated using a non-redundant dataset of 348 ligand-bound and ligand-unbound protein structure pairs, constructed by filtering entries in a ligand-binding site structure database, LigASite. Ligand-bound structure prediction (bound prediction) indicated that 74.0 % of predicted ligand-binding sites overlapped with real ligand-binding sites by over 25 % of their volume. Ligand-unbound structure prediction (unbound prediction) indicated that 73.9 % of predicted ligand-binding residues overlapped with real ligand-binding residues. The amino acid conservation score improved the average prediction accuracy by 17.0 and 17.6 points for the bound and unbound predictions, respectively. These results demonstrate the effectiveness of the combined use of the interaction energy and amino acid conservation in the ligand-binding site prediction. PMID:27400687

  3. Contamination-controlled high-throughput whole genome sequencing for influenza A viruses using the MiSeq sequencer

    PubMed Central

    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

  4. 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

  5. Interview-based sighting histories can inform regional conservation prioritization for highly threatened cryptic species

    PubMed Central

    Turvey, Samuel T; Trung, Cao Tien; Quyet, Vo Dai; Nhu, Hoang Van; Thoai, Do Van; Tuan, Vo Cong Anh; Hoa, Dang Thi; Kacha, Kouvang; Sysomphone, Thongsay; Wallate, Sousakhone; Hai, Chau Thi Thanh; Thanh, Nguyen Van; Wilkinson, Nicholas M

    2015-01-01

    The use of robust ecological data to make evidence-based management decisions is frequently prevented by limited data quantity or quality, and local ecological knowledge (LEK) is increasingly seen as an important source of information for conservation. However, there has been little assessment of LEK's usefulness for informing prioritization and management of landscapes for threatened species, or assessing comparative species status across landscapes. A large-scale interview survey in the Annamite Mountains (Vietnam and Lao PDR) compiled the first systematic LEK data set for saola Pseudoryx nghetinhensis, one of the world's rarest mammals, and eight other ungulates. Saola conservation is hindered by uncertainty over continued presence across much of its proposed distribution. We analysed comparative LEK-based last-sighting data across three landscapes to determine whether regional sighting histories support previous suggestions of landscape importance for saola conservation (Hue-Quang Nam: top-priority Vietnamese landscape; Pu Mat: lower priority Vietnamese landscape; Viengthong: high-priority Lao landscape) and whether they constitute an effective spatial prioritization tool for cryptic species management. Wild pig and red muntjac may be the only Annamite ungulates with stable populations; the regional status of all other species appears to be worse. Saola have declined more severely and/or are significantly rarer than most other ungulates and have been seen by relatively few respondents. Saola were also frequently considered locally rarest or declining, and never as species that had not declined. In contrast to other species, there are no regional differences in saola sighting histories, with continued persistence in all landscapes challenging suggestions that regional status differs greatly. Remnant populations persist in Vietnam despite heavy hunting, but even remote landscapes in Lao may be under intense pressure. Synthesis and applications. Our local

  6. Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation.

    PubMed

    Kondo, Hidemasa; Hanada, Yuichi; Sugimoto, Hiroshi; Hoshino, Tamotsu; Garnham, Christopher P; Davies, Peter L; Tsuda, Sakae

    2012-06-12

    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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. High-throughput, high-fidelity HLA genotyping with deep sequencing.

    PubMed

    Wang, Chunlin; Krishnakumar, Sujatha; Wilhelmy, Julie; Babrzadeh, Farbod; Stepanyan, Lilit; Su, Laura F; Levinson, Douglas; Fernandez-Viña, Marcelo A; Davis, Ronald W; Davis, Mark M; Mindrinos, Michael

    2012-05-29

    Human leukocyte antigen (HLA) genes are the most polymorphic in the human genome. They play a pivotal role in the immune response and have been implicated in numerous human pathologies, especially autoimmunity and infectious diseases. Despite their importance, however, they are rarely characterized comprehensively because of the prohibitive cost of standard technologies and the technical challenges of accurately discriminating between these highly related genes and their many allelles. Here we demonstrate a high-resolution, and cost-effective methodology to type HLA genes by sequencing, which combines the advantage of long-range amplification, the power of high-throughput sequencing platforms, and a unique genotyping algorithm. We calibrated our method for HLA-A, -B, -C, and -DRB1 genes with both reference cell lines and clinical samples and identified several previously undescribed alleles with mismatches, insertions, and deletions. We have further demonstrated the utility of this method in a clinical setting by typing five clinical samples in an Illumina MiSeq instrument with a 5-d turnaround. Overall, this technology has the capacity to deliver low-cost, high-throughput, and accurate HLA typing by multiplexing thousands of samples in a single sequencing run, which will enable comprehensive disease-association studies with large cohorts. Furthermore, this approach can also be extended to include other polymorphic genes.

  13. High-throughput, high-fidelity HLA genotyping with deep sequencing

    PubMed Central

    Wang, Chunlin; Krishnakumar, Sujatha; Wilhelmy, Julie; Babrzadeh, Farbod; Stepanyan, Lilit; Su, Laura F.; Levinson, Douglas; Fernandez-Viña, Marcelo A.; Davis, Ronald W.; Davis, Mark M.; Mindrinos, Michael

    2012-01-01

    Human leukocyte antigen (HLA) genes are the most polymorphic in the human genome. They play a pivotal role in the immune response and have been implicated in numerous human pathologies, especially autoimmunity and infectious diseases. Despite their importance, however, they are rarely characterized comprehensively because of the prohibitive cost of standard technologies and the technical challenges of accurately discriminating between these highly related genes and their many allelles. Here we demonstrate a high-resolution, and cost-effective methodology to type HLA genes by sequencing, which combines the advantage of long-range amplification, the power of high-throughput sequencing platforms, and a unique genotyping algorithm. We calibrated our method for HLA-A, -B, -C, and -DRB1 genes with both reference cell lines and clinical samples and identified several previously undescribed alleles with mismatches, insertions, and deletions. We have further demonstrated the utility of this method in a clinical setting by typing five clinical samples in an Illumina MiSeq instrument with a 5-d turnaround. Overall, this technology has the capacity to deliver low-cost, high-throughput, and accurate HLA typing by multiplexing thousands of samples in a single sequencing run, which will enable comprehensive disease-association studies with large cohorts. Furthermore, this approach can also be extended to include other polymorphic genes. PMID:22589303

  14. Conservation of Plastid Sequences in the Plant Nuclear Genome for Millions of Years Facilitates Endosymbiotic Evolution1[W][OA

    PubMed Central

    Rousseau-Gueutin, Mathieu; Ayliffe, Michael A.; Timmis, Jeremy N.

    2011-01-01

    The nuclear genome of eukaryotes contains large amounts of cytoplasmic organelle DNA (nuclear integrants of organelle DNA [norgs]). The recent sequencing of many mitochondrial and chloroplast genomes has enabled investigation of the potential role of norgs in endosymbiotic evolution. In this article, we describe a new polymerase chain reaction-based method that allows the identification and evolutionary study of recent and older norgs in a range of eukaryotes. We tested this method in the genus Nicotiana and obtained sequences from seven nuclear integrants of plastid DNA (nupts) totaling 25 kb in length. These nupts were estimated to have been transferred 0.033 to 5.81 million years ago. The spectrum of mutations present in the potential protein-coding sequences compared with the noncoding sequences of each nupt revealed that nupts evolve in a nuclear-specific manner and are under neutral evolution. Indels were more frequent in noncoding regions than in potential coding sequences of former chloroplastic DNA, most probably due to the presence of a higher number of homopolymeric sequences. Unexpectedly, some potential protein-coding sequences within the nupts still contained intact open reading frames for up to 5.81 million years. These results suggest that chloroplast genes transferred to the nucleus have in some cases several millions of years to acquire nuclear regulatory elements and become functional. The different factors influencing this time frame and the potential role of nupts in endosymbiotic gene transfer are discussed. PMID:22034627

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. The Effects of Stimulus Familiarity on Conservation-Type Responses to Tone Sequences: A Cross Cultural Study.

    ERIC Educational Resources Information Center

    Hargreaves, David J.; And Others

    1986-01-01

    Compares the performances of equivalent groups of six- and eight- year-olds in the United Kingdom and the United States on pitch transposition and rhythmic inversion music conservation tasks. Results showed older students outperformed younger. Maintains Piagetian explanations of results are inadequate. (Author/JDH)

  3. Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing

    PubMed Central

    2012-01-01

    Background MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression. MiRNAs play essential roles in almost all plant biological processes. Currently, few miRNAs have been identified in the model food legume Phaseolus vulgaris (common bean). Recent advances in next generation sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used Illumina's sequencing by synthesis (SBS) technology to identify and characterize the miRNA population of Phaseolus vulgaris. Results Small RNA libraries were generated from roots, flowers, leaves, and seedlings of P. vulgaris. Based on similarity to previously reported plant miRNAs,114 miRNAs belonging to 33 conserved miRNA families were identified. Stem-loop precursors and target gene sequences for several conserved common bean miRNAs were determined from publicly available databases. Less conserved miRNA families and species-specific common bean miRNA isoforms were also characterized. Moreover, novel miRNAs based on the small RNAs were found and their potential precursors were predicted. In addition, new target candidates for novel and conserved miRNAs were proposed. Finally, we studied organ-specific miRNA family expression levels through miRNA read frequencies. Conclusions This work represents the first massive-scale RNA sequencing study performed in Phaseolus vulgaris to identify and characterize its miRNA population. It significantly increases the number of miRNAs, precursors, and targets identified in this agronomically important species. The miRNA expression analysis provides a foundation for understanding common bean miRNA organ-specific expression patterns. The present study offers an expanded picture of P. vulgaris miRNAs in relation to those of other legumes. PMID:22394504

  4. 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.

  5. 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

  6. 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…

  7. 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

  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. Identification of microRNAs in PCV2 subclinically infected pigs by high throughput sequencing.

    PubMed

    Núñez-Hernández, Fernando; Pérez, Lester J; Muñoz, Marta; Vera, Gonzalo; Tomás, Anna; Egea, Raquel; Córdoba, Sarai; Segalés, Joaquim; Sánchez, Armand; Núñez, José I

    2015-01-01

    Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of PCV2-systemic disease and has been associated with other swine diseases, all of them collectively known as porcine circovirus diseases. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. miRNAs play an increasing role in many biological processes. The study of miRNA-mediated host-pathogen interactions has emerged in the last decade due to the important role that miRNAs play in antiviral defense. The objective of this study was to identify the miRNA expression pattern in PCV2 subclinically infected and non-infected pigs. For this purpose an experimental PCV2 infection was carried out and small-RNA libraries were constructed from tonsil and mediastinal lymph node (MLN) of infected and non-infected pigs. High throughput sequencing determined differences in miRNA expression in MLN between infected and non-infected while, in tonsil, a very conserved pattern was observed. In MLN, miRNA 126-3p, miRNA 126-5p, let-7d-3p, mir-129a and mir-let-7b-3p were up-regulated whereas mir-193a-5p, mir-574-5p and mir-34a down-regulated. Prediction of functional analysis showed that these miRNAs can be involved in pathways related to immune system and in processes related to the pathogenesis of PCV2, although functional assays are needed to support these predictions. This is the first study on miRNA gene expression in pigs infected with PCV2 using a high throughput sequencing approach in which several host miRNAs were differentially expressed in response to PCV2 infection. PMID:25879589

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. Buffalo (Bubalus bubalis) interleukin-2: sequence analysis reveals high nucleotide and amino acid identity with interleukin-2 of cattle and other ruminants.

    PubMed

    Sreekumar, E; Premraj, A; Saravanakumar, M; Rasool, T J

    2002-08-01

    A 4400-bp genomic sequence and a 332-bp truncated cDNA sequence of the interleukin-2 (IL-2) gene of Indian water buffalo (Bubalus bubalis) were amplified by polymerase chain reaction and cloned. The coding sequence of the buffalo IL-2 gene was assembled from the 5' end of the genomic clone and the truncated cDNA clone. This sequence had 98.5% nucleotide identity and 98% amino acid identity with cattle IL-2. Three amino acid substitutions were observed at positions 63, 124 and 135. Comparison of the predicted protein structure of buffalo IL-2 with that of human and cattle IL-2 did not reveal significant differences. The putative amino acids responsible for IL-2 receptor binding were conserved in buffalo, cattle and human IL-2. The amino acid sequence of buffalo IL-2 also showed very high identity with that of other ruminants, indicating functional cross-reactivity.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L.

    PubMed

    Watanabe, M; Ito, A; Takada, Y; Ninomiya, C; Kakizaki, T; Takahata, Y; Hatakeyama, K; Hinata, K; Suzuki, G; Takasaki, T; Satta, Y; Shiba, H; Takayama, S; Isogai, A

    2000-05-12

    Self-incompatibility (SI) enables flowering plants to discriminate between self- and non-self-pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class-I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co-evolution of the genes encoding the male and female determinants.

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. Co-ordinate expression of the two threonyl-tRNA synthetase genes in Bacillus subtilis: control by transcriptional antitermination involving a conserved regulatory sequence.

    PubMed Central

    Putzer, H; Gendron, N; Grunberg-Manago, M

    1992-01-01

    In Bacillus subtilis, two genes, thrS and thrZ, encode distinct threonyl-tRNA synthetase enzymes. Normally, only the thrS gene is expressed. Here we show that either gene, thrS or thrZ, is sufficient for normal cell growth and sporulation. Reducing the intracellular ThrS protein concentration induces thrZ expression in a dose-compensatory manner. Starvation for threonine simultaneously induces thrZ and stimulates thrS expression. The 5'-leader sequences of thrS and thrZ contain, respectively, one and three transcription terminators preceded by a conserved sequence. We show that this sequence is essential for the regulation of thrS via a transcriptional antitermination mechanism. We propose that both genes, thrS and thrZ, are regulated by the same mechanism such that the additional regulatory domains present before thrZ account for its non-expression. In contrast to Escherichia coli, structurally similar regulatory domains, i.e. the consensus sequence preceding a terminator structure, are found in the leader regions of most aminoacyl-tRNA synthetase genes of Gram-positive bacteria. This suggests that they are regulated by a common mechanism. Images PMID:1379177

  9. A comparative analysis of distribution and conservation of microsatellites in the transcripts of sequenced Fusarium species and development of genic-SSR markers for polymorphism analysis.

    PubMed

    Mahfooz, Sahil; Srivastava, Arpita; Srivastava, Alok K; Arora, Dilip K

    2015-09-01

    We used an in silico approach to survey and compare microsatellites in transcript sequences of four sequenced members of genus Fusarium. G + C content of transcripts was found to be positively correlated with the frequency of SSRs. Our analysis revealed that, in all the four transcript sequences studied, the occurrence, relative abundance and density of microsatellites varied and was not influenced by transcript sizes. No correlation between relative abundance and transcript sizes was observed. The relative abundance and density of microsatellites were highest in the transcripts of Fusarium solani when compared with F. graminearum, F. verticillioides and F. oxysporum. The maximum frequency of SSRs among all four sequence sets was of trinucleotide repeats (67.8%), whereas the dinucleotide repeat represents <1%. Among all classes of repeats, 36.5% motifs were found conserved within Fusarium species. In order to study polymorphism within Fusarium isolates, 11 polymorphic genic-SSR markers were developed. Of the 11 markers, 5 were from F. oxysporum and remaining 6 belongs to F. solani. SSR markers from F. oxysporum were found to be more polymorphic (38%) as compared to F. solani (26%). Eleven polymorphic markers obtained in this study clearly demonstrate the utility of newly developed SSR markers in establishing genetic relationships among different isolates of Fusarium.

  10. 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