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Sample records for e7 sequence compared

  1. Comparing triage algorithms using HPV DNA genotyping, HPV E7 mRNA detection and cytology in high-risk HPV DNA-positive women.

    PubMed

    Luttmer, Roosmarijn; Berkhof, Johannes; Dijkstra, Maaike G; van Kemenade, Folkert J; Snijders, Peter J F; Heideman, Daniëlle A M; Meijer, Chris J L M

    2015-06-01

    High-risk human papillomavirus (hrHPV) DNA positive women require triage testing to identify those with high-grade cervical intraepithelial neoplasia or cancer (≥CIN2). Comparing three triage algorithms (1) E7 mRNA testing following HPV16/18/31/33/45/52/58 genotyping (E7 mRNA test), (2) HPV16/18 DNA genotyping and (3) cytology, for ≥CIN2 detection in hrHPV DNA-positive women. hrHPV DNA-positive women aged 18-63 years visiting gynecology outpatient clinics were included in a prospective observational cohort study. From these women a cervical scrape and colposcopy-directed biopsies were obtained. Cervical scrapes were evaluated by cytology, HPV DNA genotyping by bead-based multiplex genotyping of GP5+6+-PCR-products, and presence of HPV16/18/31/33/45/52/58 E7 mRNA using nucleic acid sequence-based amplification (NASBA) in DNA positive women for respective HPV types. Sensitivities and specificities for ≥CIN2 were compared between E7 mRNA test and HPV16/18 DNA genotyping in the total group (n=348), and E7 mRNA test and cytology in a subgroup of women referred for non-cervix-related gynecological complaints (n=133). Sensitivity for ≥CIN2 of the E7 mRNA test was slightly higher than that of HPV16/18 DNA genotyping (66.9% versus 60.9%; ratio 1.10, 95% CI: 1.0002-1.21), at similar specificity (54.8% versus 52.3%; ratio 1.05, 95% CI: 0.93-1.18). Neither sensitivity nor specificity of the E7 mRNA test differed significantly from that of cytology (sensitivity: 68.8% versus 75.0%; ratio 0.92, 95% CI: 0.72-1.17; specificity: 59.4% versus 65.3%; ratio 0.91, 95% CI: 0.75-1.10). For detection of ≥CIN2 in hrHPV DNA-positive women, an algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58 DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Determination of malathion and diazinon resistance by sequencing the Md alpha E7 gene from Guatemala, Colombia, Manhattan, and Thailand housefly (Musca domestica L.) strains.

    PubMed

    Taşkin, Vatan; Kence, Meral; Göçmen, Belgin

    2004-04-01

    Organophosphate (OP) insecticides (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of alpha E7 gene, which is a member of alpha-esterase gene cluster, is probably playing a role in detoxification of the xenobiotic esters. In parathion/diazinon resistant M. domestica species Gly137 to Asp substitution was found in the active center of the product of alpha E7 gene. In malathion (an OP) resistant M. domestica strains Trp251 to Ser substitution was identified in the active center of the Md alpha E7. In our research, to understand the allelic diversity of the Md alpha E7, the gene was partially sequenced from four different housefly strains from different localities (Guatemala, Manhattan (USA), Colombia (USA) and Thailand). It was found out that; in Thailand strain one allele has Cys residue at the position of 251, the other allele contains a Trp for the same site. In Colombia strain, one allele has Asp137, the other allele contains a Gly residue at this point. The Manhattan and Guatemala strains have Asp137 and Trp251 residues on their both alleles at these two different positions.

  3. Sequencing and comparing whole mitochondrial genomes ofanimals

    SciT

    Boore, Jeffrey L.; Macey, J. Robert; Medina, Monica

    2005-04-22

    Comparing complete animal mitochondrial genome sequences is becoming increasingly common for phylogenetic reconstruction and as a model for genome evolution. Not only are they much more informative than shorter sequences of individual genes for inferring evolutionary relatedness, but these data also provide sets of genome-level characters, such as the relative arrangements of genes, that can be especially powerful. We describe here the protocols commonly used for physically isolating mtDNA, for amplifying these by PCR or RCA, for cloning,sequencing, assembly, validation, and gene annotation, and for comparing both sequences and gene arrangements. On several topics, we offer general observations based onmore » our experiences to date with determining and comparing complete mtDNA sequences.« less

  4. Sequence Diversity Diagram for comparative analysis of multiple sequence alignments.

    PubMed

    Sakai, Ryo; Aerts, Jan

    2014-01-01

    The sequence logo is a graphical representation of a set of aligned sequences, commonly used to depict conservation of amino acid or nucleotide sequences. Although it effectively communicates the amount of information present at every position, this visual representation falls short when the domain task is to compare between two or more sets of aligned sequences. We present a new visual presentation called a Sequence Diversity Diagram and validate our design choices with a case study. Our software was developed using the open-source program called Processing. It loads multiple sequence alignment FASTA files and a configuration file, which can be modified as needed to change the visualization. The redesigned figure improves on the visual comparison of two or more sets, and it additionally encodes information on sequential position conservation. In our case study of the adenylate kinase lid domain, the Sequence Diversity Diagram reveals unexpected patterns and new insights, for example the identification of subgroups within the protein subfamily. Our future work will integrate this visual encoding into interactive visualization tools to support higher level data exploration tasks.

  5. Comparative modeling without implicit sequence alignments.

    PubMed

    Kolinski, Andrzej; Gront, Dominik

    2007-10-01

    The number of known protein sequences is about thousand times larger than the number of experimentally solved 3D structures. For more than half of the protein sequences a close or distant structural analog could be identified. The key starting point in a classical comparative modeling is to generate the best possible sequence alignment with a template or templates. With decreasing sequence similarity, the number of errors in the alignments increases and these errors are the main causes of the decreasing accuracy of the molecular models generated. Here we propose a new approach to comparative modeling, which does not require the implicit alignment - the model building phase explores geometric, evolutionary and physical properties of a template (or templates). The proposed method requires prior identification of a template, although the initial sequence alignment is ignored. The model is built using a very efficient reduced representation search engine CABS to find the best possible superposition of the query protein onto the template represented as a 3D multi-featured scaffold. The criteria used include: sequence similarity, predicted secondary structure consistency, local geometric features and hydrophobicity profile. For more difficult cases, the new method qualitatively outperforms existing schemes of comparative modeling. The algorithm unifies de novo modeling, 3D threading and sequence-based methods. The main idea is general and could be easily combined with other efficient modeling tools as Rosetta, UNRES and others.

  6. Comparative sequence analyses of sixteen reptilian paramyxoviruses

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

    1999-01-01

    Viral genomic RNA of Fer-de-Lance virus (FDLV), a paramyxovirus highly pathogenic for reptiles, was reverse transcribed and cloned. Plasmids with significant sequence similarities to the hemagglutinin-neuraminidase (HN) and polymerase (L) genes of mammalian paramyxoviruses were identified by BLAST search. Partial sequences of the FDLV genes were used to design primers for amplification by nested polymerase chain reaction (PCR) and sequencing of 518-bp L gene and 352-bp HN gene fragments from a collection of 15 previously uncharacterized reptilian paramyxoviruses. Phylogenetic analyses of the partial L and HN sequences produced similar trees in which there were two distinct subgroups of isolates that were supported with maximum bootstrap values, and several intermediate isolates. Within each subgroup the nucleotide divergence values were less than 2.5%, while the divergence between the two subgroups was 20-22%. This indicated that the two subgroups represent distinct virus species containing multiple virus strains. The five intermediate isolates had nucleotide divergence values of 11-20% and may represent additional distinct species. In addition to establishing diversity among reptilian paramyxoviruses, the phylogenetic groupings showed some correlation with geographic location, and clearly demonstrated a low level of host species-specificity within these viruses. Copyright (C) 1999 Elsevier Science B.V.

  7. Comparing Stellar Populations Across the Hubble Sequence

    NASA Astrophysics Data System (ADS)

    Loeffler, Shane; Kaleida, Catherine C.; Parkash, Vaishali

    2015-01-01

    Previous work (Jansen et al., 2000, Taylor et al., 2005) has revealed trends in the optical wavelength radial profiles of galaxies across the Hubble Sequence. Radial profiles offer insight into stellar populations, metallicity, and dust concentrations, aspects which are deeply tied to the individual evolution of a galaxy. The Nearby Field Galaxy Survey (NFGS) provides a sampling of nearby galaxies that spans the range of morphological types, luminosities, and masses. Currently available NFGS data includes optical radial surface profiles and spectra of 196 nearby galaxies. We aim to look for trends in the infrared portion of the spectrum for these galaxies, but find that existing 2MASS data is not sufficiently deep. Herein, we expand the available data for the NGFS galaxy IC1639 deeper into the infrared using new data taken with the Infrared Sideport Imager (ISPI) on the 4-m Blanco Telescope at the Cerro Tololo Inter-American Observatory (CTIO) in Chile. Images taken in J, H, and Ks were reduced using standard IRAF and IDL procedures. Photometric calibrations were completed by using the highest quality (AAA) 2MASS stars in the field. Aperture photometry was then performed on the galaxy and radial profiles of surface brightness, J-H color, and H-Ks color were produced. For IC1639, the new ISPI data reveals flat color gradients and surface brightness gradients that decrease with radius. These trends reveal an archetypal elliptical galaxy, with a relatively homogeneous stellar population, stellar density decreasing with radius, and little-to-no obscuration by dust. We have obtained ISPI images for an additional 8 galaxies, and further reduction and analysis of these data will allow for investigation of radial trends in the infrared for galaxies across the Hubble Sequence.

  8. Swallow Event Sequencing: Comparing Healthy Older and Younger Adults.

    PubMed

    Herzberg, Erica G; Lazarus, Cathy L; Steele, Catriona M; Molfenter, Sonja M

    2018-04-23

    Previous research has established that a great deal of variation exists in the temporal sequence of swallowing events for healthy adults. Yet, the impact of aging on swallow event sequence is not well understood. Kendall et al. (Dysphagia 18(2):85-91, 2003) suggested there are 4 obligatory paired-event sequences in swallowing. We directly compared adherence to these sequences, as well as event latencies, and quantified the percentage of unique sequences in two samples of healthy adults: young (< 45) and old (> 65). The 8 swallowing events that contribute to the sequences were reliably identified from videofluoroscopy in a sample of 23 healthy seniors (10 male, mean age 74.7) and 20 healthy young adults (10 male, mean age 31.5) with no evidence of penetration-aspiration or post-swallow residue. Chi-square analyses compared the proportions of obligatory pairs and unique sequences by age group. Compared to the older subjects, younger subjects had significantly lower adherence to two obligatory sequences: Upper Esophageal Sphincter (UES) opening occurs before (or simultaneous with) the bolus arriving at the UES and UES maximum distention occurs before maximum pharyngeal constriction. The associated latencies were significantly different between age groups as well. Further, significantly fewer unique swallow sequences were observed in the older group (61%) compared with the young (82%) (χ 2  = 31.8; p < 0.001). Our findings suggest that paired swallow event sequences may not be robust across the age continuum and that variation in swallow sequences appears to decrease with aging. These findings provide normative references for comparisons to older individuals with dysphagia.

  9. Initial sequencing and comparative analysis of the mouse genome

    SciT

    Waterston, Robert H.; Lindblad-Toh, Kerstin; Birney, Ewan

    2002-12-15

    The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of themore » genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism.« less

  10. eShadow: A tool for comparing closely related sequences

    SciT

    Ovcharenko, Ivan; Boffelli, Dario; Loots, Gabriela G.

    2004-01-15

    Primate sequence comparisons are difficult to interpret due to the high degree of sequence similarity shared between such closely related species. Recently, a novel method, phylogenetic shadowing, has been pioneered for predicting functional elements in the human genome through the analysis of multiple primate sequence alignments. We have expanded this theoretical approach to create a computational tool, eShadow, for the identification of elements under selective pressure in multiple sequence alignments of closely related genomes, such as in comparisons of human to primate or mouse to rat DNA. This tool integrates two different statistical methods and allows for the dynamic visualizationmore » of the resulting conservation profile. eShadow also includes a versatile optimization module capable of training the underlying Hidden Markov Model to differentially predict functional sequences. This module grants the tool high flexibility in the analysis of multiple sequence alignments and in comparing sequences with different divergence rates. Here, we describe the eShadow comparative tool and its potential uses for analyzing both multiple nucleotide and protein alignments to predict putative functional elements. The eShadow tool is publicly available at http://eshadow.dcode.org/« less

  11. Initial sequence and comparative analysis of the cat genome

    PubMed Central

    Pontius, Joan U.; Mullikin, James C.; Smith, Douglas R.; Lindblad-Toh, Kerstin; Gnerre, Sante; Clamp, Michele; Chang, Jean; Stephens, Robert; Neelam, Beena; Volfovsky, Natalia; Schäffer, Alejandro A.; Agarwala, Richa; Narfström, Kristina; Murphy, William J.; Giger, Urs; Roca, Alfred L.; Antunes, Agostinho; Menotti-Raymond, Marilyn; Yuhki, Naoya; Pecon-Slattery, Jill; Johnson, Warren E.; Bourque, Guillaume; Tesler, Glenn; O’Brien, Stephen J.

    2007-01-01

    The genome sequence (1.9-fold coverage) of an inbred Abyssinian domestic cat was assembled, mapped, and annotated with a comparative approach that involved cross-reference to annotated genome assemblies of six mammals (human, chimpanzee, mouse, rat, dog, and cow). The results resolved chromosomal positions for 663,480 contigs, 20,285 putative feline gene orthologs, and 133,499 conserved sequence blocks (CSBs). Additional annotated features include repetitive elements, endogenous retroviral sequences, nuclear mitochondrial (numt) sequences, micro-RNAs, and evolutionary breakpoints that suggest historic balancing of translocation and inversion incidences in distinct mammalian lineages. Large numbers of single nucleotide polymorphisms (SNPs), deletion insertion polymorphisms (DIPs), and short tandem repeats (STRs), suitable for linkage or association studies were characterized in the context of long stretches of chromosome homozygosity. In spite of the light coverage capturing ∼65% of euchromatin sequence from the cat genome, these comparative insights shed new light on the tempo and mode of gene/genome evolution in mammals, promise several research applications for the cat, and also illustrate that a comparative approach using more deeply covered mammals provides an informative, preliminary annotation of a light (1.9-fold) coverage mammal genome sequence. PMID:17975172

  12. Sequencing and comparative analyses of the genomes of zoysiagrasses.

    PubMed

    Tanaka, Hidenori; Hirakawa, Hideki; Kosugi, Shunichi; Nakayama, Shinobu; Ono, Akiko; Watanabe, Akiko; Hashiguchi, Masatsugu; Gondo, Takahiro; Ishigaki, Genki; Muguerza, Melody; Shimizu, Katsuya; Sawamura, Noriko; Inoue, Takayasu; Shigeki, Yuichi; Ohno, Naoki; Tabata, Satoshi; Akashi, Ryo; Sato, Shusei

    2016-04-01

    Zoysiais a warm-season turfgrass, which comprises 11 allotetraploid species (2n= 4x= 40), each possessing different morphological and physiological traits. To characterize the genetic systems of Zoysia plants and to analyse their structural and functional differences in individual species and accessions, we sequenced the genomes of Zoysia species using HiSeq and MiSeq platforms. As a reference sequence of Zoysia species, we generated a high-quality draft sequence of the genome of Z. japonica accession 'Nagirizaki' (334 Mb) in which 59,271 protein-coding genes were predicted. In parallel, draft genome sequences of Z. matrella 'Wakaba' and Z. pacifica 'Zanpa' were also generated for comparative analyses. To investigate the genetic diversity among the Zoysia species, genome sequence reads of three additional accessions, Z. japonica'Kyoto', Z. japonica'Miyagi' and Z. matrella'Chiba Fair Green', were accumulated, and aligned against the reference genome of 'Nagirizaki' along with those from 'Wakaba' and 'Zanpa'. As a result, we detected 7,424,163 single-nucleotide polymorphisms and 852,488 short indels among these species. The information obtained in this study will be valuable for basic studies on zoysiagrass evolution and genetics as well as for the breeding of zoysiagrasses, and is made available in the 'Zoysia Genome Database' at http://zoysia.kazusa.or.jp. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  13. Sequencing and comparative analyses of the genomes of zoysiagrasses

    PubMed Central

    Tanaka, Hidenori; Hirakawa, Hideki; Kosugi, Shunichi; Nakayama, Shinobu; Ono, Akiko; Watanabe, Akiko; Hashiguchi, Masatsugu; Gondo, Takahiro; Ishigaki, Genki; Muguerza, Melody; Shimizu, Katsuya; Sawamura, Noriko; Inoue, Takayasu; Shigeki, Yuichi; Ohno, Naoki; Tabata, Satoshi; Akashi, Ryo; Sato, Shusei

    2016-01-01

    Zoysia is a warm-season turfgrass, which comprises 11 allotetraploid species (2n = 4x = 40), each possessing different morphological and physiological traits. To characterize the genetic systems of Zoysia plants and to analyse their structural and functional differences in individual species and accessions, we sequenced the genomes of Zoysia species using HiSeq and MiSeq platforms. As a reference sequence of Zoysia species, we generated a high-quality draft sequence of the genome of Z. japonica accession ‘Nagirizaki’ (334 Mb) in which 59,271 protein-coding genes were predicted. In parallel, draft genome sequences of Z. matrella ‘Wakaba’ and Z. pacifica ‘Zanpa’ were also generated for comparative analyses. To investigate the genetic diversity among the Zoysia species, genome sequence reads of three additional accessions, Z. japonica ‘Kyoto’, Z. japonica ‘Miyagi’ and Z. matrella ‘Chiba Fair Green’, were accumulated, and aligned against the reference genome of ‘Nagirizaki’ along with those from ‘Wakaba’ and ‘Zanpa’. As a result, we detected 7,424,163 single-nucleotide polymorphisms and 852,488 short indels among these species. The information obtained in this study will be valuable for basic studies on zoysiagrass evolution and genetics as well as for the breeding of zoysiagrasses, and is made available in the ‘Zoysia Genome Database’ at http://zoysia.kazusa.or.jp. PMID:26975196

  14. Isolation and characterization of an RNA aptamer for the HPV-16 E7 oncoprotein.

    PubMed

    Toscano-Garibay, Julia D; Benítez-Hess, María L; Alvarez-Salas, Luis M

    2011-02-01

    Cervical cancer is a common neoplastic disease affecting women worldwide. Expression of human papillomavirus type 16 (HPV-16) E6/E7 genes is frequently associated with cervical cancer, representing ideal targets for diagnostic and therapeutic strategies. Aptamers are oligonucleotide ligands capable of binding with high affinity and specificity to relevant markers in therapeutics and disease detection. The aim of the study was to isolate an RNA aptamer specific for the HPV-16 E7 protein. Aptamers were selected from a randomized oligonucleotide library using a modified SELEX method and recombinant HPV-16 E7 protein. Isolated aptamers were cloned and sequenced for in silico analysis. Interaction and electromobility shift assays (EMSA) were performed to establish aptamer specificity and affinity for E7. RNase footprinting and serial deletions of the aptamer and the E7 protein were made to characterize the aptamer-protein complex. Sandwich slot-blot assays were used for K(D) determination. After several rounds of SELEX, an aptamer (G5α3N.4) exhibited specificity for E7 using cell-free and protein extracts. G5α3N.4 binding yielded a K(D) comparable to aptamers directed to other small targets. Enzymatic and genetic analysis of G5α3N.4 binding showed a secondary structure with two stem-loop domains joined by single-stranded region contacting E7 in a clamp-like manner. The G5α3N.4 aptamer also produced specific complexes in HPV-positive cervical carcinoma cells. The affinity and specificity of G5α3N.4 binding domains for the HPV-16 E7 protein may be used for the detection of papillomavirus infection and cervical cancer. Copyright © 2011 IMSS. Published by Elsevier Inc. All rights reserved.

  15. Statistical method to compare massive parallel sequencing pipelines.

    PubMed

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

    2017-03-01

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

  16. Comparative Analysis of Genome Sequences Covering the Seven Cronobacter Species

    PubMed Central

    Cummings, Craig A.; Shih, Rita; Degoricija, Lovorka; Rico, Alain; Brzoska, Pius; Hamby, Stephen E.; Masood, Naqash; Hariri, Sumyya; Sonbol, Hana; Chuzhanova, Nadia; McClelland, Michael; Furtado, Manohar R.; Forsythe, Stephen J.

    2012-01-01

    Background Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Clinical cases are associated with three species, C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. Thus, it is plausible that virulence determinants have evolved in certain lineages. Methodology/Principal Findings We generated high quality sequence drafts for eleven Cronobacter genomes representing the seven Cronobacter species, including an ST4 strain of C. sakazakii. Comparative analysis of these genomes together with the two publicly available genomes revealed Cronobacter has over 6,000 genes in one or more strains and over 2,000 genes shared by all Cronobacter. Considerable variation in the presence of traits such as type six secretion systems, metal resistance (tellurite, copper and silver), and adhesins were found. C. sakazakii is unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have clinical significance. The C. sakazakii ST4 strain 701 contained additional genes as compared to other C. sakazakii but none of them were known specific virulence-related genes. Conclusions/Significance Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species, and also suggested various degrees of divergence. Sets of universal core genes and accessory genes unique to each strain were identified. These gene sequences can be used for designing genus/species specific detection assays. Genes encoding adhesins, T6SS, and metal resistance genes as well as prophages are found in only subsets of genomes and have contributed considerably to the variation of

  17. Dominant Role of HPV16 E7 in Anal Carcinogenesis

    PubMed Central

    Thomas, Marie K.; Pitot, Henry C.; Liem, Amy; Lambert, Paul F.

    2011-01-01

    Ninety percent of anal cancer is associated with human papilloma viruses (HPVs). Using our previously established HPV transgenic mouse model for anal cancer, we tested the role of the individual oncogenes E6 and E7. K14E6 and K14E7 transgenic mice were treated with dimethylbenz[a]anthracene (DMBA) to the anal canal and compared to matched nontransgenic and doubly transgenic K14E6/E7 mice. K14E7 and K14E6/E7 transgenic mice developed anal tumors (papillomas, atypias and carcinomas combined) at significantly higher rates (88% and 100%, respectively) than either K14E6 or NTG mice (18% and 19%, respectively). Likewise, K14E7 and K14E6/E7 transgenic mice developed frank cancer (carcinomas) at significantly higher rates (85% and 85%, respectively) than either K14E6 or NTG mice (18% and 10%, respectively). These findings indicate that E7 is the more potent oncogene in anal cancer caused by HPVs. PMID:21999991

  18. Dominant role of HPV16 E7 in anal carcinogenesis.

    PubMed

    Thomas, Marie K; Pitot, Henry C; Liem, Amy; Lambert, Paul F

    2011-12-20

    Ninety percent of anal cancer is associated with human papilloma viruses (HPVs). Using our previously established HPV transgenic mouse model for anal cancer, we tested the role of the individual oncogenes E6 and E7. K14E6 and K14E7 transgenic mice were treated with dimethylbenz[a]anthracene (DMBA) to the anal canal and compared to matched nontransgenic and doubly transgenic K14E6/E7 mice. K14E7 and K14E6/E7 transgenic mice developed anal tumors (papillomas, atypias and carcinomas combined) at significantly higher rates (88% and 100%, respectively) than either K14E6 or NTG mice (18% and 19%, respectively). Likewise, K14E7 and K14E6/E7 transgenic mice developed frank cancer (carcinomas) at significantly higher rates (85% and 85%, respectively) than either K14E6 or NTG mice (18% and 10%, respectively). These findings indicate that E7 is the more potent oncogene in anal cancer caused by HPVs. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Low-pass sequencing for microbial comparative genomics

    PubMed Central

    Goo, Young Ah; Roach, Jared; Glusman, Gustavo; Baliga, Nitin S; Deutsch, Kerry; Pan, Min; Kennedy, Sean; DasSarma, Shiladitya; Victor Ng, Wailap; Hood, Leroy

    2004-01-01

    Background We studied four extremely halophilic archaea by low-pass shotgun sequencing: (1) the metabolically versatile Haloarcula marismortui; (2) the non-pigmented Natrialba asiatica; (3) the psychrophile Halorubrum lacusprofundi and (4) the Dead Sea isolate Halobaculum gomorrense. Approximately one thousand single pass genomic sequences per genome were obtained. The data were analyzed by comparative genomic analyses using the completed Halobacterium sp. NRC-1 genome as a reference. Low-pass shotgun sequencing is a simple, inexpensive, and rapid approach that can readily be performed on any cultured microbe. Results As expected, the four archaeal halophiles analyzed exhibit both bacterial and eukaryotic characteristics as well as uniquely archaeal traits. All five halophiles exhibit greater than sixty percent GC content and low isoelectric points (pI) for their predicted proteins. Multiple insertion sequence (IS) elements, often involved in genome rearrangements, were identified in H. lacusprofundi and H. marismortui. The core biological functions that govern cellular and genetic mechanisms of H. sp. NRC-1 appear to be conserved in these four other halophiles. Multiple TATA box binding protein (TBP) and transcription factor IIB (TFB) homologs were identified from most of the four shotgunned halophiles. The reconstructed molecular tree of all five halophiles shows a large divergence between these species, but with the closest relationship being between H. sp. NRC-1 and H. lacusprofundi. Conclusion Despite the diverse habitats of these species, all five halophiles share (1) high GC content and (2) low protein isoelectric points, which are characteristics associated with environmental exposure to UV radiation and hypersalinity, respectively. Identification of multiple IS elements in the genome of H. lacusprofundi and H. marismortui suggest that genome structure and dynamic genome reorganization might be similar to that previously observed in the IS-element rich

  20. Comparative RNA sequencing reveals substantial genetic variation in endangered primates

    PubMed Central

    Perry, George H.; Melsted, Páll; Marioni, John C.; Wang, Ying; Bainer, Russell; Pickrell, Joseph K.; Michelini, Katelyn; Zehr, Sarah; Yoder, Anne D.; Stephens, Matthew; Pritchard, Jonathan K.; Gilad, Yoav

    2012-01-01

    Comparative genomic studies in primates have yielded important insights into the evolutionary forces that shape genetic diversity and revealed the likely genetic basis for certain species-specific adaptations. To date, however, these studies have focused on only a small number of species. For the majority of nonhuman primates, including some of the most critically endangered, genome-level data are not yet available. In this study, we have taken the first steps toward addressing this gap by sequencing RNA from the livers of multiple individuals from each of 16 mammalian species, including humans and 11 nonhuman primates. Of the nonhuman primate species, five are lemurs and two are lorisoids, for which little or no genomic data were previously available. To analyze these data, we developed a method for de novo assembly and alignment of orthologous gene sequences across species. We assembled an average of 5721 gene sequences per species and characterized diversity and divergence of both gene sequences and gene expression levels. We identified patterns of variation that are consistent with the action of positive or directional selection, including an 18-fold enrichment of peroxisomal genes among genes whose regulation likely evolved under directional selection in the ancestral primate lineage. Importantly, we found no relationship between genetic diversity and endangered status, with the two most endangered species in our study, the black and white ruffed lemur and the Coquerel's sifaka, having the highest genetic diversity among all primates. Our observations imply that many endangered lemur populations still harbor considerable genetic variation. Timely efforts to conserve these species alongside their habitats have, therefore, strong potential to achieve long-term success. PMID:22207615

  1. CloVR-Comparative: automated, cloud-enabled comparative microbial genome sequence analysis pipeline.

    PubMed

    Agrawal, Sonia; Arze, Cesar; Adkins, Ricky S; Crabtree, Jonathan; Riley, David; Vangala, Mahesh; Galens, Kevin; Fraser, Claire M; Tettelin, Hervé; White, Owen; Angiuoli, Samuel V; Mahurkar, Anup; Fricke, W Florian

    2017-04-27

    The benefit of increasing genomic sequence data to the scientific community depends on easy-to-use, scalable bioinformatics support. CloVR-Comparative combines commonly used bioinformatics tools into an intuitive, automated, and cloud-enabled analysis pipeline for comparative microbial genomics. CloVR-Comparative runs on annotated complete or draft genome sequences that are uploaded by the user or selected via a taxonomic tree-based user interface and downloaded from NCBI. CloVR-Comparative runs reference-free multiple whole-genome alignments to determine unique, shared and core coding sequences (CDSs) and single nucleotide polymorphisms (SNPs). Output includes short summary reports and detailed text-based results files, graphical visualizations (phylogenetic trees, circular figures), and a database file linked to the Sybil comparative genome browser. Data up- and download, pipeline configuration and monitoring, and access to Sybil are managed through CloVR-Comparative web interface. CloVR-Comparative and Sybil are distributed as part of the CloVR virtual appliance, which runs on local computers or the Amazon EC2 cloud. Representative datasets (e.g. 40 draft and complete Escherichia coli genomes) are processed in <36 h on a local desktop or at a cost of <$20 on EC2. CloVR-Comparative allows anybody with Internet access to run comparative genomics projects, while eliminating the need for on-site computational resources and expertise.

  2. E6 and E7 Gene Polymorphisms in Human Papillomavirus Types-58 and 33 Identified in Southwest China

    PubMed Central

    Wen, Qiang; Wang, Tao; Mu, Xuemei; Chenzhang, Yuwei; Cao, Man

    2017-01-01

    Cancer of the cervix is associated with infection by certain types of human papillomavirus (HPV). The gene variants differ in immune responses and oncogenic potential. The E6 and E7 proteins encoded by high-risk HPV play a key role in cellular transformation. HPV-33 and HPV-58 types are highly prevalent among Chinese women. To study the gene intratypic variations, polymorphisms and positive selections of HPV-33 and HPV-58 E6/E7 in southwest China, HPV-33 (E6, E7: n = 216) and HPV-58 (E6, E7: n = 405) E6 and E7 genes were sequenced and compared to others submitted to GenBank. Phylogenetic trees were constructed by Maximum-likelihood and the Kimura 2-parameters methods by MEGA 6 (Molecular Evolutionary Genetics Analysis version 6.0). The diversity of secondary structure was analyzed by PSIPred software. The selection pressures acting on the E6/E7 genes were estimated by PAML 4.8 (Phylogenetic Analyses by Maximun Likelihood version4.8) software. The positive sites of HPV-33 and HPV-58 E6/E7 were contrasted by ClustalX 2.1. Among 216 HPV-33 E6 sequences, 8 single nucleotide mutations were observed with 6/8 non-synonymous and 2/8 synonymous mutations. The 216 HPV-33 E7 sequences showed 3 single nucleotide mutations that were non-synonymous. The 405 HPV-58 E6 sequences revealed 8 single nucleotide mutations with 4/8 non-synonymous and 4/8 synonymous mutations. Among 405 HPV-58 E7 sequences, 13 single nucleotide mutations were observed with 10/13 non-synonymous mutations and 3/13 synonymous mutations. The selective pressure analysis showed that all HPV-33 and 4/6 HPV-58 E6/E7 major non-synonymous mutations were sites of positive selection. All variations were observed in sites belonging to major histocompatibility complex and/or B-cell predicted epitopes. K93N and R145 (I/N) were observed in both HPV-33 and HPV-58 E6. PMID:28141822

  3. Monitoring HPV-16 E7 phosphorylation events

    SciT

    Nogueira, Marcela O.; Hošek, Tomáš; Calçada, Eduar

    HPV-16 E7 is one of the key proteins that, by interfering with the host metabolism through many protein-protein interactions, hijacks cell regulation and contributes to malignancy. Here we report the high resolution investigation of the CR3 region of HPV-16 E7, both as an isolated domain and in the full-length protein. This opens the way to the atomic level study of the many interactions in which HPV-16 E7 is involved. Along these lines we show here the effect of one of the key post-translational modifications of HPV-16 E7, the phosphorylation by casein kinase II.

  4. International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons

    PubMed Central

    Olson, Nathan D.; Lund, Steven P.; Zook, Justin M.; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S.; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B.

    2015-01-01

    This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing®, or Ion Torrent PGM®. The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies. PMID:27077030

  5. Tinkertoys for the E 7 theory

    NASA Astrophysics Data System (ADS)

    Chacaltana, Oscar; Distler, Jacques; Trimm, Anderson; Zhu, Yinan

    2018-05-01

    We classify the class S theories of type E 7. These are four-dimensional N=2 superconformal field theories arising from the compactification of the E 7 (2, 0) theory on a punctured Riemann surface, C. The classification is given by listing all 3-punctured spheres ("fixtures"), and connecting cylinders, which can arise in a pants-decomposition of C. We find exactly 11,000 fixtures with three regular punctures, and an additional 48 with one "irregular puncture" (in the sense used in our previous works). To organize this large number of theories, we have created a web application at https://golem.ph.utexas.edu/class-S/E7/. Among these theories, we find 10 new ones with a simple exceptional global symmetry group, as well as a new rank-2 SCFT and several new rank-3 SCFTs. As an application, we study the strong-coupling limit of the E 7 gauge theory with 3 hypermultiplets in the 56. Using our results, we also verify recent conjectures that the T 2 compactification of certain 6 d (1, 0) theories can alternatively be realized in class S as fixtures in the E 7 or E 8 theories.

  6. [Comparative results of preimplantation genetic screening by array comparative genomic hybridization and new-generation sequencing].

    PubMed

    Aleksandrova, N V; Shubina, E S; Ekimov, A N; Kodyleva, T A; Mukosey, I S; Makarova, N P; Kulakova, E V; Levkov, L A; Barkov, I Yu; Trofimov, D Yu; Sukhikh, G T

    2017-01-01

    Aneuploidies as quantitative chromosome abnormalities are a main cause of failed development of morphologically normal embryos, implantation failures, and early reproductive losses. Preimplantation genetic screening (PGS) allows a preselection of embryos with a normal karyotype, thus increasing the implantation rate and reducing the frequency of early pregnancy loss after IVF. Modern PGS technologies are based on a genome-wide analysis of the embryo. The first pilot study in Russia was performed to assess the possibility of using semiconductor new-generation sequencing (NGS) as a PGS method. NGS data were collected for 38 biopsied embryos and compared with the data from array comparative genomic hybridization (array-CGH). The concordance between the NGS and array-CGH data was 94.8%. Two samples showed the karyotype 47,XXY by array-CGH and a normal karyotype by NGS. The discrepancies may be explained by loss of efficiency of array-CGH amplicon labeling.

  7. Sequencing and comparative genomic analysis of 1227 Felis catus cDNA sequences enriched for developmental, clinical and nutritional phenotypes

    PubMed Central

    2012-01-01

    Background The feline genome is valuable to the veterinary and model organism genomics communities because the cat is an obligate carnivore and a model for endangered felids. The initial public release of the Felis catus genome assembly provided a framework for investigating the genomic basis of feline biology. However, the entire set of protein coding genes has not been elucidated. Results We identified and characterized 1227 protein coding feline sequences, of which 913 map to public sequences and 314 are novel. These sequences have been deposited into NCBI's genbank database and complement public genomic resources by providing additional protein coding sequences that fill in some of the gaps in the feline genome assembly. Through functional and comparative genomic analyses, we gained an understanding of the role of these sequences in feline development, nutrition and health. Specifically, we identified 104 orthologs of human genes associated with Mendelian disorders. We detected negative selection within sequences with gene ontology annotations associated with intracellular trafficking, cytoskeleton and muscle functions. We detected relatively less negative selection on protein sequences encoding extracellular networks, apoptotic pathways and mitochondrial gene ontology annotations. Additionally, we characterized feline cDNA sequences that have mouse orthologs associated with clinical, nutritional and developmental phenotypes. Together, this analysis provides an overview of the value of our cDNA sequences and enhances our understanding of how the feline genome is similar to, and different from other mammalian genomes. Conclusions The cDNA sequences reported here expand existing feline genomic resources by providing high-quality sequences annotated with comparative genomic information providing functional, clinical, nutritional and orthologous gene information. PMID:22257742

  8. Comparative Analysis of Single-Cell RNA Sequencing Methods.

    PubMed

    Ziegenhain, Christoph; Vieth, Beate; Parekh, Swati; Reinius, Björn; Guillaumet-Adkins, Amy; Smets, Martha; Leonhardt, Heinrich; Heyn, Holger; Hellmann, Ines; Enard, Wolfgang

    2017-02-16

    Single-cell RNA sequencing (scRNA-seq) offers new possibilities to address biological and medical questions. However, systematic comparisons of the performance of diverse scRNA-seq protocols are lacking. We generated data from 583 mouse embryonic stem cells to evaluate six prominent scRNA-seq methods: CEL-seq2, Drop-seq, MARS-seq, SCRB-seq, Smart-seq, and Smart-seq2. While Smart-seq2 detected the most genes per cell and across cells, CEL-seq2, Drop-seq, MARS-seq, and SCRB-seq quantified mRNA levels with less amplification noise due to the use of unique molecular identifiers (UMIs). Power simulations at different sequencing depths showed that Drop-seq is more cost-efficient for transcriptome quantification of large numbers of cells, while MARS-seq, SCRB-seq, and Smart-seq2 are more efficient when analyzing fewer cells. Our quantitative comparison offers the basis for an informed choice among six prominent scRNA-seq methods, and it provides a framework for benchmarking further improvements of scRNA-seq protocols. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Binocular video ophthalmoscope for simultaneous recording of sequences of the human retina to compare dynamic parameters

    NASA Astrophysics Data System (ADS)

    Tornow, Ralf P.; Milczarek, Aleksandra; Odstrcilik, Jan; Kolar, Radim

    2017-07-01

    A parallel video ophthalmoscope was developed to acquire short video sequences (25 fps, 250 frames) of both eyes simultaneously with exact synchronization. Video sequences were registered off-line to compensate for eye movements. From registered video sequences dynamic parameters like cardiac cycle induced reflection changes and eye movements can be calculated and compared between eyes.

  10. Comparative mapping of human alphoid centromeric sequences in great apes

    SciT

    Archidiacono, N.; Antonacci, R.; Marzella, R.

    1994-09-01

    Metaphase spreads from chimpanzees (Pan troglodytes and Pan paniscus) and gorilla (Gorilla gorilla) have been hybridized in situ with 27 alphoid DNA probes specific for the centromere of human chromosomes, to investigate the evolutionary relationship between centromeric regions of human and great apes. The results showed that most human probes do not recognize their corresponding homologs in great apes. Chromosome X is the only chromosome showing localization consistency in all the four species. Each suprachromosomal family (SCF) exhibits a distinct and peculiar evolutionary history. SCF1 (chromosomes 1, 3, 6, 7, 19, 12, 16) is very heterogeneous: some probes gave intensemore » signals, but always on non-homologous chromosomes; others did not produce any hybridization signal. All probes localized on SCF2 (chromosomes 2, 4, 8, 9, 13, 14, 15, 18, 20, 21, and 22) recognize a single chromosome: chromosome 11 (phylogenetic IX) in PTR and PPA; chromosome 4 (phylogenetic V) in GGO. SCF3 subsets (chromosomes 1, 11, 17, X) are substantially conserved in PTR and PPA, but not in GGO, with the exception restricted to chromosome X. No signals have been detected on PPA chromosomes I, III, IV, V, VI and in PTR chromosomes V, suggesting that the centromeric region of some chromsomes have probably lost homology with human alphoid sequences.« less

  11. Comprehensive comparative analysis of 5'-end RNA-sequencing methods.

    PubMed

    Adiconis, Xian; Haber, Adam L; Simmons, Sean K; Levy Moonshine, Ami; Ji, Zhe; Busby, Michele A; Shi, Xi; Jacques, Justin; Lancaster, Madeline A; Pan, Jen Q; Regev, Aviv; Levin, Joshua Z

    2018-06-04

    Specialized RNA-seq methods are required to identify the 5' ends of transcripts, which are critical for studies of gene regulation, but these methods have not been systematically benchmarked. We directly compared six such methods, including the performance of five methods on a single human cellular RNA sample and a new spike-in RNA assay that helps circumvent challenges resulting from uncertainties in annotation and RNA processing. We found that the 'cap analysis of gene expression' (CAGE) method performed best for mRNA and that most of its unannotated peaks were supported by evidence from other genomic methods. We applied CAGE to eight brain-related samples and determined sample-specific transcription start site (TSS) usage, as well as a transcriptome-wide shift in TSS usage between fetal and adult brain.

  12. Genetic variability in E6, E7 and L1 genes of Human Papillomavirus 62 and its prevalence in Mexico.

    PubMed

    Artaza-Irigaray, Cristina; Flores-Miramontes, María Guadalupe; Olszewski, Dominik; Magaña-Torres, María Teresa; López-Cardona, María Guadalupe; Leal-Herrera, Yelda Aurora; Piña-Sánchez, Patricia; Jave-Suárez, Luis Felipe; Aguilar-Lemarroy, Adriana

    2017-01-01

    Human papillomavirus (HPV) is the main etiological agent of cervical cancer, the third most common cancer among women globally and the second most frequent in Mexico. Persistent infection with high-risk HPV genotypes is associated with premalignant lesions and cervical cancer development. HPVs considered as low risk or not yet classified, are often found in coinfection with different HPV genotypes. Indeed, HPV62 is one of the most prevalent HPV detected in some countries, but there is limited information about its prevalence in other regions and there are no HPV62 variants currently described. The aim of this study was to determine the prevalence of HPV62 in cervical samples from Mexican women and to identify mutations in the L1, E6 and E7 genes, which have never been reported in our population. HPV screening was performed by Cobas HPV Test in women who attended prevention health programs and dysplasia clinics. All HPV positive samples ( n  = 491) and 87 additional cervical cancer samples were then genotyped with Linear Array HPV Genotyping test. Some samples were selected to corroborate genotyping by Next-Generation sequencing. On the other hand, nucleotide changes in L1, E6 and E7 genes were determined using PCR, Sanger sequencing and analysis with the CLC-MainWorkbench 7.6.1 software. L1 protein structure was predicted with the I-TASSER server. Using Linear Array, HPV62 prevalence was 7.6% in general population, 8% in Cervical Intraepithelial Neoplasia grade 1 (CIN1) samples and 4.6% in cervical samples. The presence of HPV62 was confirmed with Next-Generation sequencing. Regarding L1 gene, novel sequence variations were detected, but they did not alter the tertiary structure of the protein. Moreover, several nucleotide substitutions were found in E6 and E7 genes compared to reference HPV62 genomic sequence. Specifically, three non-synonymous sequence variations were detected, two in E6 and one in E7. HPV62 is a frequent HPV genotype found mainly in general

  13. Comparative performance of the BGISEQ-500 vs Illumina HiSeq2500 sequencing platforms for palaeogenomic sequencing

    PubMed Central

    Mak, Sarah Siu Tze; Gopalakrishnan, Shyam; Carøe, Christian; Geng, Chunyu; Liu, Shanlin; Sinding, Mikkel-Holger S; Kuderna, Lukas F K; Zhang, Wenwei; Fu, Shujin; Vieira, Filipe G; Germonpré, Mietje; Bocherens, Hervé; Fedorov, Sergey; Petersen, Bent; Sicheritz-Pontén, Thomas; Marques-Bonet, Tomas; Zhang, Guojie; Jiang, Hui; Gilbert, M Thomas P

    2017-01-01

    Abstract Ancient DNA research has been revolutionized following development of next-generation sequencing platforms. Although a number of such platforms have been applied to ancient DNA samples, the Illumina series are the dominant choice today, mainly because of high production capacities and short read production. Recently a potentially attractive alternative platform for palaeogenomic data generation has been developed, the BGISEQ-500, whose sequence output are comparable with the Illumina series. In this study, we modified the standard BGISEQ-500 library preparation specifically for use on degraded DNA, then directly compared the sequencing performance and data quality of the BGISEQ-500 to the Illumina HiSeq2500 platform on DNA extracted from 8 historic and ancient dog and wolf samples. The data generated were largely comparable between sequencing platforms, with no statistically significant difference observed for parameters including level (P = 0.371) and average sequence length (P = 0718) of endogenous nuclear DNA, sequence GC content (P = 0.311), double-stranded DNA damage rate (v. 0.309), and sequence clonality (P = 0.093). Small significant differences were found in single-strand DNA damage rate (δS; slightly lower for the BGISEQ-500, P = 0.011) and the background rate of difference from the reference genome (θ; slightly higher for BGISEQ-500, P = 0.012). This may result from the differences in amplification cycles used to polymerase chain reaction–amplify the libraries. A significant difference was also observed in the mitochondrial DNA percentages recovered (P = 0.018), although we believe this is likely a stochastic effect relating to the extremely low levels of mitochondria that were sequenced from 3 of the samples with overall very low levels of endogenous DNA. Although we acknowledge that our analyses were limited to animal material, our observations suggest that the BGISEQ-500 holds the potential to represent a valid and potentially valuable

  14. E6 and E7 gene silencing results in decreased methylation of tumor suppressor genes and induces phenotype transformation of human cervical carcinoma cell lines

    PubMed Central

    Long, Jia; Shen, Danbei; Zhou, Wuqing; Zhou, Qiyan; Yang, Jia; Jiang, Mingjun

    2015-01-01

    In SiHa and CaSki cells, E6 and E7-targeting shRNA specifically and effectively knocked down human papillomavirus (HPV) 16 E6 and E7 at the transcriptional level, reduced the E6 and E7 mRNA levels by more than 80% compared with control cells that expressed a scrambled-sequence shRNA. E6 and E7 repression resulted in down-regulation of DNA methyltransferase mRNA and protein expression, decreased DNA methylation and increased mRNA expression levels of tumor suppressor genes, induced a certain apoptosis and inhibited proliferation in E6 and E7 shRNA-infected SiHa and CaSki cells compared with the uninfected cells. Repression of E6 and E7 oncogenes resulted in restoration of DNA methyltransferase suppressor pathways and induced apoptosis in HPV16-positive cervical carcinoma cell lines. Our findings suggest that the potential carcinogenic mechanism of HPV16 through influencing DNA methylation pathway to activate the development of cervical cancer exist, and maybe as a candidate therapeutic strategy for cervical and other HPV-associated cancers. PMID:26329329

  15. Whole-genome sequencing for comparative genomics and de novo genome assembly.

    PubMed

    Benjak, Andrej; Sala, Claudia; Hartkoorn, Ruben C

    2015-01-01

    Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as de novo genome assembly following sequencing using Pacific Biosciences (PacBio).

  16. Codon Optimization of the Human Papillomavirus E7 Oncogene Induces a CD8+ T Cell Response to a Cryptic Epitope Not Harbored by Wild-Type E7

    PubMed Central

    Lorenz, Felix K. M.; Wilde, Susanne; Voigt, Katrin; Kieback, Elisa; Mosetter, Barbara; Schendel, Dolores J.; Uckert, Wolfgang

    2015-01-01

    Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine. PMID:25799237

  17. Diagnosis of 25 genotypes of human papillomaviruses for their physical statuses in cervical precancerous/cancerous lesions: a comparison of E2/E6E7 ratio-based vs. multiple E1-L1/E6E7 ratio-based detection techniques.

    PubMed

    Zhang, Rong; He, Yi-feng; Chen, Mo; Chen, Chun-mei; Zhu, Qiu-jing; Lu, Huan; Wei, Zhen-hong; Li, Fang; Zhang, Xiao-xin; Xu, Cong-jian; Yu, Long

    2014-10-02

    Cervical lesions caused by integrated human papillomavirus (HPV) infection are highly dangerous because they can quickly develop into invasive cancers. However, clinicians are currently hampered by the lack of a quick, convenient and precise technique to detect integrated/mixed infections of various genotypes of HPVs in the cervix. This study aimed to develop a practical tool to determine the physical status of different HPVs and evaluate its clinical significance. The target population comprised 1162 women with an HPV infection history of > six months and an abnormal cervical cytological finding. The multiple E1-L1/E6E7 ratio analysis, a novel technique, was developed based on determining the ratios of E1/E6E7, E2/E6E7, E4E5/E6E7, L2/E6E7 and L1/E6E7 within the viral genome. Any imbalanced ratios indicate integration. Its diagnostic and predictive performances were compared with those of E2/E6E7 ratio analysis. The detection accuracy of both techniques was evaluated using the gold-standard technique "detection of integrated papillomavirus sequences" (DIPS). To realize a multigenotypic detection goal, a primer and probe library was established. The integration rate of a particular genotype of HPV was correlated with its tumorigenic potential and women with higher lesion grades often carried lower viral loads. The E1-L1/E6E7 ratio analysis achieved 92.7% sensitivity and 99.0% specificity in detecting HPV integration, while the E2/E6E7 ratio analysis showed a much lower sensitivity (75.6%) and a similar specificity (99.3%). Interference due to episomal copies was observed in both techniques, leading to false-negative results. However, some positive results of E1-L1/E6E7 ratio analysis were missed by DIPS due to its stochastic detection nature. The E1-L1/E6E7 ratio analysis is more efficient than E2/E6E7 ratio analysis and DIPS in predicting precancerous/cancerous lesions, in which both positive predictive values (36.7%-82.3%) and negative predictive values (75

  18. Optimal choice of word length when comparing two Markov sequences using a χ 2-statistic.

    PubMed

    Bai, Xin; Tang, Kujin; Ren, Jie; Waterman, Michael; Sun, Fengzhu

    2017-10-03

    Alignment-free sequence comparison using counts of word patterns (grams, k-tuples) has become an active research topic due to the large amount of sequence data from the new sequencing technologies. Genome sequences are frequently modelled by Markov chains and the likelihood ratio test or the corresponding approximate χ 2 -statistic has been suggested to compare two sequences. However, it is not known how to best choose the word length k in such studies. We develop an optimal strategy to choose k by maximizing the statistical power of detecting differences between two sequences. Let the orders of the Markov chains for the two sequences be r 1 and r 2 , respectively. We show through both simulations and theoretical studies that the optimal k= max(r 1 ,r 2 )+1 for both long sequences and next generation sequencing (NGS) read data. The orders of the Markov chains may be unknown and several methods have been developed to estimate the orders of Markov chains based on both long sequences and NGS reads. We study the power loss of the statistics when the estimated orders are used. It is shown that the power loss is minimal for some of the estimators of the orders of Markov chains. Our studies provide guidelines on choosing the optimal word length for the comparison of Markov sequences.

  19. Comparative analysis of expressed sequence tags of conifers and angiosperms reveals sequences specifically conserved in conifers.

    PubMed

    Ujino-Ihara, Tokuko; Kanamori, Hiroyuki; Yamane, Hiroko; Taguchi, Yuriko; Namiki, Nobukazu; Mukai, Yuzuru; Yoshimura, Kensuke; Tsumura, Yoshihiko

    2005-12-01

    To identify and characterize lineage-specific genes of conifers, two sets of ESTs (with 12791 and 5902 ESTs, representing 5373 and 3018 gene transcripts, respectively) were generated from the Cupressaceae species Cryptomeria japonica and Chamaecyparis obtusa. These transcripts were compared with non-redundant sets of genes generated from Pinaceae species, other gymnosperms and angiosperms. About 6% of tentative unique genes (Unigenes) of C. japonica and C. obtusa had homologs in other conifers but not angiosperms, and about 70% had apparent homologs in angiosperms. The calculated GC contents of orthologous genes showed that GC contents of coniferous genes are likely to be lower than those of angiosperms. Comparisons of the numbers of homologous genes in each species suggest that copy numbers of genes may be correlated between diverse seed plants. This correlation suggests that the multiplicity of such genes may have arisen before the divergence of gymnosperms and angiosperms.

  20. Comparative performance of high-density oligonucleotide sequencing and dideoxynucleotide sequencing of HIV type 1 pol from clinical samples.

    PubMed

    Günthard, H F; Wong, J K; Ignacio, C C; Havlir, D V; Richman, D D

    1998-07-01

    The performance of the high-density oligonucleotide array methodology (GeneChip) in detecting drug resistance mutations in HIV-1 pol was compared with that of automated dideoxynucleotide sequencing (ABI) of clinical samples, viral stocks, and plasmid-derived NL4-3 clones. Sequences from 29 clinical samples (plasma RNA, n = 17; lymph node RNA, n = 5; lymph node DNA, n = 7) from 12 patients, from 6 viral stock RNA samples, and from 13 NL4-3 clones were generated by both methods. Editing was done independently by a different investigator for each method before comparing the sequences. In addition, NL4-3 wild type (WT) and mutants were mixed in varying concentrations and sequenced by both methods. Overall, a concordance of 99.1% was found for a total of 30,865 bases compared. The comparison of clinical samples (plasma RNA and lymph node RNA and DNA) showed a slightly lower match of base calls, 98.8% for 19,831 nucleotides compared (protease region, 99.5%, n = 8272; RT region, 98.3%, n = 11,316), than for viral stocks and NL4-3 clones (protease region, 99.8%; RT region, 99.5%). Artificial mixing experiments showed a bias toward calling wild-type bases by GeneChip. Discordant base calls are most likely due to differential detection of mixtures. The concordance between GeneChip and ABI was high and appeared dependent on the nature of the templates (directly amplified versus cloned) and the complexity of mixes.

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

    PubMed Central

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

    2000-01-01

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

  2. Comparative sequence analysis of Sordaria macrospora and Neurospora crassa as a means to improve genome annotation.

    PubMed

    Nowrousian, Minou; Würtz, Christian; Pöggeler, Stefanie; Kück, Ulrich

    2004-03-01

    One of the most challenging parts of large scale sequencing projects is the identification of functional elements encoded in a genome. Recently, studies of genomes of up to six different Saccharomyces species have demonstrated that a comparative analysis of genome sequences from closely related species is a powerful approach to identify open reading frames and other functional regions within genomes [Science 301 (2003) 71, Nature 423 (2003) 241]. Here, we present a comparison of selected sequences from Sordaria macrospora to their corresponding Neurospora crassa orthologous regions. Our analysis indicates that due to the high degree of sequence similarity and conservation of overall genomic organization, S. macrospora sequence information can be used to simplify the annotation of the N. crassa genome.

  3. Comparative analysis of myostatin gene and promoter sequences of Qinchuan and Red Angus cattle.

    PubMed

    He, Y L; Wu, Y H; Quan, F S; Liu, Y G; Zhang, Y

    2013-09-04

    To better understand the function of the myostatin gene and its promoter region in bovine, we amplified and sequenced the myostatin gene and promoter from the blood of Qinchuan and Red Angus cattle by using polymerase chain reaction. The sequences of Qinchuan and Red Angus cattle were compared with those of other cattle breeds available in GenBank. Exon splice sites were confirmed by mRNA sequencing. Compared to the published sequence (GenBank accession No. AF320998), 69 single nucleotide polymorphisms (SNPs) were identified in the Qinchuan myostatin gene, only one of which was an insertion mutation in Qinchuan cattle. There was a 16-bp insertion in the first 705-bp intron in 3 Qinchuan cattle. A total of 7 SNPs were identified in exon 3, in which the mutation occurred in the third base of the codon and was synonymous. On comparing the Qinchuan myostatin gene sequence to that of Red Angus cattle, a total of 50 SNPs were identified in the first and third exons. In addition, there were 18 SNPs identified in the Qinchuan cattle promoter region compared with those of other cattle compared to the Red Angus cattle myostatin promoter region. breeds (GenBank accession No. AF348479), but only 14 SNPs when compared to the Red Angus cattle myostatin promoter region.

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

    PubMed Central

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

    2005-01-01

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

  5. Plastome Sequence Determination and Comparative Analysis for Members of the Lolium-Festuca Grass Species Complex

    PubMed Central

    Hand, Melanie L.; Spangenberg, German C.; Forster, John W.; Cogan, Noel O. I.

    2013-01-01

    Chloroplast genome sequences are of broad significance in plant biology, due to frequent use in molecular phylogenetics, comparative genomics, population genetics, and genetic modification studies. The present study used a second-generation sequencing approach to determine and assemble the plastid genomes (plastomes) of four representatives from the agriculturally important Lolium-Festuca species complex of pasture grasses (Lolium multiflorum, Festuca pratensis, Festuca altissima, and Festuca ovina). Total cellular DNA was extracted from either roots or leaves, was sequenced, and the output was filtered for plastome-related reads. A comparison between sources revealed fewer plastome-related reads from root-derived template but an increase in incidental bacterium-derived sequences. Plastome assembly and annotation indicated high levels of sequence identity and a conserved organization and gene content between species. However, frequent deletions within the F. ovina plastome appeared to contribute to a smaller plastid genome size. Comparative analysis with complete plastome sequences from other members of the Poaceae confirmed conservation of most grass-specific features. Detailed analysis of the rbcL–psaI intergenic region, however, revealed a “hot-spot” of variation characterized by independent deletion events. The evolutionary implications of this observation are discussed. The complete plastome sequences are anticipated to provide the basis for potential organelle-specific genetic modification of pasture grasses. PMID:23550121

  6. Comparative analysis of complete chloroplast genome sequence and inversion variation in Lasthenia burkei (Madieae, Asteraceae).

    PubMed

    Walker, Joseph F; Zanis, Michael J; Emery, Nancy C

    2014-04-01

    Complete chloroplast genome studies can help resolve relationships among large, complex plant lineages such as Asteraceae. We present the first whole plastome from the Madieae tribe and compare its sequence variation to other chloroplast genomes in Asteraceae. We used high throughput sequencing to obtain the Lasthenia burkei chloroplast genome. We compared sequence structure and rates of molecular evolution in the small single copy (SSC), large single copy (LSC), and inverted repeat (IR) regions to those for eight Asteraceae accessions and one Solanaceae accession. The chloroplast sequence of L. burkei is 150 746 bp and contains 81 unique protein coding genes and 4 coding ribosomal RNA sequences. We identified three major inversions in the L. burkei chloroplast, all of which have been found in other Asteraceae lineages, and a previously unreported inversion in Lactuca sativa. Regions flanking inversions contained tRNA sequences, but did not have particularly high G + C content. Substitution rates varied among the SSC, LSC, and IR regions, and rates of evolution within each region varied among species. Some observed differences in rates of molecular evolution may be explained by the relative proportion of coding to noncoding sequence within regions. Rates of molecular evolution vary substantially within and among chloroplast genomes, and major inversion events may be promoted by the presence of tRNAs. Collectively, these results provide insight into different mechanisms that may promote intramolecular recombination and the inversion of large genomic regions in the plastome.

  7. Improving de novo sequence assembly using machine learning and comparative genomics for overlap correction.

    PubMed

    Palmer, Lance E; Dejori, Mathaeus; Bolanos, Randall; Fasulo, Daniel

    2010-01-15

    With the rapid expansion of DNA sequencing databases, it is now feasible to identify relevant information from prior sequencing projects and completed genomes and apply it to de novo sequencing of new organisms. As an example, this paper demonstrates how such extra information can be used to improve de novo assemblies by augmenting the overlapping step. Finding all pairs of overlapping reads is a key task in many genome assemblers, and to this end, highly efficient algorithms have been developed to find alignments in large collections of sequences. It is well known that due to repeated sequences, many aligned pairs of reads nevertheless do not overlap. But no overlapping algorithm to date takes a rigorous approach to separating aligned but non-overlapping read pairs from true overlaps. We present an approach that extends the Minimus assembler by a data driven step to classify overlaps as true or false prior to contig construction. We trained several different classification models within the Weka framework using various statistics derived from overlaps of reads available from prior sequencing projects. These statistics included percent mismatch and k-mer frequencies within the overlaps as well as a comparative genomics score derived from mapping reads to multiple reference genomes. We show that in real whole-genome sequencing data from the E. coli and S. aureus genomes, by providing a curated set of overlaps to the contigging phase of the assembler, we nearly doubled the median contig length (N50) without sacrificing coverage of the genome or increasing the number of mis-assemblies. Machine learning methods that use comparative and non-comparative features to classify overlaps as true or false can be used to improve the quality of a sequence assembly.

  8. Distribution of human papilloma virus type 16 E6/E7 gene mutation in cervical precancer or cancer: A case control study in Guizhou Province, China.

    PubMed

    Yang, Yingjie; Ren, Jie; Zhang, Qizhu

    2016-02-01

    HPV-16 varies geographically and is correlated with cervical cancer genesis and progression. This study aimed to determine the distribution of HPV-16 E6/E7 genetic variation in patients with invasive cervical cancer or precancer in Guizhou Province, China. A case-control study was designed, and the distribution of HPV-16 E6/E7 genetic variation was compared among women with cervical cancer, precancer, and sexually active without cervical lesion. HPV infection was detected through flow-through hybridization and gene chip techniques to determine the prevalence of HPV 16 E6/E7 genetic variation. Among 90 specimens (30 cervical cancer, 30 precancer, 30 controls), 81 were subjected to HPV-16 E6/E7 gene sequencing. The rates of DNA sequence mutation and amino acid mutation were 76.5% (62/81) and 66.7% (54/81), respectively. Both E6 and E7 genes showed higher mutation rate than their prototypes. The prevalence of E6/E7 mutation significantly differed between the cervical cancer and the controls (P < 0.05) and between the cervical precancer and the controls (P < 0.05). Mutations were simultaneously detected at the E6-D32E (T96A) and E7-M28V (A82G)/L94P (T281C) sites of the amino acid sequence. The most common genetic variation was D32E/M28V/L94P, which accounted for 35.8% of the cases (29/81). D32E/M28V/L94P mutation was higher in the cervical cancer and precancer compared with the prototype. HPV-16 E6/E7 genetic variations, such as D32E/M28V/L94P, are more prevalent in cervical cancer or precancer than those in the controls. The possible correlation between genetic variation and cancerigenesis may be used to design an HPV vaccine for cervical carcinoma. © 2015 Wiley Periodicals, Inc.

  9. Comparative analysis of the prion protein gene sequences in African lion.

    PubMed

    Wu, Chang-De; Pang, Wan-Yong; Zhao, De-Ming

    2006-10-01

    The prion protein gene of African lion (Panthera Leo) was first cloned and polymorphisms screened. The results suggest that the prion protein gene of eight African lions is highly homogenous. The amino acid sequences of the prion protein (PrP) of all samples tested were identical. Four single nucleotide polymorphisms (C42T, C81A, C420T, T600C) in the prion protein gene (Prnp) of African lion were found, but no amino acid substitutions. Sequence analysis showed that the higher homology is observed to felis catus AF003087 (96.7%) and to sheep number M31313.1 (96.2%) Genbank accessed. With respect to all the mammalian prion protein sequences compared, the African lion prion protein sequence has three amino acid substitutions. The homology might in turn affect the potential intermolecular interactions critical for cross species transmission of prion disease.

  10. Sequencing, annotation and comparative analysis of nine BACs of giant panda (Ailuropoda melanoleuca).

    PubMed

    Zheng, Yang; Cai, Jing; Li, JianWen; Li, Bo; Lin, Runmao; Tian, Feng; Wang, XiaoLing; Wang, Jun

    2010-01-01

    A 10-fold BAC library for giant panda was constructed and nine BACs were selected to generate finish sequences. These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of giant panda newly generated by the Illumina GA sequencing technology. Complete sanger sequencing, assembly, annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb. Homologue search and de novo prediction methods were used to annotate genes and repeats. Twelve protein coding genes were predicted, seven of which could be functionally annotated. The seven genes have an average gene size of about 41 kb, an average coding size of about 1.2 kb and an average exon number of 6 per gene. Besides, seven tRNA genes were found. About 27 percent of the BAC sequence is composed of repeats. A phylogenetic tree was constructed using neighbor-join algorithm across five species, including giant panda, human, dog, cat and mouse, which reconfirms dog as the most related species to giant panda. Our results provide detailed sequence and structure information for new genes and repeats of giant panda, which will be helpful for further studies on the giant panda.

  11. Comparing Sanger sequencing and high-throughput metabarcoding for inferring photobiont diversity in lichens.

    PubMed

    Paul, Fiona; Otte, Jürgen; Schmitt, Imke; Dal Grande, Francesco

    2018-06-05

    The implementation of HTS (high-throughput sequencing) approaches is rapidly changing our understanding of the lichen symbiosis, by uncovering high bacterial and fungal diversity, which is often host-specific. Recently, HTS methods revealed the presence of multiple photobionts inside a single thallus in several lichen species. This differs from Sanger technology, which typically yields a single, unambiguous algal sequence per individual. Here we compared HTS and Sanger methods for estimating the diversity of green algal symbionts within lichen thalli using 240 lichen individuals belonging to two species of lichen-forming fungi. According to HTS data, Sanger technology consistently yielded the most abundant photobiont sequence in the sample. However, if the second most abundant photobiont exceeded 30% of the total HTS reads in a sample, Sanger sequencing generally failed. Our results suggest that most lichen individuals in the two analyzed species, Lasallia hispanica and L. pustulata, indeed contain a single, predominant green algal photobiont. We conclude that Sanger sequencing is a valid approach to detect the dominant photobionts in lichen individuals and populations. We discuss which research areas in lichen ecology and evolution will continue to benefit from Sanger sequencing, and which areas will profit from HTS approaches to assessing symbiont diversity.

  12. Genomic Sequence around Butterfly Wing Development Genes: Annotation and Comparative Analysis

    PubMed Central

    Conceição, Inês C.; Long, Anthony D.; Gruber, Jonathan D.; Beldade, Patrícia

    2011-01-01

    Background Analysis of genomic sequence allows characterization of genome content and organization, and access beyond gene-coding regions for identification of functional elements. BAC libraries, where relatively large genomic regions are made readily available, are especially useful for species without a fully sequenced genome and can increase genomic coverage of phylogenetic and biological diversity. For example, no butterfly genome is yet available despite the unique genetic and biological properties of this group, such as diversified wing color patterns. The evolution and development of these patterns is being studied in a few target species, including Bicyclus anynana, where a whole-genome BAC library allows targeted access to large genomic regions. Methodology/Principal Findings We characterize ∼1.3 Mb of genomic sequence around 11 selected genes expressed in B. anynana developing wings. Extensive manual curation of in silico predictions, also making use of a large dataset of expressed genes for this species, identified repetitive elements and protein coding sequence, and highlighted an expansion of Alcohol dehydrogenase genes. Comparative analysis with orthologous regions of the lepidopteran reference genome allowed assessment of conservation of fine-scale synteny (with detection of new inversions and translocations) and of DNA sequence (with detection of high levels of conservation of non-coding regions around some, but not all, developmental genes). Conclusions The general properties and organization of the available B. anynana genomic sequence are similar to the lepidopteran reference, despite the more than 140 MY divergence. Our results lay the groundwork for further studies of new interesting findings in relation to both coding and non-coding sequence: 1) the Alcohol dehydrogenase expansion with higher similarity between the five tandemly-repeated B. anynana paralogs than with the corresponding B. mori orthologs, and 2) the high conservation of non

  13. Comparative transcriptome analysis in Sclerotinia sclerotiorum and S. trifoliorum by 454 Titanium RNA sequencing

    Sclerotinia sclerotiorum and S. trifoliorum are two closely related devastating plant pathogens. Extensive research has been conducted on S. sclerotiorum and its genome sequences are available. To take advantages of the genomic information of S. sclerotiorum, we compared the transcriptome of S. tr...

  14. A Comparative Study of Sequence of Instruction When Introducing Golf Skills to Beginners.

    ERIC Educational Resources Information Center

    Kraft, Robert E.

    Three instructional methods of club sequence for introducing golf skills to beginning golfers were compared: (1) full swing; (2) putter and short approach; and (3) freedom of choice. Sixty-eight male and female college students participated in golf lessons twice weekly for 12 weeks, receiving small group and individual instruction. Two forms of…

  15. Sequence Search and Comparative Genomic Analysis of SUMO-Activating Enzymes Using CoGe.

    PubMed

    Carretero-Paulet, Lorenzo; Albert, Victor A

    2016-01-01

    The growing number of genome sequences completed during the last few years has made necessary the development of bioinformatics tools for the easy access and retrieval of sequence data, as well as for downstream comparative genomic analyses. Some of these are implemented as online platforms that integrate genomic data produced by different genome sequencing initiatives with data mining tools as well as various comparative genomic and evolutionary analysis possibilities.Here, we use the online comparative genomics platform CoGe ( http://www.genomevolution.org/coge/ ) (Lyons and Freeling. Plant J 53:661-673, 2008; Tang and Lyons. Front Plant Sci 3:172, 2012) (1) to retrieve the entire complement of orthologous and paralogous genes belonging to the SUMO-Activating Enzymes 1 (SAE1) gene family from a set of species representative of the Brassicaceae plant eudicot family with genomes fully sequenced, and (2) to investigate the history, timing, and molecular mechanisms of the gene duplications driving the evolutionary expansion and functional diversification of the SAE1 family in Brassicaceae.

  16. Microbiological profile of chicken carcasses: A comparative analysis using shotgun metagenomic sequencing

    PubMed Central

    Cesare, Alessandra De; Palma, Federica; Lucchi, Alex; Pasquali, Frederique; Manfreda, Gerardo

    2018-01-01

    In the last few years metagenomic and 16S rRNA sequencing have completly changed the microbiological investigations of food products. In this preliminary study, the microbiological profile of chicken carcasses collected from animals fed with different diets were tested by using shotgun metagenomic sequencing. A total of 15 carcasses have been collected at the slaughetrhouse at the end of the refrigeration tunnel from chickens reared for 35 days and fed with a control diet (n=5), a diet supplemented with 1500 FTU/kg of commercial phytase (n=5) and a diet supplemented with 1500 FTU/kg of commercial phytase and 3g/kg of inositol (n=5). Ten grams of neck and breast skin were obtained from each carcass and submited to total DNA extraction by using the DNeasy Blood & Tissue Kit (Qiagen). Sequencing libraries have been prepared by using the Nextera XT DNA Library Preparation Kit (Illumina) and sequenced in a HiScanSQ (Illumina) at 100 bp in paired ends. A number of sequences ranging between 5 and 9 million was obtained for each sample. Sequence analysis showed that Proteobacteria and Firmicutes represented more than 98% of whole bacterial populations associated to carcass skin in all groups but their abundances were different between groups. Moraxellaceae and other degradative bacteria showed a significantly higher abundance in the control compared to the treated groups. Furthermore, Clostridium perfringens showed a relative frequency of abundance significantly higher in the group fed with phytase and Salmonella enterica in the group fed with phytase plus inositol. The results of this preliminary study showed that metagenome sequencing is suitable to investigate and monitor carcass microbiota in order to detect specific pathogenic and/or degradative populations. PMID:29732327

  17. Comparative study of IDH1 mutations in gliomas by immunohistochemistry and DNA sequencing.

    PubMed

    Agarwal, Shipra; Sharma, Mehar Chand; Jha, Prerana; Pathak, Pankaj; Suri, Vaishali; Sarkar, Chitra; Chosdol, Kunzang; Suri, Ashish; Kale, Shashank Sharad; Mahapatra, Ashok Kumar; Jha, Pankaj

    2013-06-01

    Mutations involving isocitrate dehydrogenase 1 (IDH 1) occur in a high proportion of diffuse gliomas, with implications on diagnosis and prognosis. About 90% involve exon 4 at codon 132, replacing amino acid arginine with histidine (R132H). Rarer ones include R132C, R132S, R132G, R132L, R132V, and R132P. Most authors have used DNA-based methods to assess IDH1 status. Preliminary studies comparing imunohistochemistry (IHC) with IDH1-R132H mutation-specific antibodies have shown concordance with DNA sequencing and no cross-reactivity with wild-type IDH1 or other mutant proteins. The present study compares results of IHC with DNA sequencing in diffuse gliomas. Fifty diffuse gliomas with frozen tissue samples for DNA sequencing and adequate tissue in paraffin blocks for IHC using IDH1-R132H specific antibody were assessed for IDH1 mutations. Concordance of findings between IHC and DNA sequencing was noted in 88% (44/50) cases. All 6 cases with discrepancy were immunopositive with DIA-H09 antibody. While in 3 of these 6 cases, DNA sequencing failed to reveal any mutations, R132L (arginine replaced by leucine) mutation was found in the rest 3 cases. Interestingly, of the immunopositive cases, 46.6% (14/30) showed immunostaining in only a fraction of tumor cells. IHC is an easy and quick method of detecting IDH1-R132H mutations, but there may be some discrepancies between IHC and DNA sequencing. Although there were no false-negative cases, cross-reactivity with IDH1-R132L was seen in 3, a finding not reported thus far. Because of more universal availability of IHC over genetic testing, cross-reactivity and staining heterogeneity may have bearing over its use in detecting IDH1-R132H mutation in gliomas.

  18. Comparative study of IDH1 mutations in gliomas by immunohistochemistry and DNA sequencing

    PubMed Central

    Agarwal, Shipra; Sharma, Mehar Chand; Jha, Prerana; Pathak, Pankaj; Suri, Vaishali; Sarkar, Chitra; Chosdol, Kunzang; Suri, Ashish; Kale, Shashank Sharad; Mahapatra, Ashok Kumar; Jha, Pankaj

    2013-01-01

    Background Mutations involving isocitrate dehydrogenase 1 (IDH 1) occur in a high proportion of diffuse gliomas, with implications on diagnosis and prognosis. About 90% involve exon 4 at codon 132, replacing amino acid arginine with histidine (R132H). Rarer ones include R132C, R132S, R132G, R132L, R132V, and R132P. Most authors have used DNA-based methods to assess IDH1 status. Preliminary studies comparing imunohistochemistry (IHC) with IDH1-R132H mutation-specific antibodies have shown concordance with DNA sequencing and no cross-reactivity with wild-type IDH1 or other mutant proteins. The present study compares results of IHC with DNA sequencing in diffuse gliomas. Materials and methods Fifty diffuse gliomas with frozen tissue samples for DNA sequencing and adequate tissue in paraffin blocks for IHC using IDH1-R132H specific antibody were assessed for IDH1 mutations. Results Concordance of findings between IHC and DNA sequencing was noted in 88% (44/50) cases. All 6 cases with discrepancy were immunopositive with DIA-H09 antibody. While in 3 of these 6 cases, DNA sequencing failed to reveal any mutations, R132L (arginine replaced by leucine) mutation was found in the rest 3 cases. Interestingly, of the immunopositive cases, 46.6% (14/30) showed immunostaining in only a fraction of tumor cells. Conclusions IHC is an easy and quick method of detecting IDH1-R132H mutations, but there may be some discrepancies between IHC and DNA sequencing. Although there were no false-negative cases, cross-reactivity with IDH1-R132L was seen in 3, a finding not reported thus far. Because of more universal availability of IHC over genetic testing, cross-reactivity and staining heterogeneity may have bearing over its use in detecting IDH1-R132H mutation in gliomas. PMID:23486690

  19. openSputnik--a database to ESTablish comparative plant genomics using unsaturated sequence collections.

    PubMed

    Rudd, Stephen

    2005-01-01

    The public expressed sequence tag collections are continually being enriched with high-quality sequences that represent an ever-expanding range of taxonomically diverse plant species. While these sequence collections provide biased insight into the populations of expressed genes available within individual species and their associated tissues, the information is conceivably of wider relevance in a comparative context. When we consider the available expressed sequence tag (EST) collections of summer 2004, most of the major plant taxonomic clades are at least superficially represented. Investigation of the five million available plant ESTs provides a wealth of information that has applications in modelling the routes of plant genome evolution and the identification of lineage-specific genes and gene families. Over four million ESTs from over 50 distinct plant species have been collated within an EST analysis pipeline called openSputnik. The ESTs were resolved down into approximately one million unigene sequences. These have been annotated using orthology-based annotation transfer from reference plant genomes and using a variety of contemporary bioinformatics methods to assign peptide, structural and functional attributes. The openSputnik database is available at http://sputnik.btk.fi.

  20. Complete chloroplast genome sequence and comparative analysis of loblolly pine (Pinus taeda L.) with related species

    PubMed Central

    Khan, Abdul Latif; Khan, Muhammad Aaqil; Shahzad, Raheem; Lubna; Kang, Sang Mo; Al-Harrasi, Ahmed; Al-Rawahi, Ahmed; Lee, In-Jung

    2018-01-01

    Pinaceae, the largest family of conifers, has a diversified organization of chloroplast (cp) genomes with two typical highly reduced inverted repeats (IRs). In the current study, we determined the complete sequence of the cp genome of an economically and ecologically important conifer tree, the loblolly pine (Pinus taeda L.), using Illumina paired-end sequencing and compared the sequence with those of other pine species. The results revealed a genome size of 121,531 base pairs (bp) containing a pair of 830-bp IR regions, distinguished by a small single copy (42,258 bp) and large single copy (77,614 bp) region. The chloroplast genome of P. taeda encodes 120 genes, comprising 81 protein-coding genes, four ribosomal RNA genes, and 35 tRNA genes, with 151 randomly distributed microsatellites. Approximately 6 palindromic, 34 forward, and 22 tandem repeats were found in the P. taeda cp genome. Whole cp genome comparison with those of other Pinus species exhibited an overall high degree of sequence similarity, with some divergence in intergenic spacers. Higher and lower numbers of indels and single-nucleotide polymorphism substitutions were observed relative to P. contorta and P. monophylla, respectively. Phylogenomic analyses based on the complete genome sequence revealed that 60 shared genes generated trees with the same topologies, and P. taeda was closely related to P. contorta in the subgenus Pinus. Thus, the complete P. taeda genome provided valuable resources for population and evolutionary studies of gymnosperms and can be used to identify related species. PMID:29596414

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

    PubMed Central

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

    2015-01-01

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

  2. Draft sequencing and comparative genomics of Xylella fastidiosa strains reveal novel biological insights.

    PubMed

    Bhattacharyya, Anamitra; Stilwagen, Stephanie; Reznik, Gary; Feil, Helene; Feil, William S; Anderson, Iain; Bernal, Axel; D'Souza, Mark; Ivanova, Natalia; Kapatral, Vinayak; Larsen, Niels; Los, Tamara; Lykidis, Athanasios; Selkov, Eugene; Walunas, Theresa L; Purcell, Alexander; Edwards, Rob A; Hawkins, Trevor; Haselkorn, Robert; Overbeek, Ross; Kyrpides, Nikos C; Predki, Paul F

    2002-10-01

    Draft sequencing is a rapid and efficient method for determining the near-complete sequence of microbial genomes. Here we report a comparative analysis of one complete and two draft genome sequences of the phytopathogenic bacterium, Xylella fastidiosa, which causes serious disease in plants, including citrus, almond, and oleander. We present highlights of an in silico analysis based on a comparison of reconstructions of core biological subsystems. Cellular pathway reconstructions have been used to identify a small number of genes, which are likely to reside within the draft genomes but are not captured in the draft assembly. These represented only a small fraction of all genes and were predominantly large and small ribosomal subunit protein components. By using this approach, some of the inherent limitations of draft sequence can be significantly reduced. Despite the incomplete nature of the draft genomes, it is possible to identify several phage-related genes, which appear to be absent from the draft genomes and not the result of insufficient sequence sampling. This region may therefore identify potential host-specific functions. Based on this first functional reconstruction of a phytopathogenic microbe, we spotlight an unusual respiration machinery as a potential target for biological control. We also predicted and developed a new defined growth medium for Xylella.

  3. N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation Sequence Analysis.

    PubMed

    Chang, Elizabeth; Pourmal, Sergei; Zhou, Chun; Kumar, Rupesh; Teplova, Marianna; Pavletich, Nikola P; Marians, Kenneth J; Erdjument-Bromage, Hediye

    2016-07-01

    In recent history, alternative approaches to Edman sequencing have been investigated, and to this end, the Association of Biomolecular Resource Facilities (ABRF) Protein Sequencing Research Group (PSRG) initiated studies in 2014 and 2015, looking into bottom-up and top-down N-terminal (Nt) dimethyl derivatization of standard quantities of intact proteins with the aim to determine Nt sequence information. We have expanded this initiative and used low picomole amounts of myoglobin to determine the efficiency of Nt-dimethylation. Application of this approach on protein domains, generated by limited proteolysis of overexpressed proteins, confirms that it is a universal labeling technique and is very sensitive when compared with Edman sequencing. Finally, we compared Edman sequencing and Nt-dimethylation of the same polypeptide fragments; results confirm that there is agreement in the identity of the Nt amino acid sequence between these 2 methods.

  4. The combinational effect of E6/E7 siRNA and anti-miR-182 on apoptosis induction in HPV16-positive cervical cells.

    PubMed

    Javadi, Hamidreza; Lotfi, Abbas Sahebghadam; Hosseinkhani, Saman; Mehrani, Hossein; Amani, Jafar; Soheili, Zahra Soheila; Hojati, Zahra; Kamali, Mehdi

    2018-06-06

    In the present research, we assumed that reducing the amounts of E6 and E7 oncoproteins by a specific siRNA sequence and recovering p53 and RB proteins, along with the recovery of the FOXO1 protein by applying anti-miR-182, would increase apoptosis and reduce proliferation rate in cancer cells. The HPV16-positive CaSki cervical cancer cell line was used. 48 hours after transfection of siRNA for targeting E6 and E7 oncoproteins and anti-miR-182, expression of its cellular targets p53, p21 and FOXO1 was assessed by real-time PCR, western blot analysis and immunocytofluorescence staining. In all treatments, apoptosis rate and viability were evaluated using Annexin-V-FITC apoptosis detection kits and MTT assays, respectively. Among the designed siRNAs, E6-1 and E7-2 proved the most effective in reducing E6 and E7 expressions by increasing the apoptotic rates to 12.4% and 16%, respectively, after 48 hours. Also, using anti-miR-182 increased apoptotic rate to 12.7% 48 hours after transfection of cervical cancer cells. The combinational use of either E6-1 or E7-2 siRNAs with anti-miR-182 resulted in a rise in apoptosis to 19.3% and 26%, respectively, higher than those obtained from the individual application of either without anti-miR-182. The simultaneous use of siRNA E6-1 and siRNA E7-2 with cisplatin increased sensitivity to cisplatin and reduced the viability of the cancer cells as compared to the use of cisplatin alone. The simultaneous use of cisplatin and anti-miR-182 had no considerable effect on viability or apoptosis rate compared to cisplatin alone.

  5. Yellow lupin (Lupinus luteus L.) transcriptome sequencing: molecular marker development and comparative studies

    PubMed Central

    2012-01-01

    Background Yellow lupin (Lupinus luteus L.) is a minor legume crop characterized by its high seed protein content. Although grown in several temperate countries, its orphan condition has limited the generation of genomic tools to aid breeding efforts to improve yield and nutritional quality. In this study, we report the construction of 454-expresed sequence tag (EST) libraries, carried out comparative studies between L. luteus and model legume species, developed a comprehensive set of EST-simple sequence repeat (SSR) markers, and validated their utility on diversity studies and transferability to related species. Results Two runs of 454 pyrosequencing yielded 205 Mb and 530 Mb of sequence data for L1 (young leaves, buds and flowers) and L2 (immature seeds) EST- libraries. A combined assembly (L1L2) yielded 71,655 contigs with an average contig length of 632 nucleotides. L1L2 contigs were clustered into 55,309 isotigs. 38,200 isotigs translated into proteins and 8,741 of them were full length. Around 57% of L. luteus sequences had significant similarity with at least one sequence of Medicago, Lotus, Arabidopsis, or Glycine, and 40.17% showed positive matches with all of these species. L. luteus isotigs were also screened for the presence of SSR sequences. A total of 2,572 isotigs contained at least one EST-SSR, with a frequency of one SSR per 17.75 kbp. Empirical evaluation of the EST-SSR candidate markers resulted in 222 polymorphic EST-SSRs. Two hundred and fifty four (65.7%) and 113 (30%) SSR primer pairs were able to amplify fragments from L. hispanicus and L. mutabilis DNA, respectively. Fifty polymorphic EST-SSRs were used to genotype a sample of 64 L. luteus accessions. Neighbor-joining distance analysis detected the existence of several clusters among L. luteus accessions, strongly suggesting the existence of population subdivisions. However, no clear clustering patterns followed the accession’s origin. Conclusion L. luteus deep transcriptome

  6. Yellow lupin (Lupinus luteus L.) transcriptome sequencing: molecular marker development and comparative studies.

    PubMed

    Parra-González, Lorena B; Aravena-Abarzúa, Gabriela A; Navarro-Navarro, Cristell S; Udall, Joshua; Maughan, Jeff; Peterson, Louis M; Salvo-Garrido, Haroldo E; Maureira-Butler, Iván J

    2012-08-24

    Yellow lupin (Lupinus luteus L.) is a minor legume crop characterized by its high seed protein content. Although grown in several temperate countries, its orphan condition has limited the generation of genomic tools to aid breeding efforts to improve yield and nutritional quality. In this study, we report the construction of 454-expresed sequence tag (EST) libraries, carried out comparative studies between L. luteus and model legume species, developed a comprehensive set of EST-simple sequence repeat (SSR) markers, and validated their utility on diversity studies and transferability to related species. Two runs of 454 pyrosequencing yielded 205 Mb and 530 Mb of sequence data for L1 (young leaves, buds and flowers) and L2 (immature seeds) EST- libraries. A combined assembly (L1L2) yielded 71,655 contigs with an average contig length of 632 nucleotides. L1L2 contigs were clustered into 55,309 isotigs. 38,200 isotigs translated into proteins and 8,741 of them were full length. Around 57% of L. luteus sequences had significant similarity with at least one sequence of Medicago, Lotus, Arabidopsis, or Glycine, and 40.17% showed positive matches with all of these species. L. luteus isotigs were also screened for the presence of SSR sequences. A total of 2,572 isotigs contained at least one EST-SSR, with a frequency of one SSR per 17.75 kbp. Empirical evaluation of the EST-SSR candidate markers resulted in 222 polymorphic EST-SSRs. Two hundred and fifty four (65.7%) and 113 (30%) SSR primer pairs were able to amplify fragments from L. hispanicus and L. mutabilis DNA, respectively. Fifty polymorphic EST-SSRs were used to genotype a sample of 64 L. luteus accessions. Neighbor-joining distance analysis detected the existence of several clusters among L. luteus accessions, strongly suggesting the existence of population subdivisions. However, no clear clustering patterns followed the accession's origin. L. luteus deep transcriptome sequencing will facilitate the further

  7. Comparing sequencing assays and human-machine analyses in actionable genomics for glioblastoma.

    PubMed

    Wrzeszczynski, Kazimierz O; Frank, Mayu O; Koyama, Takahiko; Rhrissorrakrai, Kahn; Robine, Nicolas; Utro, Filippo; Emde, Anne-Katrin; Chen, Bo-Juen; Arora, Kanika; Shah, Minita; Vacic, Vladimir; Norel, Raquel; Bilal, Erhan; Bergmann, Ewa A; Moore Vogel, Julia L; Bruce, Jeffrey N; Lassman, Andrew B; Canoll, Peter; Grommes, Christian; Harvey, Steve; Parida, Laxmi; Michelini, Vanessa V; Zody, Michael C; Jobanputra, Vaidehi; Royyuru, Ajay K; Darnell, Robert B

    2017-08-01

    To analyze a glioblastoma tumor specimen with 3 different platforms and compare potentially actionable calls from each. Tumor DNA was analyzed by a commercial targeted panel. In addition, tumor-normal DNA was analyzed by whole-genome sequencing (WGS) and tumor RNA was analyzed by RNA sequencing (RNA-seq). The WGS and RNA-seq data were analyzed by a team of bioinformaticians and cancer oncologists, and separately by IBM Watson Genomic Analytics (WGA), an automated system for prioritizing somatic variants and identifying drugs. More variants were identified by WGS/RNA analysis than by targeted panels. WGA completed a comparable analysis in a fraction of the time required by the human analysts. The development of an effective human-machine interface in the analysis of deep cancer genomic datasets may provide potentially clinically actionable calls for individual patients in a more timely and efficient manner than currently possible. NCT02725684.

  8. Role of WDHD1 in Human Papillomavirus-Mediated Oncogenesis Identified by Transcriptional Profiling of E7-Expressing Cells

    PubMed Central

    Zhou, Yunying; Zhang, Qishu; Gao, Ge; Zhang, Xiaoli; Liu, Yafei; Yuan, Shoudao

    2016-01-01

    ABSTRACT The E7 oncoprotein of the high-risk human papillomavirus (HPV) plays a major role in HPV-induced carcinogenesis. E7 abrogates the G1 cell cycle checkpoint and induces genomic instability, but the mechanism is not fully understood. In this study, we performed RNA sequencing (RNA-seq) to characterize the transcriptional profile of keratinocytes expressing HPV 16 (HPV-16) E7. At the transcriptome level, 236 genes were differentially expressed between E7 and vector control cells. A subset of the differentially expressed genes, most of them novel to E7-expressing cells, was further confirmed by real-time PCR. Of interest, the activities of multiple transcription factors were altered in E7-expressing cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were investigated. The upregulated genes were enriched in cell cycle and DNA replication, as well as in the DNA metabolic process, transcription, DNA damage, DNA repair, and nucleotide metabolism. Specifically, we focused our studies on the gene encoding WDHD1 (WD repeat and high mobility group [HMG]-box DNA-binding protein), one of the genes that was upregulated in E7-expressing cells. WDHD1 is a component of the replisome that regulates DNA replication. Recent studies suggest that WDHD1 may also function as a DNA replication initiation factor as well as a G1 checkpoint regulator. We found that in E7-expressing cells, the steady-state level of WDHD1 protein was increased along with the half-life. Moreover, downregulation of WDHD1 reduced E7-induced G1 checkpoint abrogation and rereplication, demonstrating a novel function for WDHD1. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications. IMPORTANCE The high-risk HPV types induce cervical cancer and encode an E7 oncoprotein that plays a major role in HPV-induced carcinogenesis. However, the mechanism by which E7 induces carcinogenesis is not fully understood; specific anti

  9. A comparative analysis of whole genome sequencing of esophageal adenocarcinoma pre- and post-chemotherapy

    PubMed Central

    Noorani, Ayesha; Lynch, Andy G.; Achilleos, Achilleas; Eldridge, Matthew; Bower, Lawrence; Weaver, Jamie M.J.; Crawte, Jason; Ong, Chin-Ann; Shannon, Nicholas; MacRae, Shona; Grehan, Nicola; Nutzinger, Barbara; O'Donovan, Maria; Hardwick, Richard; Tavaré, Simon; Fitzgerald, Rebecca C.

    2017-01-01

    The scientific community has avoided using tissue samples from patients that have been exposed to systemic chemotherapy to infer the genomic landscape of a given cancer. Esophageal adenocarcinoma is a heterogeneous, chemoresistant tumor for which the availability and size of pretreatment endoscopic samples are limiting. This study compares whole-genome sequencing data obtained from chemo-naive and chemo-treated samples. The quality of whole-genomic sequencing data is comparable across all samples regardless of chemotherapy status. Inclusion of samples collected post-chemotherapy increased the proportion of late-stage tumors. When comparing matched pre- and post-chemotherapy samples from 10 cases, the mutational signatures, copy number, and SNV mutational profiles reflect the expected heterogeneity in this disease. Analysis of SNVs in relation to allele-specific copy-number changes pinpoints the common ancestor to a point prior to chemotherapy. For cases in which pre- and post-chemotherapy samples do show substantial differences, the timing of the divergence is near-synchronous with endoreduplication. Comparison across a large prospective cohort (62 treatment-naive, 58 chemotherapy-treated samples) reveals no significant differences in the overall mutation rate, mutation signatures, specific recurrent point mutations, or copy-number events in respect to chemotherapy status. In conclusion, whole-genome sequencing of samples obtained following neoadjuvant chemotherapy is representative of the genomic landscape of esophageal adenocarcinoma. Excluding these samples reduces the material available for cataloging and introduces a bias toward the earlier stages of cancer. PMID:28465312

  10. Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh

    PubMed Central

    Begum, Rabeya; Alam, Sheikh Shamimul; Menzel, Gerhard; Schmidt, Thomas

    2009-01-01

    Background and Aims Dendrobium species show tremendous morphological diversity and have broad geographical distribution. As repetitive sequence analysis is a useful tool to investigate the evolution of chromosomes and genomes, the aim of the present study was the characterization of repetitive sequences from Dendrobium moschatum for comparative molecular and cytogenetic studies in the related species Dendrobium aphyllum, Dendrobium aggregatum and representatives from other orchid genera. Methods In order to isolate highly repetitive sequences, a c0t-1 DNA plasmid library was established. Repeats were sequenced and used as probes for Southern hybridization. Sequence divergence was analysed using bioinformatic tools. Repetitive sequences were localized along orchid chromosomes by fluorescence in situ hybridization (FISH). Key Results Characterization of the c0t-1 library resulted in the detection of repetitive sequences including the (GA)n dinucleotide DmoO11, numerous Arabidopsis-like telomeric repeats and the highly amplified dispersed repeat DmoF14. The DmoF14 repeat is conserved in six Dendrobium species but diversified in representative species of three other orchid genera. FISH analyses showed the genome-wide distribution of DmoF14 in D. moschatum, D. aphyllum and D. aggregatum. Hybridization with the telomeric repeats demonstrated Arabidopsis-like telomeres at the chromosome ends of Dendrobium species. However, FISH using the telomeric probe revealed two pairs of chromosomes with strong intercalary signals in D. aphyllum. FISH showed the terminal position of 5S and 18S–5·8S–25S rRNA genes and a characteristic number of rDNA sites in the three Dendrobium species. Conclusions The repeated sequences isolated from D. moschatum c0t-1 DNA constitute major DNA families of the D. moschatum, D. aphyllum and D. aggregatum genomes with DmoF14 representing an ancient component of orchid genomes. Large intercalary telomere-like arrays suggest chromosomal

  11. Complete Genome Sequence and Comparative Analysis of the Fish Pathogen Lactococcus garvieae

    PubMed Central

    Oshima, Kenshiro; Yoshizaki, Mariko; Kawanishi, Michiko; Nakaya, Kohei; Suzuki, Takehito; Miyauchi, Eiji; Ishii, Yasuo; Tanabe, Soichi; Murakami, Masaru; Hattori, Masahira

    2011-01-01

    Lactococcus garvieae causes fatal haemorrhagic septicaemia in fish such as yellowtail. The comparative analysis of genomes of a virulent strain Lg2 and a non-virulent strain ATCC 49156 of L. garvieae revealed that the two strains shared a high degree of sequence identity, but Lg2 had a 16.5-kb capsule gene cluster that is absent in ATCC 49156. The capsule gene cluster was composed of 15 genes, of which eight genes are highly conserved with those in exopolysaccharide biosynthesis gene cluster often found in Lactococcus lactis strains. Sequence analysis of the capsule gene cluster in the less virulent strain L. garvieae Lg2-S, Lg2-derived strain, showed that two conserved genes were disrupted by a single base pair deletion, respectively. These results strongly suggest that the capsule is crucial for virulence of Lg2. The capsule gene cluster of Lg2 may be a genomic island from several features such as the presence of insertion sequences flanked on both ends, different GC content from the chromosomal average, integration into the locus syntenic to other lactococcal genome sequences, and distribution in human gut microbiomes. The analysis also predicted other potential virulence factors such as haemolysin. The present study provides new insights into understanding of the virulence mechanisms of L. garvieae in fish. PMID:21829716

  12. The complete chloroplast genome sequence of Dodonaea viscosa: comparative and phylogenetic analyses.

    PubMed

    Saina, Josphat K; Gichira, Andrew W; Li, Zhi-Zhong; Hu, Guang-Wan; Wang, Qing-Feng; Liao, Kuo

    2018-02-01

    The plant chloroplast (cp) genome is a highly conserved structure which is beneficial for evolution and systematic research. Currently, numerous complete cp genome sequences have been reported due to high throughput sequencing technology. However, there is no complete chloroplast genome of genus Dodonaea that has been reported before. To better understand the molecular basis of Dodonaea viscosa chloroplast, we used Illumina sequencing technology to sequence its complete genome. The whole length of the cp genome is 159,375 base pairs (bp), with a pair of inverted repeats (IRs) of 27,099 bp separated by a large single copy (LSC) 87,204 bp, and small single copy (SSC) 17,972 bp. The annotation analysis revealed a total of 115 unique genes of which 81 were protein coding, 30 tRNA, and four ribosomal RNA genes. Comparative genome analysis with other closely related Sapindaceae members showed conserved gene order in the inverted and single copy regions. Phylogenetic analysis clustered D. viscosa with other species of Sapindaceae with strong bootstrap support. Finally, a total of 249 SSRs were detected. Moreover, a comparison of the synonymous (Ks) and nonsynonymous (Ka) substitution rates in D. viscosa showed very low values. The availability of cp genome reported here provides a valuable genetic resource for comprehensive further studies in genetic variation, taxonomy and phylogenetic evolution of Sapindaceae family. In addition, SSR markers detected will be used in further phylogeographic and population structure studies of the species in this genus.

  13. Complete Sequence and Comparative Analysis of the Chloroplast Genome of Coconut Palm (Cocos nucifera)

    PubMed Central

    Huang, Ya-Yi; Matzke, Antonius J. M.; Matzke, Marjori

    2013-01-01

    Coconut, a member of the palm family (Arecaceae), is one of the most economically important trees used by mankind. Despite its diverse morphology, coconut is recognized taxonomically as only a single species (Cocos nucifera L.). There are two major coconut varieties, tall and dwarf, the latter of which displays traits resulting from selection by humans. We report here the complete chloroplast (cp) genome of a dwarf coconut plant, and describe the gene content and organization, inverted repeat fluctuations, repeated sequence structure, and occurrence of RNA editing. Phylogenetic relationships of monocots were inferred based on 47 chloroplast protein-coding genes. Potential nodes for events of gene duplication and pseudogenization related to inverted repeat fluctuation were mapped onto the tree using parsimony criteria. We compare our findings with those from other palm species for which complete cp genome sequences are available. PMID:24023703

  14. Complete sequence and comparative analysis of the chloroplast genome of coconut palm (Cocos nucifera).

    PubMed

    Huang, Ya-Yi; Matzke, Antonius J M; Matzke, Marjori

    2013-01-01

    Coconut, a member of the palm family (Arecaceae), is one of the most economically important trees used by mankind. Despite its diverse morphology, coconut is recognized taxonomically as only a single species (Cocos nucifera L.). There are two major coconut varieties, tall and dwarf, the latter of which displays traits resulting from selection by humans. We report here the complete chloroplast (cp) genome of a dwarf coconut plant, and describe the gene content and organization, inverted repeat fluctuations, repeated sequence structure, and occurrence of RNA editing. Phylogenetic relationships of monocots were inferred based on 47 chloroplast protein-coding genes. Potential nodes for events of gene duplication and pseudogenization related to inverted repeat fluctuation were mapped onto the tree using parsimony criteria. We compare our findings with those from other palm species for which complete cp genome sequences are available.

  15. Comparative genomics and repetitive sequence divergence in the species of diploid Nicotiana section Alatae.

    PubMed

    Lim, K Yoong; Kovarik, Ales; Matyasek, Roman; Chase, Mark W; Knapp, Sandra; McCarthy, Elizabeth; Clarkson, James J; Leitch, Andrew R

    2006-12-01

    Combining phylogenetic reconstructions of species relationships with comparative genomic approaches is a powerful way to decipher evolutionary events associated with genome divergence. Here, we reconstruct the history of karyotype and tandem repeat evolution in species of diploid Nicotiana section Alatae. By analysis of plastid DNA, we resolved two clades with high bootstrap support, one containing N. alata, N. langsdorffii, N. forgetiana and N. bonariensis (called the n = 9 group) and another containing N. plumbaginifolia and N. longiflora (called the n = 10 group). Despite little plastid DNA sequence divergence, we observed, via fluorescent in situ hybridization, substantial chromosomal repatterning, including altered chromosome numbers, structure and distribution of repeats. Effort was focussed on 35S and 5S nuclear ribosomal DNA (rDNA) and the HRS60 satellite family of tandem repeats comprising the elements HRS60, NP3R and NP4R. We compared divergence of these repeats in diploids and polyploids of Nicotiana. There are dramatic shifts in the distribution of the satellite repeats and complete replacement of intergenic spacers (IGSs) of 35S rDNA associated with divergence of the species in section Alatae. We suggest that sequence homogenization has replaced HRS60 family repeats at sub-telomeric regions, but that this process may not occur, or occurs more slowly, when the repeats are found at intercalary locations. Sequence homogenization acts more rapidly (at least two orders of magnitude) on 35S rDNA than 5S rDNA and sub-telomeric satellite sequences. This rapid rate of divergence is analogous to that found in polyploid species, and is therefore, in plants, not only associated with polyploidy.

  16. Some novel insights on HPV16 related cervical cancer pathogenesis based on analyses of LCR methylation, viral load, E7 and E2/E4 expressions.

    PubMed

    Das Ghosh, Damayanti; Bhattacharjee, Bornali; Sen, Shrinka; Premi, Laikangbam; Mukhopadhyay, Indranil; Chowdhury, Rahul Roy; Roy, Sudipta; Sengupta, Sharmila

    2012-01-01

    This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, (iii) expression of the transcript (E7-E1/E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1/E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 C(T)/E4 C(T). Relative quantification based on comparative C(T) (threshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 C(T) (p = 0.007) and E2 C(T) (p<0.0001), respectively, each normalized with ACTB C(T), among episomal cases only. The k-means clustering analysis considering E7 C(T) from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical

  17. Papillomavirus E7 Oncoproteins Share Functions with Polyomavirus Small T Antigens

    PubMed Central

    White, Elizabeth A.; Kramer, Rebecca E.; Hwang, Justin H.; Pores Fernando, Arun T.; Naetar, Nana; Hahn, William C.; Roberts, Thomas M.; Schaffhausen, Brian S.; Livingston, David M.

    2014-01-01

    ABSTRACT Many of the small DNA tumor viruses encode transforming proteins that function by targeting critical cellular pathways involved in cell proliferation and survival. In this study, we have examined whether some of the functions of the polyomavirus small T antigens (ST) are shared by the E6 and E7 oncoproteins of two oncogenic papillomaviruses. Using three different assays, we have found that E7 can provide some simian virus 40 (SV40) or murine polyomavirus (PyV) ST functions. Both human papillomavirus 16 (HPV16) and bovine papillomavirus (BPV1) E7 proteins are capable of partially substituting for SV40 ST in a transformation assay that also includes SV40 large T antigen, the catalytic subunit of cellular telomerase, and oncogenic Ras. Like SV40 ST, HPV16 E7 has the ability to override a quiescence block induced by mitogen deprivation. Like PyV ST, it also has the ability to inhibit myoblast differentiation. At least two of these activities are dependent upon the interaction of HPV16 E7 with retinoblastoma protein family members. For small T antigens, interaction with PP2A is needed for each of these functions. Even though there is no strong evidence that E6 or E7 share the ability of small T to interact with PP2A, E7 provides these functions related to cellular transformation. IMPORTANCE DNA tumor viruses have provided major insights into how cancers develop. Some viruses, like the human papillomaviruses, can cause cancer directly. Both the papillomaviruses and the polyomaviruses have served as tools for understanding pathways that are often perturbed in cancer. Here, we have compared the functions of transforming proteins from several DNA tumor viruses, including two papillomaviruses and two polyomaviruses. We tested the papillomavirus E6 and E7 oncoproteins in three functional assays and found that E7 can provide some or all of the functions of the SV40 small T antigen, another well-characterized oncoprotein, in two of these assays. In a third assay

  18. Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica.

    PubMed

    Hoffmann, Maria; Pettengill, James B; Gonzalez-Escalona, Narjol; Miller, John; Ayers, Sherry L; Zhao, Shaohua; Allard, Marc W; McDermott, Patrick F; Brown, Eric W; Monday, Steven R

    2017-01-01

    Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in

  19. Genome sequence and comparative analysis of a putative entomopathogenic Serratia isolated from Caenorhabditis briggsae.

    PubMed

    Abebe-Akele, Feseha; Tisa, Louis S; Cooper, Vaughn S; Hatcher, Philip J; Abebe, Eyualem; Thomas, W Kelley

    2015-07-18

    Entomopathogenic associations between nematodes in the genera Steinernema and Heterorhabdus with their cognate bacteria from the bacterial genera Xenorhabdus and Photorhabdus, respectively, are extensively studied for their potential as biological control agents against invasive insect species. These two highly coevolved associations were results of convergent evolution. Given the natural abundance of bacteria, nematodes and insects, it is surprising that only these two associations with no intermediate forms are widely studied in the entomopathogenic context. Discovering analogous systems involving novel bacterial and nematode species would shed light on the evolutionary processes involved in the transition from free living organisms to obligatory partners in entomopathogenicity. We report the complete genome sequence of a new member of the enterobacterial genus Serratia that forms a putative entomopathogenic complex with Caenorhabditis briggsae. Analysis of the 5.04 MB chromosomal genome predicts 4599 protein coding genes, seven sets of ribosomal RNA genes, 84 tRNA genes and a 64.8 KB plasmid encoding 74 genes. Comparative genomic analysis with three of the previously sequenced Serratia species, S. marcescens DB11 and S. proteamaculans 568, and Serratia sp. AS12, revealed that these four representatives of the genus share a core set of ~3100 genes and extensive structural conservation. The newly identified species shares a more recent common ancestor with S. marcescens with 99% sequence identity in rDNA sequence and orthology across 85.6% of predicted genes. Of the 39 genes/operons implicated in the virulence, symbiosis, recolonization, immune evasion and bioconversion, 21 (53.8%) were present in Serratia while 33 (84.6%) and 35 (89%) were present in Xenorhabdus and Photorhabdus EPN bacteria respectively. The majority of unique sequences in Serratia sp. SCBI (South African Caenorhabditis briggsae Isolate) are found in ~29 genomic islands of 5 to 65 genes and are

  20. RNAcode: Robust discrimination of coding and noncoding regions in comparative sequence data

    PubMed Central

    Washietl, Stefan; Findeiß, Sven; Müller, Stephan A.; Kalkhof, Stefan; von Bergen, Martin; Hofacker, Ivo L.; Stadler, Peter F.; Goldman, Nick

    2011-01-01

    With the availability of genome-wide transcription data and massive comparative sequencing, the discrimination of coding from noncoding RNAs and the assessment of coding potential in evolutionarily conserved regions arose as a core analysis task. Here we present RNAcode, a program to detect coding regions in multiple sequence alignments that is optimized for emerging applications not covered by current protein gene-finding software. Our algorithm combines information from nucleotide substitution and gap patterns in a unified framework and also deals with real-life issues such as alignment and sequencing errors. It uses an explicit statistical model with no machine learning component and can therefore be applied “out of the box,” without any training, to data from all domains of life. We describe the RNAcode method and apply it in combination with mass spectrometry experiments to predict and confirm seven novel short peptides in Escherichia coli and to analyze the coding potential of RNAs previously annotated as “noncoding.” RNAcode is open source software and available for all major platforms at http://wash.github.com/rnacode. PMID:21357752

  1. RNAcode: robust discrimination of coding and noncoding regions in comparative sequence data.

    PubMed

    Washietl, Stefan; Findeiss, Sven; Müller, Stephan A; Kalkhof, Stefan; von Bergen, Martin; Hofacker, Ivo L; Stadler, Peter F; Goldman, Nick

    2011-04-01

    With the availability of genome-wide transcription data and massive comparative sequencing, the discrimination of coding from noncoding RNAs and the assessment of coding potential in evolutionarily conserved regions arose as a core analysis task. Here we present RNAcode, a program to detect coding regions in multiple sequence alignments that is optimized for emerging applications not covered by current protein gene-finding software. Our algorithm combines information from nucleotide substitution and gap patterns in a unified framework and also deals with real-life issues such as alignment and sequencing errors. It uses an explicit statistical model with no machine learning component and can therefore be applied "out of the box," without any training, to data from all domains of life. We describe the RNAcode method and apply it in combination with mass spectrometry experiments to predict and confirm seven novel short peptides in Escherichia coli and to analyze the coding potential of RNAs previously annotated as "noncoding." RNAcode is open source software and available for all major platforms at http://wash.github.com/rnacode.

  2. Draft Genome Sequencing and Comparative Analysis of Aspergillus sojae NBRC4239

    PubMed Central

    Sato, Atsushi; Oshima, Kenshiro; Noguchi, Hideki; Ogawa, Masahiro; Takahashi, Tadashi; Oguma, Tetsuya; Koyama, Yasuji; Itoh, Takehiko; Hattori, Masahira; Hanya, Yoshiki

    2011-01-01

    We conducted genome sequencing of the filamentous fungus Aspergillus sojae NBRC4239 isolated from the koji used to prepare Japanese soy sauce. We used the 454 pyrosequencing technology and investigated the genome with respect to enzymes and secondary metabolites in comparison with other Aspergilli sequenced. Assembly of 454 reads generated a non-redundant sequence of 39.5-Mb possessing 13 033 putative genes and 65 scaffolds composed of 557 contigs. Of the 2847 open reading frames with Pfam domain scores of >150 found in A. sojae NBRC4239, 81.7% had a high degree of similarity with the genes of A. oryzae. Comparative analysis identified serine carboxypeptidase and aspartic protease genes unique to A. sojae NBRC4239. While A. oryzae possessed three copies of α-amyalse gene, A. sojae NBRC4239 possessed only a single copy. Comparison of 56 gene clusters for secondary metabolites between A. sojae NBRC4239 and A. oryzae revealed that 24 clusters were conserved, whereas 32 clusters differed between them that included a deletion of 18 508 bp containing mfs1, mao1, dmaT, and pks-nrps for the cyclopiazonic acid (CPA) biosynthesis, explaining the no productivity of CPA in A. sojae. The A. sojae NBRC4239 genome data will be useful to characterize functional features of the koji moulds used in Japanese industries. PMID:21659486

  3. Estimating population genetic parameters and comparing model goodness-of-fit using DNA sequences with error

    PubMed Central

    Liu, Xiaoming; Fu, Yun-Xin; Maxwell, Taylor J.; Boerwinkle, Eric

    2010-01-01

    It is known that sequencing error can bias estimation of evolutionary or population genetic parameters. This problem is more prominent in deep resequencing studies because of their large sample size n, and a higher probability of error at each nucleotide site. We propose a new method based on the composite likelihood of the observed SNP configurations to infer population mutation rate θ = 4Neμ, population exponential growth rate R, and error rate ɛ, simultaneously. Using simulation, we show the combined effects of the parameters, θ, n, ɛ, and R on the accuracy of parameter estimation. We compared our maximum composite likelihood estimator (MCLE) of θ with other θ estimators that take into account the error. The results show the MCLE performs well when the sample size is large or the error rate is high. Using parametric bootstrap, composite likelihood can also be used as a statistic for testing the model goodness-of-fit of the observed DNA sequences. The MCLE method is applied to sequence data on the ANGPTL4 gene in 1832 African American and 1045 European American individuals. PMID:19952140

  4. Comparing the white dwarf cooling sequences in 47 Tuc and NGC 6397

    SciT

    Richer, Harvey B.; Goldsbury, Ryan; Heyl, Jeremy

    2013-12-01

    Using deep Hubble Space Telescope imaging, color-magnitude diagrams are constructed for the globular clusters 47 Tuc and NGC 6397. As expected, because of its lower metal abundance, the main sequence of NGC 6397 lies well to the blue of that of 47 Tuc. A comparison of the white dwarf cooling sequences of the two clusters, however, demonstrates that these sequences are indistinguishable over most of their loci—a consequence of the settling out of heavy elements in the dense white dwarf atmosphere and the near equality of their masses. Lower quality data on M4 continues this trend to a third clustermore » whose metallicity is intermediate between these two. While the path of the white dwarfs in the color-magnitude diagram is nearly identical in 47 Tuc and NGC 6397, the numbers of white dwarfs along the path are not. This results from the relatively rapid relaxation in NGC 6397 compared to 47 Tuc and provides a cautionary note that simply counting objects in star clusters in random locations as a method of testing stellar evolutionary theory is likely dangerous unless dynamical considerations are included.« less

  5. Complete sequence and comparative analysis of the chloroplast genome of Plinia trunciflora

    PubMed Central

    Eguiluz, Maria; Yuyama, Priscila Mary; Guzman, Frank; Rodrigues, Nureyev Ferreira; Margis, Rogerio

    2017-01-01

    Abstract Plinia trunciflora is a Brazilian native fruit tree from the Myrtaceae family, also known as jaboticaba. This species has great potential by its fruit production. Due to the high content of essential oils in their leaves and of anthocyanins in the fruits, there is also an increasing interest by the pharmaceutical industry. Nevertheless, there are few studies focusing on its molecular biology and genetic characterization. We herein report the complete chloroplast (cp) genome of P. trunciflora using high-throughput sequencing and compare it to other previously sequenced Myrtaceae genomes. The cp genome of P. trunciflora is 159,512 bp in size, comprising inverted repeats of 26,414 bp and single-copy regions of 88,097 bp (LSC) and 18,587 bp (SSC). The genome contains 111 single-copy genes (77 protein-coding, 30 tRNA and four rRNA genes). Phylogenetic analysis using 57 cp protein-coding genes demonstrated that P. trunciflora, Eugenia uniflora and Acca sellowiana form a cluster with closer relationship to Syzygium cumini than with Eucalyptus. The complete cp sequence reported here can be used in evolutionary and population genetics studies, contributing to resolve the complex taxonomy of this species and fill the gap in genetic characterization. PMID:29111566

  6. A comparative study of ChIP-seq sequencing library preparation methods.

    PubMed

    Sundaram, Arvind Y M; Hughes, Timothy; Biondi, Shea; Bolduc, Nathalie; Bowman, Sarah K; Camilli, Andrew; Chew, Yap C; Couture, Catherine; Farmer, Andrew; Jerome, John P; Lazinski, David W; McUsic, Andrew; Peng, Xu; Shazand, Kamran; Xu, Feng; Lyle, Robert; Gilfillan, Gregor D

    2016-10-21

    ChIP-seq is the primary technique used to investigate genome-wide protein-DNA interactions. As part of this procedure, immunoprecipitated DNA must undergo "library preparation" to enable subsequent high-throughput sequencing. To facilitate the analysis of biopsy samples and rare cell populations, there has been a recent proliferation of methods allowing sequencing library preparation from low-input DNA amounts. However, little information exists on the relative merits, performance, comparability and biases inherent to these procedures. Notably, recently developed single-cell ChIP procedures employing microfluidics must also employ library preparation reagents to allow downstream sequencing. In this study, seven methods designed for low-input DNA/ChIP-seq sample preparation (Accel-NGS® 2S, Bowman-method, HTML-PCR, SeqPlex™, DNA SMART™, TELP and ThruPLEX®) were performed on five replicates of 1 ng and 0.1 ng input H3K4me3 ChIP material, and compared to a "gold standard" reference PCR-free dataset. The performance of each method was examined for the prevalence of unmappable reads, amplification-derived duplicate reads, reproducibility, and for the sensitivity and specificity of peak calling. We identified consistent high performance in a subset of the tested reagents, which should aid researchers in choosing the most appropriate reagents for their studies. Furthermore, we expect this work to drive future advances by identifying and encouraging use of the most promising methods and reagents. The results may also aid judgements on how comparable are existing datasets that have been prepared with different sample library preparation reagents.

  7. A Comparative Analysis of Three Monocular Passive Ranging Methods on Real Infrared Sequences

    NASA Astrophysics Data System (ADS)

    Bondžulić, Boban P.; Mitrović, Srđan T.; Barbarić, Žarko P.; Andrić, Milenko S.

    2013-09-01

    Three monocular passive ranging methods are analyzed and tested on the real infrared sequences. The first method exploits scale changes of an object in successive frames, while other two use Beer-Lambert's Law. Ranging methods are evaluated by comparing with simultaneously obtained reference data at the test site. Research is addressed on scenarios where multiple sensor views or active measurements are not possible. The results show that these methods for range estimation can provide the fidelity required for object tracking. Maximum values of relative distance estimation errors in near-ideal conditions are less than 8%.

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

    PubMed Central

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

    2017-01-01

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

  9. Chromosomal structures and repetitive sequences divergence in Cucumis species revealed by comparative cytogenetic mapping.

    PubMed

    Zhang, Yunxia; Cheng, Chunyan; Li, Ji; Yang, Shuqiong; Wang, Yunzhu; Li, Ziang; Chen, Jinfeng; Lou, Qunfeng

    2015-09-25

    Differentiation and copy number of repetitive sequences affect directly chromosome structure which contributes to reproductive isolation and speciation. Comparative cytogenetic mapping has been verified an efficient tool to elucidate the differentiation and distribution of repetitive sequences in genome. In present study, the distinct chromosomal structures of five Cucumis species were revealed through genomic in situ hybridization (GISH) technique and comparative cytogenetic mapping of major satellite repeats. Chromosome structures of five Cucumis species were investigated using GISH and comparative mapping of specific satellites. Southern hybridization was employed to study the proliferation of satellites, whose structural characteristics were helpful for analyzing chromosome evolution. Preferential distribution of repetitive DNAs at the subtelomeric regions was found in C. sativus, C hystrix and C. metuliferus, while majority was positioned at the pericentromeric heterochromatin regions in C. melo and C. anguria. Further, comparative GISH (cGISH) through using genomic DNA of other species as probes revealed high homology of repeats between C. sativus and C. hystrix. Specific satellites including 45S rDNA, Type I/II, Type III, Type IV, CentM and telomeric repeat were then comparatively mapped in these species. Type I/II and Type IV produced bright signals at the subtelomeric regions of C. sativus and C. hystrix simultaneously, which might explain the significance of their amplification in the divergence of Cucumis subgenus from the ancient ancestor. Unique positioning of Type III and CentM only at the centromeric domains of C. sativus and C. melo, respectively, combining with unique southern bands, revealed rapid evolutionary patterns of centromeric DNA in Cucumis. Obvious interstitial telomeric repeats were observed in chromosomes 1 and 2 of C. sativus, which might provide evidence of the fusion hypothesis of chromosome evolution from x = 12 to x = 7 in

  10. HPV E6/E7 mRNA versus HPV DNA biomarker in cervical cancer screening of a group of Macedonian women.

    PubMed

    Duvlis, Sotirija; Popovska-Jankovic, Katerina; Arsova, Zorica Sarafinovska; Memeti, Shaban; Popeska, Zaneta; Plaseska-Karanfilska, Dijana

    2015-09-01

    High risk types of human papillomaviruses E6/E7 oncogenes and their association with tumor suppressor genes products are the key factors of cervical carcinogenesis. This study proposed them as specific markers for cervical dysplasia screening. The aim of the study is to compare the clinical and prognostic significance of HPV E6/E7 mRNA as an early biomarker versus HPV DNA detection and cytology in triage of woman for cervical cancer. The study group consists of 413 women: 258 NILM, 26 ASC-US, 81 LSIL, 41 HSIL, and 7 unsatisfactory cytology. HPV4AACE screening, real-time multiplex PCR and MY09/11 consensus PCR primers methods were used for the HPV DNA detection. The real-time multiplex nucleic acid sequence-based assay (NucliSENS EasyQ HPV assay) was used for HPV E6/E7 mRNA detection of the five most common high risk HPV types in cervical cancer (16, 18, 31, 33, and 45). The results show that HPV E6/E7 mRNA testing had a higher specificity 50% (95% CI 32-67) and positive predictive value (PPV) 62% (95% CI 46-76) for CIN2+ compared to HPV DNA testing that had specificity of 18% (95% CI 7-37) and PPV 52% (95% CI 39-76) respectively. The higher specificity and PPV of HPV E6/E7 mRNA testing are valuable in predicting insignificant HPV DNA infection among cases with borderline cytological finding. It can help in avoiding aggressive procedures (biopsies and over-referral of transient HPV infections) as well as lowering patient's anxiety and follow up period. © 2015 Wiley Periodicals, Inc.

  11. Comparing sequencing assays and human-machine analyses in actionable genomics for glioblastoma

    PubMed Central

    Wrzeszczynski, Kazimierz O.; Frank, Mayu O.; Koyama, Takahiko; Rhrissorrakrai, Kahn; Robine, Nicolas; Utro, Filippo; Emde, Anne-Katrin; Chen, Bo-Juen; Arora, Kanika; Shah, Minita; Vacic, Vladimir; Norel, Raquel; Bilal, Erhan; Bergmann, Ewa A.; Moore Vogel, Julia L.; Bruce, Jeffrey N.; Lassman, Andrew B.; Canoll, Peter; Grommes, Christian; Harvey, Steve; Parida, Laxmi; Michelini, Vanessa V.; Zody, Michael C.; Jobanputra, Vaidehi; Royyuru, Ajay K.

    2017-01-01

    Objective: To analyze a glioblastoma tumor specimen with 3 different platforms and compare potentially actionable calls from each. Methods: Tumor DNA was analyzed by a commercial targeted panel. In addition, tumor-normal DNA was analyzed by whole-genome sequencing (WGS) and tumor RNA was analyzed by RNA sequencing (RNA-seq). The WGS and RNA-seq data were analyzed by a team of bioinformaticians and cancer oncologists, and separately by IBM Watson Genomic Analytics (WGA), an automated system for prioritizing somatic variants and identifying drugs. Results: More variants were identified by WGS/RNA analysis than by targeted panels. WGA completed a comparable analysis in a fraction of the time required by the human analysts. Conclusions: The development of an effective human-machine interface in the analysis of deep cancer genomic datasets may provide potentially clinically actionable calls for individual patients in a more timely and efficient manner than currently possible. ClinicalTrials.gov identifier: NCT02725684. PMID:28740869

  12. Complete Chloroplast Genome Sequences of Four Meliaceae Species and Comparative Analyses

    PubMed Central

    Mader, Malte; Pakull, Birte; Blanc-Jolivet, Céline; Paulini-Drewes, Maike; Bouda, Zoéwindé Henri-Noël; Degen, Bernd; Small, Ian

    2018-01-01

    The Meliaceae family mainly consists of trees and shrubs with a pantropical distribution. In this study, the complete chloroplast genomes of four Meliaceae species were sequenced and compared with each other and with the previously published Azadirachta indica plastome. The five plastomes are circular and exhibit a quadripartite structure with high conservation of gene content and order. They include 130 genes encoding 85 proteins, 37 tRNAs and 8 rRNAs. Inverted repeat expansion resulted in a duplication of rps19 in the five Meliaceae species, which is consistent with that in many other Sapindales, but different from many other rosids. Compared to Azadirachta indica, the four newly sequenced Meliaceae individuals share several large deletions, which mainly contribute to the decreased genome sizes. A whole-plastome phylogeny supports previous findings that the four species form a monophyletic sister clade to Azadirachta indica within the Meliaceae. SNPs and indels identified in all complete Meliaceae plastomes might be suitable targets for the future development of genetic markers at different taxonomic levels. The extended analysis of SNPs in the matK gene led to the identification of four potential Meliaceae-specific SNPs as a basis for future validation and marker development. PMID:29494509

  13. Comparative genomic sequence analysis of strawberry and other rosids reveals significant microsynteny

    PubMed Central

    2010-01-01

    Background Fragaria belongs to the Rosaceae, an economically important family that includes a number of important fruit producing genera such as Malus and Prunus. Using genomic sequences from 50 Fragaria fosmids, we have examined the microsynteny between Fragaria and other plant models. Results In more than half of the strawberry fosmids, we found syntenic regions that are conserved in Populus, Vitis, Medicago and/or Arabidopsis with Populus containing the greatest number of syntenic regions with Fragaria. The longest syntenic region was between LG VIII of the poplar genome and the strawberry fosmid 72E18, where seven out of twelve predicted genes were collinear. We also observed an unexpectedly high level of conserved synteny between Fragaria (rosid I) and Vitis (basal rosid). One of the strawberry fosmids, 34E24, contained a cluster of R gene analogs (RGAs) with NBS and LRR domains. We detected clusters of RGAs with high sequence similarity to those in 34E24 in all the genomes compared. In the phylogenetic tree we have generated, all the NBS-LRR genes grouped together with Arabidopsis CNL-A type NBS-LRR genes. The Fragaria RGA grouped together with those of Vitis and Populus in the phylogenetic tree. Conclusions Our analysis shows considerable microsynteny between Fragaria and other plant genomes such as Populus, Medicago, Vitis, and Arabidopsis to a lesser degree. We also detected a cluster of NBS-LRR type genes that are conserved in all the genomes compared. PMID:20565715

  14. Estimating and comparing microbial diversity in the presence of sequencing errors

    PubMed Central

    Chiu, Chun-Huo

    2016-01-01

    Estimating and comparing microbial diversity are statistically challenging due to limited sampling and possible sequencing errors for low-frequency counts, producing spurious singletons. The inflated singleton count seriously affects statistical analysis and inferences about microbial diversity. Previous statistical approaches to tackle the sequencing errors generally require different parametric assumptions about the sampling model or about the functional form of frequency counts. Different parametric assumptions may lead to drastically different diversity estimates. We focus on nonparametric methods which are universally valid for all parametric assumptions and can be used to compare diversity across communities. We develop here a nonparametric estimator of the true singleton count to replace the spurious singleton count in all methods/approaches. Our estimator of the true singleton count is in terms of the frequency counts of doubletons, tripletons and quadrupletons, provided these three frequency counts are reliable. To quantify microbial alpha diversity for an individual community, we adopt the measure of Hill numbers (effective number of taxa) under a nonparametric framework. Hill numbers, parameterized by an order q that determines the measures’ emphasis on rare or common species, include taxa richness (q = 0), Shannon diversity (q = 1, the exponential of Shannon entropy), and Simpson diversity (q = 2, the inverse of Simpson index). A diversity profile which depicts the Hill number as a function of order q conveys all information contained in a taxa abundance distribution. Based on the estimated singleton count and the original non-singleton frequency counts, two statistical approaches (non-asymptotic and asymptotic) are developed to compare microbial diversity for multiple communities. (1) A non-asymptotic approach refers to the comparison of estimated diversities of standardized samples with a common finite sample size or sample completeness. This

  15. Immunization with mutant HPV16 E7 protein inhibits the growth of TC-1 cells in tumor-bearing mice.

    PubMed

    Li, Yan-Li; Ma, Zhong-Liang; Zhao, Yue; Zhang, Jing

    2015-04-01

    Two human papillomavirus (HPV) 16 oncogenic proteins, E6 and E7, are co-expressed in the majority of HPV16-induced cervical cancer cells. Thus, the E6 and E7 proteins are good targets for developing therapeutic vaccines for cervical cancer. In the present study, immunization with the mutant non-transforming HPV16 E7 (mE7) protein was demonstrated to inhibit the growth of TC-1 cells in the TC-1 mouse model. The HPV16 mE7 gene was amplified by splicing overlap extension polymerase chain reaction using pET-28a(+)-E7 as a template, and the gene was cloned into pET-28a(+) to form pET-28a(+)-mE7. Compared with the E7 protein, mE7 lacks amino acid residues 94-98, and at residue 24, there is a Cys to Gly substitution. pET-28a(+)-mE7 was then introduced into Escherichia coli Rosetta. The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside. The mE7 protein was purified using Ni-NTA agarose and detected by SDS-PAGE and western blot analysis. In the tumor prevention model, no tumor was detected in the mice vaccinated with the mE7 protein. After 40 days, the tumor-free mice and control mice were challenged with 2×10 5 TC-1 cells. All control mice developed tumors six days later, but mE7 immunized mice were tumor free until 90 days. In the tumor therapy model, the TC-1 cells were initially injected subcutaneously, and the mice were subsequently vaccinated. Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control. These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.

  16. Whole Genome Sequencing Increases Molecular Diagnostic Yield Compared with Current Diagnostic Testing for Inherited Retinal Disease.

    PubMed

    Ellingford, Jamie M; Barton, Stephanie; Bhaskar, Sanjeev; Williams, Simon G; Sergouniotis, Panagiotis I; O'Sullivan, James; Lamb, Janine A; Perveen, Rahat; Hall, Georgina; Newman, William G; Bishop, Paul N; Roberts, Stephen A; Leach, Rick; Tearle, Rick; Bayliss, Stuart; Ramsden, Simon C; Nemeth, Andrea H; Black, Graeme C M

    2016-05-01

    To compare the efficacy of whole genome sequencing (WGS) with targeted next-generation sequencing (NGS) in the diagnosis of inherited retinal disease (IRD). Case series. A total of 562 patients diagnosed with IRD. We performed a direct comparative analysis of current molecular diagnostics with WGS. We retrospectively reviewed the findings from a diagnostic NGS DNA test for 562 patients with IRD. A subset of 46 of 562 patients (encompassing potential clinical outcomes of diagnostic analysis) also underwent WGS, and we compared mutation detection rates and molecular diagnostic yields. In addition, we compared the sensitivity and specificity of the 2 techniques to identify known single nucleotide variants (SNVs) using 6 control samples with publically available genotype data. Diagnostic yield of genomic testing. Across known disease-causing genes, targeted NGS and WGS achieved similar levels of sensitivity and specificity for SNV detection. However, WGS also identified 14 clinically relevant genetic variants through WGS that had not been identified by NGS diagnostic testing for the 46 individuals with IRD. These variants included large deletions and variants in noncoding regions of the genome. Identification of these variants confirmed a molecular diagnosis of IRD for 11 of the 33 individuals referred for WGS who had not obtained a molecular diagnosis through targeted NGS testing. Weighted estimates, accounting for population structure, suggest that WGS methods could result in an overall 29% (95% confidence interval, 15-45) uplift in diagnostic yield. We show that WGS methods can detect disease-causing genetic variants missed by current NGS diagnostic methodologies for IRD and thereby demonstrate the clinical utility and additional value of WGS. Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

  17. Comparative analyses of two Geraniaceae transcriptomes using next-generation sequencing.

    PubMed

    Zhang, Jin; Ruhlman, Tracey A; Mower, Jeffrey P; Jansen, Robert K

    2013-12-29

    Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with broad coverage. In addition

  18. Comparative analyses of two Geraniaceae transcriptomes using next-generation sequencing

    PubMed Central

    2013-01-01

    Background Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. Results Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. Conclusions The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with

  19. Comparative Transcriptomes and EVO-DEVO Studies Depending on Next Generation Sequencing.

    PubMed

    Liu, Tiancheng; Yu, Lin; Liu, Lei; Li, Hong; Li, Yixue

    2015-01-01

    High throughput technology has prompted the progressive omics studies, including genomics and transcriptomics. We have reviewed the improvement of comparative omic studies, which are attributed to the high throughput measurement of next generation sequencing technology. Comparative genomics have been successfully applied to evolution analysis while comparative transcriptomics are adopted in comparison of expression profile from two subjects by differential expression or differential coexpression, which enables their application in evolutionary developmental biology (EVO-DEVO) studies. EVO-DEVO studies focus on the evolutionary pressure affecting the morphogenesis of development and previous works have been conducted to illustrate the most conserved stages during embryonic development. Old measurements of these studies are based on the morphological similarity from macro view and new technology enables the micro detection of similarity in molecular mechanism. Evolutionary model of embryo development, which includes the "funnel-like" model and the "hourglass" model, has been evaluated by combination of these new comparative transcriptomic methods with prior comparative genomic information. Although the technology has promoted the EVO-DEVO studies into a new era, technological and material limitation still exist and further investigations require more subtle study design and procedure.

  20. Dynamic Evolution of Pathogenicity Revealed by Sequencing and Comparative Genomics of 19 Pseudomonas syringae Isolates

    PubMed Central

    Romanchuk, Artur; Chang, Jeff H.; Mukhtar, M. Shahid; Cherkis, Karen; Roach, Jeff; Grant, Sarah R.; Jones, Corbin D.; Dangl, Jeffery L.

    2011-01-01

    Closely related pathogens may differ dramatically in host range, but the molecular, genetic, and evolutionary basis for these differences remains unclear. In many Gram- negative bacteria, including the phytopathogen Pseudomonas syringae, type III effectors (TTEs) are essential for pathogenicity, instrumental in structuring host range, and exhibit wide diversity between strains. To capture the dynamic nature of virulence gene repertoires across P. syringae, we screened 11 diverse strains for novel TTE families and coupled this nearly saturating screen with the sequencing and assembly of 14 phylogenetically diverse isolates from a broad collection of diseased host plants. TTE repertoires vary dramatically in size and content across all P. syringae clades; surprisingly few TTEs are conserved and present in all strains. Those that are likely provide basal requirements for pathogenicity. We demonstrate that functional divergence within one conserved locus, hopM1, leads to dramatic differences in pathogenicity, and we demonstrate that phylogenetics-informed mutagenesis can be used to identify functionally critical residues of TTEs. The dynamism of the TTE repertoire is mirrored by diversity in pathways affecting the synthesis of secreted phytotoxins, highlighting the likely role of both types of virulence factors in determination of host range. We used these 14 draft genome sequences, plus five additional genome sequences previously reported, to identify the core genome for P. syringae and we compared this core to that of two closely related non-pathogenic pseudomonad species. These data revealed the recent acquisition of a 1 Mb megaplasmid by a sub-clade of cucumber pathogens. This megaplasmid encodes a type IV secretion system and a diverse set of unknown proteins, which dramatically increases both the genomic content of these strains and the pan-genome of the species. PMID:21799664

  1. Multilocus Sequence Typing Compared to Pulsed-Field Gel Electrophoresis for Molecular Typing of Pseudomonas aeruginosa▿

    PubMed Central

    Johnson, Jennifer K.; Arduino, Sonia M.; Stine, O. Colin; Johnson, Judith A.; Harris, Anthony D.

    2007-01-01

    For hospital epidemiologists, determining a system of typing that is discriminatory is essential for measuring the effectiveness of infection control measures. In situations in which the incidence of resistant Pseudomonas aeruginosa is increasing, the ability to discern whether it is due to patient-to-patient transmission versus an increase in patient endogenous strains is often made on the basis of molecular typing. The present study compared the discriminatory abilities of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for 90 P. aeruginosa isolates obtained from cultures of perirectal surveillance swabs from patients in an intensive care unit. PFGE identified 85 distinct types and 76 distinct groups when similarity cutoffs of 100% and 87%, respectively, were used. By comparison, MLST identified 60 sequence types that could be clustered into 11 clonal complexes and 32 singletons. By using the Simpson index of diversity (D), PFGE had a greater discriminatory ability than MLST for P. aeruginosa isolates (D values, 0.999 versus 0.975, respectively). Thus, while MLST was better for detecting genetic relatedness, we determined that PFGE was more discriminatory than MLST for determining genetic differences in P. aeruginosa. PMID:17881548

  2. Comparative Analyses of DNA Methylation and Sequence Evolution Using Nasonia Genomes

    PubMed Central

    Park, Jungsun; Peng, Zuogang; Zeng, Jia; Elango, Navin; Park, Taesung; Wheeler, Dave; Werren, John H.; Yi, Soojin V.

    2011-01-01

    The functional and evolutionary significance of DNA methylation in insect genomes remains to be resolved. Nasonia is well situated for comparative analyses of DNA methylation and genome evolution, since the genomes of a moderately distant outgroup species as well as closely related sibling species are available. Using direct sequencing of bisulfite-converted DNA, we uncovered a substantial level of DNA methylation in 17 of 18 Nasonia vitripennis genes and a strong correlation between methylation level and CpG depletion. Notably, in the sex-determining locus transformer, the exon that is alternatively spliced between the sexes is heavily methylated in both males and females, whereas other exons are only sparsely methylated. Orthologous genes of the honeybee and Nasonia show highly similar relative levels of CpG depletion, despite ∼190 My divergence. Densely and sparsely methylated genes in these species also exhibit similar functional enrichments. We found that the degree of CpG depletion is negatively correlated with substitution rates between closely related Nasonia species for synonymous, nonsynonymous, and intron sites. This suggests that mutation rates increase with decreasing levels of germ line methylation. Thus, DNA methylation is prevalent in the Nasonia genome, may participate in regulatory processes such as sex determination and alternative splicing, and is correlated with several aspects of genome and sequence evolution. PMID:21693438

  3. Comparative study of methods for recognition of an unknown person's action from a video sequence

    NASA Astrophysics Data System (ADS)

    Hori, Takayuki; Ohya, Jun; Kurumisawa, Jun

    2009-02-01

    This paper proposes a Tensor Decomposition Based method that can recognize an unknown person's action from a video sequence, where the unknown person is not included in the database (tensor) used for the recognition. The tensor consists of persons, actions and time-series image features. For the observed unknown person's action, one of the actions stored in the tensor is assumed. Using the motion signature obtained from the assumption, the unknown person's actions are synthesized. The actions of one of the persons in the tensor are replaced by the synthesized actions. Then, the core tensor for the replaced tensor is computed. This process is repeated for the actions and persons. For each iteration, the difference between the replaced and original core tensors is computed. The assumption that gives the minimal difference is the action recognition result. For the time-series image features to be stored in the tensor and to be extracted from the observed video sequence, the human body silhouette's contour shape based feature is used. To show the validity of our proposed method, our proposed method is experimentally compared with Nearest Neighbor rule and Principal Component analysis based method. Experiments using 33 persons' seven kinds of action show that our proposed method achieves better recognition accuracies for the seven actions than the other methods.

  4. Comparative genomic sequence analysis of novel Helicoverpa armigera nucleopolyhedrovirus (NPV) isolated from Kenya and three other previously sequenced Helicoverpa spp. NPVs.

    PubMed

    Ogembo, Javier Gordon; Caoili, Barbara L; Shikata, Masamitsu; Chaeychomsri, Sudawan; Kobayashi, Michihiro; Ikeda, Motoko

    2009-10-01

    A newly cloned Helicoverpa armigera nucleopolyhedrovirus (HearNPV) from Kenya, HearNPV-NNg1, has a higher insecticidal activity than HearNPV-G4, which also exhibits lower insecticidal activity than HearNPV-C1. In the search for genes and/or nucleotide sequences that might be involved in the observed virulence differences among Helicoverpa spp. NPVs, the entire genome of NNg1 was sequenced and compared with previously sequenced genomes of G4, C1 and Helicoverpa zea single-nucleocapsid NPV (Hz). The NNg1 genome was 132,425 bp in length, with a total of 143 putative open reading frames (ORFs), and shared high levels of overall amino acid and nucleotide sequence identities with G4, C1 and Hz. Three NNg1 ORFs, ORF5, ORF100 and ORF124, which were shared with C1, were absent in G4 and Hz, while NNg1 and C1 were missing a homologue of G4/Hz ORF5. Another three ORFs, ORF60 (bro-b), ORF119 and ORF120, and one direct repeat sequence (dr) were unique to NNg1. Relative to the overall nucleotide sequence identity, lower sequence identities were observed between NNg1 hrs and the homologous hrs in the other three Helicoverpa spp. NPVs, despite containing the same number of hrs located at essentially the same positions on the genomes. Differences were also observed between NNg1 and each of the other three Helicoverpa spp. NPVs in the diversity of bro genes encoded on the genomes. These results indicate several putative genes and nucleotide sequences that may be responsible for the virulence differences observed among Helicoverpa spp., yet the specific genes and/or nucleotide sequences responsible have not been identified.

  5. Comparative studies of copy number variation detection methods for next-generation sequencing technologies.

    PubMed

    Duan, Junbo; Zhang, Ji-Gang; Deng, Hong-Wen; Wang, Yu-Ping

    2013-01-01

    Copy number variation (CNV) has played an important role in studies of susceptibility or resistance to complex diseases. Traditional methods such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH) suffer from low resolution of genomic regions. Following the emergence of next generation sequencing (NGS) technologies, CNV detection methods based on the short read data have recently been developed. However, due to the relatively young age of the procedures, their performance is not fully understood. To help investigators choose suitable methods to detect CNVs, comparative studies are needed. We compared six publicly available CNV detection methods: CNV-seq, FREEC, readDepth, CNVnator, SegSeq and event-wise testing (EWT). They are evaluated both on simulated and real data with different experiment settings. The receiver operating characteristic (ROC) curve is employed to demonstrate the detection performance in terms of sensitivity and specificity, box plot is employed to compare their performances in terms of breakpoint and copy number estimation, Venn diagram is employed to show the consistency among these methods, and F-score is employed to show the overlapping quality of detected CNVs. The computational demands are also studied. The results of our work provide a comprehensive evaluation on the performances of the selected CNV detection methods, which will help biological investigators choose the best possible method.

  6. Next Generation Semiconductor Based Sequencing of the Donkey (Equus asinus) Genome Provided Comparative Sequence Data against the Horse Genome and a Few Millions of Single Nucleotide Polymorphisms

    PubMed Central

    Bertolini, Francesca; Scimone, Concetta; Geraci, Claudia; Schiavo, Giuseppina; Utzeri, Valerio Joe; Chiofalo, Vincenzo; Fontanesi, Luca

    2015-01-01

    Few studies investigated the donkey (Equus asinus) at the whole genome level so far. Here, we sequenced the genome of two male donkeys using a next generation semiconductor based sequencing platform (the Ion Proton sequencer) and compared obtained sequence information with the available donkey draft genome (and its Illumina reads from which it was originated) and with the EquCab2.0 assembly of the horse genome. Moreover, the Ion Torrent Personal Genome Analyzer was used to sequence reduced representation libraries (RRL) obtained from a DNA pool including donkeys of different breeds (Grigio Siciliano, Ragusano and Martina Franca). The number of next generation sequencing reads aligned with the EquCab2.0 horse genome was larger than those aligned with the draft donkey genome. This was due to the larger N50 for contigs and scaffolds of the horse genome. Nucleotide divergence between E. caballus and E. asinus was estimated to be ~ 0.52-0.57%. Regions with low nucleotide divergence were identified in several autosomal chromosomes and in the whole chromosome X. These regions might be evolutionally important in equids. Comparing Y-chromosome regions we identified variants that could be useful to track donkey paternal lineages. Moreover, about 4.8 million of single nucleotide polymorphisms (SNPs) in the donkey genome were identified and annotated combining sequencing data from Ion Proton (whole genome sequencing) and Ion Torrent (RRL) runs with Illumina reads. A higher density of SNPs was present in regions homologous to horse chromosome 12, in which several studies reported a high frequency of copy number variants. The SNPs we identified constitute a first resource useful to describe variability at the population genomic level in E. asinus and to establish monitoring systems for the conservation of donkey genetic resources. PMID:26151450

  7. Next Generation Semiconductor Based Sequencing of the Donkey (Equus asinus) Genome Provided Comparative Sequence Data against the Horse Genome and a Few Millions of Single Nucleotide Polymorphisms.

    PubMed

    Bertolini, Francesca; Scimone, Concetta; Geraci, Claudia; Schiavo, Giuseppina; Utzeri, Valerio Joe; Chiofalo, Vincenzo; Fontanesi, Luca

    2015-01-01

    Few studies investigated the donkey (Equus asinus) at the whole genome level so far. Here, we sequenced the genome of two male donkeys using a next generation semiconductor based sequencing platform (the Ion Proton sequencer) and compared obtained sequence information with the available donkey draft genome (and its Illumina reads from which it was originated) and with the EquCab2.0 assembly of the horse genome. Moreover, the Ion Torrent Personal Genome Analyzer was used to sequence reduced representation libraries (RRL) obtained from a DNA pool including donkeys of different breeds (Grigio Siciliano, Ragusano and Martina Franca). The number of next generation sequencing reads aligned with the EquCab2.0 horse genome was larger than those aligned with the draft donkey genome. This was due to the larger N50 for contigs and scaffolds of the horse genome. Nucleotide divergence between E. caballus and E. asinus was estimated to be ~ 0.52-0.57%. Regions with low nucleotide divergence were identified in several autosomal chromosomes and in the whole chromosome X. These regions might be evolutionally important in equids. Comparing Y-chromosome regions we identified variants that could be useful to track donkey paternal lineages. Moreover, about 4.8 million of single nucleotide polymorphisms (SNPs) in the donkey genome were identified and annotated combining sequencing data from Ion Proton (whole genome sequencing) and Ion Torrent (RRL) runs with Illumina reads. A higher density of SNPs was present in regions homologous to horse chromosome 12, in which several studies reported a high frequency of copy number variants. The SNPs we identified constitute a first resource useful to describe variability at the population genomic level in E. asinus and to establish monitoring systems for the conservation of donkey genetic resources.

  8. Papillomavirus E7 protein binding to the retinoblastoma protein is not required for viral induction of warts.

    PubMed Central

    Defeo-Jones, D; Vuocolo, G A; Haskell, K M; Hanobik, M G; Kiefer, D M; McAvoy, E M; Ivey-Hoyle, M; Brandsma, J L; Oliff, A; Jones, R E

    1993-01-01

    Human papillomaviruses (HPVs) are the etiologic agents responsible for benign epithelial proliferative disorders including genital warts and are a contributory factor in the pathogenesis of cervical cancer. HPVs demonstrate strict species and cell-type specificity, which is manifested by the inability of these viruses to induce disease in any species other than humans. The natural history of HPV infection in humans is closely mimicked by cottontail rabbit papillomavirus (CRPV) infection in domestic laboratory rabbits. The CRPV E7 gene is known to play an essential role in virus-mediated induction of papillomas. We now show by mutational analysis that the CRPV E7 protein's biochemical and biological properties, including binding to the retinoblastoma suppressor protein (pRB), transcription factor E2F transactivation of the adenovirus E2 promoter, disruption of pRB-E2F complexes, and cellular transformation as measured by growth in soft agar, mimic those of the HPV E7 protein. Intradermal injection of CRPV DNA lacking E7 gene sequences critical for the binding of the CRPV E7 protein to pRB induced papillomas in rabbits. These studies indicate that E7 protein binding to pRB is not required in the molecular pathogenesis of virally induced warts and suggest that other properties intrinsic to the E7 protein are necessary for papilloma formation. Images PMID:8380462

  9. Authentication Markers for Five Major Panax Species Developed via Comparative Analysis of Complete Chloroplast Genome Sequences.

    PubMed

    Nguyen, Van Binh; Park, Hyun-Seung; Lee, Sang-Choon; Lee, Junki; Park, Jee Young; Yang, Tae-Jin

    2017-08-02

    Ginseng represents a set of high-value medicinal plants of different species: Panax ginseng (Asian ginseng), Panax quinquefolius (American ginseng), Panax notoginseng (Chinese ginseng), Panax japonicus (Bamboo ginseng), and Panax vietnamensis (Vietnamese ginseng). Each species is pharmacologically and economically important, with differences in efficacy and price. Accordingly, an authentication system is needed to combat economically motivated adulteration of Panax products. We conducted comparative analysis of the chloroplast genome sequences of these five species, identifying 34-124 InDels and 141-560 SNPs. Fourteen InDel markers were developed to authenticate the Panax species. Among these, eight were species-unique markers that successfully differentiated one species from the others. We generated at least one species-unique marker for each of the five species, and any of the species can be authenticated by selection among these markers. The markers are reliable, easily detectable, and valuable for applications in the ginseng industry as well as in related research.

  10. Phylogenetic and Comparative Sequence Analysis of Thermostable Alpha Amylases of kingdom Archea, Prokaryotes and Eukaryotes.

    PubMed

    Huma, Tayyaba; Maryam, Arooma; Rehman, Shahid Ur; Qamar, Muhammad Tahir Ul; Shaheen, Tayyaba; Haque, Asma; Shaheen, Bushra

    2014-01-01

    Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase α-(1, 4) or α-(1, 6) glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family, nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis (70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm. Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and productive for industry.

  11. Comparative Sequence Analysis of the X-Inactivation Center Region in Mouse, Human, and Bovine

    PubMed Central

    Chureau, Corinne; Prissette, Marine; Bourdet, Agnès; Barbe, Valérie; Cattolico, Laurence; Jones, Louis; Eggen, André; Avner, Philip; Duret, Laurent

    2002-01-01

    We have sequenced to high levels of accuracy 714-kb and 233-kb regions of the mouse and bovine X-inactivation centers (Xic), respectively, centered on the Xist gene. This has provided the basis for a fully annotated comparative analysis of the mouse Xic with the 2.3-Mb orthologous region in human and has allowed a three-way species comparison of the core central region, including the Xist gene. These comparisons have revealed conserved genes, both coding and noncoding, conserved CpG islands and, more surprisingly, conserved pseudogenes. The distribution of repeated elements, especially LINE repeats, in the mouse Xic region when compared to the rest of the genome does not support the hypothesis of a role for these repeat elements in the spreading of X inactivation. Interestingly, an asymmetric distribution of LINE elements on the two DNA strands was observed in the three species, not only within introns but also in intergenic regions. This feature is suggestive of important transcriptional activity within these intergenic regions. In silico prediction followed by experimental analysis has allowed four new genes, Cnbp2, Ftx, Jpx, and Ppnx, to be identified and novel, widespread, complex, and apparently noncoding transcriptional activity to be characterized in a region 5′ of Xist that was recently shown to attract histone modification early after the onset of X inactivation. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AJ421478, AJ421479, AJ421480, and AJ421481. Online supplemental data are available at http://pbil.univ-lyon1.fr/datasets/Xic2002/data.html and www.genome.org.] PMID:12045143

  12. [Profiles of cell proliferation and apoptosis in the mouse epithelial regeneration model K6b-E6/E7].

    PubMed

    Bonilla-Delgado, José; Rodríguez-Uribe, Genaro; Cortés-Malagón, Enoc Mariano; Sierra Martínez, Mónica; Acosta-Altamirano, Gustavo; Gariglio-Vidal, Patricio

    2012-01-01

    Mammals have limited epithelial regeneration capacity. The K6b-E6/E7 mice model has been described as useful for the study of epithelial regeneration. The objective of this study is to compare the expression of E6/E7 oncogenes with those of cell proliferation and apoptosis during epithelization. The hypothesis of this study is that alterations in cell proliferation and apoptosis in K6b-E6/E7 mice will only occur during epithelization. Deep 2 mm punches were performed in the middle of transgenic and control mice's ears. A biopsy was collected from the epithelization zone 72 hours and 2 weeks post-injury. Assays for cell proliferation and apoptosis were carried out by immunohistochemistry and TUNEL techniques, respectively. RT-PCR in situ was performed to compare E6/E7 expressions in the areas studied. Transgenic strain K6b-E6/E7 presented more proliferative cells and less apoptotic cells in epithelizated zones. This effect was limited to suprabasal stratum only, and correlates with E6/E7 oncogenes expression. Two weeks post-injury, cell proliferation and apoptosis were similar in both samples as the E6/E7 expression went down. K6b-E6/E7 mouse model is useful for epithelial regeneration. Its mechanisms should be considered for the treatment of deep wounds.

  13. Proteomic analysis of the gamma human papillomavirus type 197 E6 and E7 associated cellular proteins

    PubMed Central

    Grace, Miranda; Munger, Karl

    2016-01-01

    Gamma HPV197 was the most frequently identified HPV when human skin cancer specimens were analyzed by deep sequencing. To gain insight into the biological activities of HPV197, we investigated the cellular interactomes of HPV197 E6 and E7. HPV197 E6 protein interacts with a broad spectrum of cellular LXXLL domain proteins, including UBE3A and MAML1. HPV197 E6 also binds and inhibits the TP53 tumor suppressor and interacts with the CCR4-NOT ubiquitin ligase and deadenylation complex. Despite lacking a canonical retinoblastoma (RB1) tumor suppressor binding site, HPV197 E7 binds RB1 and activates E2F transcription. Hence, HPV197 E6 and E7 proteins interact with a similar set of cellular proteins as E6 and E7 proteins encoded by HPVs that have been linked to human carcinogenesis and/or have transforming activities in vitro. PMID:27771561

  14. Genome Sequencing and Comparative Genomics of the Broad Host-Range Pathogen Rhizoctonia solani AG8

    PubMed Central

    Hane, James K.; Anderson, Jonathan P.; Williams, Angela H.; Sperschneider, Jana; Singh, Karam B.

    2014-01-01

    Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle which is classified into fourteen reproductively incompatible anastomosis groups (AGs). One of these, AG8, is a devastating pathogen causing bare patch of cereals, brassicas and legumes. R. solani is a multinucleate heterokaryon containing significant heterozygosity within a single cell. This complexity posed significant challenges for the assembly of its genome. We present a high quality genome assembly of R. solani AG8 and a manually curated set of 13,964 genes supported by RNA-seq. The AG8 genome assembly used novel methods to produce a haploid representation of its heterokaryotic state. The whole-genomes of AG8, the rice pathogen AG1-IA and the potato pathogen AG3 were observed to be syntenic and co-linear. Genes and functions putatively relevant to pathogenicity were highlighted by comparing AG8 to known pathogenicity genes, orthology databases spanning 197 phytopathogenic taxa and AG1-IA. We also observed SNP-level “hypermutation” of CpG dinucleotides to TpG between AG8 nuclei, with similarities to repeat-induced point mutation (RIP). Interestingly, gene-coding regions were widely affected along with repetitive DNA, which has not been previously observed for RIP in mononuclear fungi of the Pezizomycotina. The rate of heterozygous SNP mutations within this single isolate of AG8 was observed to be higher than SNP mutation rates observed across populations of most fungal species compared. Comparative analyses were combined to predict biological processes relevant to AG8 and 308 proteins with effector-like characteristics, forming a valuable resource for further study of this pathosystem. Predicted effector-like proteins had elevated levels of non-synonymous point mutations relative to synonymous mutations (dN/dS), suggesting that they may be under diversifying selection pressures. In addition, the distant relationship to sequenced necrotrophs of the Ascomycota suggests the

  15. Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test

    PubMed Central

    Lionel, Anath C; Costain, Gregory; Monfared, Nasim; Walker, Susan; Reuter, Miriam S; Hosseini, S Mohsen; Thiruvahindrapuram, Bhooma; Merico, Daniele; Jobling, Rebekah; Nalpathamkalam, Thomas; Pellecchia, Giovanna; Sung, Wilson W L; Wang, Zhuozhi; Bikangaga, Peter; Boelman, Cyrus; Carter, Melissa T; Cordeiro, Dawn; Cytrynbaum, Cheryl; Dell, Sharon D; Dhir, Priya; Dowling, James J; Heon, Elise; Hewson, Stacy; Hiraki, Linda; Inbar-Feigenberg, Michal; Klatt, Regan; Kronick, Jonathan; Laxer, Ronald M; Licht, Christoph; MacDonald, Heather; Mercimek-Andrews, Saadet; Mendoza-Londono, Roberto; Piscione, Tino; Schneider, Rayfel; Schulze, Andreas; Silverman, Earl; Siriwardena, Komudi; Snead, O Carter; Sondheimer, Neal; Sutherland, Joanne; Vincent, Ajoy; Wasserman, Jonathan D; Weksberg, Rosanna; Shuman, Cheryl; Carew, Chris; Szego, Michael J; Hayeems, Robin Z; Basran, Raveen; Stavropoulos, Dimitri J; Ray, Peter N; Bowdin, Sarah; Meyn, M Stephen; Cohn, Ronald D; Scherer, Stephen W; Marshall, Christian R

    2018-01-01

    Purpose Genetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use. Methods We prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing. Results WGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24% P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A. Conclusion WGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort. PMID:28771251

  16. Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test.

    PubMed

    Lionel, Anath C; Costain, Gregory; Monfared, Nasim; Walker, Susan; Reuter, Miriam S; Hosseini, S Mohsen; Thiruvahindrapuram, Bhooma; Merico, Daniele; Jobling, Rebekah; Nalpathamkalam, Thomas; Pellecchia, Giovanna; Sung, Wilson W L; Wang, Zhuozhi; Bikangaga, Peter; Boelman, Cyrus; Carter, Melissa T; Cordeiro, Dawn; Cytrynbaum, Cheryl; Dell, Sharon D; Dhir, Priya; Dowling, James J; Heon, Elise; Hewson, Stacy; Hiraki, Linda; Inbar-Feigenberg, Michal; Klatt, Regan; Kronick, Jonathan; Laxer, Ronald M; Licht, Christoph; MacDonald, Heather; Mercimek-Andrews, Saadet; Mendoza-Londono, Roberto; Piscione, Tino; Schneider, Rayfel; Schulze, Andreas; Silverman, Earl; Siriwardena, Komudi; Snead, O Carter; Sondheimer, Neal; Sutherland, Joanne; Vincent, Ajoy; Wasserman, Jonathan D; Weksberg, Rosanna; Shuman, Cheryl; Carew, Chris; Szego, Michael J; Hayeems, Robin Z; Basran, Raveen; Stavropoulos, Dimitri J; Ray, Peter N; Bowdin, Sarah; Meyn, M Stephen; Cohn, Ronald D; Scherer, Stephen W; Marshall, Christian R

    2018-04-01

    PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.MethodsWe prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing.ResultsWGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24%; P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A.ConclusionWGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.

  17. In vitro biological activities of the E6 and E7 genes vary among human papillomaviruses of different oncogenic potential.

    PubMed Central

    Barbosa, M S; Vass, W C; Lowy, D R; Schiller, J T

    1991-01-01

    Human papillomavirus type 16 (HPV-16) and HPV-18 are often detected in cervical carcinomas, while HPV-6, although frequently present in benign genital lesions, is only rarely present in cancers of the cervix. Therefore, infections with HPV-16 and HPV-18 are considered high risk and infection with HPV-6 is considered low risk. We found, by using a heterologous promoter system, that expression of the E7 transforming protein differs between high- and low-risk HPVs. In high-risk HPV-16, E7 is expressed from constructs containing the complete upstream E6 open reading frame. In contrast, HPV-6 E7 was efficiently translated only when E6 was deleted. By using clones in which the coding regions of HPV-6, HPV-16, and HPV-18 E7s were preceded by identical leader sequences, we found that the ability of the E7 gene products to induce anchorage-independent growth in rodent fibroblasts correlated directly with the oncogenic association of the HPV types. By using an immortalization assay of normal human keratinocytes that requires complementation of E6 and E7, we found that both E6 and E7 of HPV-18 could complement the corresponding gene from HPV-16. However, neither E6 nor E7 from HPV-6 was able to substitute for the corresponding gene of HPV-16 or HPV-18. Our results suggest that multiple factors, including lower intrinsic biological activity of E6 and E7 and differences in the regulation of their expression, account for the low activity of HPV-6, in comparison with HPV-16 and HPV-18, in in vitro assays. These same factors may, in part, account for the apparent difference in oncogenic potential between these viruses. Images PMID:1845889

  18. Identification and characterization of earthquake clusters: a comparative analysis for selected sequences in Italy

    NASA Astrophysics Data System (ADS)

    Peresan, Antonella; Gentili, Stefania

    2017-04-01

    Identification and statistical characterization of seismic clusters may provide useful insights about the features of seismic energy release and their relation to physical properties of the crust within a given region. Moreover, a number of studies based on spatio-temporal analysis of main-shocks occurrence require preliminary declustering of the earthquake catalogs. Since various methods, relying on different physical/statistical assumptions, may lead to diverse classifications of earthquakes into main events and related events, we aim to investigate the classification differences among different declustering techniques. Accordingly, a formal selection and comparative analysis of earthquake clusters is carried out for the most relevant earthquakes in North-Eastern Italy, as reported in the local OGS-CRS bulletins, compiled at the National Institute of Oceanography and Experimental Geophysics since 1977. The comparison is then extended to selected earthquake sequences associated with a different seismotectonic setting, namely to events that occurred in the region struck by the recent Central Italy destructive earthquakes, making use of INGV data. Various techniques, ranging from classical space-time windows methods to ad hoc manual identification of aftershocks, are applied for detection of earthquake clusters. In particular, a statistical method based on nearest-neighbor distances of events in space-time-energy domain, is considered. Results from clusters identification by the nearest-neighbor method turn out quite robust with respect to the time span of the input catalogue, as well as to minimum magnitude cutoff. The identified clusters for the largest events reported in North-Eastern Italy since 1977 are well consistent with those reported in earlier studies, which were aimed at detailed manual aftershocks identification. The study shows that the data-driven approach, based on the nearest-neighbor distances, can be satisfactorily applied to decompose the seismic

  19. Genome Sequencing and Comparative Analysis of the Biocontrol Agent Trichoderma harzianum sensu stricto TR274

    SciT

    Steindorff, Andrei S.; Noronha, Elilane F.; Ulhoa, Cirano J.

    2015-03-17

    Biological control is a complex process which requires many mechanisms and a high diversity of biochemical pathways. The species of Trichoderma harzianum are well known for their biocontrol activity against many plant pathogens. To gain new insights into the biocontrol mechanism used by T. harzianum, we sequenced the isolate TR274 genome using Illumina. The assembly was performed using AllPaths-LG with a maximum coverage of 100x. The assembly resulted in 2282 contigs with a N50 of 37033bp. The genome size generated was 40.8 Mb and the GC content was 47.7%, similar to other Trichoderma genomes. Using the JGI Annotation Pipeline wemore » predicted 13,932 genes with a high transcriptome support. CEGMA tests suggested 100% genome completeness and 97.9% of RNA-SEQ reads were mapped to the genome. The phylogenetic comparison using orthologous proteins with all Trichoderma genomes sequenced at JGI, corroborates the Trichoderma (T. asperellum and T. atroviride), Longibrachiatum (T. reesei and T. longibrachiatum) and Pachibasium (T. harzianum and T. virens) section division described previously. The comparison between two Trichoderma harzianum species suggests a high genome similarity but some strain-specific expansions. Analyses of the secondary metabolites, CAZymes, transporters, proteases, transcription factors were performed. The Pachybasium section expanded virtually all categories analyzed compared with the other sections, specially Longibrachiatum section, that shows a clear contraction. These results suggests that these proteins families have an important role in their respective phenotypes. Future analysis will improve the understanding of this complex genus and give some insights about its lifestyle and the interactions with the environment.« less

  20. Sequencing and Comparative Analysis of the Straw Mushroom (Volvariella volvacea) Genome

    PubMed Central

    Bao, Dapeng; Gong, Ming; Zheng, Huajun; Chen, Mingjie; Zhang, Liang; Wang, Hong; Jiang, Jianping; Wu, Lin; Zhu, Yongqiang; Zhu, Gang; Zhou, Yan; Li, Chuanhua; Wang, Shengyue; Zhao, Yan; Zhao, Guoping; Tan, Qi

    2013-01-01

    Volvariella volvacea, the edible straw mushroom, is a highly nutritious food source that is widely cultivated on a commercial scale in many parts of Asia using agricultural wastes (rice straw, cotton wastes) as growth substrates. However, developments in V. volvacea cultivation have been limited due to a low biological efficiency (i.e. conversion of growth substrate to mushroom fruit bodies), sensitivity to low temperatures, and an unclear sexuality pattern that has restricted the breeding of improved strains. We have now sequenced the genome of V. volvacea and assembled it into 62 scaffolds with a total genome size of 35.7 megabases (Mb), containing 11,084 predicted gene models. Comparative analyses were performed with the model species in basidiomycete on mating type system, carbohydrate active enzymes, and fungal oxidative lignin enzymes. We also studied transcriptional regulation of the response to low temperature (4°C). We found that the genome of V. volvacea has many genes that code for enzymes, which are involved in the degradation of cellulose, hemicellulose, and pectin. The molecular genetics of the mating type system in V. volvacea was also found to be similar to the bipolar system in basidiomycetes, suggesting that it is secondary homothallism. Sensitivity to low temperatures could be due to the lack of the initiation of the biosynthesis of unsaturated fatty acids, trehalose and glycogen biosyntheses in this mushroom. Genome sequencing of V. volvacea has improved our understanding of the biological characteristics related to the degradation of the cultivating compost consisting of agricultural waste, the sexual reproduction mechanism, and the sensitivity to low temperatures at the molecular level which in turn will enable us to increase the industrial production of this mushroom. PMID:23526973

  1. Genome Sequencing and Comparative Analysis of Saccharomyces cerevisiae Strains of the Peterhof Genetic Collection

    PubMed Central

    Drozdova, Polina B.; Tarasov, Oleg V.; Matveenko, Andrew G.; Radchenko, Elina A.; Sopova, Julia V.; Polev, Dmitrii E.; Inge-Vechtomov, Sergey G.; Dobrynin, Pavel V.

    2016-01-01

    The Peterhof genetic collection of Saccharomyces cerevisiae strains (PGC) is a large laboratory stock that has accumulated several thousands of strains for over than half a century. It originated independently of other common laboratory stocks from a distillery lineage (race XII). Several PGC strains have been extensively used in certain fields of yeast research but their genomes have not been thoroughly explored yet. Here we employed whole genome sequencing to characterize five selected PGC strains including one of the closest to the progenitor, 15V-P4, and several strains that have been used to study translation termination and prions in yeast (25-25-2V-P3982, 1B-D1606, 74-D694, and 6P-33G-D373). The genetic distance between the PGC progenitor and S288C is comparable to that between two geographically isolated populations. The PGC seems to be closer to two bakery strains than to S288C-related laboratory stocks or European wine strains. In genomes of the PGC strains, we found several loci which are absent from the S288C genome; 15V-P4 harbors a rare combination of the gene cluster characteristic for wine strains and the RTM1 cluster. We closely examined known and previously uncharacterized gene variants of particular strains and were able to establish the molecular basis for known phenotypes including phenylalanine auxotrophy, clumping behavior and galactose utilization. Finally, we made sequencing data and results of the analysis available for the yeast community. Our data widen the knowledge about genetic variation between Saccharomyces cerevisiae strains and can form the basis for planning future work in PGC-related strains and with PGC-derived alleles. PMID:27152522

  2. De novo Sequencing and Comparative Transcriptomics of Floral Development of the Distylous Species Lithospermum multiflorum

    PubMed Central

    Cohen, James I.

    2016-01-01

    Genes controlling the morphological, micromorphological, and physiological components of the breeding system distyly have been hypothesized, but many of the genes have not been investigated throughout development of the two floral morphs. To this end, the present study is an examination of comparative transcriptomes from three stages of development for the floral organs of the morphs of Lithospermum multiflorum. Transcriptomes of flowers of the two morphs, from various stages of development, were sequenced using an Illumina HiSeq 2000. The floral transcriptome of L. multiflorum was assembled, and differential gene expression (DE) was identified between morphs, throughout development. Additionally, Gene Ontology (GO) terms for DE genes were determined. Fewer genes were DE early in development compared to later in development, with more genes highly expressed in the gynoecium of the SS morph and the corolla and androecium of the LS morph. A reciprocal pattern was observed later in development, and many more genes were DE during this latter stage. During early development, DE genes appear to be involved in growth and floral development, and during later development, DE genes seem to affect physiological functions. Interestingly, many genes involved in response to stress were identified as DE between morphs. PMID:28066486

  3. De novo Sequencing and Comparative Transcriptomics of Floral Development of the Distylous Species Lithospermum multiflorum.

    PubMed

    Cohen, James I

    2016-01-01

    Genes controlling the morphological, micromorphological, and physiological components of the breeding system distyly have been hypothesized, but many of the genes have not been investigated throughout development of the two floral morphs. To this end, the present study is an examination of comparative transcriptomes from three stages of development for the floral organs of the morphs of Lithospermum multiflorum . Transcriptomes of flowers of the two morphs, from various stages of development, were sequenced using an Illumina HiSeq 2000. The floral transcriptome of L. multiflorum was assembled, and differential gene expression (DE) was identified between morphs, throughout development. Additionally, Gene Ontology (GO) terms for DE genes were determined. Fewer genes were DE early in development compared to later in development, with more genes highly expressed in the gynoecium of the SS morph and the corolla and androecium of the LS morph. A reciprocal pattern was observed later in development, and many more genes were DE during this latter stage. During early development, DE genes appear to be involved in growth and floral development, and during later development, DE genes seem to affect physiological functions. Interestingly, many genes involved in response to stress were identified as DE between morphs.

  4. Comparative sequence analysis of the X-inactivation center region in mouse, human, and bovine.

    PubMed

    Chureau, Corinne; Prissette, Marine; Bourdet, Agnès; Barbe, Valérie; Cattolico, Laurence; Jones, Louis; Eggen, André; Avner, Philip; Duret, Laurent

    2002-06-01

    We have sequenced to high levels of accuracy 714-kb and 233-kb regions of the mouse and bovine X-inactivation centers (Xic), respectively, centered on the Xist gene. This has provided the basis for a fully annotated comparative analysis of the mouse Xic with the 2.3-Mb orthologous region in human and has allowed a three-way species comparison of the core central region, including the Xist gene. These comparisons have revealed conserved genes, both coding and noncoding, conserved CpG islands and, more surprisingly, conserved pseudogenes. The distribution of repeated elements, especially LINE repeats, in the mouse Xic region when compared to the rest of the genome does not support the hypothesis of a role for these repeat elements in the spreading of X inactivation. Interestingly, an asymmetric distribution of LINE elements on the two DNA strands was observed in the three species, not only within introns but also in intergenic regions. This feature is suggestive of important transcriptional activity within these intergenic regions. In silico prediction followed by experimental analysis has allowed four new genes, Cnbp2, Ftx, Jpx, and Ppnx, to be identified and novel, widespread, complex, and apparently noncoding transcriptional activity to be characterized in a region 5' of Xist that was recently shown to attract histone modification early after the onset of X inactivation.

  5. Leveraging the rice genome sequence for monocot comparative and translational genomics.

    PubMed

    Lohithaswa, H C; Feltus, F A; Singh, H P; Bacon, C D; Bailey, C D; Paterson, A H

    2007-07-01

    Common genome anchor points across many taxa greatly facilitate translational and comparative genomics and will improve our understanding of the Tree of Life. To add to the repertoire of genomic tools applicable to the study of monocotyledonous plants in general, we aligned Allium and Musa ESTs to Oryza BAC sequences and identified candidate Allium-Oryza and Musa-Oryza conserved intron-scanning primers (CISPs). A random sampling of 96 CISP primer pairs, representing loci from 11 of the 12 chromosomes in rice, were tested on seven members of the order Poales and on representatives of the Arecales, Asparagales, and Zingiberales monocot orders. The single-copy amplification success rates of Allium (31.3%), Cynodon (31.4%), Hordeum (30.2%), Musa (37.5%), Oryza (61.5%), Pennisetum (33.3%), Sorghum (47.9%), Zea (33.3%), Triticum (30.2%), and representatives of the palm family (32.3%) suggest that subsets of these primers will provide DNA markers suitable for comparative and translational genomics in orphan crops, as well as for applications in conservation biology, ecology, invasion biology, population biology, systematic biology, and related fields.

  6. De Novo Sequencing of a Sparassis latifolia Genome and Its Associated Comparative Analyses

    PubMed Central

    Ma, Lu; Yang, Chi; Ying, Zhenghe; Jiang, Xiaoling

    2018-01-01

    Known to be rich in β-glucan, Sparassis latifolia (S. latifolia) is a valuable edible fungus cultivated in East Asia. A few studies have suggested that S. latifolia is effective on antidiabetic, antihypertension, antitumor, and antiallergen medications. However, it is still unclear genetically why the fungus has these medical effects, which has become a key bottleneck for its further applications. To provide a better understanding of this fungus, we sequenced its whole genome, which has a total size of 48.13 megabases (Mb) and contains 12,471 predicted gene models. We then performed comparative and phylogenetic analyses, which indicate that S. latifolia is closely related to a few species in the antrodia clade including Fomitopsis pinicola, Wolfiporia cocos, Postia placenta, and Antrodia sinuosa. Finally, we annotated the predicted genes. Interestingly, the S. latifolia genome encodes most enzymes involved in carbohydrate and glycoconjugate metabolism and is also enriched in genes encoding enzymes critical to secondary metabolite biosynthesis and involved in indole, terpene, and type I polyketide pathways. As a conclusion, the genome content of S. latifolia sheds light on its genetic basis of the reported medicinal properties and could also be used as a reference genome for comparative studies on fungi. PMID:29682127

  7. Globalization and sustainable development an E7 point of view

    SciT

    Strassburg, W.

    1998-07-01

    The energy sector is a crucial feedstock to economic development. One example of this industry sector's contribution towards sustainable development is the so-called E7 initiative on sustainable energy development of some of the biggest electric utilities. E7 members from Canada, France, Germany, Italy, Japan and the US are demonstrating the functioning of the requested globally synchronized approach between developed and developing countries. E7 members focus their activities on (1) the efficient use of primary energy resources, (2) maximizing the use of renewable energy resources, (3) maximizing efficiency in the generation, delivery, and use of electricity, (4) minimizing environmental impacts ofmore » energy production and use and, (5) implementing innovative options to promote win-win benefits for the environment and the economy through their network of expertise on a pro bono basis. A main emphasis lies in the consistent Joint Implementation given the fact that innovative and efficient Green House Gas reduction measures will be the core of future E7 activities. Especially commercial projects will provide valuable contributions to Green House Gas reductions as well as to economic development of the recipient country. Other instruments, such Emission Trading and Clean Development Mechanism will have to be investigated appropriately in terms of their practicability for Green House Gas reductions so that their will be a notification by the international climate protection regime. Therefore Emission Trading and Clean Development Mechanism are of importance for E7 members' future operations, too. With respect to Sustainable Development industry, energy and environmental policy in leading industry countries must be more closely aligned to a global approach than has so far been the case. The electricity sector is called on to make a major contribution.« less

  8. Analysis of human papillomavirus 16 E6, E7 genes and Long Control Region in cervical samples from Uruguayan women.

    PubMed

    Ramas, Viviana; Mirazo, Santiago; Bonilla, Sylvia; Ruchansky, Dora; Arbiza, Juan

    2018-05-15

    This study aims to investigate the HPV16 variant distribution by sequence analyses of E6, E7 oncogenes and the Long Control Region (LCR), from cervical cells collected from Uruguayan women, and to reconstruct the phylogenetic relationships among variants. Forty-seven HPV16 variants, obtained from women with HSIL, LSIL, ASCUS and NILM cytological classes were analyzed for LCR and 12 were further studied for E6 and E7. Detailed sequence comparison, genetic heterogeneity analyses and phylogenetic reconstruction were performed. A high variability was observed among LCR sequences, which were distributed in 18 different variants. E6 and E7 sequences exhibited novel non-synonymous substitutions. Uruguayan sequences mainly belonged to the European lineage, and only 5 sequences clustered in non-European branches; 3 of them in the Asian-American and North-American linage and 2 in an African branch. Additionally, 6 new variants from European and African clusters were identified. HPV16 isolates mainly belonged to the European lineage, though strains from African and Asian-American lineages were also identified. Herein is reported for the first time the distribution and molecular characterization of HPV16 variants from Uruguay, providing novel insights on the molecular epidemiology of this infectious disease in the South America. A high variability among HPV 16 isolates mainly belonged to European lineage, provides an extensive sequence dataset from a country with high burden of cervical cancer. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. The polymorphisms of LCR, E6, and E7 of HPV-58 isolates in Yunnan, Southwest China.

    PubMed

    Xi, Juemin; Chen, Junying; Xu, Miaoling; Yang, Hongying; Wen, Songjiao; Pan, Yue; Wang, Xiaodan; Ye, Chao; Qiu, Lijuan; Sun, Qiangming

    2018-04-25

    Variations in HPV LCR/E6/E7 have been shown to be associated with the viral persistence and cervical cancer development. So far, there are few reports about the polymorphisms of the HPV-58 LCR/E6/E7 sequences in Southwest China. This study aims to characterize the gene polymorphisms of the HPV-58 LCR/E6/E7 sequences in women of Southwest China, and assess the effects of variations on the immune recognition of viral E6 and E7 antigens. Twelve LCR/E6/E7 of the HPV-58 isolates were amplified and sequenced. A neighbor-joining phylogenetic tree was constructed by MEGA 7.0, followed by the secondary structure prediction of the related proteins using PSIPRED v3.3. The selection pressure acting on the HPV-58 E6 and E7 coding regions was estimated by Bayes empirical Bayes analysis of PAML 4.8. Meanwhile, the MHC class-I and II binding peptides were predicted by the ProPred-I server and ProPred server. The transcription factor binding sites in the HPV-58 LCR were analyzed using the JASPAR database. Twenty nine SNPs (20 in the LCR, 3 in the E6, 6 in the E7) were identified at 27 nucleotide sites across the HPV-58 LCR/E6/E7. From the most variable to the least variable, the nucleotide variations were LCR > E7 > E6. The combinations of all the SNPs resulted in 11 unique sequences, which were clustered into the A lineage (7 belong to A1, 2 belong to A2, and 2 belong to A3). An insertion (TGTCAGTTTCCT) was found between the nucleotide sites 7280 and 7281 in 2 variants, and a deletion (TTTAT) was found between 7429 and 7433 in 1 variant. The most common non-synonymous substitution V77A in the E7 was observed in the sequences encoding the α-helix. 63G in the E7 was determined to be the only one positively selected site in the HPV-58 E6/E7 sequences. Six non-synonymous amino acid substitutions (including S71F and K93 N in the E6, and T20I, G41R, G63S/D, and V77A in the E7) were affecting multiple putative epitopes for both CD4 + and CD8 + T-cells. In the LCR, C7265G and C

  10. Inference of Markovian properties of molecular sequences from NGS data and applications to comparative genomics.

    PubMed

    Ren, Jie; Song, Kai; Deng, Minghua; Reinert, Gesine; Cannon, Charles H; Sun, Fengzhu

    2016-04-01

    Next-generation sequencing (NGS) technologies generate large amounts of short read data for many different organisms. The fact that NGS reads are generally short makes it challenging to assemble the reads and reconstruct the original genome sequence. For clustering genomes using such NGS data, word-count based alignment-free sequence comparison is a promising approach, but for this approach, the underlying expected word counts are essential.A plausible model for this underlying distribution of word counts is given through modeling the DNA sequence as a Markov chain (MC). For single long sequences, efficient statistics are available to estimate the order of MCs and the transition probability matrix for the sequences. As NGS data do not provide a single long sequence, inference methods on Markovian properties of sequences based on single long sequences cannot be directly used for NGS short read data. Here we derive a normal approximation for such word counts. We also show that the traditional Chi-square statistic has an approximate gamma distribution ,: using the Lander-Waterman model for physical mapping. We propose several methods to estimate the order of the MC based on NGS reads and evaluate those using simulations. We illustrate the applications of our results by clustering genomic sequences of several vertebrate and tree species based on NGS reads using alignment-free sequence dissimilarity measures. We find that the estimated order of the MC has a considerable effect on the clustering results ,: and that the clustering results that use a N: MC of the estimated order give a plausible clustering of the species. Our implementation of the statistics developed here is available as R package 'NGS.MC' at http://www-rcf.usc.edu/∼fsun/Programs/NGS-MC/NGS-MC.html fsun@usc.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  11. Comparative Analysis of Four Buckwheat Species Based on Morphology and Complete Chloroplast Genome Sequences.

    PubMed

    Wang, Cheng-Long; Ding, Meng-Qi; Zou, Chen-Yan; Zhu, Xue-Mei; Tang, Yu; Zhou, Mei-Liang; Shao, Ji-Rong

    2017-07-26

    Buckwheat is a nutritional and economically crop belonging to Polygonaceae, Fagopyrum. To better understand the mutation patterns and evolution trend in the chloroplast (cp) genome of buckwheat, and found sufficient number of variable regions to explore the phylogenetic relationships of this genus, two complete cp genomes of buckwheat including Fagopyrum dibotrys (F. dibotrys) and Fagopyrum luojishanense (F. luojishanense) were sequenced, and other two Fagopyrum cp genomes were used for comparative analysis. After morphological analysis, the main difference among these buckwheat were height, leaf shape, seeds and flower type. F. luojishanense was distinguishable from the cultivated species easily. Although the F. dibotrys and two cultivated species has some similarity, they different in habit and component contents. The cp genome of F. dibotrys was 159,320 bp while the F. luojishanense was 159,265 bp. 48 and 61 SSRs were found in F. dibotrys and F. luojishanense respectively. Meanwhile, 10 highly variable regions among these buckwheat species were located precisely. The phylogenetic relationships among four Fagopyrum species based on complete cp genomes was showed. The results suggested that F. dibotrys is more closely related to Fagopyrum tataricum. These data provided valuable genetic information for Fagopyrum species identification, taxonomy, phylogenetic study and molecular breeding.

  12. Comparative genome-wide polymorphic microsatellite markers in Antarctic penguins through next generation sequencing

    PubMed Central

    Vianna, Juliana A.; Noll, Daly; Mura-Jornet, Isidora; Valenzuela-Guerra, Paulina; González-Acuña, Daniel; Navarro, Cristell; Loyola, David E.; Dantas, Gisele P. M.

    2017-01-01

    Abstract Microsatellites are valuable molecular markers for evolutionary and ecological studies. Next generation sequencing is responsible for the increasing number of microsatellites for non-model species. Penguins of the Pygoscelis genus are comprised of three species: Adélie (P. adeliae), Chinstrap (P. antarcticus) and Gentoo penguin (P. papua), all distributed around Antarctica and the sub-Antarctic. The species have been affected differently by climate change, and the use of microsatellite markers will be crucial to monitor population dynamics. We characterized a large set of genome-wide microsatellites and evaluated polymorphisms in all three species. SOLiD reads were generated from the libraries of each species, identifying a large amount of microsatellite loci: 33,677, 35,265 and 42,057 for P. adeliae, P. antarcticus and P. papua, respectively. A large number of dinucleotide (66,139), trinucleotide (29,490) and tetranucleotide (11,849) microsatellites are described. Microsatellite abundance, diversity and orthology were characterized in penguin genomes. We evaluated polymorphisms in 170 tetranucleotide loci, obtaining 34 polymorphic loci in at least one species and 15 polymorphic loci in all three species, which allow to perform comparative studies. Polymorphic markers presented here enable a number of ecological, population, individual identification, parentage and evolutionary studies of Pygoscelis, with potential use in other penguin species. PMID:28898354

  13. Comparative sequence analysis of acid sensitive/resistance proteins in Escherichia coli and Shigella flexneri

    PubMed Central

    Manikandan, Selvaraj; Balaji, Seetharaaman; Kumar, Anil; Kumar, Rita

    2007-01-01

    The molecular basis for the survival of bacteria under extreme conditions in which growth is inhibited is a question of great current interest. A preliminary study was carried out to determine residue pattern conservation among the antiporters of enteric bacteria, responsible for extreme acid sensitivity especially in Escherichia coli and Shigella flexneri. Here we found the molecular evidence that proved the relationship between E. coli and S. flexneri. Multiple sequence alignment of the gadC coded acid sensitive antiporter showed many conserved residue patterns at regular intervals at the N-terminal region. It was observed that as the alignment approaches towards the C-terminal, the number of conserved residues decreases, indicating that the N-terminal region of this protein has much active role when compared to the carboxyl terminal. The motif, FHLVFFLLLGG, is well conserved within the entire gadC coded protein at the amino terminal. The motif is also partially conserved among other antiporters (which are not coded by gadC) but involved in acid sensitive/resistance mechanism. Phylogenetic cluster analysis proves the relationship of Escherichia coli and Shigella flexneri. The gadC coded proteins are converged as a clade and diverged from other antiporters belongs to the amino acid-polyamine-organocation (APC) superfamily. PMID:21670792

  14. Comparative transcriptome analysis of rumen papillae in suckling and weaned Japanese Black calves using RNA sequencing.

    PubMed

    Nishihara, Koki; Kato, Daichi; Suzuki, Yutaka; Kim, Dahye; Nakano, Misato; Yajima, Yu; Haga, Satoshi; Nakano, Miwa; Ishizaki, Hiroshi; Kawahara-Miki, Ryouka; Kono, Tomohiro; Katoh, Kazuo; Roh, Sang-Gun

    2018-06-04

    The length and density of rumen papillae starts to increase during weaning and growth of ruminants. This significant development increases the intraruminal surface area and the efficiency of VFA (acetate, propionate, butyrate, etc.) uptake. Thus, it is important to investigate the factors controlling the growth and development of rumen papillae during weaning. This study aimed to compare the transcriptomes of rumen papillae in suckling and weaned calves. Total RNA was extracted from the rumen papillae of 10 male Japanese Black calves (5 suckling calves, 5 wk old; 5 weaned calves, 15 wk old) and used in RNA-sequencing. Transcript abundance was estimated and differentially expressed genes were identified and these data were then used in Ingenuity Pathway Analysis (IPA) to predict the major canonical pathways and upstream regulators. Among the 871 differentially expressed genes screened by IPA, 466 genes were upregulated and 405 were downregulated in the weaned group. Canonical pathway analysis showed that "atherosclerosis" was the most significant pathway, and "tretinoin," a derivative of vitamin A, was predicted as the most active upstream regulator during weaning. Analyses also predicted IgG, lipopolysaccharides, and tumor-necrosis factor-α as regulators of the microbe-epithelium interaction that activates rumen-related immune responses. The functional category and the up-regulators found in this study provide a valuable resource for studying new candidate genes related to the proliferation and development of rumen papillae from suckling to weaning Japanese Black calves.

  15. Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution.

    PubMed

    2004-12-09

    We present here a draft genome sequence of the red jungle fowl, Gallus gallus. Because the chicken is a modern descendant of the dinosaurs and the first non-mammalian amniote to have its genome sequenced, the draft sequence of its genome--composed of approximately one billion base pairs of sequence and an estimated 20,000-23,000 genes--provides a new perspective on vertebrate genome evolution, while also improving the annotation of mammalian genomes. For example, the evolutionary distance between chicken and human provides high specificity in detecting functional elements, both non-coding and coding. Notably, many conserved non-coding sequences are far from genes and cannot be assigned to defined functional classes. In coding regions the evolutionary dynamics of protein domains and orthologous groups illustrate processes that distinguish the lineages leading to birds and mammals. The distinctive properties of avian microchromosomes, together with the inferred patterns of conserved synteny, provide additional insights into vertebrate chromosome architecture.

  16. Evidence of Divergent Amino Acid Usage in Comparative Analyses of R5- and X4-Associated HIV-1 Vpr Sequences

    PubMed Central

    Antell, Gregory C.; Zhong, Wen; Kercher, Katherine; Passic, Shendra; Williams, Jean; Liu, Yucheng; James, Tony; Jacobson, Jeffrey M.; Szep, Zsofia

    2017-01-01

    Vpr is an HIV-1 accessory protein that plays numerous roles during viral replication, and some of which are cell type dependent. To test the hypothesis that HIV-1 tropism extends beyond the envelope into the vpr gene, studies were performed to identify the associations between coreceptor usage and Vpr variation in HIV-1-infected patients. Colinear HIV-1 Env-V3 and Vpr amino acid sequences were obtained from the LANL HIV-1 sequence database and from well-suppressed patients in the Drexel/Temple Medicine CNS AIDS Research and Eradication Study (CARES) Cohort. Genotypic classification of Env-V3 sequences as X4 (CXCR4-utilizing) or R5 (CCR5-utilizing) was used to group colinear Vpr sequences. To reveal the sequences associated with a specific coreceptor usage genotype, Vpr amino acid sequences were assessed for amino acid diversity and Jensen-Shannon divergence between the two groups. Five amino acid alphabets were used to comprehensively examine the impact of amino acid substitutions involving side chains with similar physiochemical properties. Positions 36, 37, 41, 89, and 96 of Vpr were characterized by statistically significant divergence across multiple alphabets when X4 and R5 sequence groups were compared. In addition, consensus amino acid switches were found at positions 37 and 41 in comparisons of the R5 and X4 sequence populations. These results suggest an evolutionary link between Vpr and gp120 in HIV-1-infected patients. PMID:28620613

  17. Gene discovery in the hamster: a comparative genomics approach for gene annotation by sequencing of hamster testis cDNAs

    PubMed Central

    Oduru, Sreedhar; Campbell, Janee L; Karri, SriTulasi; Hendry, William J; Khan, Shafiq A; Williams, Simon C

    2003-01-01

    Background Complete genome annotation will likely be achieved through a combination of computer-based analysis of available genome sequences combined with direct experimental characterization of expressed regions of individual genomes. We have utilized a comparative genomics approach involving the sequencing of randomly selected hamster testis cDNAs to begin to identify genes not previously annotated on the human, mouse, rat and Fugu (pufferfish) genomes. Results 735 distinct sequences were analyzed for their relatedness to known sequences in public databases. Eight of these sequences were derived from previously unidentified genes and expression of these genes in testis was confirmed by Northern blotting. The genomic locations of each sequence were mapped in human, mouse, rat and pufferfish, where applicable, and the structure of their cognate genes was derived using computer-based predictions, genomic comparisons and analysis of uncharacterized cDNA sequences from human and macaque. Conclusion The use of a comparative genomics approach resulted in the identification of eight cDNAs that correspond to previously uncharacterized genes in the human genome. The proteins encoded by these genes included a new member of the kinesin superfamily, a SET/MYND-domain protein, and six proteins for which no specific function could be predicted. Each gene was expressed primarily in testis, suggesting that they may play roles in the development and/or function of testicular cells. PMID:12783626

  18. Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions.

    PubMed

    Brannon, A Rose; Vakiani, Efsevia; Sylvester, Brooke E; Scott, Sasinya N; McDermott, Gregory; Shah, Ronak H; Kania, Krishan; Viale, Agnes; Oschwald, Dayna M; Vacic, Vladimir; Emde, Anne-Katrin; Cercek, Andrea; Yaeger, Rona; Kemeny, Nancy E; Saltz, Leonard B; Shia, Jinru; D'Angelica, Michael I; Weiser, Martin R; Solit, David B; Berger, Michael F

    2014-08-28

    Colorectal cancer is the second leading cause of cancer death in the United States, with over 50,000 deaths estimated in 2014. Molecular profiling for somatic mutations that predict absence of response to anti-EGFR therapy has become standard practice in the treatment of metastatic colorectal cancer; however, the quantity and type of tissue available for testing is frequently limited. Further, the degree to which the primary tumor is a faithful representation of metastatic disease has been questioned. As next-generation sequencing technology becomes more widely available for clinical use and additional molecularly targeted agents are considered as treatment options in colorectal cancer, it is important to characterize the extent of tumor heterogeneity between primary and metastatic tumors. We performed deep coverage, targeted next-generation sequencing of 230 key cancer-associated genes for 69 matched primary and metastatic tumors and normal tissue. Mutation profiles were 100% concordant for KRAS, NRAS, and BRAF, and were highly concordant for recurrent alterations in colorectal cancer. Additionally, whole genome sequencing of four patient trios did not reveal any additional site-specific targetable alterations. Colorectal cancer primary tumors and metastases exhibit high genomic concordance. As current clinical practices in colorectal cancer revolve around KRAS, NRAS, and BRAF mutation status, diagnostic sequencing of either primary or metastatic tissue as available is acceptable for most patients. Additionally, consistency between targeted sequencing and whole genome sequencing results suggests that targeted sequencing may be a suitable strategy for clinical diagnostic applications.

  19. Human papillomavirus type 16 E7 oncoprotein engages but does not abrogate the mitotic spindle assembly checkpoint

    SciT

    Yu, Yueyang; Munger, Karl, E-mail: kmunger@rics.bwh.harvard.edu

    2012-10-10

    The mitotic spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during mitosis by censoring kinetochore-microtubule interactions. It is frequently rendered dysfunctional during carcinogenesis causing chromosome missegregation and genomic instability. There are conflicting reports whether the HPV16 E7 oncoprotein drives chromosomal instability by abolishing the SAC. Here we report that degradation of mitotic cyclins is impaired in cells with HPV16 E7 expression. RNAi-mediated depletion of Mad2 or BubR1 indicated the involvement of the SAC, suggesting that HPV16 E7 expression causes sustained SAC engagement. Mutational analyses revealed that HPV16 E7 sequences that are necessary for retinoblastoma tumor suppressor protein binding as wellmore » as sequences previously implicated in binding the nuclear and mitotic apparatus (NuMA) protein and in delocalizing dynein from the mitotic spindle contribute to SAC engagement. Importantly, however, HPV16 E7 does not markedly compromise the SAC response to microtubule poisons.« less

  20. Whole genome sequence and comparative analysis of Borrelia burgdorferi MM1

    PubMed Central

    Jabbari, Neda; Reddy, Panga Jaipal; Hood, Leroy

    2018-01-01

    Lyme disease is caused by spirochaetes of the Borrelia burgdorferi sensu lato genospecies. Complete genome assemblies are available for fewer than ten strains of Borrelia burgdorferi sensu stricto, the primary cause of Lyme disease in North America. MM1 is a sensu stricto strain originally isolated in the midwestern United States. Aside from a small number of genes, the complete genome sequence of this strain has not been reported. Here we present the complete genome sequence of MM1 in relation to other sensu stricto strains and in terms of its Multi Locus Sequence Typing. Our results indicate that MM1 is a new sequence type which contains a conserved main chromosome and 15 plasmids. Our results include the first contiguous 28.5 kb assembly of lp28-8, a linear plasmid carrying the vls antigenic variation system, from a Borrelia burgdorferi sensu stricto strain. PMID:29889842

  1. Comparative analysis of ribosomal protein L5 sequences from bacteria of the genus Thermus.

    PubMed

    Jahn, O; Hartmann, R K; Boeckh, T; Erdmann, V A

    1991-06-01

    The genes for the ribosomal 5S rRNA binding protein L5 have been cloned from three extremely thermophilic eubacteria, Thermus flavus, Thermus thermophilus HB8 and Thermus aquaticus (Jahn et al, submitted). Genes for protein L5 from the three Thermus strains display 95% G/C in third positions of codons. Amino acid sequences deduced from the DNA sequence were shown to be identical for T flavus and T thermophilus, although the corresponding DNA sequences differed by two T to C transitions in the T thermophilus gene. Protein L5 sequences from T flavus and T thermophilus are 95% homologous to L5 from T aquaticus and 56.5% homologous to the corresponding E coli sequence. The lowest degrees of homology were found between the T flavus/T thermophilus L5 proteins and those of yeast L16 (27.5%), Halobacterium marismortui (34.0%) and Methanococcus vannielii (36.6%). From sequence comparison it becomes clear that thermostability of Thermus L5 proteins is achieved by an increase in hydrophobic interactions and/or by restriction of steric flexibility due to the introduction of amino acids with branched aliphatic side chains such as leucine. Alignment of the nine protein sequences equivalent to Thermus L5 proteins led to identification of a conserved internal segment, rich in acidic amino acids, which shows homology to subsequences of E coli L18 and L25. The occurrence of conserved sequence elements in 5S rRNA binding proteins and ribosomal proteins in general is discussed in terms of evolution and function.

  2. Genome Sequencing and Comparative Transcriptomics of the Model Entomopathogenic Fungi Metarhizium anisopliae and M. acridum

    PubMed Central

    Shang, Yanfang; Duan, Zhibing; Hu, Xiao; Xie, Xue-Qin; Zhou, Gang; Peng, Guoxiong; Luo, Zhibing; Huang, Wei; Wang, Bing; Fang, Weiguo; Wang, Sibao; Zhong, Yi; Ma, Li-Jun; St. Leger, Raymond J.; Zhao, Guo-Ping; Pei, Yan; Feng, Ming-Guang; Xia, Yuxian; Wang, Chengshu

    2011-01-01

    Metarhizium spp. are being used as environmentally friendly alternatives to chemical insecticides, as model systems for studying insect-fungus interactions, and as a resource of genes for biotechnology. We present a comparative analysis of the genome sequences of the broad-spectrum insect pathogen Metarhizium anisopliae and the acridid-specific M. acridum. Whole-genome analyses indicate that the genome structures of these two species are highly syntenic and suggest that the genus Metarhizium evolved from plant endophytes or pathogens. Both M. anisopliae and M. acridum have a strikingly larger proportion of genes encoding secreted proteins than other fungi, while ∼30% of these have no functionally characterized homologs, suggesting hitherto unsuspected interactions between fungal pathogens and insects. The analysis of transposase genes provided evidence of repeat-induced point mutations occurring in M. acridum but not in M. anisopliae. With the help of pathogen-host interaction gene database, ∼16% of Metarhizium genes were identified that are similar to experimentally verified genes involved in pathogenicity in other fungi, particularly plant pathogens. However, relative to M. acridum, M. anisopliae has evolved with many expanded gene families of proteases, chitinases, cytochrome P450s, polyketide synthases, and nonribosomal peptide synthetases for cuticle-degradation, detoxification, and toxin biosynthesis that may facilitate its ability to adapt to heterogenous environments. Transcriptional analysis of both fungi during early infection processes provided further insights into the genes and pathways involved in infectivity and specificity. Of particular note, M. acridum transcribed distinct G-protein coupled receptors on cuticles from locusts (the natural hosts) and cockroaches, whereas M. anisopliae transcribed the same receptor on both hosts. This study will facilitate the identification of virulence genes and the development of improved biocontrol strains

  3. Genome Sequence of Cronobacter sakazakii BAA-894 and Comparative Genomic Hybridization Analysis with Other Cronobacter Species

    PubMed Central

    Kucerova, Eva; Clifton, Sandra W.; Xia, Xiao-Qin; Long, Fred; Porwollik, Steffen; Fulton, Lucinda; Fronick, Catrina; Minx, Patrick; Kyung, Kim; Warren, Wesley; Fulton, Robert; Feng, Dongyan; Wollam, Aye; Shah, Neha; Bhonagiri, Veena; Nash, William E.; Hallsworth-Pepin, Kymberlie; Wilson, Richard K.

    2010-01-01

    Background The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified. Methodology/Principal Findings We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 kb (51% GC) and 131 kb (56% GC). The genome was used to construct a 387,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10–17% absence of genes. Conclusions/Significance CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from

  4. Efficient use of unlabeled data for protein sequence classification: a comparative study

    PubMed Central

    Kuksa, Pavel; Huang, Pai-Hsi; Pavlovic, Vladimir

    2009-01-01

    Background Recent studies in computational primary protein sequence analysis have leveraged the power of unlabeled data. For example, predictive models based on string kernels trained on sequences known to belong to particular folds or superfamilies, the so-called labeled data set, can attain significantly improved accuracy if this data is supplemented with protein sequences that lack any class tags–the unlabeled data. In this study, we present a principled and biologically motivated computational framework that more effectively exploits the unlabeled data by only using the sequence regions that are more likely to be biologically relevant for better prediction accuracy. As overly-represented sequences in large uncurated databases may bias the estimation of computational models that rely on unlabeled data, we also propose a method to remove this bias and improve performance of the resulting classifiers. Results Combined with state-of-the-art string kernels, our proposed computational framework achieves very accurate semi-supervised protein remote fold and homology detection on three large unlabeled databases. It outperforms current state-of-the-art methods and exhibits significant reduction in running time. Conclusion The unlabeled sequences used under the semi-supervised setting resemble the unpolished gemstones; when used as-is, they may carry unnecessary features and hence compromise the classification accuracy but once cut and polished, they improve the accuracy of the classifiers considerably. PMID:19426450

  5. Efficient use of unlabeled data for protein sequence classification: a comparative study.

    PubMed

    Kuksa, Pavel; Huang, Pai-Hsi; Pavlovic, Vladimir

    2009-04-29

    Recent studies in computational primary protein sequence analysis have leveraged the power of unlabeled data. For example, predictive models based on string kernels trained on sequences known to belong to particular folds or superfamilies, the so-called labeled data set, can attain significantly improved accuracy if this data is supplemented with protein sequences that lack any class tags-the unlabeled data. In this study, we present a principled and biologically motivated computational framework that more effectively exploits the unlabeled data by only using the sequence regions that are more likely to be biologically relevant for better prediction accuracy. As overly-represented sequences in large uncurated databases may bias the estimation of computational models that rely on unlabeled data, we also propose a method to remove this bias and improve performance of the resulting classifiers. Combined with state-of-the-art string kernels, our proposed computational framework achieves very accurate semi-supervised protein remote fold and homology detection on three large unlabeled databases. It outperforms current state-of-the-art methods and exhibits significant reduction in running time. The unlabeled sequences used under the semi-supervised setting resemble the unpolished gemstones; when used as-is, they may carry unnecessary features and hence compromise the classification accuracy but once cut and polished, they improve the accuracy of the classifiers considerably.

  6. Chromosome arm-specific BAC end sequences permit comparative analysis of homoeologous chromosomes and genomes of polyploid wheat

    PubMed Central

    2012-01-01

    Background Bread wheat, one of the world’s staple food crops, has the largest, highly repetitive and polyploid genome among the cereal crops. The wheat genome holds the key to crop genetic improvement against challenges such as climate change, environmental degradation, and water scarcity. To unravel the complex wheat genome, the International Wheat Genome Sequencing Consortium (IWGSC) is pursuing a chromosome- and chromosome arm-based approach to physical mapping and sequencing. Here we report on the use of a BAC library made from flow-sorted telosomic chromosome 3A short arm (t3AS) for marker development and analysis of sequence composition and comparative evolution of homoeologous genomes of hexaploid wheat. Results The end-sequencing of 9,984 random BACs from a chromosome arm 3AS-specific library (TaaCsp3AShA) generated 11,014,359 bp of high quality sequence from 17,591 BAC-ends with an average length of 626 bp. The sequence represents 3.2% of t3AS with an average DNA sequence read every 19 kb. Overall, 79% of the sequence consisted of repetitive elements, 1.38% as coding regions (estimated 2,850 genes) and another 19% of unknown origin. Comparative sequence analysis suggested that 70-77% of the genes present in both 3A and 3B were syntenic with model species. Among the transposable elements, gypsy/sabrina (12.4%) was the most abundant repeat and was significantly more frequent in 3A compared to homoeologous chromosome 3B. Twenty novel repetitive sequences were also identified using de novo repeat identification. BESs were screened to identify simple sequence repeats (SSR) and transposable element junctions. A total of 1,057 SSRs were identified with a density of one per 10.4 kb, and 7,928 junctions between transposable elements (TE) and other sequences were identified with a density of one per 1.39 kb. With the objective of enhancing the marker density of chromosome 3AS, oligonucleotide primers were successfully designed from 758 SSRs and 695

  7. Comparative analysis of Campylobacter isolates from wild birds and chickens using MALDI-TOF MS, biochemical testing, and DNA sequencing.

    PubMed

    Lawton, Samantha J; Weis, Allison M; Byrne, Barbara A; Fritz, Heather; Taff, Conor C; Townsend, Andrea K; Weimer, Bart C; Mete, Aslı; Wheeler, Sarah; Boyce, Walter M

    2018-05-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared to conventional biochemical testing methods and nucleic acid analyses (16S rDNA sequencing, hippurate hydrolysis gene testing, whole genome sequencing [WGS]) for species identification of Campylobacter isolates obtained from chickens ( Gallus gallus domesticus, n = 8), American crows ( Corvus brachyrhynchos, n = 17), a mallard duck ( Anas platyrhynchos, n = 1), and a western scrub-jay ( Aphelocoma californica, n = 1). The test results for all 27 isolates were in 100% agreement between MALDI-TOF MS, the combined results of 16S rDNA sequencing, and the hippurate hydrolysis gene PCR ( p = 0.0027, kappa = 1). Likewise, the identifications derived from WGS from a subset of 14 isolates were in 100% agreement with the MALDI-TOF MS identification. In contrast, biochemical testing misclassified 5 isolates of C. jejuni as C. coli, and 16S rDNA sequencing alone was not able to differentiate between C. coli and C. jejuni for 11 sequences ( p = 0.1573, kappa = 0.0857) when compared to MALDI-TOF MS and WGS. No agreement was observed between MALDI-TOF MS dendrograms and the phylogenetic relationships revealed by rDNA sequencing or WGS. Our results confirm that MALDI-TOF MS is a fast and reliable method for identifying Campylobacter isolates to the species level from wild birds and chickens, but not for elucidating phylogenetic relationships among Campylobacter isolates.

  8. Comparing K-mer based methods for improved classification of 16S sequences.

    PubMed

    Vinje, Hilde; Liland, Kristian Hovde; Almøy, Trygve; Snipen, Lars

    2015-07-01

    The need for precise and stable taxonomic classification is highly relevant in modern microbiology. Parallel to the explosion in the amount of sequence data accessible, there has also been a shift in focus for classification methods. Previously, alignment-based methods were the most applicable tools. Now, methods based on counting K-mers by sliding windows are the most interesting classification approach with respect to both speed and accuracy. Here, we present a systematic comparison on five different K-mer based classification methods for the 16S rRNA gene. The methods differ from each other both in data usage and modelling strategies. We have based our study on the commonly known and well-used naïve Bayes classifier from the RDP project, and four other methods were implemented and tested on two different data sets, on full-length sequences as well as fragments of typical read-length. The difference in classification error obtained by the methods seemed to be small, but they were stable and for both data sets tested. The Preprocessed nearest-neighbour (PLSNN) method performed best for full-length 16S rRNA sequences, significantly better than the naïve Bayes RDP method. On fragmented sequences the naïve Bayes Multinomial method performed best, significantly better than all other methods. For both data sets explored, and on both full-length and fragmented sequences, all the five methods reached an error-plateau. We conclude that no K-mer based method is universally best for classifying both full-length sequences and fragments (reads). All methods approach an error plateau indicating improved training data is needed to improve classification from here. Classification errors occur most frequent for genera with few sequences present. For improving the taxonomy and testing new classification methods, the need for a better and more universal and robust training data set is crucial.

  9. Comparative sequence analysis of B5R gene of zoonotic buffalo pox virus isolates with other orthopoxviruses.

    PubMed

    Chandranaik, B M; Singh, Raj Kumar; Hosamani, Mahusudan; Krishnappa, Giriappa; Harish, Balur R; Chethana, C S; Renukaprasad, C

    2011-02-01

    The present paper describes the isolation of buffalo pox virus from scab lesions and its molecular characterization through B5R gene sequencing. During our study, pustular pox lesions were observed on the teats and mammary parenchyma of cattle and buffaloes, and the disease was of significant zoonotic importance since similar lesions were produced on the hands, legs, and face of people in close contact with the affected animals. The collected scab materials were subjected for virus isolation in 9-11-day-old chicken embryos by the chorioallontoic membrane route and in the Vero cell line. The virus was confirmed by a sensitive and rapid diagnostic polymerase chain reaction using the primers that amplify "A type inclusion" gene, and further, B5R gene of the virus was sequenced and compared with the corresponding sequences of other orthopoxviruses. The results showed high sequence homology of our isolates with other orthopoxviruses.

  10. Mitochondrial genome sequences and comparative genomics ofPhytophthora ramorum and P. sojae

    SciT

    Martin, Frank N.; Douda, Bensasson; Tyler, Brett M.

    The complete sequences of the mitochondrial genomes of theoomycetes of Phytophthora ramorum and P. sojae were determined during thecourse of their complete nuclear genome sequencing (Tyler, et al. 2006).Both are circular, with sizes of 39,314 bp for P. ramorum and 42,975 bpfor P. sojae. Each contains a total of 37 identifiable protein-encodinggenes, 25 or 26 tRNAs (P. sojae and P. ramorum, respectively)specifying19 amino acids, and a variable number of ORFs (7 for P. ramorum and 12for P. sojae) which are potentially additional functional genes.Non-coding regions comprise approximately 11.5 percent and 18.4 percentof the genomes of P. ramorum and P. sojae,more » respectively. Relative to P.sojae, there is an inverted repeat of 1,150 bp in P. ramorum thatincludes an unassigned unique ORF, a tRNA gene, and adjacent non-codingsequences, but otherwise the gene order in both species is identical.Comparisons of these genomes with published sequences of the P. infestansmitochondrial genome reveals a number of similarities, but the gene orderin P. infestans differs in two adjacent locations due to inversions.Sequence alignments of the three genomes indicated sequence conservationranging from 75 to 85 percent and that specific regions were morevariable than others.« less

  11. Cluster Intradermal DNA Vaccination Rapidly Induces E7-specific CD8+ T Cell Immune Responses Leading to Therapeutic Antitumor Effects

    PubMed Central

    Peng, Shiwen; Trimble, Cornelia; Alvarez, Ronald D.; Huh, Warner K.; Lin, Zhenhua; Monie, Archana; Hung, Chien-Fu; Wu, T.-C.

    2010-01-01

    Intradermal administration of DNA vaccines via a gene gun represents a feasible strategy to deliver DNA directly into the professional antigen-presenting cells (APCs) in the skin. This helps to facilitate the enhancement of DNA vaccine potency via strategies that modify the properties of APCs. We have previously demonstrated that DNA vaccines encoding human papillomavirus type 16 (HPV-16) E7 antigen linked to calreticulin (CRT) are capable of enhancing the E7-specific CD8+ T cell immune responses and antitumor effects against E7-expressing tumors. It has also been shown that cluster (short-interval) DNA vaccination regimen generates potent immune responses in a minimal timeframe. Thus, in the current study we hypothesize that the cluster intradermal CRT/E7 DNA vaccination will generate significant antigen-specific CD8+ T cell infiltrates in E7-expressing tumors in tumor-bearing mice, leading to an increase in apoptotic tumor cell death. We found that cluster intradermal CRT/E7 DNA vaccination is capable of rapidly generating a significant number of E7-specific CD8+ T cells, resulting in significant therapeutic antitumor effects in vaccinated mice. We also observed that cluster intradermal CRT/E7 DNA vaccination in the presence of tumor generates significantly higher E7-specific CD8+ T cell immune responses in the systemic circulation as well as in the tumors. In addition, this vaccination regimen also led to significantly lower levels of CD4+Foxp3+ T regulatory cells and myeloid suppressor cells compared to vaccination with CRT DNA in peripheral blood and in tumor infiltrating lymphocytes, resulting in an increase in apoptotic tumor cell death. Thus, our study has significant potential for future clinical translation. PMID:18401437

  12. Solving the problem of comparing whole bacterial genomes across different sequencing platforms.

    PubMed

    Kaas, Rolf S; Leekitcharoenphon, Pimlapas; Aarestrup, Frank M; Lund, Ole

    2014-01-01

    Whole genome sequencing (WGS) shows great potential for real-time monitoring and identification of infectious disease outbreaks. However, rapid and reliable comparison of data generated in multiple laboratories and using multiple technologies is essential. So far studies have focused on using one technology because each technology has a systematic bias making integration of data generated from different platforms difficult. We developed two different procedures for identifying variable sites and inferring phylogenies in WGS data across multiple platforms. The methods were evaluated on three bacterial data sets and sequenced on three different platforms (Illumina, 454, Ion Torrent). We show that the methods are able to overcome the systematic biases caused by the sequencers and infer the expected phylogenies. It is concluded that the cause of the success of these new procedures is due to a validation of all informative sites that are included in the analysis. The procedures are available as web tools.

  13. Genome sequence, comparative analysis and haplotype structure of the domestic dog.

    PubMed

    Lindblad-Toh, Kerstin; Wade, Claire M; Mikkelsen, Tarjei S; Karlsson, Elinor K; Jaffe, David B; Kamal, Michael; Clamp, Michele; Chang, Jean L; Kulbokas, Edward J; Zody, Michael C; Mauceli, Evan; Xie, Xiaohui; Breen, Matthew; Wayne, Robert K; Ostrander, Elaine A; Ponting, Chris P; Galibert, Francis; Smith, Douglas R; DeJong, Pieter J; Kirkness, Ewen; Alvarez, Pablo; Biagi, Tara; Brockman, William; Butler, Jonathan; Chin, Chee-Wye; Cook, April; Cuff, James; Daly, Mark J; DeCaprio, David; Gnerre, Sante; Grabherr, Manfred; Kellis, Manolis; Kleber, Michael; Bardeleben, Carolyne; Goodstadt, Leo; Heger, Andreas; Hitte, Christophe; Kim, Lisa; Koepfli, Klaus-Peter; Parker, Heidi G; Pollinger, John P; Searle, Stephen M J; Sutter, Nathan B; Thomas, Rachael; Webber, Caleb; Baldwin, Jennifer; Abebe, Adal; Abouelleil, Amr; Aftuck, Lynne; Ait-Zahra, Mostafa; Aldredge, Tyler; Allen, Nicole; An, Peter; Anderson, Scott; Antoine, Claudel; Arachchi, Harindra; Aslam, Ali; Ayotte, Laura; Bachantsang, Pasang; Barry, Andrew; Bayul, Tashi; Benamara, Mostafa; Berlin, Aaron; Bessette, Daniel; Blitshteyn, Berta; Bloom, Toby; Blye, Jason; Boguslavskiy, Leonid; Bonnet, Claude; Boukhgalter, Boris; Brown, Adam; Cahill, Patrick; Calixte, Nadia; Camarata, Jody; Cheshatsang, Yama; Chu, Jeffrey; Citroen, Mieke; Collymore, Alville; Cooke, Patrick; Dawoe, Tenzin; Daza, Riza; Decktor, Karin; DeGray, Stuart; Dhargay, Norbu; Dooley, Kimberly; Dooley, Kathleen; Dorje, Passang; Dorjee, Kunsang; Dorris, Lester; Duffey, Noah; Dupes, Alan; Egbiremolen, Osebhajajeme; Elong, Richard; Falk, Jill; Farina, Abderrahim; Faro, Susan; Ferguson, Diallo; Ferreira, Patricia; Fisher, Sheila; FitzGerald, Mike; Foley, Karen; Foley, Chelsea; Franke, Alicia; Friedrich, Dennis; Gage, Diane; Garber, Manuel; Gearin, Gary; Giannoukos, Georgia; Goode, Tina; Goyette, Audra; Graham, Joseph; Grandbois, Edward; Gyaltsen, Kunsang; Hafez, Nabil; Hagopian, Daniel; Hagos, Birhane; Hall, Jennifer; Healy, Claire; Hegarty, Ryan; Honan, Tracey; Horn, Andrea; Houde, Nathan; Hughes, Leanne; Hunnicutt, Leigh; Husby, M; Jester, Benjamin; Jones, Charlien; Kamat, Asha; Kanga, Ben; Kells, Cristyn; Khazanovich, Dmitry; Kieu, Alix Chinh; Kisner, Peter; Kumar, Mayank; Lance, Krista; Landers, Thomas; Lara, Marcia; Lee, William; Leger, Jean-Pierre; Lennon, Niall; Leuper, Lisa; LeVine, Sarah; Liu, Jinlei; Liu, Xiaohong; Lokyitsang, Yeshi; Lokyitsang, Tashi; Lui, Annie; Macdonald, Jan; Major, John; Marabella, Richard; Maru, Kebede; Matthews, Charles; McDonough, Susan; Mehta, Teena; Meldrim, James; Melnikov, Alexandre; Meneus, Louis; Mihalev, Atanas; Mihova, Tanya; Miller, Karen; Mittelman, Rachel; Mlenga, Valentine; Mulrain, Leonidas; Munson, Glen; Navidi, Adam; Naylor, Jerome; Nguyen, Tuyen; Nguyen, Nga; Nguyen, Cindy; Nguyen, Thu; Nicol, Robert; Norbu, Nyima; Norbu, Choe; Novod, Nathaniel; Nyima, Tenchoe; Olandt, Peter; O'Neill, Barry; O'Neill, Keith; Osman, Sahal; Oyono, Lucien; Patti, Christopher; Perrin, Danielle; Phunkhang, Pema; Pierre, Fritz; Priest, Margaret; Rachupka, Anthony; Raghuraman, Sujaa; Rameau, Rayale; Ray, Verneda; Raymond, Christina; Rege, Filip; Rise, Cecil; Rogers, Julie; Rogov, Peter; Sahalie, Julie; Settipalli, Sampath; Sharpe, Theodore; Shea, Terrance; Sheehan, Mechele; Sherpa, Ngawang; Shi, Jianying; Shih, Diana; Sloan, Jessie; Smith, Cherylyn; Sparrow, Todd; Stalker, John; Stange-Thomann, Nicole; Stavropoulos, Sharon; Stone, Catherine; Stone, Sabrina; Sykes, Sean; Tchuinga, Pierre; Tenzing, Pema; Tesfaye, Senait; Thoulutsang, Dawa; Thoulutsang, Yama; Topham, Kerri; Topping, Ira; Tsamla, Tsamla; Vassiliev, Helen; Venkataraman, Vijay; Vo, Andy; Wangchuk, Tsering; Wangdi, Tsering; Weiand, Michael; Wilkinson, Jane; Wilson, Adam; Yadav, Shailendra; Yang, Shuli; Yang, Xiaoping; Young, Geneva; Yu, Qing; Zainoun, Joanne; Zembek, Lisa; Zimmer, Andrew; Lander, Eric S

    2005-12-08

    Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.

  14. Comparative Sequence Analysis of Plasmids from Lactobacillus delbrueckii and Construction of a Shuttle Cloning Vector▿

    PubMed Central

    Lee, Ju-Hoon; Halgerson, Jamie S.; Kim, Jeong-Hwan; O'Sullivan, Daniel J.

    2007-01-01

    While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three—a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria. PMID:17526779

  15. The complete chloroplast DNA sequence of Eleutherococcus senticosus (Araliaceae); comparative evolutionary analyses with other three asterids.

    PubMed

    Yi, Dong-Keun; Lee, Hae-Lim; Sun, Byung-Yun; Chung, Mi Yoon; Kim, Ki-Joong

    2012-05-01

    This study reports the complete chloroplast (cp) DNA sequence of Eleutherococcus senticosus (GenBank: JN 637765), an endangered endemic species. The genome is 156,768 bp in length, and contains a pair of inverted repeat (IR) regions of 25,930 bp each, a large single copy (LSC) region of 86,755 bp and a small single copy (SSC) region of 18,153 bp. The structural organization, gene and intron contents, gene order, AT content, codon usage, and transcription units of the E. senticosus chloroplast genome are similar to that of typical land plant cp DNA. We aligned and analyzed the sequences of 86 coding genes, 19 introns and 113 intergenic spacers (IGS) in three different taxonomic hierarchies; Eleutherococcus vs. Panax, Eleutherococcus vs. Daucus, and Eleutherococcus vs. Nicotiana. The distribution of indels, the number of polymorphic sites and nucleotide diversity indicate that positional constraint is more important than functional constraint for the evolution of cp genome sequences in Asterids. For example, the intron sequences in the LSC region exhibited base substitution rates 5-11-times higher than that of the IR regions, while the intron sequences in the SSC region evolved 7-14-times faster than those in the IR region. Furthermore, the Ka/Ks ratio of the gene coding sequences supports a stronger evolutionary constraint in the IR region than in the LSC or SSC regions. Therefore, our data suggest that selective sweeps by base collection mechanisms more frequently eliminate polymorphisms in the IR region than in other regions. Chloroplast genome regions that have high levels of base substitutions also show higher incidences of indels. Thirty-five simple sequence repeat (SSR) loci were identified in the Eleutherococcus chloroplast genome. Of these, 27 are homopolymers, while six are di-polymers and two are tri-polymers. In addition to the SSR loci, we also identified 18 medium size repeat units ranging from 22 to 79 bp, 11 of which are distributed in the IGS or

  16. Comparative Chloroplast Genomics of Gossypium Species: Insights Into Repeat Sequence Variations and Phylogeny

    PubMed Central

    Wu, Ying; Liu, Fang; Yang, Dai-Gang; Li, Wei; Zhou, Xiao-Jian; Pei, Xiao-Yu; Liu, Yan-Gai; He, Kun-Lun; Zhang, Wen-Sheng; Ren, Zhong-Ying; Zhou, Ke-Hai; Ma, Xiong-Feng; Li, Zhong-Hu

    2018-01-01

    Cotton is one of the most economically important fiber crop plants worldwide. The genus Gossypium contains a single allotetraploid group (AD) and eight diploid genome groups (A–G and K). However, the evolution of repeat sequences in the chloroplast genomes and the phylogenetic relationships of Gossypium species are unclear. Thus, we determined the variations in the repeat sequences and the evolutionary relationships of 40 cotton chloroplast genomes, which represented the most diverse in the genus, including five newly sequenced diploid species, i.e., G. nandewarense (C1-n), G. armourianum (D2-1), G. lobatum (D7), G. trilobum (D8), and G. schwendimanii (D11), and an important semi-wild race of upland cotton, G. hirsutum race latifolium (AD1). The genome structure, gene order, and GC content of cotton species were similar to those of other higher plant plastid genomes. In total, 2860 long sequence repeats (>10 bp in length) were identified, where the F-genome species had the largest number of repeats (G. longicalyx F1: 108) and E-genome species had the lowest (G. stocksii E1: 53). Large-scale repeat sequences possibly enrich the genetic information and maintain genome stability in cotton species. We also identified 10 divergence hotspot regions, i.e., rpl33-rps18, psbZ-trnG (GCC), rps4-trnT (UGU), trnL (UAG)-rpl32, trnE (UUC)-trnT (GGU), atpE, ndhI, rps2, ycf1, and ndhF, which could be useful molecular genetic markers for future population genetics and phylogenetic studies. Site-specific selection analysis showed that some of the coding sites of 10 chloroplast genes (atpB, atpE, rps2, rps3, petB, petD, ccsA, cemA, ycf1, and rbcL) were under protein sequence evolution. Phylogenetic analysis based on the whole plastomes suggested that the Gossypium species grouped into six previously identified genetic clades. Interestingly, all 13 D-genome species clustered into a strong monophyletic clade. Unexpectedly, the cotton species with C, G, and K-genomes were admixed and

  17. The complete chloroplast genome sequence of Epipremnum aureum and its comparative analysis among eight Araceae species

    PubMed Central

    Han, Limin; Chen, Chen; Wang, Zhezhi

    2018-01-01

    Epipremnum aureum is an important foliage plant in the Araceae family. In this study, we have sequenced the complete chloroplast genome of E. aureum by using Illumina Hiseq sequencing platforms. This genome is a double-stranded circular DNA sequence of 164,831 bp that contains 35.8% GC. The two inverted repeats (IRa and IRb; 26,606 bp) are spaced by a small single-copy region (22,868 bp) and a large single-copy region (88,751 bp). The chloroplast genome has 131 (113 unique) functional genes, including 86 (79 unique) protein-coding genes, 37 (30 unique) tRNA genes, and eight (four unique) rRNA genes. Tandem repeats comprise the majority of the 43 long repetitive sequences. In addition, 111 simple sequence repeats are present, with mononucleotides being the most common type and di- and tetranucleotides being infrequent events. Positive selection pressure on rps12 in the E. aureum chloroplast has been demonstrated via synonymous and nonsynonymous substitution rates and selection pressure sites analyses. Ycf15 and infA are pseudogenes in this species. We constructed a Maximum Likelihood phylogenetic tree based on the complete chloroplast genomes of 38 species from 13 families. Those results strongly indicated that E. aureum is positioned as the sister of Colocasia esculenta within the Araceae family. This work may provide information for further study of the molecular phylogenetic relationships within Araceae, as well as molecular markers and breeding novel varieties by chloroplast genetic-transformation of E. aureum in particular. PMID:29529038

  18. Five Complete Chloroplast Genome Sequences from Diospyros: Genome Organization and Comparative Analysis.

    PubMed

    Fu, Jianmin; Liu, Huimin; Hu, Jingjing; Liang, Yuqin; Liang, Jinjun; Wuyun, Tana; Tan, Xiaofeng

    2016-01-01

    Diospyros is the largest genus in Ebenaceae, comprising more than 500 species with remarkable economic value, especially Diospyros kaki Thunb., which has traditionally been an important food resource in China, Korea, and Japan. Complete chloroplast (cp) genomes from D. kaki, D. lotus L., D. oleifera Cheng., D. glaucifolia Metc., and Diospyros 'Jinzaoshi' were sequenced using Illumina sequencing technology. This is the first cp genome reported in Ebenaceae. The cp genome sequences of Diospyros ranged from 157,300 to 157,784 bp in length, presenting a typical quadripartite structure with two inverted repeats each separated by one large and one small single-copy region. For each cp genome, 134 genes were annotated, including 80 protein-coding, 31 tRNA, and 4 rRNA unique genes. In all, 179 repeats and 283 single sequence repeats were identified. Four hypervariable regions, namely, intergenic region of trnQ_rps16, trnV_ndhC, and psbD_trnT, and intron of ndhA, were identified in the Diospyros genomes. Phylogenetic analyses based on the whole cp genome, protein-coding, and intergenic and intron sequences indicated that D. oleifera is closely related to D. kaki and could be used as a model plant for future research on D. kaki; to our knowledge, this is proposed for the first time. Further, these analyses together with two large deletions (301 and 140 bp) in the cp genome of D. 'Jinzaoshi', support its placement as a new species in Diospyros. Both maximum parsimony and likelihood analyses for 19 taxa indicated the basal position of Ericales in asterids and suggested that Ebenaceae is monophyletic in Ericales.

  19. Five Complete Chloroplast Genome Sequences from Diospyros: Genome Organization and Comparative Analysis

    PubMed Central

    Hu, Jingjing; Liang, Yuqin; Liang, Jinjun; Wuyun, Tana; Tan, Xiaofeng

    2016-01-01

    Diospyros is the largest genus in Ebenaceae, comprising more than 500 species with remarkable economic value, especially Diospyros kaki Thunb., which has traditionally been an important food resource in China, Korea, and Japan. Complete chloroplast (cp) genomes from D. kaki, D. lotus L., D. oleifera Cheng., D. glaucifolia Metc., and Diospyros ‘Jinzaoshi’ were sequenced using Illumina sequencing technology. This is the first cp genome reported in Ebenaceae. The cp genome sequences of Diospyros ranged from 157,300 to 157,784 bp in length, presenting a typical quadripartite structure with two inverted repeats each separated by one large and one small single-copy region. For each cp genome, 134 genes were annotated, including 80 protein-coding, 31 tRNA, and 4 rRNA unique genes. In all, 179 repeats and 283 single sequence repeats were identified. Four hypervariable regions, namely, intergenic region of trnQ_rps16, trnV_ndhC, and psbD_trnT, and intron of ndhA, were identified in the Diospyros genomes. Phylogenetic analyses based on the whole cp genome, protein-coding, and intergenic and intron sequences indicated that D. oleifera is closely related to D. kaki and could be used as a model plant for future research on D. kaki; to our knowledge, this is proposed for the first time. Further, these analyses together with two large deletions (301 and 140 bp) in the cp genome of D. ‘Jinzaoshi’, support its placement as a new species in Diospyros. Both maximum parsimony and likelihood analyses for 19 taxa indicated the basal position of Ericales in asterids and suggested that Ebenaceae is monophyletic in Ericales. PMID:27442423

  20. Comparative characterization of random-sequence proteins consisting of 5, 12, and 20 kinds of amino acids

    PubMed Central

    Tanaka, Junko; Doi, Nobuhide; Takashima, Hideaki; Yanagawa, Hiroshi

    2010-01-01

    Screening of functional proteins from a random-sequence library has been used to evolve novel proteins in the field of evolutionary protein engineering. However, random-sequence proteins consisting of the 20 natural amino acids tend to aggregate, and the occurrence rate of functional proteins in a random-sequence library is low. From the viewpoint of the origin of life, it has been proposed that primordial proteins consisted of a limited set of amino acids that could have been abundantly formed early during chemical evolution. We have previously found that members of a random-sequence protein library constructed with five primitive amino acids show high solubility (Doi et al., Protein Eng Des Sel 2005;18:279–284). Although such a library is expected to be appropriate for finding functional proteins, the functionality may be limited, because they have no positively charged amino acid. Here, we constructed three libraries of 120-amino acid, random-sequence proteins using alphabets of 5, 12, and 20 amino acids by preselection using mRNA display (to eliminate sequences containing stop codons and frameshifts) and characterized and compared the structural properties of random-sequence proteins arbitrarily chosen from these libraries. We found that random-sequence proteins constructed with the 12-member alphabet (including five primitive amino acids and positively charged amino acids) have higher solubility than those constructed with the 20-member alphabet, though other biophysical properties are very similar in the two libraries. Thus, a library of moderate complexity constructed from 12 amino acids may be a more appropriate resource for functional screening than one constructed from 20 amino acids. PMID:20162614

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    Bacillus subtilis is the main component in the fermentation of soybeans. To investigate the genetics of the soybean-fermenting B. subtilis strains and its relationship with the productivity of extracellular poly-γ-glutamic acid (γPGA), we sequenced the whole genome of eight B. subtilis stains isolated from non-salted fermented soybean foods in Southeast Asia. Assembled nucleotide sequences were compared with those of a natto (fermented soybean food) starter strain B. subtilis BEST195 and the laboratory standard strain B. subtilis 168 that is incapable of γPGA production. Detected variants were investigated in terms of insertion sequences, biotin synthesis, production of subtilisin NAT, and regulatory genes for γPGA synthesis, which were related to fermentation process. Comparing genome sequences, we found that the strains that produce γPGA have a deletion in a protein that constitutes the flagellar basal body, and this deletion was not found in the non-producing strains. We further identified diversity in variants of the bio operon, which is responsible for the biotin auxotrophism of the natto starter strains. Phylogenetic analysis using multilocus sequencing typing revealed that the B. subtilis strains isolated from the non-salted fermented soybeans were not clustered together, while the natto-fermenting strains were tightly clustered; this analysis also suggested that the strain isolated from “Tua Nao” of Thailand traces a different evolutionary process from other strains. PMID:26505996

  3. Phenotype–genotype correlation in Hirschsprung disease is illuminated by comparative analysis of the RET protein sequence

    PubMed Central

    Kashuk, Carl S.; Stone, Eric A.; Grice, Elizabeth A.; Portnoy, Matthew E.; Green, Eric D.; Sidow, Arend; Chakravarti, Aravinda; McCallion, Andrew S.

    2005-01-01

    The ability to discriminate between deleterious and neutral amino acid substitutions in the genes of patients remains a significant challenge in human genetics. The increasing availability of genomic sequence data from multiple vertebrate species allows inclusion of sequence conservation and physicochemical properties of residues to be used for functional prediction. In this study, the RET receptor tyrosine kinase serves as a model disease gene in which a broad spectrum (≥116) of disease-associated mutations has been identified among patients with Hirschsprung disease and multiple endocrine neoplasia type 2. We report the alignment of the human RET protein sequence with the orthologous sequences of 12 non-human vertebrates (eight mammalian, one avian, and three teleost species), their comparative analysis, the evolutionary topology of the RET protein, and predicted tolerance for all published missense mutations. We show that, although evolutionary conservation alone provides significant information to predict the effect of a RET mutation, a model that combines comparative sequence data with analysis of physiochemical properties in a quantitative framework provides far greater accuracy. Although the ability to discern the impact of a mutation is imperfect, our analyses permit substantial discrimination between predicted functional classes of RET mutations and disease severity even for a multigenic disease such as Hirschsprung disease. PMID:15956201

  4. Identification of RNA Aptamers that Internalize into HPV-16 E6/E7 Transformed Tonsillar Epithelial Cells

    PubMed Central

    Gourronc, Francoise A.; Rockey, William M.; Thiel, William H.; Giangrande, Paloma H.; Klingelhutz, Aloysius J.

    2013-01-01

    Human papillomavirus type 16 (HPV-16) associated oropharyngeal cancers are on a significant increase and better therapeutic strategies are needed. The HPV-16 oncogenes E6 and E7 are expressed in HPV-associated cancers and are able to transform human tonsillar epithelial cells (HTECs). We used cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to select for RNA aptamers that entered into HPV-16 E6/E7-HTECs. After 12 rounds of cell-SELEX, a pool of aptamers was obtained that had significantly greater internalization capacity (~5-fold) into E6/E7-HTECs as compared to primary HTECs or fibroblasts. Analysis of individual aptamers from the pool indicated variable internalization into E6/E7-HTECs (1 to 8-fold as compared to a negative control). Most of the individual aptamers internalized into E6/E7 and primary HTECs with similar efficiency, while one aptamer exhibited ~3-fold better internalization into E6/E7-HTECs. Aptamers that internalize into cells may be useful for delivering therapeutic agents to HPV-16 associated malignancies. PMID:24074596

  5. G-Anchor: a novel approach for whole-genome comparative mapping utilizing evolutionary conserved DNA sequences.

    PubMed

    Lenis, Vasileios Panagiotis E; Swain, Martin; Larkin, Denis M

    2018-05-01

    Cross-species whole-genome sequence alignment is a critical first step for genome comparative analyses, ranging from the detection of sequence variants to studies of chromosome evolution. Animal genomes are large and complex, and whole-genome alignment is a computationally intense process, requiring expensive high-performance computing systems due to the need to explore extensive local alignments. With hundreds of sequenced animal genomes available from multiple projects, there is an increasing demand for genome comparative analyses. Here, we introduce G-Anchor, a new, fast, and efficient pipeline that uses a strictly limited but highly effective set of local sequence alignments to anchor (or map) an animal genome to another species' reference genome. G-Anchor makes novel use of a databank of highly conserved DNA sequence elements. We demonstrate how these elements may be aligned to a pair of genomes, creating anchors. These anchors enable the rapid mapping of scaffolds from a de novo assembled genome to chromosome assemblies of a reference species. Our results demonstrate that G-Anchor can successfully anchor a vertebrate genome onto a phylogenetically related reference species genome using a desktop or laptop computer within a few hours and with comparable accuracy to that achieved by a highly accurate whole-genome alignment tool such as LASTZ. G-Anchor thus makes whole-genome comparisons accessible to researchers with limited computational resources. G-Anchor is a ready-to-use tool for anchoring a pair of vertebrate genomes. It may be used with large genomes that contain a significant fraction of evolutionally conserved DNA sequences and that are not highly repetitive, polypoid, or excessively fragmented. G-Anchor is not a substitute for whole-genome aligning software but can be used for fast and accurate initial genome comparisons. G-Anchor is freely available and a ready-to-use tool for the pairwise comparison of two genomes.

  6. The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis.

    PubMed

    Yao, Xiaohong; Tang, Ping; Li, Zuozhou; Li, Dawei; Liu, Yifei; Huang, Hongwen

    2015-01-01

    Actinidia chinensis is an important economic plant belonging to the basal lineage of the asterids. Availability of a complete Actinidia chloroplast genome sequence is crucial to understanding phylogenetic relationships among major lineages of angiosperms and facilitates kiwifruit genetic improvement. We report here the complete nucleotide sequences of the chloroplast genomes for Actinidia chinensis and A. chinensis var deliciosa obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. The total genome size ranges from 155,446 to 157,557 bp, with an inverted repeat (IR) of 24,013 to 24,391 bp, a large single copy region (LSC) of 87,984 to 88,337 bp and a small single copy region (SSC) of 20,332 to 20,336 bp. The genome encodes 113 different genes, including 79 unique protein-coding genes, 30 tRNA genes and 4 ribosomal RNA genes, with 16 duplicated in the inverted repeats, and a tRNA gene (trnfM-CAU) duplicated once in the LSC region. Comparisons of IR boundaries among four asterid species showed that IR/LSC borders were extended into the 5' portion of the psbA gene and IR contraction occurred in Actinidia. The clap gene has been lost from the chloroplast genome in Actinidia, and may have been transferred to the nucleus during chloroplast evolution. Twenty-seven polymorphic simple sequence repeat (SSR) loci were identified in the Actinidia chloroplast genome. Maximum parsimony analyses of a 72-gene, 16 taxa angiosperm dataset strongly support the placement of Actinidiaceae in Ericales within the basal asterids.

  7. A 1463 Gene Cattle–Human Comparative Map With Anchor Points Defined by Human Genome Sequence Coordinates

    PubMed Central

    Everts-van der Wind, Annelie; Kata, Srinivas R.; Band, Mark R.; Rebeiz, Mark; Larkin, Denis M.; Everts, Robin E.; Green, Cheryl A.; Liu, Lei; Natarajan, Shreedhar; Goldammer, Tom; Lee, Jun Heon; McKay, Stephanie; Womack, James E.; Lewin, Harris A.

    2004-01-01

    A second-generation 5000 rad radiation hybrid (RH) map of the cattle genome was constructed primarily using cattle ESTs that were targeted to gaps in the existing cattle–human comparative map, as well as to sparsely populated map intervals. A total of 870 targeted markers were added, bringing the number of markers mapped on the RH5000 panel to 1913. Of these, 1463 have significant BLASTN hits (E < e–5) against the human genome sequence. A cattle–human comparative map was created using human genome sequence coordinates of the paired orthologs. One-hundred and ninety-five conserved segments (defined by two or more genes) were identified between the cattle and human genomes, of which 31 are newly discovered and 34 were extended singletons on the first-generation map. The new map represents an improvement of 20% genome-wide comparative coverage compared with the first-generation map. Analysis of gene content within human genome regions where there are gaps in the comparative map revealed gaps with both significantly greater and significantly lower gene content. The new, more detailed cattle–human comparative map provides an improved resource for the analysis of mammalian chromosome evolution, the identification of candidate genes for economically important traits, and for proper alignment of sequence contigs on cattle chromosomes. PMID:15231756

  8. Comparative and Evolutionary Analyses of Meloidogyne spp. Based on Mitochondrial Genome Sequences

    PubMed Central

    García, Laura Evangelina; Sánchez-Puerta, M. Virginia

    2015-01-01

    Molecular taxonomy and evolution of nematodes have been recently the focus of several studies. Mitochondrial sequences were proposed as an alternative for precise identification of Meloidogyne species, to study intraspecific variability and to follow maternal lineages. We characterized the mitochondrial genomes (mtDNAs) of the root knot nematodes M. floridensis, M. hapla and M. incognita. These were AT rich (81–83%) and highly compact, encoding 12 proteins, 2 rRNAs, and 22 tRNAs. Comparisons with published mtDNAs of M. chitwoodi, M. incognita (another strain) and M. graminicola revealed that they share protein and rRNA gene order but differ in the order of tRNAs. The mtDNAs of M. floridensis and M. incognita were strikingly similar (97–100% identity for all coding regions). In contrast, M. floridensis, M. chitwoodi, M. hapla and M. graminicola showed 65–84% nucleotide identity for coding regions. Variable mitochondrial sequences are potentially useful for evolutionary and taxonomic studies. We developed a molecular taxonomic marker by sequencing a highly-variable ~2 kb mitochondrial region, nad5-cox1, from 36 populations of root-knot nematodes to elucidate relationships within the genus Meloidogyne. Isolates of five species formed monophyletic groups and showed little intraspecific variability. We also present a thorough analysis of the mitochondrial region cox2-rrnS. Phylogenies based on either mitochondrial region had good discrimination power but could not discriminate between M. arenaria, M. incognita and M. floridensis. PMID:25799071

  9. Artemis and ACT: viewing, annotating and comparing sequences stored in a relational database.

    PubMed

    Carver, Tim; Berriman, Matthew; Tivey, Adrian; Patel, Chinmay; Böhme, Ulrike; Barrell, Barclay G; Parkhill, Julian; Rajandream, Marie-Adèle

    2008-12-01

    Artemis and Artemis Comparison Tool (ACT) have become mainstream tools for viewing and annotating sequence data, particularly for microbial genomes. Since its first release, Artemis has been continuously developed and supported with additional functionality for editing and analysing sequences based on feedback from an active user community of laboratory biologists and professional annotators. Nevertheless, its utility has been somewhat restricted by its limitation to reading and writing from flat files. Therefore, a new version of Artemis has been developed, which reads from and writes to a relational database schema, and allows users to annotate more complex, often large and fragmented, genome sequences. Artemis and ACT have now been extended to read and write directly to the Generic Model Organism Database (GMOD, http://www.gmod.org) Chado relational database schema. In addition, a Gene Builder tool has been developed to provide structured forms and tables to edit coordinates of gene models and edit functional annotation, based on standard ontologies, controlled vocabularies and free text. Artemis and ACT are freely available (under a GPL licence) for download (for MacOSX, UNIX and Windows) at the Wellcome Trust Sanger Institute web sites: http://www.sanger.ac.uk/Software/Artemis/ http://www.sanger.ac.uk/Software/ACT/

  10. Comparative study of the effectiveness and limitations of current methods for detecting sequence coevolution.

    PubMed

    Mao, Wenzhi; Kaya, Cihan; Dutta, Anindita; Horovitz, Amnon; Bahar, Ivet

    2015-06-15

    With rapid accumulation of sequence data on several species, extracting rational and systematic information from multiple sequence alignments (MSAs) is becoming increasingly important. Currently, there is a plethora of computational methods for investigating coupled evolutionary changes in pairs of positions along the amino acid sequence, and making inferences on structure and function. Yet, the significance of coevolution signals remains to be established. Also, a large number of false positives (FPs) arise from insufficient MSA size, phylogenetic background and indirect couplings. Here, a set of 16 pairs of non-interacting proteins is thoroughly examined to assess the effectiveness and limitations of different methods. The analysis shows that recent computationally expensive methods designed to remove biases from indirect couplings outperform others in detecting tertiary structural contacts as well as eliminating intermolecular FPs; whereas traditional methods such as mutual information benefit from refinements such as shuffling, while being highly efficient. Computations repeated with 2,330 pairs of protein families from the Negatome database corroborated these results. Finally, using a training dataset of 162 families of proteins, we propose a combined method that outperforms existing individual methods. Overall, the study provides simple guidelines towards the choice of suitable methods and strategies based on available MSA size and computing resources. Software is freely available through the Evol component of ProDy API. © The Author 2015. Published by Oxford University Press.

  11. Comparative transcriptome sequencing and de novo analysis of Vaccinium corymbosum during fruit and color development.

    PubMed

    Li, Lingli; Zhang, Hehua; Liu, Zhongshuai; Cui, Xiaoyue; Zhang, Tong; Li, Yanfang; Zhang, Lingyun

    2016-10-12

    Blueberry is an economically important fruit crop in Ericaceae family. The substantial quantities of flavonoids in blueberry have been implicated in a broad range of health benefits. However, the information regarding fruit development and flavonoid metabolites based on the transcriptome level is still limited. In the present study, the transcriptome and gene expression profiling over berry development, especially during color development were initiated. A total of approximately 13.67 Gbp of data were obtained and assembled into 186,962 transcripts and 80,836 unigenes from three stages of blueberry fruit and color development. A large number of simple sequence repeats (SSRs) and candidate genes, which are potentially involved in plant development, metabolic and hormone pathways, were identified. A total of 6429 sequences containing 8796 SSRs were characterized from 15,457 unigenes and 1763 unigenes contained more than one SSR. The expression profiles of key genes involved in anthocyanin biosynthesis were also studied. In addition, a comparison between our dataset and other published results was carried out. Our high quality reads produced in this study are an important advancement and provide a new resource for the interpretation of high-throughput data for blueberry species whether regarding sequencing data depth or species extension. The use of this transcriptome data will serve as a valuable public information database for the studies of blueberry genome and would greatly boost the research of fruit and color development, flavonoid metabolisms and regulation and breeding of more healthful blueberries.

  12. Comparative oncology DNA sequencing of canine T cell lymphoma via human hotspot panel

    PubMed Central

    Beheshti, Afshin; Pilichowska, Monika; Burgess, Kristine; Ricks-Santi, Luisel; McNiel, Elizabeth; London, Cheryl B.; Ravi, Dashnamoorthy; Evens, Andrew M.

    2018-01-01

    T-cell lymphoma (TCL) is an uncommon and aggressive form of human cancer. Lymphoma is the most common hematopoietic tumor in canines (companion animals), with TCL representing approximately 30% of diagnoses. Collectively, the canine is an appealing model for cancer research given the spontaneous occurrence of cancer, intact immune system, and phytogenetic proximity to humans. We sought to establish mutational congruence of the canine with known human TCL mutations in order to identify potential actionable oncogenic pathways. Following pathologic confirmation, DNA was sequenced in 16 canine TCL (cTCL) cases using a custom Human Cancer Hotspot Panel of 68 genes commonly mutated in human TCL. Sequencing identified 4,527,638 total reads with average length of 229 bases containing 346 unique variants and 1,474 total variants; each sample had an average of 92 variants. Among these, there were 258 germline and 32 somatic variants. Among the 32 somatic variants there were 8 missense variants, 1 splice junction variant and the remaining were intron or synonymous variants. A frequency of 4 somatic mutations per sample were noted with >7 mutations detected in MET, KDR, STK11 and BRAF. Expression of these associated proteins were also detected via Western blot analyses. In addition, Sanger sequencing confirmed three variants of high quality (MYC, MET, and TP53 missense mutation). Taken together, the mutational spectrum and protein analyses showed mutations in signaling pathways similar to human TCL and also identified novel mutations that may serve as drug targets as well as potential biomarkers. PMID:29854308

  13. Comparative Sequence Analysis of the Plasmid-Encoded Regulator of Enteropathogenic Escherichia coli Strains

    PubMed Central

    Okeke, Iruka N.; Borneman, Jade A.; Shin, Sooan; Mellies, Jay L.; Quinn, Laura E.; Kaper, James B.

    2001-01-01

    Enteropathogenic Escherichia coli (EPEC) strains that carry the EPEC adherence factor (EAF) plasmid were screened for the presence of different EAF sequences, including those of the plasmid-encoded regulator (per). Considerable variation in gene content of EAF plasmids from different strains was seen. However, bfpA, the gene encoding the structural subunit for the bundle-forming pilus, bundlin, and per genes were found in 96.8% of strains. Sequence analysis of the per operon and its promoter region from 15 representative strains revealed that it is highly conserved. Most of the variation occurs in the 5′ two-thirds of the perA gene. In contrast, the C-terminal portion of the predicted PerA protein that contains the DNA-binding helix-turn-helix motif is 100% conserved in all strains that possess a full-length gene. In a minority of strains including the O119:H2 and canine isolates and in a subset of O128:H2 and O142:H6 strains, frameshift mutations in perA leading to premature truncation and consequent inactivation of the gene were identified. Cloned perA, -B, and -C genes from these strains, unlike those from strains with a functional operon, failed to activate the LEE1 operon and bfpA transcriptional fusions or to complement a per mutant in reference strain E2348/69. Furthermore, O119, O128, and canine strains that carry inactive per operons were deficient in virulence protein expression. The context in which the perABC operon occurs on the EAF plasmid varies. The sequence upstream of the per promoter region in EPEC reference strains E2348/69 and B171-8 was present in strains belonging to most serogroups. In a subset of O119:H2, O128:H2, and O142:H6 strains and in the canine isolate, this sequence was replaced by an IS1294-homologous sequence. PMID:11500429

  14. Comparative high-throughput transcriptome sequencing and development of SiESTa, the Silene EST annotation database

    PubMed Central

    2011-01-01

    Background The genus Silene is widely used as a model system for addressing ecological and evolutionary questions in plants, but advances in using the genus as a model system are impeded by the lack of available resources for studying its genome. Massively parallel sequencing cDNA has recently developed into an efficient method for characterizing the transcriptomes of non-model organisms, generating massive amounts of data that enable the study of multiple species in a comparative framework. The sequences generated provide an excellent resource for identifying expressed genes, characterizing functional variation and developing molecular markers, thereby laying the foundations for future studies on gene sequence and gene expression divergence. Here, we report the results of a comparative transcriptome sequencing study of eight individuals representing four Silene and one Dianthus species as outgroup. All sequences and annotations have been deposited in a newly developed and publicly available database called SiESTa, the Silene EST annotation database. Results A total of 1,041,122 EST reads were generated in two runs on a Roche GS-FLX 454 pyrosequencing platform. EST reads were analyzed separately for all eight individuals sequenced and were assembled into contigs using TGICL. These were annotated with results from BLASTX searches and Gene Ontology (GO) terms, and thousands of single-nucleotide polymorphisms (SNPs) were characterized. Unassembled reads were kept as singletons and together with the contigs contributed to the unigenes characterized in each individual. The high quality of unigenes is evidenced by the proportion (49%) that have significant hits in similarity searches with the A. thaliana proteome. The SiESTa database is accessible at http://www.siesta.ethz.ch. Conclusion The sequence collections established in the present study provide an important genomic resource for four Silene and one Dianthus species and will help to further develop Silene as a

  15. Complete Chloroplast Genome Sequence of Tartary Buckwheat (Fagopyrum tataricum) and Comparative Analysis with Common Buckwheat (F. esculentum)

    PubMed Central

    Cho, Kwang-Soo; Yun, Bong-Kyoung; Yoon, Young-Ho; Hong, Su-Young; Mekapogu, Manjulatha; Kim, Kyung-Hee; Yang, Tae-Jin

    2015-01-01

    We report the chloroplast (cp) genome sequence of tartary buckwheat (Fagopyrum tataricum) obtained by next-generation sequencing technology and compared this with the previously reported common buckwheat (F. esculentum ssp. ancestrale) cp genome. The cp genome of F. tataricum has a total sequence length of 159,272 bp, which is 327 bp shorter than the common buckwheat cp genome. The cp gene content, order, and orientation are similar to those of common buckwheat, but with some structural variation at tandem and palindromic repeat frequencies and junction areas. A total of seven InDels (around 100 bp) were found within the intergenic sequences and the ycf1 gene. Copy number variation of the 21-bp tandem repeat varied in F. tataricum (four repeats) and F. esculentum (one repeat), and the InDel of the ycf1 gene was 63 bp long. Nucleotide and amino acid have highly conserved coding sequence with about 98% homology and four genes—rpoC2, ycf3, accD, and clpP—have high synonymous (Ks) value. PCR based InDel markers were applied to diverse genetic resources of F. tataricum and F. esculentum, and the amplicon size was identical to that expected in silico. Therefore, these InDel markers are informative biomarkers to practically distinguish raw or processed buckwheat products derived from F. tataricum and F. esculentum. PMID:25966355

  16. Cigarette Smoking Promotes Infection of Cervical Cells by High-Risk Human Papillomaviruses, but not Subsequent E7 Oncoprotein Expression

    PubMed Central

    Vryzas, Dimos; Chatzaki, Ekaterini; Koch, Isabel; Soutschek, Erwin; Boecher, Oliver; Tsertanidou, Athena; Maglaveras, Nikolaos; Jansen-Duerr, Pidder; Agorastos, Theodoros

    2018-01-01

    Persistent cervical infection with high-risk human papillomaviruses (hrHPVs) is a necessary, but not sufficient, condition for the development of cervical cancer. Therefore, there are other co-factors facilitating the hrHPV carcinogenic process, one of which is smoking. To assess the effect of smoking on high-risk (hr) HPV DNA positivity and on the expression of HPV E7 oncoprotein, as a surrogate of persistent hrHPV infection, we used data from women recruited for the PIPAVIR project, which examined the role of E7 protein detection in cervical cancer screening. Women were tested for hrHPV DNA, using Multiplex Genotyping (MPG), and E7 protein, using a novel sandwich ELISA method, and gave information on their smoking habits. Among 1473 women, hrHPV prevalence was 19.1%. The odds ratio (OR) for hrHPV positivity of smokers compared to non-smokers was 1.785 (95% confidence intervals (CI): 1.365–2.332, p < 0.001). The ORs for E7 positivity, concerning hrHPV positive women, ranged from 0.720 to 1.360 depending on the E7 detection assay used, but this was not statistically significant. Smoking increases the probability of hrHPV infection, and smoking intensity is positively associated to this increase. Smoking is not related to an increased probability of E7 protein positivity for hrHPV positive women. PMID:29385075

  17. Comparative sequence analysis of Mycobacterium leprae and the new leprosy-causing Mycobacterium lepromatosis.

    PubMed

    Han, Xiang Y; Sizer, Kurt C; Thompson, Erika J; Kabanja, Juma; Li, Jun; Hu, Peter; Gómez-Valero, Laura; Silva, Francisco J

    2009-10-01

    Mycobacterium lepromatosis is a newly discovered leprosy-causing organism. Preliminary phylogenetic analysis of its 16S rRNA gene and a few other gene segments revealed significant divergence from Mycobacterium leprae, a well-known cause of leprosy, that justifies the status of M. lepromatosis as a new species. In this study we analyzed the sequences of 20 genes and pseudogenes (22,814 nucleotides). Overall, the level of matching of these sequences with M. leprae sequences was 90.9%, which substantiated the species-level difference; the levels of matching for the 16S rRNA genes and 14 protein-encoding genes were 98.0% and 93.1%, respectively, but the level of matching for five pseudogenes was only 79.1%. Five conserved protein-encoding genes were selected to construct phylogenetic trees and to calculate the numbers of synonymous substitutions (dS values) and nonsynonymous substitutions (dN values) in the two species. Robust phylogenetic trees constructed using concatenated alignment of these genes placed M. lepromatosis and M. leprae in a tight cluster with long terminal branches, implying that the divergence occurred long ago. The dS and dN values were also much higher than those for other closest pairs of mycobacteria. The dS values were 14 to 28% of the dS values for M. leprae and Mycobacterium tuberculosis, a more divergent pair of species. These results thus indicate that M. lepromatosis and M. leprae diverged approximately 10 million years ago. The M. lepromatosis pseudogenes analyzed that were also pseudogenes in M. leprae showed nearly neutral evolution, and their relative ages were similar to those of M. leprae pseudogenes, suggesting that they were pseudogenes before divergence. Taken together, the results described above indicate that M. lepromatosis and M. leprae diverged from a common ancestor after the massive gene inactivation event described previously for M. leprae.

  18. Whole Genome Sequence and Comparative Genomics of the Novel Lyme Borreliosis Causing Pathogen, Borrelia mayonii

    PubMed Central

    Batra, Dhwani; Replogle, Adam; Rowe, Lori A.; Pritt, Bobbi S.; Petersen, Jeannine M.

    2016-01-01

    Borrelia mayonii, a Borrelia burgdorferi sensu lato (Bbsl) genospecies, was recently identified as a cause of Lyme borreliosis (LB) among patients from the upper midwestern United States. By microscopy and PCR, spirochete/genome loads in infected patients were estimated at 105 to 106 per milliliter of blood. Here, we present the full chromosome and plasmid sequences of two B. mayonii isolates, MN14-1420 and MN14-1539, cultured from blood of two of these patients. Whole genome sequencing and assembly was conducted using PacBio long read sequencing (Pacific Biosciences RSII instrument) followed by hierarchical genome-assembly process (HGAP). The B. mayonii genome is ~1.31 Mbp in size (26.9% average GC content) and is comprised of a linear chromosome, 8 linear and 7 circular plasmids. Consistent with its taxonomic designation as a new Bbsl genospecies, the B. mayonii linear chromosome shares only 93.83% average nucleotide identity with other genospecies. Both B. mayonii genomes contain plasmids similar to B. burgdorferi sensu stricto lp54, lp36, lp28-3, lp28-4, lp25, lp17, lp5, 5 cp32s, cp26, and cp9. The vls locus present on lp28-10 of B. mayonii MN14-1420 is remarkably long, being comprised of 24 silent vls cassettes. Genetic differences between the two B. mayonii genomes are limited and include 15 single nucleotide variations as well as 7 fewer silent vls cassettes and a lack of the lp5 plasmid in MN14-1539. Notably, 68 homologs to proteins present in B. burgdorferi sensu stricto appear to be lacking from the B. mayonii genomes. These include the complement inhibitor, CspZ (BB_H06), the fibronectin binding protein, BB_K32, as well as multiple lipoproteins and proteins of unknown function. This study shows the utility of long read sequencing for full genome assembly of Bbsl genomes, identifies putative genome regions of B. mayonii that may be linked to clinical manifestation or tissue tropism, and provides a valuable resource for pathogenicity, diagnostic and

  19. Whole Genome Sequence and Comparative Genomics of the Novel Lyme Borreliosis Causing Pathogen, Borrelia mayonii.

    PubMed

    Kingry, Luke C; Batra, Dhwani; Replogle, Adam; Rowe, Lori A; Pritt, Bobbi S; Petersen, Jeannine M

    2016-01-01

    Borrelia mayonii, a Borrelia burgdorferi sensu lato (Bbsl) genospecies, was recently identified as a cause of Lyme borreliosis (LB) among patients from the upper midwestern United States. By microscopy and PCR, spirochete/genome loads in infected patients were estimated at 105 to 106 per milliliter of blood. Here, we present the full chromosome and plasmid sequences of two B. mayonii isolates, MN14-1420 and MN14-1539, cultured from blood of two of these patients. Whole genome sequencing and assembly was conducted using PacBio long read sequencing (Pacific Biosciences RSII instrument) followed by hierarchical genome-assembly process (HGAP). The B. mayonii genome is ~1.31 Mbp in size (26.9% average GC content) and is comprised of a linear chromosome, 8 linear and 7 circular plasmids. Consistent with its taxonomic designation as a new Bbsl genospecies, the B. mayonii linear chromosome shares only 93.83% average nucleotide identity with other genospecies. Both B. mayonii genomes contain plasmids similar to B. burgdorferi sensu stricto lp54, lp36, lp28-3, lp28-4, lp25, lp17, lp5, 5 cp32s, cp26, and cp9. The vls locus present on lp28-10 of B. mayonii MN14-1420 is remarkably long, being comprised of 24 silent vls cassettes. Genetic differences between the two B. mayonii genomes are limited and include 15 single nucleotide variations as well as 7 fewer silent vls cassettes and a lack of the lp5 plasmid in MN14-1539. Notably, 68 homologs to proteins present in B. burgdorferi sensu stricto appear to be lacking from the B. mayonii genomes. These include the complement inhibitor, CspZ (BB_H06), the fibronectin binding protein, BB_K32, as well as multiple lipoproteins and proteins of unknown function. This study shows the utility of long read sequencing for full genome assembly of Bbsl genomes, identifies putative genome regions of B. mayonii that may be linked to clinical manifestation or tissue tropism, and provides a valuable resource for pathogenicity, diagnostic and

  20. Whole genomic DNA sequencing and comparative genomic analysis of Arthrospira platensis: high genome plasticity and genetic diversity

    PubMed Central

    Xu, Teng; Qin, Song; Hu, Yongwu; Song, Zhijian; Ying, Jianchao; Li, Peizhen; Dong, Wei; Zhao, Fangqing; Yang, Huanming; Bao, Qiyu

    2016-01-01

    Arthrospira platensis is a multi-cellular and filamentous non-N2-fixing cyanobacterium that is capable of performing oxygenic photosynthesis. In this study, we determined the nearly complete genome sequence of A. platensis YZ. A. platensis YZ genome is a single, circular chromosome of 6.62 Mb in size. Phylogenetic and comparative genomic analyses revealed that A. platensis YZ was more closely related to A. platensis NIES-39 than Arthrospira sp. PCC 8005 and A. platensis C1. Broad gene gains were identified between A. platensis YZ and three other Arthrospira speices, some of which have been previously demonstrated that can be laterally transferred among different species, such as restriction-modification systems-coding genes. Moreover, unprecedented extensive chromosomal rearrangements among different strains were observed. The chromosomal rearrangements, particularly the chromosomal inversions, were analysed and estimated to be closely related to palindromes that involved long inverted repeat sequences and the extensively distributed type IIR restriction enzyme in the Arthrospira genome. In addition, species from genus Arthrospira unanimously contained the highest rate of repetitive sequence compared with the other species of order Oscillatoriales, suggested that sequence duplication significantly contributed to Arthrospira genome phylogeny. These results provided in-depth views into the genomic phylogeny and structural variation of A. platensis, as well as provide a valuable resource for functional genomics studies. PMID:27330141

  1. Long-read whole genome sequencing and comparative analysis of six strains of the human pathogen Orientia tsutsugamushi.

    PubMed

    Batty, Elizabeth M; Chaemchuen, Suwittra; Blacksell, Stuart; Richards, Allen L; Paris, Daniel; Bowden, Rory; Chan, Caroline; Lachumanan, Ramkumar; Day, Nicholas; Donnelly, Peter; Chen, Swaine; Salje, Jeanne

    2018-06-01

    Orientia tsutsugamushi is a clinically important but neglected obligate intracellular bacterial pathogen of the Rickettsiaceae family that causes the potentially life-threatening human disease scrub typhus. In contrast to the genome reduction seen in many obligate intracellular bacteria, early genetic studies of Orientia have revealed one of the most repetitive bacterial genomes sequenced to date. The dramatic expansion of mobile elements has hampered efforts to generate complete genome sequences using short read sequencing methodologies, and consequently there have been few studies of the comparative genomics of this neglected species. We report new high-quality genomes of O. tsutsugamushi, generated using PacBio single molecule long read sequencing, for six strains: Karp, Kato, Gilliam, TA686, UT76 and UT176. In comparative genomics analyses of these strains together with existing reference genomes from Ikeda and Boryong strains, we identify a relatively small core genome of 657 genes, grouped into core gene islands and separated by repeat regions, and use the core genes to infer the first whole-genome phylogeny of Orientia. Complete assemblies of multiple Orientia genomes verify initial suggestions that these are remarkable organisms. They have larger genomes compared with most other Rickettsiaceae, with widespread amplification of repeat elements and massive chromosomal rearrangements between strains. At the gene level, Orientia has a relatively small set of universally conserved genes, similar to other obligate intracellular bacteria, and the relative expansion in genome size can be accounted for by gene duplication and repeat amplification. Our study demonstrates the utility of long read sequencing to investigate complex bacterial genomes and characterise genomic variation.

  2. Cell penetrating peptides: a comparative transport analysis for 474 sequence motifs.

    PubMed

    Ramaker, Katrin; Henkel, Maik; Krause, Thorsten; Röckendorf, Niels; Frey, Andreas

    2018-11-01

    Delivering reagents into cells is a key demand in molecular medicine. The vehicle of choice is often cell penetrating peptides (CPPs), which can ferry conjugated cargo across membranes. Although numerous peptides have been shown to promote such uptake events, there has been no comprehensive comparison of individual performance under standardized conditions. We have devised a method to rapidly analyze the ability of a multitude of CPP conjugates to carry a model cargo into HeLa cells. Sequence information for 474 CPPs was collected from literature sources, and the respective peptides were synthesized and modified with carboxyfluorescein (FAM) as model cargo. All candidates were evaluated in an identical uptake test, and transport was quantified using cellular fluorescence intensities. Substantial differences in the ability to carry the fluorophore into the cells were observed, with transport performance differing by a factor of 70 between the best CPP investigated and cargo alone. Strong correlations were observed between uptake efficiency and both sequence length and the presence of positive net charge. A compilation of the 20 top performers with regard to cargo delivery performance and cell compatibility is provided.

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

    PubMed

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

    2006-04-01

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

  4. Capacity for patterns and sequences in Kanerva's SDM as compared to other associative memory models

    NASA Technical Reports Server (NTRS)

    Keeler, James D.

    1987-01-01

    The information capacity of Kanerva's Sparse Distributed Memory (SDM) and Hopfield-type neural networks is investigated. Under the approximations used, it is shown that the total information stored in these systems is proportional to the number connections in the network. The proportionality constant is the same for the SDM and Hopfield-type models independent of the particular model, or the order of the model. The approximations are checked numerically. This same analysis can be used to show that the SDM can store sequences of spatiotemporal patterns, and the addition of time-delayed connections allows the retrieval of context dependent temporal patterns. A minor modification of the SDM can be used to store correlated patterns.

  5. No more non-model species: the promise of next generation sequencing for comparative immunology.

    PubMed

    Dheilly, Nolwenn M; Adema, Coen; Raftos, David A; Gourbal, Benjamin; Grunau, Christoph; Du Pasquier, Louis

    2014-07-01

    Next generation sequencing (NGS) allows for the rapid, comprehensive and cost effective analysis of entire genomes and transcriptomes. NGS provides approaches for immune response gene discovery, profiling gene expression over the course of parasitosis, studying mechanisms of diversification of immune receptors and investigating the role of epigenetic mechanisms in regulating immune gene expression and/or diversification. NGS will allow meaningful comparisons to be made between organisms from different taxa in an effort to understand the selection of diverse strategies for host defence under different environmental pathogen pressures. At the same time, it will reveal the shared and unique components of the immunological toolkit and basic functional aspects that are essential for immune defence throughout the living world. In this review, we argue that NGS will revolutionize our understanding of immune responses throughout the animal kingdom because the depth of information it provides will circumvent the need to concentrate on a few "model" species. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Comparing microarrays and next-generation sequencing technologies for microbial ecology research.

    PubMed

    Roh, Seong Woon; Abell, Guy C J; Kim, Kyoung-Ho; Nam, Young-Do; Bae, Jin-Woo

    2010-06-01

    Recent advances in molecular biology have resulted in the application of DNA microarrays and next-generation sequencing (NGS) technologies to the field of microbial ecology. This review aims to examine the strengths and weaknesses of each of the methodologies, including depth and ease of analysis, throughput and cost-effectiveness. It also intends to highlight the optimal application of each of the individual technologies toward the study of a particular environment and identify potential synergies between the two main technologies, whereby both sample number and coverage can be maximized. We suggest that the efficient use of microarray and NGS technologies will allow researchers to advance the field of microbial ecology, and importantly, improve our understanding of the role of microorganisms in their various environments.

  7. Genetic variability in E6, E7, and L1 genes of human papillomavirus genotype 52 from Southwest China.

    PubMed

    Zhang, Yiwen; Cao, Man; Wang, Mengting; Ding, Xianping; Jing, Yaling; Chen, Zuyi; Ma, Tengjiao; Chen, Honghan

    2016-07-01

    Human papillomavirus (HPV) is the major causative agent of cervical cancer, which accounts for the second highest cancer burden in women worldwide. HPV-52, the prevalent subtype in Asia, especially in southwest China, was analyzed in this study. To analyze polymorphisms, intratypic variants, and genetic variability in the E6-E7 (n=26) and L1 (n=53) genes of HPV-52, these genes were sequenced and the sequences were submitted to GenBank. Phylogenetic trees were constructed using the neighbor-joining and Kimura 2-parameters methods, followed by analysis of the diversity of secondary structure. Finally, we estimated the selection pressures acting on the E6-E7 and L1 genes. Fifty-one novel variants of HPV-52 L1, and two novel variants of HPV-52 E6-E7 were identified in this study. Thirty single nucleotide changes were observed in HPV-52 E6-E7 sequences with 19/30 non-synonymous mutations and 11/30 synonymous mutations (five in the alpha helix and five in the beta sheet). Fifty-five single nucleotide changes were observed in HPV-52 L1 sequences with 17/55 non-synonymous mutations (seven in the alpha helix and fourteen in the beta sheet) and 38/55 synonymous mutations. Selective pressure analysis predicted that most of these mutations reflect positive selection. Identifying new variants in HPV-52 may inform the rational design of new vaccines specifically for women in southwest China. Knowledge of genetic variation in HPV may be useful as an epidemiologic correlate of cervical cancer risk, or may even provide critical information for developing diagnostic probes. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Complete Genome Sequence of Acinetobacter baumannii CIP 70.10, a Susceptible Reference Strain for Comparative Genome Analyses.

    PubMed

    Krahn, Thomas; Wibberg, Daniel; Maus, Irena; Winkler, Anika; Pühler, Alfred; Poirel, Laurent; Schlüter, Andreas

    2015-07-30

    The complete genome sequence for the reference strain Acinetobacter baumannii CIP 70.10 (ATCC 15151) was established. The strain was isolated in France in 1970, is susceptible to most antimicrobial compounds, and is therefore of importance for comparative genome analyses with clinical multidrug-resistant (MDR) A. baumannii strains to study resistance development and acquisition in this emerging human pathogen. Copyright © 2015 Krahn et al.

  9. The European sea bass Dicentrarchus labrax genome puzzle: comparative BAC-mapping and low coverage shotgun sequencing

    PubMed Central

    2010-01-01

    Background Food supply from the ocean is constrained by the shortage of domesticated and selected fish. Development of genomic models of economically important fishes should assist with the removal of this bottleneck. European sea bass Dicentrarchus labrax L. (Moronidae, Perciformes, Teleostei) is one of the most important fishes in European marine aquaculture; growing genomic resources put it on its way to serve as an economic model. Results End sequencing of a sea bass genomic BAC-library enabled the comparative mapping of the sea bass genome using the three-spined stickleback Gasterosteus aculeatus genome as a reference. BAC-end sequences (102,690) were aligned to the stickleback genome. The number of mappable BACs was improved using a two-fold coverage WGS dataset of sea bass resulting in a comparative BAC-map covering 87% of stickleback chromosomes with 588 BAC-contigs. The minimum size of 83 contigs covering 50% of the reference was 1.2 Mbp; the largest BAC-contig comprised 8.86 Mbp. More than 22,000 BAC-clones aligned with both ends to the reference genome. Intra-chromosomal rearrangements between sea bass and stickleback were identified. Size distributions of mapped BACs were used to calculate that the genome of sea bass may be only 1.3 fold larger than the 460 Mbp stickleback genome. Conclusions The BAC map is used for sequencing single BACs or BAC-pools covering defined genomic entities by second generation sequencing technologies. Together with the WGS dataset it initiates a sea bass genome sequencing project. This will allow the quantification of polymorphisms through resequencing, which is important for selecting highly performing domesticated fish. PMID:20105308

  10. Genome sequencing and comparative genomics of honey bee microsporidia, Nosema apis reveal novel insights into host-parasite interactions.

    PubMed

    Chen, Yan ping; Pettis, Jeffery S; Zhao, Yan; Liu, Xinyue; Tallon, Luke J; Sadzewicz, Lisa D; Li, Renhua; Zheng, Huoqing; Huang, Shaokang; Zhang, Xuan; Hamilton, Michele C; Pernal, Stephen F; Melathopoulos, Andony P; Yan, Xianghe; Evans, Jay D

    2013-07-05

    The microsporidia parasite Nosema contributes to the steep global decline of honey bees that are critical pollinators of food crops. There are two species of Nosema that have been found to infect honey bees, Nosema apis and N. ceranae. Genome sequencing of N. apis and comparative genome analysis with N. ceranae, a fully sequenced microsporidia species, reveal novel insights into host-parasite interactions underlying the parasite infections. We applied the whole-genome shotgun sequencing approach to sequence and assemble the genome of N. apis which has an estimated size of 8.5 Mbp. We predicted 2,771 protein- coding genes and predicted the function of each putative protein using the Gene Ontology. The comparative genomic analysis led to identification of 1,356 orthologs that are conserved between the two Nosema species and genes that are unique characteristics of the individual species, thereby providing a list of virulence factors and new genetic tools for studying host-parasite interactions. We also identified a highly abundant motif in the upstream promoter regions of N. apis genes. This motif is also conserved in N. ceranae and other microsporidia species and likely plays a role in gene regulation across the microsporidia. The availability of the N. apis genome sequence is a significant addition to the rapidly expanding body of microsprodian genomic data which has been improving our understanding of eukaryotic genome diversity and evolution in a broad sense. The predicted virulent genes and transcriptional regulatory elements are potential targets for innovative therapeutics to break down the life cycle of the parasite.

  11. Complete chloroplast genome sequence of MD-2 pineapple and its comparative analysis among nine other plants from the subclass Commelinidae.

    PubMed

    Redwan, R M; Saidin, A; Kumar, S V

    2015-08-12

    Pineapple (Ananas comosus var. comosus) is known as the king of fruits for its crown and is the third most important tropical fruit after banana and citrus. The plant, which is indigenous to South America, is the most important species in the Bromeliaceae family and is largely traded for fresh fruit consumption. Here, we report the complete chloroplast sequence of the MD-2 pineapple that was sequenced using the PacBio sequencing technology. In this study, the high error rate of PacBio long sequence reads of A. comosus's total genomic DNA were improved by leveraging on the high accuracy but short Illumina reads for error-correction via the latest error correction module from Novocraft. Error corrected long PacBio reads were assembled by using a single tool to produce a contig representing the pineapple chloroplast genome. The genome of 159,636 bp in length is featured with the conserved quadripartite structure of chloroplast containing a large single copy region (LSC) with a size of 87,482 bp, a small single copy region (SSC) with a size of 18,622 bp and two inverted repeat regions (IRA and IRB) each with the size of 26,766 bp. Overall, the genome contained 117 unique coding regions and 30 were repeated in the IR region with its genes contents, structure and arrangement similar to its sister taxon, Typha latifolia. A total of 35 repeats structure were detected in both the coding and non-coding regions with a majority being tandem repeats. In addition, 205 SSRs were detected in the genome with six protein-coding genes contained more than two SSRs. Comparative chloroplast genomes from the subclass Commelinidae revealed a conservative protein coding gene albeit located in a highly divergence region. Analysis of selection pressure on protein-coding genes using Ka/Ks ratio showed significant positive selection exerted on the rps7 gene of the pineapple chloroplast with P less than 0.05. Phylogenetic analysis confirmed the recent taxonomical relation among the member of

  12. Heterogeneous genetic diversity pattern in Plasmodium vivax genes encoding merozoite surface proteins (MSP) -7E, -7F and -7L.

    PubMed

    Garzón-Ospina, Diego; Forero-Rodríguez, Johanna; Patarroyo, Manuel A

    2014-12-13

    The msp-7 gene has become differentially expanded in the Plasmodium genus; Plasmodium vivax has the highest copy number of this gene, several of which encode antigenic proteins in merozoites. DNA sequences from thirty-six Colombian clinical isolates from P. vivax (pv) msp-7E, -7F and -7L genes were analysed for characterizing and studying the genetic diversity of these pvmsp-7 members which are expressed during the intra-erythrocyte stage; natural selection signals producing the variation pattern so observed were evaluated. The pvmsp-7E gene was highly polymorphic compared to pvmsp-7F and pvmsp-7L which were seen to have limited genetic diversity; pvmsp-7E polymorphism was seen to have been maintained by different types of positive selection. Even though these copies seemed to be species-specific duplications, a search in the Plasmodium cynomolgi genome (P. vivax sister taxon) showed that both species shared the whole msp-7 repertoire. This led to exploring the long-term effect of natural selection by comparing the orthologous sequences which led to finding signatures for lineage-specific positive selection. The results confirmed that the P. vivax msp-7 family has a heterogeneous genetic diversity pattern; some members are highly conserved whilst others are highly diverse. The results suggested that the 3'-end of these genes encode MSP-7 proteins' functional region whilst the central region of pvmsp-7E has evolved rapidly. The lineage-specific positive selection signals found suggested that mutations occurring in msp-7s genes during host switch may have succeeded in adapting the ancestral P. vivax parasite population to humans.

  13. The complete chloroplast genome sequences of Lychnis wilfordii and Silene capitata and comparative analyses with other Caryophyllaceae genomes.

    PubMed

    Kang, Jong-Soo; Lee, Byoung Yoon; Kwak, Myounghai

    2017-01-01

    The complete chloroplast genomes of Lychnis wilfordii and Silene capitata were determined and compared with ten previously reported Caryophyllaceae chloroplast genomes. The chloroplast genome sequences of L. wilfordii and S. capitata contain 152,320 bp and 150,224 bp, respectively. The gene contents and orders among 12 Caryophyllaceae species are consistent, but several microstructural changes have occurred. Expansion of the inverted repeat (IR) regions at the large single copy (LSC)/IRb and small single copy (SSC)/IR boundaries led to partial or entire gene duplications. Additionally, rearrangements of the LSC region were caused by gene inversions and/or transpositions. The 18 kb inversions, which occurred three times in different lineages of tribe Sileneae, were thought to be facilitated by the intermolecular duplicated sequences. Sequence analyses of the L. wilfordii and S. capitata genomes revealed 39 and 43 repeats, respectively, including forward, palindromic, and reverse repeats. In addition, a total of 67 and 56 simple sequence repeats were discovered in the L. wilfordii and S. capitata chloroplast genomes, respectively. Finally, we constructed phylogenetic trees of the 12 Caryophyllaceae species and two Amaranthaceae species based on 73 protein-coding genes using both maximum parsimony and likelihood methods.

  14. The genome sequence of the biocontrol fungus Metarhizium anisopliae and comparative genomics of Metarhizium species.

    PubMed

    Pattemore, Julie A; Hane, James K; Williams, Angela H; Wilson, Bree A L; Stodart, Ben J; Ash, Gavin J

    2014-08-07

    Metarhizium anisopliae is an important fungal biocontrol agent of insect pests of agricultural crops. Genomics can aid the successful commercialization of biopesticides by identification of key genes differentiating closely related species, selection of virulent microbial isolates which are amenable to industrial scale production and formulation and through the reduction of phenotypic variability. The genome of Metarhizium isolate ARSEF23 was recently published as a model for M. anisopliae, however phylogenetic analysis has since re-classified this isolate as M. robertsii. We present a new annotated genome sequence of M. anisopliae (isolate Ma69) and whole genome comparison to M. robertsii (ARSEF23) and M. acridum (CQMa 102). Whole genome analysis of M. anisopliae indicates significant macrosynteny with M. robertsii but with some large genomic inversions. In comparison to M. acridum, the genome of M. anisopliae shares lower sequence homology. While alignments overall are co-linear, the genome of M. acridum is not contiguous enough to conclusively observe macrosynteny. Mating type gene analysis revealed both MAT1-1 and MAT1-2 genes present in M. anisopliae suggesting putative homothallism, despite having no known teleomorph, in contrast with the putatively heterothallic M. acridum isolate CQMa 102 (MAT1-2) and M. robertsii isolate ARSEF23 (altered MAT1-1). Repetitive DNA and RIP analysis revealed M. acridum to have twice the repetitive content of the other two species and M. anisopliae to be five times more RIP affected than M. robertsii. We also present an initial bioinformatic survey of candidate pathogenicity genes in M. anisopliae. The annotated genome of M. anisopliae is an important resource for the identification of virulence genes specific to M. anisopliae and development of species- and strain- specific assays. New insight into the possibility of homothallism and RIP affectedness has important implications for the development of M. anisopliae as a

  15. Characterization and Comparative Analysis of the Milk Transcriptome in Two Dairy Sheep Breeds using RNA Sequencing.

    PubMed

    Suárez-Vega, Aroa; Gutiérrez-Gil, Beatriz; Klopp, Christophe; Robert-Granie, Christèle; Tosser-Klopp, Gwenola; Arranz, Juan José

    2015-12-18

    This study presents a dynamic characterization of the sheep milk transcriptome aiming at achieving a better understanding of the sheep lactating mammary gland. Transcriptome sequencing (RNA-seq) was performed on total RNA extracted from milk somatic cells from ewes on days 10, 50, 120 and 150 after lambing. The experiment was performed in Spanish Churra and Assaf breeds, which differ in their milk production traits. Nearly 67% of the annotated genes in the reference genome (Oar_v3.1) were expressed in ovine milk somatic cells. For the two breeds and across the four lactation stages studied, the most highly expressed genes encoded caseins and whey proteins. We detected 573 differentially expressed genes (DEGs) across lactation points, with the largest differences being found, between day 10 and day 150. Upregulated GO terms at late lactation stages were linked mainly to developmental processes linked to extracellular matrix remodeling. A total of 256 annotated DEGs were detected in the Assaf and Churra comparison. Some genes selectively upregulated in the Churra breed grouped under the endopeptidase and channel activity GO terms. These genes could be related to the higher cheese yield of this breed. Overall, this study provides the first integrated overview on sheep milk gene expression.

  16. Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia coli Strain 042

    PubMed Central

    Chaudhuri, Roy R.; Sebaihia, Mohammed; Hobman, Jon L.; Webber, Mark A.; Leyton, Denisse L.; Goldberg, Martin D.; Cunningham, Adam F.; Scott-Tucker, Anthony; Ferguson, Paul R.; Thomas, Christopher M.; Frankel, Gad; Tang, Christoph M.; Dudley, Edward G.; Roberts, Ian S.; Rasko, David A.; Pallen, Mark J.; Parkhill, Julian; Nataro, James P.; Thomson, Nicholas R.; Henderson, Ian R.

    2010-01-01

    Background Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation. Methods In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog™ Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12. Conclusion This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies. PMID:20098708

  17. Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

    PubMed Central

    2011-01-01

    Background Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma. Results Here we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei. Conclusions The data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants. PMID:21501500

  18. Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma.

    PubMed

    Kubicek, Christian P; Herrera-Estrella, Alfredo; Seidl-Seiboth, Verena; Martinez, Diego A; Druzhinina, Irina S; Thon, Michael; Zeilinger, Susanne; Casas-Flores, Sergio; Horwitz, Benjamin A; Mukherjee, Prasun K; Mukherjee, Mala; Kredics, László; Alcaraz, Luis D; Aerts, Andrea; Antal, Zsuzsanna; Atanasova, Lea; Cervantes-Badillo, Mayte G; Challacombe, Jean; Chertkov, Olga; McCluskey, Kevin; Coulpier, Fanny; Deshpande, Nandan; von Döhren, Hans; Ebbole, Daniel J; Esquivel-Naranjo, Edgardo U; Fekete, Erzsébet; Flipphi, Michel; Glaser, Fabian; Gómez-Rodríguez, Elida Y; Gruber, Sabine; Han, Cliff; Henrissat, Bernard; Hermosa, Rosa; Hernández-Oñate, Miguel; Karaffa, Levente; Kosti, Idit; Le Crom, Stéphane; Lindquist, Erika; Lucas, Susan; Lübeck, Mette; Lübeck, Peter S; Margeot, Antoine; Metz, Benjamin; Misra, Monica; Nevalainen, Helena; Omann, Markus; Packer, Nicolle; Perrone, Giancarlo; Uresti-Rivera, Edith E; Salamov, Asaf; Schmoll, Monika; Seiboth, Bernhard; Shapiro, Harris; Sukno, Serenella; Tamayo-Ramos, Juan Antonio; Tisch, Doris; Wiest, Aric; Wilkinson, Heather H; Zhang, Michael; Coutinho, Pedro M; Kenerley, Charles M; Monte, Enrique; Baker, Scott E; Grigoriev, Igor V

    2011-01-01

    Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma. Here we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei. The data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants. © 2011 Kubicek et al.; licensee BioMed Central Ltd.

  19. Characterization and Comparative Analysis of the Milk Transcriptome in Two Dairy Sheep Breeds using RNA Sequencing

    PubMed Central

    Suárez-Vega, Aroa; Gutiérrez-Gil, Beatriz; Klopp, Christophe; Robert-Granie, Christèle; Tosser-Klopp, Gwenola; Arranz, Juan José

    2015-01-01

    This study presents a dynamic characterization of the sheep milk transcriptome aiming at achieving a better understanding of the sheep lactating mammary gland. Transcriptome sequencing (RNA-seq) was performed on total RNA extracted from milk somatic cells from ewes on days 10, 50, 120 and 150 after lambing. The experiment was performed in Spanish Churra and Assaf breeds, which differ in their milk production traits. Nearly 67% of the annotated genes in the reference genome (Oar_v3.1) were expressed in ovine milk somatic cells. For the two breeds and across the four lactation stages studied, the most highly expressed genes encoded caseins and whey proteins. We detected 573 differentially expressed genes (DEGs) across lactation points, with the largest differences being found, between day 10 and day 150. Upregulated GO terms at late lactation stages were linked mainly to developmental processes linked to extracellular matrix remodeling. A total of 256 annotated DEGs were detected in the Assaf and Churra comparison. Some genes selectively upregulated in the Churra breed grouped under the endopeptidase and channel activity GO terms. These genes could be related to the higher cheese yield of this breed. Overall, this study provides the first integrated overview on sheep milk gene expression. PMID:26677795

  20. Connective tissue growth factor linked to the E7 tumor antigen generates potent antitumor immune responses mediated by an antiapoptotic mechanism.

    PubMed

    Cheng, W-F; Chang, M-C; Sun, W-Z; Lee, C-N; Lin, H-W; Su, Y-N; Hsieh, C-Y; Chen, C-A

    2008-07-01

    A novel method for generating an antigen-specific cancer vaccine and immunotherapy has emerged using a DNA vaccine. However, antigen-presenting cells (APCs) have a limited life span, which hinders their long-term ability to prime antigen-specific T cells. Connective tissue growth factor (CTGF) has a role in cell survival. This study explored the intradermal administration of DNA encoding CTGF with a model tumor antigen, human papilloma virus type 16 E7. Mice vaccinated with CTGF/E7 DNA exhibited a dramatic increase in E7-specific CD4(+) and CD8(+) T-cell precursors. They also showed an impressive antitumor effect against E7-expressing tumors compared with mice vaccinated with the wild-type E7 DNA. The delivery of DNA encoding CTGF and E7 or CTGF alone could prolong the survival of transduced dendritic cells (DCs) in vivo. In addition, CTGF/E7-transduced DCs could enhance a higher number of E7-specific CD8(+) T cells than E7-transduced DCs. By prolonging the survival of APCs, DNA vaccine encoding CTGF linked to a tumor antigen represents an innovative approach to enhance DNA vaccine potency and holds promise for cancer prophylaxis and immunotherapy.

  1. E6^E7, a Novel Splice Isoform Protein of Human Papillomavirus 16, Stabilizes Viral E6 and E7 Oncoproteins via HSP90 and GRP78

    PubMed Central

    Ajiro, Masahiko

    2015-01-01

    ABSTRACT Transcripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41 N-terminal amino acid (aa) residues of E6 and the 38 C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy. PMID:25691589

  2. Discovery of human inversion polymorphisms by comparative analysis of human and chimpanzee DNA sequence assemblies.

    PubMed

    Feuk, Lars; MacDonald, Jeffrey R; Tang, Terence; Carson, Andrew R; Li, Martin; Rao, Girish; Khaja, Razi; Scherer, Stephen W

    2005-10-01

    With a draft genome-sequence assembly for the chimpanzee available, it is now possible to perform genome-wide analyses to identify, at a submicroscopic level, structural rearrangements that have occurred between chimpanzees and humans. The goal of this study was to investigate chromosomal regions that are inverted between the chimpanzee and human genomes. Using the net alignments for the builds of the human and chimpanzee genome assemblies, we identified a total of 1,576 putative regions of inverted orientation, covering more than 154 mega-bases of DNA. The DNA segments are distributed throughout the genome and range from 23 base pairs to 62 mega-bases in length. For the 66 inversions more than 25 kilobases (kb) in length, 75% were flanked on one or both sides by (often unrelated) segmental duplications. Using PCR and fluorescence in situ hybridization we experimentally validated 23 of 27 (85%) semi-randomly chosen regions; the largest novel inversion confirmed was 4.3 mega-bases at human Chromosome 7p14. Gorilla was used as an out-group to assign ancestral status to the variants. All experimentally validated inversion regions were then assayed against a panel of human samples and three of the 23 (13%) regions were found to be polymorphic in the human genome. These polymorphic inversions include 730 kb (at 7p22), 13 kb (at 7q11), and 1 kb (at 16q24) fragments with a 5%, 30%, and 48% minor allele frequency, respectively. Our results suggest that inversions are an important source of variation in primate genome evolution. The finding of at least three novel inversion polymorphisms in humans indicates this type of structural variation may be a more common feature of our genome than previously realized.

  3. Comparative Analysis of Predicted Plastid-Targeted Proteomes of Sequenced Higher Plant Genomes

    PubMed Central

    Schaeffer, Scott; Harper, Artemus; Raja, Rajani; Jaiswal, Pankaj; Dhingra, Amit

    2014-01-01

    Plastids are actively involved in numerous plant processes critical to growth, development and adaptation. They play a primary role in photosynthesis, pigment and monoterpene synthesis, gravity sensing, starch and fatty acid synthesis, as well as oil, and protein storage. We applied two complementary methods to analyze the recently published apple genome (Malus × domestica) to identify putative plastid-targeted proteins, the first using TargetP and the second using a custom workflow utilizing a set of predictive programs. Apple shares roughly 40% of its 10,492 putative plastid-targeted proteins with that of the Arabidopsis (Arabidopsis thaliana) plastid-targeted proteome as identified by the Chloroplast 2010 project and ∼57% of its entire proteome with Arabidopsis. This suggests that the plastid-targeted proteomes between apple and Arabidopsis are different, and interestingly alludes to the presence of differential targeting of homologs between the two species. Co-expression analysis of 2,224 genes encoding putative plastid-targeted apple proteins suggests that they play a role in plant developmental and intermediary metabolism. Further, an inter-specific comparison of Arabidopsis, Prunus persica (Peach), Malus × domestica (Apple), Populus trichocarpa (Black cottonwood), Fragaria vesca (Woodland Strawberry), Solanum lycopersicum (Tomato) and Vitis vinifera (Grapevine) also identified a large number of novel species-specific plastid-targeted proteins. This analysis also revealed the presence of alternatively targeted homologs across species. Two separate analyses revealed that a small subset of proteins, one representing 289 protein clusters and the other 737 unique protein sequences, are conserved between seven plastid-targeted angiosperm proteomes. Majority of the novel proteins were annotated to play roles in stress response, transport, catabolic processes, and cellular component organization. Our results suggest that the current state of knowledge regarding

  4. Comparative description of ten transcriptomes of newly sequenced invertebrates and efficiency estimation of genomic sampling in non-model taxa

    PubMed Central

    2012-01-01

    Introduction Traditionally, genomic or transcriptomic data have been restricted to a few model or emerging model organisms, and to a handful of species of medical and/or environmental importance. Next-generation sequencing techniques have the capability of yielding massive amounts of gene sequence data for virtually any species at a modest cost. Here we provide a comparative analysis of de novo assembled transcriptomic data for ten non-model species of previously understudied animal taxa. Results cDNA libraries of ten species belonging to five animal phyla (2 Annelida [including Sipuncula], 2 Arthropoda, 2 Mollusca, 2 Nemertea, and 2 Porifera) were sequenced in different batches with an Illumina Genome Analyzer II (read length 100 or 150 bp), rendering between ca. 25 and 52 million reads per species. Read thinning, trimming, and de novo assembly were performed under different parameters to optimize output. Between 67,423 and 207,559 contigs were obtained across the ten species, post-optimization. Of those, 9,069 to 25,681 contigs retrieved blast hits against the NCBI non-redundant database, and approximately 50% of these were assigned with Gene Ontology terms, covering all major categories, and with similar percentages in all species. Local blasts against our datasets, using selected genes from major signaling pathways and housekeeping genes, revealed high efficiency in gene recovery compared to available genomes of closely related species. Intriguingly, our transcriptomic datasets detected multiple paralogues in all phyla and in nearly all gene pathways, including housekeeping genes that are traditionally used in phylogenetic applications for their purported single-copy nature. Conclusions We generated the first study of comparative transcriptomics across multiple animal phyla (comparing two species per phylum in most cases), established the first Illumina-based transcriptomic datasets for sponge, nemertean, and sipunculan species, and generated a tractable

  5. PARRoT- a homology-based strategy to quantify and compare RNA-sequencing from non-model organisms.

    PubMed

    Gan, Ruei-Chi; Chen, Ting-Wen; Wu, Timothy H; Huang, Po-Jung; Lee, Chi-Ching; Yeh, Yuan-Ming; Chiu, Cheng-Hsun; Huang, Hsien-Da; Tang, Petrus

    2016-12-22

    Next-generation sequencing promises the de novo genomic and transcriptomic analysis of samples of interests. However, there are only a few organisms having reference genomic sequences and even fewer having well-defined or curated annotations. For transcriptome studies focusing on organisms lacking proper reference genomes, the common strategy is de novo assembly followed by functional annotation. However, things become even more complicated when multiple transcriptomes are compared. Here, we propose a new analysis strategy and quantification methods for quantifying expression level which not only generate a virtual reference from sequencing data, but also provide comparisons between transcriptomes. First, all reads from the transcriptome datasets are pooled together for de novo assembly. The assembled contigs are searched against NCBI NR databases to find potential homolog sequences. Based on the searched result, a set of virtual transcripts are generated and served as a reference transcriptome. By using the same reference, normalized quantification values including RC (read counts), eRPKM (estimated RPKM) and eTPM (estimated TPM) can be obtained that are comparable across transcriptome datasets. In order to demonstrate the feasibility of our strategy, we implement it in the web service PARRoT. PARRoT stands for Pipeline for Analyzing RNA Reads of Transcriptomes. It analyzes gene expression profiles for two transcriptome sequencing datasets. For better understanding of the biological meaning from the comparison among transcriptomes, PARRoT further provides linkage between these virtual transcripts and their potential function through showing best hits in SwissProt, NR database, assigning GO terms. Our demo datasets showed that PARRoT can analyze two paired-end transcriptomic datasets of approximately 100 million reads within just three hours. In this study, we proposed and implemented a strategy to analyze transcriptomes from non-reference organisms which offers

  6. A Proteomic Workflow Using High-Throughput De Novo Sequencing Towards Complementation of Genome Information for Improved Comparative Crop Science.

    PubMed

    Turetschek, Reinhard; Lyon, David; Desalegn, Getinet; Kaul, Hans-Peter; Wienkoop, Stefanie

    2016-01-01

    The proteomic study of non-model organisms, such as many crop plants, is challenging due to the lack of comprehensive genome information. Changing environmental conditions require the study and selection of adapted cultivars. Mutations, inherent to cultivars, hamper protein identification and thus considerably complicate the qualitative and quantitative comparison in large-scale systems biology approaches. With this workflow, cultivar-specific mutations are detected from high-throughput comparative MS analyses, by extracting sequence polymorphisms with de novo sequencing. Stringent criteria are suggested to filter for confidential mutations. Subsequently, these polymorphisms complement the initially used database, which is ready to use with any preferred database search algorithm. In our example, we thereby identified 26 specific mutations in two cultivars of Pisum sativum and achieved an increased number (17 %) of peptide spectrum matches.

  7. Complete genome sequences and comparative genome analysis of Lactobacillus plantarum strain 5-2 isolated from fermented soybean.

    PubMed

    Liu, Chen-Jian; Wang, Rui; Gong, Fu-Ming; Liu, Xiao-Feng; Zheng, Hua-Jun; Luo, Yi-Yong; Li, Xiao-Ran

    2015-12-01

    Lactobacillus plantarum is an important probiotic and is mostly isolated from fermented foods. We sequenced the genome of L. plantarum strain 5-2, which was derived from fermented soybean isolated from Yunnan province, China. The strain was determined to contain 3114 genes. Fourteen complete insertion sequence (IS) elements were found in 5-2 chromosome. There were 24 DNA replication proteins and 76 DNA repair proteins in the 5-2 genome. Consistent with the classification of L. plantarum as a facultative heterofermentative lactobacillus, the 5-2 genome encodes key enzymes required for the EMP (Embden-Meyerhof-Parnas) and phosphoketolase (PK) pathways. Several components of the secretion machinery are found in the 5-2 genome, which was compared with L. plantarum ST-III, JDM1 and WCFS1. Most of the specific proteins in the four genomes appeared to be related to their prophage elements. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Cleavage of HPV-16 E6/E7 mRNA mediated by modified 10-23 deoxyribozymes.

    PubMed

    Reyes-Gutiérrez, Pablo; Alvarez-Salas, Luis M

    2009-09-01

    Deoxyribozymes (DXZs) are small oligodeoxynucleotides capable of mediating phosphodiester bond cleavage of a target RNA in a sequence-specific manner. These molecules are a new generation of artificial catalytic nucleic acids currently used to silence many disease-related genes. The present study describes a DXZ (Dz1023-434) directed against the polycistronic mRNA from the E6 and E7 genes of human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer. Dz1023-434 showed efficient cleavage against a bona fide antisense window at nt 410-445 within HPV-16 E6/E7 mRNA even in low [Mg(2+)] conditions. Using a genetic analysis as guidance, we introduced diverse chemical modifications within Dz1023-434 catalytic core to produce a stable locked nucleic acid (LNA)-modified DXZ (Dz434-LNA) with significant cleavage activity of full E6/E7 transcripts. Cell culture testing of Dz434-LNA produced a sharp decrement of E6/E7 mRNA levels in HPV-16-positive cells resulting in decreased proliferation and considerable cell death in a specific and dose-dependent manner. No significant effects were observed with inactive or scrambled control DXZs nor from using HPV-negative cells, suggesting catalysis-dependent effect and high specificity. The biological effects of Dz434-LNA suggest a potential use for the treatment of cervical cancer.

  9. Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae).

    PubMed

    Begum, Rabeya; Zakrzewski, Falk; Menzel, Gerhard; Weber, Beatrice; Alam, Sheikh Shamimul; Schmidt, Thomas

    2013-07-01

    The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100-500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S-5·8S-25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

  10. Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae)

    PubMed Central

    Begum, Rabeya; Zakrzewski, Falk; Menzel, Gerhard; Weber, Beatrice; Alam, Sheikh Shamimul; Schmidt, Thomas

    2013-01-01

    Background and Aims The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. Methods A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100–500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Key Results Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S–5·8S–25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. Conclusions The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species. PMID:23666888

  11. Complete Genome Sequence and Comparative Genomics of a Novel Myxobacterium Myxococcus hansupus

    PubMed Central

    Sharma, Gaurav; Narwani, Tarun; Subramanian, Srikrishna

    2016-01-01

    Myxobacteria, a group of Gram-negative aerobes, belong to the class δ-proteobacteria and order Myxococcales. Unlike anaerobic δ-proteobacteria, they exhibit several unusual physiogenomic properties like gliding motility, desiccation-resistant myxospores and large genomes with high coding density. Here we report a 9.5 Mbp complete genome of Myxococcus hansupus that encodes 7,753 proteins. Phylogenomic and genome-genome distance based analysis suggest that Myxococcus hansupus is a novel member of the genus Myxococcus. Comparative genome analysis with other members of the genus Myxococcus was performed to explore their genome diversity. The variation in number of unique proteins observed across different species is suggestive of diversity at the genus level while the overrepresentation of several Pfam families indicates the extent and mode of genome expansion as compared to non-Myxococcales δ-proteobacteria. PMID:26900859

  12. Comparative systems biology between human and animal models based on next-generation sequencing methods.

    PubMed

    Zhao, Yu-Qi; Li, Gong-Hua; Huang, Jing-Fei

    2013-04-01

    Animal models provide myriad benefits to both experimental and clinical research. Unfortunately, in many situations, they fall short of expected results or provide contradictory results. In part, this can be the result of traditional molecular biological approaches that are relatively inefficient in elucidating underlying molecular mechanism. To improve the efficacy of animal models, a technological breakthrough is required. The growing availability and application of the high-throughput methods make systematic comparisons between human and animal models easier to perform. In the present study, we introduce the concept of the comparative systems biology, which we define as "comparisons of biological systems in different states or species used to achieve an integrated understanding of life forms with all their characteristic complexity of interactions at multiple levels". Furthermore, we discuss the applications of RNA-seq and ChIP-seq technologies to comparative systems biology between human and animal models and assess the potential applications for this approach in the future studies.

  13. [Construction and eukaryotic expression of PVAX1-hPV58mE6E7fcGB composite gene vaccine].

    PubMed

    Wang, He; Yu, Jiyun; Li, Li

    2013-10-01

    To construct and express a composite gene vaccine for human papillomavirus 58(HPV58)-associated cervical cancer, we inserted HPV58mE6E7 fusion gene into pCI-Fc-GPI eukaryotic expression vector, constructing a recombinant plasmid named pCI-sig-HPV58mE6E7-Fc-GPI. Then we further inserted fragment of sig-HPV58mE6E7Fc-GPI into the novel vaccine vector PVAX1-IRES-GM/B7, constructing PVAX1-HPV58mE6E7FcGB composite gene vaccine. PVAX1-HPV58mE6E7FcGB vaccine was successfully constructed and identified by restriction endonuclease and sequencing analysis. Eukaryotic expression of fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular ad-juvant GM-CSF and B7. 1 were proved to be realized at the same time by flow cytometry and immunofluorescence. So PVAX1-HPV58mE6E7FcGB can be taken as a candidate of therapeutic vaccine for HPV58-associated tumors and their precancerous transformations.

  14. Complete genome sequence and comparative genomics of the probiotic yeast Saccharomyces boulardii.

    PubMed

    Khatri, Indu; Tomar, Rajul; Ganesan, K; Prasad, G S; Subramanian, Srikrishna

    2017-03-23

    The probiotic yeast, Saccharomyces boulardii (Sb) is known to be effective against many gastrointestinal disorders and antibiotic-associated diarrhea. To understand molecular basis of probiotic-properties ascribed to Sb we determined the complete genomes of two strains of Sb i.e. Biocodex and unique28 and the draft genomes for three other Sb strains that are marketed as probiotics in India. We compared these genomes with 145 strains of S. cerevisiae (Sc) to understand genome-level similarities and differences between these yeasts. A distinctive feature of Sb from other Sc is absence of Ty elements Ty1, Ty3, Ty4 and associated LTR. However, we could identify complete Ty2 and Ty5 elements in Sb. The genes for hexose transporters HXT11 and HXT9, and asparagine-utilization are absent in all Sb strains. We find differences in repeat periods and copy numbers of repeats in flocculin genes that are likely related to the differential adhesion of Sb as compared to Sc. Core-proteome based taxonomy places Sb strains along with wine strains of Sc. We find the introgression of five genes from Z. bailii into the chromosome IV of Sb and wine strains of Sc. Intriguingly, genes involved in conferring known probiotic properties to Sb are conserved in most Sc strains.

  15. Comparative genome sequencing reveals genomic signature of extreme desiccation tolerance in the anhydrobiotic midge

    PubMed Central

    Gusev, Oleg; Suetsugu, Yoshitaka; Cornette, Richard; Kawashima, Takeshi; Logacheva, Maria D.; Kondrashov, Alexey S.; Penin, Aleksey A.; Hatanaka, Rie; Kikuta, Shingo; Shimura, Sachiko; Kanamori, Hiroyuki; Katayose, Yuichi; Matsumoto, Takashi; Shagimardanova, Elena; Alexeev, Dmitry; Govorun, Vadim; Wisecaver, Jennifer; Mikheyev, Alexander; Koyanagi, Ryo; Fujie, Manabu; Nishiyama, Tomoaki; Shigenobu, Shuji; Shibata, Tomoko F.; Golygina, Veronika; Hasebe, Mitsuyasu; Okuda, Takashi; Satoh, Nori; Kikawada, Takahiro

    2014-01-01

    Anhydrobiosis represents an extreme example of tolerance adaptation to water loss, where an organism can survive in an ametabolic state until water returns. Here we report the first comparative analysis examining the genomic background of extreme desiccation tolerance, which is exclusively found in larvae of the only anhydrobiotic insect, Polypedilum vanderplanki. We compare the genomes of P. vanderplanki and a congeneric desiccation-sensitive midge P. nubifer. We determine that the genome of the anhydrobiotic species specifically contains clusters of multi-copy genes with products that act as molecular shields. In addition, the genome possesses several groups of genes with high similarity to known protective proteins. However, these genes are located in distinct paralogous clusters in the genome apart from the classical orthologues of the corresponding genes shared by both chironomids and other insects. The transcripts of these clustered paralogues contribute to a large majority of the mRNA pool in the desiccating larvae and most likely define successful anhydrobiosis. Comparison of expression patterns of orthologues between two chironomid species provides evidence for the existence of desiccation-specific gene expression systems in P. vanderplanki. PMID:25216354

  16. 42 CFR 52e.7 - What are the terms and conditions of awards?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false What are the terms and conditions of awards? 52e.7 Section 52e.7 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.7 What are the terms...

  17. 42 CFR 52e.7 - What are the terms and conditions of awards?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false What are the terms and conditions of awards? 52e.7 Section 52e.7 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.7 What are the terms...

  18. 42 CFR 52e.7 - What are the terms and conditions of awards?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false What are the terms and conditions of awards? 52e.7 Section 52e.7 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.7 What are the terms...

  19. 42 CFR 52e.7 - What are the terms and conditions of awards?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false What are the terms and conditions of awards? 52e.7 Section 52e.7 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.7 What are the terms...

  20. 42 CFR 52e.7 - What are the terms and conditions of awards?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false What are the terms and conditions of awards? 52e.7 Section 52e.7 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.7 What are the terms...

  1. Genome sequencing and comparative genomics reveal a repertoire of putative pathogenicity genes in chilli anthracnose fungus Colletotrichum truncatum.

    PubMed

    Rao, Soumya; Nandineni, Madhusudan R

    2017-01-01

    Colletotrichum truncatum, a major fungal phytopathogen, causes the anthracnose disease on an economically important spice crop chilli (Capsicum annuum), resulting in huge economic losses in tropical and sub-tropical countries. It follows a subcuticular intramural infection strategy on chilli with a short, asymptomatic, endophytic phase, which contrasts with the intracellular hemibiotrophic lifestyle adopted by most of the Colletotrichum species. However, little is known about the molecular determinants and the mechanism of pathogenicity in this fungus. A high quality whole genome sequence and gene annotation based on transcriptome data of an Indian isolate of C. truncatum from chilli has been obtained. Analysis of the genome sequence revealed a rich repertoire of pathogenicity genes in C. truncatum encoding secreted proteins, effectors, plant cell wall degrading enzymes, secondary metabolism associated proteins, with potential roles in the host-specific infection strategy, placing it next only to the Fusarium species. The size of genome assembly, number of predicted genes and some of the functional categories were similar to other sequenced Colletotrichum species. The comparative genomic analyses with other species and related fungi identified some unique genes and certain highly expanded gene families of CAZymes, proteases and secondary metabolism associated genes in the genome of C. truncatum. The draft genome assembly and functional annotation of potential pathogenicity genes of C. truncatum provide an important genomic resource for understanding the biology and lifestyle of this important phytopathogen and will pave the way for designing efficient disease control regimens.

  2. A comparative analysis of MC4R gene sequence, polymorphism, and chromosomal localization in Chinese raccoon dog and Arctic fox.

    PubMed

    Skorczyk, Anna; Flisikowski, Krzysztof; Switonski, Marek

    2012-05-01

    Numerous mutations of the human melanocortin receptor type 4 (MC4R) gene are responsible for monogenic obesity, and some of them appear to be associated with predisposition or resistance to polygenic obesity. Thus, this gene is considered a functional candidate for fat tissue accumulation and body weight in domestic mammals. The aim of the study was comparative analysis of chromosome localization, nucleotide sequence, and polymorphism of the MC4R gene in two farmed species of the Canidae family, namely the Chinese raccoon dog (Nycterutes procyonoides procyonoides) and the arctic fox (Alopex lagopus). The whole coding sequence, including fragments of 3'UTR and 5'UTR, shows 89% similarity between the arctic fox (1276 bp) and Chinese raccoon dog (1213 bp). Altogether, 30 farmed Chinese raccoon dogs and 30 farmed arctic foxes were searched for polymorphisms. In the Chinese raccoon dog, only one silent substitution in the coding sequence was identified; whereas in the arctic fox, four InDels and two single-nucleotide polymorphisms (SNPs) in the 5'UTR and six silent SNPs in the exon were found. The studied gene was mapped by FISH to the Chinese raccoon dog chromosome 9 (NPP9q1.2) and arctic fox chromosome 24 (ALA24q1.2-1.3). The obtained results are discussed in terms of genome evolution of species belonging to the family Canidae and their potential use in animal breeding.

  3. Molecular Characterization and Comparative Sequence Analysis of Defense-Related Gene, Oryza rufipogon Receptor-Like Protein Kinase 1

    PubMed Central

    Law, Yee-Song; Gudimella, Ranganath; Song, Beng-Kah; Ratnam, Wickneswari; Harikrishna, Jennifer Ann

    2012-01-01

    Many of the plant leucine rich repeat receptor-like kinases (LRR-RLKs) have been found to regulate signaling during plant defense processes. In this study, we selected and sequenced an LRR-RLK gene, designated as Oryza rufipogon receptor-like protein kinase 1 (OrufRPK1), located within yield QTL yld1.1 from the wild rice Oryza rufipogon (accession IRGC105491). A 2055 bp coding region and two exons were identified. Southern blotting determined OrufRPK1 to be a single copy gene. Sequence comparison with cultivated rice orthologs (OsI219RPK1, OsI9311RPK1 and OsJNipponRPK1, respectively derived from O. sativa ssp. indica cv. MR219, O. sativa ssp. indica cv. 9311 and O. sativa ssp. japonica cv. Nipponbare) revealed the presence of 12 single nucleotide polymorphisms (SNPs) with five non-synonymous substitutions, and 23 insertion/deletion sites. The biological role of the OrufRPK1 as a defense related LRR-RLK is proposed on the basis of cDNA sequence characterization, domain subfamily classification, structural prediction of extra cellular domains, cluster analysis and comparative gene expression. PMID:22942769

  4. KRAS mutation detection in colorectal cancer by a commercially available gene chip array compares well with Sanger sequencing.

    PubMed

    French, Deborah; Smith, Andrew; Powers, Martin P; Wu, Alan H B

    2011-08-17

    Binding of a ligand to the epidermal growth factor receptor (EGFR) stimulates various intracellular signaling pathways resulting in cell cycle progression, proliferation, angiogenesis and apoptosis inhibition. KRAS is involved in signaling pathways including RAF/MAPK and PI3K and mutations in this gene result in constitutive activation of these pathways, independent of EGFR activation. Seven mutations in codons 12 and 13 of KRAS comprise around 95% of the observed human mutations, rendering monoclonal antibodies against EGFR (e.g. cetuximab and panitumumab) useless in treatment of colorectal cancer. KRAS mutation testing by two different methodologies was compared; Sanger sequencing and AutoGenomics INFINITI® assay, on DNA extracted from colorectal cancers. Out of 29 colorectal tumor samples tested, 28 were concordant between the two methodologies for the KRAS mutations that were detected in both assays with the INFINITI® assay detecting a mutation in one sample that was indeterminate by Sanger sequencing and a third methodology; single nucleotide primer extension. This study indicates the utility of the AutoGenomics INFINITI® methodology in a clinical laboratory setting where technical expertise or access to equipment for DNA sequencing does not exist. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Genome sequencing and comparative genomics reveal a repertoire of putative pathogenicity genes in chilli anthracnose fungus Colletotrichum truncatum

    PubMed Central

    Rao, Soumya

    2017-01-01

    Colletotrichum truncatum, a major fungal phytopathogen, causes the anthracnose disease on an economically important spice crop chilli (Capsicum annuum), resulting in huge economic losses in tropical and sub-tropical countries. It follows a subcuticular intramural infection strategy on chilli with a short, asymptomatic, endophytic phase, which contrasts with the intracellular hemibiotrophic lifestyle adopted by most of the Colletotrichum species. However, little is known about the molecular determinants and the mechanism of pathogenicity in this fungus. A high quality whole genome sequence and gene annotation based on transcriptome data of an Indian isolate of C. truncatum from chilli has been obtained. Analysis of the genome sequence revealed a rich repertoire of pathogenicity genes in C. truncatum encoding secreted proteins, effectors, plant cell wall degrading enzymes, secondary metabolism associated proteins, with potential roles in the host-specific infection strategy, placing it next only to the Fusarium species. The size of genome assembly, number of predicted genes and some of the functional categories were similar to other sequenced Colletotrichum species. The comparative genomic analyses with other species and related fungi identified some unique genes and certain highly expanded gene families of CAZymes, proteases and secondary metabolism associated genes in the genome of C. truncatum. The draft genome assembly and functional annotation of potential pathogenicity genes of C. truncatum provide an important genomic resource for understanding the biology and lifestyle of this important phytopathogen and will pave the way for designing efficient disease control regimens. PMID:28846714

  6. Genome Sequencing and Comparative Analysis of Stenotrophomonas acidaminiphila Reveal Evolutionary Insights Into Sulfamethoxazole Resistance.

    PubMed

    Huang, Yao-Ting; Chen, Jia-Min; Ho, Bing-Ching; Wu, Zong-Yen; Kuo, Rita C; Liu, Po-Yu

    2018-01-01

    Stenotrophomonas acidaminiphila is an aerobic, glucose non-fermentative, Gram-negative bacterium that been isolated from various environmental sources, particularly aquatic ecosystems. Although resistance to multiple antimicrobial agents has been reported in S. acidaminiphila , the mechanisms are largely unknown. Here, for the first time, we report the complete genome and antimicrobial resistome analysis of a clinical isolate S. acidaminiphila SUNEO which is resistant to sulfamethoxazole. Comparative analysis among closely related strains identified common and strain-specific genes. In particular, comparison with a sulfamethoxazole-sensitive strain identified a mutation within the sulfonamide-binding site of folP in SUNEO, which may reduce the binding affinity of sulfamethoxazole. Selection pressure analysis indicated folP in SUNEO is under purifying selection, which may be owing to long-term administration of sulfonamide against Stenotrophomonas .

  7. Unclassified renal cell carcinoma: a clinicopathological, comparative genomic hybridization, and whole-genome exon sequencing study.

    PubMed

    Hu, Zhen-Yan; Pang, Li-Juan; Qi, Yan; Kang, Xue-Ling; Hu, Jian-Ming; Wang, Lianghai; Liu, Kun-Peng; Ren, Yuan; Cui, Mei; Song, Li-Li; Li, Hong-An; Zou, Hong; Li, Feng

    2014-01-01

    Unclassified renal cell carcinoma (URCC) is a rare variant of RCC, accounting for only 3-5% of all cases. Studies on the molecular genetics of URCC are limited, and hence, we report on 2 cases of URCC analyzed using comparative genome hybridization (CGH) and the genome-wide human exon GeneChip technique to identify the genomic alterations of URCC. Both URCC patients (mean age, 72 years) presented at an advanced stage and died within 30 months post-surgery. Histologically, the URCCs were composed of undifferentiated, multinucleated, giant cells with eosinophilic cytoplasm. Immunostaining revealed that both URCC cases had strong p53 protein expression and partial expression of cluster of differentiation-10 and cytokeratin. The CGH profiles showed chromosomal imbalances in both URCC cases: gains were observed in chromosomes 1p11-12, 1q12-13, 2q20-23, 3q22-23, 8p12, and 16q11-15, whereas losses were detected on chromosomes 1q22-23, 3p12-22, 5p30-ter, 6p, 11q, 16q18-22, 17p12-14, and 20p. Compared with 18 normal renal tissues, 40 mutated genes were detected in the URCC tissues, including 32 missense and 8 silent mutations. Functional enrichment analysis revealed that the missense mutation genes were involved in 11 different biological processes and pathways, including cell cycle regulation, lipid localization and transport, neuropeptide signaling, organic ether metabolism, and ATP-binding cassette transporter signaling. Our findings indicate that URCC may be a highly aggressive cancer, and the genetic alterations identified herein may provide clues regarding the tumorigenesis of URCC and serve as a basis for the development of targeted therapies against URCC in the future.

  8. Unclassified renal cell carcinoma: a clinicopathological, comparative genomic hybridization, and whole-genome exon sequencing study

    PubMed Central

    Hu, Zhen-Yan; Pang, Li-Juan; Qi, Yan; Kang, Xue-Ling; Hu, Jian-Ming; Wang, Lianghai; Liu, Kun-Peng; Ren, Yuan; Cui, Mei; Song, Li-Li; Li, Hong-An; Zou, Hong; Li, Feng

    2014-01-01

    Unclassified renal cell carcinoma (URCC) is a rare variant of RCC, accounting for only 3-5% of all cases. Studies on the molecular genetics of URCC are limited, and hence, we report on 2 cases of URCC analyzed using comparative genome hybridization (CGH) and the genome-wide human exon GeneChip technique to identify the genomic alterations of URCC. Both URCC patients (mean age, 72 years) presented at an advanced stage and died within 30 months post-surgery. Histologically, the URCCs were composed of undifferentiated, multinucleated, giant cells with eosinophilic cytoplasm. Immunostaining revealed that both URCC cases had strong p53 protein expression and partial expression of cluster of differentiation-10 and cytokeratin. The CGH profiles showed chromosomal imbalances in both URCC cases: gains were observed in chromosomes 1p11-12, 1q12-13, 2q20-23, 3q22-23, 8p12, and 16q11-15, whereas losses were detected on chromosomes 1q22-23, 3p12-22, 5p30-ter, 6p, 11q, 16q18-22, 17p12-14, and 20p. Compared with 18 normal renal tissues, 40 mutated genes were detected in the URCC tissues, including 32 missense and 8 silent mutations. Functional enrichment analysis revealed that the missense mutation genes were involved in 11 different biological processes and pathways, including cell cycle regulation, lipid localization and transport, neuropeptide signaling, organic ether metabolism, and ATP-binding cassette transporter signaling. Our findings indicate that URCC may be a highly aggressive cancer, and the genetic alterations identified herein may provide clues regarding the tumorigenesis of URCC and serve as a basis for the development of targeted therapies against URCC in the future. PMID:25120763

  9. Expressed sequence tags from poplar wood tissues--a comparative analysis from multiple libraries.

    PubMed

    Déjardin, A; Leplé, J-C; Lesage-Descauses, M-C; Costa, G; Pilate, G

    2004-01-01

    Xylogenesis involves successive developmental processes--cambial division, cell expansion and differentiation, cell death--each occurring along a gradient from the cambium to the pith of the stem. Taking advantage of the high level of organisation of wood tissues, we isolated cambial zone (CZ), differentiating xylem (DX) and mature xylem (MX) from both tension wood (TW) and opposite wood (OW) of bent poplars. Four different cDNA libraries were then constructed and used to generate 10,062 EST, reflecting the genes expressed in the different wood tissues. For the most abundant clusters, the EST distributions were compared between libraries in order to identify genes specific or over-represented at some specific developmental stages. They clearly showed a developmental shift between CZ and DX, whereas there is a continuity of development between DX and MX. CZ was mainly characterized by clusters of genes involved in cell cycle, protein synthesis and fate. Interestingly, two clusters with no assigned function were found specific to the cambial zone. In DX and MX, clusters were mostly involved in methylation of lignin precursors and microtubule cytoskeleton. In addition, in DX, EST from TW and OW were compared: five clusters of arabinogalactan proteins, one for sucrose synthase and one for fructokinase were specific or over-represented in TW. Moreover, a putative transcription factor and a cluster of unknown function were also identified in DX-TW. The informative comparison of multiple libraries prepared from wood tissues led to the identification of genes--some with still unknown functions--putatively involved in xylogenesis and tension wood formation.

  10. Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure

    PubMed Central

    Coyne, Robert S; Thiagarajan, Mathangi; Jones, Kristie M; Wortman, Jennifer R; Tallon, Luke J; Haas, Brian J; Cassidy-Hanley, Donna M; Wiley, Emily A; Smith, Joshua J; Collins, Kathleen; Lee, Suzanne R; Couvillion, Mary T; Liu, Yifan; Garg, Jyoti; Pearlman, Ronald E; Hamilton, Eileen P; Orias, Eduardo; Eisen, Jonathan A; Methé, Barbara A

    2008-01-01

    Background Tetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing. Results We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified. Conclusion We report here significant progress in genome closure and reannotation of Tetrahymena thermophila. Our experience to date

  11. Comparative Genomic Analysis of Two Clonally Related Multidrug Resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing.

    PubMed

    Leung, Kenneth Siu-Sing; Siu, Gilman Kit-Hang; Tam, Kingsley King-Gee; To, Sabrina Wai-Chi; Rajwani, Rahim; Ho, Pak-Leung; Wong, Samson Sai-Yin; Zhao, Wei W; Ma, Oliver Chiu-Kit; Yam, Wing-Cheong

    2017-01-01

    Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB under in vivo challenge of anti-TB drugs. Result: Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations at rpoB S531L, inhA C-15T, and embB M306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 of rv0888 in pre-treatment strain, and a missense mutation at rv3303c ( lpdA) V44I and a 6-bp inframe deletion at codon 67-68 in rv2071c ( cobM) in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function. Conclusion: This is the first study that compared the full genomes of two clonally-related MDR-TB clinical

  12. Gene Discovery in the Apicomplexa as Revealed by EST Sequencing and Assembly of a Comparative Gene Database

    PubMed Central

    Li, Li; Brunk, Brian P.; Kissinger, Jessica C.; Pape, Deana; Tang, Keliang; Cole, Robert H.; Martin, John; Wylie, Todd; Dante, Mike; Fogarty, Steven J.; Howe, Daniel K.; Liberator, Paul; Diaz, Carmen; Anderson, Jennifer; White, Michael; Jerome, Maria E.; Johnson, Emily A.; Radke, Jay A.; Stoeckert, Christian J.; Waterston, Robert H.; Clifton, Sandra W.; Roos, David S.; Sibley, L. David

    2003-01-01

    Large-scale EST sequencing projects for several important parasites within the phylum Apicomplexa were undertaken for the purpose of gene discovery. Included were several parasites of medical importance (Plasmodium falciparum, Toxoplasma gondii) and others of veterinary importance (Eimeria tenella, Sarcocystis neurona, and Neospora caninum). A total of 55,192 ESTs, deposited into dbEST/GenBank, were included in the analyses. The resulting sequences have been clustered into nonredundant gene assemblies and deposited into a relational database that supports a variety of sequence and text searches. This database has been used to compare the gene assemblies using BLAST similarity comparisons to the public protein databases to identify putative genes. Of these new entries, ∼15%–20% represent putative homologs with a conservative cutoff of p < 10−9, thus identifying many conserved genes that are likely to share common functions with other well-studied organisms. Gene assemblies were also used to identify strain polymorphisms, examine stage-specific expression, and identify gene families. An interesting class of genes that are confined to members of this phylum and not shared by plants, animals, or fungi, was identified. These genes likely mediate the novel biological features of members of the Apicomplexa and hence offer great potential for biological investigation and as possible therapeutic targets. [The sequence data from this study have been submitted to dbEST division of GenBank under accession nos.: Toxoplasma gondii: –, –, –, –, – , –, –, –, –. Plasmodium falciparum: –, –, –, –. Sarcocystis neurona: , , , , , , , , , , , , , –, –, –, –, –. Eimeria tenella: –, –, –, –, –, –, –, –, – , –, –, –, –, –, –, –, –, –, –, –. Neospora caninum: –, –, , – , –, –.] PMID:12618375

  13. Comparative analysis of grapevine whole-genome gene predictions, functional annotation, categorization and integration of the predicted gene sequences

    PubMed Central

    2012-01-01

    Background The first draft assembly and gene prediction of the grapevine genome (8X base coverage) was made available to the scientific community in 2007, and functional annotation was developed on this gene prediction. Since then additional Sanger sequences were added to the 8X sequences pool and a new version of the genomic sequence with superior base coverage (12X) was produced. Results In order to more efficiently annotate the function of the genes predicted in the new assembly, it is important to build on as much of the previous work as possible, by transferring 8X annotation of the genome to the 12X version. The 8X and 12X assemblies and gene predictions of the grapevine genome were compared to answer the question, “Can we uniquely map 8X predicted genes to 12X predicted genes?” The results show that while the assemblies and gene structure predictions are too different to make a complete mapping between them, most genes (18,725) showed a one-to-one relationship between 8X predicted genes and the last version of 12X predicted genes. In addition, reshuffled genomic sequence structures appeared. These highlight regions of the genome where the gene predictions need to be taken with caution. Based on the new grapevine gene functional annotation and in-depth functional categorization, twenty eight new molecular networks have been created for VitisNet while the existing networks were updated. Conclusions The outcomes of this study provide a functional annotation of the 12X genes, an update of VitisNet, the system of the grapevine molecular networks, and a new functional categorization of genes. Data are available at the VitisNet website (http://www.sdstate.edu/ps/research/vitis/pathways.cfm). PMID:22554261

  14. A recombinant isoform of the Ole e 7 olive pollen allergen assembled by de novo mass spectrometry retains the allergenic ability of the natural allergen.

    PubMed

    Oeo-Santos, Carmen; Mas, Salvador; Benedé, Sara; López-Lucendo, María; Quiralte, Joaquín; Blanca, Miguel; Mayorga, Cristobalina; Villalba, Mayte; Barderas, Rodrigo

    2018-06-05

    The allergenic non-specific lipid transfer protein Ole e 7 from olive pollen is a major allergen associated with severe symptoms in areas with high olive pollen levels. Despite its clinical importance, its cloning and recombinant production has been unable by classical approaches. This study aimed at determining by mass-spectrometry based proteomics its complete amino acid sequence for its subsequent expression and characterization. To this end, the natural protein was in-2D-gel tryptic digested, and CID and HCD fragmentation spectra obtained by nLC-MS/MS analyzed using PEAKS software. Thirteen out of the 457 de novo sequenced peptides obtained allowed assembling its full-length amino acid sequence. Then, Ole e 7-encoding cDNA was synthesized and cloned in pPICZαA vector for its expression in Pichia pastoris yeast. The analyses by Circular Dichroism, and WB, ELISA and cell-based tests using sera and blood from olive pollen-sensitized patients showed that rOle e 7 mostly retained the structural, allergenic and antigenic properties of the natural allergen. In summary, rOle e 7 allergen assembled by de novo peptide sequencing by MS behaved immunologically similar to the natural allergen scarcely isolated from pollen. Olive pollen is an important cause of allergy. The non-specific lipid binding protein Ole e 7 is a major allergen with a high incidence and a phenotype associated to severe clinical symptoms. Despite its relevance, its cloning and recombinant expression has been unable by classical techniques. Here, we have inferred the primary amino acid sequence of Ole e 7 by mass-spectrometry. We separated Ole e 7 isolated from pollen by 2DE. After in-gel digestion with trypsin and a direct analysis by nLC-MS/MS in an LTQ-Orbitrap Velos, we got the complete de novo sequenced peptides repertoire that allowed the assembling of the primary sequence of Ole e 7. After its protein expression, purification to homogeneity, and structural and immunological characterization

  15. Comparative sequence analysis revealed altered chromosomal organization and a novel insertion sequence encoding DNA modification and potentially stress-related functions in an Escherichia coli O157:H7 foodborne isolate

    We recently described the complete genome of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain NADC 6564, an isolate of strain 86-24 linked to the 1986 disease outbreak. In the current study, we compared the chromosomal sequence of NADC 6564 to the well-characterized chromosomal sequences of ...

  16. Sequencing and Comparative Genome Analysis of Two Pathogenic Streptococcus gallolyticus Subspecies: Genome Plasticity, Adaptation and Virulence

    PubMed Central

    Teng, Yu-Ting; Wu, Hui-Lun; Liu, Yen-Ming; Wu, Keh-Ming; Chang, Chuan-Hsiung; Hsu, Ming-Ta

    2011-01-01

    Streptococcus gallolyticus infections in humans are often associated with bacteremia, infective endocarditis and colon cancers. The disease manifestations are different depending on the subspecies of S. gallolyticus causing the infection. Here, we present the complete genomes of S. gallolyticus ATCC 43143 (biotype I) and S. pasteurianus ATCC 43144 (biotype II.2). The genomic differences between the two biotypes were characterized with comparative genomic analyses. The chromosome of ATCC 43143 and ATCC 43144 are 2,36 and 2,10 Mb in length and encode 2246 and 1869 CDS respectively. The organization and genomic contents of both genomes were most similar to the recently published S. gallolyticus UCN34, where 2073 (92%) and 1607 (86%) of the ATCC 43143 and ATCC 43144 CDS were conserved in UCN34 respectively. There are around 600 CDS conserved in all Streptococcus genomes, indicating the Streptococcus genus has a small core-genome (constitute around 30% of total CDS) and substantial evolutionary plasticity. We identified eight and five regions of genome plasticity in ATCC 43143 and ATCC 43144 respectively. Within these regions, several proteins were recognized to contribute to the fitness and virulence of each of the two subspecies. We have also predicted putative cell-surface associated proteins that could play a role in adherence to host tissues, leading to persistent infections causing sub-acute and chronic diseases in humans. This study showed evidence that the S. gallolyticus still possesses genes making it suitable in a rumen environment, whereas the ability for S. pasteurianus to live in rumen is reduced. The genome heterogeneity and genetic diversity among the two biotypes, especially membrane and lipoproteins, most likely contribute to the differences in the pathogenesis of the two S. gallolyticus biotypes and the type of disease an infected patient eventually develops. PMID:21633709

  17. Oxytocin stimulates cell proliferation in vaginal cell line Vk2E6E7.

    PubMed

    Kallak, Theodora K; Uvnäs-Moberg, Kerstin

    2017-03-01

    Objective During and after menopause, the symptoms of vaginal atrophy cause great discomfort and necessitate effective treatment options. Currently, vaginally applied oxytocin is being investigated as a treatment for the symptoms of vaginal atrophy in postmenopausal women. To clarify the mechanisms behind oxytocins effects on vaginal atrophy, the present study investigated the effects of oxytocin on cell proliferation in the cells of the Vk2E6E7 line, a non-tumour vaginal cell line. The study also compared the effects of oxytocin with those of estradiol (E2). Study design The effects of both oxytocin and E2 on the proliferation of Vk2E6E7 cells were investigated using Cell Proliferation ELISA BrdU Colorimetric Assay. The expression of both oxytocin and oxytocin receptor was studied in Vk2E6E7 cells using quantitative real-time polymerase chain reaction and immunofluorescent staining. Main outcome measures Cell proliferation and gene expression. Results Oxytocin increased cell proliferation both time dependently and dose dependently. This differed from the effect pattern observed in cells treated with E2. In addition, in oxytocin-treated cells, the oxytocin receptor was found to be co-localized with caveolin-1, indicating pro-proliferative signalling within the cell. Conclusions Oxytocin stimulates cell proliferation and the co-localization of oxytocin receptor with caveolin-1 in oxytocin-treated cells, supporting the role of oxytocin signalling in cell proliferation. In addition, these findings suggest that increased cell proliferation is one mechanism by which local vaginal oxytocin treatment increases vaginal thickness and relieves vaginal symptoms in postmenopausal women with vaginal atrophy.

  18. Comparative Maps of Human 19p13.3 and Mouse Chromosome 10 Allow Identification of Sequences at Evolutionary Breakpoints

    PubMed Central

    Puttagunta, Radhika; Gordon, Laurie A.; Meyer, Gary E.; Kapfhamer, David; Lamerdin, Jane E.; Kantheti, Prameela; Portman, Kathleen M.; Chung, Wendy K.; Jenne, Dieter E.; Olsen, Anne S.; Burmeister, Margit

    2000-01-01

    A cosmid/bacterial artificial chromosome (BAC) contiguous (contig) map of human chromosome (HSA) 19p13.3 has been constructed, and over 50 genes have been localized to the contig. Genes and anonymous ESTs from ≈4000 kb of human 19p13.3 were placed on the central mouse chromosome 10 map by genetic mapping and pulsed-field gel electrophoresis (PFGE) analysis. A region of ∼2500 kb of HSA 19p13.3 is collinear to mouse chromosome (MMU) 10. In contrast, the adjacent ≈1200 kb are inverted. Two genes are located in a 50-kb region after the inversion on MMU 10, followed by a region of homology to mouse chromosome 17. The synteny breakpoint and one of the inversion breakpoints has been localized to sequenced regions in human <5 kb in size. Both breakpoints are rich in simple tandem repeats, including (TCTG)n, (CT)n, and (GTCTCT)n, suggesting that simple repeat sequences may be involved in chromosome breaks during evolution. The overall size of the region in mouse is smaller, although no large regions are missing. Comparing the physical maps to the genetic maps showed that in contrast to the higher-than-average rate of genetic recombination in gene-rich telomeric region on HSA 19p13.3, the average rate of recombination is lower than expected in the homologous mouse region. This might indicate that a hot spot of recombination may have been lost in mouse or gained in human during evolution, or that the position of sequences along the chromosome (telomeric compared to the middle of a chromosome) is important for recombination rates. PMID:10984455

  19. Genome sequences and comparative genomics of two Lactobacillus ruminis strains from the bovine and human intestinal tracts

    PubMed Central

    2011-01-01

    Background The genus Lactobacillus is characterized by an extraordinary degree of phenotypic and genotypic diversity, which recent genomic analyses have further highlighted. However, the choice of species for sequencing has been non-random and unequal in distribution, with only a single representative genome from the L. salivarius clade available to date. Furthermore, there is no data to facilitate a functional genomic analysis of motility in the lactobacilli, a trait that is restricted to the L. salivarius clade. Results The 2.06 Mb genome of the bovine isolate Lactobacillus ruminis ATCC 27782 comprises a single circular chromosome, and has a G+C content of 44.4%. In silico analysis identified 1901 coding sequences, including genes for a pediocin-like bacteriocin, a single large exopolysaccharide-related cluster, two sortase enzymes, two CRISPR loci and numerous IS elements and pseudogenes. A cluster of genes related to a putative pilin was identified, and shown to be transcribed in vitro. A high quality draft assembly of the genome of a second L. ruminis strain, ATCC 25644 isolated from humans, suggested a slightly larger genome of 2.138 Mb, that exhibited a high degree of synteny with the ATCC 27782 genome. In contrast, comparative analysis of L. ruminis and L. salivarius identified a lack of long-range synteny between these closely related species. Comparison of the L. salivarius clade core proteins with those of nine other Lactobacillus species distributed across 4 major phylogenetic groups identified the set of shared proteins, and proteins unique to each group. Conclusions The genome of L. ruminis provides a comparative tool for directing functional analyses of other members of the L. salivarius clade, and it increases understanding of the divergence of this distinct Lactobacillus lineage from other commensal lactobacilli. The genome sequence provides a definitive resource to facilitate investigation of the genetics, biochemistry and host interactions of

  20. spa Typing and Multilocus Sequence Typing Show Comparable Performance in a Macroepidemiologic Study of Staphylococcus aureus in the United States

    PubMed Central

    O'Hara, F. Patrick; Suaya, Jose A.; Ray, G. Thomas; Baxter, Roger; Brown, Megan L.; Mera, Robertino M.; Close, Nicole M.; Thomas, Elizabeth

    2016-01-01

    A number of molecular typing methods have been developed for characterization of Staphylococcus aureus isolates. The utility of these systems depends on the nature of the investigation for which they are used. We compared two commonly used methods of molecular typing, multilocus sequence typing (MLST) (and its clustering algorithm, Based Upon Related Sequence Type [BURST]) with the staphylococcal protein A (spa) typing (and its clustering algorithm, Based Upon Repeat Pattern [BURP]), to assess the utility of these methods for macroepidemiology and evolutionary studies of S. aureus in the United States. We typed a total of 366 clinical isolates of S. aureus by these methods and evaluated indices of diversity and concordance values. Our results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST. Therefore, spa typing is appropriate for use in macroepidemiology and evolutionary studies and, given its lower implementation cost, this method appears to be more efficient. The findings are robust and are consistent across different settings, patient ages, and specimen sources. Our results also support a model in which the methicillin-resistant S. aureus (MRSA) population in the United States comprises two major lineages (USA300 and USA100), which each consist of closely related variants. PMID:26669861

  1. spa Typing and Multilocus Sequence Typing Show Comparable Performance in a Macroepidemiologic Study of Staphylococcus aureus in the United States.

    PubMed

    O'Hara, F Patrick; Suaya, Jose A; Ray, G Thomas; Baxter, Roger; Brown, Megan L; Mera, Robertino M; Close, Nicole M; Thomas, Elizabeth; Amrine-Madsen, Heather

    2016-01-01

    A number of molecular typing methods have been developed for characterization of Staphylococcus aureus isolates. The utility of these systems depends on the nature of the investigation for which they are used. We compared two commonly used methods of molecular typing, multilocus sequence typing (MLST) (and its clustering algorithm, Based Upon Related Sequence Type [BURST]) with the staphylococcal protein A (spa) typing (and its clustering algorithm, Based Upon Repeat Pattern [BURP]), to assess the utility of these methods for macroepidemiology and evolutionary studies of S. aureus in the United States. We typed a total of 366 clinical isolates of S. aureus by these methods and evaluated indices of diversity and concordance values. Our results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST. Therefore, spa typing is appropriate for use in macroepidemiology and evolutionary studies and, given its lower implementation cost, this method appears to be more efficient. The findings are robust and are consistent across different settings, patient ages, and specimen sources. Our results also support a model in which the methicillin-resistant S. aureus (MRSA) population in the United States comprises two major lineages (USA300 and USA100), which each consist of closely related variants.

  2. Complete genome sequence of Enterococcus faecium strain TX16 and comparative genomic analysis of Enterococcus faecium genomes

    PubMed Central

    2012-01-01

    Background Enterococci are among the leading causes of hospital-acquired infections in the United States and Europe, with Enterococcus faecalis and Enterococcus faecium being the two most common species isolated from enterococcal infections. In the last decade, the proportion of enterococcal infections caused by E. faecium has steadily increased compared to other Enterococcus species. Although the underlying mechanism for the gradual replacement of E. faecalis by E. faecium in the hospital environment is not yet understood, many studies using genotyping and phylogenetic analysis have shown the emergence of a globally dispersed polyclonal subcluster of E. faecium strains in clinical environments. Systematic study of the molecular epidemiology and pathogenesis of E. faecium has been hindered by the lack of closed, complete E. faecium genomes that can be used as references. Results In this study, we report the complete genome sequence of the E. faecium strain TX16, also known as DO, which belongs to multilocus sequence type (ST) 18, and was the first E. faecium strain ever sequenced. Whole genome comparison of the TX16 genome with 21 E. faecium draft genomes confirmed that most clinical, outbreak, and hospital-associated (HA) strains (including STs 16, 17, 18, and 78), in addition to strains of non-hospital origin, group in the same clade (referred to as the HA clade) and are evolutionally considerably more closely related to each other by phylogenetic and gene content similarity analyses than to isolates in the community-associated (CA) clade with approximately a 3–4% average nucleotide sequence difference between the two clades at the core genome level. Our study also revealed that many genomic loci in the TX16 genome are unique to the HA clade. 380 ORFs in TX16 are HA-clade specific and antibiotic resistance genes are enriched in HA-clade strains. Mobile elements such as IS16 and transposons were also found almost exclusively in HA strains, as previously reported

  3. Parallel computation for biological sequence comparison: comparing a portable model to the native model for the Intel Hypercube.

    PubMed Central

    Nadkarni, P. M.; Miller, P. L.

    1991-01-01

    A parallel program for inter-database sequence comparison was developed on the Intel Hypercube using two models of parallel programming. One version was built using machine-specific Hypercube parallel programming commands. The other version was built using Linda, a machine-independent parallel programming language. The two versions of the program provide a case study comparing these two approaches to parallelization in an important biological application area. Benchmark tests with both programs gave comparable results with a small number of processors. As the number of processors was increased, the Linda version was somewhat less efficient. The Linda version was also run without change on Network Linda, a virtual parallel machine running on a network of desktop workstations. PMID:1807632

  4. Parallel computation for biological sequence comparison: comparing a portable model to the native model for the Intel Hypercube.

    PubMed

    Nadkarni, P M; Miller, P L

    1991-01-01

    A parallel program for inter-database sequence comparison was developed on the Intel Hypercube using two models of parallel programming. One version was built using machine-specific Hypercube parallel programming commands. The other version was built using Linda, a machine-independent parallel programming language. The two versions of the program provide a case study comparing these two approaches to parallelization in an important biological application area. Benchmark tests with both programs gave comparable results with a small number of processors. As the number of processors was increased, the Linda version was somewhat less efficient. The Linda version was also run without change on Network Linda, a virtual parallel machine running on a network of desktop workstations.

  5. Second generation DNA sequencing of the mitogenome of the Chinstrap penguin and comparative genomics of Antarctic penguins.

    PubMed

    Subramanian, Sankar; Lingala, Syamala Gowri; Swaminathan, Siva; Huynen, Leon; Lambert, David

    2014-08-01

    The complete mitochondrial genome of the Chinstrap penguin (Pygoscelis antarcticus) was sequenced and compared with other penguin mitogenomes. The genome is 15,972 bp in length with the number and order of protein coding genes and RNAs being very similar to that of other known penguin mitogenomes. Comparative nucleotide analysis showed the Chinstrap mitogenome shares 94% homology with the mitogenome of its sister species, Pygoscelis adelie (Adélie penguin). Divergence at nonsynonymous nucleotide positions was found to be up to 23 times less than that observed in synonymous positions of protein coding genes, suggesting high selection constraints. The complete mitogenome data will be useful for genetic and evolutionary studies of penguins.

  6. The HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of Polo-like kinase 4 expression

    PubMed Central

    2011-01-01

    Background Infection with high-risk human papillomaviruses (HPVs) such as HPV-16 is intimately associated with squamous cell carcinomas (SCCs) of the anogenital tract and a subset of oropharyngeal carcinomas. Such lesions, including pre-invasive precursors, frequently show multipolar mitoses and aneuploidy. The high-risk HPV-16-encoded E7 oncoprotein has been shown to rapidly induce centrosome abnormalities thereby causing the formation of supernumerary mitotic spindle poles and increasing the risk for chromosome missegregation. HPV-16 E7 has been found to rapidly induce centriole overduplication, in part, through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication). The precise molecular mechanism that underlies HPV-16 E7-induced centriole multiplication, however, remains poorly understood. Findings Here, we show that human keratinocytes engineered to stably express the HPV-16 E7 oncoprotein exhibit aberrant Polo-like kinase 4 (PLK4) protein expression at maternal centrioles. Real-time quantitative reverse transcriptase (qRT-PCR) analysis of these cells revealed an increase of PLK4 mRNA levels compared to control cells. Importantly, the ability of the HPV-16 E7 oncoprotein to induce centriole multiplication was found to correlate with its ability to activate the PLK4 promoter and to up-regulate PLK4 mRNA. Conclusions These results highlight the critical role of PLK4 transcriptional deregulation in centriole multiplication in HPV-16 E7-expressing cells. Our findings encourage further experiments to test transcriptional inhibitors or small molecules targeting PLK4 to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms and other tumors in which PLK4 regulation is disrupted. PMID:21609466

  7. Comparative Genome Analysis of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Sequence Type 131 Strains from Nepal and Japan

    PubMed Central

    Miyoshi-Akiyama, Tohru; Sherchan, Jatan Bahadur; Doi, Yohei; Nagamatsu, Maki; Sherchand, Jeevan B.; Tandukar, Sarmila; Ohmagari, Norio; Kirikae, Teruo; Ohara, Hiroshi

    2016-01-01

    ABSTRACT The global spread of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli (ESBL-E. coli) has largely been driven by the pandemic sequence type 131 (ST131). This study aimed to determine the molecular epidemiology of their spread in two Asian countries with contrasting prevalence. We conducted whole-genome sequencing (WGS) of ESBL-E. coli ST131 strains collected prospectively from Nepal and Japan, two countries in Asia with a high and low prevalence of ESBL-E. coli, respectively. We also systematically compared these genomes with those reported from other regions using publicly available WGS data for E. coli ST131 strains. Further, we conducted phylogenetic analysis of these isolates and all genome sequence data for ST131 strains to determine sequence diversity. One hundred five unique ESBL-E. coli isolates from Nepal (February 2013 to July 2013) and 76 isolates from Japan (October 2013 to September 2014) were included. Of these isolates, 54 (51%) isolates from Nepal and 11 (14%) isolates from Japan were identified as ST131 by WGS. Phylogenetic analysis based on WGS suggested that the majority of ESBL-E. coli ST131 isolates from Nepal clustered together, whereas those from Japan were more diverse. Half of the ESBL-E. coli ST131 isolates from Japan belonged to virotype C, whereas half of the isolates from Nepal belonged to a virotype other than virotype A, B, C, D, or E (A/B/C/D/E). The dominant sublineage of E. coli ST131 was H30Rx, which was most prominent in ESBL-E. coli ST131 isolates from Nepal. Our results revealed distinct phylogenetic characteristics of ESBL-E. coli ST131 spread in the two geographical areas of Asia, indicating the involvement of multiple factors in its local spread in each region. IMPORTANCE The global spread of ESBL-E. coli has been driven in large part by pandemic sequence type 131 (ST131). A recent study suggested that, within E. coli ST131, certain sublineages have disseminated worldwide with little association

  8. Complete genome sequence and comparative genome analysis of Klebsiella oxytoca HKOPL1 isolated from giant panda feces.

    PubMed

    Jiang, Jingwei; Tun, Hein Min; Mauroo, Nathalie France; Ma, Angel Po Yee; Chan, San Yuen; Leung, Frederick C

    2014-11-23

    The giant panda (Ailuropoda melanoleuca) is an endangered species well-known for ingesting bamboo as a major part of their diet despite the fact that it belongs to order Carnivora. However, the giant panda's draft genome shows no direct evidence of enzymatic genes responsible for cellulose digestion. To explore this phenomenon, we study the giant panda's gut microbiota using genomic approaches in order to better understand their physiological processes as well as any potential microbial cellulose digestion processes. A complete genome of isolated Klebsiella oxytoca HKOPL1 of 5.9 Mb has been successfully sequenced, closed and comprehensively annotated against various databases. Genome comparisons within the Klebsiella genus and K. oxytoca species have also been performed. A total of 5,772 genes were predicted, and among them, 211 potential virulence genes, 35 pathogenicity island-like regions, 1,615 potential horizontal transferring genes, 23 potential antibiotics resistant genes, a potential prophage integrated region, 8 genes in 2,3-Butanediol production pathway and 3 genes in the cellulose degradation pathway could be identified and discussed based on the comparative genomic studies between the complete genome sequence of K. oxytoca HKOPL1 and other Klebsiella strains. A functional study shows that K. oxytoca HKOPL1 can degrade cellulose within 72 hours. Phylogenomic studies indicate that K. oxytoca HKOPL1 is clustered with K. oxytoca strains 1686 and E718. K. oxytoca HKOPL1 is a gram-negative bacterium able to degrade cellulose. We report here the first complete genome sequence of K. oxytoca isolated from giant panda feces. These studies have provided further insight into the role of gut microbiota in giant panda digestive physiology. In addition, K. oxytoca HKOPL1 has the potential for biofuel application in terms of cellulose degradation and potential for the production of 2,3-Butanediol (an important industrial raw material).

  9. Complete genome sequence of the fire blight pathogen Erwinia pyrifoliae DSM 12163T and comparative genomic insights into plant pathogenicity

    PubMed Central

    2010-01-01

    Background Erwinia pyrifoliae is a newly described necrotrophic pathogen, which causes fire blight on Asian (Nashi) pear and is geographically restricted to Eastern Asia. Relatively little is known about its genetics compared to the closely related main fire blight pathogen E. amylovora. Results The genome of the type strain of E. pyrifoliae strain DSM 12163T, was sequenced using both 454 and Solexa pyrosequencing and annotated. The genome contains a circular chromosome of 4.026 Mb and four small plasmids. Based on their respective role in virulence in E. amylovora or related organisms, we identified several putative virulence factors, including type III and type VI secretion systems and their effectors, flagellar genes, sorbitol metabolism, iron uptake determinants, and quorum-sensing components. A deletion in the rpoS gene covering the most conserved region of the protein was identified which may contribute to the difference in virulence/host-range compared to E. amylovora. Comparative genomics with the pome fruit epiphyte Erwinia tasmaniensis Et1/99 showed that both species are overall highly similar, although specific differences were identified, for example the presence of some phage gene-containing regions and a high number of putative genomic islands containing transposases in the E. pyrifoliae DSM 12163T genome. Conclusions The E. pyrifoliae genome is an important addition to the published genome of E. tasmaniensis and the unfinished genome of E. amylovora providing a foundation for re-sequencing additional strains that may shed light on the evolution of the host-range and virulence/pathogenicity of this important group of plant-associated bacteria. PMID:20047678

  10. O2 and Water Migration Pathways between the Solvent and Heme Pockets of Hemoglobin with Open and Closed Conformations of the Distal HisE7.

    PubMed

    Shadrina, Maria S; Peslherbe, Gilles H; English, Ann M

    2015-09-01

    Hemoglobin transports O2 by binding the gas at its four hemes. Hydrogen bonding between the distal histidine (HisE7) and heme-bound O2 significantly increases the affinity of human hemoglobin (HbA) for this ligand. HisE7 is also proposed to regulate the release of O2 to the solvent via a transient E7 channel. To reveal the O2 escape routes controlled by HisE7 and to evaluate its role in gating heme access, we compare simulations of O2 diffusion from the distal heme pockets of the T and R states of HbA performed with HisE7 in its open (protonated) and closed (neutral) conformations. Irrespective of HisE7's conformation, we observe the same four or five escape routes leading directly from the α- or β-distal heme pockets to the solvent. Only 21-53% of O2 escapes occur via these routes, with the remainder escaping through routes that encompass multiple internal cavities in HbA. The conformation of the distal HisE7 controls the escape of O2 from the heme by altering the distal pocket architecture in a pH-dependent manner, not by gating the E7 channel. Removal of the HisE7 side chain in the GlyE7 variant exposes the distal pockets to the solvent, and the percentage of O2 escapes to the solvent directly from the α- or β-distal pockets of the mutant increases to 70-88%. In contrast to O2, the dominant water route from the bulk solvent is gated by HisE7 because protonation and opening of this residue dramatically increase the rate of influx of water into the empty distal heme pockets. The occupancy of the distal heme site by a water molecule, which functions as an additional nonprotein barrier to binding of the ligand to the heme, is also controlled by HisE7. Overall, analysis of gas and water diffusion routes in the subunits of HbA and its GlyE7 variant sheds light on the contribution of distal HisE7 in controlling polar and nonpolar ligand movement between the solvent and the hemes.

  11. Immortalization of normal human embryonic fibroblasts by introduction of either the human papillomavirus type 16 E6 or E7 gene alone.

    PubMed

    Yamamoto, Akito; Kumakura, Shin-ichi; Uchida, Minoru; Barrett, J Carl; Tsutsui, Takeki

    2003-09-01

    The ability of the human papillomavirus type 16 (HPV-16) E6 or E7 gene to induce immortalization of normal human embryonic fibroblast WHE-7 cells was examined. WHE-7 cells at 9 population doublings (PD) were infected with retrovirus vectors encoding either HPV-16 E6 or E7 alone or both E6 and E7 (E6/E7). One of 4 isolated clones carrying E6 alone became immortal and is currently at >445 PD. Four of 4 isolated clones carrying E7 alone escaped from crisis and are currently at >330 PD. Three of 5 isolated clones carrying E6/E7 were also immortalized and are currently at >268 PD. The immortal clone carrying E6 only and 2 of the 3 immortal clones carrying E6/E7 expressed a high level of E6 protein, and all the immortal clones carrying E7 alone and the other immortal clone carrying E6/E7 expressed a high level of E7 protein when compared to their mortal or precrisis clones. The immortal clones expressing a high level of E6 or E7 protein were positive for telomerase activity or an alternative mechanism of telomere maintenance, respectively, known as ALT (alternative lengthening of telomeres). All the mortal or precrisis clones were negative for both phenotypes. All the immortal clones exhibited abrogation of G1 arrest after DNA damage by X-ray irradiation. The expression of INK4a protein (p16(INK4a)) was undetectable in the E6-infected mortal and immortal clones, whereas Rb protein (pRb) was hyperphosphorylated only in the immortal clone. The p16(INK4a) protein was overexpressed in all the E7-infected immortal clones and their clones in the pre-crisis period as well as all the E6/E7-infected mortal and immortal clones, but the pRb expression was downregulated in all of these clones. These results demonstrate for the first time to our knowledge that HPV-16 E6 or E7 alone can induce immortalization of normal human embryonic fibroblasts. Inactivation of p16(INK4a)/pRb pathways in combination with activation of a telomere maintenance mechanism is suggested to be necessary for

  12. The BPS spectrum of the 4d {N}=2 SCFT's H 1, H 2, D 4, E 6, E 7, E 8

    NASA Astrophysics Data System (ADS)

    Cecotti, Sergio; Del Zotto, Michele

    2013-06-01

    Extending results of 1112.3984, we show that all rank 1 {N}=2 SCFT's in the sequence H 1, H 2, D 4 E 6, E 7, E 8 have canonical finite BPS chambers containing precisely 2 h(F) = 12(∆ - 1) hypermultiplets. The BPS spectrum of the canonical BPS chambers saturates the conformal central charge c, and satisfies some intriguing numerology.

  13. Analysis of the Complete Mitochondrial Genome Sequence of the Diploid Cotton Gossypium raimondii by Comparative Genomics Approaches

    PubMed Central

    Paterson, Andrew H.; Wang, Xuelin; Xu, Yiqing; Wu, Dongyang; Qu, Yanshu; Jiang, Anna; Ye, Qiaolin

    2016-01-01

    Cotton is one of the most important economic crops and the primary source of natural fiber and is an important protein source for animal feed. The complete nuclear and chloroplast (cp) genome sequences of G. raimondii are already available but not mitochondria. Here, we assembled the complete mitochondrial (mt) DNA sequence of G. raimondii into a circular genome of length of 676,078 bp and performed comparative analyses with other higher plants. The genome contains 39 protein-coding genes, 6 rRNA genes, and 25 tRNA genes. We also identified four larger repeats (63.9 kb, 10.6 kb, 9.1 kb, and 2.5 kb) in this mt genome, which may be active in intramolecular recombination in the evolution of cotton. Strikingly, nearly all of the G. raimondii mt genome has been transferred to nucleus on Chr1, and the transfer event must be very recent. Phylogenetic analysis reveals that G. raimondii, as a member of Malvaceae, is much closer to another cotton (G. barbadense) than other rosids, and the clade formed by two Gossypium species is sister to Brassicales. The G. raimondii mt genome may provide a crucial foundation for evolutionary analysis, molecular biology, and cytoplasmic male sterility in cotton and other higher plants. PMID:27847816

  14. Low- and high-risk human papillomavirus E7 proteins regulate p130 differently

    SciT

    Barrow-Laing, Lisa; Chen Wei; Roman, Ann, E-mail: aroman@iupui.ed

    2010-05-10

    The E7 protein of high-risk human papillomaviruses (HR HPVs) targets pRb family members (pRb, p107 and p130) for degradation; low-risk (LR) HPV E7 only targets p130 for degradation. The effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7more » or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. These results suggest that there may be divergent mechanisms by which LR and HR HPV E7 target p130 for degradation.« less

  15. Complete coding sequence characterization and comparative analysis of the putative novel human rhinovirus (HRV) species C and B

    PubMed Central

    2011-01-01

    Background Human Rhinoviruses (HRVs) are well recognized viral pathogens associated with acute respiratory tract illnesses (RTIs) abundant worldwide. Although recent studies have phylogenetically identified the new HRV species (HRV-C), data on molecular epidemiology, genetic diversity, and clinical manifestation have been limited. Result To gain new insight into HRV genetic diversity, we determined the complete coding sequences of putative new members of HRV species C (HRV-CU072 with 1% prevalence) and HRV-B (HRV-CU211) identified from clinical specimens collected from pediatric patients diagnosed with a symptom of acute lower RTI. Complete coding sequence and phylogenetic analysis revealed that the HRV-CU072 strain shared a recent common ancestor with most closely related Chinese strain (N4). Comparative analysis at the protein level showed that HRV-CU072 might accumulate substitutional mutations in structural proteins, as well as nonstructural proteins 3C and 3 D. Comparative analysis of all available HRVs and HEVs indicated that HRV-C contains a relatively high G+C content and is more closely related to HEV-D. This might be correlated to their replication and capability to adapt to the high temperature environment of the human lower respiratory tract. We herein report an infrequently occurring intra-species recombination event in HRV-B species (HRV-CU211) with a crossing over having taken place at the boundary of VP2 and VP3 genes. Moreover, we observed phylogenetic compatibility in all HRV species and suggest that dynamic mechanisms for HRV evolution seem to be related to recombination events. These findings indicated that the elementary units shaping the genetic diversity of HRV-C could be found in the nonstructural 2A and 3D genes. Conclusion This study provides information for understanding HRV genetic diversity and insight into the role of selection pressure and recombination mechanisms influencing HRV evolution. PMID:21214911

  16. Complete coding sequence characterization and comparative analysis of the putative novel human rhinovirus (HRV) species C and B.

    PubMed

    Linsuwanon, Piyada; Payungporn, Sunchai; Suwannakarn, Kamol; Chieochansin, Thaweesak; Theamboonlers, Apiradee; Poovorawan, Yong

    2011-01-07

    Human Rhinoviruses (HRVs) are well recognized viral pathogens associated with acute respiratory tract illnesses (RTIs) abundant worldwide. Although recent studies have phylogenetically identified the new HRV species (HRV-C), data on molecular epidemiology, genetic diversity, and clinical manifestation have been limited. To gain new insight into HRV genetic diversity, we determined the complete coding sequences of putative new members of HRV species C (HRV-CU072 with 1% prevalence) and HRV-B (HRV-CU211) identified from clinical specimens collected from pediatric patients diagnosed with a symptom of acute lower RTI. Complete coding sequence and phylogenetic analysis revealed that the HRV-CU072 strain shared a recent common ancestor with most closely related Chinese strain (N4). Comparative analysis at the protein level showed that HRV-CU072 might accumulate substitutional mutations in structural proteins, as well as nonstructural proteins 3C and 3 D. Comparative analysis of all available HRVs and HEVs indicated that HRV-C contains a relatively high G+C content and is more closely related to HEV-D. This might be correlated to their replication and capability to adapt to the high temperature environment of the human lower respiratory tract. We herein report an infrequently occurring intra-species recombination event in HRV-B species (HRV-CU211) with a crossing over having taken place at the boundary of VP2 and VP3 genes. Moreover, we observed phylogenetic compatibility in all HRV species and suggest that dynamic mechanisms for HRV evolution seem to be related to recombination events. These findings indicated that the elementary units shaping the genetic diversity of HRV-C could be found in the nonstructural 2A and 3D genes. This study provides information for understanding HRV genetic diversity and insight into the role of selection pressure and recombination mechanisms influencing HRV evolution.

  17. Comparative sequence analysis of domain I of Plasmodium falciparum apical membrane antigen 1 from Saudi Arabia and worldwide isolates.

    PubMed

    Al-Qahtani, Ahmed A; Abdel-Muhsin, Abdel-Muhsin A; Dajem, Saad M Bin; AlSheikh, Adel Ali H; Bohol, Marie Fe F; Al-Ahdal, Mohammed N; Putaporntip, Chaturong; Jongwutiwes, Somchai

    2016-04-01

    The apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) plays a crucial role in erythrocyte invasion and is a target of protective antibodies. Although domain I of PfAMA1 has been considered a promising vaccine component, extensive sequence diversity in this domain could compromise an effective vaccine design. To explore the extent of sequence diversity in domain I of PfAMA1, P. falciparum-infected blood samples from Saudi Arabia collected between 2007 and 2009 were analyzed and compared with those from worldwide parasite populations. Forty-six haplotypes and a novel codon change (M190V) were found among Saudi Arabian isolates. The haplotype diversity (0.948±0.004) and nucleotide diversity (0.0191±0.0008) were comparable to those from African hyperendemic countries. Positive selection in domain I of PfAMA1 among Saudi Arabian parasite population was observed because nonsynonymous nucleotide substitutions per nonsynonymous site (dN) significantly exceeded synonymous nucleotide substitutions per synonymous site (dS) and Tajima's D and its related statistics significantly deviated from neutrality in the positive direction. Despite a relatively low prevalence of malaria in Saudi Arabia, a minimum of 17 recombination events occurred in domain I. Genetic differentiation was significant between P. falciparum in Saudi Arabia and parasites from other geographic origins. Several shared or closely related haplotypes were found among parasites from different geographic areas, suggesting that vaccine derived from multiple shared epitopes could be effective across endemic countries. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Comparative Analyses of Plastid Sequences between Native and Introduced Populations of Aquatic Weeds Elodea canadensis and E. nuttallii

    PubMed Central

    Huotari, Tea; Korpelainen, Helena

    2013-01-01

    Non-indigenous species (NIS) are species living outside their historic or native range. Invasive NIS often cause severe environmental impacts, and may have large economical and social consequences. Elodea (Hydrocharitaceae) is a New World genus with at least five submerged aquatic angiosperm species living in fresh water environments. Our aim was to survey the geographical distribution of cpDNA haplotypes within the native and introduced ranges of invasive aquatic weeds Elodea canadensis and E. nuttallii and to reconstruct the spreading histories of these invasive species. In order to reveal informative chloroplast (cp) genome regions for phylogeographic analyses, we compared the plastid sequences of native and introduced individuals of E. canadensis. In total, we found 235 variable sites (186 SNPs, 47 indels and two inversions) between the two plastid sequences consisting of 112,193 bp and developed primers flanking the most variable genomic areas. These 29 primer pairs were used to compare the level and pattern of intraspecific variation within E. canadensis to interspecific variation between E. canadensis and E. nuttallii. Nine potentially informative primer pairs were used to analyze the phylogeographic structure of both Elodea species, based on 70 E. canadensis and 25 E. nuttallii individuals covering native and introduced distributions. On the whole, the level of variation between the two Elodea species was 53% higher than that within E. canadensis. In our phylogeographic analysis, only a single haplotype was found in the introduced range in both species. These haplotypes H1 (E. canadensis) and A (E. nuttallii) were also widespread in the native range, covering the majority of native populations analyzed. Therefore, we were not able to identify either the geographic origin of the introduced populations or test the hypothesis of single versus multiple introductions. The divergence between E. canadensis haplotypes was surprisingly high, and future research may

  19. Design of a colicin E7 based chimeric zinc-finger nuclease

    NASA Astrophysics Data System (ADS)

    Németh, Eszter; Schilli, Gabriella K.; Nagy, Gábor; Hasenhindl, Christoph; Gyurcsik, Béla; Oostenbrink, Chris

    2014-08-01

    Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity—offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.

  20. Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

    PubMed Central

    Ajiro, Masahiko; Jia, Rong; Zhang, Lifang; Liu, Xuefeng; Zheng, Zhi-Ming

    2012-01-01

    HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16+ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of 91QYNK94 to 91PSFW94 displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression. PMID:23056301

  1. Whole-genome sequence of the Tibetan frog Nanorana parkeri and the comparative evolution of tetrapod genomes.

    PubMed

    Sun, Yan-Bo; Xiong, Zi-Jun; Xiang, Xue-Yan; Liu, Shi-Ping; Zhou, Wei-Wei; Tu, Xiao-Long; Zhong, Li; Wang, Lu; Wu, Dong-Dong; Zhang, Bao-Lin; Zhu, Chun-Ling; Yang, Min-Min; Chen, Hong-Man; Li, Fang; Zhou, Long; Feng, Shao-Hong; Huang, Chao; Zhang, Guo-Jie; Irwin, David; Hillis, David M; Murphy, Robert W; Yang, Huan-Ming; Che, Jing; Wang, Jun; Zhang, Ya-Ping

    2015-03-17

    The development of efficient sequencing techniques has resulted in large numbers of genomes being available for evolutionary studies. However, only one genome is available for all amphibians, that of Xenopus tropicalis, which is distantly related from the majority of frogs. More than 96% of frogs belong to the Neobatrachia, and no genome exists for this group. This dearth of amphibian genomes greatly restricts genomic studies of amphibians and, more generally, our understanding of tetrapod genome evolution. To fill this gap, we provide the de novo genome of a Tibetan Plateau frog, Nanorana parkeri, and compare it to that of X. tropicalis and other vertebrates. This genome encodes more than 20,000 protein-coding genes, a number similar to that of Xenopus. Although the genome size of Nanorana is considerably larger than that of Xenopus (2.3 vs. 1.5 Gb), most of the difference is due to the respective number of transposable elements in the two genomes. The two frogs exhibit considerable conserved whole-genome synteny despite having diverged approximately 266 Ma, indicating a slow rate of DNA structural evolution in anurans. Multigenome synteny blocks further show that amphibians have fewer interchromosomal rearrangements than mammals but have a comparable rate of intrachromosomal rearrangements. Our analysis also identifies 11 Mb of anuran-specific highly conserved elements that will be useful for comparative genomic analyses of frogs. The Nanorana genome offers an improved understanding of evolution of tetrapod genomes and also provides a genomic reference for other evolutionary studies.

  2. Human papillomavirus type 16 E7 oncoprotein mediates CCNA1 promoter methylation.

    PubMed

    Chalertpet, Kanwalat; Pakdeechaidan, Watcharapong; Patel, Vyomesh; Mutirangura, Apiwat; Yanatatsaneejit, Pattamawadee

    2015-10-01

    Human papillomavirus (HPV) oncoproteins drive distinctive promoter methylation patterns in cancer. However, the underlying mechanism remains to be elucidated. Cyclin A1 (CCNA1) promoter methylation is strongly associated with HPV-associated cancer. CCNA1 methylation is found in HPV-associated cervical cancers, as well as in head and neck squamous cell cancer. Numerous pieces of evidence suggest that E7 may drive CCNA1 methylation. First, the CCNA1 promoter is methylated in HPV-positive epithelial lesions after transformation. Second, the CCNA1 promoter is methylated at a high level when HPV is integrated into the human genome. Finally, E7 has been shown to interact with DNA methyltransferase 1 (Dnmt1). Here, we sought to determine the mechanism by which E7 increases methylation in cervical cancer by using CCNA1 as a gene model. We investigated whether E7 induces CCNA1 promoter methylation, resulting in the loss of expression. Using both E7 knockdown and overexpression approaches in SiHa and C33a cells, our data showed that CCNA1 promoter methylation decreases with a corresponding increase in expression in E7 siRNA-transfected cells. By contrast, CCNA1 promoter methylation was augmented with a corresponding reduction in expression in E7-overexpressing cells. To confirm whether the binding of the E7-Dnmt1 complex to the CCNA1 promoter induced methylation and loss of expression, ChIP assays were carried out in E7-, del CR3-E7 and vector control-overexpressing C33a cells. The data showed that E7 induced CCNA1 methylation by forming a complex with Dnmt1 at the CCNA1 promoter, resulting in the subsequent reduction of expression in cancers. It is interesting to further explore the genome-wide mechanism of E7 oncoprotein-mediated DNA methylation. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  3. Double demonstration of oncogenic high risk human papilloma virus DNA and HPV-E7 protein in oral cancers.

    PubMed

    Pannone, G; Santoro, A; Carinci, F; Bufo, P; Papagerakis, S M; Rubini, C; Campisi, G; Giovannelli, L; Contaldo, M; Serpico, R; Mazzotta, M; Lo Muzio, L

    2011-01-01

    Oncogenic HPVs are necessarily involved in cervical cancer but their role in oral carcinogenesis is debated. To detect HPV in oral cancer, 38 cases of formalin fixed-paraffin embedded OSCC were studied by both DNA genotyping (MY09/11 L1 consensus primers in combination with GP5-GP6 primer pair followed by sequencing) and immunohistochemistry (monoclonal Abs against capsid protein and HPV-E7 protein, K1H8 DAKO and clone 8C9 INVITROGEN, respectively). HPV-16 tonsil cancer was used as positive control. The overall prevalence of HPV infection in OSCCs was 10.5%. Amplification of DNA samples showed single HPV DNA infection in 3 cases (HPV16; HPV53; HPV70) and double infection in one case of cheek cancer (HPV31/HPV44). The overall HR-HPV prevalence was 7.5%. E-7 antigen was immunohistochemically detected in all HPV-positive cases. HPV+ OSCC cases showed an overall better outcome than HPV negative oral cancers, as evaluated by Kaplan-Meier curves. HPVs exert their oncogenic role after DNA integration, gene expression of E5, E6 and E7 loci and p53/pRb host proteins suppression. This study showed that HPV-E7 protein inactivating pRb is expressed in oral cancer cells infected by oncogenic HPV other than classical HR-HPV-16/18. Interestingly HPV-70, considered a low risk virus with no definite collocation in oncogenic type category, gives rise to the expression of HPV-E7 protein and inactivate pRb in oral cancer. HPV-70, as proved in current literature, is able to inactivates also p53 protein, promoting cell immortalization. HPV-53, classified as a possible high risk virus, expresses E7 protein in OSCC, contributing to oral carcinogenesis. We have identified among OSCCs, a subgroup characterized by HPV infection (10.5%). Finally, we have proved the oncogenic potential of some HPV virus types, not well known in literature.

  4. A simulation study on terahertz absorption of liquid crystal mixture E7

    NASA Astrophysics Data System (ADS)

    Dong, Jian-qi; Cheng, Wen-qi; Li, Meng-ge; Wang, Kai-li; Chen, Ze-zhang; Ma, Heng

    2017-09-01

    A simulation work on a broad THz absorption of liquid crystal mixture E7 consisting of 5CB, 7CB, 8OCB and 5CT is reported. Based on the density functional theory, the molecular structures of the monomers were optimized and calculated using the Gaussian package with base set B3LYP and 6-311g. The results indicate that the simulation of the characteristic absorption spectra is accurate compared to the experimental and literature report in the infrared band. By analyzing contribution of the benzene ring, C-O and alkyl bonds on THz absorption, it is found that there are no significant effects from the cyano group and the alkyl radical. The addition of a benzene ring leads to an increase in absorption intensity and redshift. By discussing the atomic mass distribution and the structural symmetry of the monomers, a reason for the strong THz absorption of 8OCB is proposed.

  5. Comparable contributions of structural-functional constraints and expression level to the rate of protein sequence evolution

    PubMed Central

    Wolf, Maxim Y; Wolf, Yuri I; Koonin, Eugene V

    2008-01-01

    multidomain proteins or contained in distinct proteins. Substantial homogenization of evolutionary rates in multidomain proteins was, indeed, observed in both animals and plants, although highly significant differences between domain-specific rates remained. The contributions of the translation rate, as determined by the effect of the fusion of a pair of domains within a multidomain protein, and intrinsic, domain-specific structural-functional constraints appear to be comparable in magnitude. Conclusion Fusion of domains in a multidomain protein results in substantial homogenization of the domain-specific evolutionary rates but significant differences between domain-specific evolution rates remain. Thus, the rate of translation and intrinsic structural-functional constraints both exert sizable and comparable effects on sequence evolution. Reviewers This article was reviewed by Sergei Maslov, Dennis Vitkup, Claus Wilke (nominated by Orly Alter), and Allan Drummond (nominated by Joel Bader). For the full reviews, please go to the Reviewers' Reports section. PMID:18840284

  6. OrthoMaM v8: a database of orthologous exons and coding sequences for comparative genomics in mammals.

    PubMed

    Douzery, Emmanuel J P; Scornavacca, Celine; Romiguier, Jonathan; Belkhir, Khalid; Galtier, Nicolas; Delsuc, Frédéric; Ranwez, Vincent

    2014-07-01

    Comparative genomic studies extensively rely on alignments of orthologous sequences. Yet, selecting, gathering, and aligning orthologous exons and protein-coding sequences (CDS) that are relevant for a given evolutionary analysis can be a difficult and time-consuming task. In this context, we developed OrthoMaM, a database of ORTHOlogous MAmmalian Markers describing the evolutionary dynamics of orthologous genes in mammalian genomes using a phylogenetic framework. Since its first release in 2007, OrthoMaM has regularly evolved, not only to include newly available genomes but also to incorporate up-to-date software in its analytic pipeline. This eighth release integrates the 40 complete mammalian genomes available in Ensembl v73 and provides alignments, phylogenies, evolutionary descriptor information, and functional annotations for 13,404 single-copy orthologous CDS and 6,953 long exons. The graphical interface allows to easily explore OrthoMaM to identify markers with specific characteristics (e.g., taxa availability, alignment size, %G+C, evolutionary rate, chromosome location). It hence provides an efficient solution to sample preprocessed markers adapted to user-specific needs. OrthoMaM has proven to be a valuable resource for researchers interested in mammalian phylogenomics, evolutionary genomics, and has served as a source of benchmark empirical data sets in several methodological studies. OrthoMaM is available for browsing, query and complete or filtered downloads at http://www.orthomam.univ-montp2.fr/. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Complete chloroplast genome sequence of common bermudagrass (Cynodon dactylon (L.) Pers.) and comparative analysis within the family Poaceae

    PubMed Central

    Huang, Ya-Yi; Cho, Shu-Ting; Haryono, Mindia; Kuo, Chih-Horng

    2017-01-01

    Common bermudagrass (Cynodon dactylon (L.) Pers.) belongs to the subfamily Chloridoideae of the Poaceae family, one of the most important plant families ecologically and economically. This grass has a long connection with human culture but its systematics is relatively understudied. In this study, we sequenced and investigated the chloroplast genome of common bermudagrass, which is 134,297 bp in length with two single copy regions (LSC: 79,732 bp; SSC: 12,521 bp) and a pair of inverted repeat (IR) regions (21,022 bp). The annotation contains a total of 128 predicted genes, including 82 protein-coding, 38 tRNA, and 8 rRNA genes. Additionally, our in silico analyses identified 10 sets of repeats longer than 20 bp and predicted the presence of 36 RNA editing sites. Overall, the chloroplast genome of common bermudagrass resembles those from other Poaceae lineages. Compared to most angiosperms, the accD gene and the introns of both clpP and rpoC1 genes are missing. Additionally, the ycf1, ycf2, ycf15, and ycf68 genes are pseudogenized and two genome rearrangements exist. Our phylogenetic analysis based on 47 chloroplast protein-coding genes supported the placement of common bermudagrass within Chloridoideae. Our phylogenetic character mapping based on the parsimony principle further indicated that the loss of the accD gene and clpP introns, the pseudogenization of four ycf genes, and the two rearrangements occurred only once after the most recent common ancestor of the Poaceae diverged from other monocots, which could explain the unusual long branch leading to the Poaceae when phylogeny is inferred based on chloroplast sequences. PMID:28617867

  8. The complete chloroplast genome sequence of the CAM epiphyte Spanish moss (Tillandsia usneoides, Bromeliaceae) and its comparative analysis.

    PubMed

    Poczai, Péter; Hyvönen, Jaakko

    2017-01-01

    Spanish moss (Tillandsia usneoides) is an epiphytic bromeliad widely distributed throughout tropical and warm temperate America. This plant is highly adapted to extreme environmental conditions. Striking features of this species include specialized trichomes (scales) covering the surface of its shoots aiding the absorption of water and nutrients directly from the atmosphere and a specific photosynthesis using crassulacean acid metabolism (CAM). Here we report the plastid genome of Spanish moss and present the comparison of genome organization and sequence evolution within Poales. The plastome of Spanish moss has a quadripartite structure consisting of a large single copy (LSC, 87,439 bp), two inverted regions (IRa and IRb, 26,803 bp) and short single copy (SSC, 18,612 bp) region. The plastid genome had 37.2% GC content and 134 genes with 88 being unique protein-coding genes and 20 of these are duplicated in the IR, similar to other reported bromeliads. Our study shows that early diverging lineages of Poales do not have high substitution rates as compared to grasses, and plastid genomes of bromeliads show structural features considered to be ancestral in graminids. These include the loss of the introns in the clpP and rpoC1 genes and the complete loss or partial degradation of accD and ycf genes in the Graminid clade. Further structural rearrangements appeared in the graminids lacking in Spanish moss, which include a 28-kb inversion between the trnG-UCC-rps14 region and 6-kb in the trnG-UCC-psbD, followed by a third <1kb inversion in the trnT sequence.

  9. The complete chloroplast genome sequence of the CAM epiphyte Spanish moss (Tillandsia usneoides, Bromeliaceae) and its comparative analysis

    PubMed Central

    Hyvönen, Jaakko

    2017-01-01

    Spanish moss (Tillandsia usneoides) is an epiphytic bromeliad widely distributed throughout tropical and warm temperate America. This plant is highly adapted to extreme environmental conditions. Striking features of this species include specialized trichomes (scales) covering the surface of its shoots aiding the absorption of water and nutrients directly from the atmosphere and a specific photosynthesis using crassulacean acid metabolism (CAM). Here we report the plastid genome of Spanish moss and present the comparison of genome organization and sequence evolution within Poales. The plastome of Spanish moss has a quadripartite structure consisting of a large single copy (LSC, 87,439 bp), two inverted regions (IRa and IRb, 26,803 bp) and short single copy (SSC, 18,612 bp) region. The plastid genome had 37.2% GC content and 134 genes with 88 being unique protein-coding genes and 20 of these are duplicated in the IR, similar to other reported bromeliads. Our study shows that early diverging lineages of Poales do not have high substitution rates as compared to grasses, and plastid genomes of bromeliads show structural features considered to be ancestral in graminids. These include the loss of the introns in the clpP and rpoC1 genes and the complete loss or partial degradation of accD and ycf genes in the Graminid clade. Further structural rearrangements appeared in the graminids lacking in Spanish moss, which include a 28-kb inversion between the trnG-UCC–rps14 region and 6-kb in the trnG-UCC–psbD, followed by a third <1kb inversion in the trnT sequence. PMID:29095905

  10. Complete chloroplast genome sequence of common bermudagrass (Cynodon dactylon (L.) Pers.) and comparative analysis within the family Poaceae.

    PubMed

    Huang, Ya-Yi; Cho, Shu-Ting; Haryono, Mindia; Kuo, Chih-Horng

    2017-01-01

    Common bermudagrass (Cynodon dactylon (L.) Pers.) belongs to the subfamily Chloridoideae of the Poaceae family, one of the most important plant families ecologically and economically. This grass has a long connection with human culture but its systematics is relatively understudied. In this study, we sequenced and investigated the chloroplast genome of common bermudagrass, which is 134,297 bp in length with two single copy regions (LSC: 79,732 bp; SSC: 12,521 bp) and a pair of inverted repeat (IR) regions (21,022 bp). The annotation contains a total of 128 predicted genes, including 82 protein-coding, 38 tRNA, and 8 rRNA genes. Additionally, our in silico analyses identified 10 sets of repeats longer than 20 bp and predicted the presence of 36 RNA editing sites. Overall, the chloroplast genome of common bermudagrass resembles those from other Poaceae lineages. Compared to most angiosperms, the accD gene and the introns of both clpP and rpoC1 genes are missing. Additionally, the ycf1, ycf2, ycf15, and ycf68 genes are pseudogenized and two genome rearrangements exist. Our phylogenetic analysis based on 47 chloroplast protein-coding genes supported the placement of common bermudagrass within Chloridoideae. Our phylogenetic character mapping based on the parsimony principle further indicated that the loss of the accD gene and clpP introns, the pseudogenization of four ycf genes, and the two rearrangements occurred only once after the most recent common ancestor of the Poaceae diverged from other monocots, which could explain the unusual long branch leading to the Poaceae when phylogeny is inferred based on chloroplast sequences.

  11. Comparative analysis of the complete genome sequence of the California MSW strain of myxoma virus reveals potential host adaptations.

    PubMed

    Kerr, Peter J; Rogers, Matthew B; Fitch, Adam; Depasse, Jay V; Cattadori, Isabella M; Hudson, Peter J; Tscharke, David C; Holmes, Edward C; Ghedin, Elodie

    2013-11-01

    Myxomatosis is a rapidly lethal disease of European rabbits that is caused by myxoma virus (MYXV). The introduction of a South American strain of MYXV into the European rabbit population of Australia is the classic case of host-pathogen coevolution following cross-species transmission. The most virulent strains of MYXV for European rabbits are the Californian viruses, found in the Pacific states of the United States and the Baja Peninsula, Mexico. The natural host of Californian MYXV is the brush rabbit, Sylvilagus bachmani. We determined the complete sequence of the MSW strain of Californian MYXV and performed a comparative analysis with other MYXV genomes. The MSW genome is larger than that of the South American Lausanne (type) strain of MYXV due to an expansion of the terminal inverted repeats (TIRs) of the genome, with duplication of the M156R, M154L, M153R, M152R, and M151R genes and part of the M150R gene from the right-hand (RH) end of the genome at the left-hand (LH) TIR. Despite the extreme virulence of MSW, no novel genes were identified; five genes were disrupted by multiple indels or mutations to the ATG start codon, including two genes, M008.1L/R and M152R, with major virulence functions in European rabbits, and a sixth gene, M000.5L/R, was absent. The loss of these gene functions suggests that S. bachmani is a relatively recent host for MYXV and that duplication of virulence genes in the TIRs, gene loss, or sequence variation in other genes can compensate for the loss of M008.1L/R and M152R in infections of European rabbits.

  12. [Experimental study on carcinogenesis by human papillomavirus type 8 E7 gene].

    PubMed

    Nishikawa, T

    1994-05-01

    Human papillomavirus (HPV) 5 and HPV8 are often detected in skin cancers developed in patients suffering from epidermodysplasia verruciformis, as well as in skin cancers developed in immunosuppressed patients. In the present study, in order to examine the transforming activity of the HPV8E7 gene, the HPV8E7 and HPV8E6/E7 genes were cloned into the expression vector (pcD2-Y), under the SV40 enhancer/promoter to construct pcD2-8E7 and pcD2-8E6/E7, respectively. The E7 and E6/E7 genes of genital high-risk HPV16 were also cloned into pcD2-Y to construct pcD2-16E7 and pcD2-16E6/E7, respectively. They were tested for their ability to collaboratively transform primary rat embryo fibroblasts (REFs) with activated H-ras gene. Transfection experiments of REFs having an activated H-ras gene revealed that pcD2-8E7, as well as pcD2-16E7 and pcD2-16E6/E7, induced transformation of cells in G418-resistant colonies at efficiencies of 11.9%, 43.0% and 53.0%, respectively. Transformed cell lines induced by activated H-ras gene and pcD2-8E7 or pcD2-16E7 were named 8RE and 16RE cell lines, respectively. Tumor induction in syngeneic newborn rats by injected the 8RE cells was higher than that of the 16RE cells. In cytological and histological examination, the 8RE cell lines and their induced tumors were different from the 16RE cell lines and their induced tumors. The 8RE cell lines showed the characteristic transformation with efficient growth ability on plastic and colony formation in 0.3% soft agar. These results support the hypothesis that the HPV8E7 gene plays an important role in the carcinogenesis of skin cancers.

  13. Randomized comparison of next-generation sequencing and array comparative genomic hybridization for preimplantation genetic screening: a pilot study.

    PubMed

    Yang, Zhihong; Lin, James; Zhang, John; Fong, Wai Ieng; Li, Pei; Zhao, Rong; Liu, Xiaohong; Podevin, William; Kuang, Yanping; Liu, Jiaen

    2015-06-23

    Recent advances in next-generation sequencing (NGS) have provided new methods for preimplantation genetic screening (PGS) of human embryos from in vitro fertilization (IVF) cycles. However, there is still limited information about clinical applications of NGS in IVF and PGS (IVF-PGS) treatments. The present study aimed to investigate the effects of NGS screening on clinical pregnancy and implantation outcomes for PGS patients in comparison to array comparative genomic hybridization (aCGH) screening. This study was performed in two phases. Phase I study evaluated the accuracy of NGS for aneuploidy screening in comparison to aCGH. Whole-genome amplification (WGA) products (n = 164) derived from previous IVF-PGS cycles (n = 38) were retrospectively analyzed with NGS. The NGS results were then compared with those of aCGH. Phase II study further compared clinical pregnancy and implantation outcomes between NGS and aCGH for IVF-PGS patients. A total of 172 patients at mean age 35.2 ± 3.5 years were randomized into two groups: 1) NGS (Group A): patients (n = 86) had embryos screened with NGS and 2) aCGH (Group B): patients (n = 86) had embryos screened with aCGH. For both groups, blastocysts were vitrified after trophectoderm biopsy. One to two euploid blastocysts were thawed and transferred to individual patients primarily based on the PGS results. Ongoing pregnancy and implantation rates were compared between the two study groups. NGS detected all types of aneuploidies of human blastocysts accurately and provided a 100 % 24-chromosome diagnosis consistency with the highly validated aCGH method. Moreover, NGS screening identified euploid blastocysts for transfer and resulted in similarly high ongoing pregnancy rates for PGS patients compared to aCGH screening (74.7 % vs. 69.2 %, respectively, p >0.05). The observed implantation rates were also comparable between the NGS and aCGH groups (70.5 % vs. 66.2 %, respectively, p >0.05). While NGS screening

  14. Construction of Pseudomolecule Sequences of the aus Rice Cultivar Kasalath for Comparative Genomics of Asian Cultivated Rice

    PubMed Central

    Sakai, Hiroaki; Kanamori, Hiroyuki; Arai-Kichise, Yuko; Shibata-Hatta, Mari; Ebana, Kaworu; Oono, Youko; Kurita, Kanako; Fujisawa, Hiroko; Katagiri, Satoshi; Mukai, Yoshiyuki; Hamada, Masao; Itoh, Takeshi; Matsumoto, Takashi; Katayose, Yuichi; Wakasa, Kyo; Yano, Masahiro; Wu, Jianzhong

    2014-01-01

    Having a deep genetic structure evolved during its domestication and adaptation, the Asian cultivated rice (Oryza sativa) displays considerable physiological and morphological variations. Here, we describe deep whole-genome sequencing of the aus rice cultivar Kasalath by using the advanced next-generation sequencing (NGS) technologies to gain a better understanding of the sequence and structural changes among highly differentiated cultivars. The de novo assembled Kasalath sequences represented 91.1% (330.55 Mb) of the genome and contained 35 139 expressed loci annotated by RNA-Seq analysis. We detected 2 787 250 single-nucleotide polymorphisms (SNPs) and 7393 large insertion/deletion (indel) sites (>100 bp) between Kasalath and Nipponbare, and 2 216 251 SNPs and 3780 large indels between Kasalath and 93-11. Extensive comparison of the gene contents among these cultivars revealed similar rates of gene gain and loss. We detected at least 7.39 Mb of inserted sequences and 40.75 Mb of unmapped sequences in the Kasalath genome in comparison with the Nipponbare reference genome. Mapping of the publicly available NGS short reads from 50 rice accessions proved the necessity and the value of using the Kasalath whole-genome sequence as an additional reference to capture the sequence polymorphisms that cannot be discovered by using the Nipponbare sequence alone. PMID:24578372

  15. Phylogeny and polymorphism in the long control regions E6, E7, and L1 of HPV Type 56 in women from southwest China

    PubMed Central

    Jing, Yaling; Wang, Tao; Chen, Zuyi; Ding, Xianping; Xu, Jianju; Mu, Xuemei; Cao, Man; Chen, Honghan

    2018-01-01

    Globally, human papillomavirus (HPV)-56 accounts for a small proportion of all high-risk HPV types; however, HPV-56 is detected at a higher rate in Asia, particularly in southwest China. The present study analyzed polymorphisms, intratypic variants, and genetic variability in the long control regions (LCR), E6, E7, and L1 of HPV-56 (n=75). The LCRs, E6, E7 and L1 were sequenced using a polymerase chain reaction and the sequences were submitted to GenBank. Maximum-likelihood trees were constructed using Kimura's two-parameter model, followed by secondary structure analysis and protein damaging prediction. Additionally, in order to assess the effect of variations in the LCR on putative binding sites for cellular proteins, MATCH server was used. Finally, the selection pressures of the E6-E7 and L1 genes were estimated. A total of 18 point substitutions, a 42-bp deletion and a 19-bp deletion of LCR were identified. Some of those mutations are embedded in the putative binding sites for transcription factors. 18 single nucleotide changes occurred in the E6-E7 sequence, 11/18 were non-synonymous substitutions and 7/18 were synonymous mutations. A total 24 single nucleotide changes were identified in the L1 sequence, 6/24 being non-synonymous mutations and 18/24 synonymous mutations. Selective pressure analysis predicted that the majority of mutations of HPV-56 E6, E7 and L1 were of positive selection. The phylogenetic tree demonstrated that the isolates distributed in two lineages. Data on the prevalence and genetic variation of HPV-56 types in southwest China may aid future studies on viral molecular mechanisms and contribute to future investigations of diagnostic probes and therapeutic vaccines. PMID:29568922

  16. Cooperative transformation and coexpression of bovine papillomavirus type 1 E5 and E7 proteins.

    PubMed

    Bohl, J; Hull, B; Vande Pol, S B

    2001-01-01

    Productively infected bovine fibropapillomas were examined for bovine papillomavirus type 1 (BPV-1) E7 localization. BPV-1 E7 was observed in the cytoplasm of basal and lower spinous epithelial cells, coexpressed in the cytoplasm of basal cells with the E5 oncoprotein. E7 was also observed in nucleoli throughout the basal and spinous layers but not in the granular cell layer. Ectopic expression of E7 in cultured epithelial cells gave rise to localization similar to that seen in productive fibropapillomas, with cytoplasmic and nucleolar expression observed. Consistent with the coexpression of E7 and E5 in basal keratinocytes, BPV-1 E7 cooperated with E5 as well as E6 in an anchorage independence transformation assay. While E5 is expressed in both basal and superficial differentiating keratinocytes, BPV-1 E7 is only observed in basal and lower spinous epithelial cells. Therefore, BPV-1 E7 may serve to modulate the cellular response of basal epithelial cells to E5 expression.

  17. Enhanced immunogenicity of HPV 16 E7 fusion proteins in DNA vaccination.

    PubMed

    Michel, Nico; Osen, Wolfram; Gissmann, Lutz; Schumacher, Ton N M; Zentgraf, Hanswalter; Müller, Martin

    2002-03-01

    DNA vaccination is a promising approach for inducing both humoral and cellular immune responses. For immunotherapy of HPV-16-associated diseases the E7 protein is considered a prime candidate, as it is expressed in all HPV-16-positive tumors. Unfortunately, the E7 protein is a very poor inducer of a cytotoxic T-cell response, when being used as antigen in DNA vaccination. Here we demonstrate that after fusion to protein export/import signals such as the herpes simplex virus ferry protein VP22, E7 can translocate in vitro from VP22-E7-expressing cells to neighboring cells that do not carry the VP22-E7 gene. In vivo, the VP22-E7 fusion shows significantly increased efficiency in inducing a cytotoxic T-cell response. Our data suggest that the export function of VP22 plays a major role in this phenomenon, since VP22 can be replaced by classical protein export signals, without impairing the induction of the E7-specific cellular immune response. However, all E7 fusion constructs showed significantly elevated protein steady-state levels, which might also account for the observed boost in immunogenicity. (C)2002 Elsevier Science (USA).

  18. Sequences of Normative Evaluation in Two Telecollaboration Projects: A Comparative Study of Multimodal Feedback through Desktop Videoconference

    ERIC Educational Resources Information Center

    Cappellini, Marco; Azaoui, Brahim

    2017-01-01

    In our study we analyse how the same interactional dynamic is produced in two different pedagogical settings exploiting a desktop videoconference system. We propose to focus our attention on a specific type of conversational side sequence, known in the Francophone literature as sequences of normative evaluation. More particularly, we analyse data…

  19. Decreased Load on General Motor Preparation and Visual-Working Memory while Preparing Familiar as Compared to Unfamiliar Movement Sequences

    ERIC Educational Resources Information Center

    De Kleine, Elian; Van der Lubbe, Rob H. J.

    2011-01-01

    Learning movement sequences is thought to develop from an initial controlled attentive phase to a more automatic inattentive phase. Furthermore, execution of sequences becomes faster with practice, which may result from changes at a general motor processing level rather than at an effector specific motor processing level. In the current study, we…

  20. Complete Mitochondrial Genome Sequence of Acrida cinerea (Acrididae: Orthoptera) and Comparative Analysis of Mitochondrial Genomes in Orthoptera

    PubMed Central

    Liu, Nian; Huang, Yuan

    2010-01-01

    The complete 15,599-bp mitogenome of Acrida cinerea was determined and compared with that of the other 20 orthopterans. It displays characteristic gene content, genome organization, nucleotide composition, and codon usage found in other Caelifera mitogenomes. Comparison of 21 orthopteran sequences revealed that the tRNAs encoded by the H-strand appear more conserved than those by the L-stand. All tRNAs form the typical clover-leaf structure except trnS (agn), and most of the size variation among tRNAs stemmed from the length variation in the arm and loop of TΨC and the loop of DHU. The derived secondary structure models of the rrnS and rrnL from 21 orthoptera species closely resemble those from other insects on CRW except a considerably enlarged loop of helix 1399 of rrnS in Caelifera, which is a potentially autapomorphy of Caelifera. In the A+T-rich region, tandem repeats are not only conserved in the closely related mitogenome but also share some conserved motifs in the same subfamily. A stem-loop structure, 16 bp or longer, is likely to be involved in replication initiation in Caelifera and Grylloidea. A long T-stretch (>17 bp) with conserved stem-loop structure next to rrnS on the H-strand, bounded by a purine at either end, exists in the three species from Tettigoniidae. PMID:21197069

  1. A comparative study of cold- and warm-adapted Endonucleases A using sequence analyses and molecular dynamics simulations.

    PubMed

    Michetti, Davide; Brandsdal, Bjørn Olav; Bon, Davide; Isaksen, Geir Villy; Tiberti, Matteo; Papaleo, Elena

    2017-01-01

    The psychrophilic and mesophilic endonucleases A (EndA) from Aliivibrio salmonicida (VsEndA) and Vibrio cholera (VcEndA) have been studied experimentally in terms of the biophysical properties related to thermal adaptation. The analyses of their static X-ray structures was no sufficient to rationalize the determinants of their adaptive traits at the molecular level. Thus, we used Molecular Dynamics (MD) simulations to compare the two proteins and unveil their structural and dynamical differences. Our simulations did not show a substantial increase in flexibility in the cold-adapted variant on the nanosecond time scale. The only exception is a more rigid C-terminal region in VcEndA, which is ascribable to a cluster of electrostatic interactions and hydrogen bonds, as also supported by MD simulations of the VsEndA mutant variant where the cluster of interactions was introduced. Moreover, we identified three additional amino acidic substitutions through multiple sequence alignment and the analyses of MD-based protein structure networks. In particular, T120V occurs in the proximity of the catalytic residue H80 and alters the interaction with the residue Y43, which belongs to the second coordination sphere of the Mg2+ ion. This makes T120V an amenable candidate for future experimental mutagenesis.

  2. A comparative study of sequence- and structure-based features of small RNAs and other RNAs of bacteria.

    PubMed

    Barik, Amita; Das, Santasabuj

    2018-01-02

    Small RNAs (sRNAs) in bacteria have emerged as key players in transcriptional and post-transcriptional regulation of gene expression. Here, we present a statistical analysis of different sequence- and structure-related features of bacterial sRNAs to identify the descriptors that could discriminate sRNAs from other bacterial RNAs. We investigated a comprehensive and heterogeneous collection of 816 sRNAs, identified by northern blotting across 33 bacterial species and compared their various features with other classes of bacterial RNAs, such as tRNAs, rRNAs and mRNAs. We observed that sRNAs differed significantly from the rest with respect to G+C composition, normalized minimum free energy of folding, motif frequency and several RNA-folding parameters like base-pairing propensity, Shannon entropy and base-pair distance. Based on the selected features, we developed a predictive model using Random Forests (RF) method to classify the above four classes of RNAs. Our model displayed an overall predictive accuracy of 89.5%. These findings would help to differentiate bacterial sRNAs from other RNAs and further promote prediction of novel sRNAs in different bacterial species.

  3. The PTPN14 Tumor Suppressor Is a Degradation Target of Human Papillomavirus E7.

    PubMed

    Szalmás, Anita; Tomaić, Vjekoslav; Basukala, Om; Massimi, Paola; Mittal, Suruchi; Kónya, József; Banks, Lawrence

    2017-04-01

    Activation of signaling pathways ensuring cell growth is essential for the proliferative competence of human papillomavirus (HPV)-infected cells. Tyrosine kinases and phosphatases are key regulators of cellular growth control pathways. A recently identified potential cellular target of HPV E7 is the cytoplasmic protein tyrosine phosphatase PTPN14, which is a potential tumor suppressor and is linked to the control of the Hippo and Wnt/beta-catenin signaling pathways. In this study, we show that the E7 proteins of both high-risk and low-risk mucosal HPV types can interact with PTPN14. This interaction is independent of retinoblastoma protein (pRb) and involves residues in the carboxy-terminal region of E7. We also show that high-risk E7 induces proteasome-mediated degradation of PTPN14 in cells derived from cervical tumors. This degradation appears to be independent of cullin-1 or cullin-2 but most likely involves the UBR4/p600 ubiquitin ligase. The degree to which E7 downregulates PTPN14 would suggest that this interaction is important for the viral life cycle and potentially also for the development of malignancy. In support of this we find that overexpression of PTPN14 decreases the ability of HPV-16 E7 to cooperate with activated EJ-ras in primary cell transformation assays. IMPORTANCE This study links HPV E7 to the deregulation of protein tyrosine phosphatase signaling pathways. PTPN14 is classified as a potential tumor suppressor protein, and here we show that it is very susceptible to HPV E7-induced proteasome-mediated degradation. Intriguingly, this appears to use a mechanism that is different from that employed by E7 to target pRb. Therefore, this study has important implications for our understanding of the molecular basis for E7 function and also sheds important light on the potential role of PTPN14 as a tumor suppressor. Copyright © 2017 American Society for Microbiology.

  4. Comparative RNA-sequencing of the acarbose producer Actinoplanes sp. SE50/110 cultivated in different growth media.

    PubMed

    Schwientek, Patrick; Wendler, Sergej; Neshat, Armin; Eirich, Christina; Rückert, Christian; Klein, Andreas; Wehmeier, Udo F; Kalinowski, Jörn; Stoye, Jens; Pühler, Alfred

    2013-08-20

    Actinoplanes sp. SE50/110 is known as the producer of the alpha-glucosidase inhibitor acarbose, a potent drug in the treatment of type-2 diabetes mellitus. We conducted the first whole transcriptome analysis of Actinoplanes sp. SE50/110, using RNA-sequencing technology for comparative gene expression studies between cells grown in maltose minimal medium, maltose minimal medium with trace elements, and glucose complex medium. We first studied the behavior of Actinoplanes sp. SE50/110 cultivations in these three media and found that the different media had significant impact on growth rate and in particular on acarbose production. It was demonstrated that Actinoplanes sp. SE50/110 grew well in all three media, but acarbose biosynthesis was only observed in cultures grown in maltose minimal medium with and without trace elements. When comparing the expression profiles between the maltose minimal media with and without trace elements, only few significantly differentially expressed genes were found, which mainly code for uptake systems of metal ions provided in the trace element solution. In contrast, the comparison of expression profiles from maltose minimal medium and glucose complex medium revealed a large number of differentially expressed genes, of which the most conspicuous genes account for iron storage and uptake. Furthermore, the acarbose gene cluster was found to be highly expressed in maltose-containing media and almost silent in the glucose-containing medium. In addition, a putative antibiotic biosynthesis gene cluster was found to be similarly expressed as the acarbose cluster. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Comparative Genomic Sequencing and Pathogenic Properties of Equine Herpesvirus 1 KyA and RacL11

    PubMed Central

    Shakya, Akhalesh K.; O’Callaghan, Dennis J.; Kim, Seong K.

    2017-01-01

    Equine herpesvirus 1 (EHV-1) is a major pathogen affecting equines worldwide. The virus causes respiratory disease, abortion, and, in some cases, neurological disease. EHV-1 Kentucky A (KyA) is attenuated in the mouse and equine, whereas wild-type pathogenic strain RacL11 induces severe inflammatory infiltration of the lung, causing infected mice to succumb. The complete DNA sequencing of the KyA genome revealed that genes UL17 (ORF17), US6 (ORF73; gI), US7 (ORF74; gE), and US8 (ORF75; 10 K) are deleted as compared to the RacL11 and Ab4 genomes. In-frame deletions in the US1 (ORF68), US4 (ORF71; gp2), and UL63 (ORF63; EICP0) genes and point mutations in 14 different open reading frames (ORFs) were detected in the KyA genome. Interestingly, UL1 (ORF1) and UL2 (ORF2) were deleted in both KyA and RacL11. Our previous studies showed that EHV-1 glycoproteins gI, gE, and full-length gp2 contribute to the pathogenesis of the RacL11 strain. The confirmation of these gene deletions in KyA suggests their contribution to the attenuation of this virus. The growth kinetics results revealed that KyA replicates to high titers in cell culture as compared to RacL11 and Ab4, indicating that the above genomic deletions and mutations in KyA do not have an inhibitory effect on KyA replication in cells of mouse, rabbit, equine, or human origin. Studies of EHV-1 pathogenesis in CBA mice showed that KyA is attenuated whereas mice infected with RacL11 succumbed by 3–6 days post-infection, which is consistent with our previous results. PMID:29312962

  6. Comparative analysis of response to selection with three insecticides in the dengue mosquito Aedes aegypti using mRNA sequencing

    PubMed Central

    2014-01-01

    Background Mosquito control programmes using chemical insecticides are increasingly threatened by the development of resistance. Such resistance can be the consequence of changes in proteins targeted by insecticides (target site mediated resistance), increased insecticide biodegradation (metabolic resistance), altered transport, sequestration or other mechanisms. As opposed to target site resistance, other mechanisms are far from being fully understood. Indeed, insecticide selection often affects a large number of genes and various biological processes can hypothetically confer resistance. In this context, the aim of the present study was to use RNA sequencing (RNA-seq) for comparing transcription level and polymorphism variations associated with adaptation to chemical insecticides in the mosquito Aedes aegypti. Biological materials consisted of a parental susceptible strain together with three child strains selected across multiple generations with three insecticides from different classes: the pyrethroid permethrin, the neonicotinoid imidacloprid and the carbamate propoxur. Results After ten generations, insecticide-selected strains showed elevated resistance levels to the insecticides used for selection. RNA-seq data allowed detecting over 13,000 transcripts, of which 413 were differentially transcribed in insecticide-selected strains as compared to the susceptible strain. Among them, a significant enrichment of transcripts encoding cuticle proteins, transporters and enzymes was observed. Polymorphism analysis revealed over 2500 SNPs showing > 50% allele frequency variations in insecticide-selected strains as compared to the susceptible strain, affecting over 1000 transcripts. Comparing gene transcription and polymorphism patterns revealed marked differences among strains. While imidacloprid selection was linked to the over transcription of many genes, permethrin selection was rather linked to polymorphism variations. Focusing on detoxification enzymes

  7. Comparative analysis of response to selection with three insecticides in the dengue mosquito Aedes aegypti using mRNA sequencing.

    PubMed

    David, Jean-Philippe; Faucon, Frédéric; Chandor-Proust, Alexia; Poupardin, Rodolphe; Riaz, Muhammad Asam; Bonin, Aurélie; Navratil, Vincent; Reynaud, Stéphane

    2014-03-05

    Mosquito control programmes using chemical insecticides are increasingly threatened by the development of resistance. Such resistance can be the consequence of changes in proteins targeted by insecticides (target site mediated resistance), increased insecticide biodegradation (metabolic resistance), altered transport, sequestration or other mechanisms. As opposed to target site resistance, other mechanisms are far from being fully understood. Indeed, insecticide selection often affects a large number of genes and various biological processes can hypothetically confer resistance. In this context, the aim of the present study was to use RNA sequencing (RNA-seq) for comparing transcription level and polymorphism variations associated with adaptation to chemical insecticides in the mosquito Aedes aegypti. Biological materials consisted of a parental susceptible strain together with three child strains selected across multiple generations with three insecticides from different classes: the pyrethroid permethrin, the neonicotinoid imidacloprid and the carbamate propoxur. After ten generations, insecticide-selected strains showed elevated resistance levels to the insecticides used for selection. RNA-seq data allowed detecting over 13,000 transcripts, of which 413 were differentially transcribed in insecticide-selected strains as compared to the susceptible strain. Among them, a significant enrichment of transcripts encoding cuticle proteins, transporters and enzymes was observed. Polymorphism analysis revealed over 2500 SNPs showing > 50% allele frequency variations in insecticide-selected strains as compared to the susceptible strain, affecting over 1000 transcripts. Comparing gene transcription and polymorphism patterns revealed marked differences among strains. While imidacloprid selection was linked to the over transcription of many genes, permethrin selection was rather linked to polymorphism variations. Focusing on detoxification enzymes revealed that permethrin

  8. An Alignment-Free Algorithm in Comparing the Similarity of Protein Sequences Based on Pseudo-Markov Transition Probabilities among Amino Acids

    PubMed Central

    Li, Yushuang; Yang, Jiasheng; Zhang, Yi

    2016-01-01

    In this paper, we have proposed a novel alignment-free method for comparing the similarity of protein sequences. We first encode a protein sequence into a 440 dimensional feature vector consisting of a 400 dimensional Pseudo-Markov transition probability vector among the 20 amino acids, a 20 dimensional content ratio vector, and a 20 dimensional position ratio vector of the amino acids in the sequence. By evaluating the Euclidean distances among the representing vectors, we compare the similarity of protein sequences. We then apply this method into the ND5 dataset consisting of the ND5 protein sequences of 9 species, and the F10 and G11 datasets representing two of the xylanases containing glycoside hydrolase families, i.e., families 10 and 11. As a result, our method achieves a correlation coefficient of 0.962 with the canonical protein sequence aligner ClustalW in the ND5 dataset, much higher than those of other 5 popular alignment-free methods. In addition, we successfully separate the xylanases sequences in the F10 family and the G11 family and illustrate that the F10 family is more heat stable than the G11 family, consistent with a few previous studies. Moreover, we prove mathematically an identity equation involving the Pseudo-Markov transition probability vector and the amino acids content ratio vector. PMID:27918587

  9. Sequence-based prediction of protein-binding sites in DNA: comparative study of two SVM models.

    PubMed

    Park, Byungkyu; Im, Jinyong; Tuvshinjargal, Narankhuu; Lee, Wook; Han, Kyungsook

    2014-11-01

    As many structures of protein-DNA complexes have been known in the past years, several computational methods have been developed to predict DNA-binding sites in proteins. However, its inverse problem (i.e., predicting protein-binding sites in DNA) has received much less attention. One of the reasons is that the differences between the interaction propensities of nucleotides are much smaller than those between amino acids. Another reason is that DNA exhibits less diverse sequence patterns than protein. Therefore, predicting protein-binding DNA nucleotides is much harder than predicting DNA-binding amino acids. We computed the interaction propensity (IP) of nucleotide triplets with amino acids using an extensive dataset of protein-DNA complexes, and developed two support vector machine (SVM) models that predict protein-binding nucleotides from sequence data alone. One SVM model predicts protein-binding nucleotides using DNA sequence data alone, and the other SVM model predicts protein-binding nucleotides using both DNA and protein sequences. In a 10-fold cross-validation with 1519 DNA sequences, the SVM model that uses DNA sequence data only predicted protein-binding nucleotides with an accuracy of 67.0%, an F-measure of 67.1%, and a Matthews correlation coefficient (MCC) of 0.340. With an independent dataset of 181 DNAs that were not used in training, it achieved an accuracy of 66.2%, an F-measure 66.3% and a MCC of 0.324. Another SVM model that uses both DNA and protein sequences achieved an accuracy of 69.6%, an F-measure of 69.6%, and a MCC of 0.383 in a 10-fold cross-validation with 1519 DNA sequences and 859 protein sequences. With an independent dataset of 181 DNAs and 143 proteins, it showed an accuracy of 67.3%, an F-measure of 66.5% and a MCC of 0.329. Both in cross-validation and independent testing, the second SVM model that used both DNA and protein sequence data showed better performance than the first model that used DNA sequence data. To the best of

  10. E6 and E7 Antibody Levels Are Potential Biomarkers of Recurrence in Patients with Advanced-Stage Human Papillomavirus-Positive Oropharyngeal Squamous Cell Carcinoma.

    PubMed

    Spector, Matthew E; Sacco, Assuntina G; Bellile, Emily; Taylor, Jeremy M G; Jones, Tamara; Sun, Kan; Brown, William C; Birkeland, Andrew C; Bradford, Carol R; Wolf, Gregory T; Prince, Mark E; Moyer, Jeffrey S; Malloy, Kelly; Swiecicki, Paul; Eisbruch, Avraham; McHugh, Jonathan B; Chepeha, Douglas B; Rozek, Laura; Worden, Francis P

    2017-06-01

    Purpose: There is a paucity of biomarkers to predict failure in human papillomavirus-positive (HPV + ) oropharyngeal squamous cell carcinoma (OPSCC) following curative therapy. E6/E7 viral oncoproteins are constitutively expressed in HPV + tumors and highly immunogenic, resulting in readily detected serum antibodies. The purpose of this study is to determine whether serum E6 and E7 antibody levels can potentially serve as a biomarker of recurrence in patients with HPV+OPSCC. Experimental Design: We evaluated E6/E7 antibody levels in patients with previously untreated, advanced stage (III, IVa-b), HPV+OPSCC receiving definitive chemoradiation under a uniform protocol from 2003 to 2010. Baseline and longitudinal serum samples were obtained from our archived repository. E6/E7 serum levels were measured using a glutathione- S -transferase capture ELISA and quantified by approximating the area under the dilution curve, and were analyzed using ANOVA and linear mixed model for longitudinal analysis. Results: We compared 22 HPV+OPSCC patients who developed recurrence with 30 patients who remained disease-free. There were no differences in T classification, N classification, disease subsite, or smoking status between the groups. In a longitudinal analysis, recurrent patients had significantly higher E6 and E7 serum antibody levels than the nonrecurrent patients over the follow-up period ( P = 0.02 and P = 0.002, respectively). Patients who recurred had a lower clearance of E7 antibody than patients who remained disease-free ( P = 0.0016). Conclusions: Patients with HPV+OPSCC whose disease recurs have a lower clearance of E6 and E7 antibodies than patients who do not have recurrence. The ratio of E7 antibody at disease recurrence compared with baseline is potentially a clinically significant measurement of disease status in HPV+OPSCC. Clin Cancer Res; 23(11); 2723-9. ©2016 AACR . ©2016 American Association for Cancer Research.

  11. Lessons learned from the initial sequencing of the pig genome: comparative analysis of an 8 Mb region of pig chromosome 17

    PubMed Central

    Hart, Elizabeth A; Caccamo, Mario; Harrow, Jennifer L; Humphray, Sean J; Gilbert, James GR; Trevanion, Steve; Hubbard, Tim; Rogers, Jane; Rothschild, Max F

    2007-01-01

    Background We describe here the sequencing, annotation and comparative analysis of an 8 Mb region of pig chromosome 17, which provides a useful test region to assess coverage and quality for the pig genome sequencing project. We report our findings comparing the annotation of draft sequence assembled at different depths of coverage. Results Within this region we annotated 71 loci, of which 53 are orthologous to human known coding genes. When compared to the syntenic regions in human (20q13.13-q13.33) and mouse (chromosome 2, 167.5 Mb-178.3 Mb), this region was found to be highly conserved with respect to gene order. The most notable difference between the three species is the presence of a large expansion of zinc finger coding genes and pseudogenes on mouse chromosome 2 between Edn3 and Phactr3 that is absent from pig and human. All of our annotation has been made publicly available in the Vertebrate Genome Annotation browser, VEGA. We assessed the impact of coverage on sequence assembly across this region and found, as expected, that increased sequence depth resulted in fewer, longer contigs. One-third of our annotated loci could not be fully re-aligned back to the low coverage version of the sequence, principally because the transcripts are fragmented over several contigs. Conclusion We have demonstrated the considerable advantages of sequencing at increased read depths and discuss the implications that lower coverage sequence may have on subsequent comparative and functional studies, particularly those involving complex loci such as GNAS. PMID:17705864

  12. SNPs in putative regulatory regions identified by human mouse comparative sequencing and transcription factor binding site data

    SciT

    Banerjee, Poulabi; Bahlo, Melanie; Schwartz, Jody R.

    2002-01-01

    Genome wide disease association analysis using SNPs is being explored as a method for dissecting complex genetic traits and a vast number of SNPs have been generated for this purpose. As there are cost and throughput limitations of genotyping large numbers of SNPs and statistical issues regarding the large number of dependent tests on the same data set, to make association analysis practical it has been proposed that SNPs should be prioritized based on likely functional importance. The most easily identifiable functional SNPs are coding SNPs (cSNPs) and accordingly cSNPs have been screened in a number of studies. SNPs inmore » gene regulatory sequences embedded in noncoding DNA are another class of SNPs suggested for prioritization due to their predicted quantitative impact on gene expression. The main challenge in evaluating these SNPs, in contrast to cSNPs is a lack of robust algorithms and databases for recognizing regulatory sequences in noncoding DNA. Approaches that have been previously used to delineate noncoding sequences with gene regulatory activity include cross-species sequence comparisons and the search for sequences recognized by transcription factors. We combined these two methods to sift through mouse human genomic sequences to identify putative gene regulatory elements and subsequently localized SNPs within these sequences in a 1 Megabase (Mb) region of human chromosome 5q31, orthologous to mouse chromosome 11 containing the Interleukin cluster.« less

  13. DNA sequence-based comparative studies between non-extremophile and extremophile organisms with implications in exobiology

    NASA Astrophysics Data System (ADS)

    Holden, Todd; Marchese, P.; Tremberger, G., Jr.; Cheung, E.; Subramaniam, R.; Sullivan, R.; Schneider, P.; Flamholz, A.; Lieberman, D.; Cheung, T.

    2008-08-01

    We have characterized function related DNA sequences of various organisms using informatics techniques, including fractal dimension calculation, nucleotide and multi-nucleotide statistics, and sequence fluctuation analysis. Our analysis shows trends which differentiate extremophile from non-extremophile organisms, which could be reproduced in extraterrestrial life. Among the systems studied are radiation repair genes, genes involved in thermal shocks, and genes involved in drug resistance. We also evaluate sequence level changes that have occurred during short term evolution (several thousand generations) under extreme conditions.

  14. The clinical value of HPV E6/E7 and STAT3 mRNA detection in cervical cancer screening.

    PubMed

    Fan, Yibing; Shen, Zongji

    2018-05-01

    To explore the value of human papillomavirus (HPV) E6/E7 and signal transducer and activator of transcription 3 (STAT3) mRNA detection in the screening of cervical lesions. 192 patients with abnormal ThinPrep cytology test (TCT) results and/or high-risk HPV infection were screened to identify possible cervical lesions in cases. Diagnoses were confirmed by histopathology. Fluorescence in situ hybridization (FISH) was performed to detect and qualify the mRNAs of HPV E6/E7, STAT3, and Survivin in cervical exfoliated cells. In addition, the performance of separate and combined mRNA detection methods were compared with TCT, HR-HPV DNA schemes respectively. 1. Compared with HPVE6/E7 and STAT3 mRNA methods, Survivin mRNA assay had poor specificity (Sp), Youden index (YI) and concordance rate. 2. HPV E6/E7, STAT3, and STAT3 + HR-HPV methods had the best Sp, concordance rate and positive predictive value (PPV) for cervical lesions screening and atypical squamous cells of undetermined significance (ASCUS) triage. For screening of high grade squamous intraepithelial lesions or greater (HSILs+), no difference was observed in the Se of mRNA detection methods in comparison with that of TCT, HR-HPV and TCT + HR-HPV, whereas the false positive rate (FPR) decreased by 41.48%/55.99%/17.19% and the colposcopy referral rate reduced by about 20.00%/25.00%/11.17%. For triage of women with ASCUS, no difference was observed in the Se of mRNA detection methods as compared to that of HR-HPV (χ 2  = 1.05, P > 0.75), while the FPR decreased by 45.83%/37.50%/41.66% and the colposcopy referral rate reduced by 32.42%/22.60%/25.28%, respectively. The Se, YI, and PPV of the combined methods increased in comparison to each method alone. 3. Compared with the TCT + HR-HPV method, HPV E6/E7 + STAT3 method had perfect Sp (95.92%) and PPV (95.40%) for screening HSILs+, the FPR and colposcopy referral rate decreased by 31.06% and 22.48% respectively. 1. The expression of HPV E6/E

  15. The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth.

    PubMed Central

    Brehm, A; Nielsen, S J; Miska, E A; McCance, D J; Reid, J L; Bannister, A J; Kouzarides, T

    1999-01-01

    E7 is the main transforming protein of human papilloma virus type 16 (HPV16) which is implicated in the formation of cervical cancer. The transforming activity of E7 has been attributed to its interaction with the retinoblastoma (Rb) tumour suppressor. However, Rb binding is not sufficient for transformation by E7. Mutations within a zinc finger domain, which is dispensable for Rb binding, also abolish E7 transformation functions. Here we show that HPV16 E7 associates with histone deacetylase in vitro and in vivo, via its zinc finger domain. Using a genetic screen, we identify Mi2beta, a component of the recently identified NURD histone deacetylase complex, as a protein that binds directly to the E7 zinc finger. A zinc finger point mutant which is unable to bind Mi2beta and histone deacetylase but is still able to bind Rb fails to overcome cell cycle arrest in osteosarcoma cells. Our results suggest that the binding to a histone deacetylase complex is an important parameter for the growthpromoting activity of the human papilloma virus E7 protein. This provides the first indication that viral oncoproteins control cell proliferation by targeting deacetylation pathways. PMID:10228159

  16. The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth.

    PubMed

    Brehm, A; Nielsen, S J; Miska, E A; McCance, D J; Reid, J L; Bannister, A J; Kouzarides, T

    1999-05-04

    E7 is the main transforming protein of human papilloma virus type 16 (HPV16) which is implicated in the formation of cervical cancer. The transforming activity of E7 has been attributed to its interaction with the retinoblastoma (Rb) tumour suppressor. However, Rb binding is not sufficient for transformation by E7. Mutations within a zinc finger domain, which is dispensable for Rb binding, also abolish E7 transformation functions. Here we show that HPV16 E7 associates with histone deacetylase in vitro and in vivo, via its zinc finger domain. Using a genetic screen, we identify Mi2beta, a component of the recently identified NURD histone deacetylase complex, as a protein that binds directly to the E7 zinc finger. A zinc finger point mutant which is unable to bind Mi2beta and histone deacetylase but is still able to bind Rb fails to overcome cell cycle arrest in osteosarcoma cells. Our results suggest that the binding to a histone deacetylase complex is an important parameter for the growthpromoting activity of the human papilloma virus E7 protein. This provides the first indication that viral oncoproteins control cell proliferation by targeting deacetylation pathways.

  17. Complete Genomic Sequence and Comparative Analysis of the Genome Segments of Sweet Potato Chlorotic Stunt Virus in China

    PubMed Central

    Qin, Yanhong; Wang, Li; Zhang, Zhenchen; Qiao, Qi; Zhang, Desheng; Tian, Yuting; Wang, Shuang; Wang, Yongjiang; Yan, Zhaoling

    2014-01-01

    Background Sweet potato chlorotic stunt virus (family Closteroviridae, genus Crinivirus) features a large bipartite, single-stranded, positive-sense RNA genome. To date, only three complete genomic sequences of SPCSV can be accessed through GenBank. SPCSV was first detected from China in 2011, only partial genomic sequences have been determined in the country. No report on the complete genomic sequence and genome structure of Chinese SPCSV isolates or the genetic relation between isolates from China and other countries is available. Methodology/Principal Findings The complete genomic sequences of five isolates from different areas in China were characterized. This study is the first to report the complete genome sequences of SPCSV from whitefly vectors. Genome structure analysis showed that isolates of WA and EA strains from China have the same coding protein as isolates Can181-9 and m2-47, respectively. Twenty cp genes and four RNA1 partial segments were sequenced and analyzed, and the nucleotide identities of complete genomic, cp, and RNA1 partial sequences were determined. Results indicated high conservation among strains and significant differences between WA and EA strains. Genetic analysis demonstrated that, except for isolates from Guangdong Province, SPCSVs from other areas belong to the WA strain. Genome organization analysis showed that the isolates in this study lack the p22 gene. Conclusions/Significance We presented the complete genome sequences of SPCSV in China. Comparison of nucleotide identities and genome structures between these isolates and previously reported isolates showed slight differences. The nucleotide identities of different SPCSV isolates showed high conservation among strains and significant differences between strains. All nine isolates in this study lacked p22 gene. WA strains were more extensively distributed than EA strains in China. These data provide important insights into the molecular variation and genomic structure of SPCSV

  18. De novo transcriptome sequencing and comparative analysis to discover genes involved in ovarian maturity in Strongylocentrotus nudus.

    PubMed

    Jia, Zhiying; Wang, Qiai; Wu, Kaikai; Wei, Zhenlin; Zhou, Zunchun; Liu, Xiaolin

    2017-09-01

    Strongylocentrotus nudus is an edible sea urchin, mainly harvested in China. Correlation studies indicated that S. nudus with larger diameter have a prolonged marketing time and better palatability owing to their precocious gonads and extended maturation process. However, the molecular mechanism underlying this phenomenon is still unknown. Here, transcriptome sequencing was applied to study the ovaries of adult S. nudus with different shell diameters to explore the possible mechanism. In this study, four independent cDNA libraries were constructed, including two from the big size urchins and two from the small ones using a HiSeq™2500 platform. A total of 88,581 unigenes were acquired with a mean length of 1354bp, of which 66,331 (74.88%) unigenes could be annotated using six major publicly available databases. Comparative analysis revealed that 353 unigenes were differentially expressed (with log2(ratio)≥1, FDR≤0.001) between the two groups. Of these, 20 differentially expressed genes (DEGs) were selected to confirm the accuracy of RNA-seq data by quantitative real-time RT-PCR. Furthermore, gene ontology and KEGG pathway enrichment analyses were performed to find the putative genes and pathways related to ovarian maturity. Eight unigenes were identified as significant DEGs involved in reproduction related pathways; these included Mos, Cdc20, Rec8, YP30, cytochrome P450 2U1, ovoperoxidase, proteoliaisin, and rendezvin. Our research fills the gap in the studies on the S. nudus ovaries using transcriptome analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Refractive indices of liquid crystal E7 depending on temperature and wavelengths

    NASA Astrophysics Data System (ADS)

    Ma, Mingjian; Li, Shuguang; Jing, Xili; Chen, Hailiang

    2017-11-01

    The dependence of refractive indices of liquid crystal (LC) on temperature is represented by the Haller approximation model, and its dependence on the wavelength is expressed by the extended Cauchy model. We derived the refractive indices expressions of nematic LC E7 depending on temperature and wavelength simultaneously by combining these two models. Based on the obtained expressions, one can acquire the refractive indices of E7 at arbitrary temperature and wavelengths. The birefringence, variation rate of refractive indices, macroscopic order parameter Q, and orientational order parameter ⟨P2⟩ of E7 were then discussed based on the expressions.

  20. Why There Are No Elliptical Galaxies More Flattened Than E7. Thirty Years Later

    NASA Astrophysics Data System (ADS)

    Caimmi, R.

    2006-12-01

    Elliptical galaxies are modelled as homeoidally striated Jacobi ellipsoids (Caimmi and Marmo 2005) where the peculiar velocity distribution is anisotropic, or equivalently as their adjoint configurations i.e. classical Jacobi ellipsoids of equal mass and axes, in real or imaginary rotation (Caimmi 2006). Reasons for the coincidence of bifurcation points from axisymmetric to triaxial configurations in both the sequences (Caimmi 2006), contrary to earlier findings (Wiegandt 1982a,b, Caimmi and Marmo 2005) are presented and discussed. The effect of centrifugal support at the ends of the major equatorial axis is briefly outlined. The existence of a lower limit to the flattening of elliptical galaxies is investigated in dealing with a number of limiting situations. More specifically, (i) elliptical galaxies are considered as isolated systems, and an allowed region within Ellipsoidland (Hunter and de Zeeuw 1997), related to the occurrence of bifurcation points from ellipsoidal to pear-shaped configurations, is shown to be consistent with observations; (ii) elliptical galaxies are considered as embedded within dark matter haloes and, under reasonable assumptions, it is shown that tidal effects from hosting haloes have little influence on the above mentioned results; (iii) dark matter haloes and embedded elliptical galaxies, idealized as a single homeoidally striated Jacobi ellipsoid, are considered in connection with the cosmological transition from expansion to relaxation, by generalizing an earlier model (Thuan and Gott 1975), and the existence of a lower limit to the flattening of relaxed (oblate-like) configurations, is established. On the other hand, no lower limit is found to the elongation of relaxed (prolate-like) configurations, and the existence of some sort of instability is predicted, owing to the observed lack of elliptical galaxies more flattened or elongated than E7.

  1. Antitumor properties of (5E,7E) analogs of vitamin D3.

    PubMed

    Filip, B; Milczarek, M; Wietrzyk, J; Chodyński, M; Kutner, A

    2010-07-01

    Geometric isomers (5E,7E) of major active metabolites of vitamin D3 [1alpha,25(OH)2D3 and (24R)-1,24(OH)2D3] were synthesized by a new convenient procedure. Vitamin D triene system of the metabolites was first derivatized as a Diels-Alder adduct. Removal of the triene protecting group, in a key synthetic step, yielded the title compounds PRI-2208 and PRI-2209, respectively. The analogs were examined for their antiproliferative activity in vitro against human breast cancer cells (MCF-7) and promyelocytic leukemia (HL-60) cells. The activity was compared with one of the parent compounds. Both analogs examined revealed similar or higher antiproliferative activity compared to 1alpha,25(OH)2D3 or to (24R)-1,24(OH)2D3. The studies of calcemic activity in vivo showed that analogs PRI-2208 and PRI-2209 did not influence the serum calcium level in doses, in which 1alpha,25(OH)2D3 or (24R)-1,24(OH)2D3 significantly increased this level. The antitumor activity of these analogs in the LLC mice tumor model was studied. Analog PRI-2208 was found to be more active in inhibiting LLC tumor growth than 1alpha,25(OH)2D3, as well as than PRI-2191 and PRI-2209. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  2. Complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera, and comparative analyses with other grass genomes

    PubMed Central

    Saski, Christopher; Lee, Seung-Bum; Fjellheim, Siri; Guda, Chittibabu; Jansen, Robert K.; Luo, Hong; Tomkins, Jeffrey; Rognli, Odd Arne; Clarke, Jihong Liu

    2009-01-01

    Comparisons of complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera to six published grass chloroplast genomes reveal that gene content and order are similar but two microstructural changes have occurred. First, the expansion of the IR at the SSC/IRa boundary that duplicates a portion of the 5′ end of ndhH is restricted to the three genera of the subfamily Pooideae (Agrostis, Hordeum and Triticum). Second, a 6 bp deletion in ndhK is shared by Agrostis, Hordeum, Oryza and Triticum, and this event supports the sister relationship between the subfamilies Erhartoideae and Pooideae. Repeat analysis identified 19–37 direct and inverted repeats 30 bp or longer with a sequence identity of at least 90%. Seventeen of the 26 shared repeats are found in all the grass chloroplast genomes examined and are located in the same genes or intergenic spacer (IGS) regions. Examination of simple sequence repeats (SSRs) identified 16–21 potential polymorphic SSRs. Five IGS regions have 100% sequence identity among Zea mays, Saccharum officinarum and Sorghum bicolor, whereas no spacer regions were identical among Oryza sativa, Triticum aestivum, H. vulgare and A. stolonifera despite their close phylogenetic relationship. Alignment of EST sequences and DNA coding sequences identified six C–U conversions in both Sorghum bicolor and H. vulgare but only one in A. stolonifera. Phylogenetic trees based on DNA sequences of 61 protein-coding genes of 38 taxa using both maximum parsimony and likelihood methods provide moderate support for a sister relationship between the subfamilies Erhartoideae and Pooideae. PMID:17534593

  3. Whole-exome sequencing gives additional benefits compared to candidate gene sequencing in the molecular diagnosis of children with growth hormone or IGF-1 insensitivity.

    PubMed

    Shapiro, Lucy; Chatterjee, Sumana; Ramadan, Dina G; Davies, Kate M; Savage, Martin O; Metherell, Louise A; Storr, Helen L

    2017-12-01

    GH insensitivity (GHI) is characterised by short stature, IGF-1 deficiency and normal/elevated serum GH. IGF-1 insensitivity results in pre- and post-natal growth failure with normal/high IGF-1 levels. The prevalence of genetic defects is unknown. To identify the underlying genetic diagnoses in a paediatric cohort with GH or IGF-1 insensitivity using candidate gene (CGS) and whole-exome sequencing (WES) and assess factors associated with the discovery of a genetic defect. We undertook a prospective study of 132 patients with short stature and suspected GH or IGF-1 insensitivity referred to our centre for genetic analysis. 107 (96 GHI, 88 probands; 11 IGF-1 insensitivity, 9 probands) underwent CGS. WES was performed in those with no defined genetic aetiology following CGS. A genetic diagnosis was discovered 38/107 (36%) patients (32% probands) by CGS. WES revealed 11 patients with genetic variants in genes known to cause short stature. A further 2 patients had hypomethylation in the H19/IGF2 region or mUPD7 consistent with Silver-Russell Syndrome (total with genetic diagnosis 51/107, 48% or 41/97, 42% probands). WES also identified homozygous putative variants in FANCA and PHKB in 2 patients. Low height SDS and consanguinity were highly predictive for identifying a genetic defect. Comprehensive genetic testing confirms the genetic heterogeneity of GH/IGF-1 insensitivity and successfully identified the genetic aetiology in a significant proportion of cases. WES is rapid and may isolate genetic variants that have been missed by traditional clinically driven genetic testing. This emphasises the benefits of specialist diagnostic centres. © 2017 European Society of Endocrinology.

  4. Comparative effectiveness of next generation genomic sequencing for disease diagnosis: design of a randomized controlled trial in patients with colorectal cancer/polyposis syndromes.

    PubMed

    Gallego, Carlos J; Bennette, Caroline S; Heagerty, Patrick; Comstock, Bryan; Horike-Pyne, Martha; Hisama, Fuki; Amendola, Laura M; Bennett, Robin L; Dorschner, Michael O; Tarczy-Hornoch, Peter; Grady, William M; Fullerton, S Malia; Trinidad, Susan B; Regier, Dean A; Nickerson, Deborah A; Burke, Wylie; Patrick, Donald L; Jarvik, Gail P; Veenstra, David L

    2014-09-01

    Whole exome and whole genome sequencing are applications of next generation sequencing transforming clinical care, but there is little evidence whether these tests improve patient outcomes or if they are cost effective compared to current standard of care. These gaps in knowledge can be addressed by comparative effectiveness and patient-centered outcomes research. We designed a randomized controlled trial that incorporates these research methods to evaluate whole exome sequencing compared to usual care in patients being evaluated for hereditary colorectal cancer and polyposis syndromes. Approximately 220 patients will be randomized and followed for 12 months after return of genomic findings. Patients will receive findings associated with colorectal cancer in a first return of results visit, and findings not associated with colorectal cancer (incidental findings) during a second return of results visit. The primary outcome is efficacy to detect mutations associated with these syndromes; secondary outcomes include psychosocial impact, cost-effectiveness and comparative costs. The secondary outcomes will be obtained via surveys before and after each return visit. The expected challenges in conducting this randomized controlled trial include the relatively low prevalence of genetic disease, difficult interpretation of some genetic variants, and uncertainty about which incidental findings should be returned to patients. The approaches utilized in this study may help guide other investigators in clinical genomics to identify useful outcome measures and strategies to address comparative effectiveness questions about the clinical implementation of genomic sequencing in clinical care. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Comparing an accelerated 3D fast spin-echo sequence (CS-SPACE) for knee 3-T magnetic resonance imaging with traditional 3D fast spin-echo (SPACE) and routine 2D sequences.

    PubMed

    Altahawi, Faysal F; Blount, Kevin J; Morley, Nicholas P; Raithel, Esther; Omar, Imran M

    2017-01-01

    To compare a faster, new, high-resolution accelerated 3D-fast-spin-echo (3D-FSE) acquisition sequence (CS-SPACE) to traditional 2D and high-resolution 3D sequences for knee 3-T magnetic resonance imaging (MRI). Twenty patients received knee MRIs that included routine 2D (T1, PD ± FS, T2-FS; 0.5 × 0.5 × 3 mm 3 ; ∼10 min), traditional 3D FSE (SPACE-PD-FS; 0.5 × 0.5 × 0.5 mm 3 ; ∼7.5 min), and accelerated 3D-FSE prototype (CS-SPACE-PD-FS; 0.5 × 0.5 × 0.5 mm 3 ; ∼5 min) acquisitions on a 3-T MRI system (Siemens MAGNETOM Skyra). Three musculoskeletal radiologists (MSKRs) prospectively and independently reviewed the studies with graded surveys comparing image and diagnostic quality. Tissue-specific signal-to-noise ratios (SNR) and contrast-to-noise ratios (CNR) were also compared. MSKR-perceived diagnostic quality of cartilage was significantly higher for CS-SPACE than for SPACE and 2D sequences (p < 0.001). Assessment of diagnostic quality of menisci and synovial fluid was higher for CS-SPACE than for SPACE (p < 0.001). CS-SPACE was not significantly different from SPACE but had lower assessments than 2D sequences for evaluation of bones, ligaments, muscles, and fat (p ≤ 0.004). 3D sequences had higher spatial resolution, but lower overall assessed contrast (p < 0.001). Overall image quality from CS-SPACE was assessed as higher than SPACE (p = 0.007), but lower than 2D sequences (p < 0.001). Compared to SPACE, CS-SPACE had higher fluid SNR and CNR against all other tissues (all p < 0.001). The CS-SPACE prototype allows for faster isotropic acquisitions of knee MRIs over currently used protocols. High fluid-to-cartilage CNR and higher spatial resolution over routine 2D sequences may present a valuable role for CS-SPACE in the evaluation of cartilage and menisci.

  6. Phylogenetic origins of the plant mitochondrion based on a comparative analysis of 5S ribosomal RNA sequences

    NASA Technical Reports Server (NTRS)

    Villanueva, E.; Delihas, N.; Luehrsen, K. R.; Fox, G. E.; Gibson, J.

    1985-01-01

    The complete nucleotide sequences of 5S ribosomal RNAs from Rhodocyclus gelatinosa, Rhodobacter sphaeroides, and Pseudomonas cepacia were determined. Comparisons of these 5S RNA sequences show that rather than being phylogenetically related to one another, the two photosynthetic bacterial 5S RNAs share more sequence and signature homology with the RNAs of two nonphotosynthetic strains. Rhodobacter sphaeroides is specifically related to Paracoccus denitrificans and Rc. gelatinosa is related to Ps. cepacia. These results support earlier 16S ribosomal RNA studies and add two important groups to the 5S RNA data base. Unique 5S RNA structural features previously found in P. denitrificans are present also in the 5S RNA of Rb. sphaeroides; these provide the basis for subdivisional signatures. The immediate consequence of obtaining these new sequences is that it is possible to clarify the phylogenetic origins of the plant mitochondrion. In particular, a close phylogenetic relationship is found between the plant mitochondria and members of the alpha subdivision of the purple photosynthetic bacteria, namely, Rb. sphaeroides, P. denitrificans, and Rhodospirillum rubrum.

  7. Comparative phylobiomic analysis of the bacterial community of water kefir by 16S rRNA gene amplicon sequencing and ARDRA analysis.

    PubMed

    Gulitz, A; Stadie, J; Ehrmann, M A; Ludwig, W; Vogel, R F

    2013-04-01

    The aim of this study was to analyse the bacterial microbiota of water kefir using culture-independent methods. We compared four water kefirs of different origins using 16S rDNA amplicon sequencing and ARDRA. The microbiota consisted of different proportions of the genera Lactobacillus (Lact.), Leuconostoc (Leuc.), Acetobacter (Acet.) and Gluconobacter. Surprisingly, varying but consistently high numbers of sequences representing members of the genus Bifidobacterium (Bif.) were found in all kefirs. Whereas part of the bifidobacterial sequences could be assigned to Bifidobacterium psychraerophilum, a majority of sequences identical to each other could not be assigned to any known species. A nearly full-length sequence of the latter exhibited a beyond-species similarity (96.4%) with the sequence from the closest relative species Bif. psychraerophilum. A Bifidobacterium-specific ARDRA analysis reflected the abundance of the novel Bifidobacterium species by revealing its unique MboI restriction profile. Attempts to isolate the bifidobacteria were successful for Bif. psychraerophilum only. The complexity of the water kefir microbiota has been underestimated in previously studies. The occurrence of bifidobacteria as part of the consortium is novel. These data give new insights into the understanding of the complexity of food fermentations and underline the need for approaches detecting noncultivable organisms. © 2013 The Society for Applied Microbiology.

  8. Comparing diffusion weighted imaging with clinical and blood parameters, and with short tau inversion recovery sequence in detecting spinal and sacroiliac joint inflammation in axial spondyloarthritis.

    PubMed

    Chung, Ho Yin; Xu, Xiaopei; Lau, Vince Wing Hang; Ho, Grace; Lee, Ka Lai; Li, Philip Hei; Tsang, Helen Hoi Lun; Kwok, Suet Kei; Lau, Chak Sing; Wong, Chun Sing

    2017-01-01

    To investigate the usefulness of diffusion weighted imaging (DWI) by comparing with clinical features, blood parameters and traditional short tau inversion recovery (STIR) sequence in detecting spinal and sacroiliac (SI) joint inflammation in axial spondyloarthritis (axSpA) patients. One hundred and ten axSpA patients were recruited. Clinical, radiological and blood parameters were recorded. DWI and STIR MRI were performed simultaneously and results were scored according to the Spondyloarthritis Research Consortium of Canada (SPARCC) for comparison. Apparent diffusion coef cient (ADC) values were also calculated. DWI did not correlate with clinical parameters or blood parameters. It also had lowered sensitivity. When compared with STIR sequence, it correlated well with STIR sequence at the SI joint level (CC 0.76, p<0.001), but weakly at the spinal level (CC 0.23, p=0.02). At the SI joint level, the presence of inflammation on both STIR sequence and DWI was associated with an increase in maximum (B=0.24, p=0.02 in STIR; B=0.37, p<0.001 in DWI) and mean ADC values (B=0.17, p=0.003 in STIR; B=0.15, p=0.01 in DWI). Maximum (B=0.19, p=0.04) and mean spinal ADC values (B=0.18, p=0.01) were also positively associated with DWI detected spinal inflammation. Presence of Modic lesions showed positive correlation with STIR sequence (B=7.12, p=0.01) but not spinal ADC values. Despite DWI correlates with STIR sequence, it has lower sensitivity. However, ADC values appear to be independent of Modic lesions and may supplement STIR sequence to differentiate degeneration.

  9. Separation of the Components of a Commercial Analgesic Tablet: A Two-Week Sequence Comparing Purification by Two-Base Extraction and Column Chromatography

    ERIC Educational Resources Information Center

    Revell, Kevin D.

    2011-01-01

    A new laboratory experiment is described in which students compare two benchtop separation methods to isolate the three active components of the commercial analgesic Excedrin. In the two-week sequence, aspirin, acetaminophen, and caffeine are separated using either a two-base liquid-liquid extraction or silica column chromatography. Students then…

  10. [Magnetic resonance in traumatic brain injury: A comparative study of the different conventional magnetic resonance imaging sequences and their diagnostic value in diffuse axonal injury].

    PubMed

    Cicuendez, Marta; Castaño-León, Ana; Ramos, Ana; Hilario, Amaya; Gómez, Pedro A; Lagares, Alfonso

    To compare the identification capability of traumatic axonal injury (TAI) by different sequences on conventional magnetic resonance (MR) studies in traumatic brain injury (TBI) patients. We retropectevely analyzed 264 TBI patients to whom a MR had been performed in the first 60 days after trauma. All clinical variables related to prognosis were registered, as well as the data from the initial computed tomography. The MR imaging protocol consisted of a 3-plane localizer sequence T1-weighted and T2-weighted fast spin-echo, FLAIR and gradient-echo images (GRET2*). TAI lesions were classified according to Gentry and Firsching classifications. We calculated weighted kappa coefficients and the area under the ROC curve for each MR sequence. A multivariable analyses was performed to correlate MR findings in each sequence with the final outcome of the patients. TAI lesions were adequately visualized on T2, FLAIR and GRET2* sequences in more than 80% of the studies. Subcortical TAI lesions were well on FLAIR and GRET2* sequences visualized hemorrhagic TAI lesions. We saw that these MR sequences had a high inter-rater agreement for TAI diagnosis (0.8). T2 sequence presented the highest value on ROC curve in Gentry (0.68, 95%CI: 0.61-0.76, p<0.001, Nagerlkerke-R 2 0.26) and Firsching classifications (0.64, 95%CI 0.57-0.72, p<0.001, Nagerlkerke-R 2 0.19), followed by FLAIR and GRET2* sequences. Both classifications determined by each of these sequences were associated with poor outcome after performing a multivariable analyses adjusted for prognostic factors (p<0.02). We recommend to perform conventional MR study in subacute phase including T2, FLAIR and GRET2* sequences for visualize TAI lesions. These MR findings added prognostic information in TBI patients. Copyright © 2017 Sociedad Española de Neurocirugía. Publicado por Elsevier España, S.L.U. All rights reserved.

  11. Adenovirus-mediated transfer of HPV 16 E6/E7 antisense RNA combined with cisplatin inhibits cellular growth and induces apoptosis in HPV-positive head and neck cancer cells.

    PubMed

    Kojima, Yasutaka; Otsuki, Naoki; Kubo, Mie; Kitamoto, Junko; Takata, Eri; Saito, Hiroki; Kosaka, Kyoko; Morishita, Naoya; Uehara, Natsumi; Shirakawa, Toshiro; Nibu, Ken-Ich

    2018-05-24

    Human papillomavirus (HPV) infection has been identified as an etiologic factor of head and neck cancers (HNCs). We explored the potential use of antisense HPV RNA transcripts for gene therapy and its effect in combination with cisplatin (CDDP) for HPV-positive HNCs. We introduced the antisense RNA transcripts of the E6 and E7 genes of HPV type 16 into UM-SCC-47 cells harboring HPV 16 and YCU-T892 cells that were HPV-negative using a recombinant adenoviral vector, Ad-E6/E7-AS. We then analyzed the effects of the introduction of Ad-E7-AS on cell and tumor growth and the synergistic effect with CDDP in vitro and in vivo. After infection of Ad-E6/E7-AS, the cellular growth of UM-SCC-47 cells were suppressed, but not that of YCU-T892 cells. E7 protein expression was suppressed, and p53 and pRb protein expression increased after infection of Ad-E7-AS. Cell growth and tumorigenicity were greatly suppressed in combination with CDDP compared with Ad-E7-AS or CDDP treatment alone in vitro. Ad-E7-AS combined with CDDP treatment significantly reduced the volumes of established subcutaneous tumors. Transfection with HPV 16 E7 antisense RNA combined with CDDP treatment might be a potentially useful approach to the therapy of HPV 16-positive HNC.

  12. Characterization and Comparative Overview of Complete Sequences of the First Plasmids of Pandoraea across Clinical and Non-clinical Strains

    PubMed Central

    Yong, Delicia; Tee, Kok Keng; Yin, Wai-Fong; Chan, Kok-Gan

    2016-01-01

    To date, information on plasmid analysis in Pandoraea spp. is scarce. To address the gap of knowledge on this, the complete sequences of eight plasmids from Pandoraea spp. namely Pandoraea faecigallinarum DSM 23572T (pPF72-1, pPF72-2), Pandoraea oxalativorans DSM 23570T (pPO70-1, pPO70-2, pPO70-3, pPO70-4), Pandoraea vervacti NS15 (pPV15) and Pandoraea apista DSM 16535T (pPA35) were studied for the first time in this study. The information on plasmid sequences in Pandoraea spp. is useful as the sequences did not match any known plasmid sequence deposited in public databases. Replication genes were not identified in some plasmids, a situation that has led to the possibility of host interaction involvement. Some plasmids were also void of par genes and intriguingly, repA gene was also not discovered in these plasmids. This further leads to the hypothesis of host-plasmid interaction. Plasmid stabilization/stability protein-encoding genes were observed in some plasmids but were not established for participating in plasmid segregation. Toxin-antitoxin systems MazEF, VapBC, RelBE, YgiT-MqsR, HigBA, and ParDE were identified across the plasmids and their presence would improve plasmid maintenance. Conjugation genes were identified portraying the conjugation ability amongst Pandoraea plasmids. Additionally, we found a shared region amongst some of the plasmids that consists of conjugation genes. The identification of genes involved in replication, segregation, toxin-antitoxin systems and conjugation, would aid the design of drugs to prevent the survival or transmission of plasmids carrying pathogenic properties. Additionally, genes conferring virulence and antibiotic resistance were identified amongst the plasmids. The observed features in the plasmids shed light on the Pandoraea spp. as opportunistic pathogens. PMID:27790203

  13. Cost-effectiveness Analysis of Treatment Sequence Initiating With Etanercept Compared With Leflunomide in Rheumatoid Arthritis: Impact of Reduced Etanercept Cost With Patent Expiration in South Korea.

    PubMed

    Park, Sun-Kyeong; Park, Seung-Hoo; Lee, Min-Young; Park, Ji-Hyun; Jeong, Jae-Hong; Lee, Eui-Kyung

    2016-11-01

    In south Korea, the price of biologics has been decreasing owing to patent expiration and the availability of biosimilars. This study evaluated the cost-effectiveness of a treatment strategy initiated with etanercept (ETN) compared with leflunomide (LFN) after a 30% reduction in the medication cost of ETN in patients with active rheumatoid arthritis (RA) with an inadequate response to methotrexate (MTX-IR). A cohort-based Markov model was designed to evaluate the lifetime cost-effectiveness of treatment sequence initiated with ETN (A) compared with 2 sequences initiated with LFN: LFN-ETN sequence (B) and LFN sequence (C). Patients transited through the treatment sequences, which consisted of sequential biologics and palliative therapy, based on American College of Rheumatology (ACR) responses and the probability of discontinuation. A systematic literature review and a network meta-analysis were conducted to estimate ACR responses to ETN and LFN. Utility was estimated by mapping an equation for converting the Health Assessment Questionnaire-Disability Index score to utility weight. The costs comprised medications, outpatient visits, administration, dispensing, monitoring, palliative therapy, and treatment for adverse events. A subanalysis was conducted to identify the influence of the ETN price reduction compared with the unreduced price, and sensitivity analyses explored the uncertainty of model parameters and assumptions. The ETN sequence (A) was associated with higher costs and a gain in quality-adjusted life years (QALYs) compared with both sequences initiated with LFN (B, C) throughout the lifetime of patients with RA and MTX-IR. The incremental cost-effectiveness ratio (ICER) for strategy A versus B was ₩13,965,825 (US$1726) per QALY and that for strategy A versus C was ₩9,587,983 (US$8050) per QALY. The results indicated that strategy A was cost-effective based on the commonly cited ICER threshold of ₩20,000,000 (US$16,793) per QALY in South Korea. The

  14. Comparative In silico Study of Sex-Determining Region Y (SRY) Protein Sequences Involved in Sex-Determining.

    PubMed

    Vakili Azghandi, Masoume; Nasiri, Mohammadreza; Shamsa, Ali; Jalali, Mohsen; Shariati, Mohammad Mahdi

    2016-04-01

    The SRY gene (SRY) provides instructions for making a transcription factor called the sex-determining region Y protein. The sex-determining region Y protein causes a fetus to develop as a male. In this study, SRY of 15 spices included of human, chimpanzee, dog, pig, rat, cattle, buffalo, goat, sheep, horse, zebra, frog, urial, dolphin and killer whale were used for determine of bioinformatic differences. Nucleotide sequences of SRY were retrieved from the NCBI databank. Bioinformatic analysis of SRY is done by CLC Main Workbench version 5.5 and ClustalW (http:/www.ebi.ac.uk/clustalw/) and MEGA6 softwares. The multiple sequence alignment results indicated that SRY protein sequences from Orcinus orca (killer whale) and Tursiopsaduncus (dolphin) have least genetic distance of 0.33 in these 15 species and are 99.67% identical at the amino acid level. Homosapiens and Pantroglodytes (chimpanzee) have the next lowest genetic distance of 1.35 and are 98.65% identical at the amino acid level. These findings indicate that the SRY proteins are conserved in the 15 species, and their evolutionary relationships are similar.

  15. The complete chloroplast genome sequence of Aconitum coreanum and Aconitum carmichaelii and comparative analysis with other Aconitum species

    PubMed Central

    Park, Inkyu; Kim, Wook-jin; Yang, Sungyu; Yeo, Sang-Min; Li, Hulin

    2017-01-01

    Aconitum species (belonging to the Ranunculaceae) are well known herbaceous medicinal ingredients and have great economic value in Asian countries. However, there are still limited genomic resources available for Aconitum species. In this study, we sequenced the chloroplast (cp) genomes of two Aconitum species, A. coreanum and A. carmichaelii, using the MiSeq platform. The two Aconitum chloroplast genomes were 155,880 and 157,040 bp in length, respectively, and exhibited LSC and SSC regions separated by a pair of inverted repeat regions. Both cp genomes had 38% GC content and contained 131 unique functional genes including 86 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The gene order, content, and orientation of the two Aconitum cp genomes exhibited the general structure of angiosperms, and were similar to those of other Aconitum species. Comparison of the cp genome structure and gene order with that of other Aconitum species revealed general contraction and expansion of the inverted repeat regions and single copy boundary regions. Divergent regions were also identified. In phylogenetic analysis, Aconitum species positon among the Ranunculaceae was determined with other family cp genomes in the Ranunculales. We obtained a barcoding target sequence in a divergent region, ndhC–trnV, and successfully developed a SCAR (sequence characterized amplified region) marker for discrimination of A. coreanum. Our results provide useful genetic information and a specific barcode for discrimination of Aconitum species. PMID:28863163

  16. The complete chloroplast genome sequence of Aconitum coreanum and Aconitum carmichaelii and comparative analysis with other Aconitum species.

    PubMed

    Park, Inkyu; Kim, Wook-Jin; Yang, Sungyu; Yeo, Sang-Min; Li, Hulin; Moon, Byeong Cheol

    2017-01-01

    Aconitum species (belonging to the Ranunculaceae) are well known herbaceous medicinal ingredients and have great economic value in Asian countries. However, there are still limited genomic resources available for Aconitum species. In this study, we sequenced the chloroplast (cp) genomes of two Aconitum species, A. coreanum and A. carmichaelii, using the MiSeq platform. The two Aconitum chloroplast genomes were 155,880 and 157,040 bp in length, respectively, and exhibited LSC and SSC regions separated by a pair of inverted repeat regions. Both cp genomes had 38% GC content and contained 131 unique functional genes including 86 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The gene order, content, and orientation of the two Aconitum cp genomes exhibited the general structure of angiosperms, and were similar to those of other Aconitum species. Comparison of the cp genome structure and gene order with that of other Aconitum species revealed general contraction and expansion of the inverted repeat regions and single copy boundary regions. Divergent regions were also identified. In phylogenetic analysis, Aconitum species positon among the Ranunculaceae was determined with other family cp genomes in the Ranunculales. We obtained a barcoding target sequence in a divergent region, ndhC-trnV, and successfully developed a SCAR (sequence characterized amplified region) marker for discrimination of A. coreanum. Our results provide useful genetic information and a specific barcode for discrimination of Aconitum species.

  17. Quantivirus® HPV E6/E7 RNA 3.0 assay (bDNA) is as sensitive, but less specific than Hybrid Capture 2 test.

    PubMed

    Shen, Yong; Gong, Jiaomei; He, Yanxia; Cheng, Guomei; Okunieff, Paul; Li, Xiaofu

    2013-02-01

    Human papillomavirus (HPV) infection is the primary cause of cervical cancer. The Quantivirus(®) HPV E6/E7 RNA 3.0 assay (DiaCarta, CA, USA) detects E6/E7 mRNA of 13 high risk subtypes and 6 low risk subtypes. Cervical specimens collected in PreservCyt were processed for HPV detection. Cervical biopsies were taken only from those women with abnormal colposcopy. 200 out of 272 (73.5%) cases were mRNA positive. The percentage of HPV E6/E7 mRNA positive samples increases with the severity of the cytological diagnosis, but not in histological diagnosis. In 146 patients with both tests, the E6/E7 mRNA assay had significant higher positivity rate than the Hybrid Capture 2 assay (75.3% versus 62.3%). The HPV mRNA assay and the HC2 assay had the same sensitivity of high grade cervical intraepithelial neoplasia (CIN 2+), 82.4% (14/17) (95% confidence interval [CI], 64.3, 100). However, the specificity of CIN 2+ for the HPV mRNA assay was significantly lower than HC2 assay. Receiver operating characteristic curve analysis was used to compare the diagnostic performance of the E6/E7 mRNA and HC2. E6/E7 mRNA achieved 58.8% sensitivity with 74.1% specificity, HC2, achieved 47.1% sensitivity with 70.7% specificity. The overall performance of HPV E6/E7 mRNA assay for detecting CIN 2+ was lower than HC2. This study does not support the use of this assay in screening for cervical cancer prevention alone. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Comparative analysis of the complete genome of an epidemic hospital sequence type 203 clone of vancomycin-resistant Enterococcus faecium

    PubMed Central

    2013-01-01

    Background In this report we have explored the genomic and microbiological basis for a sustained increase in bloodstream infections at a major Australian hospital caused by Enterococcus faecium multi-locus sequence type (ST) 203, an outbreak strain that has largely replaced a predecessor ST17 sequence type. Results To establish a ST203 reference sequence we fully assembled and annotated the genome of Aus0085, a 2009 vancomycin-resistant Enterococcus faecium (VREfm) bloodstream isolate, and the first example of a completed ST203 genome. Aus0085 has a 3.2 Mb genome, comprising a 2.9 Mb circular chromosome and six circular plasmids (2 kb–130 kb). Twelve percent of the 3222 coding sequences (CDS) in Aus0085 are not present in ST17 E. faecium Aus0004 and ST18 E. faecium TX16. Extending this comparison to an additional 12 ST17 and 14 ST203 E. faecium hospital isolate genomes revealed only six genomic regions spanning 41 kb that were present in all ST203 and absent from all ST17 genomes. The 40 CDS have predicted functions that include ion transport, riboflavin metabolism and two phosphotransferase systems. Comparison of the vancomycin resistance-conferring Tn1549 transposon between Aus0004 and Aus0085 revealed differences in transposon length and insertion site, and van locus sequence variation that correlated with a higher vancomycin MIC in Aus0085. Additional phenotype comparisons between ST17 and ST203 isolates showed that while there were no differences in biofilm-formation and killing of Galleria mellonella, ST203 isolates grew significantly faster and out-competed ST17 isolates in growth assays. Conclusions Here we have fully assembled and annotated the first ST203 genome, and then characterized the genomic differences between ST17 and ST203 E. faecium. We also show that ST203 E. faecium are faster growing and can out-compete ST17 E. faecium. While a causal genetic basis for these phenotype differences is not provided here, this study revealed conserved genetic

  19. Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi

    PubMed Central

    2011-01-01

    Background Rust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology. Results To support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar

  20. Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection.

    PubMed

    Ruan, Yi Jun; Wei, Chia Lin; Ee, Ai Ling; Vega, Vinsensius B; Thoreau, Herve; Su, Se Thoe Yun; Chia, Jer-Ming; Ng, Patrick; Chiu, Kuo Ping; Lim, Landri; Zhang, Tao; Peng, Chan Kwai; Lin, Ean Oon Lynette; Lee, Ng Mah; Yee, Sin Leo; Ng, Lisa F P; Chee, Ren Ee; Stanton, Lawrence W; Long, Philip M; Liu, Edison T

    2003-05-24

    The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavirus. Whole genome sequence analysis of various isolates might provide an indication of potential strain differences of this new virus. Moreover, mutation analysis will help to develop effective vaccines. We sequenced the entire SARS viral genome of cultured isolates from the index case (SIN2500) presenting in Singapore, from three primary contacts (SIN2774, SIN2748, and SIN2677), and one secondary contact (SIN2679). These sequences were compared with the isolates from Canada (TOR2), Hong Kong (CUHK-W1 and HKU39849), Hanoi (URBANI), Guangzhou (GZ01), and Beijing (BJ01, BJ02, BJ03, BJ04). We identified 129 sequence variations among the 14 isolates, with 16 recurrent variant sequences. Common variant sequences at four loci define two distinct genotypes of the SARS virus. One genotype was linked with infections originating in Hotel M in Hong Kong, the second contained isolates from Hong Kong, Guangzhou, and Beijing with no association with Hotel M (p<0.0001). Moreover, other common sequence variants further distinguished the geographical origins of the isolates, especially between Singapore and Beijing. Despite the recent onset of the SARS epidemic, genetic signatures are emerging that partition the worldwide SARS viral isolates into groups on the basis of contact source history and geography. These signatures can be used to trace sources of infection. In addition, a common variant associated with a non-conservative aminoacid change in the S1 region of the spike protein, suggests that immunological pressures might be starting to influence the evolution of the SARS virus in human populations.

  1. Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks.

    PubMed

    Smoot, James C; Barbian, Kent D; Van Gompel, Jamie J; Smoot, Laura M; Chaussee, Michael S; Sylva, Gail L; Sturdevant, Daniel E; Ricklefs, Stacy M; Porcella, Stephen F; Parkins, Larye D; Beres, Stephen B; Campbell, David S; Smith, Todd M; Zhang, Qing; Kapur, Vivek; Daly, Judy A; Veasy, L George; Musser, James M

    2002-04-02

    Acute rheumatic fever (ARF), a sequelae of group A Streptococcus (GAS) infection, is the most common cause of preventable childhood heart disease worldwide. The molecular basis of ARF and the subsequent rheumatic heart disease are poorly understood. Serotype M18 GAS strains have been associated for decades with ARF outbreaks in the U.S. As a first step toward gaining new insight into ARF pathogenesis, we sequenced the genome of strain MGAS8232, a serotype M18 organism isolated from a patient with ARF. The genome is a circular chromosome of 1,895,017 bp, and it shares 1.7 Mb of closely related genetic material with strain SF370 (a sequenced serotype M1 strain). Strain MGAS8232 has 178 ORFs absent in SF370. Phages, phage-like elements, and insertion sequences are the major sources of variation between the genomes. The genomes of strain MGAS8232 and SF370 encode many of the same proven or putative virulence factors. Importantly, strain MGAS8232 has genes encoding many additional secreted proteins involved in human-GAS interactions, including streptococcal pyrogenic exotoxin A (scarlet fever toxin) and two uncharacterized pyrogenic exotoxin homologues, all phage-associated. DNA microarray analysis of 36 serotype M18 strains from diverse localities showed that most regions of variation were phages or phage-like elements. Two epidemics of ARF occurring 12 years apart in Salt Lake City, UT, were caused by serotype M18 strains that were genetically identical, or nearly so. Our analysis provides a critical foundation for accelerated research into ARF pathogenesis and a molecular framework to study the plasticity of GAS genomes.

  2. Complete Genome Sequence of the Broad-Host-Range Vibriophage KVP40: Comparative Genomics of a T4-Related Bacteriophage

    PubMed Central

    Miller, Eric S.; Heidelberg, John F.; Eisen, Jonathan A.; Nelson, William C.; Durkin, A. Scott; Ciecko, Ann; Feldblyum, Tamara V.; White, Owen; Paulsen, Ian T.; Nierman, William C.; Lee, Jong; Szczypinski, Bridget; Fraser, Claire M.

    2003-01-01

    The complete genome sequence of the T4-like, broad-host-range vibriophage KVP40 has been determined. The genome sequence is 244,835 bp, with an overall G+C content of 42.6%. It encodes 386 putative protein-encoding open reading frames (CDSs), 30 tRNAs, 33 T4-like late promoters, and 57 potential rho-independent terminators. Overall, 92.1% of the KVP40 genome is coding, with an average CDS size of 587 bp. While 65% of the CDSs were unique to KVP40 and had no known function, the genome sequence and organization show specific regions of extensive conservation with phage T4. At least 99 KVP40 CDSs have homologs in the T4 genome (Blast alignments of 45 to 68% amino acid similarity). The shared CDSs represent 36% of all T4 CDSs but only 26% of those from KVP40. There is extensive representation of the DNA replication, recombination, and repair enzymes as well as the viral capsid and tail structural genes. KVP40 lacks several T4 enzymes involved in host DNA degradation, appears not to synthesize the modified cytosine (hydroxymethyl glucose) present in T-even phages, and lacks group I introns. KVP40 likely utilizes the T4-type sigma-55 late transcription apparatus, but features of early- or middle-mode transcription were not identified. There are 26 CDSs that have no viral homolog, and many did not necessarily originate from Vibrio spp., suggesting an even broader host range for KVP40. From these latter CDSs, an NAD salvage pathway was inferred that appears to be unique among bacteriophages. Features of the KVP40 genome that distinguish it from T4 are presented, as well as those, such as the replication and virion gene clusters, that are substantially conserved. PMID:12923095

  3. Comparative Sequence and X-Inactivation Analyses of a Domain of Escape in Human Xp11.2 and the Conserved Segment in Mouse

    PubMed Central

    Tsuchiya, Karen D.; Greally, John M.; Yi, Yajun; Noel, Kevin P.; Truong, Jean-Pierre; Disteche, Christine M.

    2004-01-01

    We have performed X-inactivation and sequence analyses on 350 kb of sequence from human Xp11.2, a region shown previously to contain a cluster of genes that escape X inactivation, and we compared this region with the region of conserved synteny in mouse. We identified several new transcripts from this region in human and in mouse, which defined the full extent of the domain escaping X inactivation in both species. In human, escape from X inactivation involves an uninterrupted 235-kb domain of multiple genes. Despite highly conserved gene content and order between the two species, Smcx is the only mouse gene from the conserved segment that escapes inactivation. As repetitive sequences are believed to facilitate spreading of X inactivation along the chromosome, we compared the repetitive sequence composition of this region between the two species. We found that long terminal repeats (LTRs) were decreased in the human domain of escape, but not in the majority of the conserved mouse region adjacent to Smcx in which genes were subject to X inactivation, suggesting that these repeats might be excluded from escape domains to prevent spreading of silencing. Our findings indicate that genomic context, as well as gene-specific regulatory elements, interact to determine expression of a gene from the inactive X-chromosome. PMID:15197169

  4. Exploring a Nonmodel Teleost Genome Through RAD Sequencing-Linkage Mapping in Common Pandora, Pagellus erythrinus and Comparative Genomic Analysis.

    PubMed

    Manousaki, Tereza; Tsakogiannis, Alexandros; Taggart, John B; Palaiokostas, Christos; Tsaparis, Dimitris; Lagnel, Jacques; Chatziplis, Dimitrios; Magoulas, Antonios; Papandroulakis, Nikos; Mylonas, Constantinos C; Tsigenopoulos, Costas S

    2015-12-29

    Common pandora (Pagellus erythrinus) is a benthopelagic marine fish belonging to the teleost family Sparidae, and a newly recruited species in Mediterranean aquaculture. The paucity of genetic information relating to sparids, despite their growing economic value for aquaculture, provides the impetus for exploring the genomics of this fish group. Genomic tool development, such as genetic linkage maps provision, lays the groundwork for linking genotype to phenotype, allowing fine-mapping of loci responsible for beneficial traits. In this study, we applied ddRAD methodology to identify polymorphic markers in a full-sib family of common pandora. Employing the Illumina MiSeq platform, we sampled and sequenced a size-selected genomic fraction of 99 individuals, which led to the identification of 920 polymorphic loci. Downstream mapping analysis resulted in the construction of 24 robust linkage groups, corresponding to the karyotype of the species. The common pandora linkage map showed varying degrees of conserved synteny with four other teleost genomes, namely the European seabass (Dicentrarchus labrax), Nile tilapia (Oreochromis niloticus), stickleback (Gasterosteus aculeatus), and medaka (Oryzias latipes), suggesting a conserved genomic evolution in Sparidae. Our work exploits the possibilities of genotyping by sequencing to gain novel insights into genome structure and evolution. Such information will boost the study of cultured species and will set the foundation for a deeper understanding of the complex evolutionary history of teleosts. Copyright © 2016 Manousaki et al.

  5. Comparative Analysis of the Gut Microbial Communities in Forest and Alpine Musk Deer Using High-Throughput Sequencing

    PubMed Central

    Hu, Xiaolong; Liu, Gang; Shafer, Aaron B. A.; Wei, Yuting; Zhou, Juntong; Lin, Shaobi; Wu, Haibin; Zhou, Mi; Hu, Defu; Liu, Shuqiang

    2017-01-01

    The gut ecosystem is characterized by dynamic and reciprocal interactions between the host and bacteria. Although characterizing microbiota for herbivores has become recognized as important tool for gauging species health, no study to date has investigated the bacterial communities and evaluated the age-related bacterial dynamics of musk deer. Moreover, gastrointestinal diseases have been hypothesized to be a limiting factor of population growth in captive musk deer. Here, high-throughput sequencing of the bacterial 16S rRNA gene was used to profile the fecal bacterial communities in juvenile and adult alpine and forest musk deer. The two musk deer species harbored similar bacterial communities at the phylum level, whereas the key genera for the two species were distinct. The bacterial communities were dominated by Firmicutes and Bacteroidetes, with the bacterial diversity being higher in forest musk deer. The Firmicutes to Bacteroidetes ratio also increased from juvenile to adult, while the bacterial diversity, within-group and between-group similarity, all increased with age. This work serves as the first sequence-based analysis of variation in bacterial communities within and between musk deer species, and demonstrates how the gut microbial community dynamics vary among closely related species and shift with age. As gastrointestinal diseases have been observed in captive populations, this study provides valuable data that might benefit captive management and future reintroduction programs. PMID:28421061

  6. A Comparative Genomics Strategy for Targeted Discovery of Single-Nucleotide Polymorphisms and Conserved-Noncoding Sequences in Orphan Crops1[W

    PubMed Central

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

    2006-01-01

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

  7. Capacity for patterns and sequences in Kanerva's SDM as compared to other associative memory models. [Sparse, Distributed Memory

    NASA Technical Reports Server (NTRS)

    Keeler, James D.

    1988-01-01

    The information capacity of Kanerva's Sparse Distributed Memory (SDM) and Hopfield-type neural networks is investigated. Under the approximations used here, it is shown that the total information stored in these systems is proportional to the number connections in the network. The proportionality constant is the same for the SDM and Hopfield-type models independent of the particular model, or the order of the model. The approximations are checked numerically. This same analysis can be used to show that the SDM can store sequences of spatiotemporal patterns, and the addition of time-delayed connections allows the retrieval of context dependent temporal patterns. A minor modification of the SDM can be used to store correlated patterns.

  8. Comparative sequencing in the genus Lycopersicon. Implications for the evolution of fruit size in the domestication of cultivated tomatoes.

    PubMed Central

    Nesbitt, T Clint; Tanksley, Steven D

    2002-01-01

    Sequence variation was sampled in cultivated and related wild forms of tomato at fw2.2--a fruit weight QTL key to the evolution of domesticated tomatoes. Variation at fw2.2 was contrasted with variation at four other loci not involved in fruit weight determination. Several conclusions could be reached: (1) Fruit weight variation attributable to fw2.2 is not caused by variation in the FW2.2 protein sequence; more likely, it is due to transcriptional variation associated with one or more of eight nucleotide changes unique to the promoter of large-fruit alleles; (2) fw2.2 and loci not involved in fruit weight have not evolved at distinguishably different rates in cultivated and wild tomatoes, despite the fact that fw2.2 was likely a target of selection during domestication; (3) molecular-clock-based estimates suggest that the large-fruit allele of fw2.2, now fixed in most cultivated tomatoes, arose in tomato germplasm long before domestication; (4) extant accessions of L. esculentum var. cerasiforme, the subspecies thought to be the most likely wild ancestor of domesticated tomatoes, appear to be an admixture of wild and cultivated tomatoes rather than a transitional step from wild to domesticated tomatoes; and (5) despite the fact that cerasiforme accessions are polymorphic for large- and small-fruit alleles at fw2.2, no significant association was detected between fruit size and fw2.2 genotypes in the subspecies--as tested by association genetic studies in the relatively small sample studied--suggesting the role of other fruit weight QTL in fruit weight variation in cerasiforme. PMID:12242247

  9. 77 FR 9948 - International Conference on Harmonisation; Guidance on E7 Studies in Support of Special...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-21

    ...] International Conference on Harmonisation; Guidance on E7 Studies in Support of Special Populations; Geriatrics... in Support of Special Populations: Geriatrics; Questions and Answers.'' The guidance was prepared... well as observers from the World Health Organization, Health Canada, and the European Free Trade Area...

  10. Differentiated evolutionary relationships among chordates from comparative alignments of multiple sequences of MyoD and MyoG myogenic regulatory factors.

    PubMed

    Oliani, L C; Lidani, K C F; Gabriel, J E

    2015-10-16

    MyoD and MyoG are transcription factors that have essential roles in myogenic lineage determination and muscle differentiation. The purpose of this study was to compare multiple amino acid sequences of myogenic regulatory proteins to infer evolutionary relationships among chordates. Protein sequences from Mus musculus (P10085 and P12979), human Homo sapiens (P15172 and P15173), bovine Bos taurus (Q7YS82 and Q7YS81), wild pig Sus scrofa (P49811 and P49812), quail Coturnix coturnix (P21572 and P34060), chicken Gallus gallus (P16075 and P17920), rat Rattus norvegicus (Q02346 and P20428), domestic water buffalo Bubalus bubalis (D2SP11 and A7L034), and sheep Ovis aries (Q90477 and D3YKV7) were searched from a non-redundant protein sequence database UniProtKB/Swiss-Prot, and subsequently analyzed using the Mega6.0 software. MyoD evolutionary analyses revealed the presence of three main clusters with all mammals branched in one cluster, members of the order Rodentia (mouse and rat) in a second branch linked to the first, and birds of the order Galliformes (chicken and quail) remaining isolated in a third. MyoG evolutionary analyses aligned sequences in two main clusters, all mammalian specimens grouped in different sub-branches, and birds clustered in a second branch. These analyses suggest that the evolution of MyoD and MyoG was driven by different pathways.

  11. Assembly of the Lactuca sativa, L. cv. Tizian draft genome sequence reveals differences within major resistance complex 1 as compared to the cv. Salinas reference genome.

    PubMed

    Verwaaijen, Bart; Wibberg, Daniel; Nelkner, Johanna; Gordin, Miriam; Rupp, Oliver; Winkler, Anika; Bremges, Andreas; Blom, Jochen; Grosch, Rita; Pühler, Alfred; Schlüter, Andreas

    2018-02-10

    Lettuce (Lactuca sativa, L.) is an important annual plant of the family Asteraceae (Compositae). The commercial lettuce cultivar Tizian has been used in various scientific studies investigating the interaction of the plant with phytopathogens or biological control agents. Here, we present the de novo draft genome sequencing and gene prediction for this specific cultivar derived from transcriptome sequence data. The assembled scaffolds amount to a size of 2.22 Gb. Based on RNAseq data, 31,112 transcript isoforms were identified. Functional predictions for these transcripts were determined within the GenDBE annotation platform. Comparison with the cv. Salinas reference genome revealed a high degree of sequence similarity on genome and transcriptome levels, with an average amino acid identity of 99%. Furthermore, it was observed that two large regions are either missing or are highly divergent within the cv. Tizian genome compared to cv. Salinas. One of these regions covers the major resistance complex 1 region of cv. Salinas. The cv. Tizian draft genome sequence provides a valuable resource for future functional and transcriptome analyses focused on this lettuce cultivar. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Molecular mobility of nematic E7 confined to molecular sieves with a low filling degree.

    PubMed

    Brás, A R; Frunza, S; Guerreiro, L; Fonseca, I M; Corma, A; Frunza, L; Dionísio, M; Schönhals, A

    2010-06-14

    The nematic liquid crystalline mixture E7 was confined with similar filling degrees to molecular sieves with constant composition but different pore diameters (from 2.8 to 6.8 nm). Fourier transform infrared analysis proved that the E7 molecules interact via the cyanogroup with the pore walls of the molecular sieves. The molecular dynamics of the system was investigated by broadband dielectric spectroscopy (10(-2)-10(9) Hz) covering a wide temperature range of approximately 200 K from temperatures well above the isotropic-nematic transition down to the glass transition of bulk E7. A variety of relaxation processes is observed including two modes that are located close to the bulk behavior in its temperature dependence. For all confined samples, two relaxation processes, at frequencies lower than the processes observed for the bulk, were detected. At lower temperatures, their relaxation rates have different temperature dependencies whereas at higher temperatures, they seem to collapse into one chart. The temperature dependence of the slowest process (S-process) obeys the Vogel-Fulcher-Tammann law indicating a glassy dynamics of the E7 molecules anchored to the pore surface. The pore size dependence of both the Vogel temperature and fragility revealed a steplike transition around 4 nm pore size, which indicates a transition from a strong to a fragile behavior. The process with a relaxation rate in between the bulklike and the S-process (I-process) shows no dependence on the pore size. The agreement of the I-process with the behavior of a 5CB surface layer adsorbed on nonporous silica leads to the assignment of E7 molecules anchored at the outer surface of the microcrystals of the molecular sieves.

  13. 1E7-03, a low MW compound targeting host protein phosphatase-1, inhibits HIV-1 transcription

    PubMed Central

    Ammosova, Tatyana; Platonov, Maxim; Ivanov, Andrei; Kont, Yasemin Saygideğer; Kumari, Namita; Kehn-Hall, Kylene; Jerebtsova, Marina; Kulkarni, Amol A; Üren, Aykut; Kovalskyy, Dmytro; Nekhai, Sergei

    2014-01-01

    Background and Purpose HIV-1 transcription is activated by the Tat protein which recruits the cyclin-dependent kinase CDK9/cyclin T1 to TAR RNA. Tat binds to protein phosphatase-1 (PP1) through the Q35VCF38 sequence and translocates PP1 to the nucleus. PP1 dephosphorylates CDK9 and activates HIV-1 transcription. We have synthesized a low MW compound 1H4, that targets PP1 and prevents HIV-1 Tat interaction with PP1 and inhibits HIV-1 gene transcription. Here, we report our further work with the 1H4-derived compounds and analysis of their mechanism of action. Experimental Approach Using the 1H4-PP1 complex as a model, we iteratively designed and synthesized follow-up libraries that were analysed for the inhibition of HIV-1 transcription and toxicity. We also confirmed the mechanism of action of the PP1-targeting molecules by determining the affinity of binding of these molecules to PP1, by analysing their effects on PP1 activity, disruption of PP1 binding to Tat and shuttling of PP1 to the nucleus. Key Results We identified a tetrahydroquinoline derivative, compound 7, which disrupted the interaction of Tat with PP1. We further optimized compound 7 and obtained compound 7c, renamed 1E7-03, which inhibited HIV-1 with low IC50 (fivefold lower than the previously reported compound, 1H4), showed no cytotoxicity and displayed a plasma half-life greater than 8 h in mice. 1E7-03 bound to PP1 in vitro and prevented shuttling of PP1 into the nucleus. Conclusions and Implications Our study shows that low MW compounds that functionally mimic the PP1-binding RVxF peptide can inhibit HIV-1 transcription by deregulating PP1. PMID:25073485

  14. [Comparative studies of serological typing and HLA-A, B antigen genotyping with PCR using sequence-specific primers].

    PubMed

    Wu, Da-lin; Ling, Han-xin; Tang, Hao

    2004-11-01

    To evaluate the accuracy of PCR with sequence-specific primers (PCR-SSP) for HLA-I genotyping and analyze the causes of the errors occurring in the genotyping. DNA samples and were obtained from 34 clinical patients, and serological typing with monoclonal antibody (mAb) and HLA-A and, B antigen genotyping with PCR-SSP were performed. HLA-A and, B alleles were successfully typed in 34 clinical samples by mAb and PCR-SSP. No false positive or false negative results were found, and the erroneous and missed diagnosis rates were obviously higher in serological detection, being 23.5% for HLA-A and 26.5% for HLA-B. Error or confusion was more likely to occur in the antigens of A2 and A68, A32 and A33, B5, B60 and B61. DNA typing for HLA-I class (A, B antigens) by PCR-SSP has high resolution, high specificity, and good reproducibility, which is more suitable for clinical application than serological typing. PCR-SSP may accurately detect the alleles that are easily missed or mistaken in serological typing.

  15. Comparative RNA-Sequence Transcriptome Analysis of Phenolic Acid Metabolism in Salvia miltiorrhiza, a Traditional Chinese Medicine Model Plant

    PubMed Central

    Song, Zhenqiao; Guo, Linlin; Liu, Tian; Lin, Caicai; Wang, Jianhua

    2017-01-01

    Salvia miltiorrhiza Bunge is an important traditional Chinese medicine (TCM). In this study, two S. miltiorrhiza genotypes (BH18 and ZH23) with different phenolic acid concentrations were used for de novo RNA sequencing (RNA-seq). A total of 170,787 transcripts and 56,216 unigenes were obtained. There were 670 differentially expressed genes (DEGs) identified between BH18 and ZH23, 250 of which were upregulated in ZH23, with genes involved in the phenylpropanoid biosynthesis pathway being the most upregulated genes. Nine genes involved in the lignin biosynthesis pathway were upregulated in BH18 and thus result in higher lignin content in BH18. However, expression profiles of most genes involved in the core common upstream phenylpropanoid biosynthesis pathway were higher in ZH23 than that in BH18. These results indicated that genes involved in the core common upstream phenylpropanoid biosynthesis pathway might play an important role in downstream secondary metabolism and demonstrated that lignin biosynthesis was a putative partially competing pathway with phenolic acid biosynthesis. The results of this study expanded our understanding of the regulation of phenolic acid biosynthesis in S. miltiorrhiza. PMID:28194403

  16. Insights into the evolution of Darwin’s finches from comparative analysis of the Geospiza magnirostris genome sequence

    PubMed Central

    2013-01-01

    Background A classical example of repeated speciation coupled with ecological diversification is the evolution of 14 closely related species of Darwin’s (Galápagos) finches (Thraupidae, Passeriformes). Their adaptive radiation in the Galápagos archipelago took place in the last 2–3 million years and some of the molecular mechanisms that led to their diversification are now being elucidated. Here we report evolutionary analyses of genome of the large ground finch, Geospiza magnirostris. Results 13,291 protein-coding genes were predicted from a 991.0 Mb G. magnirostris genome assembly. We then defined gene orthology relationships and constructed whole genome alignments between the G. magnirostris and other vertebrate genomes. We estimate that 15% of genomic sequence is functionally constrained between G. magnirostris and zebra finch. Genic evolutionary rate comparisons indicate that similar selective pressures acted along the G. magnirostris and zebra finch lineages suggesting that historical effective population size values have been similar in both lineages. 21 otherwise highly conserved genes were identified that each show evidence for positive selection on amino acid changes in the Darwin's finch lineage. Two of these genes (Igf2r and Pou1f1) have been implicated in beak morphology changes in Darwin’s finches. Five of 47 genes showing evidence of positive selection in early passerine evolution have cilia related functions, and may be examples of adaptively evolving reproductive proteins. Conclusions These results provide insights into past evolutionary processes that have shaped G. magnirostris genes and its genome, and provide the necessary foundation upon which to build population genomics resources that will shed light on more contemporaneous adaptive and non-adaptive processes that have contributed to the evolution of the Darwin’s finches. PMID:23402223

  17. Cofactor-binding sites in proteins of deviating sequence: comparative analysis and clustering in torsion angle, cavity, and fold space.

    PubMed

    Stegemann, Björn; Klebe, Gerhard

    2012-02-01

    Small molecules are recognized in protein-binding pockets through surface-exposed physicochemical properties. To optimize binding, they have to adopt a conformation corresponding to a local energy minimum within the formed protein-ligand complex. However, their conformational flexibility makes them competent to bind not only to homologous proteins of the same family but also to proteins of remote similarity with respect to the shape of the binding pockets and folding pattern. Considering drug action, such observations can give rise to unexpected and undesired cross reactivity. In this study, datasets of six different cofactors (ADP, ATP, NAD(P)(H), FAD, and acetyl CoA, sharing an adenosine diphosphate moiety as common substructure), observed in multiple crystal structures of protein-cofactor complexes exhibiting sequence identity below 25%, have been analyzed for the conformational properties of the bound ligands, the distribution of physicochemical properties in the accommodating protein-binding pockets, and the local folding patterns next to the cofactor-binding site. State-of-the-art clustering techniques have been applied to group the different protein-cofactor complexes in the different spaces. Interestingly, clustering in cavity (Cavbase) and fold space (DALI) reveals virtually the same data structuring. Remarkable relationships can be found among the different spaces. They provide information on how conformations are conserved across the host proteins and which distinct local cavity and fold motifs recognize the different portions of the cofactors. In those cases, where different cofactors are found to be accommodated in a similar fashion to the same fold motifs, only a commonly shared substructure of the cofactors is used for the recognition process. Copyright © 2011 Wiley Periodicals, Inc.

  18. Human papillomavirus type 16 E6 and E7 oncoproteins act synergistically to cause head and neck cancer in mice.

    PubMed

    Jabbar, Sean; Strati, Katerina; Shin, Myeong Kyun; Pitot, Henry C; Lambert, Paul F

    2010-11-10

    High-risk human papillomaviruses (HPVs) contribute to cervical and other anogenital cancers, and they are also linked etiologically to a subset of head and neck squamous cell carcinomas (HNSCC). We previously established a model for HPV-associated HNSCC in which we treated transgenic mice expressing the papillomaviral oncoproteins with the chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO). We found that the HPV-16 E7 oncoprotein was highly potent in causing HNSCC, and its dominance masked any potential oncogenic contribution of E6, a second papillomaviral oncoprotein commonly expressed in human cancers. In the current study, we shortened the duration of treatment with 4-NQO to reduce the incidence of cancers and discovered a striking synergy between E6 and E7 in causing HNSCC. Comparing the oncogenic properties of wild-type versus mutant E6 genes in this model for HNSCC uncovered a role for some but not other cellular targets of E6 previously shown to contribute to cervical cancer. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. Human Papillomavirus Type 16 E6 and E7 Oncoproteins Act Synergistically to Cause Head and Neck Cancer in Mice

    PubMed Central

    Jabbar, Sean; Strati, Katerina; Shin, Myeong Kyun; Pitot, Henry C.; Lambert, Paul F.

    2010-01-01

    High-risk human papillomaviruses (HPVs) contribute to cervical and other anogenital cancers, and they are also linked etiologically to a subset of head and neck squamous cell carcinomas (HNSCC). We previously established a model for HPV-associated HNSCC in which we treated transgenic mice expressing the papillomaviral oncoproteins with the chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO). We found that the HPV-16 E7 oncoprotein was highly potent in causing HNSCC, and its dominance masked any potential oncogenic contribution of E6, a second papillomaviral oncoprotein commonly expressed in human cancers. In the current study, we shortened the duration of treatment with 4-NQO to reduce the incidence of cancers and discovered a striking synergy between E6 and E7 in causing HNSCC. Comparing the oncogenic properties of wild-type versus mutant E6 genes in this model for HNSCC uncovered a role for some but not other cellular targets of E6 previously shown to contribute to cervical cancer. PMID:20797753

  20. Kakusan4 and Aminosan: two programs for comparing nonpartitioned, proportional and separate models for combined molecular phylogenetic analyses of multilocus sequence data.

    PubMed

    Tanabe, Akifumi S

    2011-09-01

    Proportional and separate models able to apply different combination of substitution rate matrix (SRM) and among-site rate variation model (ASRVM) to each locus are frequently used in phylogenetic studies of multilocus data. A proportional model assumes that branch lengths are proportional among partitions and a separate model assumes that each partition has an independent set of branch lengths. However, the selection from among nonpartitioned (i.e., a common combination of models is applied to all-loci concatenated sequences), proportional and separate models is usually based on the researcher's preference rather than on any information criteria. This study describes two programs, 'Kakusan4' (for DNA sequences) and 'Aminosan' (for amino-acid sequences), which allow the selection of evolutionary models based on several types of information criteria. The programs can handle both multilocus and single-locus data, in addition to providing an easy-to-use wizard interface and a noninteractive command line interface. In the case of multilocus data, SRMs and ASRVMs are compared at each locus and at all-loci concatenated sequences, after which nonpartitioned, proportional and separate models are compared based on information criteria. The programs also provide model configuration files for mrbayes, paup*, phyml, raxml and Treefinder to support further phylogenetic analysis using a selected model. When likelihoods are optimized by Treefinder, the best-fit models were found to differ depending on the data set. Furthermore, differences in the information criteria among nonpartitioned, proportional and separate models were much larger than those among the nonpartitioned models. These findings suggest that selecting from nonpartitioned, proportional and separate models results in a better phylogenetic tree. Kakusan4 and Aminosan are available at http://www.fifthdimension.jp/. They are licensed under gnugpl Ver.2, and are able to run on Windows, MacOS X and Linux. © 2011

  1. ocsESTdb: a database of oil crop seed EST sequences for comparative analysis and investigation of a global metabolic network and oil accumulation metabolism.

    PubMed

    Ke, Tao; Yu, Jingyin; Dong, Caihua; Mao, Han; Hua, Wei; Liu, Shengyi

    2015-01-21

    Oil crop seeds are important sources of fatty acids (FAs) for human and animal nutrition. Despite their importance, there is a lack of an essential bioinformatics resource on gene transcription of oil crops from a comparative perspective. In this study, we developed ocsESTdb, the first database of expressed sequence tag (EST) information on seeds of four large-scale oil crops with an emphasis on global metabolic networks and oil accumulation metabolism that target the involved unigenes. A total of 248,522 ESTs and 106,835 unigenes were collected from the cDNA libraries of rapeseed (Brassica napus), soybean (Glycine max), sesame (Sesamum indicum) and peanut (Arachis hypogaea). These unigenes were annotated by a sequence similarity search against databases including TAIR, NR protein database, Gene Ontology, COG, Swiss-Prot, TrEMBL and Kyoto Encyclopedia of Genes and Genomes (KEGG). Five genome-scale metabolic networks that contain different numbers of metabolites and gene-enzyme reaction-association entries were analysed and constructed using Cytoscape and yEd programs. Details of unigene entries, deduced amino acid sequences and putative annotation are available from our database to browse, search and download. Intuitive and graphical representations of EST/unigene sequences, functional annotations, metabolic pathways and metabolic networks are also available. ocsESTdb will be updated regularly and can be freely accessed at http://ocri-genomics.org/ocsESTdb/ . ocsESTdb may serve as a valuable and unique resource for comparative analysis of acyl lipid synthesis and metabolism in oilseed plants. It also may provide vital insights into improving oil content in seeds of oil crop species by transcriptional reconstruction of the metabolic network.

  2. The human papillomavirus type 58 E7 oncoprotein modulates cell cycle regulatory proteins and abrogates cell cycle checkpoints

    SciT

    Zhang Weifang; Department of Microbiology, School of Medicine, Shandong University, Jinan, Shandong; Li Jing

    2010-02-05

    HPV type 58 (HPV-58) is the third most common HPV type in cervical cancer from Eastern Asia, yet little is known about how it promotes carcinogenesis. In this study, we demonstrate that HPV-58 E7 significantly promoted the proliferation and extended the lifespan of primary human keratinocytes (PHKs). HPV-58 E7 abrogated the G1 and the postmitotic checkpoints, although less efficiently than HPV-16 E7. Consistent with these observations, HPV-58 E7 down-regulated the cellular tumor suppressor pRb to a lesser extent than HPV-16 E7. Similar to HPV-16 E7 expressing PHKs, Cdk2 remained active in HPV-58 E7 expressing PHKs despite the presence of elevatedmore » levels of p53 and p21. Interestingly, HPV-58 E7 down-regulated p130 more efficiently than HPV-16 E7. Our study demonstrates a correlation between the ability of down-regulating pRb/p130 and abrogating cell cycle checkpoints by HPV-58 E7, which also correlates with the biological risks of cervical cancer progression associated with HPV-58 infection.« less

  3. Comparative analyses of simple sequence repeats (SSRs) in 23 mosquito species genomes: Identification, characterization and distribution (Diptera: Culicidae).

    PubMed

    Wang, Xiao-Ting; Zhang, Yu-Juan; Qiao, Liang; Chen, Bin

    2018-02-27

    Simple sequence repeats (SSRs) exist in both eukaryotic and prokaryotic genomes and are the most popular genetic markers, but the SSRs of mosquito genomes are still not well understood. In this study, we identified and analyzed the SSRs in 23 mosquito species using Drosophila melanogaster as reference at the whole-genome level. The results show that SSR numbers (33 076-560 175/genome) and genome sizes (574.57-1342.21 Mb) are significantly positively correlated (R 2 = 0.8992, P < 0.01), but the correlation in individual species varies in these mosquito species. In six types of SSR, mono- to trinucleotide SSRs are dominant with cumulative percentages of 95.14%-99.00% and densities of 195.65/Mb-787.51/Mb, whereas tetra- to hexanucleotide SSRs are rare with 1.12%-4.22% and 3.76/Mb-40.23/Mb. The (A/T)n, (AC/GT)n and (AGC/GCT)n are the most frequent motifs in mononucleotide, dinucleotide and trinucleotide SSRs, respectively, and the motif frequencies of tetra- to hexanucleotide SSRs appear to be species-specific. The 10-20 bp length of SSRs are dominant with the number of 110 561 ± 93 482 and the frequency of 87.25% ± 5.73% on average, and the number and frequency decline with the increase of length. Most SSRs (83.34% ± 7.72%) are located in intergenic regions, followed by intron regions (11.59% ± 5.59%), exon regions (3.74% ± 1.95%), and untranslated regions (1.32% ± 1.39%). The mono-, di- and trinucleotide SSRs are the main SSRs in both gene regions (98.55% ± 0.85%) and exon regions (99.27% ± 0.52%). An average of 42.52% of total genes contains SSRs, and the preference for SSR occurrence in different gene subcategories are species-specific. The study provides useful insights into the SSR diversity, characteristics and distribution in 23 mosquito species of genomes. © 2018 Institute of Zoology, Chinese Academy of Sciences.

  4. Comparative transcriptome analysis of the biocontrol strain Bacillus amyloliquefaciens FZB42 as response to biofilm formation analyzed by RNA sequencing.

    PubMed

    Kröber, Magdalena; Verwaaijen, Bart; Wibberg, Daniel; Winkler, Anika; Pühler, Alfred; Schlüter, Andreas

    2016-08-10

    The strain Bacillus amyloliquefaciens FZB42 is a plant growth promoting rhizobacterium (PGPR) and biocontrol agent known to keep infections of lettuce (Lactuca sativa) by the phytopathogen Rhizoctonia solani down. Several mechanisms, including the production of secondary metabolites possessing antimicrobial properties and induction of the host plant's systemic resistance (ISR), were proposed to explain the biocontrol effect of the strain. B. amyloliquefaciens FZB42 is able to form plaques (biofilm-like structures) on plant roots and this feature was discussed to be associated with its biocontrol properties. For this reason, formation of B. amyloliquefaciens biofilms was studied at the transcriptional level using high-throughput sequencing of whole transcriptome cDNA libraries from cells grown under biofilm-forming conditions vs. planktonic growth. Comparison of the transcriptional profiles of B. amyloliquefaciens FZB42 under these growth conditions revealed a common set of highly transcribed genes mostly associated with basic cellular functions. The lci gene, encoding an antimicrobial peptide (AMP), was among the most highly transcribed genes of cells under both growth conditions suggesting that AMP production may contribute to biocontrol. In contrast, gene clusters coding for synthesis of secondary metabolites with antimicrobial properties were only moderately transcribed and not induced in biofilm-forming cells. Differential gene expression revealed that 331 genes were significantly up-regulated and 230 genes were down-regulated in the transcriptome of B. amyloliquefaciens FZB42 under biofilm-forming conditions in comparison to planktonic cells. Among the most highly up-regulated genes, the yvqHI operon, coding for products involved in nisin (class I bacteriocin) resistance, was identified. In addition, an operon whose products play a role in fructosamine metabolism was enhanced in its transcription. Moreover, genes involved in the production of the extracellular

  5. Complete nucleotide sequence of the Cryptomeria japonica D. Don. chloroplast genome and comparative chloroplast genomics: diversified genomic structure of coniferous species.

    PubMed

    Hirao, Tomonori; Watanabe, Atsushi; Kurita, Manabu; Kondo, Teiji; Takata, Katsuhiko

    2008-06-23

    The recent determination of complete chloroplast (cp) genomic sequences of various plant species has enabled numerous comparative analyses as well as advances in plant and genome evolutionary studies. In angiosperms, the complete cp genome sequences of about 70 species have been determined, whereas those of only three gymnosperm species, Cycas taitungensis, Pinus thunbergii, and Pinus koraiensis have been established. The lack of information regarding the gene content and genomic structure of gymnosperm cp genomes may severely hamper further progress of plant and cp genome evolutionary studies. To address this need, we report here the complete nucleotide sequence of the cp genome of Cryptomeria japonica, the first in the Cupressaceae sensu lato of gymnosperms, and provide a comparative analysis of their gene content and genomic structure that illustrates the unique genomic features of gymnosperms. The C. japonica cp genome is 131,810 bp in length, with 112 single copy genes and two duplicated (trnI-CAU, trnQ-UUG) genes that give a total of 116 genes. Compared to other land plant cp genomes, the C. japonica cp has lost one of the relevant large inverted repeats (IRs) found in angiosperms, fern, liverwort, and gymnosperms, such as Cycas and Gingko, and additionally has completely lost its trnR-CCG, partially lost its trnT-GGU, and shows diversification of accD. The genomic structure of the C. japonica cp genome also differs significantly from those of other plant species. For example, we estimate that a minimum of 15 inversions would be required to transform the gene organization of the Pinus thunbergii cp genome into that of C. japonica. In the C. japonica cp genome, direct repeat and inverted repeat sequences are observed at the inversion and translocation endpoints, and these sequences may be associated with the genomic rearrangements. The observed differences in genomic structure between C. japonica and other land plants, including pines, strongly support the

  6. Regression of Established Human Papillomavirus Type 16 (HPV-16) Immortalized Tumors In Vivo by Vaccinia Viruses Expressing Different Forms of HPV-16 E7 Correlates with Enhanced CD8+ T-Cell Responses That Home to the Tumor Site

    PubMed Central

    Lamikanra, Abigail; Pan, Zhen-Kun; Isaacs, Stuart N.; Wu, Tzyy-Choou; Paterson, Yvonne

    2001-01-01

    Using vaccinia virus as a live vector, we show that the expression of human papillomavirus type 16 (HPV-16) E7 fused to a nonhemolytic portion of the Listeria monocytogenes virulence factor, listeriolysin O (LLO), induces an immune response that causes the regression of established HPV-16 immortalized tumors in C57BL/6 mice. The vaccinia virus construct expressing LLO fused to E7 (VacLLOE7) was compared with two previously described vaccinia virus constructs: one that expresses unmodified E7 (VacE7) and another that expresses E7 in a form designed to direct it to intracellular lysosomal compartments and improve major histocompatibility complex class II-restricted responses (VacSigE7LAMP-1). C57BL/6 mice bearing established HPV-16 immortalized tumors of 5 or 8 mm were treated with each of these vaccines. Fifty percent of the mice treated with VacLLOE7 remained tumor free 2 months after tumor inoculation, whereas 12 to 25% of the mice were tumor free after treatment with VacSigE7LAMP-1 (depending on the size of the tumor). No mice were tumor free in the group given VacE7. Compared to VacE7, VacSigE7LAMP-1 and VacLLOE7 resulted in increased numbers of H2-Db-specific tetramer-positive CD8+ T cells in mouse spleens that produced gamma interferon and tumor necrosis factor alpha upon stimulation with RAHYNIVTF peptide. In addition, the highest frequency of tetramer-positive T cells was seen in the tumor sites of mice treated with VacLLOE7. An increased efficiency of E7-specific lysis by splenocytes from mice immunized with VacLLOE7 was also observed. These results indicate that the fusion of E7 with LLO not only enhances antitumor therapy by improving the tumoricidal function of E7-specific CD8+ T cells but may also increase the number of antigen-specific CD8+ T cells in the tumor, the principle site of antigen expression. PMID:11559797

  7. Modulation of DNA methylation by human papillomavirus E6 and E7 oncoproteins in cervical cancer

    PubMed Central

    Sen, Prakriti; Ganguly, Pooja; Ganguly, Niladri

    2018-01-01

    Human papillomaviruses (HPVs) are double stranded circular DNA viruses that infect cutaneous and mucosal epithelial cells. Almost 99% of cervical cancer has a HPV infection. The early oncoproteins E6 and E7 are important in this cellular transformation process. Epigenetic mechanisms have long been known to result in decisive alterations in DNA, leading to alterations in DNA-protein interactions, alterations in chromatin structure and compaction and significant alterations in gene expression. The enzymes responsible for these epigenetic modifications are DNA methyl transferases (DNMTs), histone acetylases and deacetylases. Epigenetics has an important role in cancer development by modifying the cellular micro environment. In this review, the authors discuss the role of HPV oncoproteins E6 and E7 in modulating the epigenetic mechanisms inside the host cell. The oncoproteins induce the expression of DNMTs which lead to aberrant DNA methylations and disruption of the normal epigenetic processes. The E7 oncoprotein may additionally directly bind and induce methyl transferase activity of the enzyme. These modulations lead to altered gene expression levels, particularly the genes involved in apoptosis, cell cycle and cell adhesion. In addition, the present review discusses how epigenetic mechanisms may be targeted for possible therapeutic interventions for HPV mediated cervical cancer. PMID:29285184

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

    PubMed Central

    2009-01-01

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

  9. RNA Sequencing Reveals Differential Expression of Mitochondrial and Oxidation Reduction Genes in the Long-Lived Naked Mole-Rat When Compared to Mice

    PubMed Central

    Holmes, Andrew; Szafranski, Karol; Faulkes, Chris G.; Coen, Clive W.; Buffenstein, Rochelle; Platzer, Matthias; de Magalhães, João Pedro; Church, George M.

    2011-01-01

    The naked mole-rat (Heterocephalus glaber) is a long-lived, cancer resistant rodent and there is a great interest in identifying the adaptations responsible for these and other of its unique traits. We employed RNA sequencing to compare liver gene expression profiles between naked mole-rats and wild-derived mice. Our results indicate that genes associated with oxidoreduction and mitochondria were expressed at higher relative levels in naked mole-rats. The largest effect is nearly 300-fold higher expression of epithelial cell adhesion molecule (Epcam), a tumour-associated protein. Also of interest are the protease inhibitor, alpha2-macroglobulin (A2m), and the mitochondrial complex II subunit Sdhc, both ageing-related genes found strongly over-expressed in the naked mole-rat. These results hint at possible candidates for specifying species differences in ageing and cancer, and in particular suggest complex alterations in mitochondrial and oxidation reduction pathways in the naked mole-rat. Our differential gene expression analysis obviated the need for a reference naked mole-rat genome by employing a combination of Illumina/Solexa and 454 platforms for transcriptome sequencing and assembling transcriptome contigs of the non-sequenced species. Overall, our work provides new research foci and methods for studying the naked mole-rat's fascinating characteristics. PMID:22073188

  10. A Bioinformatics Approach for Integrated Transcriptomic and Proteomic Comparative Analyses of Model and Non-sequenced Anopheline Vectors of Human Malaria Parasites*

    PubMed Central

    Mohien, Ceereena Ubaida; Colquhoun, David R.; Mathias, Derrick K.; Gibbons, John G.; Armistead, Jennifer S.; Rodriguez, Maria C.; Rodriguez, Mario Henry; Edwards, Nathan J.; Hartler, Jürgen; Thallinger, Gerhard G.; Graham, David R.; Martinez-Barnetche, Jesus; Rokas, Antonis; Dinglasan, Rhoel R.

    2013-01-01

    Malaria morbidity and mortality caused by both Plasmodium falciparum and Plasmodium vivax extend well beyond the African continent, and although P. vivax causes between 80 and 300 million severe cases each year, vivax transmission remains poorly understood. Plasmodium parasites are transmitted by Anopheles mosquitoes, and the critical site of interaction between parasite and host is at the mosquito's luminal midgut brush border. Although the genome of the “model” African P. falciparum vector, Anopheles gambiae, has been sequenced, evolutionary divergence limits its utility as a reference across anophelines, especially non-sequenced P. vivax vectors such as Anopheles albimanus. Clearly, technologies and platforms that bridge this substantial scientific gap are required in order to provide public health scientists with key transcriptomic and proteomic information that could spur the development of novel interventions to combat this disease. To our knowledge, no approaches have been published that address this issue. To bolster our understanding of P. vivax–An. albimanus midgut interactions, we developed an integrated bioinformatic-hybrid RNA-Seq-LC-MS/MS approach involving An. albimanus transcriptome (15,764 contigs) and luminal midgut subproteome (9,445 proteins) assembly, which, when used with our custom Diptera protein database (685,078 sequences), facilitated a comparative proteomic analysis of the midgut brush borders of two important malaria vectors, An. gambiae and An. albimanus. PMID:23082028

  11. A bioinformatics approach for integrated transcriptomic and proteomic comparative analyses of model and non-sequenced anopheline vectors of human malaria parasites.

    PubMed

    Ubaida Mohien, Ceereena; Colquhoun, David R; Mathias, Derrick K; Gibbons, John G; Armistead, Jennifer S; Rodriguez, Maria C; Rodriguez, Mario Henry; Edwards, Nathan J; Hartler, Jürgen; Thallinger, Gerhard G; Graham, David R; Martinez-Barnetche, Jesus; Rokas, Antonis; Dinglasan, Rhoel R

    2013-01-01

    Malaria morbidity and mortality caused by both Plasmodium falciparum and Plasmodium vivax extend well beyond the African continent, and although P. vivax causes between 80 and 300 million severe cases each year, vivax transmission remains poorly understood. Plasmodium parasites are transmitted by Anopheles mosquitoes, and the critical site of interaction between parasite and host is at the mosquito's luminal midgut brush border. Although the genome of the "model" African P. falciparum vector, Anopheles gambiae, has been sequenced, evolutionary divergence limits its utility as a reference across anophelines, especially non-sequenced P. vivax vectors such as Anopheles albimanus. Clearly, technologies and platforms that bridge this substantial scientific gap are required in order to provide public health scientists with key transcriptomic and proteomic information that could spur the development of novel interventions to combat this disease. To our knowledge, no approaches have been published that address this issue. To bolster our understanding of P. vivax-An. albimanus midgut interactions, we developed an integrated bioinformatic-hybrid RNA-Seq-LC-MS/MS approach involving An. albimanus transcriptome (15,764 contigs) and luminal midgut subproteome (9,445 proteins) assembly, which, when used with our custom Diptera protein database (685,078 sequences), facilitated a comparative proteomic analysis of the midgut brush borders of two important malaria vectors, An. gambiae and An. albimanus.

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

    PubMed

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

    2009-10-12

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

  13. The Complete Chloroplast Genome Sequence of a Relict Conifer Glyptostrobus pensilis: Comparative Analysis and Insights into Dynamics of Chloroplast Genome Rearrangement in Cupressophytes and Pinaceae

    PubMed Central

    Zheng, Renhua; Xu, Haibin; Zhou, Yanwei; Li, Meiping; Lu, Fengjuan; Dong, Yini; Liu, Xin; Chen, Jinhui; Shi, Jisen

    2016-01-01

    Glyptostrobus pensilis, belonging to the monotypic genus Glyptostrobus (Family: Cupressaceae), is an ancient conifer that is naturally distributed in low-lying wet areas. Here, we report the complete chloroplast (cp) genome sequence (132,239 bp) of G. pensilis. The G. pensilis cp genome is similar in gene content, organization and genome structure to the sequenced cp genomes from other cupressophytes, especially with respect to the loss of the inverted repeat region A (IRA). Through phylogenetic analysis, we demonstrated that the genus Glyptostrobus is closely related to the genus Cryptomeria, supporting previous findings based on physiological characteristics. Since IRs play an important role in stabilize cp genome and conifer cp genomes lost different IR regions after splitting in two clades (cupressophytes and Pinaceae), we performed cp genome rearrangement analysis and found more extensive cp genome rearrangements among the species of cupressophytes relative to Pinaceae. Additional repeat analysis indicated that cupressophytes cp genomes contained less potential functional repeats, especially in Cupressaceae, compared with Pinaceae. These results suggested that dynamics of cp genome rearrangement in conifers differed since the two clades, Pinaceae and cupressophytes, lost IR copies independently and developed different repeats to complement the residual IRs. In addition, we identified 170 perfect simple sequence repeats that will be useful in future research focusing on the evolution of genetic diversity and conservation of genetic variation for this endangered species in the wild. PMID:27560965

  14. Complete genome sequence and comparative analysis of Acetobacter pasteurianus 386B, a strain well-adapted to the cocoa bean fermentation ecosystem.

    PubMed

    Illeghems, Koen; De Vuyst, Luc; Weckx, Stefan

    2013-08-01

    Acetobacter pasteurianus 386B, an acetic acid bacterium originating from a spontaneous cocoa bean heap fermentation, proved to be an ideal functional starter culture for coca bean fermentations. It is able to dominate the fermentation process, thereby resisting high acetic acid concentrations and temperatures. However, the molecular mechanisms underlying its metabolic capabilities and niche adaptations are unknown. In this study, whole-genome sequencing and comparative genome analysis was used to investigate this strain's mechanisms to dominate the cocoa bean fermentation process. The genome sequence of A. pasteurianus 386B is composed of a 2.8-Mb chromosome and seven plasmids. The annotation of 2875 protein-coding sequences revealed important characteristics, including several metabolic pathways, the occurrence of strain-specific genes such as an endopolygalacturonase, and the presence of mechanisms involved in tolerance towards various stress conditions. Furthermore, the low number of transposases in the genome and the absence of complete phage genomes indicate that this strain might be more genetically stable compared with other A. pasteurianus strains, which is an important advantage for the use of this strain as a functional starter culture. Comparative genome analysis with other members of the Acetobacteraceae confirmed the functional properties of A. pasteurianus 386B, such as its thermotolerant nature and unique genetic composition. Genome analysis of A. pasteurianus 386B provided detailed insights into the underlying mechanisms of its metabolic features, niche adaptations, and tolerance towards stress conditions. Combination of these data with previous experimental knowledge enabled an integrated, global overview of the functional characteristics of this strain. This knowledge will enable improved fermentation strategies and selection of appropriate acetic acid bacteria strains as functional starter culture for cocoa bean fermentation processes.

  15. Comparative and genetic analysis of the four sequenced Paenibacillus polymyxa genomes reveals a diverse metabolism and conservation of genes relevant to plant-growth promotion and competitiveness.

    PubMed

    Eastman, Alexander W; Heinrichs, David E; Yuan, Ze-Chun

    2014-10-03

    Members of the genus Paenibacillus are important plant growth-promoting rhizobacteria that can serve as bio-reactors. Paenibacillus polymyxa promotes the growth of a variety of economically important crops. Our lab recently completed the genome sequence of Paenibacillus polymyxa CR1. As of January 2014, four P. polymyxa genomes have been completely sequenced but no comparative genomic analyses have been reported. Here we report the comparative and genetic analyses of four sequenced P. polymyxa genomes, which revealed a significantly conserved core genome. Complex metabolic pathways and regulatory networks were highly conserved and allow P. polymyxa to rapidly respond to dynamic environmental cues. Genes responsible for phytohormone synthesis, phosphate solubilization, iron acquisition, transcriptional regulation, σ-factors, stress responses, transporters and biomass degradation were well conserved, indicating an intimate association with plant hosts and the rhizosphere niche. In addition, genes responsible for antimicrobial resistance and non-ribosomal peptide/polyketide synthesis are present in both the core and accessory genome of each strain. Comparative analyses also reveal variations in the accessory genome, including large plasmids present in strains M1 and SC2. Furthermore, a considerable number of strain-specific genes and genomic islands are irregularly distributed throughout each genome. Although a variety of plant-growth promoting traits are encoded by all strains, only P. polymyxa CR1 encodes the unique nitrogen fixation cluster found in other Paenibacillus sp. Our study revealed that genomic loci relevant to host interaction and ecological fitness are highly conserved within the P. polymyxa genomes analysed, despite variations in the accessory genome. This work suggets that plant-growth promotion by P. polymyxa is mediated largely through phytohormone production, increased nutrient availability and bio-control mechanisms. This study provides an in

  16. Whole-Genome Sequencing and Comparative Analysis of Mycobacterium brisbanense Reveals a Possible Soil Origin and Capability in Fertiliser Synthesis.

    PubMed

    Wee, Wei Yee; Tan, Tze King; Jakubovics, Nicholas S; Choo, Siew Woh

    2016-01-01

    Mycobacterium brisbanense is a member of Mycobacterium fortuitum third biovariant complex, which includes rapidly growing Mycobacterium spp. that normally inhabit soil, dust and water, and can sometimes cause respiratory tract infections in humans. We present the first whole-genome analysis of M. brisbanense UM_WWY which was isolated from a 70-year-old Malaysian patient. Molecular phylogenetic analyses confirmed the identification of this strain as M. brisbanense and showed that it has an unusually large genome compared with related mycobacteria. The large genome size of M. brisbanense UM_WWY (~7.7Mbp) is consistent with further findings that this strain has a highly variable genome structure that contains many putative horizontally transferred genomic islands and prophage. Comparative analysis showed that M. brisbanense UM_WWY is the only Mycobacterium species that possesses a complete set of genes encoding enzymes involved in the urea cycle, suggesting that this soil bacterium is able to synthesize urea for use as plant fertilizers. It is likely that M. brisbanense UM_WWY is adapted to live in soil as its primary habitat since the genome contains many genes associated with nitrogen metabolism. Nevertheless, a large number of predicted virulence genes were identified in M. brisbanense UM_WWY that are mostly shared with well-studied mycobacterial pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. These findings are consistent with the role of M. brisbanense as an opportunistic pathogen of humans. The whole-genome study of UM_WWY has provided the basis for future work of M. brisbanense.

  17. Comparative genomic analysis of single-molecule sequencing and hybrid approaches for finishing the Clostridium autoethanogenum JA1-1 strain DSM 10061 genome

    SciT

    Brown, Steven D; Nagaraju, Shilpa; Utturkar, Sagar M

    Background Clostridium autoethanogenum strain JA1-1 (DSM 10061) is an acetogen capable of fermenting CO, CO2 and H2 (e.g. from syngas or waste gases) into biofuel ethanol and commodity chemicals such as 2,3-butanediol. A draft genome sequence consisting of 100 contigs has been published. Results A closed, high-quality genome sequence for C. autoethanogenum DSM10061 was generated using only the latest single-molecule DNA sequencing technology and without the need for manual finishing. It is assigned to the most complex genome classification based upon genome features such as repeats, prophage, nine copies of the rRNA gene operons. It has a low G +more » C content of 31.1%. Illumina, 454, Illumina/454 hybrid assemblies were generated and then compared to the draft and PacBio assemblies using summary statistics, CGAL, QUAST and REAPR bioinformatics tools and comparative genomic approaches. Assemblies based upon shorter read DNA technologies were confounded by the large number repeats and their size, which in the case of the rRNA gene operons were ~5 kb. CRISPR (Clustered Regularly Interspaced Short Paloindromic Repeats) systems among biotechnologically relevant Clostridia were classified and related to plasmid content and prophages. Potential associations between plasmid content and CRISPR systems may have implications for historical industrial scale Acetone-Butanol-Ethanol (ABE) fermentation failures and future large scale bacterial fermentations. While C. autoethanogenum contains an active CRISPR system, no such system is present in the closely related Clostridium ljungdahlii DSM 13528. A common prophage inserted into the Arg-tRNA shared between the strains suggests a common ancestor. However, C. ljungdahlii contains several additional putative prophages and it has more than double the amount of prophage DNA compared to C. autoethanogenum. Other differences include important metabolic genes for central metabolism (as an additional hydrogenase and the absence of a

  18. Comparative venom gland transcriptomics of Naja kaouthia (monocled cobra) from Malaysia and Thailand: elucidating geographical venom variation and insights into sequence novelty

    PubMed Central

    Chanhome, Lawan; Tan, Nget Hong

    2017-01-01

    Background The monocled cobra (Naja kaouthia) is a medically important venomous snake in Southeast Asia. Its venom has been shown to vary geographically in relation to venom composition and neurotoxic activity, indicating vast diversity of the toxin genes within the species. To investigate the polygenic trait of the venom and its locale-specific variation, we profiled and compared the venom gland transcriptomes of N. kaouthia from Malaysia (NK-M) and Thailand (NK-T) applying next-generation sequencing (NGS) technology. Methods The transcriptomes were sequenced on the Illumina HiSeq platform, assembled and followed by transcript clustering and annotations for gene expression and function. Pairwise or multiple sequence alignments were conducted on the toxin genes expressed. Substitution rates were studied for the major toxins co-expressed in NK-M and NK-T. Results and discussion The toxin transcripts showed high redundancy (41–82% of the total mRNA expression) and comprised 23 gene families expressed in NK-M and NK-T, respectively (22 gene families were co-expressed). Among the venom genes, three-finger toxins (3FTxs) predominated in the expression, with multiple sequences noted. Comparative analysis and selection study revealed that 3FTxs are genetically conserved between the geographical specimens whilst demonstrating distinct differential expression patterns, implying gene up-regulation for selected principal toxins, or alternatively, enhanced transcript degradation or lack of transcription of certain traits. One of the striking features that elucidates the inter-geographical venom variation is the up-regulation of α-neurotoxins (constitutes ∼80.0% of toxin’s fragments per kilobase of exon model per million mapped reads (FPKM)), particularly the long-chain α-elapitoxin-Nk2a (48.3%) in NK-T but only 1.7% was noted in NK-M. Instead, short neurotoxin isoforms were up-regulated in NK-M (46.4%). Another distinct transcriptional pattern observed is the

  19. Magnetic resonance imaging of the sacroiliac joints in the early detection of spondyloarthritis: no added value of gadolinium compared with short tau inversion recovery sequence.

    PubMed

    de Hooge, Manouk; van den Berg, Rosaline; Navarro-Compán, Victoria; van Gaalen, Floris; van der Heijde, Désirée; Huizinga, Tom; Reijnierse, Monique

    2013-07-01

    To investigate the additional value of T1 fat-saturated after gadolinium (T1/Gd) compared with T1 and short tau inversion recovery (STIR) sequence in detecting active lesions of the SI joints typical of axial SpA (axSpA) in a prospective cohort study, the SpondyloArthritis Caught Early (SPACE) cohort, and to assess its influence on final MRI diagnosis of the SI joint (MRI-SIJ) based on the Assessment of Spondyloarthritis International Society (ASAS) definition of active sacroiliitis. Patients in the SPACE cohort received baseline and 3-month follow-up MRI-SIJ with coronal oblique T1, STIR and T1/Gd sequences. Bone marrow oedema (BME), capsulitis/enthesitis and synovitis and active sacroiliitis according to the ASAS definition were evaluated by three blinded readers. A total of 127 patients received an MRI-SIJ at baseline and 67 patients also received an MRI-SIJ at 3 months follow-up since the Gd protocol was added some months after the start of the SPACE project. Twenty-five of the 127 patients (19.7%) with a baseline MRI-SIJ and 14 of 67 patients (20.6%) with a follow-up MRI-SIJ presented BME on the STIR sequence sufficient to fulfill the ASAS definition for a positive MRI-SIJ. In eight patients, additional synovitis and/or capsulitis/enthesitis was observed; however, no additional BME was visualized on T1/Gd. One patient, without clinical diagnosis of axSpA, showed synovitis as an isolated finding. Synovitis and capsulitis/enthesitis are detectable with the administration of Gd. However, they are always observed in the presence of BME. Therefore T1 and STIR sequence alone are sufficient in the MRI assessment that, among others, is used for diagnosing patients with early axSpA.

  20. The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells.

    PubMed Central

    Halbert, C L; Demers, G W; Galloway, D A

    1992-01-01

    Previous studies have shown that the E7 gene of human papillomavirus (HPV) type 16 or 18 alone was sufficient for immortalization of human foreskin epithelial cells (HFE) and that the efficiency was increased in cooperation with the respective E6 gene, whereas the HPV6 E6 or E7 gene was not active in HFE. To detect weak immortalizing activities of the HPV6 genes, cells were infected with recombinant retroviruses containing HPV genes, alone and in homologous and heterologous combinations. The HPV6 genes, alone or together (HPV6 E6 plus HPV6 E7), were not able to immortalize cells. However the HPV6 E6 gene, in concert with HPV16 E7, increased the frequency of immortalization threefold over that obtained with HPV16 E7 alone. Interestingly, 6 of 20 clones containing the HPV16 E6 gene and the HPV6 E7 gene were immortalized, whereas neither gene alone was sufficient. Thus, the HPV6 E6 and E7 genes have weak immortalizing activities which can be detected in cooperation with the more active transforming genes of HPV16. Acute expression of the HPV6 and HPV16 E6 and E7 genes revealed that only HPV16 E7 was able to stimulate the proliferation of cells in organotypic culture, resulting in increased expression of the proliferative cell nuclear antigen and the formation of a disorganized epithelial layer. Additionally, combinations of genes that immortalized HFE cells (HPV16 E6 plus HPV16 E7, HPV16 E6 plus HPV6 E7, and HPV6 E6 plus HPV16 E7) also stimulated proliferation. Images PMID:1312623

  1. Local motion compensation in image sequences degraded by atmospheric turbulence: a comparative analysis of optical flow vs. block matching methods

    NASA Astrophysics Data System (ADS)

    Huebner, Claudia S.

    2016-10-01

    As a consequence of fluctuations in the index of refraction of the air, atmospheric turbulence causes scintillation, spatial and temporal blurring as well as global and local image motion creating geometric distortions. To mitigate these effects many different methods have been proposed. Global as well as local motion compensation in some form or other constitutes an integral part of many software-based approaches. For the estimation of motion vectors between consecutive frames simple methods like block matching are preferable to more complex algorithms like optical flow, at least when challenged with near real-time requirements. However, the processing power of commercially available computers continues to increase rapidly and the more powerful optical flow methods have the potential to outperform standard block matching methods. Therefore, in this paper three standard optical flow algorithms, namely Horn-Schunck (HS), Lucas-Kanade (LK) and Farnebäck (FB), are tested for their suitability to be employed for local motion compensation as part of a turbulence mitigation system. Their qualitative performance is evaluated and compared with that of three standard block matching methods, namely Exhaustive Search (ES), Adaptive Rood Pattern Search (ARPS) and Correlation based Search (CS).

  2. Whole genome sequencing and comparative genomics of closely related Fusarium Head Blight fungi: Fusarium graminearum, F. meridionale and F. asiaticum.

    PubMed

    Walkowiak, Sean; Rowland, Owen; Rodrigue, Nicolas; Subramaniam, Rajagopal

    2016-12-09

    The Fusarium graminearum species complex is composed of many distinct fungal species that cause several diseases in economically important crops, including Fusarium Head Blight of wheat. Despite being closely related, these species and individuals within species have distinct phenotypic differences in toxin production and pathogenicity, with some isolates reported as non-pathogenic on certain hosts. In this report, we compare genomes and gene content of six new isolates from the species complex, including the first available genomes of F. asiaticum and F. meridionale, with four other genomes reported in previous studies. A comparison of genome structure and gene content revealed a 93-99% overlap across all ten genomes. We identified more than 700 k base pairs (kb) of single nucleotide polymorphisms (SNPs), insertions, and deletions (indels) within common regions of the genome, which validated the species and genetic populations reported within species. We constructed a non-redundant pan gene list containing 15,297 genes from the ten genomes and among them 1827 genes or 12% were absent in at least one genome. These genes were co-localized in telomeric regions and select regions within chromosomes with a corresponding increase in SNPs and indels. Many are also predicted to encode for proteins involved in secondary metabolism and other functions associated with disease. Genes that were common between isolates contained high levels of nucleotide variation and may be pseudogenes, allelic, or under diversifying selection. The genomic resources we have contributed will be useful for the identification of genes that contribute to the phenotypic variation and niche specialization that have been reported among members of the F. graminearum species complex.

  3. Comparative Analyses by Sequencing of Transcriptomes during Skeletal Muscle Development between Pig Breeds Differing in Muscle Growth Rate and Fatness

    PubMed Central

    Li, Anning; Gong, Wen; Xiao, Shuqi; Zhang, Yue; Qin, Limei; Niu, Yuna; Guo, Yunxue; Liu, Xiaohong; Cong, Peiqing; He, Zuyong; Wang, Chong; Li, Jiaqi; Chen, Yaosheng

    2011-01-01

    Understanding the dynamics of muscle transcriptome during development and between breeds differing in muscle growth is necessary to uncover the complex mechanism underlying muscle development. Herein, we present the first transcriptome-wide longissimus dorsi muscle development research concerning Lantang (LT, obese) and Landrace (LR, lean) pig breeds during 10 time-points from 35 days-post-coitus (dpc) to 180 days-post-natum (dpn) using Solexa/Illumina's Genome Analyzer. The data demonstrated that myogenesis was almost completed before 77 dpc, but the muscle phenotypes were still changed from 77 dpc to 28 dpn. Comparative analysis of the two breeds suggested that myogenesis started earlier but progressed more slowly in LT than in LR, the stages ranging from 49 dpc to 77 dpc are critical for formation of different muscle phenotypes. 595 differentially expressed myogenesis genes were identified, and their roles in myogenesis were discussed. Furthermore, GSK3B, IKBKB, ACVR1, ITGA and STMN1 might contribute to later myogenesis and more muscle fibers in LR than LT. Some myogenesis inhibitors (ID1, ID2, CABIN1, MSTN, SMAD4, CTNNA1, NOTCH2, GPC3 and HMOX1) were higher expressed in LT than in LR, which might contribute to more slow muscle differentiation in LT than in LR. We also identified several genes which might contribute to intramuscular adipose differentiation. Most important, we further proposed a novel model in which MyoD and MEF2A controls the balance between intramuscular adipogenesis and myogenesis by regulating CEBP family; Myf5 and MEF2C are essential during the whole myogenesis process while MEF2D affects muscle growth and maturation. The MRFs and MEF2 families are also critical for the phenotypic differences between the two pig breeds. Overall, this study contributes to elucidating the mechanism underlying muscle development, which could provide valuable information for pig meat quality improvement. The raw data have been submitted to Gene Expression

  4. Comparative analyses by sequencing of transcriptomes during skeletal muscle development between pig breeds differing in muscle growth rate and fatness.

    PubMed

    Zhao, Xiao; Mo, Delin; Li, Anning; Gong, Wen; Xiao, Shuqi; Zhang, Yue; Qin, Limei; Niu, Yuna; Guo, Yunxue; Liu, Xiaohong; Cong, Peiqing; He, Zuyong; Wang, Chong; Li, Jiaqi; Chen, Yaosheng

    2011-01-01

    Understanding the dynamics of muscle transcriptome during development and between breeds differing in muscle growth is necessary to uncover the complex mechanism underlying muscle development. Herein, we present the first transcriptome-wide longissimus dorsi muscle development research concerning Lantang (LT, obese) and Landrace (LR, lean) pig breeds during 10 time-points from 35 days-post-coitus (dpc) to 180 days-post-natum (dpn) using Solexa/Illumina's Genome Analyzer. The data demonstrated that myogenesis was almost completed before 77 dpc, but the muscle phenotypes were still changed from 77 dpc to 28 dpn. Comparative analysis of the two breeds suggested that myogenesis started earlier but progressed more slowly in LT than in LR, the stages ranging from 49 dpc to 77 dpc are critical for formation of different muscle phenotypes. 595 differentially expressed myogenesis genes were identified, and their roles in myogenesis were discussed. Furthermore, GSK3B, IKBKB, ACVR1, ITGA and STMN1 might contribute to later myogenesis and more muscle fibers in LR than LT. Some myogenesis inhibitors (ID1, ID2, CABIN1, MSTN, SMAD4, CTNNA1, NOTCH2, GPC3 and HMOX1) were higher expressed in LT than in LR, which might contribute to more slow muscle differentiation in LT than in LR. We also identified several genes which might contribute to intramuscular adipose differentiation. Most important, we further proposed a novel model in which MyoD and MEF2A controls the balance between intramuscular adipogenesis and myogenesis by regulating CEBP family; Myf5 and MEF2C are essential during the whole myogenesis process while MEF2D affects muscle growth and maturation. The MRFs and MEF2 families are also critical for the phenotypic differences between the two pig breeds. Overall, this study contributes to elucidating the mechanism underlying muscle development, which could provide valuable information for pig meat quality improvement. The raw data have been submitted to Gene Expression

  5. The expression of miR-21 and miR-143 is deregulated by the HPV16 E7 oncoprotein and 17β-estradiol.

    PubMed

    Gómez-Gómez, Yazmín; Organista-Nava, Jorge; Ocadiz-Delgado, Rodolfo; García-Villa, Enrique; Leyva-Vazquez, Marco Antonio; Illades-Aguiar, Berenice; Lambert, Paul F; García-Carrancá, Alejandro; Gariglio, Patricio

    2016-08-01

    MicroRNAs (miRNAs) are a class of non-coding RNAs that negatively regulate their target mRNAs at a posttranscriptional level, thereby affecting crucial processes in cancer development. However, little is known about the molecular events that control expression of miRNAs in cervical cancer (CC). HPV16 E7 oncoprotein in conjunction with estrogen are sufficient to produce high grade cervical dysplasia and invasive cervical malignancies in a mouse model. In the present study, we determined the potential role that the E7 oncoprotein and 17β-estradiol (E2) play in the deregulation of miR-21 and miR-143 expression levels by these two risk factors. We found that, while the expression of miR-21 was upregulated and the expression of miR-143 was downregulated by the HPV16 E7 oncoprotein in vivo, and in vitro and that E2 treatment is also implicated in the deregulation of these important miRNAs in vivo. Sustained upregulation of miR-21 resulted in suppression of PTEN expression, and repression of miR-143 increased the mRNA and protein levels from Bcl-2. These results suggested that HPV type 16 E7 oncoprotein and E2 play an important role in regulating miR-21 and miR-143 expression. We have observed similar results in CC patients containing HPV16 sequences, suggesting that these miRNAs could serve as diagnostic biomarkers in CC. The present study highlights the roles of miRNAs in cervical tissue and implicates these important molecules in cervical carcinogenesis.

  6. Characterization, sequencing and comparative genomic analysis of vB_AbaM-IME-AB2, a novel lytic bacteriophage that infects multidrug-resistant Acinetobacter baumannii clinical isolates.

    PubMed

    Peng, Fan; Mi, Zhiqiang; Huang, Yong; Yuan, Xin; Niu, Wenkai; Wang, Yahui; Hua, Yuhui; Fan, Huahao; Bai, Changqing; Tong, Yigang

    2014-07-05

    With the use of broad-spectrum antibiotics, immunosuppressive drugs, and glucocorticoids, multidrug-resistant Acinetobacter baumannii (MDR-AB) has become a major nosocomial pathogen species. The recent renaissance of bacteriophage therapy may provide new treatment strategies for combatting drug-resistant bacterial infections. In this study, we isolated a lytic bacteriophage vB_AbaM-IME-AB2 has a short latent period and a small burst size, which clear its host's suspension quickly, was selected for characterization and a complete genomic comparative study. The isolated bacteriophage vB_AbaM-IME-AB2 has an icosahedral head and displays morphology resembling Myoviridae family. Gel separation assays showed that the phage particle contains at least nine protein bands with molecular weights ranging 15-100 kDa. vB_AbaM-IME-AB2 could adsorb its host cells in 9 min with an adsorption rate more than 99% and showed a short latent period (20 min) and a small burst size (62 pfu/cell). It could form clear plaques in the double-layer assay and clear its host's suspension in just 4 hours. Whole genome of vB_AbaM-IME-AB2 was sequenced and annotated and the results showed that its genome is a double-stranded DNA molecule consisting of 43,665 nucleotides. The genome has a G + C content of 37.5% and 82 putative coding sequences (CDSs). We compared the characteristics and complete genome sequence of all known Acinetobacter baumannii bacteriophages. There are only three that have been sequenced Acinetobacter baumannii phages AB1, AP22, and phiAC-1, which have a relatively high similarity and own a coverage of 65%, 50%, 8% respectively when compared with our phage vB_AbaM-IME-AB2. A nucleotide alignment of the four Acinetobacter baumannii phages showed that some CDSs are similar, with no significant rearrangements observed. Yet some sections of these strains of phage are nonhomologous. vB_AbaM-IME-AB2 was a novel and unique A. baumannii bacteriophage. These findings suggest a common

  7. Colorimetric and dynamic light scattering detection of DNA sequences by using positively charged gold nanospheres: a comparative study with gold nanorods

    NASA Astrophysics Data System (ADS)

    Pylaev, T. E.; Khanadeev, V. A.; Khlebtsov, B. N.; Dykman, L. A.; Bogatyrev, V. A.; Khlebtsov, N. G.

    2011-07-01

    We introduce a new genosensing approach employing CTAB (cetyltrimethylammonium bromide)-coated positively charged colloidal gold nanoparticles (GNPs) to detect target DNA sequences by using absorption spectroscopy and dynamic light scattering. The approach is compared with a previously reported method employing unmodified CTAB-coated gold nanorods (GNRs). Both approaches are based on the observation that whereas the addition of probe and target ssDNA to CTAB-coated particles results in particle aggregation, no aggregation is observed after addition of probe and nontarget DNA sequences. Our goal was to compare the feasibility and sensitivity of both methods. A 21-mer ssDNA from the human immunodeficiency virus type 1 HIV-1 U5 long terminal repeat (LTR) sequence and a 23-mer ssDNA from the Bacillus anthracis cryptic protein and protective antigen precursor (pagA) genes were used as ssDNA models. In the case of GNRs, unexpectedly, the colorimetric test failed with perfect cigar-like particles but could be performed with dumbbell and dog-bone rods. By contrast, our approach with cationic CTAB-coated GNPs is easy to implement and possesses excellent feasibility with retention of comparable sensitivity—a 0.1 nM concentration of target cDNA can be detected with the naked eye and 10 pM by dynamic light scattering (DLS) measurements. The specificity of our method is illustrated by successful DLS detection of one-three base mismatches in cDNA sequences for both DNA models. These results suggest that the cationic GNPs and DLS can be used for genosensing under optimal DNA hybridization conditions without any chemical modifications of the particle surface with ssDNA molecules and signal amplification. Finally, we discuss a more than two-three-order difference in the reported estimations of the detection sensitivity of colorimetric methods (0.1 to 10-100 pM) to show that the existing aggregation models are inconsistent with the detection limits of about 0.1-1 pM DNA and that

  8. The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli

    PubMed Central

    2011-01-01

    Background Escherichia coli is a model prokaryote, an important pathogen, and a key organism for industrial biotechnology. E. coli W (ATCC 9637), one of four strains designated as safe for laboratory purposes, has not been sequenced. E. coli W is a fast-growing strain and is the only safe strain that can utilize sucrose as a carbon source. Lifecycle analysis has demonstrated that sucrose from sugarcane is a preferred carbon source for industrial bioprocesses. Results We have sequenced and annotated the genome of E. coli W. The chromosome is 4,900,968 bp and encodes 4,764 ORFs. Two plasmids, pRK1 (102,536 bp) and pRK2 (5,360 bp), are also present. W has unique features relative to other sequenced laboratory strains (K-12, B and Crooks): it has a larger genome and belongs to phylogroup B1 rather than A. W also grows on a much broader range of carbon sources than does K-12. A genome-scale reconstruction was developed and validated in order to interrogate metabolic properties. Conclusions The genome of W is more similar to commensal and pathogenic B1 strains than phylogroup A strains, and therefore has greater utility for comparative analyses with these strains. W should therefore be the strain of choice, or 'type strain' for group B1 comparative analyses. The genome annotation and tools created here are expected to allow further utilization and development of E. coli W as an industrial organism for sucrose-based bioprocesses. Refinements in our E. coli metabolic reconstruction allow it to more accurately define E. coli metabolism relative to previous models. PMID:21208457

  9. Global MLST of Salmonella Typhi Revisited in Post-genomic Era: Genetic Conservation, Population Structure, and Comparative Genomics of Rare Sequence Types.

    PubMed

    Yap, Kien-Pong; Ho, Wing S; Gan, Han M; Chai, Lay C; Thong, Kwai L

    2016-01-01

    Typhoid fever, caused by Salmonella enterica serovar Typhi, remains an important public health burden in Southeast Asia and other endemic countries. Various genotyping methods have been applied to study the genetic variations of this human-restricted pathogen. Multilocus sequence typing (MLST) is one of the widely accepted methods, and recently, there is a growing interest in the re-application of MLST in the post-genomic era. In this study, we provide the global MLST distribution of S. Typhi utilizing both publicly available 1,826 S. Typhi genome sequences in addition to performing conventional MLST on S. Typhi strains isolated from various endemic regions spanning over a century. Our global MLST analysis confirms the predominance of two sequence types (ST1 and ST2) co-existing in the endemic regions. Interestingly, S. Typhi strains with ST8 are currently confined within the African continent. Comparative genomic analyses of ST8 and other rare STs with genomes of ST1/ST2 revealed unique mutations in important virulence genes such as flhB, sipC, and tviD that may explain the variations that differentiate between seemingly successful (widespread) and unsuccessful (poor dissemination) S. Typhi populations. Large scale whole-genome phylogeny demonstrated evidence of phylogeographical structuring and showed that ST8 may have diverged from the earlier ancestral population of ST1 and ST2, which later lost some of its fitness advantages, leading to poor worldwide dissemination. In response to the unprecedented increase in genomic data, this study demonstrates and highlights the utility of large-scale genome-based MLST as a quick and effective approach to narrow the scope of in-depth comparative genomic analysis and consequently provide new insights into the fine scale of pathogen evolution and population structure.

  10. Comparative Genomics of Completely Sequenced Lactobacillus helveticus Genomes Provides Insights into Strain-Specific Genes and Resolves Metagenomics Data Down to the Strain Level.

    PubMed

    Schmid, Michael; Muri, Jonathan; Melidis, Damianos; Varadarajan, Adithi R; Somerville, Vincent; Wicki, Adrian; Moser, Aline; Bourqui, Marc; Wenzel, Claudia; Eugster-Meier, Elisabeth; Frey, Juerg E; Irmler, Stefan; Ahrens, Christian H

    2018-01-01

    Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus -to our knowledge-identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus . Notably, the functional Clusters of Orthologous Groups of proteins categories "cell wall/membrane biogenesis" and "defense mechanisms" were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be

  11. Comparative Genomics of Completely Sequenced Lactobacillus helveticus Genomes Provides Insights into Strain-Specific Genes and Resolves Metagenomics Data Down to the Strain Level

    PubMed Central

    Schmid, Michael; Muri, Jonathan; Melidis, Damianos; Varadarajan, Adithi R.; Somerville, Vincent; Wicki, Adrian; Moser, Aline; Bourqui, Marc; Wenzel, Claudia; Eugster-Meier, Elisabeth; Frey, Juerg E.; Irmler, Stefan; Ahrens, Christian H.

    2018-01-01

    Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus—to our knowledge—identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus. Notably, the functional Clusters of Orthologous Groups of proteins categories “cell wall/membrane biogenesis” and “defense mechanisms” were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences

  12. Eradication of large tumors expressing human papillomavirus E7 protein by therapeutic vaccination with E7 fused to the extra domain a from fibronectin.

    PubMed

    Mansilla, Cristina; Berraondo, Pedro; Durantez, Maika; Martínez, Marta; Casares, Noelia; Arribillaga, Laura; Rudilla, Francesc; Fioravanti, Jessica; Lozano, Teresa; Villanueva, Lorea; Sarobe, Pablo; Borrás, Francisco; Leclerc, Claude; Prieto, Jesús; Lasarte, Juan José

    2012-08-01

    Cervical carcinoma is one of the most common cancers in women worldwide. It is well established that chronic infection of the genital tract by various mucosatropic human papillomavirus (HPV) types causes cervical cancer. Cellular immunity to E7 protein from HPV (HPVE7) has been associated with clinical and cytologic resolution of HPV-induced lesions. Thus, we decided to test if targeting of HPVE7 to dendritic cells using a fusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for TLR4, and HPVE7 (EDA-HPVE7) might be an efficient vaccine for the treatment of cervical carcinoma. We found that EDA-HPVE7 fusion protein was efficiently captured by bone marrow derived dendritic cells in vitro and induced their maturation, with the upregulation of maturation markers and the production of IL-12. Immunization of mice with EDA-HPVE7 fusion protein induced antitumor CD8(+) T cell responses in the absence of additional adjuvants. Repeated intratumoral administration of EDA-HPVE7 in saline was able to cure established TC-1 tumors of 5-7 mm in diameter. More importantly, intravenous injection with EDA-HPVE7 in combination with the TLR ligand polyinosinic-polycytidylic acid (pIC), or with low doses of cyclophosphamide and the TLR9 ligand CpG-B complexed in cationic lipids, were able to eradicate large established TC-1 tumors (1.2 cm in diameter). Thus, therapeutic vaccination with EDA-HPVE7 fusion protein may be effective in the treatment of human cervical carcinoma. Copyright © 2011 UICC.

  13. [Comparative analysis of real-time quantitative PCR-Sanger sequencing method and TaqMan probe method for detection of KRAS/BRAF mutation in colorectal carcinomas].

    PubMed

    Zhang, Xun; Wang, Yuehua; Gao, Ning; Wang, Jinfen

    2014-02-01

    To compare the application values of real-time quantitative PCR-Sanger sequencing and TaqMan probe method in the detection of KRAS and BRAF mutations, and to correlate KRAS/BRAF mutations with the clinicopathological characteristics in colorectal carcinomas. Genomic DNA of the tumor cells was extracted from formalin fixed paraffin embedded (FFPE) tissue samples of 344 colorectal carcinomas by microdissection. Real-time quantitative PCR-Sanger sequencing and TaqMan probe method were performed to detect the KRAS/BRAF mutations. The frequency and types of KRAS/BRAF mutations, clinicopathological characteristics and survival time were analyzed. KRAS mutations were detected in 39.8% (137/344) and 38.7% (133/344) of 344 colorectal carcinomas by using real-time quantitative PCR-Sanger sequencing and TaqMan probe method, respectively. BRAF mutation was detected in 4.7% (16/344) and 4.1% (14/344), respectively. There was no significant correlation between the two methods. The frequency of the KRAS mutation in female was higher than that in male (P < 0.05). The frequency of the BRAF mutation in colon was higher than that in rectum. The frequency of the BRAF mutation in stage III-IV cases was higher than that in stageI-II cases. The frequency of the BRAF mutation in signet ring cell carcinoma was higher than that in mucinous carcinoma and nonspecific adenocarcinoma had the lowest mutation rate. The frequency of the BRAF mutation in grade III cases was higher than that in grade II cases (P < 0.05). The overall concordance for the two methods of KRAS/BRAF mutation detection was 98.8% (kappa = 0.976). There was statistic significance between BRAF and KRAS mutations for the survival time of colorectal carcinomas (P = 0.039). There were no statistic significance between BRAF mutation type and BRAF/KRAS wild type (P = 0.058). (1) Compared with real-time quantitative PCR-Sanger sequencing, TaqMan probe method is better with regard to handling time, efficiency, repeatability, cost

  14. A Comparative Evaluation of Accuracy of the Dies Affected by Tray Type, Material Viscosity, and Pouring Sequence of Dual and Single Arch Impressions- An In vitro Study

    PubMed Central

    Kulkarni, Rahul S.; Shah, Rupal J.; Chhajlani, Rahul; Saklecha, Bhuwan; Maru, Kavita

    2017-01-01

    Introduction The clinician’s skill, impression techniques, and materials play a very important role in recording fine details in an impression for accuracy of fixed partial denture prosthesis. Impression of prepared teeth and of the opposing arch can be recorded simultaneously by dual-arch trays, while the full arch metal trays are used for impressions of prepared teeth in one arch. Aim To measure and compare the accuracy of working dies made from impressions with metal and plastic dual arch trays and metal full arch trays, for two viscosities of impression material and by changing the sequence of pour of working and non-working sides. Materials and Methods A balanced design with independent samples was used to study the three variables (tray type, impression material viscosity, and pouring sequence). An impression made by dual arch trays and single arch trays were divided in to three groups (Group A-plastic dual arch tray, Group B-metal dual arch tray, Group C-full arch metal stock tray). Out of these three groups, two groups (Group A and B) were subdivided in to four subgroups each and one group (Group C) was subdivided in to two subgroups. A sample size of 30 was used in each subgroup yielding a total 300 impressions in three groups or ten subgroups. Impressions were made of a machined circular stainless steel die. All three dimensions (Occlusogingival, Mesiodistal, and Buccolingual) of the working dies as well as stainless steel standard die were measured three times, and the mean was used for the three standard sample values to which all working dies means were compared. Statistical analysis used for this study was a 3-factor analysis of variance with hypothesis testing at α =0.05. Results With respect to the selection of impression material viscosity statistically significant differences were found in the dies for the buccolingual and mesiodistal dimensions. Metal dual arch trays were slightly more accurate in the mesiodistal dimension in comparison to the

  15. A Non-oncogenic HPV 16 E6/E7 Vaccine Enhances Treatment of HPV Expressing Tumors

    PubMed Central

    Wieking, Bryant G.; Vermeer, Daniel W.; Spanos, William C.; Lee, Kimberly M.; Vermeer, Paola; Lee, Walter T.; Xu, Younong; Gabitzsch, Elizabeth S.; Balcaitis, Stephanie; Balint, Joseph P.; Jones, Frank R.; Lee, John H.

    2012-01-01

    Human papillomaviruses (HPVs) are the causative factor for greater than 90% of cervical cancers and 25% of head and neck cancers. The incidence of HPV positive (+) head and neck squamous cell carcinomas (HNSCCs) has greatly increased in the last 30 years. E6 and E7 are the two key viral oncoproteins that induce and propagate cellular transformation. An immune response generated during cisplatin/radiation therapy improves tumor clearance of HPV(+) cancers. Augmenting this induced response during therapy with an adenoviral HPV16 E6/E7 vaccine improves long term survival in preclinical models. Here we describe the generation of an HPV16 E6/E7 construct, which contains mutations that render E6/E7 non-oncogenic, while preserving antigenicity. These mutations do not allow E6/E7 to degrade p53, pRb, PTPN13, or activate telomerase. Non-oncogenic E6/E7 (E6Δ/E7Δ) expressed as a stable integrant, or in the [E1-, E2b-] adenovirus, lacks the ability to transform human cells while retaining the ability to induce an HPV specific immune response. Moreover, E6Δ/E7Δ plus chemotherapy/radiation statistically enhances clearance of established HPV(+) cancer in vivo. PMID:22918471

  16. HPV16 E7 protein associates with the protein kinase p33CDK2 and cyclin A.

    PubMed

    Tommasino, M; Adamczewski, J P; Carlotti, F; Barth, C F; Manetti, R; Contorni, M; Cavalieri, F; Hunt, T; Crawford, L

    1993-01-01

    E7 is the major transforming protein of human papillomavirus type 16 (HPV16). It has been found to associate with the retinoblastoma protein Rb1. We investigated whether HPV16 E7 protein was associated with other cellular proteins, in particular with those involved in cell cycle control. Immunoprecipitates from CaSki cell extracts with an anti E7 monoclonal antibody contained a histone H1 kinase. Recombinant E7, synthesized in yeast, when mixed with protein extracts from epithelial cells bound histone H1 kinase activity in vitro. The in vivo and the in vitro-formed E7-kinase complex had the same periodicity of activity during the cell cycle, being most active in S and G2/M. Immunoblotting of E7 immunoprecipitates with an antibody raised against the p33CDK2, revealed a 33 kDa protein band not detected by an anti-p34cdc2 antibody, suggesting that the E7-associated kinase activity is due to the p33CDK2. The interaction appears to be via cyclin A, since probing of similar immunoblots showed a 50 kDa band corresponding to cyclin A. The association of E7 with cyclin A appeared to be direct, not involving Rb 1 or other proteins.

  17. [Comparative analysis on the complete genome sequence of mumps epidemic strain and mumps vaccine strain S79 isolated in Zhejiang province, China between year 2005 and 2010].

    PubMed

    Zhang, Dong-Yan; Feng, Yan; Zhong, Shu-Ling; Lu, Yi-Yu; Zhuang, Fang-Cheng; Xu, Chang-Ping

    2012-03-01

    To compare the differences in the complete genome sequence between mumps epidemic strain and mumps vaccine strain S79 isolated in Zhejiang province. A total of 4 mumps epidemic strains, which were separated from Zhejiang province during 2005 to 2010, named as ZJ05-1, ZJ06-3, ZJ08-1 and ZJ10-1 were selected in the study. The complete genome sequences were amplified using RT-PCR. The genetic differences between vaccine strain S79 and other genotype strains were compared; while the genetic-distance was calculated and the evolution was analyzed. The biggest difference between the 4 epidemic strains and the vaccine strain S79 was found on the membrane associated protein gene; whose average nucleotide differential number was 42.5 +/- 3.0 and the average variant ratio was 13.6%; while the mean amino acid differential number was 12.8 +/- 1.5 and the average variant ratio was 22.4%. The smallest difference among the 4 epidemic strains and the vaccine strain was found in stromatin genes, whose average nucleotide differential number was 73.8 +/- 2.5 and the average variant ratio was 5.9%; while the mean amino acid differential number was 3.0 +/- 0.8 and the average variant ratio was 0.8%. The dn/ds value of the stromatin genes of the 4 epidemic strains reached the highest, as 0.6526; but without any positive pressure (dn/ds < 1, chi2 = 0.87, P > 0.05). There were mutations happened on the known antigen epitope, as 8th amino acid of membrane associated protein genes and on the 336th and 356th amino acid of hemagglutinin/neuraminidase proteins. Compared with the vaccine strain, the glycosylation sites of ZJ05-1, ZJ06-3, ZJ08-1 and ZJ10-1 increased 1, 1, 2 and 2 respectively. The complete amino acid sequence of all strains showed that there were 17 characteristic sites found on the genotype-F mumps strain. Within the complete genome, the genetic-distance between epidemic strains and vaccine strains in Zhejiang province (0.071) was significantly larger than the genetic

  18. The association of mammalian DREAM complex and HPV16 E7 proteins

    PubMed Central

    Rashid, Nurshamimi Nor; Rothan, Hussin A; Yusoff, Mohd Shahrizal Mohd

    2015-01-01

    The mammalian DREAM (Drosophila, RB, E2F, and Myb) complex was discovered in 2004 by several research groups. It was initially identified in Drosophila followed by Caenorhaditis elegans and later in mammalian cells. The composition of DREAM is temporally regulated during cell cycle; being associated with E2F-4 and either p107 or p130 in G0/G1 (repressive DREAM complexes) and with B-myb transcription factor in S/G2 (activator DREAM complex). High risk human papillomavirus (HPV) E6 and E7 oncoproteins expression are important for malignant transformation of cervical cancer cells. In particular, the E7 of high risk HPV binds to pRB family members (pRB, p107 and p130) for degradation. It has recently been discovered that the p107 and p130 ‘pocket proteins’ are members of mammalian DREAM complexes. With this understanding, we would like to hypothesise the mammalian DREAM complex could plays a critical role for malignant transformation in cervical cancer cells. PMID:26885443

  19. Establishment of immunoassay for detecting HPV16 E6 and E7 RNA

    PubMed Central

    Ding, Sen; Qian, Steven Y.; Zhang, Yang; Wu, Wenlei; Lu, Gensheng; Lu, Yan; Feng, Xiujing; Li, Li; Shen, Pingping

    2015-01-01

    Cervical carcinoma is the most prevalent malignancy second only to breast cancer among women worldwide. Since more than 99% of cervical cancers are caused by human papilloma virus (HPV), measurement of HPV (HPV test) was commonly used in screening risk and/or early stage of cervical cancer as well as assessing the efficacies of the treatments that can decrease the incidence of cervical cancer. Many approaches that diagnose HPV infections have been developed, while most of them have distinct shortcomings. We here established a novel immunoassay method in which the pairs of unlabeled DNA probes firstly bind to HPV16 E6 and E7 RNAs to form the DNA-RNA hybrids, and the hybrids will subsequently be identified by S9.6 antibody. The sensitivity of this highly specific method can reach ~0.923 pg/mL and ~0.424 pg/mL of in vitro transcribed HPV16 E6 and E7 RNA, respectively, and reverse transcription and polymerase chain reaction (PCR) amplification were no longer needed. Thus, our immunoassay approaches can precisely reflect the actually viral load that is related to the course of HPV infection. In addition, it has also fast and low cost characteristic feature. PMID:26333509

  20. Motif-independent prediction of a secondary metabolism gene cluster using comparative genomics: application to sequenced genomes of Aspergillus and ten other filamentous fungal species.

    PubMed

    Takeda, Itaru; Umemura, Myco; Koike, Hideaki; Asai, Kiyoshi; Machida, Masayuki

    2014-08-01

    Despite their biological importance, a significant number of genes for secondary metabolite biosynthesis (SMB) remain undetected due largely to the fact that they are highly diverse and are not expressed under a variety of cultivation conditions. Several software tools including SMURF and antiSMASH have been developed to predict fungal SMB gene clusters by finding core genes encoding polyketide synthase, nonribosomal peptide synthetase and dimethylallyltryptophan synthase as well as several others typically present in the cluster. In this work, we have devised a novel comparative genomics method to identify SMB gene clusters that is independent of motif information of the known SMB genes. The method detects SMB gene clusters by searching for a similar order of genes and their presence in nonsyntenic blocks. With this method, we were able to identify many known SMB gene clusters with the core genes in the genomic sequences of 10 filamentous fungi. Furthermore, we have also detected SMB gene clusters without core genes, including the kojic acid biosynthesis gene cluster of Aspergillus oryzae. By varying the detection parameters of the method, a significant difference in the sequence characteristics was detected between the genes residing inside the clusters and those outside the clusters. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  1. The preferred nucleotide contexts of the AID/APOBEC cytidine deaminases have differential effects when mutating retrotransposon and virus sequences compared to host genes

    PubMed Central

    Chen, Jeffrey

    2017-01-01

    The AID / APOBEC genes are a family of cytidine deaminases that have evolved in vertebrates, and particularly mammals, to mutate RNA and DNA at distinct preferred nucleotide contexts (or “hotspots”) on foreign genomes such as viruses and retrotransposons. These enzymes play a pivotal role in intrinsic immunity defense mechanisms, often deleteriously mutating invading retroviruses or retrotransposons and, in the case of AID, changing antibody sequences to drive affinity maturation. We investigate the strength of various hotspots on their known biological targets by evaluating the potential impact of mutations on the DNA coding sequences of these targets, and compare these results to hypothetical hotspots that did not evolve. We find that the existing AID / APOBEC hotspots have a large impact on retrotransposons and non-mammalian viruses while having a much smaller effect on vital mammalian genes, suggesting co-evolution with AID / APOBECs may have had an impact on the genomes of the viruses we analyzed. We determine that GC content appears to be a significant, but not sole, factor in resistance to deaminase activity. We discuss possible mechanisms AID and APOBEC viral targets have adopted to escape the impacts of deamination activity, including changing the GC content of the genome. PMID:28362825

  2. Study of canine parvovirus evolution: comparative analysis of full-length VP2 gene sequences from Argentina and international field strains.

    PubMed

    Gallo Calderón, Marina; Wilda, Maximiliano; Boado, Lorena; Keller, Leticia; Malirat, Viviana; Iglesias, Marcela; Mattion, Nora; La Torre, Jose

    2012-02-01

    The continuous emergence of new strains of canine parvovirus (CPV), poorly protected by current vaccination, is a concern among breeders, veterinarians, and dog owners around the world. Therefore, the understanding of the genetic variation in emerging CPV strains is crucial for the design of disease control strategies, including vaccines. In this paper, we obtained the sequences of the full-length gene encoding for the main capsid protein (VP2) of 11 canine parvovirus type 2 (CPV-2) Argentine representative field strains, selected from a total of 75 positive samples studied in our laboratory in the last 9 years. A comparative sequence analysis was performed on 9 CPV-2c, one CPV-2a, and one CPV-2b Argentine strains with respect to international strains reported in the GenBank database. In agreement with previous reports, a high degree of identity was found among CPV-2c Argentine strains (99.6-100% and 99.7-100% at nucleotide and amino acid levels, respectively). However, the appearance of a new substitution in the 440 position (T440A) in four CPV-2c Argentine strains obtained after the year 2009 gives support to the variability observed for this position located within the VP2, three-fold spike. This is the first report on the genetic characterization of the full-length VP2 gene of emerging CPV strains in South America and shows that all the Argentine CPV-2c isolates cluster together with European and North American CPV-2c strains.

  3. De novo sequencing and comparative analysis of leaf transcriptomes of diverse condensed tannin-containing lines of underutilized Psophocarpus tetragonolobus (L.) DC

    PubMed Central

    Singh, Vinayak; Goel, Ridhi; Pande, Veena; Asif, Mehar Hasan; Mohanty, Chandra Sekhar

    2017-01-01

    Condensed tannin (CT) or proanthocyanidin (PA) is a unique group of phenolic metabolite with high molecular weight with specific structure. It is reported that, the presence of high-CT in the legumes adversely affect the nutrients in the plant and impairs the digestibility upon consumption by animals. Winged bean (Psophocarpus tetragonolobus (L.) DC.) is one of the promising underutilized legume with high protein and oil-content. One of the reasons for its underutilization is due to the presence of CT. Transcriptome sequencing of leaves of two diverse CT-containing lines of P. tetragonolobus was carried out on Illumina Nextseq 500 sequencer to identify the underlying genes and contigs responsible for CT-biosynthesis. RNA-Seq data generated 102586 and 88433 contigs for high (HCTW) and low CT (LCTW) lines of P. tetragonolobus, respectively. Based on the similarity searches against gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) database revealed 5210 contigs involved in 229 different pathways. A total of 1235 contigs were detected to differentially express between HCTW and LCTW lines. This study along with its findings will be helpful in providing information for functional and comparative genomic analysis of condensed tannin biosynthesis in this plant in specific and legumes in general. PMID:28322296

  4. Characterization of fungi in office dust: Comparing results of microbial secondary metabolites, fungal internal transcribed spacer region sequencing, viable culture and other microbial indices.

    PubMed

    Park, J-H; Sulyok, M; Lemons, A R; Green, B J; Cox-Ganser, J M

    2018-05-04

    Recent developments in molecular and chemical methods have enabled the analysis of fungal DNA and secondary metabolites, often produced during fungal growth, in environmental samples. We compared 3 fungal analytical methods by analysing floor dust samples collected from an office building for fungi using viable culture, internal transcribed spacer (ITS) sequencing and secondary metabolites using liquid chromatography-tandem mass spectrometry. Of the 32 metabolites identified, 29 had a potential link to fungi with levels ranging from 0.04 (minimum for alternariol monomethylether) to 5700 ng/g (maximum for neoechinulin A). The number of fungal metabolites quantified per sample ranged from 8 to 16 (average = 13/sample). We identified 216 fungal operational taxonomic units (OTUs) with the number per sample ranging from 6 to 29 (average = 18/sample). We identified 37 fungal species using culture, and the number per sample ranged from 2 to 13 (average = 8/sample). Agreement in identification between ITS sequencing and culturing was weak (kappa = -0.12 to 0.27). The number of cultured fungal species poorly correlated with OTUs, which did not correlate with the number of metabolites. These suggest that using multiple measurement methods may provide an improved understanding of fungal exposures in indoor environments and that secondary metabolites may be considered as an additional source of exposure. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. A Streamlined Protocol for Molecular Testing of the DMD Gene within a Diagnostic Laboratory: A Combination of Array Comparative Genomic Hybridization and Bidirectional Sequence Analysis

    PubMed Central

    Marquis-Nicholson, Renate; Lai, Daniel; Love, Jennifer M.; Love, Donald R.

    2013-01-01

    Purpose. The aim of this study was to develop a streamlined mutation screening protocol for the DMD gene in order to confirm a clinical diagnosis of Duchenne or Becker muscular dystrophy in affected males and to clarify the carrier status of female family members. Methods. Sequence analysis and array comparative genomic hybridization (aCGH) were used to identify mutations in the dystrophin DMD gene. We analysed genomic DNA from six individuals with a range of previously characterised mutations and from eight individuals who had not previously undergone any form of molecular analysis. Results. We successfully identified the known mutations in all six patients. A molecular diagnosis was also made in three of the four patients with a clinical diagnosis who had not undergone prior genetic screening, and testing for familial mutations was successfully completed for the remaining four patients. Conclusion. The mutation screening protocol described here meets best practice guidelines for molecular testing of the DMD gene in a diagnostic laboratory. The aCGH method is a superior alternative to more conventional assays such as multiplex ligation-dependent probe amplification (MLPA). The combination of aCGH and sequence analysis will detect mutations in 98% of patients with the Duchenne or Becker muscular dystrophy. PMID:23476807

  6. An Expressed Sequence Tag (EST)-enriched genetic map of turbot (Scophthalmus maximus): a useful framework for comparative genomics across model and farmed teleosts

    PubMed Central

    2012-01-01

    Background The turbot (Scophthalmus maximus) is a relevant species in European aquaculture. The small turbot genome provides a source for genomics strategies to use in order to understand the genetic basis of productive traits, particularly those related to sex, growth and pathogen resistance. Genetic maps represent essential genomic screening tools allowing to localize quantitative trait loci (QTL) and to identify candidate genes through comparative mapping. This information is the backbone to develop marker-assisted selection (MAS) programs in aquaculture. Expressed sequenced tag (EST) resources have largely increased in turbot, thus supplying numerous type I markers suitable for extending the previous linkage map, which was mostly based on anonymous loci. The aim of this study was to construct a higher-resolution turbot genetic map using EST-linked markers, which will turn out to be useful for comparative mapping studies. Results A consensus gene-enriched genetic map of the turbot was constructed using 463 SNP and microsatellite markers in nine reference families. This map contains 438 markers, 180 EST-linked, clustered at 24 linkage groups. Linkage and comparative genomics evidences suggested additional linkage group fusions toward the consolidation of turbot map according to karyotype information. The linkage map showed a total length of 1402.7 cM with low average intermarker distance (3.7 cM; ~2 Mb). A global 1.6:1 female-to-male recombination frequency (RF) ratio was observed, although largely variable among linkage groups and chromosome regions. Comparative sequence analysis revealed large macrosyntenic patterns against model teleost genomes, significant hits decreasing from stickleback (54%) to zebrafish (20%). Comparative mapping supported particular chromosome rearrangements within Acanthopterygii and aided to assign unallocated markers to specific turbot linkage groups. Conclusions The new gene-enriched high-resolution turbot map represents a

  7. De novo sequencing and comparative transcriptome analysis of the male and hermaphroditic flowers provide insights into the regulation of flower formation in andromonoecious taihangia rupestris.

    PubMed

    Li, Weiguo; Zhang, Lihui; Ding, Zhan; Wang, Guodong; Zhang, Yandi; Gong, Hongmei; Chang, Tianjun; Zhang, Yanwen

    2017-02-28

    Taihangia rupestris, an andromonoecious plant species, bears both male and hermaphroditic flowers within the same individual. However, the establishment and development of male and hermaphroditic flowers in andromonoecious Taihangia remain poorly understood, due to the limited genetic and sequence information. To investigate the potential molecular mechanism in the regulation of Taihangia flower formation, we used de novo RNA sequencing to compare the transcriptome profiles of male and hermaphroditic flowers at early and late developmental stages. Four cDNA libraries, including male floral bud, hermaphroditic floral bud, male flower, and hermaphroditic flower, were constructed and sequenced by using the Illumina RNA-Seq method. Totally, 84,596,426 qualified Illumina reads were obtained and then assembled into 59,064 unigenes, of which 24,753 unigenes were annotated in the NCBI non-redundant protein database. In addition, 12,214, 7,153, and 8,115 unigenes were assigned into 53 Gene Ontology (GO) functional groups, 25 Clusters of Orthologous Group (COG) categories, and 126 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. By pairwise comparison of unigene abundance between the samples, we identified 1,668 differential expressed genes (DEGs), including 176 transcription factors (TFs) between the male and hermaphroditic flowers. At the early developmental stage, we found 263 up-regulated genes and 436 down-regulated genes expressed in hermaphroditic floral buds, while 844 up-regulated genes and 314 down-regulated genes were detected in hermaphroditic flowers at the late developmental stage. GO and KEGG enrichment analyses showed that a large number of DEGs were associated with a wide range of functions, including cell cycle, epigenetic processes, flower development, and biosynthesis of unsaturated fatty acid pathway. Finally, real-time quantitative PCR was conducted to validate the DEGs identified in the present study. In this study, transcriptome

  8. Comparative genomics and prediction of conditionally dispensable sequences in legume-infecting Fusarium oxysporum formae speciales facilitates identification of candidate effectors.

    PubMed

    Williams, Angela H; Sharma, Mamta; Thatcher, Louise F; Azam, Sarwar; Hane, James K; Sperschneider, Jana; Kidd, Brendan N; Anderson, Jonathan P; Ghosh, Raju; Garg, Gagan; Lichtenzveig, Judith; Kistler, H Corby; Shea, Terrance; Young, Sarah; Buck, Sally-Anne G; Kamphuis, Lars G; Saxena, Rachit; Pande, Suresh; Ma, Li-Jun; Varshney, Rajeev K; Singh, Karam B

    2016-03-05

    Soil-borne fungi of the Fusarium oxysporum species complex cause devastating wilt disease on many crops including legumes that supply human dietary protein needs across many parts of the globe. We present and compare draft genome assemblies for three legume-infecting formae speciales (ff. spp.): F. oxysporum f. sp. ciceris (Foc-38-1) and f. sp. pisi (Fop-37622), significant pathogens of chickpea and pea respectively, the world's second and third most important grain legumes, and lastly f. sp. medicaginis (Fom-5190a) for which we developed a model legume pathosystem utilising Medicago truncatula. Focusing on the identification of pathogenicity gene content, we leveraged the reference genomes of Fusarium pathogens F. oxysporum f. sp. lycopersici (tomato-infecting) and F. solani (pea-infecting) and their well-characterised core and dispensable chromosomes to predict genomic organisation in the newly sequenced legume-infecting isolates. Dispensable chromosomes are not essential for growth and in Fusarium species are known to be enriched in host-specificity and pathogenicity-associated genes. Comparative genomics of the publicly available Fusarium species revealed differential patterns of sequence conservation across F. oxysporum formae speciales, with legume-pathogenic formae speciales not exhibiting greater sequence conservation between them relative to non-legume-infecting formae speciales, possibly indicating the lack of a common ancestral source for legume pathogenicity. Combining predicted dispensable gene content with in planta expression in the model legume-infecting isolate, we identified small conserved regions and candidate effectors, four of which shared greatest similarity to proteins from another legume-infecting ff. spp. We demonstrate that distinction of core and potential dispensable genomic regions of novel F. oxysporum genomes is an effective tool to facilitate effector discovery and the identification of gene content possibly linked to host

  9. Interaction of colicin E7 with the major coat protein (g8p) may confer limited protection on colicinogenic Escherichia coli against M13 bacteriophage infection.

    PubMed

    Chen, Yuh-Ren; Yang, Tsung-Yeh; Lei, Guang-Sheng; Liao, Chen-Chung; Chak, Kin-Fu

    2010-11-01

    Colicin release provides producer strains with a competitive advantage under certain circumstances. We found that propagation of M13 bacteriophage in cells producing colicin E7 is impaired, without alteration in the efficiency of bacteriophage adsorption, as compared with non-producing cells. In contrast to the protective effect of the colicin against M13 bacteriophage infection, the endogenously expressed colicin does not confer limited protection against transfection with M13 bacteriophage DNA. Furthermore, it was found that the translocation-receptor-binding domain and toxicity domain of the colicin are able to interact with the M13 major coat protein, g8p, during bacteriophage infection. Based on these observations, we propose that interaction between colicin E7 and g8p during infection interferes with g8p depolymerizing into the cytoplasmic membrane during bacteriophage DNA penetration, thus resulting in the limited protection against M13 bacteriophage infection.

  10. Differential gene expression between skin and cervix induced by the E7 oncoprotein in a transgenic mouse model

    PubMed Central

    Ibarra Sierra, E; Díaz Chávez, J; Cortés-Malagón, EM; Uribe-Figueroa, L; Hidalgo-Miranda, A; Lambert, PF; Gariglio, P

    2013-01-01

    HPV16 E7 oncoprotein expression in K14E7 transgenic mice induces cervical cancer after 6 months of treatment with the co-carcinogen 17β-estradiol. In untreated mice, E7 also induces skin tumors late in life albeit at low penetrance. These findings indicate that E7 alters cellular functions in cervix and skin so as to predispose these organs to tumorigenesis. Using microarrays, we determined the global genes expression profile in cervical and skin tissue of young adult K14E7 transgenic mice without estrogen treatment. In these tissues, the E7 oncoprotein altered the transcriptional pattern of genes involved in several biological processes including signal transduction, transport, metabolic process, cell adhesion, apoptosis, cell differentiation, immune response and inflammatory response. Among the E7-dysregulated genes were ones not previously known to be involved in cervical neoplasia including DMBT1, GLI1 and 17βHSD2 in cervix, as well as MMP2, 12, 14, 19 and 27 in skin. PMID:22980503

  11. Differential gene expression between skin and cervix induced by the E7 oncoprotein in a transgenic mouse model.

    PubMed

    Ibarra Sierra, E; Díaz Chávez, J; Cortés-Malagón, E M; Uribe-Figueroa, L; Hidalgo-Miranda, A; Lambert, P F; Gariglio, P

    2012-11-25

    HPV16 E7 oncoprotein expression in K14E7 transgenic mice induces cervical cancer after 6 months of treatment with the co-carcinogen 17β-estradiol. In untreated mice, E7 also induces skin tumors late in life albeit at low penetrance. These findings indicate that E7 alters cellular functions in cervix and skin so as to predispose these organs to tumorigenesis. Using microarrays, we determined the global genes expression profile in cervical and skin tissue of young adult K14E7 transgenic mice without estrogen treatment. In these tissues, the E7 oncoprotein altered the transcriptional pattern of genes involved in several biological processes including signal transduction, transport, metabolic process, cell adhesion, apoptosis, cell differentiation, immune response and inflammatory response. Among the E7-dysregulated genes were ones not previously known to be involved in cervical neoplasia including DMBT1, GLI1 and 17βHSD2 in cervix, as well as MMP2, 12, 14, 19 and 27 in skin. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Immunomodulatory activity of a plant extract containing human papillomavirus 16-E7 protein in human monocyte-derived dendritic cells.

    PubMed

    Di Bonito, P; Grasso, F; Mangino, G; Massa, S; Illiano, E; Franconi, R; Fanales-Belasio, E; Falchi, M; Affabris, E; Giorgi, C

    2009-01-01

    This study reports the immunomodulatory activity on human monocyte derived dendritic cells (MDDCs) of a vaccine preparation shown to be effective against an HPV16-related tumour in an animal model. The vaccine is composed of extract from Nicotiana benthamiana leaves containing HPV16 E7 protein expressed by a potato virus X-derived vector (NbPVX-E7). The effect of the extract was evaluated on MDDC differentiation and maturation by monitoring the phenotypic expression of specific markers. The results show that NbPVX-E7 does not induce monocyte differentiation to dendritic cells, but does induce MDDC maturation. Plant extract does not influence MDDC-uptake of E7-FITC while it significantly improves the Ovalbumin-FITC uptake, considered as a model antigen. Importantly, NbPVX-E7-pulsed MDDCs/PBMCs are able to prime human blood-derived lymphocytes from healthy individuals to induce HPV16 E7-specific cytotoxic activity. This is a propaedeutic study for a possible use of E7-containing plant extract in human immunotherapy of HPV-related lesions.

  13. Intracellular human papillomavirus E6, E7 mRNA quantification predicts CIN 2+ in cervical biopsies better than Papanicolaou screening for women regardless of age.

    PubMed

    Pierry, Deirdre; Weiss, Gerald; Lack, Benjamin; Chen, Victor; Fusco, Judy

    2012-08-01

    Cervical cancer screening in women younger than 30 years relies on cervical cytology because of the poor performance of human papillomavirus (HPV) DNA testing in this age group. To determine the performance of in-cell HPV E6, E7 mRNA quantification (HPV OncoTect) for the detection of high-grade cervical intraepithelial neoplasia in women younger than 30 years. We analyzed 3133 cytology specimens from a screening population of women aged 19-75 years investigate HPV OncoTect as a triage/secondary screening test for atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) cytology in women younger than 30 years. Test results were compared to histology in 246 cases. The sensitivity of E6, E7 mRNA was 89% for CIN 2+ and 100% for CIN 3+ lesions in women 30 years and older. In women younger than 30 years, the sensitivity of E6, E7 mRNA for CIN 2+ lesions was 88% for CIN 2+ and 92% for CIN 3+ lesions. Abnormal cytology (≥ASCUS) exhibited a sensitivity of 89% for CIN 2+ and 100% for CIN 3+ in women 30 years and older and 96% sensitivity for CIN 2+ and 93% sensitivity for CIN 3+ in women younger than 30. The specificity of E6, E7 mRNA was >80% for CIN 2+ and CIN 3+ in both groups of women compared to a specificity of abnormal cytology of <10% for CIN 2+ and CIN 3+ in both groups. HPV OncoTect demonstrates a performance that would be effective for ASCUS/LSIL triage in women including those younger than 30 years.

  14. Comparing species tree estimation with large anchored phylogenomic and small Sanger-sequenced molecular datasets: an empirical study on Malagasy pseudoxyrhophiine snakes.

    PubMed

    Ruane, Sara; Raxworthy, Christopher J; Lemmon, Alan R; Lemmon, Emily Moriarty; Burbrink, Frank T

    2015-10-12

    Using molecular data generated by high throughput next generation sequencing (NGS) platforms to infer phylogeny is becoming common as costs go down and the ability to capture loci from across the genome goes up. While there is a general consensus that greater numbers of independent loci should result in more robust phylogenetic estimates, few studies have compared phylogenies resulting from smaller datasets for commonly used genetic markers with the large datasets captured using NGS. Here, we determine how a 5-locus Sanger dataset compares with a 377-locus anchored genomics dataset for understanding the evolutionary history of the pseudoxyrhophiine snake radiation centered in Madagascar. The Pseudoxyrhophiinae comprise ~86 % of Madagascar's serpent diversity, yet they are poorly known with respect to ecology, behavior, and systematics. Using the 377-locus NGS dataset and the summary statistics species-tree methods STAR and MP-EST, we estimated a well-supported species tree that provides new insights concerning intergeneric relationships for the pseudoxyrhophiines. We also compared how these and other methods performed with respect to estimating tree topology using datasets with varying numbers of loci. Using Sanger sequencing and an anchored phylogenomics approach, we sequenced datasets comprised of 5 and 377 loci, respectively, for 23 pseudoxyrhophiine taxa. For each dataset, we estimated phylogenies using both gene-tree (concatenation) and species-tree (STAR, MP-EST) approaches. We determined the similarity of resulting tree topologies from the different datasets using Robinson-Foulds distances. In addition, we examined how subsets of these data performed compared to the complete Sanger and anchored datasets for phylogenetic accuracy using the same tree inference methodologies, as well as the program *BEAST to determine if a full coalescent model for species tree estimation could generate robust results with fewer loci compared to the summary statistics species

  15. Immortalization of human prostate epithelial cells by HPV 16 E6/E7 open reading frames.

    PubMed

    Choo, C K; Ling, M T; Chan, K W; Tsao, S W; Zheng, Z; Zhang, D; Chan, L C; Wong, Y C

    1999-08-01

    The exact pathogenesis for prostate cancer is not known. Progress made in prostate cancer research has been slow, largely due to the lack of suitable in vitro models. Here, we report our work on the immortalization of a human prostate epithelial cell line and show that it can be used as a model to study prostate tumorigenesis. Replication-defective retrovirus harboring the human papillomavirus (HPV) type 16 E6 and E7 open reading frames was used to infect primary human prostate epithelial cells. Polymerase chain reaction, followed by Southern hybridization for the HPV 16 E6/E7, Western blot for prostatic acid phosphatase, telomeric repeat amplification protocol assay for telomerase activity, two-dimensional gels for cytokeratins, and cytogenetic analysis were undertaken to characterized the infected cells. The retrovirus-infected cell line, HPr-1, continued to grow in culture for more than 80 successive passages. Normal primary cells failed to proliferate after passage 6. HPr-1 cells bore close resemblance to normal primary prostate epithelial cells, both morphologically and biochemically. However, they possessed telomerase activity and proliferated indefinitely. Cytogenetic analysis of HPr-1 cells revealed a human male karyotype with clonal abnormalities and the appearance of multiple double minutes. The HPr-1 cells expressed prostatic acid phosphatase and cytokeratins K8 and K18, proving that they were prostate epithelial cells. They were benign in nude mice tumor formation and soft agar colony formation assay. The HPr-1 cell line is an in vitro representation of early prostate neoplastic progression. Copyright 1999 Wiley-Liss, Inc.

  16. Comparative analysis of single-cell RNA sequencing data from mouse spermatogonial and mesenchymal stem cells to identify differentially expressed genes and transcriptional regulators of germline cells.

    PubMed

    Sisakhtnezhad, Sajjad; Heshmati, Parvin

    2018-07-01

    Identifying effective internal factors for regulating germline commitment during development and for maintaining spermatogonial stem cells (SSCs) self-renewal is important to understand the molecular basis of spermatogenesis process, and to develop new protocols for the production of the germline cells from other cell sources. Therefore, this study was designed to investigate single-cell RNA-sequencing data for identification of differentially expressed genes (DEGs) in 12 mouse-derived single SSCs (mSSCs) in compare with 16 mouse-derived single mesenchymal stem cells. We also aimed to find transcriptional regulators of DEGs. Collectively, 1,584 up-regulated DEGs were identified that are associated with 32 biological processes. Moreover, investigation of the expression profiles of genes including in spermatogenesis process revealed that Dazl, Ddx4, Sall4, Fkbp6, Tex15, Tex19.1, Rnf17, Piwil2, Taf7l, Zbtb16, and Cadm1 are presented in the first 30 up-regulated DEGs. We also found 12 basal transcription factors (TFs) and three sequence-specific TFs that control the expression of DEGs. Our findings also indicated that MEIS1, SMC3, TAF1, KAT2A, STAT3, GTF3C2, SIN3A, BDP1, PHC1, and EGR1 are the main central regulators of DEGs in mSSCs. In addition, we collectively detected two significant protein complexes in the protein-protein interactions network for DEGs regulators. Finally, this study introduces the major upstream kinases for the main central regulators of DEGs and the components of core protein complexes. In conclusion, this study provides a molecular blueprint to uncover the molecular mechanisms behind the biology of SSCs and offers a list of candidate factors for cell type conversion approaches and production of germ cells. © 2017 Wiley Periodicals, Inc.

  17. Genotyping-by-sequencing highlights original diversity patterns within a European collection of 1191 maize flint lines, as compared to the maize USDA genebank.

    PubMed

    Gouesnard, Brigitte; Negro, Sandra; Laffray, Amélie; Glaubitz, Jeff; Melchinger, Albrecht; Revilla, Pedro; Moreno-Gonzalez, Jesus; Madur, Delphine; Combes, Valérie; Tollon-Cordet, Christine; Laborde, Jacques; Kermarrec, Dominique; Bauland, Cyril; Moreau, Laurence; Charcosset, Alain; Nicolas, Stéphane

    2017-10-01

    Genotyping by sequencing is suitable for analysis of global diversity in maize. We showed the distinctiveness of flint maize inbred lines of interest to enrich the diversity of breeding programs. Genotyping-by-sequencing (GBS) is a highly cost-effective procedure that permits the analysis of large collections of inbred lines. We used it to characterize diversity in 1191 maize flint inbred lines from the INRA collection, the European Cornfed association panel, and lines recently derived from landraces. We analyzed the properties of GBS data obtained with different imputation methods, through comparison with a 50 K SNP array. We identified seven ancestral groups within the Flint collection (dent, Northern flint, Italy, Pyrenees-Galicia, Argentina, Lacaune, Popcorn) in agreement with breeding knowledge. Analysis highlighted many crosses between different origins and the improvement of flint germplasm with dent germplasm. We performed association studies on different agronomic traits, revealing SNPs associated with cob color, kernel color, and male flowering time variation. We compared the diversity of both our collection and the USDA collection which has been previously analyzed by GBS. The population structure of the 4001 inbred lines confirmed the influence of the historical inbred lines (B73, A632, Oh43, Mo17, W182E, PH207, and Wf9) within the dent group. It showed distinctly different tropical and popcorn groups, a sweet-Northern flint group and a flint group sub-structured in Italian and European flint (Pyrenees-Galicia and Lacaune) groups. Interestingly, we identified several selective sweeps between dent, flint, and tropical inbred lines that co-localized with SNPs associated with flowering time variation. The joint analysis of collections by GBS offers opportunities for a global diversity analysis of maize inbred lines.

  18. Comparative Analysis of Syntenic Genes in Grass Genomes Reveals Accelerated Rates of Gene Structure and Coding Sequence Evolution in Polyploid Wheat1[W][OA

    PubMed Central

    Akhunov, Eduard D.; Sehgal, Sunish; Liang, Hanquan; Wang, Shichen; Akhunova, Alina R.; Kaur, Gaganpreet; Li, Wanlong; Forrest, Kerrie L.; See, Deven; Šimková, Hana; Ma, Yaqin; Hayden, Matthew J.; Luo, Mingcheng; Faris, Justin D.; Doležel, Jaroslav; Gill, Bikram S.

    2013-01-01

    Cycles of whole-genome duplication (WGD) and diploidization are hallmarks of eukaryotic genome evolution and speciation. Polyploid wheat (Triticum aestivum) has had a massive increase in genome size largely due to recent WGDs. How these processes may impact the dynamics of gene evolution was studied by comparing the patterns of gene structure changes, alternative splicing (AS), and codon substitution rates among wheat and model grass genomes. In orthologous gene sets, significantly more acquired and lost exonic sequences were detected in wheat than in model grasses. In wheat, 35% of these gene structure rearrangements resulted in frame-shift mutations and premature termination codons. An increased codon mutation rate in the wheat lineage compared with Brachypodium distachyon was found for 17% of orthologs. The discovery of premature termination codons in 38% of expressed genes was consistent with ongoing pseudogenization of the wheat genome. The rates of AS within the individual wheat subgenomes (21%–25%) were similar to diploid plants. However, we uncovered a high level of AS pattern divergence between the duplicated homeologous copies of genes. Our results are consistent with the accelerated accumulation of AS isoforms, nonsynonymous mutations, and gene structure rearrangements in the wheat lineage, likely due to genetic redundancy created by WGDs. Whereas these processes mostly contribute to the degeneration of a duplicated genome and its diploidization, they have the potential to facilitate the origin of new functional variations, which, upon selection in the evolutionary lineage, may play an important role in the origin of novel traits. PMID:23124323

  19. Phase I safety and antigenicity of TA-GW: a recombinant HPV6 L2E7 vaccine for the treatment of genital warts.

    PubMed

    Thompson, H S; Davies, M L; Holding, F P; Fallon, R E; Mann, A E; O'Neill, T; Roberts, J S

    1999-01-01

    A phase I double-blind, randomized, placebo-controlled study was carried out in healthy subjects to assess the safety and immunogenicity of TA-GW, a recombinant HPV6 L2E7 fusion protein vaccine for the treatment of genital warts. Forty-two healthy male volunteers were randomised to receive three intramuscular injections of either 0, 3, 30 or 300 microg of recombinant L2E7 adsorbed onto Alhydrogel. Two vaccination schedules were compared: weeks 0, 1 and 4 (accelerated schedule) and weeks 0, 4 and 8 (classical schedule). Subjects were monitored for adverse events throughout. Immunogenicity was assessed by measuring L2E7 specific in vitro T cell proliferative responses, production of IFNgamma and IL-5 and serum antibodies. Dose-dependent and long-lived T and B cell immune responses were elicited by TA-GW with both vaccination schedules. In conclusion, TA-GW is both safe, well-tolerated and immunogenic. The results allow the selection of the 300-microg vaccine formulation and accelerated vaccination schedule for phase II trials in patients with genital warts.

  20. Antitumor HPV E7-specific CTL activity elicited by in vivo engineered exosomes produced through DNA inoculation.

    PubMed

    Di Bonito, Paola; Chiozzini, Chiara; Arenaccio, Claudia; Anticoli, Simona; Manfredi, Francesco; Olivetta, Eleonora; Ferrantelli, Flavia; Falcone, Emiliana; Ruggieri, Anna; Federico, Maurizio

    2017-01-01

    We recently proved that exosomes engineered in vitro to deliver high amounts of HPV E7 upon fusion with the Nef mut exosome-anchoring protein elicit an efficient anti-E7 cytotoxic T lymphocyte immune response. However, in view of a potential clinic application of this finding, our exosome-based immunization strategy was faced with possible technical difficulties including industrial manufacturing, cost of production, and storage. To overcome these hurdles, we designed an as yet unproven exosome-based immunization strategy relying on delivery by intramuscular inoculation of a DNA vector expressing Nef mut fused with HPV E7. In this way, we predicted that the expression of the Nef mut /E7 vector in muscle cells would result in a continuous source of endogenous (ie, produced by the inoculated host) engineered exosomes able to induce an E7-specific immune response. To assess this hypothesis, we first demonstrated that the injection of a Nef mut /green fluorescent protein-expressing vector led to the release of fluorescent exosomes, as detected in plasma of inoculated mice. Then, we observed that mice inoculated intramuscularly with a vector expressing Nef mut /E7 developed a CD8 + T-cell immune response against both Nef and E7. Conversely, no CD8 + T-cell responses were detected upon injection of vectors expressing either the wild-type Nef isoform of E7 alone, most likely a consequence of their inefficient exosome incorporation. The production of immunogenic exosomes in the DNA-injected mice was formally demonstrated by the E7-specific CD8 + T-cell immune response we detected in mice inoculated with exosomes isolated from plasma of mice inoculated with the Nef mut /E7 vector. Finally, we provide evidence that the injection of Nef mut /E7 DNA led to the generation of effective antigen-specific cytotoxic T lymphocytes whose activity was likely part of the potent, therapeutic antitumor effect we observed in mice implanted with TC-1 tumor cells. In summary, we established

  1. Wogonin induces apoptosis by suppressing E6 and E7 expressions and activating intrinsic signaling pathways in HPV-16 cervical cancer cells.

    PubMed

    Kim, Man Sub; Bak, Yesol; Park, Yun Sun; Lee, Dong Hun; Kim, Jung Hee; Kang, Jeong Woo; Song, Hyuk-Hwan; Oh, Sei-Ryang; Yoon, Do Young

    2013-08-01

    Wogonin is a flavonoid compound extracted from Scutellaria baicalensis and is well known as a benzodiazepine receptor ligand with anxiolytic effects. Many recent studies have demonstrated that wogonin modulates angiogenesis, proliferation, invasion, and tumor progress in various cancer tissues. We further explored the mechanism of action of wogonin on cervical cancer cells that contain or lack human papillomavirus (HPV) DNA. Wogonin was cytotoxic to HPV 16 (+) cervical cancer cells, SiHa and CaSki, but not to HPV-negative cells. We demonstrated that wogonin induced apoptosis by suppressing the expressions of the E6 and E7 viral oncogenes in HPV-infected cervical cancer CaSki and SiHa cells. The modulation of p53 and protein retinoblastoma (pRb) were also triggered by the suppression of E6 and E7 expressions. However, p53 was not altered in HPV-negative cervical cancer C33A cells. Moreover, wogonin modulated the mitochondrial membrane potential and the expression of pro- and anti-apoptotic factors such as Bax and Bcl-2. Wogonin also provoked the cleavage of caspase-3, caspase-9, and poly ADP ribose polymerase. After transfection of siRNAs to target E6 and E7, additional restoration of p53 and pRb was not induced, but processing of caspases and PARP was increased compared with wogonin treatment alone. Together, our findings demonstrated that wogonin effectively promotes apoptosis by downregulating E6 and E7 expressions and promoting intrinsic apoptosis in human cervical cancer cells.

  2. Biosynthesis of the Cyanogenic Glucosides Linamarin and Lotaustralin in Cassava: Isolation, Biochemical Characterization, and Expression Pattern of CYP71E7, the Oxime-Metabolizing Cytochrome P450 Enzyme1[OA

    PubMed Central

    Jørgensen, Kirsten; Morant, Anne Vinther; Morant, Marc; Jensen, Niels Bjerg; Olsen, Carl Erik; Kannangara, Rubini; Motawia, Mohammed Saddik; Møller, Birger Lindberg; Bak, Søren

    2011-01-01

    Cassava (Manihot esculenta) is a eudicotyledonous plant that produces the valine- and isoleucine-derived cyanogenic glucosides linamarin and lotaustralin with the corresponding oximes and cyanohydrins as key intermediates. CYP79 enzymes catalyzing amino acid-to-oxime conversion in cyanogenic glucoside biosynthesis are known from several plants including cassava. The enzyme system converting oxime into cyanohydrin has previously only been identified in the monocotyledonous plant great millet (Sorghum bicolor). Using this great millet CYP71E1 sequence as a query in a Basic Local Alignment Search Tool-p search, a putative functional homolog that exhibited an approximately 50% amino acid sequence identity was found in cassava. The corresponding full-length cDNA clone was obtained from a plasmid library prepared from cassava shoot tips and was assigned CYP71E7. Heterologous expression of CYP71E7 in yeast afforded microsomes converting 2-methylpropanal oxime (valine-derived oxime) and 2-methylbutanal oxime (isoleucine-derived oxime) to the corresponding cyanohydrins, which dissociate into acetone and 2-butanone, respectively, and hydrogen cyanide. The volatile ketones were detected as 2.4-dinitrophenylhydrazone derivatives by liquid chromatography-mass spectrometry. A KS of approximately 0.9 μm was determined for 2-methylbutanal oxime based on substrate-binding spectra. CYP71E7 exhibits low specificity for the side chain of the substrate and catalyzes the conversion of aliphatic and aromatic oximes with turnovers of approximately 21, 17, 8, and 1 min−1 for the oximes derived from valine, isoleucine, tyrosine, and phenylalanine, respectively. A second paralog of CYP71E7 was identified by database searches and showed approximately 90% amino acid sequence identity. In tube in situ polymerase chain reaction showed that in nearly unfolded leaves, the CYP71E7 paralogs are preferentially expressed in specific cells in the endodermis and in most cells in the first cortex cell

  3. 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

  4. The binding of histone deacetylases and the integrity of zinc finger-like motifs of the E7 protein are essential for the life cycle of human papillomavirus type 31.

    PubMed

    Longworth, Michelle S; Laimins, Laimonis A

    2004-04-01

    The E7 oncoprotein of high-risk human papillomaviruses (HPVs) binds to and alters the action of cell cycle regulatory proteins such as members of the retinoblastoma (Rb) family of proteins as well as the histone deacetylases (HDACs). To examine the significance of the binding of E7 to HDACs in the viral life cycle, a mutational analysis of the E7 open reading frame was performed in the context of the complete HP