Non-repetitive Regions Research Articles

A-to-I RNA editing promotes developmental stage–specific gene and lncRNA expression

A-to-I RNA editing is a conserved widespread phenomenon in which adenosine (A) is converted to inosine (I) by adenosine deaminases (ADARs) in double-stranded RNA regions, mainly noncoding. Mutations in ADAR enzymes in Caenorhabditis elegans cause defects in normal development but are not lethal as in human and mouse. Previous studies in C. elegans indicated competition between RNA interference (RNAi) and RNA editing mechanisms, based on the observation that worms that lack both mechanisms do not exhibit defects, in contrast to the developmental defects observed when only RNA editing is absent. To study the effects of RNA editing on gene expression and function, we established a novel screen that enabled us to identify thousands of RNA editing sites in nonrepetitive regions in the genome. These include dozens of genes that are edited at their 3′ UTR region. We found that these genes are mainly germline and neuronal genes, and that they are down-regulated in the absence of ADAR enzymes. Moreover, we discovered that almost half of these genes are edited in a developmental-specific manner, indicating that RNA editing is a highly regulated process. We found that many pseudogenes and other lncRNAs are also extensively down-regulated in the absence of ADARs in the embryo but not in the fourth larval (L4) stage. This down-regulation is not observed upon additional knockout of RNAi. Furthermore, levels of siRNAs aligned to pseudogenes in ADAR mutants are enhanced. Taken together, our results suggest a role for RNA editing in normal growth and development by regulating silencing via RNAi.

High-Throughput Yeast Strain Sequencing.

The original yeast genome sequencing project was a monumental task, spanning several years, which resulted in the first sequenced eukaryotic genome. The 12 Mbp reference sequence was generated from yeast strain S288c and was of extremely high quality. In the years since it was published, sequencing technology has advanced apace, such that it is within the reach of most labs to sequence yeast strains of interest almost as a matter of standard practice, either via core facilities at their institution or through commercial sequencing services. Because of the availability of the high-quality reference sequence (which itself has received approximately 1500 updates derived from high-throughput sequencing data), reliable identification of differences between a strain of interest and the reference is relatively straightforward, at least for the nonrepetitive regions of the genome. In this introduction, we describe current high-throughput sequencing technology and methods for analysis of the resulting data.

Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors.

Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins.

Potentially functional polypeptides for modification of biomaterials

Event Abstract Back to Event Potentially functional polypeptides for modification of biomaterials Jiannan Wang1, Mingyang Wu1, Yunfei Liu1 and Fangfang Tu1 1 Soochow University, National Engineering Laboratory for Modern Silk, China Potentially functional polypeptides for modification of biomaterials Mingyang Wu, Yunfei Liu, Fangfang Tu, Jiannan Wang* National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No. 199 Ren-ai Road, Suzhou Industrial Park, Suzhou, Jiangsu Province, 215123, China *Corresponding author: Jiannan Wang, wangjn@suda.edu.cn Silk fibroin is a native protein with regular amino acid sequence. Such as, Bombyx mori silk fibroin is composed of 12 repetitive and 11 non-repetitive regions, with the non-repetitive domain consisting of a hydrophilic polypeptide chain. The RGD tripeptides distribute in Antheraea yamamai and Antheraea pernyi silk fibroins up to 14 and 12 repeats, respectively. In order to determine the biomedical function of these typical peptides or potentially use them to modify materials, we cloned and extended a gene motif (f(1)) encoding the non-repetitive domain of Bombyx mori silk fibroin and a gene motif (-rgd-) encoding RGD-contained domain of Antheraea yamamai or Antheraea pernyi silk fibroins. These motif and their multimers were inserted into a glutathione S-transferase (GST)-tagged fusion-protein expression vector. Motif (f(1)) and multimers (f(4), f(8), (-rgd-)4, and (-rgd-)8) were expressed in Escherichia coli BL21 cells following isopropyl β-D-1-thiogalactopyranoside induction, purified by GST-affinity chromatography. All express products (GST-F(1), GST-F(4), GST-F(8), GST-(-RGD-)4, and GST-(-RGD-)8) were successfully expressed confirmed by SDS-PAGE, mass spectrometry (MS), amino acid composition and charge assay. The peptides F(1), F(4), F(8), (-RGD-)4, and (-RGD-)8 were all cleaved clearly from the GST-fusion tag following thrombin digestion. The molecular weight by MS, amino acid composition and isoelectric points of released peptides were all consistent with the predicted values. The peptides F(1), F(4), and F(8) were used to modify dacron membrane. Results showed F(1), F(4), and F(8) significantly improved water contact angle of dacron, and increased rapid adhesion and proliferation ability of cells. Cell adhesion and cell proliferation on (-RGD-)4 and (-RGD-)8 modified Bombyx mori silk fibroin films were better than that on unmodified film. The study indicated that the express products of F(1), F(4), and F(8) could improve the hydrophilicity of hydrophobe materials for better application in biomaterials. F(1), F(4), and F(8) exhibited a negative ζ-potential and the measured pI was about 3.3, 3.2 and 3.0, respectively, expecting to be used in vascular tissue engineering. The express products (-RGD-)4 and (-RGD-)8 could be used to modify cellular interface materials to improve cell adhesion and growth on materials surface. Acknowledgements This work was supported by National Natural Science Foundation of China (Nos. 51173125 and 51473108), Natural Science Foundation of Jiangsu Province of China (No. BK2012633), College Natural Science Research Project of Jiangsu Province of China (No. 12KJA43004). Refences [1] Y. X. Yang, H. Y. Huang, Z. F. Tian, et al. Textile Bioengineering and Informatics Symposium Proceedings, 2012, 70-75. [2] H. Y. Huang, Z. F. Tian, Y. X. Yang, et al. Journal of Donghua University (English Edition ), 2012, 29: 26-29. [3] H. R. Zhao, Y. X. Yang, H. G. Yi, et al. Bio-Medical Materials and Engineering, 2014, 24: 2057–2064. Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Adhesive biomaterials Citation: Wang J, Wu M, Liu Y and Tu F (2016). Potentially functional polypeptides for modification of biomaterials. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02647 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jiannan Wang Mingyang Wu Yunfei Liu Fangfang Tu Google Jiannan Wang Mingyang Wu Yunfei Liu Fangfang Tu Google Scholar Jiannan Wang Mingyang Wu Yunfei Liu Fangfang Tu PubMed Jiannan Wang Mingyang Wu Yunfei Liu Fangfang Tu Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Perm-seq: Mapping Protein-DNA Interactions in Segmental Duplication and Highly Repetitive Regions of Genomes with Prior-Enhanced Read Mapping.

Segmental duplications and other highly repetitive regions of genomes contribute significantly to cells’ regulatory programs. Advancements in next generation sequencing enabled genome-wide profiling of protein-DNA interactions by chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq). However, interactions in highly repetitive regions of genomes have proven difficult to map since short reads of 50–100 base pairs (bps) from these regions map to multiple locations in reference genomes. Standard analytical methods discard such multi-mapping reads and the few that can accommodate them are prone to large false positive and negative rates. We developed Perm-seq, a prior-enhanced read allocation method for ChIP-seq experiments, that can allocate multi-mapping reads in highly repetitive regions of the genomes with high accuracy. We comprehensively evaluated Perm-seq, and found that our prior-enhanced approach significantly improves multi-read allocation accuracy over approaches that do not utilize additional data types. The statistical formalism underlying our approach facilitates supervising of multi-read allocation with a variety of data sources including histone ChIP-seq. We applied Perm-seq to 64 ENCODE ChIP-seq datasets from GM12878 and K562 cells and identified many novel protein-DNA interactions in segmental duplication regions. Our analysis reveals that although the protein-DNA interactions sites are evolutionarily less conserved in repetitive regions, they share the overall sequence characteristics of the protein-DNA interactions in non-repetitive regions.

Biosynthesis and characterization of a non-repetitive polypeptide derived from silk fibroin heavy chain

Silk fibroin heavy chain is the major protein component of Bombyx mori silk fibroin and is composed of 12 repetitive and 11 non-repetitive regions, with the non-repetitive domain consisting of a hydrophilic polypeptide chain. In order to determine the biomedical function of the non-repetitive domain or potentially use it to modify hydrophobic biomaterials, high-purity isolation is necessary. Previously, we cloned and extended a gene motif (f(1)) encoding the non-repetitive domain. Here, this motif and its multimers are inserted into a glutathione S-transferase (GST)-tagged fusion-protein expression vector. Motif f(1) and multimers f(4) and f(8) were expressed in Escherichia coli BL21 cells following isopropyl β-D-1-thiogalactopyranoside induction, purified by GST-affinity chromatography, and single bands of purified fusion proteins GST-F(1), GST-F(4), and GST-F(8), were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Target polypeptides F(1), F(4), and F(8), were cleaved clearly from the GST-fusion tag following thrombin digestion. Mass spectrometry results indicate that the molecular weights associated with fusion proteins GST-F(1), GST-F(4), and GST-F(8) are 31.5, 43.8, and 59.0kDa, respectively, and with the cleaved polypeptides F(1), F(4), and F(8) are 4.8, 16.8, and 32.8kDa, respectively. The F(1), F(4), and F(8) polypeptide chains are negatively charged with isoelectric points (pI) of 3.3, 3.2, and 3.0, respectively. The molecular weight and pI values of the polypeptide chains are consistent with the predicted values and the amino acid compositions similar to predicted sequences. FTIR and CD results show the molecular conformation of F(1) was mainly random coil, and more stable α-helix structure formed in longer molecular chain.

Abstract 4602: Exome sequencing of UKLS lung cancer CT screened early stage cancers

Abstract INTRODUCTION Developments in both low-dose CT-screening within populations at high risk of lung cancer and sequencing analysis of lung tumours have improved the prospects for early detection and clinical management of lung cancer. Such approaches potentially could provide biological insight into the early stages of lung cancer development, which until now have been refractory to genomic analysis, since lung tumours are rarely identified at a presymptomatic timepoint. BRIEF DESCRIPTION OF PERTINENT EXPERIMENTAL PROCEDURES Individuals with at least a 5% risk of developing lung cancer within 5 years (according to the LLPv2 lung cancer risk score) were recruited to the UK Lung low dose CT Screening (UKLS) trial. Ten of the early stage NSCLC UKLS tumours were prepared for exome sequencing. Tumour DNA was extracted from FFPE material and DNA was also extracted from matched blood samples. Tumour and blood libraries were prepared using an Agilent SureSelect exome-capture kit and sequenced to 100x and 50x, depth respectively, on the Illumina HiSeq platform (Oxford Gene Technology, UK). Sequence data were aligned against the GRCh37 human reference and matched samples were subject to local realignment in pairs. Somatic variants were identified using a combination of EBCall, MuTect and VarScan2 and were post-filtered to remove false positive variants (alignment artifacts and FFPE-hypersensitive sites) by comparison between samples. TCGA (The Cancer Genome Atlas) lung adenocarcinoma and squamous cell carcinoma samples from smokers and former smokers (who had complete data for a minimal set of clinical and epidemiological variables) were compared to the UKLS samples described above. To ensure comparable genomic regions were studied, both the UKLS and TCGA somatic variants were restricted to protein-coding regions covered by the Agilent SureSelect array but disjoint from simple-repetitive regions. SUMMARY OF THE NEW, UNPUBLISHED DATA Despite detection at an early stage, somatic mutations were identified in all CT-detected tumours, including non-synonymous variants in known driver genes. Between 71 and 471 variants were detected within non-repetitive protein-coding regions, which is comparable to, if somewhat lower than, the number of variants in the same regions in the TCGA ever-smoker samples (p = 0.17; Kruskal-Wallis). The results suggest that the majority of somatic variants exist in lung tumours prior to the evolution of symptoms and are consistent with recent studies of lung cancer heterogeneity wherein the majority of mutations occur prior to subclonal branching. STATEMENT OF CONCLUSIONS Somatic variations in the host genome are readily detected in FFPE material from pre-symptomatic lung tumours. Mutational burden is comparable to lung adenocarcinoma and squamous carcinoma from mature symptomatic individuals providing further evidence for the long latency of lung tumour development. Citation Format: Russell Hyde, Michael Davies, Martin Ledson, John A. Holemans, Richard D. Page, John Gosney, David R. Baldwin, Anand Devaraj, David M. Hansell, Stephen W. Duffy, John K. Field. Exome sequencing of UKLS lung cancer CT screened early stage cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4602. doi:10.1158/1538-7445.AM2015-4602

Development of a novel molecular detection method for clustered regularly interspaced short palindromic repeats (CRISPRs) in Taylorella organisms.

Contagious equine metritis is a bacterial infectious disease of horses caused by Taylorella equigenitalis, a Gram-negative eubacterium. The disease has been described in several continents, including Europe, North America and Asia. A novel molecular method was developed to detect clustered regularly interspaced short palindromic repeats (CRISPRs), which were separated by non-repetitive unique spacer regions (NRUSRs) of similar length, in the Taylorella equigenitalis EQ59 strain using a primer pair, f-/r-TeCRISPR-ladder, by PCR amplification. In total, 31 Taylorella isolates (17 T. equigenitalis and 14 Taylorella asinigenitalis) were examined. The T. equigenitalis isolates came from thoroughbred and cold-blooded horses from nine countries during 1980-1996, whilst the T. asinigenitalis isolates all originated from donkey jacks in France and the USA during 1997-2006. PAGE fractionated all of the 13 CRISPRs separated by 12 NRUSRs in T. equigenitalis EQ59. Permutation examples of CRISPRs, which were separated by NRUSRs for small-sized ladders, consisting of two doublet bands were shown. Putative CRISPRs separated by NRUSRs were amplified with 14/17 (82.4 %) geographically disparate T. equigenitalis isolates using the newly designed primer pair. Approximately 82.4 % of the T. equigenitalis isolates had CRISPRs separated by NRUSRs. The CRISPR locus was also found in the French T. asinigenitalis strain MCE3. Putative CRISPRs separated by NRUSRs were detected similarly in 4/14 (28.6 %) T. asinigenitalis isolates. Overall, a more detailed understanding of the molecular biology of CRISPRs within Taylorella organisms may help elucidate the pathogenic virulence and transmission mechanisms associated with this important equine pathogen.

Knowledge in the Investigation of A-to-I RNA Editing Signals

RNA editing is a post-transcriptional alteration of RNA sequences that is able to affect protein structure as well as RNA and protein expression. Adenosine-to-inosine (A-to-I) RNA editing is the most frequent and common post-transcriptional modification in human, where adenosine (A) deamination produces its conversion into inosine (I), which in turn is interpreted by the translation and splicing machineries as guanosine (G). The disruption of the editing machinery has been associated to various human diseases such as cancer or neurodegenerative diseases. This biological phenomenon is catalyzed by members of the adenosine deaminase acting on RNA (ADAR) family of enzymes and occurs on dsRNA structures. Despite the enormous efforts made in the last decade, the real biological function underlying such a phenomenon, as well as ADAR’s substrate features still remain unknown. In this work, we summarize the major computational aspects of predicting and understanding RNA editing events. We also investigate the detection of short motif sequences potentially characterizing RNA editing signals and the use of a logistic regression technique to model a predictor of RNA editing events. The latter, named AIRlINER, an algorithmic approach to assessment of A-to-I RNA editing sites in non-repetitive regions, is available as a web app at: http://alpha.dmi.unict.it/airliner/. Results and comparisons with the existing methods encourage our findings on both aspects.

P15 Directional Next Generation Whole Transcriptome Sequencing Of Primary Human Pulmonary Microvascular Endothelial Cells

Introduction and objectives To improve our understanding of the dynamic interplay between non-coding and coding RNA species, we developed solution-based methods to capture the entire endothelial transcriptome. In contrast to microarrays and CHIP-based methodologies, there was no pre-specification of RNA target sequences, and the identity of the DNA strand of origin was preserved. Methods Triplicate confluent primary human microvascular endothelial cells (Promocell GmbH) were cultured in the presence and absence of TGF-beta stimuli relevant to pulmonary arteriovenous malformations and hereditary haemorrhagic telangiectasia (HHT). Following rRNA-depletion of total RNA, seven libraries were prepared using Illumina reagents, and 8 pM of each library used for cluster generation and sequencing on Genome Analyser II. Algorithms for aligning reads to NCBI36/hg18 and GRCh37/hg19 included Bowtie, TopHat and Seqmap. Validations were performed using quantitative rt-PCR. Results More than 2 Gigabases of sequence was generated. Transcriptome-wide profiles were similar between libraries, with sixteen types of RNA species detected including 146 micro (mi)RNA families (47 broadly conserved), and 10,749 protein-encoding mRNAs representing ~5.5% of mapped reads. Alignments to endothelial mRNAs/miRNAs were substantially higher than to gene loci for non-endothelial mRNAs/miRNAs. mRNA exon alignments demonstrated sharp exon boundary delineation, but replicate alignments to non-repetitive intronic regions involved in multi-exon deletions in HHT patients. There was an inverse relationship between alignments depths and qt-PCT cycle thresholds (Ct), where single alignments were detectable, and Ct values of 20 generated by 0.02 nM spiked RNA. Across all experiments in replicate donor/treatment RNAs, for a panel of single open reading frame miRNA genes, RNASeq alignments (gene strand read counts normalised to the total number of valid reads and exon/locus size) explained 72% of the variance of qt-PCR cycle threshold (p Conclusions These novel directional next generation RNA sequencing methods provide new insights for mutational mechanisms, and a systems approach to dissection of regulatory and target RNA networks relevant to human disease.

G-Quadruplex (G4) Motifs in the Maize (Zea mays L.) Genome Are Enriched at Specific Locations in Thousands of Genes Coupled to Energy Status, Hypoxia, Low Sugar, and Nutrient Deprivation

The G-quadruplex (G4) elements comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This G4 DNA can form within or across single-stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The reversibility and structural diversity of G4s make them highly versatile genetic structures, as demonstrated by their roles in various functions including telomere metabolism, genome maintenance, immunoglobulin gene diversification, transcription, and translation. Sequence motifs capable of forming G4 DNA are typically located in telomere repeat DNA and other non-telomeric genomic loci. To investigate their potential roles in a large-genome model plant species, we computationally identified 149,988 non-telomeric G4 motifs in maize (Zea mays L., B73 AGPv2), 29% of which were in non-repetitive genomic regions. G4 motif hotspots exhibited non-random enrichment in genes at two locations on the antisense strand, one in the 5′ UTR and the other at the 5′ end of the first intron. Several genic G4 motifs were shown to adopt sequence-specific and potassium-dependent G4 DNA structures in vitro. The G4 motifs were prevalent in key regulatory genes associated with hypoxia (group VII ERFs), oxidative stress (DJ-1/GATase1), and energy status (AMPK/SnRK) pathways. They also showed statistical enrichment for genes in metabolic pathways that function in glycolysis, sugar degradation, inositol metabolism, and base excision repair. Collectively, the maize G4 motifs may represent conditional regulatory elements that can aid in energy status gene responses. Such a network of elements could provide a mechanistic basis for linking energy status signals to gene regulation in maize, a model genetic system and major world crop species for feed, food, and fuel.

Diversity arrays technology (DArT) for studying the genetic polymorphism of flue-cured tobacco (Nicotiana tabacum)

Diversity arrays technology (DArT) is a microarray-based marker system that achieves high throughput by reducing the complexity of the genome. A DArT chip has recently been developed for tobacco. In this study, we genotyped 267 flue-cured cultivars/landraces, including 121 Chinese accessions over five decades from widespread geographic regions in China, 103 from the Americas, and 43 other foreign cultivars, using the newly developed chip. Three hundred and thirty polymorphic DArT makers were selected and used for a phylogenetic analysis, which suggested that the 267 accessions could be classified into two subgroups, which could each be further divided into 2‒4 sections. Eight elite cultivars, which account for 83% of the area of Chinese tobacco production, were all found in one subgroup. Two high-quality cultivars, HHDJY and Cuibi1, were grouped together in one section, while six other high-yield cultivars were grouped into another section. The 330 DArT marker clones were sequenced and close to 95% of them are within non-repetitive regions. Finally, the implications of this study for Chinese flue-cured tobacco breeding and production programs were discussed.

Structural and Functional Analysis of the N-terminal Domain of the Streptococcus gordonii Adhesin Sgo0707

The commensal Streptococcus gordonii expresses numerous surface adhesins with which it interacts with other microorganisms, host cells and salivary proteins to initiate dental plaque formation. However, this Gram-positive bacterium can also spread to non-oral sites such as the heart valves and cause infective endocarditis. One of its surface adhesins, Sgo0707, is a large protein composed of a non-repetitive N-terminal region followed by several C-terminal repeat domains and a cell wall sorting motif. Here we present the crystal structure of the Sgo0707 N-terminal domains, refined to 2.1 Å resolution. The model consists of two domains, N1 and N2. The largest domain, N1, comprises a putative binding cleft with a single cysteine located in its centre and exhibits an unexpected structural similarity to the variable domains of the streptococcal Antigen I/II adhesins. The N2-domain has an IgG-like fold commonly found among Gram-positive surface adhesins. Binding studies performed on S. gordonii wild-type and a Sgo0707 deficient mutant show that the Sgo0707 adhesin is involved in binding to type-1 collagen and to oral keratinocytes.

Adaptive Mutations and Replacements of Virulence Traits in the Escherichia coli O104:H4 Outbreak Population

The sequencing of highly virulent Escherichia coli O104:H4 strains isolated during the outbreak of bloody diarrhea and hemolytic uremic syndrome in Europe in 2011 revealed a genome that contained a Shiga toxin encoding prophage and a plasmid encoding enteroaggregative fimbriae. Here, we present the draft genome sequence of a strain isolated in Sweden from a patient who had travelled to Tunisia in 2010 (E112/10) and was found to differ from the outbreak strains by only 38 SNPs in non-repetitive regions, 16 of which were mapped to the branch to the outbreak strain. We identified putatively adaptive mutations in genes for transporters, outer surface proteins and enzymes involved in the metabolism of carbohydrates. A comparative analysis with other historical strains showed that E112/10 contained Shiga toxin prophage genes of the same genotype as the outbreak strain, while these genes have been replaced by a different genotype in two otherwise very closely related strains isolated in the Republic of Georgia in 2009. We also present the genome sequences of two enteroaggregative E. coli strains affiliated with phylogroup A (C43/90 and C48/93) that contain the agg genes for the AAF/I-type fimbriae characteristic of the outbreak population. Interestingly, C43/90 also contained a tet/mer antibiotic resistance island that was nearly identical in sequence to that of the outbreak strain, while the corresponding island in the Georgian strains was most similar to E. coli strains of other serotypes. We conclude that the pan-genome of the outbreak population is shared with strains of the A phylogroup and that its evolutionary history is littered with gene replacement events, including most recently independent acquisitions of antibiotic resistance genes in the outbreak strains and its nearest neighbors. The results are summarized in a refined evolutionary model for the emergence of the O104:H4 outbreak population.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in Campylobacter lari

PCR amplifications using primers for the clustered regularly interspaced short palindromic repeats (CRISPRs)-associated gene 1 (cas1), cas2, putative (p)-cas and CRISPRs genes generated cas1, cas2, p-cas and CRISPRs genes segments with 9–28 of 28 urease-positive thermophilic Campylobacter (UPTC) isolates, respectively. The p-cas and CRISPRs genes segments were amplified with 10 of 11 and 0 of 11 urease-negative (UN) Campylobacter lari isolates, respectively. When the nucleotide sequences of the CRISPRs consensus sequence repeats of each 33–37 base pairs from the 18 Campylobacter jejuni isolates were aligned, as well as from the four C. jejuni reference and UPTC CF89-12 strains, the repeats were identified as being almost identical. Although a total of all 18 C. jejuni isolates examined gave PCR-positive signals for the CRISPRs genes, it was, interestingly, suggested that many numbers of C. lari and C. jejuni isolates may possibly carry cas but not CRISPRs genes within their CRISPRs loci. In addition, PCR amplification by using a novel primer pair of f-ClCRISPR-ladder and ClCRISPRs-R, which were novel to this study, with the UPTC CF89-12 strain was shown to be useful for the detection of the putative CRISPRs separated by the non-repetitive unique spacer regions, with the electrophoretic ladder DNA profile following 5.0 % polyacrylamide gel electrophoresis. Secondary structure models of the CRISPRs repeats were predicted with UPTC CF89-12 and two C. jejuni strains.

Abstract 2020: Next-generation sequencing for mutation discovery in Ewing sarcoma.

Abstract Background: Ewing sarcoma (EWS) is the second most common type of primary bone tumor to affect children and adolescent and accounts for 2.9% of all childhood cancers. Despite advances in multidisciplinary treatment leading to improved outcomes over time, long term survival still remains less than 50% for patients older than 15 years of age and only 65% in younger patients. In an effort to identify novel prognostic and therapeutic targets, we utilized next generation sequencing techniques to evaluate EWS samples for somatic mutation. Methods: Whole genome paired-end sequencing using the Complete Genomics method was performed on the DNA from 6 EWS matched tumor-normal samples. Somatic mutations were identified and classified using standard bioinformatics techniques and mutations of interest were verified using capillary sequencing. To extend discoveries and evaluate for mutational recurrence, targeted sequencing was performed on a validation cohort of 61 EWS tumors and 22 EWS cell lines using a custom multiplex PCR design followed by high-coverage sequencing on the Ion Torrent system. Results: In the discovery cohort, we detected an average of 361 somatic mutations per tumor in non-repetitive regions and an average of 6 somatic mutations per tumor in protein coding regions (=0.15 mutations/Mb of coding sequence). The well-established FLI1-EWSR1 gene fusion was detected by the whole genome sequencing in all 6 samples. Additional previously reported genetic events were also seen including frequent chromosomal trisomy and deletion of the CDKN2A containing locus on chromosomal region 9p21 in 2 of 6 samples. In the validation cohort of tumors, known recurrent mutations such as in TP53 (3/61 = 4.9%) were seen at rates similar to that previously reported in EWS. Continued efforts are underway to verify novel recurrent mutations. Conclusion: Next-generation sequencing provides a powerful tool to characterize the genetic landscape and evaluate for recurrent mutations in cancer. We utilize this tool in Ewing sarcoma in a project that provides both a comprehensive genomic evaluation from whole genome sequencing data and as well evaluates for recurrent mutations using a targeted sequencing approach. Citation Format: Andrew S. Brohl, Young Song, Laura Hurd, Hongling Liao, Li Chen, Rajesh Patidar, John F. Shern, Jun S. Wei, Javed Khan. Next-generation sequencing for mutation discovery in Ewing sarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2020. doi:10.1158/1538-7445.AM2013-2020

Prediction of constitutive A-to-I editing sites from human transcriptomes in the absence of genomic sequences

BackgroundAdenosine-to-inosine (A-to-I) RNA editing is recognized as a cellular mechanism for generating both RNA and protein diversity. Inosine base pairs with cytidine during reverse transcription and therefore appears as guanosine during sequencing of cDNA. Current approaches of RNA editing identification largely depend on the comparison between transcriptomes and genomic DNA (gDNA) sequencing datasets from the same individuals, and it has been challenging to identify editing candidates from transcriptomes in the absence of gDNA information.ResultsWe have developed a new strategy to accurately predict constitutive RNA editing sites from publicly available human RNA-seq datasets in the absence of relevant genomic sequences. Our approach establishes new parameters to increase the ability to map mismatches and to minimize sequencing/mapping errors and unreported genome variations. We identified 695 novel constitutive A-to-I editing sites that appear in clusters (named “editing boxes”) in multiple samples and which exhibit spatial and dynamic regulation across human tissues. Some of these editing boxes are enriched in non-repetitive regions lacking inverted repeat structures and contain an extremely high conversion frequency of As to Is. We validated a number of editing boxes in multiple human cell lines and confirmed that ADAR1 is responsible for the observed promiscuous editing events in non-repetitive regions, further expanding our knowledge of the catalytic substrate of A-to-I RNA editing by ADAR enzymes.ConclusionsThe approach we present here provides a novel way of identifying A-to-I RNA editing events by analyzing only RNA-seq datasets. This method has allowed us to gain new insights into RNA editing and should also aid in the identification of more constitutive A-to-I editing sites from additional transcriptomes.

Mosaic Evolution of Silk Genes in Aliatypus Trapdoor Spiders (Mygalomorphae, Antrodiaetidae)

Spider silk genes are composed mostly of repetitive sequence that is flanked by non-repetitive terminal regions. Inferences about the evolutionary processes that influenced silk genes have largely been made from analyses using distantly related taxa and ancient silk gene duplicates. These studies have relied on comparisons across the conserved non-repetitive terminal regions to determine orthologous and paralogous relationships, as well as the influence of selection on silk genes. While the repetitive region heavily influences silk fiber mechanical properties, few molecular evolutionary analyses have been conducted on this region due to difficulty in determining homology. Here, we sample internal repetitive and carboxy terminal regions from all extant species of the trapdoor spider genus, Aliatypus. Aliatypus spiders are highly dispersal limited and rely on their silk lined burrow for protection. We determine positional homology across species for the carboxy terminal regions and relative positional homology for the internal repetitive regions. Gene trees based on each of these regions are in good agreement with the Aliatypus species tree, which indicates we sampled single spidroin orthologs in each species. In addition, we find that purifying selection and concerted evolution have acted to conserve Aliatypus spidroin internal repetitive regions. In contrast, selection testing identifies evidence of sites that evolved under positive selection and amino acid replacements that result in radical physicochemical changes in the carboxy terminal region. These findings indicate that comparison of spidroin orthologs across a comprehensive sample of congenerics reveal molecular evolutionary patterns obscured from studies using higher-level sampling of silk encoding genes.

Full-Length Minor Ampullate Spidroin Gene Sequence

Spider silk includes seven protein based fibers and glue-like substances produced by glands in the spider's abdomen. Minor ampullate silk is used to make the auxiliary spiral of the orb-web and also for wrapping prey, has a high tensile strength and does not supercontract in water. So far, only partial cDNA sequences have been obtained for minor ampullate spidroins (MiSps). Here we describe the first MiSp full-length gene sequence from the spider species Araneus ventricosus, using a multidimensional PCR approach. Comparative analysis of the sequence reveals regulatory elements, as well as unique spidroin gene and protein architecture including the presence of an unusually large intron. The spliced full-length transcript of MiSp gene is 5440 bp in size and encodes 1766 amino acid residues organized into conserved nonrepetitive N- and C-terminal domains and a central predominantly repetitive region composed of four units that are iterated in a non regular manner. The repeats are more conserved within A. ventricosus MiSp than compared to repeats from homologous proteins, and are interrupted by two nonrepetitive spacer regions, which have 100% identity even at the nucleotide level.

LIN28 Binds Messenger RNAs at GGAGA Motifs and Regulates Splicing Factor Abundance

LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.