Protein Crystal Eco R1 Endonulease-DNA Complex

NASA Technical Reports Server (NTRS)

1998-01-01

Type II restriction enzymes, such as Eco R1 endonulease, present a unique advantage for the study of sequence-specific recognition because they leave a record of where they have been in the form of the cleaved ends of the DNA sites where they were bound. The differential behavior of a sequence -specific protein at sites of differing base sequence is the essence of the sequence-specificity; the core question is how do these proteins discriminate between different DNA sequences especially when the two sequences are very similar. Principal Investigator: Dan Carter/New Century Pharmaceuticals

PHOTOSYNTHETIC PIGMENT AND GENETIC DIFFERENCES BETWEEN TWO SOUTHERN OCEAN MORPHOTYPES OF EMILIANIA HUXLEYI (HAPTOPHYTA)1.

PubMed

Cook, Suellen S; Whittock, Lucy; Wright, Simon W; Hallegraeff, Gustaaf M

2011-06-01

The widespread coccolithophorid Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler plays a pivotal role in the carbon pump and is known to exhibit significant morphological, genetic, and physiological diversity. In this study, we compared photosynthetic pigments and morphology of triplicate strains of Southern Ocean types A and B/C. The two morphotypes differed in width of coccolith distal shield elements (0.11-0.24 μm, type A; 0.06-0.12 μm, type B/C) and morphology of distal shield central area (grill of curved rods in type A; thin plain plate in type B/C) and showed differences in carotenoid composition. The mean 19'-hexanoyloxyfucoxanthin (Hex):chl a ratio in type B/C was >1, whereas the type A ratio was <1. The Hex:fucoxanthin (fuc) ratio for type B/C was 11 times greater than that for type A, and the proportion of fuc in type A was 6 times higher than that in type B/C. The fuc derivative 4-keto-19'-hexanoyloxyfucoxanthin (4-keto-hex) was present in type A but undetected in B/C. DNA sequencing of tufA distinguished morphotypes A, B/C (indistinguishable from B), and R, while little variation was observed within morphotypes. Thirty single nucleotide polymorphisms were identified in the 710 bp tufA sequence, of which 10 alleles were unique to B/C and B morphotypes, seven alleles were unique to type A, and six alleles were unique to type R. We propose that the morphologically, physiologically, and genetically distinct Southern Ocean type B/C sensu Young et al. (2003) be classified as E. huxleyi var. aurorae var. nov. S. S. Cook et Hallegr. © 2011 Phycological Society of America.

Ketide Synthase (KS) Domain Prediction and Analysis of Iterative Type II PKS Gene in Marine Sponge-Associated Actinobacteria Producing Biosurfactants and Antimicrobial Agents

PubMed Central

Selvin, Joseph; Sathiyanarayanan, Ganesan; Lipton, Anuj N.; Al-Dhabi, Naif Abdullah; Valan Arasu, Mariadhas; Kiran, George S.

2016-01-01

The important biological macromolecules, such as lipopeptide and glycolipid biosurfactant producing marine actinobacteria were analyzed and their potential linkage between type II polyketide synthase (PKS) genes was explored. A unique feature of type II PKS genes is their high amino acid (AA) sequence homology and conserved gene organization. These enzymes mediate the biosynthesis of polyketide natural products with enormous structural complexity and chemical nature by combinatorial use of various domains. Therefore, deciphering the order of AA sequence encoded by PKS domains tailored the chemical structure of polyketide analogs still remains a great challenge. The present work deals with an in vitro and in silico analysis of PKS type II genes from five actinobacterial species to correlate KS domain architecture and structural features. Our present analysis reveals the unique protein domain organization of iterative type II PKS and KS domain of marine actinobacteria. The findings of this study would have implications in metabolic pathway reconstruction and design of semi-synthetic genomes to achieve rational design of novel natural products. PMID:26903957

Molecular identification of Armillaria gallica from the Niobrara Valley Preserve in Nebraska

Treesearch

Mee-Sook Kim; Ned B. Klopfenstein

2011-01-01

Armillaria isolates were collected from a unique forest ecosystem in the Niobrara Valley Preserve in Nebraska, USA, which comprises a glacial and early postglacial refugium in the central plains of North America. The isolates were collected from diverse forest trees representing a unique mixture of forest types. Combined methods of rDNA sequencing and flow cytometric...

Structure and Genetic Content of the Megaplasmids of Neurotoxigenic Clostridium butyricum Type E Strains from Italy

PubMed Central

Iacobino, Angelo; Scalfaro, Concetta; Franciosa, Giovanna

2013-01-01

We determined the genetic maps of the megaplasmids of six neutoroxigenic Clostridium butyricum type E strains from Italy using molecular and bioinformatics techniques. The megaplasmids are circular, not linear as we had previously proposed. The differently-sized megaplasmids share a genetic region that includes structural, metabolic and regulatory genes. In addition, we found that a 168 kb genetic region is present only in the larger megaplasmids of two tested strains, whereas it is absent from the smaller megaplasmids of the four remaining strains. The genetic region unique to the larger megaplasmids contains, among other features, a locus for clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (cas) genes, i.e. a bacterial adaptive immune system providing sequence-specific protection from invading genetic elements. Some CRISPR spacer sequences of the neurotoxigenic C. butyricum type E strains showed homology to prophage, phage and plasmid sequences from closely related clostridia species or from distant species, all sharing the intestinal habitat, suggesting that the CRISPR locus might be involved in the microorganism adaptation to the human or animal intestinal environment. Besides, we report here that each of four distinct CRISPR spacers partially matched DNA sequences of different prophages and phages, at identical nucleotide locations. This suggests that, at least in neurotoxigenic C. butyricum type E, the CRISPR locus is potentially able to recognize the same conserved DNA sequence of different invading genetic elements, besides targeting sequences unique to previously encountered invading DNA, as currently predicted for a CRISPR locus. Thus, the results of this study introduce the possibility that CRISPR loci can provide resistance to a wider range of invading DNA elements than previously appreciated. Whether it is more advantageous for the peculiar neurotoxigenic C. butyricum type E strains to maintain or to lose the CRISPR-cas system remains an open question. PMID:23967192

The Physics and Mathematics of MRI

NASA Astrophysics Data System (ADS)

Ansorge, Richard; Graves, Martin

2016-10-01

Magnetic Resonance Imaging is a very important clinical imaging tool. It combines different fields of physics and engineering in a uniquely complex way. MRI is also surprisingly versatile, `pulse sequences' can be designed to yield many different types of contrast. This versatility is unique to MRI. This short book gives both an in depth account of the methods used for the operation and construction of modern MRI systems and also the principles of sequence design and many examples of applications. An important additional feature of this book is the detailed discussion of the mathematical principles used in building optimal MRI systems and for sequence design. The mathematical discussion is very suitable for undergraduates attending medical physics courses. It is also more complete than usually found in alternative books for physical scientists or more clinically orientated works.

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

International Life Science Institute North America Cronobacter (Formerly Enterobacter sakazakii) isolate set.

PubMed

Ivy, Reid A; Farber, Jeffrey M; Pagotto, Franco; Wiedmann, Martin

2013-01-01

Foodborne pathogen isolate collections are important for the development of detection methods, for validation of intervention strategies, and to develop an understanding of pathogenesis and virulence. We have assembled a publicly available Cronobacter (formerly Enterobacter sakazakii) isolate set that consists of (i) 25 Cronobacter sakazakii isolates, (ii) two Cronobacter malonaticus isolates, (iii) one Cronobacter muytjensii isolate, which displays some atypical phenotypic characteristics, biochemical profiles, and colony color on selected differential media, and (iv) two nonclinical Enterobacter asburiae isolates, which show some phenotypic characteristics similar to those of Cronobacter spp. The set consists of human (n = 10), food (n = 11), and environmental (n = 9) isolates. Analysis of partial 16S rDNA sequence and seven-gene multilocus sequence typing data allowed for reliable identification of these isolates to species and identification of 14 isolates as sequence type 4, which had previously been shown to be the most common C. sakazakii sequence type associated with neonatal meningitis. Phenotypic characterization was carried out with API 20E and API 32E test strips and streaking on two selective chromogenic agars; isolates were also assessed for sorbitol fermentation and growth at 45°C. Although these strategies typically produced the same classification as sequence-based strategies, based on a panel of four biochemical tests, one C. sakazakii isolate yielded inconclusive data and one was classified as C. malonaticus. EcoRI automated ribotyping and pulsed-field gel electrophoresis (PFGE) with XbaI separated the set into 23 unique ribotypes and 30 unique PFGE types, respectively, indicating subtype diversity within the set. Subtype and source data for the collection are publicly available in the PathogenTracker database (www. pathogentracker. net), which allows for continuous updating of information on the set, including links to publications that include information on isolates from this collection.

Diversity of 16S rRNA genes of new Ehrlichia strains isolated from horses with clinical signs of Potomac horse fever.

PubMed

Wen, B; Rikihisa, Y; Fuerst, P A; Chaichanasiriwithaya, W

1995-04-01

Ehrlichia risticii is the causative agent of Potomac horse fever. Variations among the major antigens of different local E. risticii strains have been detected previously. To further assess genetic variability in this species or species complex, the sequences of the 16S rRNA genes of several isolates obtained from sick horses diagnosed as having Potomac horse fever were determined. The sequences of six isolates obtained from Ohio and three isolates obtained from Kentucky were amplified by PCR. Three groups of sequences were identified. The sequences of five of the Ohio isolates were identical to the sequence of the type strain of E. risticii, the Illinois strain. The sequence of one Ohio isolate, isolate 081, was unique; this sequence differed in 10 nucleotides from the sequence of the type strain (level of similarity, 99.3%). The sequences of the three Kentucky isolates were identical to each other, but differed by five bases from the sequence of the type strain (level of similarity, 99.6%). The levels of sequence similarity of isolate 081, the Kentucky isolates, and the type strain to the next most closely related Ehrlichia sp., Ehrlichia sennetsu, were 99.3, 99.2, and 99.2%, respectively. On the basis of the distinct antigenic profiles and the levels of 16S rRNA sequence divergence, isolate 081 is as divergent from the type strain of E. risticii as E. sennetsu is. Therefore, we suggest that strain 081 and the Kentucky isolates may represent two new distinct Ehrlichia species.

Analysis and functional annotation of expressed sequence tags from in vitro cell lines of elasmobranchs: spiny dogfish shark (Squalus acanthias) and little skate (Leucoraja erinacea)

PubMed Central

Parton, Angela; Bayne, Christopher J.; Barnes, David W.

2010-01-01

Elasmobranchs are the most commonly used experimental models among the jawed, cartilaginous fish (Chondrichthyes). Previously we developed cell lines from embryos of two elasmobranchs, Squalus acanthias the spiny dogfish shark (SAE line), and Leucoraja erinacea the little skate (LEE-1 line). From these lines cDNA libraries were derived and expressed sequence tags (ESTs) generated. From the SAE cell line 4303 unique transcripts were identified, with 1848 of these representing unknown sequences (showing no BLASTX identification). From the LEE-1 cell line, 3660 unique transcripts were identified, and unknown, unique sequences totaled 1333. Gene Ontology (GO) annotation showed that GO assignments for the two cell lines were in general similar. These results suggest that the procedures used to derive the cell lines led to isolation of cell types of the same general embryonic origin from both species. The LEE-1 transcripts included GO categories “envelope” and “oxidoreductase activity” but the SAE transcripts did not. GO analysis of SAE transcripts identified the category “anatomical structure formation” that was not present in LEE-1 cells. Increased organelle compartments may exist within LEE-1 cells compared to SAE cells, and the higher oxidoreductase activity in LEE-1 cells may indicate a role for these cells in responses associated with innate immunity or in steroidogenesis. These EST libraries from elasmobranch cell lines provide information for assembly of genomic sequences and are useful in revealing gene diversity, new genes and molecular markers, as well as in providing means for elucidation of full-length cDNAs and probes for gene array analyses. This is the first study of this type with members of the Chondrichthyes. PMID:20471924

Analysis and functional annotation of expressed sequence tags from in vitro cell lines of elasmobranchs: Spiny dogfish shark (Squalus acanthias) and little skate (Leucoraja erinacea).

PubMed

Parton, Angela; Bayne, Christopher J; Barnes, David W

2010-09-01

Elasmobranchs are the most commonly used experimental models among the jawed, cartilaginous fish (Chondrichthyes). Previously we developed cell lines from embryos of two elasmobranchs, Squalus acanthias the spiny dogfish shark (SAE line), and Leucoraja erinacea the little skate (LEE-1 line). From these lines cDNA libraries were derived and expressed sequence tags (ESTs) generated. From the SAE cell line 4303 unique transcripts were identified, with 1848 of these representing unknown sequences (showing no BLASTX identification). From the LEE-1 cell line, 3660 unique transcripts were identified, and unknown, unique sequences totaled 1333. Gene Ontology (GO) annotation showed that GO assignments for the two cell lines were in general similar. These results suggest that the procedures used to derive the cell lines led to isolation of cell types of the same general embryonic origin from both species. The LEE-1 transcripts included GO categories "envelope" and "oxidoreductase activity" but the SAE transcripts did not. GO analysis of SAE transcripts identified the category "anatomical structure formation" that was not present in LEE-1 cells. Increased organelle compartments may exist within LEE-1 cells compared to SAE cells, and the higher oxidoreductase activity in LEE-1 cells may indicate a role for these cells in responses associated with innate immunity or in steroidogenesis. These EST libraries from elasmobranch cell lines provide information for assembly of genomic sequences and are useful in revealing gene diversity, new genes and molecular markers, as well as in providing means for elucidation of full-length cDNAs and probes for gene array analyses. This is the first study of this type with members of the Chondrichthyes. Copyright 2010 Elsevier Inc. All rights reserved.

``Sequence space soup'' of proteins and copolymers

NASA Astrophysics Data System (ADS)

Chan, Hue Sun; Dill, Ken A.

1991-09-01

To study the protein folding problem, we use exhaustive computer enumeration to explore ``sequence space soup,'' an imaginary solution containing the ``native'' conformations (i.e., of lowest free energy) under folding conditions, of every possible copolymer sequence. The model is of short self-avoiding chains of hydrophobic (H) and polar (P) monomers configured on the two-dimensional square lattice. By exhaustive enumeration, we identify all native structures for every possible sequence. We find that random sequences of H/P copolymers will bear striking resemblance to known proteins: Most sequences under folding conditions will be approximately as compact as known proteins, will have considerable amounts of secondary structure, and it is most probable that an arbitrary sequence will fold to a number of lowest free energy conformations that is of order one. In these respects, this simple model shows that proteinlike behavior should arise simply in copolymers in which one monomer type is highly solvent averse. It suggests that the structures and uniquenesses of native proteins are not consequences of having 20 different monomer types, or of unique properties of amino acid monomers with regard to special packing or interactions, and thus that simple copolymers might be designable to collapse to proteinlike structures and properties. A good strategy for designing a sequence to have a minimum possible number of native states is to strategically insert many P monomers. Thus known proteins may be marginally stable due to a balance: More H residues stabilize the desired native state, but more P residues prevent simultaneous stabilization of undesired native states.

Highly conserved intragenic HSV-2 sequences: Results from next-generation sequencing of HSV-2 UL and US regions from genital swabs collected from 3 continents.

PubMed

Johnston, Christine; Magaret, Amalia; Roychoudhury, Pavitra; Greninger, Alexander L; Cheng, Anqi; Diem, Kurt; Fitzgibbon, Matthew P; Huang, Meei-Li; Selke, Stacy; Lingappa, Jairam R; Celum, Connie; Jerome, Keith R; Wald, Anna; Koelle, David M

2017-10-01

Understanding the variability in circulating herpes simplex virus type 2 (HSV-2) genomic sequences is critical to the development of HSV-2 vaccines. Genital lesion swabs containing ≥ 10 7 log 10 copies HSV DNA collected from Africa, the USA, and South America underwent next-generation sequencing, followed by K-mer based filtering and de novo genomic assembly. Sites of heterogeneity within coding regions in unique long and unique short (U L _U S ) regions were identified. Phylogenetic trees were created using maximum likelihood reconstruction. Among 46 samples from 38 persons, 1468 intragenic base-pair substitutions were identified. The maximum nucleotide distance between strains for concatenated U L_ U S segments was 0.4%. Phylogeny did not reveal geographic clustering. The most variable proteins had non-synonymous mutations in < 3% of amino acids. Unenriched HSV-2 DNA can undergo next-generation sequencing to identify intragenic variability. The use of clinical swabs for sequencing expands the information that can be gathered directly from these specimens. Copyright © 2017 Elsevier Inc. All rights reserved.

Effectiveness of the standard and an alternative set of Streptococcus pneumoniae multi locus sequence typing primers.

PubMed

Adamiak, Paul; Vanderkooi, Otto G; Kellner, James D; Schryvers, Anthony B; Bettinger, Julie A; Alcantara, Joenel

2014-06-03

Multi-locus sequence typing (MLST) is a portable, broadly applicable method for classifying bacterial isolates at an intra-species level. This methodology provides clinical and scientific investigators with a standardized means of monitoring evolution within bacterial populations. MLST uses the DNA sequences from a set of genes such that each unique combination of sequences defines an isolate's sequence type. In order to reliably determine the sequence of a typing gene, matching sequence reads for both strands of the gene must be obtained. This study assesses the ability of both the standard, and an alternative set of, Streptococcus pneumoniae MLST primers to completely sequence, in both directions, the required typing alleles. The results demonstrated that for five (aroE, recP, spi, xpt, ddl) of the seven S. pneumoniae typing alleles, the standard primers were unable to obtain the complete forward and reverse sequences. This is due to the standard primers annealing too closely to the target regions, and current sequencing technology failing to sequence the bases that are too close to the primer. The alternative primer set described here, which includes a combination of primers proposed by the CDC and several designed as part of this study, addresses this limitation by annealing to highly conserved segments further from the target region. This primer set was subsequently employed to sequence type 105 S. pneumoniae isolates collected by the Canadian Immunization Monitoring Program ACTive (IMPACT) over a period of 18 years. The inability of several of the standard S. pneumoniae MLST primers to fully sequence the required region was consistently observed and is the result of a shift in sequencing technology occurring after the original primers were designed. The results presented here introduce clear documentation describing this phenomenon into the literature, and provide additional guidance, through the introduction of a widely validated set of alternative primers, to research groups seeking to undertake S. pneumoniae MLST based studies.

Multilocus sequence typing scheme versus pulsed-field gel electrophoresis for typing Mycobacterium abscessus isolates.

PubMed

Machado, Gabriel Esquitini; Matsumoto, Cristianne Kayoko; Chimara, Erica; Duarte, Rafael da Silva; de Freitas, Denise; Palaci, Moises; Hadad, David Jamil; Lima, Karla Valéria Batista; Lopes, Maria Luiza; Ramos, Jesus Pais; Campos, Carlos Eduardo; Caldas, Paulo César; Heym, Beate; Leão, Sylvia Cardoso

2014-08-01

Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Whole-genome sequencing reveals clonal expansion of multiresistant Staphylococcus haemolyticus in European hospitals.

PubMed

Cavanagh, Jorunn Pauline; Hjerde, Erik; Holden, Matthew T G; Kahlke, Tim; Klingenberg, Claus; Flægstad, Trond; Parkhill, Julian; Bentley, Stephen D; Sollid, Johanna U Ericson

2014-11-01

Staphylococcus haemolyticus is an emerging cause of nosocomial infections, primarily affecting immunocompromised patients. A comparative genomic analysis was performed on clinical S. haemolyticus isolates to investigate their genetic relationship and explore the coding sequences with respect to antimicrobial resistance determinants and putative hospital adaptation. Whole-genome sequencing was performed on 134 isolates of S. haemolyticus from geographically diverse origins (Belgium, 2; Germany, 10; Japan, 13; Norway, 54; Spain, 2; Switzerland, 43; UK, 9; USA, 1). Each genome was individually assembled. Protein coding sequences (CDSs) were predicted and homologous genes were categorized into three types: Type I, core genes, homologues present in all strains; Type II, unique core genes, homologues shared by only a subgroup of strains; and Type III, unique genes, strain-specific CDSs. The phylogenetic relationship between the isolates was built from variable sites in the form of single nucleotide polymorphisms (SNPs) in the core genome and used to construct a maximum likelihood phylogeny. SNPs in the genome core regions divided the isolates into one major group of 126 isolates and one minor group of isolates with highly diverse genomes. The major group was further subdivided into seven clades (A-G), of which four (A-D) encompassed isolates only from Europe. Antimicrobial multiresistance was observed in 77.7% of the collection. High levels of homologous recombination were detected in genes involved in adherence, staphylococcal host adaptation and bacterial cell communication. The presence of several successful and highly resistant clones underlines the adaptive potential of this opportunistic pathogen. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

Whole-genome sequencing reveals clonal expansion of multiresistant Staphylococcus haemolyticus in European hospitals

PubMed Central

Cavanagh, Jorunn Pauline; Hjerde, Erik; Holden, Matthew T. G.; Kahlke, Tim; Klingenberg, Claus; Flægstad, Trond; Parkhill, Julian; Bentley, Stephen D.; Sollid, Johanna U. Ericson

2014-01-01

Objectives Staphylococcus haemolyticus is an emerging cause of nosocomial infections, primarily affecting immunocompromised patients. A comparative genomic analysis was performed on clinical S. haemolyticus isolates to investigate their genetic relationship and explore the coding sequences with respect to antimicrobial resistance determinants and putative hospital adaptation. Methods Whole-genome sequencing was performed on 134 isolates of S. haemolyticus from geographically diverse origins (Belgium, 2; Germany, 10; Japan, 13; Norway, 54; Spain, 2; Switzerland, 43; UK, 9; USA, 1). Each genome was individually assembled. Protein coding sequences (CDSs) were predicted and homologous genes were categorized into three types: Type I, core genes, homologues present in all strains; Type II, unique core genes, homologues shared by only a subgroup of strains; and Type III, unique genes, strain-specific CDSs. The phylogenetic relationship between the isolates was built from variable sites in the form of single nucleotide polymorphisms (SNPs) in the core genome and used to construct a maximum likelihood phylogeny. Results SNPs in the genome core regions divided the isolates into one major group of 126 isolates and one minor group of isolates with highly diverse genomes. The major group was further subdivided into seven clades (A–G), of which four (A–D) encompassed isolates only from Europe. Antimicrobial multiresistance was observed in 77.7% of the collection. High levels of homologous recombination were detected in genes involved in adherence, staphylococcal host adaptation and bacterial cell communication. Conclusions The presence of several successful and highly resistant clones underlines the adaptive potential of this opportunistic pathogen. PMID:25038069

A disruptive sequencer meets disruptive publishing.

PubMed

Loman, Nick; Goodwin, Sarah; Jansen, Hans; Loose, Matt

2015-01-01

Nanopore sequencing was recently made available to users in the form of the Oxford Nanopore MinION. Released to users through an early access programme, the MinION is made unique by its tiny form factor and ability to generate very long sequences from single DNA molecules. The platform is undergoing rapid evolution with three distinct nanopore types and five updates to library preparation chemistry in the last 18 months. To keep pace with the rapid evolution of this sequencing platform, and to provide a space where new analysis methods can be openly discussed, we present a new F1000Research channel devoted to updates to and analysis of nanopore sequence data.

Optimization of sequence alignment for simple sequence repeat regions.

PubMed

Jighly, Abdulqader; Hamwieh, Aladdin; Ogbonnaya, Francis C

2011-07-20

Microsatellites, or simple sequence repeats (SSRs), are tandemly repeated DNA sequences, including tandem copies of specific sequences no longer than six bases, that are distributed in the genome. SSR has been used as a molecular marker because it is easy to detect and is used in a range of applications, including genetic diversity, genome mapping, and marker assisted selection. It is also very mutable because of slipping in the DNA polymerase during DNA replication. This unique mutation increases the insertion/deletion (INDELs) mutation frequency to a high ratio - more than other types of molecular markers such as single nucleotide polymorphism (SNPs).SNPs are more frequent than INDELs. Therefore, all designed algorithms for sequence alignment fit the vast majority of the genomic sequence without considering microsatellite regions, as unique sequences that require special consideration. The old algorithm is limited in its application because there are many overlaps between different repeat units which result in false evolutionary relationships. To overcome the limitation of the aligning algorithm when dealing with SSR loci, a new algorithm was developed using PERL script with a Tk graphical interface. This program is based on aligning sequences after determining the repeated units first, and the last SSR nucleotides positions. This results in a shifting process according to the inserted repeated unit type.When studying the phylogenic relations before and after applying the new algorithm, many differences in the trees were obtained by increasing the SSR length and complexity. However, less distance between different linage had been observed after applying the new algorithm. The new algorithm produces better estimates for aligning SSR loci because it reflects more reliable evolutionary relations between different linages. It reduces overlapping during SSR alignment, which results in a more realistic phylogenic relationship.

Molecular epidemiology demonstrated three emerging clusters of human immunodeficiency virus type 1 subtype B infection in Hong Kong.

PubMed

Leung, Tommy W C; Mak, Darwin; Wong, K H; Wang, Y; Song, Y H; Tsang, D N C; Wong, C; Shao, Y M; Lim, W L

2008-07-01

We conducted a molecular epidemiological study on newly diagnosed human immunodeficiency virus type 1 (HIV-1)-infected patients in Hong Kong to identify the epidemiological linkage of HIV-1 infection in the locality. Reverse transcription polymerase chain reaction (RT-PCR) for HIV-1 was performed on newly diagnosed HIV-1-positive sera collected from January 2002 to December 2006. PCR products correspond to the env C2V3V4 region and gag p17/p24 junction of the HIV-1 genome were nucleotide sequenced. Phylogenetic analyses performed on the acquired nucleotide sequences revealed that CRF01_AE and subtype B were the two dominant HIV-1 subtypes. Analyses also demonstrated the presence of three emerging HIV-1 clusters among the subtype B sequences in Hong Kong. Individual cluster possesses a unique cluster-specific amino acid signature for identification. Data show that one of the clusters (Cluster I) is rapidly expanding. In addition to the unique cluster-specific amino acid signature, the majority of sequences in Cluster I harbor a 6-amino acid insertion at the gag p17/p24 junction in a region that is thought to be closely associated with HIV-1 infectivity.

Clonal Relatedness of Enterotoxigenic Escherichia coli (ETEC) Strains Expressing LT and CS17 Isolated from Children with Diarrhoea in La Paz, Bolivia

PubMed Central

Rodas, Claudia; Klena, John D.; Nicklasson, Matilda; Iniguez, Volga; Sjöling, Åsa

2011-01-01

Background Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. Methodology/Principal Findings In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNPbol in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNPbol) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. Conclusion/Significance The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors. PMID:22140423

Clonal relatedness of enterotoxigenic Escherichia coli (ETEC) strains expressing LT and CS17 isolated from children with diarrhoea in La Paz, Bolivia.

PubMed

Rodas, Claudia; Klena, John D; Nicklasson, Matilda; Iniguez, Volga; Sjöling, Asa

2011-01-01

Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNP(bol) in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNP(bol)) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors.

Comparative analysis of mitochondrial genomes between a wheat K-type cytoplasmic male sterility (CMS) line and its maintainer line.

PubMed

Liu, Huitao; Cui, Peng; Zhan, Kehui; Lin, Qiang; Zhuo, Guoyin; Guo, Xiaoli; Ding, Feng; Yang, Wenlong; Liu, Dongcheng; Hu, Songnian; Yu, Jun; Zhang, Aimin

2011-03-29

Plant mitochondria, semiautonomous organelles that function as manufacturers of cellular ATP, have their own genome that has a slow rate of evolution and rapid rearrangement. Cytoplasmic male sterility (CMS), a common phenotype in higher plants, is closely associated with rearrangements in mitochondrial DNA (mtDNA), and is widely used to produce F1 hybrid seeds in a variety of valuable crop species. Novel chimeric genes deduced from mtDNA rearrangements causing CMS have been identified in several plants, such as rice, sunflower, pepper, and rapeseed, but there are very few reports about mtDNA rearrangements in wheat. In the present work, we describe the mitochondrial genome of a wheat K-type CMS line and compare it with its maintainer line. The complete mtDNA sequence of a wheat K-type (with cytoplasm of Aegilops kotschyi) CMS line, Ks3, was assembled into a master circle (MC) molecule of 647,559 bp and found to harbor 34 known protein-coding genes, three rRNAs (18 S, 26 S, and 5 S rRNAs), and 16 different tRNAs. Compared to our previously published sequence of a K-type maintainer line, Km3, we detected Ks3-specific mtDNA (> 100 bp, 11.38%) and repeats (> 100 bp, 29 units) as well as genes that are unique to each line: rpl5 was missing in Ks3 and trnH was absent from Km3. We also defined 32 single nucleotide polymorphisms (SNPs) in 13 protein-coding, albeit functionally irrelevant, genes, and predicted 22 unique ORFs in Ks3, representing potential candidates for K-type CMS. All these sequence variations are candidates for involvement in CMS. A comparative analysis of the mtDNA of several angiosperms, including those from Ks3, Km3, rice, maize, Arabidopsis thaliana, and rapeseed, showed that non-coding sequences of higher plants had mostly divergent multiple reorganizations during the mtDNA evolution of higher plants. The complete mitochondrial genome of the wheat K-type CMS line Ks3 is very different from that of its maintainer line Km3, especially in non-coding sequences. Sequence rearrangement has produced novel chimeric ORFs, which may be candidate genes for CMS. Comparative analysis of several angiosperm mtDNAs indicated that non-coding sequences are the most frequently reorganized during mtDNA evolution in higher plants.

An improved divergent synthesis of comb-type branched oligodeoxyribonucleotides (bDNA) containing multiple secondary sequences.

PubMed

Horn, T; Chang, C A; Urdea, M S

1997-12-01

The divergent synthesis of branched DNA (bDNA) comb structures is described. This new type of bDNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branch network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb structures were assembled on a solid support and several synthesis parameters were investigated and optimized. The bDNA comb molecules were characterized by polyacrylamide gel electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The developed chemistry allows synthesis of bDNA comb molecules containing multiple secondary sequences. In the accompanying article we describe the synthesis and characterization of large bDNA combs containing all four deoxynucleotides for use as signal amplifiers in nucleic acid quantification assays.

An improved divergent synthesis of comb-type branched oligodeoxyribonucleotides (bDNA) containing multiple secondary sequences.

PubMed Central

Horn, T; Chang, C A; Urdea, M S

1997-01-01

The divergent synthesis of branched DNA (bDNA) comb structures is described. This new type of bDNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branch network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb structures were assembled on a solid support and several synthesis parameters were investigated and optimized. The bDNA comb molecules were characterized by polyacrylamide gel electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The developed chemistry allows synthesis of bDNA comb molecules containing multiple secondary sequences. In the accompanying article we describe the synthesis and characterization of large bDNA combs containing all four deoxynucleotides for use as signal amplifiers in nucleic acid quantification assays. PMID:9365265

Draft Genome Sequences of Mycobacterium setense Type Strain DSM-45070 and the Nonpathogenic Strain Manresensis, Isolated from the Bank of the Cardener River in Manresa, Catalonia, Spain

PubMed Central

Vilaplana, Cristina; Velasco, Juan; Pluvinet, Raquel; Santín, Sheila; Prat, Cristina; Julián, Esther; Alcaide, Fernando; Comas, Iñaki; Sumoy, Lauro; Cardona, Pere-Joan

2015-01-01

We present here the draft genome sequences of two Mycobacterium setense strains. One of them corresponds to the M. setense type strain DSM-45070, originally isolated from a patient with a posttraumatic chronic skin abscess. The other one corresponds to the nonpathogenic M. setense strain Manresensis, isolated from the Cardener River crossing Manresa, Catalonia, Spain. A comparative genomic analysis shows a smaller genome size and fewer genes in M. setense strain Manresensis relative to those of the type strain, and it shows the genome segments unique to each strain. PMID:25657273

Added Value of Next-Generation Sequencing for Multilocus Sequence Typing Analysis of a Pneumocystis jirovecii Pneumonia Outbreak1.

PubMed

Charpentier, Elena; Garnaud, Cécile; Wintenberger, Claire; Bailly, Sébastien; Murat, Jean-Benjamin; Rendu, John; Pavese, Patricia; Drouet, Thibault; Augier, Caroline; Malvezzi, Paolo; Thiébaut-Bertrand, Anne; Mallaret, Marie-Reine; Epaulard, Olivier; Cornet, Muriel; Larrat, Sylvie; Maubon, Danièle

2017-08-01

Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus.

Added Value of Next-Generation Sequencing for Multilocus Sequence Typing Analysis of a Pneumocystis jirovecii Pneumonia Outbreak1

PubMed Central

Charpentier, Elena; Garnaud, Cécile; Wintenberger, Claire; Bailly, Sébastien; Murat, Jean-Benjamin; Rendu, John; Pavese, Patricia; Drouet, Thibault; Augier, Caroline; Malvezzi, Paolo; Thiébaut-Bertrand, Anne; Mallaret, Marie-Reine; Epaulard, Olivier; Cornet, Muriel; Larrat, Sylvie

2017-01-01

Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus. PMID:28726611

The complete genome sequence of human adenovirus 84, a highly recombinant new Human mastadenovirus D type with a unique fiber gene.

PubMed

Kaján, Győző L; Kajon, Adriana E; Pinto, Alexis Castillo; Bartha, Dániel; Arnberg, Niklas

2017-10-15

A novel human adenovirus was isolated from a pediatric case of acute respiratory disease in Panama City, Panama in 2011. The clinical isolate was initially identified as an intertypic recombinant based on hexon and fiber gene sequencing. Based on the analysis of its complete genome sequence, the novel complex recombinant Human mastadenovirus D (HAdV-D) strain was classified into a new HAdV type: HAdV-84, and it was designated Adenovirus D human/PAN/P309886/2011/84[P43H17F84]. HAdV-D types possess usually an ocular or gastrointestinal tropism, and respiratory association is scarcely reported. The virus has a novel fiber type, most closely related to, but still clearly distant from that of HAdV-36. The predicted fiber is hypothesised to bind sialic acid with lower affinity compared to HAdV-37. Bioinformatic analysis of the complete genomic sequence of HAdV-84 revealed multiple homologous recombination events and provided deeper insight into HAdV evolution. Copyright © 2017 Elsevier B.V. All rights reserved.

DNA methylation assessment from human slow- and fast-twitch skeletal muscle fibers

PubMed Central

Begue, Gwénaëlle; Raue, Ulrika; Jemiolo, Bozena

2017-01-01

A new application of the reduced representation bisulfite sequencing method was developed using low-DNA input to investigate the epigenetic profile of human slow- and fast-twitch skeletal muscle fibers. Successful library construction was completed with as little as 15 ng of DNA, and high-quality sequencing data were obtained with 32 ng of DNA. Analysis identified 143,160 differentially methylated CpG sites across 14,046 genes. In both fiber types, selected genes predominantly expressed in slow or fast fibers were hypomethylated, which was supported by the RNA-sequencing analysis. These are the first fiber type-specific methylation data from human skeletal muscle and provide a unique platform for future research. NEW & NOTEWORTHY This study validates a low-DNA input reduced representation bisulfite sequencing method for human muscle biopsy samples to investigate the methylation patterns at a fiber type-specific level. These are the first fiber type-specific methylation data reported from human skeletal muscle and thus provide initial insight into basal state differences in myosin heavy chain I and IIa muscle fibers among young, healthy men. PMID:28057818

Bifidobacterium animalis subsp. lactis ATCC 27673 Is a Genomically Unique Strain within Its Conserved Subspecies

PubMed Central

Loquasto, Joseph R.; Barrangou, Rodolphe; Dudley, Edward G.; Stahl, Buffy; Chen, Chun

2013-01-01

Many strains of Bifidobacterium animalis subsp. lactis are considered health-promoting probiotic microorganisms and are commonly formulated into fermented dairy foods. Analyses of previously sequenced genomes of B. animalis subsp. lactis have revealed little genetic diversity, suggesting that it is a monomorphic subspecies. However, during a multilocus sequence typing survey of Bifidobacterium, it was revealed that B. animalis subsp. lactis ATCC 27673 gave a profile distinct from that of the other strains of the subspecies. As part of an ongoing study designed to understand the genetic diversity of this subspecies, the genome of this strain was sequenced and compared to other sequenced genomes of B. animalis subsp. lactis and B. animalis subsp. animalis. The complete genome of ATCC 27673 was 1,963,012 bp, contained 1,616 genes and 4 rRNA operons, and had a G+C content of 61.55%. Comparative analyses revealed that the genome of ATCC 27673 contained six distinct genomic islands encoding 83 open reading frames not found in other strains of the same subspecies. In four islands, either phage or mobile genetic elements were identified. In island 6, a novel clustered regularly interspaced short palindromic repeat (CRISPR) locus which contained 81 unique spacers was identified. This type I-E CRISPR-cas system differs from the type I-C systems previously identified in this subspecies, representing the first identification of a different system in B. animalis subsp. lactis. This study revealed that ATCC 27673 is a strain of B. animalis subsp. lactis with novel genetic content and suggests that the lack of genetic variability observed is likely due to the repeated sequencing of a limited number of widely distributed commercial strains. PMID:23995933

Genomics of an emerging clone of Salmonella serovar Typhimurium ST313 from Nigeria and the Democratic Republic of Congo.

PubMed

Leekitcharoenphon, Pimlapas; Friis, Carsten; Zankari, Ea; Svendsen, Christina Aaby; Price, Lance B; Rahmani, Maral; Herrero-Fresno, Ana; Fashae, Kayode; Vandenberg, Olivier; Aarestrup, Frank M; Hendriksen, Rene S

2013-10-15

Salmonella enterica serovar Typhimurium ST313 is an invasive and phylogenetically distinct lineage present in sub-Saharan Africa. We report the presence of S. Typhimurium ST313 from patients in the Democratic Republic of Congo and Nigeria. Eighteen S. Typhimurium ST313 isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Additionally, six of the isolates were characterized by whole genome sequence typing (WGST). The presence of a putative virulence determinant was examined in 177 Salmonella isolates belonging to 57 different serovars. All S. Typhimurium ST313 isolates harbored resistant genes encoded by blaTEM1b, catA1, strA/B, sul1, and dfrA1. Additionally, aac(6')1aa gene was detected. Phylogenetic analyses revealed close genetic relationships among Congolese and Nigerian isolates from both blood and stool. Comparative genomic analyses identified a putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and S. Dublin. We showed in a limited number of isolates that S. Typhimurium ST313 is a prevalent sequence-type causing gastrointestinal diseases and septicemia in patients from Nigeria and DRC. We found three distinct phylogenetic clusters based on the origin of isolation suggesting some spatial evolution. Comparative genomics showed an interesting putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and invasive S. Dublin.

CRISPR regulation of intraspecies diversification by limiting IS transposition and intercellular recombination.

PubMed

Watanabe, Takayasu; Nozawa, Takashi; Aikawa, Chihiro; Amano, Atsuo; Maruyama, Fumito; Nakagawa, Ichiro

2013-01-01

Mobile genetic elements (MGEs) and genetic rearrangement are considered as major driving forces of bacterial diversification. Previous comparative genome analysis of Porphyromonas gingivalis, a pathogen related to periodontitis, implied such an important relationship. As a counterpart system to MGEs, clustered regularly interspaced short palindromic repeats (CRISPRs) in bacteria may be useful for genetic typing. We found that CRISPR typing could be a reasonable alternative to conventional methods for characterizing phylogenetic relationships among 60 highly diverse P. gingivalis isolates. Examination of genetic recombination along with multilocus sequence typing suggests the importance of such events between different isolates. MGEs appear to be strategically located at the breakpoint gaps of complicated genome rearrangements. Of these MGEs, insertion sequences (ISs) were found most frequently. CRISPR analysis identified 2,150 spacers that were clustered into 1,187 unique ones. Most of these spacers exhibited no significant nucleotide similarity to known sequences (97.6%: 1,158/1,187). Surprisingly, CRISPR spacers exhibiting high nucleotide similarity to regions of P. gingivalis genomes including ISs were predominant. The proportion of such spacers to all the unique spacers (1.6%: 19/1,187) was the highest among previous studies, suggesting novel functions for these CRISPRs. These results indicate that P. gingivalis is a bacterium with high intraspecies diversity caused by frequent insertion sequence (IS) transposition, whereas both the introduction of foreign DNA, primarily from other P. gingivalis cells, and IS transposition are limited by CRISPR interference. It is suggested that P. gingivalis CRISPRs could be an important source for understanding the role of CRISPRs in the development of bacterial diversity.

Structural features of the rice chromosome 4 centromere.

PubMed

Zhang, Yu; Huang, Yuchen; Zhang, Lei; Li, Ying; Lu, Tingting; Lu, Yiqi; Feng, Qi; Zhao, Qiang; Cheng, Zhukuan; Xue, Yongbiao; Wing, Rod A; Han, Bin

2004-01-01

A complete sequence of a chromosome centromere is necessary for fully understanding centromere function. We reported the sequence structures of the first complete rice chromosome centromere through sequencing a large insert bacterial artificial chromosome clone-based contig, which covered the rice chromosome 4 centromere. Complete sequencing of the 124-kb rice chromosome 4 centromere revealed that it consisted of 18 tracts of 379 tandemly arrayed repeats known as CentO and a total of 19 centromeric retroelements (CRs) but no unique sequences were detected. Four tracts, composed of 65 CentO repeats, were located in the opposite orientation, and 18 CentO tracts were flanked by 19 retroelements. The CRs were classified into four types, and the type I retroelements appeared to be more specific to rice centromeres. The preferential insert of the CRs among CentO repeats indicated that the centromere-specific retroelements may contribute to centromere expansion during evolution. The presence of three intact retrotransposons in the centromere suggests that they may be responsible for functional centromere initiation through a transcription-mediated mechanism.

Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities

PubMed Central

Drobni, Mirva; Hallberg, Kristina; Öhman, Ulla; Birve, Anna; Persson, Karina; Johansson, Ingegerd; Strömberg, Nicklas

2006-01-01

Background Actinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galβ binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking. Results Here we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4) and genospecies 2 (n = 4), both of which harboured variant Galβ-dependent hemagglutination (HA) types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8–66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (>97.2%) within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained a signal peptide, pilin motif, E box, proline-rich segment and an LPXTG sorting motif among other conserved segments for secretion, assembly and sorting of fimbrial proteins. The highly conserved pilin, E box and LPXTG motifs are present in fimbriae proteins from other Gram-positive bacteria. Moreover, only strains of genospecies 1 were agglutinated with type-2 fimbriae antisera derived from A. naeslundii genospecies 1 strain 12104, emphasizing that the overall folding of FimA may generate different functionalities. Western blot analyses with FimA antisera revealed monomers and oligomers of FimA in whole cell protein extracts and a purified recombinant FimA preparation, indicating a sortase-independent oligomerization of FimA. Conclusion The genus Actinomyces involves a diversity of unique FimA proteins with conserved pilin, E box and LPXTG motifs, depending on subspecies and associated binding specificity. In addition, a sortase independent oligomerization of FimA subunit proteins in solution was indicated. PMID:16686953

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 genomic content. Differences in gene content likely contribute to differences in the clinical and environmental distribution of species and sequence types. PMID:23166675

Vibrio cholerae typing phage N4: genome sequence and its relatedness to T7 viral supergroup.

PubMed

Das, Mayukh; Nandy, R K; Bhowmick, Tushar Suvra; Yamasaki, S; Ghosh, A; Nair, G B; Sarkar, B L

2012-01-01

In countries where cholera is endemic, Vibrio cholerae O1 bacteriophages have been detected in sewage water. These have been used to serve not only as strain markers, but also for the typing of V. cholerae strains. Vibriophage N4 (ATCC 51352-B1) occupies a unique position in the new phage-typing scheme and can infect a larger number of V. cholerae O1 biotype El Tor strains. Here we characterized the complete genome sequence of this typing vibriophage. The complete DNA sequence of the N4 genome was determined by using a shotgun sequencing approach. Complete genome sequence explored that phage N4 is comprised of one circular, double-stranded chromosome of 38,497 bp with an overall GC content of 42.8%. A total of 47 open reading frames were identified and functions could be assigned to 30 of them. Further, a close relationship with another vibriophage, VP4, and the enterobacteriophage T7 could be established. DNA-DNA hybridization among V. cholerae O1 and O139 phages revealed homology among O1 vibriophages at their genomic level. This study indicates two evolutionary distinctive branches of the possible phylogenetic origin of O1 and O139 vibriophages and provides an unveiled collection of information on viral gene products of typing vibriophages. Copyright © 2011 S. Karger AG, Basel.

Population Structure in Nontypeable Haemophilus influenzae

PubMed Central

LaCross, Nathan C.; Marrs, Carl F.; Gilsdorf, Janet R.

2013-01-01

Nontypeable Haemophilus influenzae (NTHi) frequently colonize the human pharynx asymptomatically, and are an important cause of otitis media in children. Past studies have identified typeable H. influenzae as being clonal, but the population structure of NTHi has not been extensively characterized. The research presented here investigated the diversity and population structure in a well-characterized collection of NTHi isolated from the middle ears of children with otitis media or the pharynges of healthy children in three disparate geographic regions. Multilocus sequence typing identified 109 unique sequence types among 170 commensal and otitis media-associated NTHi isolates from Finland, Israel, and the US. The largest clonal complex contained only five sequence types, indicating a high level of genetic diversity. The eBURST v3, ClonalFrame 1.1, and structure 2.3.3 programs were used to further characterize diversity and population structure from the sequence typing data. Little clustering was apparent by either disease state (otitis media or commensalism) or geography in the ClonalFrame phylogeny. Population structure was clearly evident, with support for eight populations when all 170 isolates were analyzed. Interestingly, one population contained only commensal isolates, while two others consisted solely of otitis media isolates, suggesting associations between population structure and disease. PMID:23266487

Bacterial CRISPR Regions: General Features and their Potential for Epidemiological Molecular Typing Studies.

PubMed

Karimi, Zahra; Ahmadi, Ali; Najafi, Ali; Ranjbar, Reza

2018-01-01

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci as novel and applicable regions in prokaryotic genomes have gained great attraction in the post genomics era. These unique regions are diverse in number and sequence composition in different pathogenic bacteria and thereby can be a suitable candidate for molecular epidemiology and genotyping studies. Results:Furthermore, the arrayed structure of CRISPR loci (several unique repeats spaced with the variable sequence) and associated cas genes act as an active prokaryotic immune system against viral replication and conjugative elements. This property can be used as a tool for RNA editing in bioengineering studies. The aim of this review was to survey some details about the history, nature, and potential applications of CRISPR arrays in both genetic engineering and bacterial genotyping studies.

DNA-based differentiation of the Ecuadorian cocoa types CCN-51 and Arriba based on sequence differences in the chloroplast genome.

PubMed

Herrmann, Luise; Haase, Ilka; Blauhut, Maike; Barz, Nadine; Fischer, Markus

2014-12-17

Two cocoa types, Arriba and CCN-51, are being cultivated in Ecuador. With regard to the unique aroma, Arriba is considered a fine cocoa type, while CCN-51 is a bulk cocoa because of its weaker aroma. Because it is being assumed that Arriba is mixed with CCN-51, there is an interest in the analytical differentiation of the two types. Two methods to identify CCN-51 adulterations in Arriba cocoa were developed on the basis of differences in the chloroplast DNA. On the one hand, a different repeat of the sequence TAAAG in the inverted repeat region results in a different length of amplicons for the two cocoa types, which can be detected by agarose gel electrophoresis, capillary gel electrophoresis, and denaturing high-performance liquid chromatography. On the other hand, single nucleotide polymorphisms (SNPs) between the CCN-51 and Arriba sequences represent restriction sites, which can be used for restriction fragment length polymorphism analysis. A semi-quantitative analysis based on these SNPs is feasible. A method for an exact quantitation based on these results is not realizable. These sequence variations were confirmed for a comprehensive cultivar collection of Arriba and CCN-51, for both bean and leaf samples.

mtDNA control-region sequence variation suggests multiple independent origins of an "Asian-specific" 9-bp deletion in sub-Saharan Africans.

PubMed Central

Soodyall, H.; Vigilant, L.; Hill, A. V.; Stoneking, M.; Jenkins, T.

1996-01-01

The intergenic COII/tRNA(Lys) 9-bp deletion in human mtDNA, which is found at varying frequencies in Asia, Southeast Asia, Polynesia, and the New World, was also found in 81 of 919 sub-Saharan Africans. Using mtDNA control-region sequence data from a subset of 41 individuals with the deletion, we identified 22 unique mtDNA types associated with the deletion in Africa. A comparison of the unique mtDNA types from sub-Saharan Africans and Asians with the 9-bp deletion revealed that sub-Saharan Africans and Asians have sequence profiles that differ in the locations and frequencies of variant sites. Both phylogenetic and mismatch-distribution analysis suggest that 9-bp deletion arose independently in sub-Saharan Africa and Asia and that the deletion has arisen more than once in Africa. Within Africa, the deletion was not found among Khoisan peoples and was rare to absent in western and southwestern African populations, but it did occur in Pygmy and Negroid populations from central Africa and in Malawi and southern African Bantu-speakers. The distribution of the 9-bp deletion in Africa suggests that the deletion could have arisen in central Africa and was then introduced to southern Africa via the recent "Bantu expansion." PMID:8644719

Draft Genome Sequence of Mycobacterium chimaera Type ...

EPA Pesticide Factsheets

We report the draft genome sequence of the type strain Mycobacterium chimaera Fl-0169T, a member of the Mycobacterium avium complex (MAC). M. chimaera Fl-0169T was isolated from a patient in Italy and is highly similar to strains of M. chimaera isolated in Ireland, though Fl-0169T possesses unique virulence genes. Evidence suggests that M. avium, M. intracellulare, and M. chimaera are differently virulent and a comparative genomic analysis is critically needed to identify diagnostic targets that reliably differentiate species of MAC. With treatment costs for Mycobacterium infections estimated to be >$1.8 B annually in the U.S., correct species identification will result in improved treatment selection, lower costs, and improved patient outcomes.

Identification of a novel astrovirus in domestic sheep in Hungary.

PubMed

Reuter, Gábor; Pankovics, Péter; Delwart, Eric; Boros, Ákos

2012-02-01

The family Astroviridae consists of two genera, Avastrovirus and Mamastrovirus, whose members are associated with gastroenteritis in avian and mammalian hosts, respectively. We serendipitously identified a novel ovine astrovirus in a fecal specimen from a domestic sheep (Ovis aries) in Hungary by viral metagenomic analysis. Sequencing of the fragment indicated that it was an ORF1b/ORF2/3'UTR sequence, and it has been submitted to the GenBank database as ovine astrovirus type 2 (OAstV-2/Hungary/2009) with accession number JN592482. The unique sequence characteristics and the phylogenetic position of OAstV-2 suggest that genetically divergent lineages of astroviruses exist in sheep.

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 Cucumis species. Besides, the significant correlation was found between gene density along chromosome and GISH band intensity in C. sativus and C. melo. In summary, comparative cytogenetic mapping of major satellites and GISH revealed the distinct differentiation of chromosome structure during species formation. The evolution of repetitive sequences was the main force for the divergence of Cucumis species from common ancestor.

Full genome sequence analysis of a novel adenovirus of rhesus macaque origin indicates a new simian adenovirus type and species.

PubMed

Malouli, Daniel; Howell, Grant L; Legasse, Alfred W; Kahl, Christoph; Axthelm, Michael K; Hansen, Scott G; Früh, Klaus

2014-09-01

Multiple novel simian adenoviruses have been isolated over the past years and their potential to cross the species barrier and infect the human population is an ever present threat. Here we describe the isolation and full genome sequencing of a novel simian adenovirus (SAdV) isolated from the urine of two independent, never co-housed, late stage simian immunodeficiency virus (SIV)-infected rhesus macaques. The viral genome sequences revealed a novel type with a unique genome length, GC content, E3 region and DNA polymerase amino acid sequence that is sufficiently distinct from all currently known human- or simian adenovirus species to warrant classifying these isolates as a novel species of simian adenovirus. This new species, termed Simian mastadenovirus D (SAdV-D), displays the standard genome organization for the genus Mastadenovirus containing only one copy of the fiber gene which sets it apart from the old world monkey adenovirus species HAdV-G, SAdV-B and SAdV-C.

Discovery of "Escherichia coli" CRISPR Sequences in an Undergraduate Laboratory

ERIC Educational Resources Information Center

Militello, Kevin T.; Lazatin, Justine C.

2017-01-01

Clustered regularly interspaced short palindromic repeats (CRISPRs) represent a novel type of adaptive immune system found in eubacteria and archaebacteria. CRISPRs have recently generated a lot of attention due to their unique ability to catalog foreign nucleic acids, their ability to destroy foreign nucleic acids in a mechanism that shares some…

Combining one-step Sanger sequencing with phasing probe hybridization for HLA class I typing yields rapid, G-group resolution predicting 99% of unique full length protein sequences.

PubMed

Tu, Bin; Masaberg, Carly; Hou, Lihua; Behm, Daniel; Brescia, Peter; Cha, Nuri; Kariyawasam, Kanthi; Lee, Jar How; Nong, Thoa; Sells, John; Tausch, Paul; Yang, Ruyan; Ng, Jennifer; Hurley, Carolyn Katovich

2017-02-01

Sanger-based DNA sequencing of exons 2+3 of HLA class I alleles from a heterozygote frequently results in two or more alternative genotypes. This study was undertaken to reduce the time and effort required to produce a single high resolution HLA genotype. Samples were typed in parallel by Sanger sequencing and oligonucleotide probe hybridization. This workflow, together with optimization of analysis software, was tested and refined during the typing of over 42,000 volunteers for an unrelated hematopoietic progenitor cell donor registry. Next generation DNA sequencing (NGS) was applied to over 1000 of these samples to identify the alleles present within the G group designations. Single genotypes at G level resolution were obtained for over 95% of the loci without additional assays. The vast majority of alleles identified (>99%) were the primary allele giving the G groups their name. Only 0.7% of the alleles identified encoded protein variants that were not detected by a focus on the antigen recognition domain (ARD)-encoding exons. Our combined method routinely provides biologically relevant typing resolution at the level of the ARD. It can be applied to both single samples or to large volume typing supporting either bone marrow or solid organ transplantation using technologies currently available in many HLA laboratories. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Bacterial CRISPR Regions: General Features and their Potential for Epidemiological Molecular Typing Studies

PubMed Central

Karimi, Zahra; Ahmadi, Ali; Najafi, Ali; Ranjbar, Reza

2018-01-01

Introduction: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci as novel and applicable regions in prokaryotic genomes have gained great attraction in the post genomics era. Methods: These unique regions are diverse in number and sequence composition in different pathogenic bacteria and thereby can be a suitable candidate for molecular epidemiology and genotyping studies. Results:Furthermore, the arrayed structure of CRISPR loci (several unique repeats spaced with the variable sequence) and associated cas genes act as an active prokaryotic immune system against viral replication and conjugative elements. This property can be used as a tool for RNA editing in bioengineering studies. Conclusion: The aim of this review was to survey some details about the history, nature, and potential applications of CRISPR arrays in both genetic engineering and bacterial genotyping studies. PMID:29755603

Complete genome sequence of Dyadobacter fermentans type strain (NS114T)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lang, Elke; Lapidus, Alla; Chertkov, Olga

Dyadobacter fermentans (Chelius MK and Triplett EW, 2000) is the type species of the genus Dyadobacter. It is of phylogenetic interest because of its location in the Cytophagaceae, a very diverse family within the order 'Sphingobacteriales'. D. fermentans has a mainly respiratory metabolism, stains Gram-negative, is non-motile and oxidase and catalase positive. It is characterized by the production of cell filaments in ageing cultures, a flexirubin-like pigment and its ability to ferment glucose, which is almost unique in the aerobically living members of this taxonomically difficult family. Here we describe the features of this organism, together with the complete genomemore » sequence, and annotation. This is the first complete genome sequence of the 'sphingobacterial' genus Dyadobacter, and this 6,967,790 bp long single replicon genome with its 5804 protein-coding and 50 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.« less

BrucellaBase: Genome information resource.

PubMed

Sankarasubramanian, Jagadesan; Vishnu, Udayakumar S; Khader, L K M Abdul; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

2016-09-01

Brucella sp. causes a major zoonotic disease, brucellosis. Brucella belongs to the family Brucellaceae under the order Rhizobiales of Alphaproteobacteria. We present BrucellaBase, a web-based platform, providing features of a genome database together with unique analysis tools. We have developed a web version of the multilocus sequence typing (MLST) (Whatmore et al., 2007) and phylogenetic analysis of Brucella spp. BrucellaBase currently contains genome data of 510 Brucella strains along with the user interfaces for BLAST, VFDB, CARD, pairwise genome alignment and MLST typing. Availability of these tools will enable the researchers interested in Brucella to get meaningful information from Brucella genome sequences. BrucellaBase will regularly be updated with new genome sequences, new features along with improvements in genome annotations. BrucellaBase is available online at http://www.dbtbrucellosis.in/brucellabase.html or http://59.99.226.203/brucellabase/homepage.html. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs

NASA Technical Reports Server (NTRS)

Pitulle, C.; Hedenstierna, K. O.; Fox, G. E.

1995-01-01

Further improvements in technology for efficient monitoring of genetically engineered microorganisms (GEMs) in the environment are needed. Technology for monitoring rRNA is well established but has not generally been applicable to GEMs because of the lack of unique rRNA target sequences. In the work described herein, it is demonstrated that a deletion mutant of a plasmid-borne Vibrio proteolyticus 5S rRNA gene continues to accumulate to high levels in Escherichia coli although it is no longer incorporated into 70S ribosomes. This deletion construct was subsequently modified by mutagenesis to create a unique recognition site for the restriction endonuclease BstEII, into which new sequences could be readily inserted. Finally, a novel 17-nucleotide identifier sequence from Pennisetum purpureum was embedded into the construct to create an RNA identification cassette. The artificial identifier RNA, expressed from this cassette in vivo, accumulated in E. coli to levels comparable to those of wild-type 5S rRNA without being seriously detrimental to cell survival in laboratory experiments and without entering the ribosomes. These results demonstrate that artificial, stable RNAs containing sequence segments remarkably different from those present in any known rRNA can be designed and that neither the deleted sequence segment nor ribosome incorporation is essential for accumulation of an RNA product.

Reconstitution of wild type viral DNA in simian cells transfected with early and late SV40 defective genomes.

PubMed

O'Neill, F J; Gao, Y; Xu, X

1993-11-01

The DNAs of polyomaviruses ordinarily exist as a single circular molecule of approximately 5000 base pairs. Variants of SV40, BKV and JCV have been described which contain two complementing defective DNA molecules. These defectives, which form a bipartite genome structure, contain either the viral early region or the late region. The defectives have the unique property of being able to tolerate variable sized reiterations of regulatory and terminus region sequences, and portions of the coding region. They can also exchange coding region sequences with other polyomaviruses. It has been suggested that the bipartite genome structure might be a stage in the evolution of polyomaviruses which can uniquely sustain genome and sequence diversity. However, it is not known if the regulatory and terminus region sequences are highly mutable. Also, it is not known if the bipartite genome structure is reversible and what the conditions might be which would favor restoration of the monomolecular genome structure. We addressed the first question by sequencing the reiterated regulatory and terminus regions of E- and L-SV40 DNAs. This revealed a large number of mutations in the regulatory regions of the defective genomes, including deletions, insertions, rearrangements and base substitutions. We also detected insertions and base substitutions in the T-antigen gene. We addressed the second question by introducing into permissive simian cells, E- and L-SV40 genomes which had been engineered to contain only a single regulatory region. Analysis of viral DNA from transfected cells demonstrated recombined genomes containing a wild type monomolecular DNA structure. However, the complete defectives, containing reiterated regulatory regions, could often compete away the wild type genomes. The recombinant monomolecular genomes were isolated, cloned and found to be infectious. All of the DNA alterations identified in one of the regulatory regions of E-SV40 DNA were present in the recombinant monomolecular genomes. These and other findings indicate that the bipartite genome state can sustain many mutations which wtSV40 cannot directly sustain. However, the mutations can later be introduced into the wild type genomes when the E- and L-SV40 DNAs recombine to generate a new monomolecular genome structure.

Distinct Circular Single-Stranded DNA Viruses Exist in Different Soil Types

PubMed Central

Swanson, Maud M.; Dawson, Lorna; Freitag, Thomas E.; Singh, Brajesh K.; Torrance, Lesley; Mushegian, Arcady R.

2015-01-01

The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors. PMID:25841004

Selection and Validation of a Multilocus Variable-Number Tandem-Repeat Analysis Panel for Typing Shigella spp.▿ †

PubMed Central

Gorgé, Olivier; Lopez, Stéphanie; Hilaire, Valérie; Lisanti, Olivier; Ramisse, Vincent; Vergnaud, Gilles

2008-01-01

The Shigella genus has historically been separated into four species, based on biochemical assays. The classification within each species relies on serotyping. Recently, genome sequencing and DNA assays, in particular the multilocus sequence typing (MLST) approach, greatly improved the current knowledge of the origin and phylogenetic evolution of Shigella spp. The Shigella and Escherichia genera are now considered to belong to a unique genomospecies. Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) provides valuable polymorphic markers for genotyping and performing phylogenetic analyses of highly homogeneous bacterial pathogens. Here, we assess the capability of MLVA for Shigella typing. Thirty-two potentially polymorphic VNTRs were selected by analyzing in silico five Shigella genomic sequences and subsequently evaluated. Eventually, a panel of 15 VNTRs was selected (i.e., MLVA15 analysis). MLVA15 analysis of 78 strains or genome sequences of Shigella spp. and 11 strains or genome sequences of Escherichia coli distinguished 83 genotypes. Shigella population cluster analysis gave consistent results compared to MLST. MLVA15 analysis showed capabilities for E. coli typing, providing classification among pathogenic and nonpathogenic E. coli strains included in the study. The resulting data can be queried on our genotyping webpage (http://mlva.u-psud.fr). The MLVA15 assay is rapid, highly discriminatory, and reproducible for Shigella and Escherichia strains, suggesting that it could significantly contribute to epidemiological trace-back analysis of Shigella infections and pathogenic Escherichia outbreaks. Typing was performed on strains obtained mostly from collections. Further studies should include strains of much more diverse origins, including all pathogenic E. coli types. PMID:18216214

Canine Parvovirus Types 2c and 2b Circulating in North American Dogs in 2006 and 2007▿

PubMed Central

Kapil, Sanjay; Cooper, Emily; Lamm, Cathy; Murray, Brandy; Rezabek, Grant; Johnston, Larry; Campbell, Gregory; Johnson, Bill

2007-01-01

Parvovirus is the most common viral cause of diarrhea in young puppies. Based on the analysis of a partial VP2 sequence of 54 samples, canine parvovirus type 2c (CPV-2c) (n = 26), CPV-2b (n = 25), and CPV-2 (n = 3) were detected in the United States. The American CPV-2b isolates have unique codons (494 and 572) in VP2. PMID:17928423

A fully automatic evolutionary classification of protein folds: Dali Domain Dictionary version 3

PubMed Central

Dietmann, Sabine; Park, Jong; Notredame, Cedric; Heger, Andreas; Lappe, Michael; Holm, Liisa

2001-01-01

The Dali Domain Dictionary (http://www.ebi.ac.uk/dali/domain) is a numerical taxonomy of all known structures in the Protein Data Bank (PDB). The taxonomy is derived fully automatically from measurements of structural, functional and sequence similarities. Here, we report the extension of the classification to match the traditional four hierarchical levels corresponding to: (i) supersecondary structural motifs (attractors in fold space), (ii) the topology of globular domains (fold types), (iii) remote homologues (functional families) and (iv) homologues with sequence identity above 25% (sequence families). The computational definitions of attractors and functional families are new. In September 2000, the Dali classification contained 10 531 PDB entries comprising 17 101 chains, which were partitioned into five attractor regions, 1375 fold types, 2582 functional families and 3724 domain sequence families. Sequence families were further associated with 99 582 unique homologous sequences in the HSSP database, which increases the number of effectively known structures several-fold. The resulting database contains the description of protein domain architecture, the definition of structural neighbours around each known structure, the definition of structurally conserved cores and a comprehensive library of explicit multiple alignments of distantly related protein families. PMID:11125048

Applying the Concept of Peptide Uniqueness to Anti-Polio Vaccination.

PubMed

Kanduc, Darja; Fasano, Candida; Capone, Giovanni; Pesce Delfino, Antonella; Calabrò, Michele; Polimeno, Lorenzo

2015-01-01

Although rare, adverse events may associate with anti-poliovirus vaccination thus possibly hampering global polio eradication worldwide. To design peptide-based anti-polio vaccines exempt from potential cross-reactivity risks and possibly able to reduce rare potential adverse events such as the postvaccine paralytic poliomyelitis due to the tendency of the poliovirus genome to mutate. Proteins from poliovirus type 1, strain Mahoney, were analyzed for amino acid sequence identity to the human proteome at the pentapeptide level, searching for sequences that (1) have zero percent of identity to human proteins, (2) are potentially endowed with an immunologic potential, and (3) are highly conserved among poliovirus strains. Sequence analyses produced a set of consensus epitopic peptides potentially able to generate specific anti-polio immune responses exempt from cross-reactivity with the human host. Peptide sequences unique to poliovirus proteins and conserved among polio strains might help formulate a specific and universal anti-polio vaccine able to react with multiple viral strains and exempt from the burden of possible cross-reactions with human proteins. As an additional advantage, using a peptide-based vaccine instead of current anti-polio DNA vaccines would eliminate the rare post-polio poliomyelitis cases and other disabling symptoms that may appear following vaccination.

Analysis of the genome-wide variations among multiple strains of the plant pathogenic bacterium Xylella fastidiosa

PubMed Central

Doddapaneni, Harshavardhan; Yao, Jiqiang; Lin, Hong; Walker, M Andrew; Civerolo, Edwin L

2006-01-01

Background The Gram-negative, xylem-limited phytopathogenic bacterium Xylella fastidiosa is responsible for causing economically important diseases in grapevine, citrus and many other plant species. Despite its economic impact, relatively little is known about the genomic variations among strains isolated from different hosts and their influence on the population genetics of this pathogen. With the availability of genome sequence information for four strains, it is now possible to perform genome-wide analyses to identify and categorize such DNA variations and to understand their influence on strain functional divergence. Results There are 1,579 genes and 194 non-coding homologous sequences present in the genomes of all four strains, representing a 76. 2% conservation of the sequenced genome. About 60% of the X. fastidiosa unique sequences exist as tandem gene clusters of 6 or more genes. Multiple alignments identified 12,754 SNPs and 14,449 INDELs in the 1528 common genes and 20,779 SNPs and 10,075 INDELs in the 194 non-coding sequences. The average SNP frequency was 1.08 × 10-2 per base pair of DNA and the average INDEL frequency was 2.06 × 10-2 per base pair of DNA. On an average, 60.33% of the SNPs were synonymous type while 39.67% were non-synonymous type. The mutation frequency, primarily in the form of external INDELs was the main type of sequence variation. The relative similarity between the strains was discussed according to the INDEL and SNP differences. The number of genes unique to each strain were 60 (9a5c), 54 (Dixon), 83 (Ann1) and 9 (Temecula-1). A sub-set of the strain specific genes showed significant differences in terms of their codon usage and GC composition from the native genes suggesting their xenologous origin. Tandem repeat analysis of the genomic sequences of the four strains identified associations of repeat sequences with hypothetical and phage related functions. Conclusion INDELs and strain specific genes have been identified as the main source of variations among strains, with individual strains showing different rates of genome evolution. Based on these genome comparisons, it appears that the Pierce's disease strain Temecula-1 genome represents the ancestral genome of the X. fastidiosa. Results of this analysis are publicly available in the form of a web database. PMID:16948851

Unique BK virus non-coding control region (NCCR) variants in hematopoietic stem cell transplant recipients with and without hemorrhagic cystitis.

PubMed

Carr, Michael J; McCormack, Grace P; Mutton, Ken J; Crowley, Brendan

2006-04-01

Hematopoietic stem cell transplant recipients frequently develop BK virus (BKV)-associated hemorrhagic cystitis, which coincides with BK viruria. However, the precise role of BKV in the etiology of hemorrhagic cystitis in hematopoietic stem cell transplant recipients remains unclear, since approximately 50% of all such adult transplant recipients excrete BKV, yet do not develop this clinical condition. In the present study, BKV were analyzed to determine if mutations in the non-coding control region (NCCR), and specific BKV sub-types defined by sequence analysis of major capsid protein VP1, were associated with development of hemorrhagic cystitis in hematopoietic stem cell transplant recipients. The regions encoding VP1 and NCCRs of BKV in urine samples collected from 15 hematopoietic stem cell transplant recipients with hemorrhagic cystitis and 20 without this illness were amplified and sequenced. Sequence variations in the NCCRs of BKV were identified in urine samples from those with and without hemorrhagic cystitis. Furthermore, five unique sequence variations within transcription factor binding sites in the canonical NCCR, O-P-Q-R-S, were identified, representing new BKV variants from a population of cloned quasi-species obtained from patients with and without hemorrhagic cystitis. Thirty-five BKV VP1 sequences were analyzed by phylogenetic analysis but no specific BKV sub-type was associated with hemorrhagic cystitis. Five previously unrecognized naturally occurring variants of the BKV are described which involve amplifications, deletions, and rearrangements of the archetypal BKV NCCRs in individuals with and without hemorrhagic cystitis. Architectural rearrangements in the NCCRs of BKV did not appear to be a prerequisite for development of hemorrhagic cystitis in hematopoietic stem cell transplant recipients. Copyright 2006 Wiley-Liss, Inc.

Genome Re-Sequencing of Semi-Wild Soybean Reveals a Complex Soja Population Structure and Deep Introgression

PubMed Central

Wu, Sanling; Wang, Ying-Ying; Ye, Chu-Yu; Bai, Xuefei; Li, Zefeng; Yan, Chenghai; Wang, Weidi; Wang, Ziqiang; Shu, Qingyao; Xie, Jiahua; Lee, Suk-Ha; Fan, Longjiang

2014-01-01

Semi-wild soybean is a unique type of soybean that retains both wild and domesticated characteristics, which provides an important intermediate type for understanding the evolution of the subgenus Soja population in the Glycine genus. In this study, a semi-wild soybean line (Maliaodou) and a wild line (Lanxi 1) collected from the lower Yangtze regions were deeply sequenced while nine other semi-wild lines were sequenced to a 3-fold genome coverage. Sequence analysis revealed that (1) no independent phylogenetic branch covering all 10 semi-wild lines was observed in the Soja phylogenetic tree; (2) besides two distinct subpopulations of wild and cultivated soybean in the Soja population structure, all semi-wild lines were mixed with some wild lines into a subpopulation rather than an independent one or an intermediate transition type of soybean domestication; (3) high heterozygous rates (0.19–0.49) were observed in several semi-wild lines; and (4) over 100 putative selective regions were identified by selective sweep analysis, including those related to the development of seed size. Our results suggested a hybridization origin for the semi-wild soybean, which makes a complex Soja population structure. PMID:25265539

A public HTLV-1 molecular epidemiology database for sequence management and data mining.

PubMed

Araujo, Thessika Hialla Almeida; Souza-Brito, Leandro Inacio; Libin, Pieter; Deforche, Koen; Edwards, Dustin; de Albuquerque-Junior, Antonio Eduardo; Vandamme, Anne-Mieke; Galvao-Castro, Bernardo; Alcantara, Luiz Carlos Junior

2012-01-01

It is estimated that 15 to 20 million people are infected with the human T-cell lymphotropic virus type 1 (HTLV-1). At present, there are more than 2,000 unique HTLV-1 isolate sequences published. A central database to aggregate sequence information from a range of epidemiological aspects including HTLV-1 infections, pathogenesis, origins, and evolutionary dynamics would be useful to scientists and physicians worldwide. Described here, we have developed a database that collects and annotates sequence data and can be accessed through a user-friendly search interface. The HTLV-1 Molecular Epidemiology Database website is available at http://htlv1db.bahia.fiocruz.br/. All data was obtained from publications available at GenBank or through contact with the authors. The database was developed using Apache Webserver 2.1.6 and SGBD MySQL. The webpage interfaces were developed in HTML and sever-side scripting written in PHP. The HTLV-1 Molecular Epidemiology Database is hosted on the Gonçalo Moniz/FIOCRUZ Research Center server. There are currently 2,457 registered sequences with 2,024 (82.37%) of those sequences representing unique isolates. Of these sequences, 803 (39.67%) contain information about clinical status (TSP/HAM, 17.19%; ATL, 7.41%; asymptomatic, 12.89%; other diseases, 2.17%; and no information, 60.32%). Further, 7.26% of sequences contain information on patient gender while 5.23% of sequences provide the age of the patient. The HTLV-1 Molecular Epidemiology Database retrieves and stores annotated HTLV-1 proviral sequences from clinical, epidemiological, and geographical studies. The collected sequences and related information are now accessible on a publically available and user-friendly website. This open-access database will support clinical research and vaccine development related to viral genotype.

A genomewide survey of basic helix–loop–helix factors in Drosophila

PubMed Central

Moore, Adrian W.; Barbel, Sandra; Jan, Lily Yeh; Jan, Yuh Nung

2000-01-01

The basic helix–loop–helix (bHLH) transcription factors play important roles in the specification of tissue type during the development of animals. We have used the information contained in the recently published genomic sequence of Drosophila melanogaster to identify 12 additional bHLH proteins. By sequence analysis we have assigned these proteins to families defined by Atonal, Hairy-Enhancer of Split, Hand, p48, Mesp, MYC/USF, and the bHLH-Per, Arnt, Sim (PAS) domain. In addition, one single protein represents a unique family of bHLH proteins. mRNA in situ analysis demonstrates that the genes encoding these proteins are expressed in several tissue types but are particularly concentrated in the developing nervous system and mesoderm. PMID:10973473

Putative and unique gene sequence utilization for the design of species specific probes as modeled by Lactobacillus plantarum

USDA-ARS?s Scientific Manuscript database

The concept of utilizing putative and unique gene sequences for the design of species specific probes was tested. The abundance profile of assigned functions within the Lactobacillus plantarum genome was used for the identification of the putative and unique gene sequence, csh. The targeted gene (cs...

Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases.

PubMed

Lohman, Jeremy R; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

2015-10-13

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.

Structural and evolutionary relationships of “AT-less” type I polyketide synthase ketosynthases

PubMed Central

Lohman, Jeremy R.; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

2015-01-01

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs. PMID:26420866

Identification of Type A, B, E, and F Botulinum Neurotoxin Genes and of Botulinum Neurotoxigenic Clostridia by Denaturing High-Performance Liquid Chromatography

PubMed Central

Franciosa, Giovanna; Pourshaban, Manoocheher; De Luca, Alessandro; Buccino, Anna; Dallapiccola, Bruno; Aureli, Paolo

2004-01-01

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism. PMID:15240298

Transcriptomic and anatomical complexity of primary, seminal, and crown roots highlight root type-specific functional diversity in maize (Zea mays L.)

PubMed Central

Tai, Huanhuan; Lu, Xin; Opitz, Nina; Marcon, Caroline; Paschold, Anja; Lithio, Andrew; Nettleton, Dan; Hochholdinger, Frank

2016-01-01

Maize develops a complex root system composed of embryonic and post-embryonic roots. Spatio-temporal differences in the formation of these root types imply specific functions during maize development. A comparative transcriptomic study of embryonic primary and seminal, and post-embryonic crown roots of the maize inbred line B73 by RNA sequencing along with anatomical studies were conducted early in development. Seminal roots displayed unique anatomical features, whereas the organization of primary and crown roots was similar. For instance, seminal roots displayed fewer cortical cell files and their stele contained more meta-xylem vessels. Global expression profiling revealed diverse patterns of gene activity across all root types and highlighted the unique transcriptome of seminal roots. While functions in cell remodeling and cell wall formation were prominent in primary and crown roots, stress-related genes and transcriptional regulators were over-represented in seminal roots, suggesting functional specialization of the different root types. Dynamic expression of lignin biosynthesis genes and histochemical staining suggested diversification of cell wall lignification among the three root types. Our findings highlight a cost-efficient anatomical structure and a unique expression profile of seminal roots of the maize inbred line B73 different from primary and crown roots. PMID:26628518

2D nanomaterials assembled from sequence-defined molecules

DOE PAGES

Mu, Peng; Zhou, Guangwen; Chen, Chun-Long

2017-10-21

Two dimensional (2D) nanomaterials have attracted broad interest owing to their unique physical and chemical properties with potential applications in electronics, chemistry, biology, medicine and pharmaceutics. Due to the current limitations of traditional 2D nanomaterials (e.g., graphene and graphene oxide) in tuning surface chemistry and compositions, 2D nanomaterials assembled from sequence-defined molecules (e.g., DNAs, proteins, peptides and peptoids) have recently been developed. They represent an emerging class of 2D nanomaterials with attractive physical and chemical properties. Here, we summarize the recent progress in the synthesis and applications of this type of sequence-defined 2D nanomaterials. We also discuss the challenges andmore » opportunities in this new field.« less

2D nanomaterials assembled from sequence-defined molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mu, Peng; Zhou, Guangwen; Chen, Chun-Long

Two dimensional (2D) nanomaterials have attracted broad interest owing to their unique physical and chemical properties with potential applications in electronics, chemistry, biology, medicine and pharmaceutics. Due to the current limitations of traditional 2D nanomaterials (e.g., graphene and graphene oxide) in tuning surface chemistry and compositions, 2D nanomaterials assembled from sequence-defined molecules (e.g., DNAs, proteins, peptides and peptoids) have recently been developed. They represent an emerging class of 2D nanomaterials with attractive physical and chemical properties. Here, we summarize the recent progress in the synthesis and applications of this type of sequence-defined 2D nanomaterials. We also discuss the challenges andmore » opportunities in this new field.« less

Phonological and Semantic Cues to Learning from Word-Types

PubMed Central

Richtsmeier, Peter

2017-01-01

Word-types represent the primary form of data for many models of phonological learning, and they often predict performance in psycholinguistic tasks. Word-types are often tacitly defined as phonologically unique words. Yet, an explicit test of this definition is lacking, and natural language patterning suggests that word meaning could also act as a cue to word-type status. This possibility was tested in a statistical phonotactic learning experiment in which phonological and semantic properties of word-types varied. During familiarization, the learning targets—word-medial consonant sequences—were instantiated either by four related word-types or by just one word-type (the experimental frequency factor). The expectation was that more word-types would lead participants to generalize the target sequences. Regarding semantic cues, related word-types were either associated with different referents or all with a single referent. Regarding phonological cues, related word-types differed from each other by one, two, or more phonemes. At test, participants rated novel wordforms for their similarity to the familiarization words. When participants heard four related word-types, they gave higher ratings to test words with the same consonant sequences, irrespective of the phonological and semantic manipulations. The results support the existing phonological definition of word-types. PMID:29187914

Novel division level bacterial diversity in a Yellowstone hot spring.

PubMed

Hugenholtz, P; Pitulle, C; Hershberger, K L; Pace, N R

1998-01-01

A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609-1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or Bacteria-specific rDNA primers and cloned. Unique rDNA types among > 300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (> or = 98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio and Thermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing delta-Proteobacteria. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that Archaea dominate hydrothermal environments. Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.

Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)

PubMed Central

Russo, James J.; Bohenzky, Roy A.; Chien, Ming-Cheng; Chen, Jing; Yan, Ming; Maddalena, Dawn; Parry, J. Preston; Peruzzi, Daniela; Edelman, Isidore S.; Chang, Yuan; Moore, Patrick S.

1996-01-01

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G+C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor. PMID:8962146

The Hydrologic Implications Of Unique Urban Soil Horizon Sequencing On The Functions Of Passive Green Infrastructure

NASA Astrophysics Data System (ADS)

Shuster, W.; Schifman, L. A.; Herrmann, D.

2017-12-01

Green infrastructure represents a broad set of site- to landscape-scale practices that can be flexibly implemented to increase sewershed retention capacity, and can thereby improve on the management of water quantity and quality. Although much green infrastructure presents as formal engineered designs, urbanized landscapes with highly-interspersed pervious surfaces (e.g., right-of-way, parks, lawns, vacant land) may offer ecosystem services as passive, infiltrative green infrastructure. Yet, infiltration and drainage processes are regulated by soil surface conditions, and then the layering of subsoil horizons, respectively. Drawing on a unique urban soil taxonomic and hydrologic dataset collected in 12 cities (each city representing a major soil order), we determined how urbanization processes altered the sequence of soil horizons (compared to pre-urbanized reference soil pedons) and modeled the hydrologic implications of these shifts in layering with an unsaturated zone code (HYDRUS2D). We found that the different layering sequences in urbanized soils render different types and extents of supporting (plant-available soil water), provisioning (productive vegetation), and regulating (runoff mitigation) ecosystem services.

Improved serial analysis of V1 ribosomal sequence tags (SARST-V1) provides a rapid, comprehensive, sequence-based characterization of bacterial diversity and community composition.

PubMed

Yu, Zhongtang; Yu, Marie; Morrison, Mark

2006-04-01

Serial analysis of ribosomal sequence tags (SARST) is a recently developed technology that can generate large 16S rRNA gene (rrs) sequence data sets from microbiomes, but there are numerous enzymatic and purification steps required to construct the ribosomal sequence tag (RST) clone libraries. We report here an improved SARST method, which still targets the V1 hypervariable region of rrs genes, but reduces the number of enzymes, oligonucleotides, reagents, and technical steps needed to produce the RST clone libraries. The new method, hereafter referred to as SARST-V1, was used to examine the eubacterial diversity present in community DNA recovered from the microbiome resident in the ovine rumen. The 190 sequenced clones contained 1055 RSTs and no less than 236 unique phylotypes (based on > or = 95% sequence identity) that were assigned to eight different eubacterial phyla. Rarefaction and monomolecular curve analyses predicted that the complete RST clone library contains 99% of the 353 unique phylotypes predicted to exist in this microbiome. When compared with ribosomal intergenic spacer analysis (RISA) of the same community DNA sample, as well as a compilation of nine previously published conventional rrs clone libraries prepared from the same type of samples, the RST clone library provided a more comprehensive characterization of the eubacterial diversity present in rumen microbiomes. As such, SARST-V1 should be a useful tool applicable to comprehensive examination of diversity and composition in microbiomes and offers an affordable, sequence-based method for diversity analysis.

Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples

PubMed Central

Nguyen, David; Valenzuela, Nicole; Takemura, Ping; Bolon, Yung-Tsi; Springer, Brianna; Saito, Katsuyuki; Zheng, Ying; Hague, Tim; Pasztor, Agnes; Horvath, Gyorgy; Rigo, Krisztina; Reed, Elaine F.; Zhang, Qiuheng

2016-01-01

Background Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA. Methods Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3’ UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing. Results Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%). Conclusion Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation. PMID:27798706

Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples.

PubMed

Yin, Yuxin; Lan, James H; Nguyen, David; Valenzuela, Nicole; Takemura, Ping; Bolon, Yung-Tsi; Springer, Brianna; Saito, Katsuyuki; Zheng, Ying; Hague, Tim; Pasztor, Agnes; Horvath, Gyorgy; Rigo, Krisztina; Reed, Elaine F; Zhang, Qiuheng

2016-01-01

Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA. Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3' UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing. Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%). Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.

Towards a physical classification of early-type galaxies. Profile of a key programme.

NASA Astrophysics Data System (ADS)

Bender, R.; Capaccioli, M.; Macchetto, F.; Nieto, J.-L.

1989-03-01

Hubble was the first who succeeded in classifying galaxies within a scheme of some physical meaning. Although it soon became clear that Hubble's tuning fork does not represent an evolutionary sequence, this essential diagram has proven to be a powerful tool especially for the understanding of late-type galaxies. On the other hand, the "early-type" sequence of elliptical (E) and SO galaxies is less satisfying, because it does not seem to reflect a unique sequence of physical properties. The SO class, although conceived to bridge the gap between disk- and disk-Iess galaxies, has often been abused to host ellipticals exhibiting peculiarities incompatible with their definition as structureless objects. For the elliptical galaxies themselves, "ellipticity" has been found to be essentially meaningless with regard to their angular momentum properties, and shows Iittle, if any, correlation with other global parameters. This fact became apparent after the first stellar kinematical measurements of luminous ellipticals (Bertola and Capaccioli 1975, IIlingworth 1977); E galaxies are not necessarily f1attened by rotation and may have anisotropie velocity dispersions (Binney 1978).

Molecular characterization of the probiotic strain Bacillus cereus var. toyoi NCIMB 40112 and differentiation from food poisoning strains.

PubMed

Klein, Günter

2011-07-01

Bacillus cereus var. toyoi strain NCIMB 40112 (Toyocerin), a probiotic authorized in the European Union as feed additive for swine, bovines, poultry, and rabbits, was characterized by DNA fingerprinting applying pulsed-field gel electrophoresis and multilocus sequence typing and was compared with reference strains (of clinical and environmental origins). The probiotic strain was clearly characterized by pulsed-field gel electrophoresis using the restriction enzymes Apa I and Sma I resulting in unique DNA patterns. The comparison to the clinical reference strain B. cereus DSM 4312 was done with the same restriction enzymes, and again a clear differentiation of the two strains was possible by the resulting DNA patterns. The use of the restriction enzymes Apa I and Sma I is recommended for further studies. Furthermore, multilocus sequence typing analysis revealed a sequence type (ST 111) that was different from all known STs of B. cereus strains from food poisoning incidents. Thus, a strain characterization and differentiation from food poisoning strains for the probiotic strain was possible. Copyright ©, International Association for Food Protection

Aircraft stress sequence development: A complex engineering process made simple

NASA Technical Reports Server (NTRS)

Schrader, K. H.; Butts, D. G.; Sparks, W. A.

1994-01-01

Development of stress sequences for critical aircraft structure requires flight measured usage data, known aircraft loads, and established relationships between aircraft flight loads and structural stresses. Resulting cycle-by-cycle stress sequences can be directly usable for crack growth analysis and coupon spectra tests. Often, an expert in loads and spectra development manipulates the usage data into a typical sequence of representative flight conditions for which loads and stresses are calculated. For a fighter/trainer type aircraft, this effort is repeated many times for each of the fatigue critical locations (FCL) resulting in expenditure of numerous engineering hours. The Aircraft Stress Sequence Computer Program (ACSTRSEQ), developed by Southwest Research Institute under contract to San Antonio Air Logistics Center, presents a unique approach for making complex technical computations in a simple, easy to use method. The program is written in Microsoft Visual Basic for the Microsoft Windows environment.

The genetic structure of the A mating-type locus of Lentinula edodes.

PubMed

Au, Chun Hang; Wong, Man Chun; Bao, Dapeng; Zhang, Meiyan; Song, Chunyan; Song, Wenhua; Law, Patrick Tik Wan; Kües, Ursula; Kwan, Hoi Shan

2014-02-10

The Shiitake mushroom, Lentinula edodes (Berk.) Pegler is a tetrapolar basidiomycete with two unlinked mating-type loci, commonly called the A and B loci. Identifying the mating-types in shiitake is important for enhancing the breeding and cultivation of this economically-important edible mushroom. Here, we identified the A mating-type locus from the first draft genome sequence of L. edodes and characterized multiple alleles from different monokaryotic strains. Two intron-length polymorphism markers were developed to facilitate rapid molecular determination of A mating-type. L. edodes sequences were compared with those of known tetrapolar and bipolar basidiomycete species. The A mating-type genes are conserved at the homeodomain region across the order Agaricales. However, we observed unique genomic organization of the locus in L. edodes which exhibits atypical gene order and multiple repetitive elements around its A locus. To our knowledge, this is the first known exception among Homobasidiomycetes, in which the mitochondrial intermediate peptidase (mip) gene is not closely linked to A locus. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular structures of centromeric heterochromatin and karyotypic evolution in the Siamese crocodile (Crocodylus siamensis) (Crocodylidae, Crocodylia).

PubMed

Kawagoshi, Taiki; Nishida, Chizuko; Ota, Hidetoshi; Kumazawa, Yoshinori; Endo, Hideki; Matsuda, Yoichi

2008-01-01

Crocodilians have several unique karyotypic features, such as small diploid chromosome numbers (30-42) and the absence of dot-shaped microchromosomes. Of the extant crocodilian species, the Siamese crocodile (Crocodylus siamensis) has no more than 2n = 30, comprising mostly bi-armed chromosomes with large centromeric heterochromatin blocks. To investigate the molecular structures of C-heterochromatin and genomic compartmentalization in the karyotype, characterized by the disappearance of tiny microchromosomes and reduced chromosome number, we performed molecular cloning of centromeric repetitive sequences and chromosome mapping of the 18S-28S rDNA and telomeric (TTAGGG)( n ) sequences. The centromeric heterochromatin was composed mainly of two repetitive sequence families whose characteristics were quite different. Two types of GC-rich CSI-HindIII family sequences, the 305 bp CSI-HindIII-S (G+C content, 61.3%) and 424 bp CSI-HindIII-M (63.1%), were localized to the intensely PI-stained centric regions of all chromosomes, except for chromosome 2 with PI-negative heterochromatin. The 94 bp CSI-DraI (G+C content, 48.9%) was tandem-arrayed satellite DNA and localized to chromosome 2 and four pairs of small-sized chromosomes. The chromosomal size-dependent genomic compartmentalization that is supposedly unique to the Archosauromorpha was probably lost in the crocodilian lineage with the disappearance of microchromosomes followed by the homogenization of centromeric repetitive sequences between chromosomes, except for chromosome 2.

Rather than by direct acquisition via lateral gene transfer, GHF5 cellulases were passed on from early Pratylenchidae to root-knot and cyst nematodes.

PubMed

Rybarczyk-Mydłowska, Katarzyna; Maboreke, Hazel Ruvimbo; van Megen, Hanny; van den Elsen, Sven; Mooyman, Paul; Smant, Geert; Bakker, Jaap; Helder, Johannes

2012-11-21

Plant parasitic nematodes are unusual Metazoans as they are equipped with genes that allow for symbiont-independent degradation of plant cell walls. Among the cell wall-degrading enzymes, glycoside hydrolase family 5 (GHF5) cellulases are relatively well characterized, especially for high impact parasites such as root-knot and cyst nematodes. Interestingly, ancestors of extant nematodes most likely acquired these GHF5 cellulases from a prokaryote donor by one or multiple lateral gene transfer events. To obtain insight into the origin of GHF5 cellulases among evolutionary advanced members of the order Tylenchida, cellulase biodiversity data from less distal family members were collected and analyzed. Single nematodes were used to obtain (partial) genomic sequences of cellulases from representatives of the genera Meloidogyne, Pratylenchus, Hirschmanniella and Globodera. Combined Bayesian analysis of ≈ 100 cellulase sequences revealed three types of catalytic domains (A, B, and C). Represented by 84 sequences, type B is numerically dominant, and the overall topology of the catalytic domain type shows remarkable resemblance with trees based on neutral (= pathogenicity-unrelated) small subunit ribosomal DNA sequences. Bayesian analysis further suggested a sister relationship between the lesion nematode Pratylenchus thornei and all type B cellulases from root-knot nematodes. Yet, the relationship between the three catalytic domain types remained unclear. Superposition of intron data onto the cellulase tree suggests that types B and C are related, and together distinct from type A that is characterized by two unique introns. All Tylenchida members investigated here harbored one or multiple GHF5 cellulases. Three types of catalytic domains are distinguished, and the presence of at least two types is relatively common among plant parasitic Tylenchida. Analysis of coding sequences of cellulases suggests that root-knot and cyst nematodes did not acquire this gene directly by lateral genes transfer. More likely, these genes were passed on by ancestors of a family nowadays known as the Pratylenchidae.

Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species.

PubMed

Wyllie, Anne L; Pannekoek, Yvonne; Bovenkerk, Sandra; van Engelsdorp Gastelaars, Jody; Ferwerda, Bart; van de Beek, Diederik; Sanders, Elisabeth A M; Trzciński, Krzysztof; van der Ende, Arie

2017-09-01

The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease ( n = 101) and from carriage ( n = 103), and on non-typeable pneumococci from asymptomatic individuals ( n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae , targeting cpsA , lytA , piaB , ply , Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae , whereas assays targeting cpsA , ply , Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates. © 2017 The Authors.

Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species

PubMed Central

Pannekoek, Yvonne; Bovenkerk, Sandra; van Engelsdorp Gastelaars, Jody; Ferwerda, Bart; van de Beek, Diederik; Sanders, Elisabeth A. M.; Trzciński, Krzysztof; van der Ende, Arie

2017-01-01

The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae. Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease (n = 101) and from carriage (n = 103), and on non-typeable pneumococci from asymptomatic individuals (n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae, targeting cpsA, lytA, piaB, ply, Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae, whereas assays targeting cpsA, ply, Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates. PMID:28931649

Novel adenoviruses detected in British mustelids, including a unique Aviadenovirus in the tissues of pine martens (Martes martes)

PubMed Central

Gregory, William F.; Turnbull, Dylan; Rocchi, Mara; Meredith, Anna L.; Philbey, Adrian W.; Sharp, Colin P.

2017-01-01

Several adenoviruses are known to cause severe disease in veterinary species. Recent evidence suggests that canine adenovirus type 1 (CAV-1) persists in the tissues of healthy red foxes (Vulpes vulpes), which may be a source of infection for susceptible species. It was hypothesized that mustelids native to the UK, including pine martens (Martes martes) and Eurasian otters (Lutra lutra), may also be persistently infected with adenoviruses. Based on high-throughput sequencing and additional Sanger sequencing, a novel Aviadenovirus, tentatively named marten adenovirus type 1 (MAdV-1), was detected in pine marten tissues. The detection of an Aviadenovirus in mammalian tissue has not been reported previously. Two mastadenoviruses, tentatively designated marten adenovirus type 2 (MAdV-2) and lutrine adenovirus type 1 (LAdV-1), were also detected in tissues of pine martens and Eurasian otters, respectively. Apparently healthy free-ranging animals may be infected with uncharacterized adenoviruses with possible implications for translocation of wildlife. PMID:28749327

Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lohman, Jeremy; Ma, Ming; Osipiuk, Jerzy

2015-10-13

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representationmore » of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.« less

Distribution of sequence-based types of legionella pneumophila serogroup 1 strains isolated from cooling towers, hot springs, and potable water systems in China.

PubMed

Qin, Tian; Zhou, Haijian; Ren, Hongyu; Guan, Hong; Li, Machao; Zhu, Bingqing; Shao, Zhujun

2014-04-01

Legionella pneumophila serogroup 1 causes Legionnaires' disease. Water systems contaminated with Legionella are the implicated sources of Legionnaires' disease. This study analyzed L. pneumophila serogroup 1 strains in China using sequence-based typing. Strains were isolated from cooling towers (n = 96), hot springs (n = 42), and potable water systems (n = 26). Isolates from cooling towers, hot springs, and potable water systems were divided into 25 sequence types (STs; index of discrimination [IOD], 0.711), 19 STs (IOD, 0.934), and 3 STs (IOD, 0.151), respectively. The genetic variation among the potable water isolates was lower than that among cooling tower and hot spring isolates. ST1 was the predominant type, accounting for 49.4% of analyzed strains (n = 81), followed by ST154. With the exception of two strains, all potable water isolates (92.3%) belonged to ST1. In contrast, 53.1% (51/96) and only 14.3% (6/42) of cooling tower and hot spring, respectively, isolates belonged to ST1. There were differences in the distributions of clone groups among the water sources. The comparisons among L. pneumophila strains isolated in China, Japan, and South Korea revealed that similar clones (ST1 complex and ST154 complex) exist in these countries. In conclusion, in China, STs had several unique allelic profiles, and ST1 was the most prevalent sequence type of environmental L. pneumophila serogroup 1 isolates, similar to its prevalence in Japan and South Korea.

Applying the Concept of Peptide Uniqueness to Anti-Polio Vaccination

PubMed Central

Kanduc, Darja; Fasano, Candida; Capone, Giovanni; Pesce Delfino, Antonella; Calabrò, Michele; Polimeno, Lorenzo

2015-01-01

Background. Although rare, adverse events may associate with anti-poliovirus vaccination thus possibly hampering global polio eradication worldwide. Objective. To design peptide-based anti-polio vaccines exempt from potential cross-reactivity risks and possibly able to reduce rare potential adverse events such as the postvaccine paralytic poliomyelitis due to the tendency of the poliovirus genome to mutate. Methods. Proteins from poliovirus type 1, strain Mahoney, were analyzed for amino acid sequence identity to the human proteome at the pentapeptide level, searching for sequences that (1) have zero percent of identity to human proteins, (2) are potentially endowed with an immunologic potential, and (3) are highly conserved among poliovirus strains. Results. Sequence analyses produced a set of consensus epitopic peptides potentially able to generate specific anti-polio immune responses exempt from cross-reactivity with the human host. Conclusion. Peptide sequences unique to poliovirus proteins and conserved among polio strains might help formulate a specific and universal anti-polio vaccine able to react with multiple viral strains and exempt from the burden of possible cross-reactions with human proteins. As an additional advantage, using a peptide-based vaccine instead of current anti-polio DNA vaccines would eliminate the rare post-polio poliomyelitis cases and other disabling symptoms that may appear following vaccination. PMID:26568962

Dynamics of actin evolution in dinoflagellates.

PubMed

Kim, Sunju; Bachvaroff, Tsvetan R; Handy, Sara M; Delwiche, Charles F

2011-04-01

Dinoflagellates have unique nuclei and intriguing genome characteristics with very high DNA content making complete genome sequencing difficult. In dinoflagellates, many genes are found in multicopy gene families, but the processes involved in the establishment and maintenance of these gene families are poorly understood. Understanding the dynamics of gene family evolution in dinoflagellates requires comparisons at different evolutionary scales. Studies of closely related species provide fine-scale information relative to species divergence, whereas comparisons of more distantly related species provides broad context. We selected the actin gene family as a highly expressed conserved gene previously studied in dinoflagellates. Of the 142 sequences determined in this study, 103 were from the two closely related species, Dinophysis acuminata and D. caudata, including full length and partial cDNA sequences as well as partial genomic amplicons. For these two Dinophysis species, at least three types of sequences could be identified. Most copies (79%) were relatively similar and in nucleotide trees, the sequences formed two bushy clades corresponding to the two species. In comparisons within species, only eight to ten nucleotide differences were found between these copies. The two remaining types formed clades containing sequences from both species. One type included the most similar sequences in between-species comparisons with as few as 12 nucleotide differences between species. The second type included the most divergent sequences in comparisons between and within species with up to 93 nucleotide differences between sequences. In all the sequences, most variation occurred in synonymous sites or the 5' UnTranslated Region (UTR), although there was still limited amino acid variation between most sequences. Several potential pseudogenes were found (approximately 10% of all sequences depending on species) with incomplete open reading frames due to frameshifts or early stop codons. Overall, variation in the actin gene family fits best with the "birth and death" model of evolution based on recent duplications, pseudogenes, and incomplete lineage sorting. Divergence between species was similar to variation within species, so that actin may be too conserved to be useful for phylogenetic estimation of closely related species.

2D nanomaterials assembled from sequence-defined molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mu, Peng; Zhou, Guangwen; Chen, Chun-Long

Two dimensional (2D) nanomaterials have attracted broad interest owing to their unique physical and chemical properties with potential applications in electronics, chemistry, biology, medicine and pharmaceutics. Due to the current limitations of traditional 2D nanomaterials (e.g., graphene and graphene oxide) in tuning surface chemistry and compositions, 2D nanomaterials assembled from sequence-defined molecules (e.g., DNAs, proteins, peptides and peptoids) have recently been developed. They represent an emerging class of 2D nanomaterials with attractive physical and chemical properties. In this mini-review, we summarize the recent progress in the synthesis and applications of this type of sequence-defined 2D nanomaterials. The challenges and opportunitiesmore » in this new field are also discussed.« less

Weissella fabaria sp. nov., from a Ghanaian cocoa fermentation.

PubMed

De Bruyne, Katrien; Camu, Nicholas; De Vuyst, Luc; Vandamme, Peter

2010-09-01

Two lactic acid bacteria, strains 257(T) and 252, were isolated from traditional heap fermentations of Ghanaian cocoa beans. 16S rRNA gene sequence analysis of these strains allocated them to the genus Weissella, showing 99.5 % 16S rRNA gene sequence similarity towards Weissella ghanensis LMG 24286(T). Whole-cell protein electrophoresis, fluorescent amplified fragment length polymorphism fingerprinting of whole genomes and biochemical tests confirmed their unique taxonomic position. DNA-DNA hybridization experiments towards their nearest phylogenetic neighbour demonstrated that the two strains represent a novel species, for which we propose the name Weissella fabaria sp. nov., with strain 257(T) (=LMG 24289(T) =DSM 21416(T)) as the type strain. Additional sequence analysis using pheS gene sequences proved useful for identification of all Weissella-Leuconostoc-Oenococcus species and for the recognition of the novel species.

Clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing

PubMed Central

Momeni, Stephanie S.; Whiddon, Jennifer; Cheon, Kyounga; Moser, Stephen A.; Childers, Noel K.

2015-01-01

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African American children was examined using MLST. Serotype and presence of collagen-binding proteins (CBP) cnm/cbm were also assessed. One hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using START2 and MEGA. Thirty-four sequence types (ST) were identified of which 27 were unique to this population. Seventy-five percent of the isolates clustered into 16 clonal groups. Serotypes observed were c (n=84), e (n=3), and k (n=11). The prevalence of S. mutans isolates serotype k was notably high at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized populations studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study is higher than reported in most populations and is the first report of S. mutans serotype k in a US population. PMID:26443288

Stereotypic and complex phrase types provide structural evidence for a multi-message display in humpback whales (Megaptera novaeangliae).

PubMed

Murray, Anita; Dunlop, Rebecca A; Noad, Michael J; Goldizen, Anne W

2018-02-01

Male humpback whales produce a mating display called "song." Behavioral studies indicate song has inter- and/or intra-sexual functionality, suggesting song may be a multi-message display. Multi-message displays often include stereotypic components that convey group membership for mate attraction and/or male-male interactions, and complex components that convey individual quality for courtship. Humpback whale song contains sounds ("units") arranged into sequences ("phrases"). Repetitions of a specific phrase create a "theme." Within a theme, imperfect phrase repetitions ("phrase variants") create variability among phrases of the same type ("phrase type"). The hypothesis that song contains stereotypic and complex phrase types, structural characteristics consistent with a multi-message display, is investigated using recordings of 17 east Australian males (8:2004, 9:2011). Phrase types are categorized as stereotypic or complex using number of unit types, number of phrase variants, and the proportion of phrases that is unique to an individual versus shared amongst males. Unit types are determined using self-organizing maps. Phrase variants are determined by Levenshtein distances between phrases. Stereotypic phrase types have smaller numbers of unit types and shared phrase variants. Complex phrase types have larger numbers of unit types and unique phrase variants. This study supports the hypothesis that song could be a multi-message display.

Linezolid-Resistant Staphylococcus aureus Strain 1128105, the First Known Clinical Isolate Possessing the cfr Multidrug Resistance Gene

PubMed Central

Zuill, Douglas E.; Scharn, Caitlyn R.; Deane, Jennifer; Sahm, Daniel F.; Denys, Gerald A.; Goering, Richard V.; Shaw, Karen J.

2014-01-01

The Cfr methyltransferase confers resistance to six classes of drugs which target the peptidyl transferase center of the 50S ribosomal subunit, including some oxazolidinones, such as linezolid (LZD). The mobile cfr gene was identified in European veterinary isolates from the late 1990s, although the earliest report of a clinical cfr-positive strain was the 2005 Colombian methicillin-resistant Staphylococcus aureus (MRSA) isolate CM05. Here, through retrospective analysis of LZDr clinical strains from a U.S. surveillance program, we identified a cfr-positive MRSA isolate, 1128105, from January 2005, predating CM05 by 5 months. Molecular typing of 1128105 revealed a unique pulsed-field gel electrophoresis (PFGE) profile most similar to that of USA100, spa type t002, and multilocus sequence type 5 (ST5). In addition to cfr, LZD resistance in 1128105 is partially attributed to the presence of a single copy of the 23S rRNA gene mutation T2500A. Transformation of the ∼37-kb conjugative p1128105 cfr-bearing plasmid from 1128105 into S. aureus ATCC 29213 background strains was successful in recapitulating the Cfr antibiogram, as well as resistance to aminoglycosides and trimethoprim. A 7-kb cfr-containing region of p1128105 possessed sequence nearly identical to that found in the Chinese veterinary Proteus vulgaris isolate PV-01 and in U.S. clinical S. aureus isolate 1900, although the presence of IS431-like sequences is unique to p1128105. The cfr gene environment in this early clinical cfr-positive isolate has now been identified in Gram-positive and Gram-negative strains of clinical and veterinary origin and has been associated with multiple mobile elements, highlighting the versatility of this multidrug resistance gene and its potential for further dissemination. PMID:25155597

Transcriptomic and anatomical complexity of primary, seminal, and crown roots highlight root type-specific functional diversity in maize (Zea mays L.).

PubMed

Tai, Huanhuan; Lu, Xin; Opitz, Nina; Marcon, Caroline; Paschold, Anja; Lithio, Andrew; Nettleton, Dan; Hochholdinger, Frank

2016-02-01

Maize develops a complex root system composed of embryonic and post-embryonic roots. Spatio-temporal differences in the formation of these root types imply specific functions during maize development. A comparative transcriptomic study of embryonic primary and seminal, and post-embryonic crown roots of the maize inbred line B73 by RNA sequencing along with anatomical studies were conducted early in development. Seminal roots displayed unique anatomical features, whereas the organization of primary and crown roots was similar. For instance, seminal roots displayed fewer cortical cell files and their stele contained more meta-xylem vessels. Global expression profiling revealed diverse patterns of gene activity across all root types and highlighted the unique transcriptome of seminal roots. While functions in cell remodeling and cell wall formation were prominent in primary and crown roots, stress-related genes and transcriptional regulators were over-represented in seminal roots, suggesting functional specialization of the different root types. Dynamic expression of lignin biosynthesis genes and histochemical staining suggested diversification of cell wall lignification among the three root types. Our findings highlight a cost-efficient anatomical structure and a unique expression profile of seminal roots of the maize inbred line B73 different from primary and crown roots. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Unique Conformation in a Natural Interruption Sequence of Type XIX Collagen Revealed by Its High-Resolution Crystal Structure.

PubMed

Xu, Tingting; Zhou, Cong-Zhao; Xiao, Jianxi; Liu, Jinsong

2018-02-20

Naturally occurring interruptions in nonfibrillar collagen play key roles in molecular flexibility, collagen degradation, and ligand binding. The structural feature of the interruption sequences and the molecular basis for their functions have not been well studied. Here, we focused on a G5G type natural interruption sequence G-POALO-G from human type XIX collagen, a homotrimer collagen, as this sequence possesses distinct properties compared with those of a pathological similar Gly mutation sequence in collagen mimic peptides. We determined the crystal structures of the host-guest peptide (GPO) 3 -GPOALO-(GPO) 4 to 1.03 Å resolution in two crystal forms. In these structures, the interruption zone brings localized disruptions to the triple helix and introduces a light 6-8° bend with the same directional preference to the whole molecule, which may correspond structurally to the first physiological kink site in type XIX collagen. Furthermore, at the G5G interruption site, the presence of Ala and Leu residues, both with free N-H groups, allows the formation of more direct and water-mediated interchain hydrogen bonds than in the related Gly → Ala structure. These could partly explain the difference in thermal stability between the different interruptions. In addition, our structures provide a detailed view of the dynamic property of such an interrupted zone with respect to hydrogen bonding topology, torsion angles, and helical parameters. Our results, for the first time, also identified the binding of zinc to the end of the triple helix. These findings will shed light on how the interruption sequence influences the conformation of the collagen molecule and provide a structural basis for further functional studies.

Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Genome-Wide Typing of Clostridium difficile.

PubMed

Bletz, Stefan; Janezic, Sandra; Harmsen, Dag; Rupnik, Maja; Mellmann, Alexander

2018-06-01

Clostridium difficile , recently renamed Clostridioides difficile , is the most common cause of antibiotic-associated nosocomial gastrointestinal infections worldwide. To differentiate endogenous infections and transmission events, highly discriminatory subtyping is necessary. Today, methods based on whole-genome sequencing data are increasingly used to subtype bacterial pathogens; however, frequently a standardized methodology and typing nomenclature are missing. Here we report a core genome multilocus sequence typing (cgMLST) approach developed for C. difficile Initially, we determined the breadth of the C. difficile population based on all available MLST sequence types with Bayesian inference (BAPS). The resulting BAPS partitions were used in combination with C. difficile clade information to select representative isolates that were subsequently used to define cgMLST target genes. Finally, we evaluated the novel cgMLST scheme with genomes from 3,025 isolates. BAPS grouping ( n = 6 groups) together with the clade information led to a total of 11 representative isolates that were included for cgMLST definition and resulted in 2,270 cgMLST genes that were present in all isolates. Overall, 2,184 to 2,268 cgMLST targets were detected in the genome sequences of 70 outbreak-associated and reference strains, and on average 99.3% cgMLST targets (1,116 to 2,270 targets) were present in 2,954 genomes downloaded from the NCBI database, underlining the representativeness of the cgMLST scheme. Moreover, reanalyzing different cluster scenarios with cgMLST were concordant to published single nucleotide variant analyses. In conclusion, the novel cgMLST is representative for the whole C. difficile population, is highly discriminatory in outbreak situations, and provides a unique nomenclature facilitating interlaboratory exchange. Copyright © 2018 American Society for Microbiology.

Pan-genome multilocus sequence typing and outbreak-specific reference-based single nucleotide polymorphism analysis to resolve two concurrent Staphylococcus aureus outbreaks in neonatal services.

PubMed

Roisin, S; Gaudin, C; De Mendonça, R; Bellon, J; Van Vaerenbergh, K; De Bruyne, K; Byl, B; Pouseele, H; Denis, O; Supply, P

2016-06-01

We used a two-step whole genome sequencing analysis for resolving two concurrent outbreaks in two neonatal services in Belgium, caused by exfoliative toxin A-encoding-gene-positive (eta+) methicillin-susceptible Staphylococcus aureus with an otherwise sporadic spa-type t209 (ST-109). Outbreak A involved 19 neonates and one healthcare worker in a Brussels hospital from May 2011 to October 2013. After a first episode interrupted by decolonization procedures applied over 7 months, the outbreak resumed concomitantly with the onset of outbreak B in a hospital in Asse, comprising 11 neonates and one healthcare worker from mid-2012 to January 2013. Pan-genome multilocus sequence typing, defined on the basis of 42 core and accessory reference genomes, and single-nucleotide polymorphisms mapped on an outbreak-specific de novo assembly were used to compare 28 available outbreak isolates and 19 eta+/spa-type t209 isolates identified by routine or nationwide surveillance. Pan-genome multilocus sequence typing showed that the outbreaks were caused by independent clones not closely related to any of the surveillance isolates. Isolates from only ten cases with overlapping stays in outbreak A, including four pairs of twins, showed no or only a single nucleotide polymorphism variation, indicating limited sequential transmission. Detection of larger genomic variation, even from the start of the outbreak, pointed to sporadic seeding from a pre-existing exogenous source, which persisted throughout the whole course of outbreak A. Whole genome sequencing analysis can provide unique fine-tuned insights into transmission pathways of complex outbreaks even at their inception, which, with timely use, could valuably guide efforts for early source identification. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Plasmid Characterization and Chromosome Analysis of Two netF+ Clostridium perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis.

PubMed

Mehdizadeh Gohari, Iman; Kropinski, Andrew M; Weese, Scott J; Parreira, Valeria R; Whitehead, Ashley E; Boerlin, Patrick; Prescott, John F

2016-01-01

The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus.

Genomic characterization of a Helicobacter pylori isolate from a patient with gastric cancer in China

PubMed Central

2014-01-01

Background Helicobacter pylori is well known for its relationship with the occurrence of several severe gastric diseases. The mechanisms of pathogenesis triggered by H. pylori are less well known. In this study, we report the genome sequence and genomic characterizations of H. pylori strain HLJ039 that was isolated from a patient with gastric cancer in the Chinese province of Heilongjiang, where there is a high incidence of gastric cancer. To investigate potential genomic features that may be involved in pathogenesis of carcinoma, the genome was compared to three previously sequenced genomes in this area. Result We obtained 42 contigs with a total length of 1,611,192 bp and predicted 1,687 coding sequences. Compared to strains isolated from gastritis and ulcers in this area, 10 different regions were identified as being unique for HLJ039; they mainly encoded type II restriction-modification enzyme, type II m6A methylase, DNA-cytosine methyltransferase, DNA methylase, and hypothetical proteins. A unique 547-bp fragment sharing 93% identity with a hypothetical protein of Helicobacter cinaedi ATCC BAA-847 was not present in any other previous H. pylori strains. Phylogenetic analysis based on core genome single nucleotide polymorphisms shows that HLJ039 is defined as hspEAsia subgroup, which belongs to the hpEastAsia group. Conclusion DNA methylations, variations of the genomic regions involved in restriction and modification systems, are the “hot” regions that may be related to the mechanism of H. pylori-induced gastric cancer. The genome sequence will provide useful information for the deep mining of potential mechanisms related to East Asian gastric cancer. PMID:24565107

Reproducibility of Illumina platform deep sequencing errors allows accurate determination of DNA barcodes in cells.

PubMed

Beltman, Joost B; Urbanus, Jos; Velds, Arno; van Rooij, Nienke; Rohr, Jan C; Naik, Shalin H; Schumacher, Ton N

2016-04-02

Next generation sequencing (NGS) of amplified DNA is a powerful tool to describe genetic heterogeneity within cell populations that can both be used to investigate the clonal structure of cell populations and to perform genetic lineage tracing. For applications in which both abundant and rare sequences are biologically relevant, the relatively high error rate of NGS techniques complicates data analysis, as it is difficult to distinguish rare true sequences from spurious sequences that are generated by PCR or sequencing errors. This issue, for instance, applies to cellular barcoding strategies that aim to follow the amount and type of offspring of single cells, by supplying these with unique heritable DNA tags. Here, we use genetic barcoding data from the Illumina HiSeq platform to show that straightforward read threshold-based filtering of data is typically insufficient to filter out spurious barcodes. Importantly, we demonstrate that specific sequencing errors occur at an approximately constant rate across different samples that are sequenced in parallel. We exploit this observation by developing a novel approach to filter out spurious sequences. Application of our new method demonstrates its value in the identification of true sequences amongst spurious sequences in biological data sets.

A curated gluten protein sequence database to support development of proteomics methods for determination of gluten in gluten-free foods.

PubMed

Bromilow, Sophie; Gethings, Lee A; Buckley, Mike; Bromley, Mike; Shewry, Peter R; Langridge, James I; Clare Mills, E N

2017-06-23

The unique physiochemical properties of wheat gluten enable a diverse range of food products to be manufactured. However, gluten triggers coeliac disease, a condition which is treated using a gluten-free diet. Analytical methods are required to confirm if foods are gluten-free, but current immunoassay-based methods can unreliable and proteomic methods offer an alternative but require comprehensive and well annotated sequence databases which are lacking for gluten. A manually a curated database (GluPro V1.0) of gluten proteins, comprising 630 discrete unique full length protein sequences has been compiled. It is representative of the different types of gliadin and glutenin components found in gluten. An in silico comparison of their coeliac toxicity was undertaken by analysing the distribution of coeliac toxic motifs. This demonstrated that whilst the α-gliadin proteins contained more toxic motifs, these were distributed across all gluten protein sub-types. Comparison of annotations observed using a discovery proteomics dataset acquired using ion mobility MS/MS showed that more reliable identifications were obtained using the GluPro V1.0 database compared to the complete reviewed Viridiplantae database. This highlights the value of a curated sequence database specifically designed to support the proteomic workflows and the development of methods to detect and quantify gluten. We have constructed the first manually curated open-source wheat gluten protein sequence database (GluPro V1.0) in a FASTA format to support the application of proteomic methods for gluten protein detection and quantification. We have also analysed the manually verified sequences to give the first comprehensive overview of the distribution of sequences able to elicit a reaction in coeliac disease, the prevalent form of gluten intolerance. Provision of this database will improve the reliability of gluten protein identification by proteomic analysis, and aid the development of targeted mass spectrometry methods in line with Codex Alimentarius Commission requirements for foods designed to meet the needs of gluten intolerant individuals. Copyright © 2017. Published by Elsevier B.V.

RECOVIR Software for Identifying Viruses

NASA Technical Reports Server (NTRS)

Chakravarty, Sugoto; Fox, George E.; Zhu, Dianhui

2013-01-01

Most single-stranded RNA (ssRNA) viruses mutate rapidly to generate a large number of strains with highly divergent capsid sequences. Determining the capsid residues or nucleotides that uniquely characterize these strains is critical in understanding the strain diversity of these viruses. RECOVIR (an acronym for "recognize viruses") software predicts the strains of some ssRNA viruses from their limited sequence data. Novel phylogenetic-tree-based databases of protein or nucleic acid residues that uniquely characterize these virus strains are created. Strains of input virus sequences (partial or complete) are predicted through residue-wise comparisons with the databases. RECOVIR uses unique characterizing residues to identify automatically strains of partial or complete capsid sequences of picorna and caliciviruses, two of the most highly diverse ssRNA virus families. Partition-wise comparisons of the database residues with the corresponding residues of more than 300 complete and partial sequences of these viruses resulted in correct strain identification for all of these sequences. This study shows the feasibility of creating databases of hitherto unknown residues uniquely characterizing the capsid sequences of two of the most highly divergent ssRNA virus families. These databases enable automated strain identification from partial or complete capsid sequences of these human and animal pathogens.

Recombination and Insertion Events Involving the Botulinum Neurotoxin Complex Genes in Clostridium botulinum Types A, B, E and F and Clostridium butyricum Type E Strains

DTIC Science & Technology

2009-10-05

to be located within a small plasmid [11]. The genomic sequence data for the Eklund 17B strain verified the presence of bont/np b within a unique...average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed...three BoNT/A1 strains (ATCC 3502, ATCC 19397, Hall) revealed that these strains are nearly identical in genomic organization ( data not shown). The

Molecular characterization of Streptococcus agalactiae and Streptococcus uberis isolates from bovine milk.

PubMed

Shome, Bibek Ranjan; Bhuvana, Mani; Mitra, Susweta Das; Krithiga, Natesan; Shome, Rajeswari; Velu, Dhanikachalam; Banerjee, Apala; Barbuddhe, Sukhadeo B; Prabhudas, Krishnamshetty; Rahman, Habibar

2012-12-01

Streptococci are one among the major mastitis pathogens which have a considerable impact on cow health, milk quality, and productivity. The aim of the present study was to investigate the occurrence and virulence characteristics of streptococci from bovine milk and to assess the molecular epidemiology and population structure of the Indian isolates using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Out of a total of 209 bovine composite milk samples screened from four herds (A-D), 30 Streptococcus spp. were isolated from 29 milk samples. Among the 30 isolates, species-specific PCR and partial 16S rRNA gene sequence analysis identified 17 Streptococcus agalactiae arising from herd A and 13 Streptococcus uberis comprising of 5, 7, and 1 isolates from herds B, C, and D respectively. PCR based screening for virulence genes revealed the presence of the cfb and the pavA genes in 17 and 1 S. agalactiae isolates, respectively. Similarly, in S. uberis isolates, cfu gene was present in six isolates from herd C, the pau A/skc gene in all the isolates from herds B, C, and D, whereas the sua gene was present in four isolates from herd B and the only isolate from herd D. On MLST analysis, all the S. agalactiae isolates were found to be of a novel sequence type (ST), ST-483, reported for the first time and is a single locus variant of the predicted subgroup founder ST-310, while the S. uberis isolates were found to be of three novel sequence types, namely ST-439, ST-474, and ST-475, all reported for the first time. ST-474 was a double locus variant of three different STs of global clonal complex ST-143 considered to be associated with clinical and subclinical mastitis, but ST-439 and ST-475 were singletons. Unique sequence types identified for both S. agalactiae and S. uberis were found to be herd specific. On PFGE analysis, identical or closely related restriction patterns for S. agalactiae ST-483 and S. uberis ST-439 in herds A and B respectively, but an unrelated restriction pattern for S. uberis ST-474 and ST-475 isolates from herds D and C respectively, were obtained. This signifies that the isolates of particular ST may exhibit related PFGE patterns suggesting detection of a faster molecular clock by PFGE than MLST. Since all the isolates of both the species belonged to novel sequence types, their epidemiological significance in global context could not be ascertained, however, evidence suggests that they have uniquely evolved in Indian conditions. Further research would be useful for understanding the role of these pathogens in bovine sub-clinical mastitis and implementing effective control strategies in India.

Computational prediction of CRISPR cassettes in gut metagenome samples from Chinese type-2 diabetic patients and healthy controls.

PubMed

Mangericao, Tatiana C; Peng, Zhanhao; Zhang, Xuegong

2016-01-11

CRISPR has been becoming a hot topic as a powerful technique for genome editing for human and other higher organisms. The original CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats coupled with CRISPR-associated proteins) is an important adaptive defence system for prokaryotes that provides resistance against invading elements such as viruses and plasmids. A CRISPR cassette contains short nucleotide sequences called spacers. These unique regions retain a history of the interactions between prokaryotes and their invaders in individual strains and ecosystems. One important ecosystem in the human body is the human gut, a rich habitat populated by a great diversity of microorganisms. Gut microbiomes are important for human physiology and health. Metagenome sequencing has been widely applied for studying the gut microbiomes. Most efforts in metagenome study has been focused on profiling taxa compositions and gene catalogues and identifying their associations with human health. Less attention has been paid to the analysis of the ecosystems of microbiomes themselves especially their CRISPR composition. We conducted a preliminary analysis of CRISPR sequences in a human gut metagenomic data set of Chinese individuals of type-2 diabetes patients and healthy controls. Applying an available CRISPR-identification algorithm, PILER-CR, we identified 3169 CRISPR cassettes in the data, from which we constructed a set of 1302 unique repeat sequences and 36,709 spacers. A more extensive analysis was made for the CRISPR repeats: these repeats were submitted to a more comprehensive clustering and classification using the web server tool CRISPRmap. All repeats were compared with known CRISPRs in the database CRISPRdb. A total of 784 repeats had matches in the database, and the remaining 518 repeats from our set are potentially novel ones. The computational analysis of CRISPR composition based contigs of metagenome sequencing data is feasible. It provides an efficient approach for finding potential novel CRISPR arrays and for analysing the ecosystem and history of human microbiomes.

Mapping the Geometric Evolution of Protein Folding Motor.

PubMed

Jerath, Gaurav; Hazam, Prakash Kishore; Shekhar, Shashi; Ramakrishnan, Vibin

2016-01-01

Polypeptide chain has an invariant main-chain and a variant side-chain sequence. How the side-chain sequence determines fold in terms of its chemical constitution has been scrutinized extensively and verified periodically. However, a focussed investigation on the directive effect of side-chain geometry may provide important insights supplementing existing algorithms in mapping the geometrical evolution of protein chains and its structural preferences. Geometrically, folding of protein structure may be envisaged as the evolution of its geometric variables: ϕ, and ψ dihedral angles of polypeptide main-chain directed by χ1, and χ2 of side chain. In this work, protein molecule is metaphorically modelled as a machine with 4 rotors ϕ, ψ, χ1 and χ2, with its evolution to the functional fold is directed by combinations of its rotor directions. We observe that differential rotor motions lead to different secondary structure formations and the combinatorial pattern is unique and consistent for particular secondary structure type. Further, we found that combination of rotor geometries of each amino acid is unique which partly explains how different amino acid sequence combinations have unique structural evolution and functional adaptation. Quantification of these amino acid rotor preferences, resulted in the generation of 3 substitution matrices, which later on plugged in the BLAST tool, for evaluating their efficiency in aligning sequences. We have employed BLOSUM62 and PAM30 as standard for primary evaluation. Generation of substitution matrices is a logical extension of the conceptual framework we attempted to build during the development of this work. Optimization of matrices following the conventional routines and possible application with biologically relevant data sets are beyond the scope of this manuscript, though it is a part of the larger project design.

The Draft Genome Sequence of a Novel High-Efficient Butanol-Producing Bacterium Clostridium Diolis Strain WST.

PubMed

Chen, Chaoyang; Sun, Chongran; Wu, Yi-Rui

2018-03-21

A wild-type solventogenic strain Clostridium diolis WST, isolated from mangrove sediments, was characterized to produce high amount of butanol and acetone with negligible level of ethanol and acids from glucose via a unique acetone-butanol (AB) fermentation pathway. Through the genomic sequencing, the assembled draft genome of strain WST is calculated to be 5.85 Mb with a GC content of 29.69% and contains 5263 genes that contribute to the annotation of 5049 protein-coding sequences. Within these annotated genes, the butanol dehydrogenase gene (bdh) was determined to be in a higher amount from strain WST compared to other Clostridial strains, which is positively related to its high-efficient production of butanol. Therefore, we present a draft genome sequence analysis of strain WST in this article that should facilitate to further understand the solventogenic mechanism of this special microorganism.

The first FDA marketing authorizations of next-generation sequencing technology and tests: challenges, solutions and impact for future assays.

PubMed

Bijwaard, Karen; Dickey, Jennifer S; Kelm, Kellie; Težak, Živana

2015-01-01

The rapid emergence and clinical translation of novel high-throughput sequencing technologies created a need to clarify the regulatory pathway for the evaluation and authorization of these unique technologies. Recently, the US FDA authorized for marketing four next generation sequencing (NGS)-based diagnostic devices which consisted of two heritable disease-specific assays, library preparation reagents and a NGS platform that are intended for human germline targeted sequencing from whole blood. These first authorizations can serve as a case study in how different types of NGS-based technology are reviewed by the FDA. In this manuscript we describe challenges associated with the evaluation of these novel technologies and provide an overview of what was reviewed. Besides making validated NGS-based devices available for in vitro diagnostic use, these first authorizations create a regulatory path for similar future instruments and assays.

Distinct Microbial Signatures Associated With Different Breast Cancer Types

PubMed Central

Banerjee, Sagarika; Tian, Tian; Wei, Zhi; Shih, Natalie; Feldman, Michael D.; Peck, Kristen N.; DeMichele, Angela M.; Alwine, James C.; Robertson, Erle S.

2018-01-01

A dysbiotic microbiome can potentially contribute to the pathogenesis of many different diseases including cancer. Breast cancer is the second leading cause of cancer death in women. Thus, we investigated the diversity of the microbiome in the four major types of breast cancer: endocrine receptor (ER) positive, triple positive, Her2 positive and triple negative breast cancers. Using a whole genome and transcriptome amplification and a pan-pathogen microarray (PathoChip) strategy, we detected unique and common viral, bacterial, fungal and parasitic signatures for each of the breast cancer types. These were validated by PCR and Sanger sequencing. Hierarchical cluster analysis of the breast cancer samples, based on their detected microbial signatures, showed distinct patterns for the triple negative and triple positive samples, while the ER positive and Her2 positive samples shared similar microbial signatures. These signatures, unique or common to the different breast cancer types, provide a new line of investigation to gain further insights into prognosis, treatment strategies and clinical outcome, as well as better understanding of the role of the micro-organisms in the development and progression of breast cancer. PMID:29867857

Tissue-Specific Transcriptomics in the Field Cricket Teleogryllus oceanicus

PubMed Central

Bailey, Nathan W.; Veltsos, Paris; Tan, Yew-Foon; Millar, A. Harvey; Ritchie, Michael G.; Simmons, Leigh W.

2013-01-01

Field crickets (family Gryllidae) frequently are used in studies of behavioral genetics, sexual selection, and sexual conflict, but there have been no studies of transcriptomic differences among different tissue types. We evaluated transcriptome variation among testis, accessory gland, and the remaining whole-body preparations from males of the field cricket, Teleogryllus oceanicus. Non-normalized cDNA libraries from each tissue were sequenced on the Roche 454 platform, and a master assembly was constructed using testis, accessory gland, and whole-body preparations. A total of 940,200 reads were assembled into 41,962 contigs, to which 36,856 singletons (reads not assembled into a contig) were added to provide a total of 78,818 sequences used in annotation analysis. A total of 59,072 sequences (75%) were unique to one of the three tissues. Testis tissue had the greatest proportion of tissue-specific sequences (62.6%), followed by general body (56.43%) and accessory gland tissue (44.16%). We tested the hypothesis that tissues expressing gene products expected to evolve rapidly as a result of sexual selection—testis and accessory gland—would yield a smaller proportion of BLASTx matches to homologous genes in the model organism Drosophila melanogaster compared with whole-body tissue. Uniquely expressed sequences in both testis and accessory gland showed a significantly lower rate of matching to annotated D. melanogaster genes compared with those from general body tissue. These results correspond with empirical evidence that genes expressed in testis and accessory gland tissue are rapidly evolving targets of selection. PMID:23390599

Tissue-specific transcriptomics in the field cricket Teleogryllus oceanicus.

PubMed

Bailey, Nathan W; Veltsos, Paris; Tan, Yew-Foon; Millar, A Harvey; Ritchie, Michael G; Simmons, Leigh W

2013-02-01

Field crickets (family Gryllidae) frequently are used in studies of behavioral genetics, sexual selection, and sexual conflict, but there have been no studies of transcriptomic differences among different tissue types. We evaluated transcriptome variation among testis, accessory gland, and the remaining whole-body preparations from males of the field cricket, Teleogryllus oceanicus. Non-normalized cDNA libraries from each tissue were sequenced on the Roche 454 platform, and a master assembly was constructed using testis, accessory gland, and whole-body preparations. A total of 940,200 reads were assembled into 41,962 contigs, to which 36,856 singletons (reads not assembled into a contig) were added to provide a total of 78,818 sequences used in annotation analysis. A total of 59,072 sequences (75%) were unique to one of the three tissues. Testis tissue had the greatest proportion of tissue-specific sequences (62.6%), followed by general body (56.43%) and accessory gland tissue (44.16%). We tested the hypothesis that tissues expressing gene products expected to evolve rapidly as a result of sexual selection--testis and accessory gland--would yield a smaller proportion of BLASTx matches to homologous genes in the model organism Drosophila melanogaster compared with whole-body tissue. Uniquely expressed sequences in both testis and accessory gland showed a significantly lower rate of matching to annotated D. melanogaster genes compared with those from general body tissue. These results correspond with empirical evidence that genes expressed in testis and accessory gland tissue are rapidly evolving targets of selection.

Next-Generation Sequencing of Coccidioides immitis Isolated during Cluster Investigation

PubMed Central

Engelthaler, David M.; Chiller, Tom; Schupp, James A.; Colvin, Joshua; Beckstrom-Sternberg, Stephen M.; Driebe, Elizabeth M.; Moses, Tracy; Tembe, Waibhav; Sinari, Shripad; Beckstrom-Sternberg, James S.; Christoforides, Alexis; Pearson, John V.; Carpten, John; Keim, Paul; Peterson, Ashley; Terashita, Dawn

2011-01-01

Next-generation sequencing enables use of whole-genome sequence typing (WGST) as a viable and discriminatory tool for genotyping and molecular epidemiologic analysis. We used WGST to confirm the linkage of a cluster of Coccidioides immitis isolates from 3 patients who received organ transplants from a single donor who later had positive test results for coccidioidomycosis. Isolates from the 3 patients were nearly genetically identical (a total of 3 single-nucleotide polymorphisms identified among them), thereby demonstrating direct descent of the 3 isolates from an original isolate. We used WGST to demonstrate the genotypic relatedness of C. immitis isolates that were also epidemiologically linked. Thus, WGST offers unique benefits to public health for investigation of clusters considered to be linked to a single source. PMID:21291593

Neisseria arctica sp. nov. isolated from nonviable eggs of greater white-fronted geese (Anser albifrons) in Arctic Alaska

USGS Publications Warehouse

Hansen, Cristina M.; Himschoot, Elizabeth; Hare, Rebekah F.; Meixell, Brandt W.; Van Hemert, Caroline R.; Hueffer, Karsten

2017-01-01

During the summers of 2013 and 2014, isolates of a novel Gram-negative coccus in the Neisseria genus were obtained from the contents of nonviable greater white-fronted goose (Anser albifrons) eggs on the Arctic Coastal Plain of Alaska. We used a polyphasic approach to determine whether these isolates represent a novel species. 16S rRNA gene sequences, 23S rRNA gene sequences, and chaperonin 60 gene sequences suggested that these Alaskan isolates are members of a distinct species that is most closely related to Neisseria canis, N. animaloris, and N. shayeganii. Analysis of the rplF gene additionally showed that our isolates are unique and most closely related to N. weaveri. Average nucleotide identity of the whole genome sequence of our type strain was between 71.5% and 74.6% compared to close relatives, further supporting designation as a novel species. Fatty acid methyl ester analysis showed a predominance of C14:0, C16:0, and C16:1ω7c fatty acids. Finally, biochemical characteristics distinguished our isolates from other Neisseria species. The name Neisseria arctica (type strain KH1503T = ATCC TSD-57T = DSM 103136T) is proposed.

Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation

NASA Astrophysics Data System (ADS)

Sheynkman, Gloria M.; Shortreed, Michael R.; Cesnik, Anthony J.; Smith, Lloyd M.

2016-06-01

Mass spectrometry-based proteomics has emerged as the leading method for detection, quantification, and characterization of proteins. Nearly all proteomic workflows rely on proteomic databases to identify peptides and proteins, but these databases typically contain a generic set of proteins that lack variations unique to a given sample, precluding their detection. Fortunately, proteogenomics enables the detection of such proteomic variations and can be defined, broadly, as the use of nucleotide sequences to generate candidate protein sequences for mass spectrometry database searching. Proteogenomics is experiencing heightened significance due to two developments: (a) advances in DNA sequencing technologies that have made complete sequencing of human genomes and transcriptomes routine, and (b) the unveiling of the tremendous complexity of the human proteome as expressed at the levels of genes, cells, tissues, individuals, and populations. We review here the field of human proteogenomics, with an emphasis on its history, current implementations, the types of proteomic variations it reveals, and several important applications.

Great expectations: patient perspectives and anticipated utility of non-diagnostic genomic-sequencing results.

PubMed

Hylind, Robyn; Smith, Maureen; Rasmussen-Torvik, Laura; Aufox, Sharon

2018-01-01

The management of secondary findings is a challenge to health-care providers relaying clinical genomic-sequencing results to patients. Understanding patients' expectations from non-diagnostic genomic sequencing could help guide this management. This study interviewed 14 individuals enrolled in the eMERGE (Electronic Medical Records and Genomics) study. Participants in eMERGE consent to undergo non-diagnostic genomic sequencing, receive results, and have results returned to their physicians. The interviews assessed expectations and intended use of results. The majority of interviewees were male (64%) and 43% identified as non-Caucasian. A unique theme identified was that many participants expressed uncertainty about the type of diseases they expected to receive results on, what results they wanted to learn about, and how they intended to use results. Participant uncertainty highlights the complex nature of deciding to undergo genomic testing and a deficiency in genomic knowledge. These results could help improve how genomic sequencing and secondary findings are discussed with patients.

Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation

PubMed Central

Sheynkman, Gloria M.; Shortreed, Michael R.; Cesnik, Anthony J.; Smith, Lloyd M.

2016-01-01

Mass spectrometry–based proteomics has emerged as the leading method for detection, quantification, and characterization of proteins. Nearly all proteomic workflows rely on proteomic databases to identify peptides and proteins, but these databases typically contain a generic set of proteins that lack variations unique to a given sample, precluding their detection. Fortunately, proteogenomics enables the detection of such proteomic variations and can be defined, broadly, as the use of nucleotide sequences to generate candidate protein sequences for mass spectrometry database searching. Proteogenomics is experiencing heightened significance due to two developments: (a) advances in DNA sequencing technologies that have made complete sequencing of human genomes and transcriptomes routine, and (b) the unveiling of the tremendous complexity of the human proteome as expressed at the levels of genes, cells, tissues, individuals, and populations. We review here the field of human proteogenomics, with an emphasis on its history, current implementations, the types of proteomic variations it reveals, and several important applications. PMID:27049631

Assessment of clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing.

PubMed

Momeni, Stephanie S; Whiddon, Jennifer; Cheon, Kyounga; Moser, Stephen A; Childers, Noel K

2015-12-01

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African-American children was examined using MLST. Serotype and the presence of collagen-binding proteins (CBPs) encoded by cnm/cbm were also assessed. One-hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using start2 and mega. Thirty-four sequence types were identified, of which 27 were unique to this population. Seventy-five per cent of the isolates clustered into 16 clonal groups. The serotypes observed were c (n = 84), e (n = 3), and k (n = 11). The prevalence of S. mutans isolates of serotype k was notably high, at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized population studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study, is higher than reported in most populations and is the first report of S. mutans serotype k in a United States population. © 2015 Eur J Oral Sci.

Kiloniella laminariae gen. nov., sp. nov., an alphaproteobacterium from the marine macroalga Laminaria saccharina.

PubMed

Wiese, Jutta; Thiel, Vera; Gärtner, Andrea; Schmaljohann, Rolf; Imhoff, Johannes F

2009-02-01

A novel alphaproteobacterium, strain LD81(T), was isolated from the marine macroalga Laminaria saccharina. The bacterium is mesophilic and shows a typical marine growth response. It is a chemoheterotrophic aerobe with the potential for denitrification. Growth optima are 25 degrees C, pH 5.5 and 3 % NaCl. Strain LD81(T) has a unique phylogenetic position, not fitting any of the known families of the Alphaproteobacteria. The 16S rRNA gene sequence revealed a distant relationship to species of several orders of the Alphaproteobacteria, with less than 90 % sequence similarity. Phylogenetically, strain LD81(T) is related to the type strains of Terasakiella pusilla (88.4 % 16S rRNA gene sequence similarity) and the three Thalassospira species (88.9-89.2 %). It forms a cluster with these bacteria and a novel as-yet undescribed isolate (KOPRI 13522; 96.6 % sequence similarity). Strain LD81(T) has a relatively low DNA G+C content (51.1 mol%) and, due to its distant phylogenetic position from all other alphaproteobacteria, strain LD81(T) (=NCIMB 14374(T) =JCM 14845(T)) is considered as the type strain of a novel species within a new genus, for which the name Kiloniella laminariae gen. nov., sp. nov. is proposed. The genus Kiloniella represents the type of the new family Kiloniellaceae fam. nov. and order Kiloniellales ord. nov.

Lactobacillus buchneri genotyping on the basis of clustered regularly interspaced short palindromic repeat (CRISPR) locus diversity.

PubMed

Briner, Alexandra E; Barrangou, Rodolphe

2014-02-01

Clustered regularly interspaced short palindromic repeats (CRISPR) in combination with associated sequences (cas) constitute the CRISPR-Cas immune system, which uptakes DNA from invasive genetic elements as novel "spacers" that provide a genetic record of immunization events. We investigated the potential of CRISPR-based genotyping of Lactobacillus buchneri, a species relevant for commercial silage, bioethanol, and vegetable fermentations. Upon investigating the occurrence and diversity of CRISPR-Cas systems in Lactobacillus buchneri genomes, we observed a ubiquitous occurrence of CRISPR arrays containing a 36-nucleotide (nt) type II-A CRISPR locus adjacent to four cas genes, including the universal cas1 and cas2 genes and the type II signature gene cas9. Comparative analysis of CRISPR spacer content in 26 L. buchneri pickle fermentation isolates associated with spoilage revealed 10 unique locus genotypes that contained between 9 and 29 variable spacers. We observed a set of conserved spacers at the ancestral end, reflecting a common origin, as well as leader-end polymorphisms, reflecting recent divergence. Some of these spacers showed perfect identity with phage sequences, and many spacers showed homology to Lactobacillus plasmid sequences. Following a comparative analysis of sequences immediately flanking protospacers that matched CRISPR spacers, we identified a novel putative protospacer-adjacent motif (PAM), 5'-AAAA-3'. Overall, these findings suggest that type II-A CRISPR-Cas systems are valuable for genotyping of L. buchneri.

Analysis of the type II-A CRISPR-Cas system of Streptococcus agalactiae reveals distinctive features according to genetic lineages

PubMed Central

Lier, Clément; Baticle, Elodie; Horvath, Philippe; Haguenoer, Eve; Valentin, Anne-Sophie; Glaser, Philippe; Mereghetti, Laurent; Lanotte, Philippe

2015-01-01

CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) are found in 90% of archaea and about 40% of bacteria. In this original system, CRISPR arrays comprise short, almost unique sequences called spacers that are interspersed with conserved palindromic repeats. These systems play a role in adaptive immunity and participate to fight non-self DNA such as integrative and conjugative elements, plasmids, and phages. In Streptococcus agalactiae, a bacterium implicated in colonization and infections in humans since the 1960s, two CRISPR-Cas systems have been described. A type II-A system, characterized by proteins Cas9, Cas1, Cas2, and Csn2, is ubiquitous, and a type I–C system, with the Cas8c signature protein, is present in about 20% of the isolates. Unlike type I–C, which appears to be non-functional, type II-A appears fully functional. Here we studied type II-A CRISPR-cas loci from 126 human isolates of S. agalactiae belonging to different clonal complexes that represent the diversity of the species and that have been implicated in colonization or infection. The CRISPR-cas locus was analyzed both at spacer and repeat levels. Major distinctive features were identified according to the phylogenetic lineages previously defined by multilocus sequence typing, especially for the sequence type (ST) 17, which is considered hypervirulent. Among other idiosyncrasies, ST-17 shows a significantly lower number of spacers in comparison with other lineages. This characteristic could reflect the peculiar virulence or colonization specificities of this lineage. PMID:26124774

Dance choreography is coordinated with song repertoire in a complex avian display.

PubMed

Dalziell, Anastasia H; Peters, Richard A; Cockburn, Andrew; Dorland, Alexandra D; Maisey, Alex C; Magrath, Robert D

2013-06-17

All human cultures have music and dance, and the two activities are so closely integrated that many languages use just one word to describe both. Recent research points to a deep cognitive connection between music and dance-like movements in humans, fueling speculation that music and dance have coevolved and prompting the need for studies of audiovisual displays in other animals. However, little is known about how nonhuman animals integrate acoustic and movement display components. One striking property of human displays is that performers coordinate dance with music by matching types of dance movements with types of music, as when dancers waltz to waltz music. Here, we show that a bird also temporally coordinates a repertoire of song types with a repertoire of dance-like movements. During displays, male superb lyrebirds (Menura novaehollandiae) sing four different song types, matching each with a unique set of movements and delivering song and dance types in a predictable sequence. Crucially, display movements are both unnecessary for the production of sound and voluntary, because males sometimes sing without dancing. Thus, the coordination of independently produced repertoires of acoustic and movement signals is not a uniquely human trait. Copyright © 2013 Elsevier Ltd. All rights reserved.

Performance of the unique-word-reverse-modulation type demodulator for mobile satellite communications

NASA Technical Reports Server (NTRS)

Dohi, Tomohiro; Nitta, Kazumasa; Ueda, Takashi

1993-01-01

This paper proposes a new type of coherent demodulator, the unique-word (UW)-reverse-modulation type demodulator, for burst signal controlled by voice operated transmitter (VOX) in mobile satellite communication channels. The demodulator has three individual circuits: a pre-detection signal combiner, a pre-detection UW detector, and a UW-reverse-modulation type demodulator. The pre-detection signal combiner combines signal sequences received by two antennas and improves bit energy-to-noise power density ratio (E(sub b)/N(sub 0)) 2.5 dB to yield 10(exp -3) average bit error rate (BER) when carrier power-to-multipath power ratio (CMR) is 15 dB. The pre-detection UW detector improves UW detection probability when the frequency offset is large. The UW-reverse-modulation type demodulator realizes a maximum pull-in frequency of 3.9 kHz, the pull-in time is 2.4 seconds and frequency error is less than 20 Hz. The performances of this demodulator are confirmed through computer simulations and its effect is clarified in real-time experiments at a bit rate of 16.8 kbps using a digital signal processor (DSP).

ChAy/Bx, a novel chimeric high-molecular-weight glutenin subunit gene apparently created by homoeologous recombination in Triticum turgidum ssp. dicoccoides.

PubMed

Guo, Xiao-Hui; Bi, Zhe-Guang; Wu, Bi-Hua; Wang, Zhen-Zhen; Hu, Ji-Liang; Zheng, You-Liang; Liu, Deng-Cai

2013-12-01

High-molecular-weight glutenin subunits (HMW-GSs) are of considerable interest, because they play a crucial role in determining dough viscoelastic properties and end-use quality of wheat flour. In this paper, ChAy/Bx, a novel chimeric HMW-GS gene from Triticum turgidum ssp. dicoccoides (AABB, 2n=4x=28) accession D129, was isolated and characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the electrophoretic mobility of the glutenin subunit encoded by ChAy/Bx was slightly faster than that of 1Dy12. The complete ORF of ChAy/Bx contained 1,671 bp encoding a deduced polypeptide of 555 amino acid residues (or 534 amino acid residues for the mature protein), making it the smallest HMW-GS gene known from Triticum species. Sequence analysis showed that ChAy/Bx was neither a conventional x-type nor a conventional y-type subunit gene, but a novel chimeric gene. Its first 1305 nt sequence was highly homologous with the corresponding sequence of 1Ay type genes, while its final 366 nt sequence was highly homologous with the corresponding sequence of 1Bx type genes. The mature ChAy/Bx protein consisted of the N-terminus of 1Ay type subunit (the first 414 amino acid residues) and the C-terminus of 1Bx type subunit (the final 120 amino acid residues). Secondary structure prediction showed that ChAy/Bx contained some domains of 1Ay subunit and some domains of 1Bx subunit. The special structure of this HMW glutenin chimera ChAy/Bx subunit might have unique effects on the end-use quality of wheat flour. Here we propose that homoeologous recombination might be a novel pathway for allelic variation or molecular evolution of HMW-GSs. © 2013.

Emergence of viral hemorrhagic septicemia virus in the North American Great Lakes region is associated with low viral genetic diversity

USGS Publications Warehouse

Thompson, T.M.; Batts, W.N.; Faisal, M.; Bowser, P.; Casey, J.W.; Phillips, K.; Garver, K.A.; Winton, J.; Kurath, G.

2011-01-01

Viral hemorrhagic septicemia virus (VHSV) is a fish rhabdovirus that causes disease in a broad range of marine and freshwater hosts. The known geographic range includes the Northern Atlantic and Pacific Oceans, and recently it has invaded the Great Lakes region of North Ame­rica. The goal of this work was to characterize genetic diversity of Great Lakes VHSV isolates at the early stage of this viral emergence by comparing a partial glycoprotein (G) gene sequence (669 nt) of 108 isolates collected from 2003 to 2009 from 31 species and at 37 sites. Phylogenetic analysis showed that all isolates fell into sub-lineage IVb within the major VHSV genetic group IV. Among these 108 isolates, genetic diversity was low, with a maximum of 1.05% within the 669 nt region. There were 11 unique sequences, designated vcG001 to vcG011. Two dominant sequence types, vcG001 and vcG002, accounted for 90% (97 of 108) of the isolates. The vcG001 isolates were most widespread. We saw no apparent association of sequence type with host or year of isolation, but we did note a spatial pattern, in which vcG002 isolates were more prevalent in the easternmost sub-regions, including inland New York state and the St. Lawrence Seaway. Different sequence types were found among isolates from single disease outbreaks, and mixtures of types were evident within 2 isolates from ­individual fish. Overall, the genetic diversity of VHSV in the Great Lakes region was found to be extremely low, consistent with an introduction of a new virus into a geographic region with ­previously naïve host populations.

Emergence of Viral hemorrhagic septicemia virus in the North American Great Lakes region is associated with low viral genetic diversity.

PubMed

Thompson, Tarin M; Batts, William N; Faisal, Mohamed; Bowser, Paul; Casey, James W; Phillips, Kenneth; Garver, Kyle A; Winton, James; Kurath, Gael

2011-08-29

Viral hemorrhagic septicemia virus (VHSV) is a fish rhabdovirus that causes disease in a broad range of marine and freshwater hosts. The known geographic range includes the Northern Atlantic and Pacific Oceans, and recently it has invaded the Great Lakes region of North America. The goal of this work was to characterize genetic diversity of Great Lakes VHSV isolates at the early stage of this viral emergence by comparing a partial glycoprotein (G) gene sequence (669 nt) of 108 isolates collected from 2003 to 2009 from 31 species and at 37 sites. Phylogenetic analysis showed that all isolates fell into sub-lineage IVb within the major VHSV genetic group IV. Among these 108 isolates, genetic diversity was low, with a maximum of 1.05% within the 669 nt region. There were 11 unique sequences, designated vcG001 to vcG011. Two dominant sequence types, vcG001 and vcG002, accounted for 90% (97 of 108) of the isolates. The vcG001 isolates were most widespread. We saw no apparent association of sequence type with host or year of isolation, but we did note a spatial pattern, in which vcG002 isolates were more prevalent in the easternmost sub-regions, including inland New York state and the St. Lawrence Seaway. Different sequence types were found among isolates from single disease outbreaks, and mixtures of types were evident within 2 isolates from individual fish. Overall, the genetic diversity of VHSV in the Great Lakes region was found to be extremely low, consistent with an introduction of a new virus into a geographic region with previously naive host populations.

Characterization and Exploitation of CRISPR Loci in Bifidobacterium longum

PubMed Central

Hidalgo-Cantabrana, Claudio; Crawley, Alexandra B.; Sanchez, Borja; Barrangou, Rodolphe

2017-01-01

Diverse CRISPR-Cas systems provide adaptive immunity in many bacteria and most archaea, via a DNA-encoded, RNA-mediated, nucleic-acid targeting mechanism. Over time, CRISPR loci expand via iterative uptake of invasive DNA sequences into the CRISPR array during the adaptation process. These genetic vaccination cards thus provide insights into the exposure of strains to phages and plasmids in space and time, revealing the historical predatory exposure of a strain. These genetic loci thus constitute a unique basis for genotyping of strains, with potential of resolution at the strain-level. Here, we investigate the occurrence and diversity of CRISPR-Cas systems in the genomes of various Bifidobacterium longum strains across three sub-species. Specifically, we analyzed the genomic content of 66 genomes belonging to B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis, and identified 25 strains that carry 29 total CRISPR-Cas systems. We identify various Type I and Type II CRISPR-Cas systems that are widespread in this species, notably I-C, I-E, and II-C. Noteworthy, Type I-C systems showed extended CRISPR arrays, with extensive spacer diversity. We show how these hypervariable loci can be used to gain insights into strain origin, evolution and phylogeny, and can provide discriminatory sequences to distinguish even clonal isolates. By investigating CRISPR spacer sequences, we reveal their origin and implicate phages and prophages as drivers of CRISPR immunity expansion in this species, with redundant targeting of select prophages. Analysis of CRISPR spacer origin also revealed novel PAM sequences. Our results suggest that CRISPR-Cas immune systems are instrumental in mounting diversified viral resistance in B. longum, and show that these sequences are useful for typing across three subspecies. PMID:29033911

Characterization and Exploitation of CRISPR Loci in Bifidobacterium longum.

PubMed

Hidalgo-Cantabrana, Claudio; Crawley, Alexandra B; Sanchez, Borja; Barrangou, Rodolphe

2017-01-01

Diverse CRISPR-Cas systems provide adaptive immunity in many bacteria and most archaea, via a DNA-encoded, RNA-mediated, nucleic-acid targeting mechanism. Over time, CRISPR loci expand via iterative uptake of invasive DNA sequences into the CRISPR array during the adaptation process. These genetic vaccination cards thus provide insights into the exposure of strains to phages and plasmids in space and time, revealing the historical predatory exposure of a strain. These genetic loci thus constitute a unique basis for genotyping of strains, with potential of resolution at the strain-level. Here, we investigate the occurrence and diversity of CRISPR-Cas systems in the genomes of various Bifidobacterium longum strains across three sub-species. Specifically, we analyzed the genomic content of 66 genomes belonging to B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis , and identified 25 strains that carry 29 total CRISPR-Cas systems. We identify various Type I and Type II CRISPR-Cas systems that are widespread in this species, notably I-C, I-E, and II-C. Noteworthy, Type I-C systems showed extended CRISPR arrays, with extensive spacer diversity. We show how these hypervariable loci can be used to gain insights into strain origin, evolution and phylogeny, and can provide discriminatory sequences to distinguish even clonal isolates. By investigating CRISPR spacer sequences, we reveal their origin and implicate phages and prophages as drivers of CRISPR immunity expansion in this species, with redundant targeting of select prophages. Analysis of CRISPR spacer origin also revealed novel PAM sequences. Our results suggest that CRISPR-Cas immune systems are instrumental in mounting diversified viral resistance in B. longum , and show that these sequences are useful for typing across three subspecies.

Size and sequence polymorphisms in the glutamate-rich protein gene of the human malaria parasite Plasmodium falciparum in Thailand.

PubMed

Pattaradilokrat, Sittiporn; Trakoolsoontorn, Chawinya; Simpalipan, Phumin; Warrit, Natapot; Kaewthamasorn, Morakot; Harnyuttanakorn, Pongchai

2018-01-22

The glutamate-rich protein (GLURP) of the malaria parasite Plasmodium falciparum is a key surface antigen that serves as a component of a clinical vaccine. Moreover, the GLURP gene is also employed routinely as a genetic marker for malarial genotyping in epidemiological studies. While extensive size polymorphisms in GLURP are well recorded, the extent of the sequence diversity of this gene is rarely investigated. The present study aimed to explore the genetic diversity of GLURP in natural populations of P. falciparum. The polymorphic C-terminal repetitive R2 region of GLURP sequences from 65 P. falciparum isolates in Thailand were generated and combined with the data from 103 worldwide isolates to generate a GLURP database. The collection was comprised of 168 alleles, encoding 105 unique GLURP subtypes, characterized by 18 types of amino acid repeat units (AAU). Of these, 28 GLURP subtypes, formed by 10 AAU types, were detected in P. falciparum in Thailand. Among them, 19 GLURP subtypes and 2 AAU types are described for the first time in the Thai parasite population. The AAU sequences were highly conserved, which is likely due to negative selection. Standard Fst analysis revealed the shared distributions of GLURP types among the P. falciparum populations, providing evidence of gene flow among the different demographic populations. Sequence diversity causing size variations in GLURP in Thai P. falciparum populations were detected, and caused by non-synonymous substitutions in repeat units and some insertion/deletion of aspartic acid or glutamic acid codons between repeat units. The P. falciparum population structure based on GLURP showed promising implications for the development of GLURP-based vaccines and for monitoring vaccine efficacy.

Identification of a Unique Amyloid Sequence in AA Amyloidosis of a Pig Associated With Streptococcus Suis Infection.

PubMed

Kamiie, J; Sugahara, G; Yoshimoto, S; Aihara, N; Mineshige, T; Uetsuka, K; Shirota, K

2017-01-01

Here we report a pig with amyloid A (AA) amyloidosis associated with Streptococcus suis infection and identification of a unique amyloid sequence in the amyloid deposits in the tissue. Tissues from the 180-day-old underdeveloped pig contained foci of necrosis and suppurative inflammation associated with S. suis infection. Congo red stain, immunohistochemistry, and electron microscopy revealed intense AA deposition in the spleen and renal glomeruli. Mass spectrometric analysis of amyloid material extracted from the spleen showed serum AA 2 (SAA2) peptide as well as a unique peptide sequence previously reported in a pig with AA amyloidosis. The common detection of the unique amyloid sequence in the current and past cases of AA amyloidosis in pigs suggests that this amyloid sequence might play a key role in the development of porcine AA amyloidosis. An in vitro fibrillation assay demonstrated that the unique AA peptide formed typically rigid, long amyloid fibrils (10 nm wide) and the N-terminus peptide of SAA2 formed zigzagged, short fibers (7 nm wide). Moreover, the SAA2 peptide formed long, rigid amyloid fibrils in the presence of sonicated amyloid fibrils formed by the unique AA peptide. These findings indicate that the N-terminus of SAA2 as well as the AA peptide mediate the development of AA amyloidosis in pigs via cross-seeding polymerization.

Microbiological Features of KPC-Producing Enterobacter Isolates Identified in a U.S. Hospital System

PubMed Central

Ahn, Chulsoo; Syed, Alveena; Hu, Fupin; O’Hara, Jessica A.; Rivera, Jesabel I.; Doi, Yohei

2014-01-01

Microbiological data regarding KPC-producing Enterobacter spp. are scarce. In this study, 11 unique KPC-producing Enterobacter isolates were identified among 44 ertapenem-non-susceptible Enterobacter isolates collected between 2009 and 2013 at a hospital system in Western Pennsylvania. All cases were healthcare-associated and occurred in medically complex patients. While pulsed-field gel electrophoresis (PFGE) showed diverse restriction patterns overall, multilocus sequence typing (MLST) identified Enterobacter cloacae isolates with sequence types (STs) 93 and 171 from two hospitals each. The levels of carbapenem minimum inhibitory concentrations were highly variable. All isolates remained susceptible to colistin, tigecycline, and the majority to amikacin and doxycycline. A blaKPC-carrying IncN plasmid conferring trimethoprim-sulfamethoxazole resistance was identified in three of the isolates. Spread of blaKPC in Enterobacter spp. appears to be due to a combination of plasmid-mediated and clonal processes. PMID:25053203

A multiplexable TALE-based binary expression system for in vivo cellular interaction studies.

PubMed

Toegel, Markus; Azzam, Ghows; Lee, Eunice Y; Knapp, David J H F; Tan, Ying; Fa, Ming; Fulga, Tudor A

2017-11-21

Binary expression systems have revolutionised genetic research by enabling delivery of loss-of-function and gain-of-function transgenes with precise spatial-temporal resolution in vivo. However, at present, each existing platform relies on a defined exogenous transcription activator capable of binding a unique recognition sequence. Consequently, none of these technologies alone can be used to simultaneously target different tissues or cell types in the same organism. Here, we report a modular system based on programmable transcription activator-like effector (TALE) proteins, which enables parallel expression of multiple transgenes in spatially distinct tissues in vivo. Using endogenous enhancers coupled to TALE drivers, we demonstrate multiplexed orthogonal activation of several transgenes carrying cognate variable activating sequences (VAS) in distinct neighbouring cell types of the Drosophila central nervous system. Since the number of combinatorial TALE-VAS pairs is virtually unlimited, this platform provides an experimental framework for highly complex genetic manipulation studies in vivo.

Talaromyces neofusisporus and T. qii, two new species of section Talaromyces isolated from plant leaves in Tibet, China.

PubMed

Wang, Qi-Ming; Zhang, Yong-Hong; Wang, Bo; Wang, Long

2016-01-04

Two new species isolated from plant leaves belonging to Talaromyces section Talaromyces are reported, namely T. neofusisporus (ex-type AS3.15415 (T) = CBS 139516 (T)) and T. qii (ex-type AS3.15414 (T) = CBS 139515 (T)). Morphologically, T. neofusisporus is featured by forming synnemata on CYA and YES, bearing appressed biverticillate penicilli and smooth-walled fusiform conidia about 3.5-4.5 × 2-2.5 μm; and T. qii is characterized by velutinous colony texture, yellowish green conidia, yellow mycelium and ovoid to subglobose echinulate conidia measuring 3-3.5 μm. Phylogenetically, T. neofusisporus is such a unique species that no close relatives are found according to CaM, BenA and ITS1-5.8S-ITS2 as well as the combined three-gene sequences; and T. qii is related to T. thailandensis according to CaM, BenA and the combined sequence matrices, whereas ITS1-5.8S-ITS2 sequences do not support the close relationship between T. qii and T. thailandensis.

Membership and Coronal Activity in the NGC 2232 and Cr 140 Open Clusters

NASA Technical Reports Server (NTRS)

Oliversen, Ronald J. (Technical Monitor); Patten, Brian M.

2004-01-01

Making use of eight archival ROSAT HRI images in the regions of the NGC 2232 and Cr 140, this project's primary focus is to identify X-ray sources and to extract net source counts for these sources in these two open clusters. These X-ray data would be combined with ground-based photometry and spectroscopy in order to identify G, K, and early-M type cluster members. Such membership data are important because, at present, no members later than spectral type approx. F5 are currently known for either cluster. With ages estimated to be approx. 25 Myr and at distances of just approx. 350 pc, the combined late-type membership of the NGC 2232 and Cr 140 clusters would yield an almost unique sample of solar-type stars in the post-T Tauri/pre-main sequence phase of evolution. These stars could be used to assess the level and dispersion of coronal activity levels, as a part of a probe of the importance of magnetic braking and the level of magnetic dynamo activity, for solar-type stars just before they reach the zero-age main sequence.

The occurrence of metacercariae of Petasiger (Digenea: Echinostomatidae) in an unusual site, within the lateral line scales of cyprinid fishes.

PubMed

Molnar, Kalman; Gibson, David I; Cech, Gabor; Papp, Melitta; Deak-Paulus, Petra; Juhasz, Lajos; Toth, Norbert; Szekely, Csaba

2015-01-01

During a regular veterinary inspection of fishes from Lake Balaton, Hungary, echinostomatid metacercariae (Digenea), with collar spines characteristic of species of the genera Petasiger Dietz, 1909 and Paryphostomum Dietz, 1909, were found in the lateral line scales of a roach Rutilus rutilus (Linnaeus), an apparently unique site. In a subsequent examination of 586 fishes from 20 different species, similar infections were found in 11 species. The infection was virtually restricted to the lateral line scales, other scales being infected only incidentally. These encysted metacercariae had 27 collar spines, including eight larger angle spines and 19 smaller dorsal spines arranged in two rows. Two types of metacercarial cyst were found. One type had a cyst diameter of 138-171 µm × 105-120 µm and three central dorsal spines that were larger than the remainder and tended to resemble the angle spines. The second type of metacercarial cyst had a diameter of 128-157 µm × 105-115 µm and all 19 dorsal spines of the metacercaria were of a similar size. ITS sequences of the second type of metacercaria exhibited a 100% similarity to sequences of two adult Petasiger phalacrocoracis (Yamaguti, 1939) specimens collected from the gut of Phalacrocorax carbo (Linnaeus) in Hungary and to P. phalacrocoracis deposited in the GenBank database. Sequences obtained from two metacercariae of the first type showed a 2.8-2.9 % difference from sequences of the second type of metacercaria and from those of adult specimens of P. phalacrocoracis from cormorants. Based on these results, the second type metacercaria is considered to be a larval stage of P. phalacrocoracis, but the identity of the first type is uncertain. The unusual location of these metacercariae in the lateral line scales is discussed in relation to their transmission.

M Protein Gene (emm Type) Analysis of Group A Beta-Hemolytic Streptococci from Ethiopia Reveals Unique Patterns

PubMed Central

Tewodros, Wezenet; Kronvall, Göran

2005-01-01

The genetic diversity of group A streptococcal (GAS) isolates obtained in 1990 from Ethiopian children with various streptococcal diseases was studied by using emm gene sequence analysis. A total of 217 GAS isolates were included: 155 and 62 isolates from throat and skin, respectively. A total of 78 different emm/st types were detected among the 217 isolates. Of these, 166 (76.5%) belonged to 52 validated reference emm types, 26 (11.9%) belonged to 16 already recognized sequence types (st types) and 25 (11.5%) belonged to 10 undocumented new sequence types. Resistance to tetracycline (148 of 217) was not correlated to emm type. Isolation rate of the classical rheumatogenic and nephritogenic strains was low from cases of acute rheumatic fever (ARF) and acute glomerulonephritis (AGN), respectively. Instead, the recently discovered st types were overrepresented among isolates from patients with ARF (3 of 7) and AGN (9 of 16) (P < 0.01) compared to isolates from subjects with tonsillitis and from healthy carriers (10 of 57 and 16 of 90, respectively). In contrast to rheumatogenic strains from the temperate regions, more than half of the isolates from ARF (four of seven) carried the genetic marker for skin preference, emm pattern D, although most of them (six of seven) were isolated from throat. Of 57 tonsillitis-associated isolates, 16 (28%) belonged to emm pattern D compared to <1% in temperate regions. As in other reports emm patterns A to C were strongly associated with throat, whereas emm pattern D did not correlate to skin. This first large-scale emm typing report from Africa has demonstrated a heterogeneous GAS population and contrasting nature of GAS epidemiology in the region. PMID:16145079

Whole genome sequencing options for bacterial strain typing and epidemiologic analysis based on single nucleotide polymorphism versus gene-by-gene-based approaches.

PubMed

Schürch, A C; Arredondo-Alonso, S; Willems, R J L; Goering, R V

2018-04-01

Whole genome sequence (WGS)-based strain typing finds increasing use in the epidemiologic analysis of bacterial pathogens in both public health as well as more localized infection control settings. This minireview describes methodologic approaches that have been explored for WGS-based epidemiologic analysis and considers the challenges and pitfalls of data interpretation. Personal collection of relevant publications. When applying WGS to study the molecular epidemiology of bacterial pathogens, genomic variability between strains is translated into measures of distance by determining single nucleotide polymorphisms in core genome alignments or by indexing allelic variation in hundreds to thousands of core genes, assigning types to unique allelic profiles. Interpreting isolate relatedness from these distances is highly organism specific, and attempts to establish species-specific cutoffs are unlikely to be generally applicable. In cases where single nucleotide polymorphism or core gene typing do not provide the resolution necessary for accurate assessment of the epidemiology of bacterial pathogens, inclusion of accessory gene or plasmid sequences may provide the additional required discrimination. As with all epidemiologic analysis, realizing the full potential of the revolutionary advances in WGS-based approaches requires understanding and dealing with issues related to the fundamental steps of data generation and interpretation. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Group B Streptococcus Vaginal Carriage in Pregnant Women as Deciphered by Clustered Regularly Interspaced Short Palindromic Repeat Analysis.

PubMed

Beauruelle, Clemence; Pastuszka, Adeline; Mereghetti, Laurent; Lanotte, Philippe

2018-06-01

We evaluated the diversity of group B Streptococcus (GBS) vaginal carriage populations in pregnant women. For this purpose, we studied each isolate present in a primary culture of a vaginal swab using a new approach based on clustered regularly interspaced short palindromic repeats (CRISPR) locus analysis. To evaluate the CRISPR array composition rapidly, a restriction fragment length polymorphism (RFLP) analysis was performed. For each different pattern observed, the CRISPR array was sequenced and capsular typing and multilocus sequence typing (MLST) were performed. A total of 970 isolates from 10 women were analyzed by CRISPR-RFLP. Each woman carrying GBS isolates presented one to five specific "personal" patterns. Five women showed similar isolates with specific and unique restriction patterns, suggesting the carriage of a single GBS clone. Different patterns were observed among isolates from the other five women. For three of these, CRISPR locus sequencing highlighted low levels of internal modifications in the locus backbone, whereas there were high levels of modifications for the last two women, suggesting the carriage of two different clones. These two clones were closely related, having the same ancestral spacer(s), the same capsular type and, in one case, the same ST, but showed different antibiotic resistance patterns in pairs. Eight of 10 women were colonized by a single GBS clone, while two of them were colonized by two strains, leading to a risk of selection of more-virulent and/or more-resistant clones during antibiotic prophylaxis. This CRISPR analysis made it possible to separate isolates belonging to a single capsular type and sequence type, highlighting the greater discriminating power of this approach. Copyright © 2018 American Society for Microbiology.

Virulence gene typing of methicillin-resistant Staphylococcus aureus as a complement in epidemiological typing.

PubMed

Nowrouzian, Forough L; Karami, Nahid; Welinder-Olsson, Christina; Ahrén, Christina

2013-06-01

Methicillin-resistant Staphylococcus aureus (MRSA) has widely spread to all parts of the world. For surveillance and effective infection control molecular typing is required. We have evaluated the utility of virulence gene determination as a complementary tool for epidemiological typing of MRSA in relation to spa-typing and pulsed-field gel electrophoresis (PFGE). We assessed 63 community-acquired MRSA (CA-MRSA) isolates detected in the West part of Sweden for 30 virulence factor genes (VF) and agr allele variations by serial polymerase chain reaction (PCR) assays. These isolates belonged to sequence types (ST) 8, 80, 45 and 30 as classified by multilocus sequence typing. The isolates in each spa-type and PFGE-type were examined over an extended time-period and constituted a varying number of PFGE-subtypes (5-14) and spa-types (3-11) within four major PFGE types. Each ST had a unique VF profile. For isolates within a major PFGE type showing high diversity both in PFGE subtypes and spa the VF profile varied as well in contrast to those with low diversity where no alterations were seen. Thus, the accuracy of each typing method does not only vary by the method per se but is rather dependent on the genetic repertoire of the typed strains and genes evaluated. For strains demonstrating high diversity VF typing may be a useful complement in the epidemiological investigations, and may highlight the accurate discriminatory power of spa or PFGE typing. Copyright © 2013 Elsevier B.V. All rights reserved.

Variants of uncertain significance in newborn screening disorders: implications for large-scale genomic sequencing.

PubMed

Narravula, Alekhya; Garber, Kathryn B; Askree, S Hussain; Hegde, Madhuri; Hall, Patricia L

2017-01-01

As exome and genome sequencing using high-throughput sequencing technologies move rapidly into the diagnostic process, laboratories and clinicians need to develop a strategy for dealing with uncertain findings. A commitment must be made to minimize these findings, and all parties may need to make adjustments to their processes. The information required to reclassify these variants is often available but not communicated to all relevant parties. To illustrate these issues, we focused on three well-characterized monogenic, metabolic disorders included in newborn screens: classic galactosemia, caused by GALT variants; phenylketonuria, caused by PAH variants; and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, caused by ACADM variants. In 10 years of clinical molecular testing, we have observed 134 unique GALT variants, 46 of which were variants of uncertain significance (VUS). In PAH, we observed 132 variants, including 17 VUS, and for ACADM, we observed 64 unique variants, of which 33 were uncertain. After this review, 17 VUS (37%; 7 in ACADM, 9 in GALT, and 1 in PAH) were reclassified from uncertain (6 to benign or likely benign and 11 to pathogenic or likely pathogenic). We identified common types of missing information that would have helped make a definitive classification and categorized this information by ease and cost to obtain.Genet Med 19 1, 77-82.

Revealing the transcriptomic complexity of switchgrass by PacBio long-read sequencing.

PubMed

Zuo, Chunman; Blow, Matthew; Sreedasyam, Avinash; Kuo, Rita C; Ramamoorthy, Govindarajan Kunde; Torres-Jerez, Ivone; Li, Guifen; Wang, Mei; Dilworth, David; Barry, Kerrie; Udvardi, Michael; Schmutz, Jeremy; Tang, Yuhong; Xu, Ying

2018-01-01

Switchgrass ( Panicum virgatum L.) is an important bioenergy crop widely used for lignocellulosic research. While extensive transcriptomic analyses have been conducted on this species using short read-based sequencing techniques, very little has been reliably derived regarding alternatively spliced (AS) transcripts. We present an analysis of transcriptomes of six switchgrass tissue types pooled together, sequenced using Pacific Biosciences (PacBio) single-molecular long-read technology. Our analysis identified 105,419 unique transcripts covering 43,570 known genes and 8795 previously unknown genes. 45,168 are novel transcripts of known genes. A total of 60,096 AS transcripts are identified, 45,628 being novel. We have also predicted 1549 transcripts of genes involved in cell wall construction and remodeling, 639 being novel transcripts of known cell wall genes. Most of the predicted transcripts are validated against Illumina-based short reads. Specifically, 96% of the splice junction sites in all the unique transcripts are validated by at least five Illumina reads. Comparisons between genes derived from our identified transcripts and the current genome annotation revealed that among the gene set predicted by both analyses, 16,640 have different exon-intron structures. Overall, substantial amount of new information is derived from the PacBio RNA data regarding both the transcriptome and the genome of switchgrass.

Wide spread of OXA-23-producing carbapenem-resistant Acinetobacter baumannii belonging to clonal complex II in different hospitals in Lebanon.

PubMed

Al Atrouni, Ahmad; Hamze, Monzer; Jisr, Tamima; Lemarié, Carole; Eveillard, Matthieu; Joly-Guillou, Marie-Laure; Kempf, Marie

2016-11-01

To investigate the molecular epidemiology of Acinetobacter baumannii strains isolated from different hospitals in Lebanon. A total of 119 non-duplicate Acinetobacter strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and partial rpoB gene sequencing. Antibiotic susceptibility testing was performed by disc diffusion method and all identified carbapenem-resistant isolates were investigated by PCR assays for the presence of the carbapenemase-encoding genes. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for molecular typing. Of the 119 A. baumannii isolates, 76.5% were resistant to carbapenems. The most common carbapenemase was the OXA-23-type, found in 82 isolates. The study of population structure using MLST revealed the presence of 30 sequence types (STs) including 18 new ones, with ST2 being the most commonly detected, accounting for 61% of the isolates typed. PFGE performed on all strains of ST2 identified a major cluster of 53 isolates, in addition to three other minor clusters and ten unique profiles. This study highlights the wide dissemination of highly related OXA-23-producing carbapenem-resistant A. baumannii belonging to the international clone II in Lebanon. Thus, appropriate infection control measures are recommended in order to control the geographical spread of this clone in this country. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

A high-throughput venom-gland transcriptome for the Eastern Diamondback Rattlesnake (Crotalus adamanteus) and evidence for pervasive positive selection across toxin classes.

PubMed

Rokyta, Darin R; Wray, Kenneth P; Lemmon, Alan R; Lemmon, Emily Moriarty; Caudle, S Brian

2011-04-01

Despite causing considerable human mortality and morbidity, animal toxins represent a valuable source of pharmacologically active macromolecules, a unique system for studying molecular adaptation, and a powerful framework for examining structure-function relationships in proteins. Snake venoms are particularly useful in the latter regard as they consist primarily of a moderate number of proteins and peptides that have been found to belong to just a handful of protein families. As these proteins and peptides are produced in dedicated glands, transcriptome sequencing has proven to be an effective approach to identifying the expressed toxin genes. We generated a venom-gland transcriptome for the Eastern Diamondback Rattlesnake (Crotalus adamanteus) using Roche 454 sequencing technology. In the current work, we focus on transcripts encoding toxins. We identified 40 unique toxin transcripts, 30 of which have full-length coding sequences, and 10 have only partial coding sequences. These toxins account for 24% of the total sequencing reads. We found toxins from 11 previously described families of snake-venom toxins and have discovered two putative, previously undescribed toxin classes. The most diverse and highly expressed toxin classes in the C. adamanteus venom-gland transcriptome are the serine proteinases, metalloproteinases, and C-type lectins. The serine proteinases are the most abundant class, accounting for 35% of the toxin sequencing reads. Metalloproteinases are the most diverse; 11 different forms have been identified. Using our sequences and those available in public databases, we detected positive selection in seven of the eight toxin families for which sufficient sequences were available for the analysis. We find that the vast majority of the genes that contribute directly to this vertebrate trait show evidence for a role for positive selection in their evolutionary history. Copyright © 2011 Elsevier Ltd. All rights reserved.

Pestoides F, an atypical Yersinia pestis strain from the former Soviet Union.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia, Emilio; Worsham, Patricia; Bearden, S.

2007-01-01

Unlike the classical Yersinia pestis strains, members of an atypical group of Y. pestis from Central Asia, denominated Y. pestis subspecies caucasica (also known as one of several pestoides types), are distinguished by a number of characteristics including their ability to ferment rhamnose and melibiose, their lack of the small plasmid encoding the plasminogen activator (pla) and pesticin, and their exceptionally large variants of the virulence plasmid pMT (encoding murine toxin and capsular antigen). We have obtained the entire genome sequence of Y. pestis Pestoides F, an isolate from the former Soviet Union that has enabled us to carryout amore » comprehensive genome-wide comparison of this organism's genomic content against the six published sequences of Y. pestis and their Y. pseudotuberculosis ancestor. Based on classical glycerol fermentation (+ve) and nitrate reduction (+ve) Y. pestis Pestoides F is an isolate that belongs to the biovar antiqua. This strain is unusual in other characteristics such as the fact that it carries a non-consensus V antigen (lcrV) sequence, and that unlike other Pla(-) strains, Pestoides F retains virulence by the parenteral and aerosol routes. The chromosome of Pestoides F is 4,517,345 bp in size comprising some 3,936 predicted coding sequences, while its pCD and pMT plasmids are 71,507 bp and 137,010 bp in size respectively. Comparison of chromosome-associated genes in Pestoides F with those in the other sequenced Y. pestis strains reveals differences ranging from strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. There is a single approximately 7 kb unique region in the chromosome not found in any of the completed Y. pestis strains sequenced to date, but which is present in the Y. pseudotuberculosis ancestor. Taken together, these findings are consistent with Pestoides F being derived from the most ancient lineage of Y. pestis yet sequenced.« less

Pestoides F, and Atypical Yersinia pestis Strain from the Former Soviet Union

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia, E; Worsham, P; Bearden, S

2007-01-05

Unlike the classical Yersinia pestis strains, members of an atypical group of Y. pestis from Central Asia, denominated Y. pestis subspecies caucasica (also known as one of several pestoides types), are distinguished by a number of characteristics including their ability to ferment rhamnose and melibiose, their lacking the small plasmid encoding the plasminogen activator (pla) and pesticin, and their exceptionally large variants of the virulence plasmid pMT (encoding murine toxin and capsular antigen). We have obtained the entire genome sequence of Y. pestis Pestoides F, an isolate from the former Soviet Union that has enabled us to carryout a comprehensivemore » genome-wide comparison of this organism's genomic content against the six published sequences of Y. pestis and their Y. pseudotuberculosis ancestor. Based on classical glycerol fermentation (+ve) and nitrate reduction (+ve) Y. pestis Pestoides F is an isolate that belongs to the biovar antiqua. This strain is unusual in other characteristics such as the fact that it carries a non-consensus V antigen (lcrV) sequence, and that unlike other Pla{sup -} strains, Pestoides F retains virulence by the parenteral and aerosol routes. The chromosome of Pestoides F is 4,517,345 bp in size comprising some 3,936 predicted coding sequences, while its pCD and pMT plasmids are 71,507 bp and 137,010 bp in size respectively. Comparison of chromosome-associated genes in Pestoides F with those in the other sequenced Y. pestis strains, reveals a series of differences ranging from strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. There is a single {approx}7 kb unique region in the chromosome not found in any of the completed Y. pestis strains sequenced to date, but which is present in the Y. pseudotuberculosis ancestor. Taken together, these findings are consistent with Pestoides F being derived from the most ancient lineage of Y. pestis yet sequenced.« less

16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.

PubMed

Hellberg, Rosalee S; Martin, Keely G; Keys, Ashley L; Haney, Christopher J; Shen, Yuelian; Smiley, R Derike

2013-12-01

Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type. Copyright © 2013 Elsevier Ltd. All rights reserved.

Unique Footprint in the scl1.3 Locus Affects Adhesion and Biofilm Formation of the Invasive M3-Type Group A Streptococcus.

PubMed

Bachert, Beth A; Choi, Soo J; LaSala, Paul R; Harper, Tiffany I; McNitt, Dudley H; Boehm, Dylan T; Caswell, Clayton C; Ciborowski, Pawel; Keene, Douglas R; Flores, Anthony R; Musser, James M; Squeglia, Flavia; Marasco, Daniela; Berisio, Rita; Lukomski, Slawomir

2016-01-01

The streptococcal collagen-like proteins 1 and 2 (Scl1 and Scl2) are major surface adhesins that are ubiquitous among group A Streptococcus (GAS). Invasive M3-type strains, however, have evolved two unique conserved features in the scl1 locus: (i) an IS1548 element insertion in the scl1 promoter region and (ii) a nonsense mutation within the scl1 coding sequence. The scl1 transcript is drastically reduced in M3-type GAS, contrasting with a high transcription level of scl1 allele in invasive M1-type GAS. This leads to a lack of Scl1 expression in M3 strains. In contrast, while scl2 transcription and Scl2 production are elevated in M3 strains, M1 GAS lack Scl2 surface expression. M3-type strains were shown to have reduced biofilm formation on inanimate surfaces coated with cellular fibronectin and laminin, and in human skin equivalents. Repair of the nonsense mutation and restoration of Scl1 expression on M3-GAS cells, restores biofilm formation on cellular fibronectin and laminin coatings. Inactivation of scl1 in biofilm-capable M28 and M41 strains results in larger skin lesions in a mouse model, indicating that lack of Scl1 adhesin promotes bacterial spread over localized infection. These studies suggest the uniquely evolved scl1 locus in the M3-type strains, which prevents surface expression of the major Scl1 adhesin, contributed to the emergence of the invasive M3-type strains. Furthermore these studies provide insight into the molecular mechanisms mediating colonization, biofilm formation, and pathogenesis of group A streptococci.

Plasmid Characterization and Chromosome Analysis of Two netF+ Clostridium perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis

PubMed Central

Mehdizadeh Gohari, Iman; Kropinski, Andrew M.; Weese, Scott J.; Parreira, Valeria R.; Whitehead, Ashley E.; Boerlin, Patrick; Prescott, John F.

2016-01-01

The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus. PMID:26859667

Complete genome sequencing and evolutionary analysis of Indian isolates of Dengue virus type 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dash, Paban Kumar, E-mail: pabandash@rediffmail.com; Sharma, Shashi; Soni, Manisha

Highlights: •Complete genome of Indian DENV-2 was deciphered for the first time in this study. •The recent Indian DENV-2 revealed presence of many unique amino acid residues. •Genotype shift (American to Cosmopolitan) characterizes evolution of DENV-2 in India. •Circulation of a unique clade of DENV-2 in South Asia was identified. -- Abstract: Dengue is the most important arboviral infection of global public health significance. It is now endemic in most parts of the South East Asia including India. Though Dengue virus type 2 (DENV-2) is predominantly associated with major outbreaks in India, complete genome information of Indian DENV-2 is notmore » available. In this study, the full-length genome of five DENV-2 isolates (four from 2001 to 2011 and one from 1960), from different parts of India was determined. The complete genome of the Indian DENV-2 was found to be 10,670 bases long with an open reading frame coding for 3391 amino acids. The recent Indian DENV-2 (2001–2011) revealed a nucleotide sequence identity of around 90% and 97% with an older Indian DENV-2 (1960) and closely related Sri Lankan and Chinese DENV-2 respectively. Presence of unique amino acid residues and non-conservative substitutions in critical amino acid residues of major structural and non-structural proteins was observed in recent Indian DENV-2. Selection pressure analysis revealed positive selection in few amino acid sites of the genes encoding for structural and non-structural proteins. The molecular phylogenetic analysis based on comparison of both complete coding region and envelope protein gene with globally diverse DENV-2 viruses classified the recent Indian isolates into a unique South Asian clade within Cosmopolitan genotype. A shift of genotype from American to Cosmopolitan in 1970s characterized the evolution of DENV-2 in India. Present study is the first report on complete genome characterization of emerging DENV-2 isolates from India and highlights the circulation of a unique clade in South Asia.« less

Infant auditory short-term memory for non-linguistic sounds.

PubMed

Ross-Sheehy, Shannon; Newman, Rochelle S

2015-04-01

This research explores auditory short-term memory (STM) capacity for non-linguistic sounds in 10-month-old infants. Infants were presented with auditory streams composed of repeating sequences of either 2 or 4 unique instruments (e.g., flute, piano, cello; 350 or 700 ms in duration) followed by a 500-ms retention interval. These instrument sequences either stayed the same for every repetition (Constant) or changed by 1 instrument per sequence (Varying). Using the head-turn preference procedure, infant listening durations were recorded for each stream type (2- or 4-instrument sequences composed of 350- or 700-ms notes). Preference for the Varying stream was taken as evidence of auditory STM because detection of the novel instrument required memory for all of the instruments in a given sequence. Results demonstrate that infants listened longer to Varying streams for 2-instrument sequences, but not 4-instrument sequences, composed of 350-ms notes (Experiment 1), although this effect did not hold when note durations were increased to 700 ms (Experiment 2). Experiment 3 replicates and extends results from Experiments 1 and 2 and provides support for a duration account of capacity limits in infant auditory STM. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons

PubMed Central

Landeweert, Renske; Leeflang, Paula; Kuyper, Thom W.; Hoffland, Ellis; Rosling, Anna; Wernars, Karel; Smit, Eric

2003-01-01

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (≥99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had ≥98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil. PMID:12514012

Incorporation of unique molecular identifiers in TruSeq adapters improves the accuracy of quantitative sequencing.

PubMed

Hong, Jungeui; Gresham, David

2017-11-01

Quantitative analysis of next-generation sequencing (NGS) data requires discriminating duplicate reads generated by PCR from identical molecules that are of unique origin. Typically, PCR duplicates are identified as sequence reads that align to the same genomic coordinates using reference-based alignment. However, identical molecules can be independently generated during library preparation. Misidentification of these molecules as PCR duplicates can introduce unforeseen biases during analyses. Here, we developed a cost-effective sequencing adapter design by modifying Illumina TruSeq adapters to incorporate a unique molecular identifier (UMI) while maintaining the capacity to undertake multiplexed, single-index sequencing. Incorporation of UMIs into TruSeq adapters (TrUMIseq adapters) enables identification of bona fide PCR duplicates as identically mapped reads with identical UMIs. Using TrUMIseq adapters, we show that accurate removal of PCR duplicates results in improved accuracy of both allele frequency (AF) estimation in heterogeneous populations using DNA sequencing and gene expression quantification using RNA-Seq.

Partial bisulfite conversion for unique template sequencing

PubMed Central

Kumar, Vijay; Rosenbaum, Julie; Wang, Zihua; Forcier, Talitha; Ronemus, Michael; Wigler, Michael

2018-01-01

Abstract We introduce a new protocol, mutational sequencing or muSeq, which uses sodium bisulfite to randomly deaminate unmethylated cytosines at a fixed and tunable rate. The muSeq protocol marks each initial template molecule with a unique mutation signature that is present in every copy of the template, and in every fragmented copy of a copy. In the sequenced read data, this signature is observed as a unique pattern of C-to-T or G-to-A nucleotide conversions. Clustering reads with the same conversion pattern enables accurate count and long-range assembly of initial template molecules from short-read sequence data. We explore count and low-error sequencing by profiling 135 000 restriction fragments in a PstI representation, demonstrating that muSeq improves copy number inference and significantly reduces sporadic sequencer error. We explore long-range assembly in the context of cDNA, generating contiguous transcript clusters greater than 3,000 bp in length. The muSeq assemblies reveal transcriptional diversity not observable from short-read data alone. PMID:29161423

Screening of differentially expressed genes in male idiopathic osteoporosis via RNA sequencing.

PubMed

Feng, Li; Wang, Yan; Zhou, Jing; Tian, Baofang; Xia, Bo

2018-05-07

As a type of osteoporosis (OP), male idiopathic OP (MIO) is a bone disorder that occurs in young males and is a public health problem worldwide. However, the detailed pathogenesis of MIO remains to be elucidated. In the present study, blood samples of patients with MIO, senile OP, postmenopausal OP and normal controls (NCs) were obtained for RNA sequencing. Compared with the NC group, differentially expressed genes (DEGs) in the three types of OP were identified. DEGs that were common among the three types of OP and the DEGs that were unique to patients with MIO were determined. Gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted. MIO‑specific and OP‑specific protein‑protein interaction (PPI) networks were constructed. Compared with NCs, a total of 519, 368 and 1,472 DEGs were identified in samples from MIO, senile OP and postmenopausal OP, respectively. Tetraspanin 5 (TSPAN5) and α‑synuclein (SNCA) were unique DEGs in MIO that were not identified in the other two types of OP compared with NCs. Furthermore, the expression of carbonic anhydrase 1 (CA1) and S100 calcium‑binding protein P (S100P) in MIO was significantly different compared with senile OP, postmenopausal OP and NC samples. 'MAPK signaling pathway', 'type I diabetes mellitus' and 'hematopoietic cell lineage' were among significantly enriched pathways of DEGs in MIO. SNCA and CDC‑like kinase 1 were the hub genes in the MIO‑specific PPI network. In conclusion, the mitogen‑activated protein kinase signaling and type I diabetes mellitus pathways may be involved in bone formation; SNCA and TSPAN5 may be associated with bone resorption. These two pathways and two genes may serve a role in MIO. CA1 and S100P may regulate the process of MIO by modulation of calcification and dysregulation of calcium binding. These findings may have provided an experimental basis for elucidating the underlying mechanisms and developing potential diagnostic biomarkers of MIO.

Expressed sequences tags of the anther smut fungus, Microbotryum violaceum, identify mating and pathogenicity genes

PubMed Central

Yockteng, Roxana; Marthey, Sylvain; Chiapello, Hélène; Gendrault, Annie; Hood, Michael E; Rodolphe, François; Devier, Benjamin; Wincker, Patrick; Dossat, Carole; Giraud, Tatiana

2007-01-01

Background The basidiomycete fungus Microbotryum violaceum is responsible for the anther-smut disease in many plants of the Caryophyllaceae family and is a model in genetics and evolutionary biology. Infection is initiated by dikaryotic hyphae produced after the conjugation of two haploid sporidia of opposite mating type. This study describes M. violaceum ESTs corresponding to nuclear genes expressed during conjugation and early hyphal production. Results A normalized cDNA library generated 24,128 sequences, which were assembled into 7,765 unique genes; 25.2% of them displayed significant similarity to annotated proteins from other organisms, 74.3% a weak similarity to the same set of known proteins, and 0.5% were orphans. We identified putative pheromone receptors and genes that in other fungi are involved in the mating process. We also identified many sequences similar to genes known to be involved in pathogenicity in other fungi. The M. violaceum EST database, MICROBASE, is available on the Web and provides access to the sequences, assembled contigs, annotations and programs to compare similarities against MICROBASE. Conclusion This study provides a basis for cloning the mating type locus, for further investigation of pathogenicity genes in the anther smut fungi, and for comparative genomics. PMID:17692127

Identification and characterization of mutant clones with enhanced propagation rates from phage-displayed peptide libraries.

PubMed

Nguyen, Kieu T H; Adamkiewicz, Marta A; Hebert, Lauren E; Zygiel, Emily M; Boyle, Holly R; Martone, Christina M; Meléndez-Ríos, Carola B; Noren, Karen A; Noren, Christopher J; Hall, Marilena Fitzsimons

2014-10-01

A target-unrelated peptide (TUP) can arise in phage display selection experiments as a result of a propagation advantage exhibited by the phage clone displaying the peptide. We previously characterized HAIYPRH, from the M13-based Ph.D.-7 phage display library, as a propagation-related TUP resulting from a G→A mutation in the Shine-Dalgarno sequence of gene II. This mutant was shown to propagate in Escherichia coli at a dramatically faster rate than phage bearing the wild-type Shine-Dalgarno sequence. We now report 27 additional fast-propagating clones displaying 24 different peptides and carrying 14 unique mutations. Most of these mutations are found either in or upstream of the gene II Shine-Dalgarno sequence, but still within the mRNA transcript of gene II. All 27 clones propagate at significantly higher rates than normal library phage, most within experimental error of wild-type M13 propagation, suggesting that mutations arise to compensate for the reduced virulence caused by the insertion of a lacZα cassette proximal to the replication origin of the phage used to construct the library. We also describe an efficient and convenient assay to diagnose propagation-related TUPS among peptide sequences selected by phage display. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Aliphatic peptides show similar self-assembly to amyloid core sequences, challenging the importance of aromatic interactions in amyloidosis.

PubMed

Lakshmanan, Anupama; Cheong, Daniel W; Accardo, Angelo; Di Fabrizio, Enzo; Riekel, Christian; Hauser, Charlotte A E

2013-01-08

The self-assembly of abnormally folded proteins into amyloid fibrils is a hallmark of many debilitating diseases, from Alzheimer's and Parkinson diseases to prion-related disorders and diabetes type II. However, the fundamental mechanism of amyloid aggregation remains poorly understood. Core sequences of four to seven amino acids within natural amyloid proteins that form toxic fibrils have been used to study amyloidogenesis. We recently reported a class of systematically designed ultrasmall peptides that self-assemble in water into cross-β-type fibers. Here we compare the self-assembly of these peptides with natural core sequences. These include core segments from Alzheimer's amyloid-β, human amylin, and calcitonin. We analyzed the self-assembly process using circular dichroism, electron microscopy, X-ray diffraction, rheology, and molecular dynamics simulations. We found that the designed aliphatic peptides exhibited a similar self-assembly mechanism to several natural sequences, with formation of α-helical intermediates being a common feature. Interestingly, the self-assembly of a second core sequence from amyloid-β, containing the diphenylalanine motif, was distinctly different from all other examined sequences. The diphenylalanine-containing sequence formed β-sheet aggregates without going through the α-helical intermediate step, giving a unique fiber-diffraction pattern and simulation structure. Based on these results, we propose a simplified aliphatic model system to study amyloidosis. Our results provide vital insight into the nature of early intermediates formed and suggest that aromatic interactions are not as important in amyloid formation as previously postulated. This information is necessary for developing therapeutic drugs that inhibit and control amyloid formation.

An unbiased study of debris discs around A-type stars with Herschel

NASA Astrophysics Data System (ADS)

Thureau, N. D.; Greaves, J. S.; Matthews, B. C.; Kennedy, G.; Phillips, N.; Booth, M.; Duchêne, G.; Horner, J.; Rodriguez, D. R.; Sibthorpe, B.; Wyatt, M. C.

2014-12-01

The Herschel DEBRIS (Disc Emission via a Bias-free Reconnaissance in the Infrared/Submillimetre) survey brings us a unique perspective on the study of debris discs around main-sequence A-type stars. Bias-free by design, the survey offers a remarkable data set with which to investigate the cold disc properties. The statistical analysis of the 100 and 160 μm data for 86 main-sequence A stars yields a lower than previously found debris disc rate. Considering better than 3σ excess sources, we find a detection rate ≥24 ± 5 per cent at 100 μm which is similar to the debris disc rate around main-sequence F/G/K-spectral type stars. While the 100 and 160 μm excesses slowly decline with time, debris discs with large excesses are found around some of the oldest A stars in our sample, evidence that the debris phenomenon can survive throughout the length of the main sequence (˜1 Gyr). Debris discs are predominantly detected around the youngest and hottest stars in our sample. Stellar properties such as metallicity are found to have no effect on the debris disc incidence. Debris discs are found around A stars in single systems and multiple systems at similar rates. While tight and wide binaries (<1 and >100 au, respectively) host debris discs with a similar frequency and global properties, no intermediate separation debris systems were detected in our sample.

Lactobacillus buchneri Genotyping on the Basis of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Locus Diversity

PubMed Central

Briner, Alexandra E.

2014-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) in combination with associated sequences (cas) constitute the CRISPR-Cas immune system, which uptakes DNA from invasive genetic elements as novel “spacers” that provide a genetic record of immunization events. We investigated the potential of CRISPR-based genotyping of Lactobacillus buchneri, a species relevant for commercial silage, bioethanol, and vegetable fermentations. Upon investigating the occurrence and diversity of CRISPR-Cas systems in Lactobacillus buchneri genomes, we observed a ubiquitous occurrence of CRISPR arrays containing a 36-nucleotide (nt) type II-A CRISPR locus adjacent to four cas genes, including the universal cas1 and cas2 genes and the type II signature gene cas9. Comparative analysis of CRISPR spacer content in 26 L. buchneri pickle fermentation isolates associated with spoilage revealed 10 unique locus genotypes that contained between 9 and 29 variable spacers. We observed a set of conserved spacers at the ancestral end, reflecting a common origin, as well as leader-end polymorphisms, reflecting recent divergence. Some of these spacers showed perfect identity with phage sequences, and many spacers showed homology to Lactobacillus plasmid sequences. Following a comparative analysis of sequences immediately flanking protospacers that matched CRISPR spacers, we identified a novel putative protospacer-adjacent motif (PAM), 5′-AAAA-3′. Overall, these findings suggest that type II-A CRISPR-Cas systems are valuable for genotyping of L. buchneri. PMID:24271175

De Novo Sequencing and Analysis of Lemongrass Transcriptome Provide First Insights into the Essential Oil Biosynthesis of Aromatic Grasses.

PubMed

Meena, Seema; Kumar, Sarma R; Venkata Rao, D K; Dwivedi, Varun; Shilpashree, H B; Rastogi, Shubhra; Shasany, Ajit K; Nagegowda, Dinesh A

2016-01-01

Aromatic grasses of the genus Cymbopogon (Poaceae family) represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavor, fragrance, cosmetic, and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step toward understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass) by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases, pyrophosphatases, alcohol dehydrogenases, aldo-keto reductases, carotenoid cleavage dioxygenases, alcohol acetyltransferases, and aldehyde dehydrogenases, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type) with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes, and acetates. Molecular modeling and docking further supported the role of identified protein sequences in aroma formation in Cymbopogon. Also, simple sequence repeats were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition.

Evidence for Sequence Scrambling and Divergent H/D Exchange Reactions of Doubly-Charged Isobaric b-Type Fragment Ions

NASA Astrophysics Data System (ADS)

Zekavat, Behrooz; Miladi, Mahsan; Al-Fdeilat, Abdullah H.; Somogyi, Arpad; Solouki, Touradj

2014-02-01

To date, only a limited number of reports are available on structural variants of multiply-charged b-fragment ions. We report on observed bimodal gas-phase hydrogen/deuterium exchange (HDX) reaction kinetics and patterns for substance P b10 2+ that point to presence of isomeric structures. We also compare HDX reactions, post-ion mobility/collision-induced dissociation (post-IM/CID), and sustained off-resonance irradiation-collision induced dissociation (SORI-CID) of substance P b10 2+ and a cyclic peptide with an identical amino acid (AA) sequence order to substance P b10. The observed HDX patterns and reaction kinetics and SORI-CID pattern for the doubly charged head-to-tail cyclized peptide were different from either of the presumed isomers of substance P b10 2+, suggesting that b10 2+ may not exist exclusively as a head-to-tail cyclized structure. Ultra-high mass measurement accuracy was used to assign identities of the observed SORI-CID fragment ions of substance P b10 2+; over 30 % of the observed SORI-CID fragment ions from substance P b10 2+ had rearranged (scrambled) AA sequences. Moreover, post-IM/CID experiments revealed the presence of two conformer types for substance P b10 2+, whereas only one conformer type was observed for the head-to-tail cyclized peptide. We also show that AA sequence scrambling from CID of doubly-charged b-fragment ions is not unique to substance P b10 2+.

Evidence for sequence scrambling and divergent H/D exchange reactions of doubly-charged isobaric b-type fragment ions.

PubMed

Zekavat, Behrooz; Miladi, Mahsan; Al-Fdeilat, Abdullah H; Somogyi, Arpad; Solouki, Touradj

2014-02-01

To date, only a limited number of reports are available on structural variants of multiply-charged b-fragment ions. We report on observed bimodal gas-phase hydrogen/deuterium exchange (HDX) reaction kinetics and patterns for substance P b10(2+) that point to presence of isomeric structures. We also compare HDX reactions, post-ion mobility/collision-induced dissociation (post-IM/CID), and sustained off-resonance irradiation-collision induced dissociation (SORI-CID) of substance P b10(2+) and a cyclic peptide with an identical amino acid (AA) sequence order to substance P b10. The observed HDX patterns and reaction kinetics and SORI-CID pattern for the doubly charged head-to-tail cyclized peptide were different from either of the presumed isomers of substance P b10(2+), suggesting that b10(2+) may not exist exclusively as a head-to-tail cyclized structure. Ultra-high mass measurement accuracy was used to assign identities of the observed SORI-CID fragment ions of substance P b10(2+); over 30% of the observed SORI-CID fragment ions from substance P b10(2+) had rearranged (scrambled) AA sequences. Moreover, post-IM/CID experiments revealed the presence of two conformer types for substance P b10(2+), whereas only one conformer type was observed for the head-to-tail cyclized peptide. We also show that AA sequence scrambling from CID of doubly-charged b-fragment ions is not unique to substance P b10(2+).

De Novo Sequencing and Analysis of Lemongrass Transcriptome Provide First Insights into the Essential Oil Biosynthesis of Aromatic Grasses

PubMed Central

Meena, Seema; Kumar, Sarma R.; Venkata Rao, D. K.; Dwivedi, Varun; Shilpashree, H. B.; Rastogi, Shubhra; Shasany, Ajit K.; Nagegowda, Dinesh A.

2016-01-01

Aromatic grasses of the genus Cymbopogon (Poaceae family) represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavor, fragrance, cosmetic, and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step toward understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass) by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases, pyrophosphatases, alcohol dehydrogenases, aldo-keto reductases, carotenoid cleavage dioxygenases, alcohol acetyltransferases, and aldehyde dehydrogenases, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type) with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes, and acetates. Molecular modeling and docking further supported the role of identified protein sequences in aroma formation in Cymbopogon. Also, simple sequence repeats were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition. PMID:27516768

A novel, privacy-preserving cryptographic approach for sharing sequencing data

PubMed Central

Cassa, Christopher A; Miller, Rachel A; Mandl, Kenneth D

2013-01-01

Objective DNA samples are often processed and sequenced in facilities external to the point of collection. These samples are routinely labeled with patient identifiers or pseudonyms, allowing for potential linkage to identity and private clinical information if intercepted during transmission. We present a cryptographic scheme to securely transmit externally generated sequence data which does not require any patient identifiers, public key infrastructure, or the transmission of passwords. Materials and methods This novel encryption scheme cryptographically protects participant sequence data using a shared secret key that is derived from a unique subset of an individual’s genetic sequence. This scheme requires access to a subset of an individual’s genetic sequence to acquire full access to the transmitted sequence data, which helps to prevent sample mismatch. Results We validate that the proposed encryption scheme is robust to sequencing errors, population uniqueness, and sibling disambiguation, and provides sufficient cryptographic key space. Discussion Access to a set of an individual’s genotypes and a mutually agreed cryptographic seed is needed to unlock the full sequence, which provides additional sample authentication and authorization security. We present modest fixed and marginal costs to implement this transmission architecture. Conclusions It is possible for genomics researchers who sequence participant samples externally to protect the transmission of sequence data using unique features of an individual’s genetic sequence. PMID:23125421

Evolution and molecular epidemiology of classical swine fever virus during a multi-annual outbreak amongst European wild boar.

PubMed

Goller, Katja V; Gabriel, Claudia; Dimna, Mireille Le; Le Potier, Marie-Frédérique; Rossi, Sophie; Staubach, Christoph; Merboth, Matthias; Beer, Martin; Blome, Sandra

2016-03-01

Classical swine fever is a viral disease of pigs that carries tremendous socio-economic impact. In outbreak situations, genetic typing is carried out for the purpose of molecular epidemiology in both domestic pigs and wild boar. These analyses are usually based on harmonized partial sequences. However, for high-resolution analyses towards the understanding of genetic variability and virus evolution, full-genome sequences are more appropriate. In this study, a unique set of representative virus strains was investigated that was collected during an outbreak in French free-ranging wild boar in the Vosges-du-Nord mountains between 2003 and 2007. Comparative sequence and evolutionary analyses of the nearly full-length sequences showed only slow evolution of classical swine fever virus strains over the years and no impact of vaccination on mutation rates. However, substitution rates varied amongst protein genes; furthermore, a spatial and temporal pattern could be observed whereby two separate clusters were formed that coincided with physical barriers.

Draft genome sequence of marine-derived Streptomyces sp. TP-A0598, a producer of anti-MRSA antibiotic lydicamycins.

PubMed

Komaki, Hisayuki; Ichikawa, Natsuko; Hosoyama, Akira; Fujita, Nobuyuki; Igarashi, Yasuhiro

2015-01-01

Streptomyces sp. TP-A0598, isolated from seawater, produces lydicamycin, structurally unique type I polyketide bearing two nitrogen-containing five-membered rings, and four congeners TPU-0037-A, -B, -C, and -D. We herein report the 8 Mb draft genome sequence of this strain, together with classification and features of the organism and generation, annotation and analysis of the genome sequence. The genome encodes 7,240 putative ORFs, of which 4,450 ORFs were assigned with COG categories. Also, 66 tRNA genes and one rRNA operon were identified. The genome contains eight gene clusters involved in the production of polyketides and nonribosomal peptides. Among them, a PKS/NRPS gene cluster was assigned to be responsible for lydicamycin biosynthesis and a plausible biosynthetic pathway was proposed on the basis of gene function prediction. This genome sequence data will facilitate to probe the potential of secondary metabolism in marine-derived Streptomyces.

The DNA sequence of the human X chromosome

PubMed Central

Ross, Mark T.; Grafham, Darren V.; Coffey, Alison J.; Scherer, Steven; McLay, Kirsten; Muzny, Donna; Platzer, Matthias; Howell, Gareth R.; Burrows, Christine; Bird, Christine P.; Frankish, Adam; Lovell, Frances L.; Howe, Kevin L.; Ashurst, Jennifer L.; Fulton, Robert S.; Sudbrak, Ralf; Wen, Gaiping; Jones, Matthew C.; Hurles, Matthew E.; Andrews, T. Daniel; Scott, Carol E.; Searle, Stephen; Ramser, Juliane; Whittaker, Adam; Deadman, Rebecca; Carter, Nigel P.; Hunt, Sarah E.; Chen, Rui; Cree, Andrew; Gunaratne, Preethi; Havlak, Paul; Hodgson, Anne; Metzker, Michael L.; Richards, Stephen; Scott, Graham; Steffen, David; Sodergren, Erica; Wheeler, David A.; Worley, Kim C.; Ainscough, Rachael; Ambrose, Kerrie D.; Ansari-Lari, M. Ali; Aradhya, Swaroop; Ashwell, Robert I. S.; Babbage, Anne K.; Bagguley, Claire L.; Ballabio, Andrea; Banerjee, Ruby; Barker, Gary E.; Barlow, Karen F.; Barrett, Ian P.; Bates, Karen N.; Beare, David M.; Beasley, Helen; Beasley, Oliver; Beck, Alfred; Bethel, Graeme; Blechschmidt, Karin; Brady, Nicola; Bray-Allen, Sarah; Bridgeman, Anne M.; Brown, Andrew J.; Brown, Mary J.; Bonnin, David; Bruford, Elspeth A.; Buhay, Christian; Burch, Paula; Burford, Deborah; Burgess, Joanne; Burrill, Wayne; Burton, John; Bye, Jackie M.; Carder, Carol; Carrel, Laura; Chako, Joseph; Chapman, Joanne C.; Chavez, Dean; Chen, Ellson; Chen, Guan; Chen, Yuan; Chen, Zhijian; Chinault, Craig; Ciccodicola, Alfredo; Clark, Sue Y.; Clarke, Graham; Clee, Chris M.; Clegg, Sheila; Clerc-Blankenburg, Kerstin; Clifford, Karen; Cobley, Vicky; Cole, Charlotte G.; Conquer, Jen S.; Corby, Nicole; Connor, Richard E.; David, Robert; Davies, Joy; Davis, Clay; Davis, John; Delgado, Oliver; DeShazo, Denise; Dhami, Pawandeep; Ding, Yan; Dinh, Huyen; Dodsworth, Steve; Draper, Heather; Dugan-Rocha, Shannon; Dunham, Andrew; Dunn, Matthew; Durbin, K. James; Dutta, Ireena; Eades, Tamsin; Ellwood, Matthew; Emery-Cohen, Alexandra; Errington, Helen; Evans, Kathryn L.; Faulkner, Louisa; Francis, Fiona; Frankland, John; Fraser, Audrey E.; Galgoczy, Petra; Gilbert, James; Gill, Rachel; Glöckner, Gernot; Gregory, Simon G.; Gribble, Susan; Griffiths, Coline; Grocock, Russell; Gu, Yanghong; Gwilliam, Rhian; Hamilton, Cerissa; Hart, Elizabeth A.; Hawes, Alicia; Heath, Paul D.; Heitmann, Katja; Hennig, Steffen; Hernandez, Judith; Hinzmann, Bernd; Ho, Sarah; Hoffs, Michael; Howden, Phillip J.; Huckle, Elizabeth J.; Hume, Jennifer; Hunt, Paul J.; Hunt, Adrienne R.; Isherwood, Judith; Jacob, Leni; Johnson, David; Jones, Sally; de Jong, Pieter J.; Joseph, Shirin S.; Keenan, Stephen; Kelly, Susan; Kershaw, Joanne K.; Khan, Ziad; Kioschis, Petra; Klages, Sven; Knights, Andrew J.; Kosiura, Anna; Kovar-Smith, Christie; Laird, Gavin K.; Langford, Cordelia; Lawlor, Stephanie; Leversha, Margaret; Lewis, Lora; Liu, Wen; Lloyd, Christine; Lloyd, David M.; Loulseged, Hermela; Loveland, Jane E.; Lovell, Jamieson D.; Lozado, Ryan; Lu, Jing; Lyne, Rachael; Ma, Jie; Maheshwari, Manjula; Matthews, Lucy H.; McDowall, Jennifer; McLaren, Stuart; McMurray, Amanda; Meidl, Patrick; Meitinger, Thomas; Milne, Sarah; Miner, George; Mistry, Shailesh L.; Morgan, Margaret; Morris, Sidney; Müller, Ines; Mullikin, James C.; Nguyen, Ngoc; Nordsiek, Gabriele; Nyakatura, Gerald; O’Dell, Christopher N.; Okwuonu, Geoffery; Palmer, Sophie; Pandian, Richard; Parker, David; Parrish, Julia; Pasternak, Shiran; Patel, Dina; Pearce, Alex V.; Pearson, Danita M.; Pelan, Sarah E.; Perez, Lesette; Porter, Keith M.; Ramsey, Yvonne; Reichwald, Kathrin; Rhodes, Susan; Ridler, Kerry A.; Schlessinger, David; Schueler, Mary G.; Sehra, Harminder K.; Shaw-Smith, Charles; Shen, Hua; Sheridan, Elizabeth M.; Shownkeen, Ratna; Skuce, Carl D.; Smith, Michelle L.; Sotheran, Elizabeth C.; Steingruber, Helen E.; Steward, Charles A.; Storey, Roy; Swann, R. Mark; Swarbreck, David; Tabor, Paul E.; Taudien, Stefan; Taylor, Tineace; Teague, Brian; Thomas, Karen; Thorpe, Andrea; Timms, Kirsten; Tracey, Alan; Trevanion, Steve; Tromans, Anthony C.; d’Urso, Michele; Verduzco, Daniel; Villasana, Donna; Waldron, Lenee; Wall, Melanie; Wang, Qiaoyan; Warren, James; Warry, Georgina L.; Wei, Xuehong; West, Anthony; Whitehead, Siobhan L.; Whiteley, Mathew N.; Wilkinson, Jane E.; Willey, David L.; Williams, Gabrielle; Williams, Leanne; Williamson, Angela; Williamson, Helen; Wilming, Laurens; Woodmansey, Rebecca L.; Wray, Paul W.; Yen, Jennifer; Zhang, Jingkun; Zhou, Jianling; Zoghbi, Huda; Zorilla, Sara; Buck, David; Reinhardt, Richard; Poustka, Annemarie; Rosenthal, André; Lehrach, Hans; Meindl, Alfons; Minx, Patrick J.; Hillier, LaDeana W.; Willard, Huntington F.; Wilson, Richard K.; Waterston, Robert H.; Rice, Catherine M.; Vaudin, Mark; Coulson, Alan; Nelson, David L.; Weinstock, George; Sulston, John E.; Durbin, Richard; Hubbard, Tim; Gibbs, Richard A.; Beck, Stephan; Rogers, Jane; Bentley, David R.

2009-01-01

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence. PMID:15772651

Massively parallel nanowell-based single-cell gene expression profiling.

PubMed

Goldstein, Leonard D; Chen, Ying-Jiun Jasmine; Dunne, Jude; Mir, Alain; Hubschle, Hermann; Guillory, Joseph; Yuan, Wenlin; Zhang, Jingli; Stinson, Jeremy; Jaiswal, Bijay; Pahuja, Kanika Bajaj; Mann, Ishminder; Schaal, Thomas; Chan, Leo; Anandakrishnan, Sangeetha; Lin, Chun-Wah; Espinoza, Patricio; Husain, Syed; Shapiro, Harris; Swaminathan, Karthikeyan; Wei, Sherry; Srinivasan, Maithreyan; Seshagiri, Somasekar; Modrusan, Zora

2017-07-07

Technological advances have enabled transcriptome characterization of cell types at the single-cell level providing new biological insights. New methods that enable simple yet high-throughput single-cell expression profiling are highly desirable. Here we report a novel nanowell-based single-cell RNA sequencing system, ICELL8, which enables processing of thousands of cells per sample. The system employs a 5,184-nanowell-containing microchip to capture ~1,300 single cells and process them. Each nanowell contains preprinted oligonucleotides encoding poly-d(T), a unique well barcode, and a unique molecular identifier. The ICELL8 system uses imaging software to identify nanowells containing viable single cells and only wells with single cells are processed into sequencing libraries. Here, we report the performance and utility of ICELL8 using samples of increasing complexity from cultured cells to mouse solid tissue samples. Our assessment of the system to discriminate between mixed human and mouse cells showed that ICELL8 has a low cell multiplet rate (< 3%) and low cross-cell contamination. We characterized single-cell transcriptomes of more than a thousand cultured human and mouse cells as well as 468 mouse pancreatic islets cells. We were able to identify distinct cell types in pancreatic islets, including alpha, beta, delta and gamma cells. Overall, ICELL8 provides efficient and cost-effective single-cell expression profiling of thousands of cells, allowing researchers to decipher single-cell transcriptomes within complex biological samples.

Identification of a Unique Fe-S Cluster Binding Site in a Glycyl-Radical Type Microcompartment Shell Protein

PubMed Central

Thompson, Michael C.; Wheatley, Nicole M.; Jorda, Julien; Sawaya, Michael R.; Gidaniyan, Soheil D.; Ahmed, Hoda; Yang, Zhongyu; McCarty, Krystal N.; Whitelegge, Julian P.; Yeates, Todd O.

2014-01-01

Recently, progress has been made toward understanding the functional diversity of bacterial microcompartment (MCP) systems, which serve as protein-based metabolic organelles in diverse microbes. New types of MCPs have been identified, including the glycyl-radical propanediol (Grp) MCP. Within these elaborate protein complexes, BMC-domain shell proteins assemble to form a polyhedral barrier that encapsulates the enzymatic contents of the MCP. Interestingly, the Grp MCP contains a number of shell proteins with unusual sequence features. GrpU is one such shell protein, whose amino acid sequence is particularly divergent from other members of the BMC-domain superfamily of proteins that effectively defines all MCPs. Expression, purification, and subsequent characterization of the protein showed, unexpectedly, that it binds an iron-sulfur cluster. We determined X-ray crystal structures of two GrpU orthologs, providing the first structural insight into the homohexameric BMC-domain shell proteins of the Grp system. The X-ray structures of GrpU, both obtained in the apo form, combined with spectroscopic analyses and computational modeling, show that the metal cluster resides in the central pore of the BMC shell protein at a position of broken 6-fold symmetry. The result is a structurally polymorphic iron-sulfur cluster binding site that appears to be unique among metalloproteins studied to date. PMID:25102080

Cloning, analysis and functional annotation of expressed sequence tags from the Earthworm Eisenia fetida

PubMed Central

Pirooznia, Mehdi; Gong, Ping; Guan, Xin; Inouye, Laura S; Yang, Kuan; Perkins, Edward J; Deng, Youping

2007-01-01

Background Eisenia fetida, commonly known as red wiggler or compost worm, belongs to the Lumbricidae family of the Annelida phylum. Little is known about its genome sequence although it has been extensively used as a test organism in terrestrial ecotoxicology. In order to understand its gene expression response to environmental contaminants, we cloned 4032 cDNAs or expressed sequence tags (ESTs) from two E. fetida libraries enriched with genes responsive to ten ordnance related compounds using suppressive subtractive hybridization-PCR. Results A total of 3144 good quality ESTs (GenBank dbEST accession number EH669363–EH672369 and EL515444–EL515580) were obtained from the raw clone sequences after cleaning. Clustering analysis yielded 2231 unique sequences including 448 contigs (from 1361 ESTs) and 1783 singletons. Comparative genomic analysis showed that 743 or 33% of the unique sequences shared high similarity with existing genes in the GenBank nr database. Provisional function annotation assigned 830 Gene Ontology terms to 517 unique sequences based on their homology with the annotated genomes of four model organisms Drosophila melanogaster, Mus musculus, Saccharomyces cerevisiae, and Caenorhabditis elegans. Seven percent of the unique sequences were further mapped to 99 Kyoto Encyclopedia of Genes and Genomes pathways based on their matching Enzyme Commission numbers. All the information is stored and retrievable at a highly performed, web-based and user-friendly relational database called EST model database or ESTMD version 2. Conclusion The ESTMD containing the sequence and annotation information of 4032 E. fetida ESTs is publicly accessible at . PMID:18047730

Gut microbial profile analysis by MiSeq sequencing of pancreatic carcinoma patients in China

PubMed Central

Xie, Haiyang; Li, Ang; Lu, Haifeng; Xu, Shaoyan; Zhou, Lin; Zhang, Hua; Cui, Guangying; Chen, Xinhua; Liu, Yuanxing; Wu, Liming; Qin, Nan; Sun, Ranran; Wang, Wei; Li, Lanjuan; Wang, Weilin; Zheng, Shusen

2017-01-01

Pancreatic carcinoma (PC) is a lethal cancer. Gut microbiota is associated with some risk factors of PC, e.g. obesity and types II diabetes. However, the specific gut microbial profile in clinical PC in China has never been reported. This prospective study collected 85 PC and 57 matched healthy controls (HC) to analyze microbial characteristics by MiSeq sequencing. The results showed that gut microbial diversity was decreased in PC with an unique microbial profile, which partly attributed to its decrease of alpha diversity. Microbial alterations in PC featured by the increase of certain pathogens and lipopolysaccharides-producing bacteria, and the decrease of probiotics and butyrate-producing bacteria. Microbial community in obstruction cases was separated from the un-obstructed cases. Streptococcus was associated with the bile. Furthermore, 23 microbial functions e.g. Leucine and LPS biosynthesis were enriched, while 13 functions were reduced in PC. Importantly, based on 40 genera associated with PC, microbial markers achieves a high classification power with AUC of 0.842. In conclusion, gut microbial profile was unique in PC, providing a microbial marker for non-invasive PC diagnosis. PMID:29221120

Allen Brain Atlas: an integrated spatio-temporal portal for exploring the central nervous system

PubMed Central

Sunkin, Susan M.; Ng, Lydia; Lau, Chris; Dolbeare, Tim; Gilbert, Terri L.; Thompson, Carol L.; Hawrylycz, Michael; Dang, Chinh

2013-01-01

The Allen Brain Atlas (http://www.brain-map.org) provides a unique online public resource integrating extensive gene expression data, connectivity data and neuroanatomical information with powerful search and viewing tools for the adult and developing brain in mouse, human and non-human primate. Here, we review the resources available at the Allen Brain Atlas, describing each product and data type [such as in situ hybridization (ISH) and supporting histology, microarray, RNA sequencing, reference atlases, projection mapping and magnetic resonance imaging]. In addition, standardized and unique features in the web applications are described that enable users to search and mine the various data sets. Features include both simple and sophisticated methods for gene searches, colorimetric and fluorescent ISH image viewers, graphical displays of ISH, microarray and RNA sequencing data, Brain Explorer software for 3D navigation of anatomy and gene expression, and an interactive reference atlas viewer. In addition, cross data set searches enable users to query multiple Allen Brain Atlas data sets simultaneously. All of the Allen Brain Atlas resources can be accessed through the Allen Brain Atlas data portal. PMID:23193282

Properties of an equine herpesvirus 1 mutant devoid of the internal inverted repeat sequence of the genomic short region

PubMed Central

Ahn, ByungChul; Zhang, Yunfei; Osterrieder, Nikolaus; O'Callaghan, Dennis J.

2010-01-01

The 150 kbp genome of equine herpesvirus -1 (EHV-1) is composed of a unique long (UL) region and a unique short (Us) segment, which is flanked by identical internal and terminal repeat (IR and TR) sequences of 12.7kbp. We constructed an EHV-1 lacking the entire IR (vL11ΔIR) and showed that the IR is dispensable for EHV-1 replication but that the vL11ΔIR exhibits a smaller plaque size and delayed growth kinetics. Western blot analyses of cells infected with vL11ΔIR showed that the synthesis of viral proteins encoded by the immediate-early, early, and late genes was reduced at immediate-early and early times, but by late stages of replication reached wild type levels. Intranasal infection of CBA mice revealed that the vL11ΔIR was significantly attenuated as mice infected with the vL11ΔIR showed a reduced lung viral titer and greater ability to survive infection compared to mice infected with parental or revertant virus. PMID:21176938

Merging mythology and morphology: the multifaceted lifestyle of Proteus mirabilis.

PubMed

Armbruster, Chelsie E; Mobley, Harry L T

2012-11-01

Proteus mirabilis, named for the Greek god who changed shape to avoid capture, has fascinated microbiologists for more than a century with its unique swarming differentiation, Dienes line formation and potent urease activity. Transcriptome profiling during both host infection and swarming motility, coupled with the availability of the complete genome sequence for P. mirabilis, has revealed the occurrence of interbacterial competition and killing through a type VI secretion system, and the reciprocal regulation of adhesion and motility, as well as the intimate connections between metabolism, swarming and virulence. This Review addresses some of the unique and recently described aspects of P. mirabilis biology and pathogenesis, and emphasizes the potential role of this bacterium in single-species and polymicrobial urinary tract infections.

Merging mythology and morphology: the multifaceted lifestyle of Proteus mirabilis

PubMed Central

Armbruster, Chelsie E.; Mobley, Harry L. T.

2013-01-01

Proteus mirabilis, named for the Greek god who changed shape to avoid capture, has fascinated microbiologists for more than a century with its unique swarming differentiation, Dienes line formation and potent urease activity. Transcriptome profiling during both host infection and swarming motility, coupled with the availability of the complete genome sequence for P. mirabilis, has revealed the occurrence of interbacterial competition and killing through a type VI secretion system, and the reciprocal regulation of adhesion and motility, as well as the intimate connections between metabolism, swarming and virulence. This Review addresses some of the unique and recently described aspects of P. mirabilis biology and pathogenesis, and emphasizes the potential role of this bacterium in single- species and polymicrobial urinary tract infections. PMID:23042564

Non-coding RNA networks in cancer.

PubMed

Anastasiadou, Eleni; Jacob, Leni S; Slack, Frank J

2018-01-01

Thousands of unique non-coding RNA (ncRNA) sequences exist within cells. Work from the past decade has altered our perception of ncRNAs from 'junk' transcriptional products to functional regulatory molecules that mediate cellular processes including chromatin remodelling, transcription, post-transcriptional modifications and signal transduction. The networks in which ncRNAs engage can influence numerous molecular targets to drive specific cell biological responses and fates. Consequently, ncRNAs act as key regulators of physiological programmes in developmental and disease contexts. Particularly relevant in cancer, ncRNAs have been identified as oncogenic drivers and tumour suppressors in every major cancer type. Thus, a deeper understanding of the complex networks of interactions that ncRNAs coordinate would provide a unique opportunity to design better therapeutic interventions.

Genetic and Chemical Profiling of Gymnema sylvestre Accessions from Central India: Its Implication for Quality Control and Therapeutic Potential of Plant

PubMed Central

Verma, Ashutosh Kumar; Dhawan, Sunita Singh; Singh, Seema; Bharati, Kumar Avinash; Jyotsana

2016-01-01

Background: Gymnema sylvestre, a vulnerable plant species, is mentioned in Indian Pharmacopeia as an antidiabetic drug Objective: Study of genetic and chemical diversity and its implications in accessions of G. sylvestre Materials and Methods: Fourteen accessions of G. sylvestre collected from Central India and assessment of their genetic and chemical diversity were carried out using ISSR (inter simple sequence repeat) and HPLC (high performance liquid chromatography) fingerprinting methods Results: Among the screened 40 ISSR primers, 15 were found polymorphic and collectively produced nine unique accession-specific bands. The maximum and minimum numbers of amplicones were noted for ISSR-15 and ISSR-11, respectively. The ISSR -11 and ISSR-13 revealed 100% polymorphism. HPLC chromatograms showed that accessions possess the secondary metabolites of mid-polarity with considerable variability. Unknown peaks with retention time 2.63, 3.41, 23.83, 24.50, and 44.67 were found universal type. Comparative hierarchical clustering analysis based on foresaid fingerprints indicates that both techniques have equal potential to discriminate accessions according to percentage gymnemic acid in their leaf tissue. Second approach was noted more efficiently for separation of accessions according to their agro-climatic/collection site Conclusion: Highly polymorphic ISSRs could be utilized as molecular probes for further selection of high gymnemic acid yielding accessions. Observed accession specific bands may be used as a descriptor for plant accessions protection and converted into sequence tagged sites markers. Identified five universal type peaks could be helpful in identification of G. sylvestre-based various herbal preparations. SUMMARY Nine accession specific unique bandsFive marker peaks for G. sylvestre.Suitability of genetic and chemical fingerprinting Abbreviations used: HPLC: High Performance Liquid Chromatography, ISSR: Inter Simple Sequence Repeats, CTAB: Cetyl Trimethylammonium Bromide, DNTP: Deoxynucleotide Triphosphates PMID:27761067

Genetic and Chemical Profiling of Gymnema sylvestre Accessions from Central India: Its Implication for Quality Control and Therapeutic Potential of Plant.

PubMed

Verma, Ashutosh Kumar; Dhawan, Sunita Singh; Singh, Seema; Bharati, Kumar Avinash; Jyotsana

2016-07-01

Gymnema sylvestre , a vulnerable plant species, is mentioned in Indian Pharmacopeia as an antidiabetic drug. Study of genetic and chemical diversity and its implications in accessions of G. sylvestre . Fourteen accessions of G. sylvestre collected from Central India and assessment of their genetic and chemical diversity were carried out using ISSR (inter simple sequence repeat) and HPLC (high performance liquid chromatography) fingerprinting methods. Among the screened 40 ISSR primers, 15 were found polymorphic and collectively produced nine unique accession-specific bands. The maximum and minimum numbers of amplicones were noted for ISSR-15 and ISSR-11, respectively. The ISSR -11 and ISSR-13 revealed 100% polymorphism. HPLC chromatograms showed that accessions possess the secondary metabolites of mid-polarity with considerable variability. Unknown peaks with retention time 2.63, 3.41, 23.83, 24.50, and 44.67 were found universal type. Comparative hierarchical clustering analysis based on foresaid fingerprints indicates that both techniques have equal potential to discriminate accessions according to percentage gymnemic acid in their leaf tissue. Second approach was noted more efficiently for separation of accessions according to their agro-climatic/collection site. Highly polymorphic ISSRs could be utilized as molecular probes for further selection of high gymnemic acid yielding accessions. Observed accession specific bands may be used as a descriptor for plant accessions protection and converted into sequence tagged sites markers. Identified five universal type peaks could be helpful in identification of G. sylvestre -based various herbal preparations. Nine accession specific unique bandsFive marker peaks for G. sylvestre .Suitability of genetic and chemical fingerprinting Abbreviations used: HPLC: High Performance Liquid Chromatography, ISSR: Inter Simple Sequence Repeats, CTAB: Cetyl Trimethylammonium Bromide, DNTP: Deoxynucleotide Triphosphates.

[Clinical value of MRI united-sequences examination in diagnosis and differentiation of morphological sub-type of hilar and extrahepatic big bile duct cholangiocarcinoma].

PubMed

Yin, Long-Lin; Song, Bin; Guan, Ying; Li, Ying-Chun; Chen, Guang-Wen; Zhao, Li-Ming; Lai, Li

2014-09-01

To investigate MRI features and associated histological and pathological changes of hilar and extrahepatic big bile duct cholangiocarcinoma with different morphological sub-types, and its value in differentiating between nodular cholangiocarcinoma (NCC) and intraductal growing cholangiocarcinoma (IDCC). Imaging data of 152 patients with pathologically confirmed hilar and extrahepatic big bile duct cholangiocarcinoma were reviewed, which included 86 periductal infiltrating cholangiocarcinoma (PDCC), 55 NCC, and 11 IDCC. Imaging features of the three morphological sub-types were compared. Each of the subtypes demonstrated its unique imaging features. Significant differences (P < 0.05) were found between NCC and IDCC in tumor shape, dynamic enhanced pattern, enhancement degree during equilibrium phase, multiplicity or singleness of tumor, changes in wall and lumen of bile duct at the tumor-bearing segment, dilatation of tumor upstream or downstream bile duct, and invasion of adjacent organs. Imaging features reveal tumor growth patterns of hilar and extrahepatic big bile duct cholangiocarcinoma. MRI united-sequences examination can accurately describe those imaging features for differentiation diagnosis.

Novel LRPPRC Mutation in a Boy With Mild Leigh Syndrome, French-Canadian Type Outside of Québec.

PubMed

Han, Velda Xinying; Tan, Teresa S; Wang, Furene S; Tay, Stacey Kiat-Hong

2017-01-01

Leigh syndrome, French-Canadian type is unique to patients from a genetic isolate in the Saguenay-Lac-Saint-Jean region of Québec. It has also been recently described in 10 patients with LRPPRC mutation outside of Québec. It is an autosomal recessive genetic disorder with fatal metabolic crisis and severe neurological morbidity in infancy caused by LRPPRC mutation. The authors report a boy with a novel LRPPRC compound heterozygous missense mutations c.3130C>T, c.3430C>T, and c.4078G>A found on whole-exome sequencing which correlated with isolated cytochrome c-oxidase deficiency found in skeletal muscle. LRPPRC mutation is a rare cause of cytochrome c-oxidase-deficient form of Leigh syndrome outside of Québec. Our patient broadens the spectrum of phenotypes of Leigh syndrome, French-Canadian type. LRPPRC mutation should be considered in children with early childhood neurodegenerative disorder, even in the absence of metabolic crisis. Early evaluation with whole-exome sequencing is useful for early diagnosis and for genetic counseling.

Signatures from Tissue-specific MPSS Libraries Identify Transcripts Preferentially Expressed in the Mouse Inner Ear

PubMed Central

Peters, Linda M.; Belyantseva, Inna A.; Lagziel, Ayala; Battey, James F.; Friedman, Thomas B.; Morell, Robert J.

2007-01-01

Specialization in cell function and morphology is influenced by the differential expression of mRNAs, many of which are expressed at low abundance and restricted to certain cell types. Detecting such transcripts in cDNA libraries may require sequencing millions of clones. Massively parallel signature sequencing (MPSS) is well-suited for identifying transcripts that are expressed in discrete cell types and in low abundance. We have made MPSS libraries from microdissections of three inner ear tissues. By comparing these MPSS libraries to those of 87 other tissues included in the Mouse Reference Transcriptome (MRT) online resource, we have identified genes that are highly enriched in, or specific to, the inner ear. We show by RT-PCR and in situ hybridization that signatures unique to the inner ear libraries identify transcripts with highly specific cell-type localizations. These transcripts serve to illustrate the utility of a resource that is available to the research community. Utilization of these resources will increase the number of known transcription units and expand our knowledge of the tissue-specific regulation of the transcriptome. PMID:17049805

Full-length VP2 gene analysis of canine parvovirus reveals emergence of newer variants in India.

PubMed

Nookala, Mangadevi; Mukhopadhyay, Hirak Kumar; Sivaprakasam, Amsaveni; Balasubramanian, Brindhalakshmi; Antony, Prabhakar Xavier; Thanislass, Jacob; Srinivas, Mouttou Vivek; Pillai, Raghavan Madhusoodanan

2016-12-01

The canine parvovirus (CPV) infection is a highly contagious and serious enteric disease of dogs with high fatality rate. The present study was taken up to characterize the full-length viral polypeptide 2 (VP2) gene of CPV of Indian origin along with the commercially available vaccines. The faecal samples from parvovirus suspected dogs were collected from various states of India for screening by PCR assay and 66.29% of samples were found positive. Six CPV-2a, three CPV-2b, and one CPV-2c types were identified by sequence analysis. Several unique and existing mutations have been noticed in CPV types analyzed indicating emergence of newer variants of CPV in India. The phylogenetic analysis revealed that all the field CPV types were grouped in different subclades within two main clades, but away from the commercial vaccine strains. CPV-2b and CPV-2c types with unique mutations were found to be establishing in India apart from the prevailing CPV-2a type. Mutations and the positive selection of the mutants were found to be the major mechanism of emergence and evolution of parvovirus. Therefore, the incorporation of local strain in the vaccine formulation may be considered for effective control of CPV infections in India.

Genome Sequence of a Canadian Vibrio parahaemolyticus Isolate with Unique Mobilizing Capacity.

PubMed

Bioteau, Audrey; Huguet, Kévin; Burrus, Vincent; Banerjee, Swapan

2018-06-14

Vibrio parahaemolyticus is a clinically significant marine bacterium implicated in gastroenteritis among consumers of raw or undercooked seafood. This report presents the whole-genome sequence of a unique strain of V. parahaemolyticus isolated from oysters harvested in Canada. © Crown copyright 2018.

Computational and experimental analysis of DNA shuffling

PubMed Central

Maheshri, Narendra; Schaffer, David V.

2003-01-01

We describe a computational model of DNA shuffling based on the thermodynamics and kinetics of this process. The model independently tracks a representative ensemble of DNA molecules and records their states at every stage of a shuffling reaction. These data can subsequently be analyzed to yield information on any relevant metric, including reassembly efficiency, crossover number, type and distribution, and DNA sequence length distributions. The predictive ability of the model was validated by comparison to three independent sets of experimental data, and analysis of the simulation results led to several unique insights into the DNA shuffling process. We examine a tradeoff between crossover frequency and reassembly efficiency and illustrate the effects of experimental parameters on this relationship. Furthermore, we discuss conditions that promote the formation of useless “junk” DNA sequences or multimeric sequences containing multiple copies of the reassembled product. This model will therefore aid in the design of optimal shuffling reaction conditions. PMID:12626764

Universal sequence map (USM) of arbitrary discrete sequences

PubMed Central

2002-01-01

Background For over a decade the idea of representing biological sequences in a continuous coordinate space has maintained its appeal but not been fully realized. The basic idea is that any sequence of symbols may define trajectories in the continuous space conserving all its statistical properties. Ideally, such a representation would allow scale independent sequence analysis – without the context of fixed memory length. A simple example would consist on being able to infer the homology between two sequences solely by comparing the coordinates of any two homologous units. Results We have successfully identified such an iterative function for bijective mappingψ of discrete sequences into objects of continuous state space that enable scale-independent sequence analysis. The technique, named Universal Sequence Mapping (USM), is applicable to sequences with an arbitrary length and arbitrary number of unique units and generates a representation where map distance estimates sequence similarity. The novel USM procedure is based on earlier work by these and other authors on the properties of Chaos Game Representation (CGR). The latter enables the representation of 4 unit type sequences (like DNA) as an order free Markov Chain transition table. The properties of USM are illustrated with test data and can be verified for other data by using the accompanying web-based tool:http://bioinformatics.musc.edu/~jonas/usm/. Conclusions USM is shown to enable a statistical mechanics approach to sequence analysis. The scale independent representation frees sequence analysis from the need to assume a memory length in the investigation of syntactic rules. PMID:11895567

Sequence quality analysis tool for HIV type 1 protease and reverse transcriptase.

PubMed

Delong, Allison K; Wu, Mingham; Bennett, Diane; Parkin, Neil; Wu, Zhijin; Hogan, Joseph W; Kantor, Rami

2012-08-01

Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature ( http://hivdb.Stanford.edu ). Nucleic acid sequences are read into SQUAT, identified, aligned, and translated. Nucleic acid sequences are flagged if with >five 1-2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations; >zero unique amino acids; or <0.5% or >15% genetic distance from another submitted sequence. Thresholds are user modifiable. SQUAT output includes a summary report with detailed comments for troubleshooting of flagged sequences, histograms of pairwise genetic distances, neighbor joining phylogenetic trees, and aligned nucleic and amino acid sequences. SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences.

A 28,000 Years Old Cro-Magnon mtDNA Sequence Differs from All Potentially Contaminating Modern Sequences

PubMed Central

Caramelli, David; Milani, Lucio; Vai, Stefania; Modi, Alessandra; Pecchioli, Elena; Girardi, Matteo; Pilli, Elena; Lari, Martina; Lippi, Barbara; Ronchitelli, Annamaria; Mallegni, Francesco; Casoli, Antonella; Bertorelle, Giorgio; Barbujani, Guido

2008-01-01

Background DNA sequences from ancient speciments may in fact result from undetected contamination of the ancient specimens by modern DNA, and the problem is particularly challenging in studies of human fossils. Doubts on the authenticity of the available sequences have so far hampered genetic comparisons between anatomically archaic (Neandertal) and early modern (Cro-Magnoid) Europeans. Methodology/Principal Findings We typed the mitochondrial DNA (mtDNA) hypervariable region I in a 28,000 years old Cro-Magnoid individual from the Paglicci cave, in Italy (Paglicci 23) and in all the people who had contact with the sample since its discovery in 2003. The Paglicci 23 sequence, determined through the analysis of 152 clones, is the Cambridge reference sequence, and cannot possibly reflect contamination because it differs from all potentially contaminating modern sequences. Conclusions/Significance: The Paglicci 23 individual carried a mtDNA sequence that is still common in Europe, and which radically differs from those of the almost contemporary Neandertals, demonstrating a genealogical continuity across 28,000 years, from Cro-Magnoid to modern Europeans. Because all potential sources of modern DNA contamination are known, the Paglicci 23 sample will offer a unique opportunity to get insight for the first time into the nuclear genes of early modern Europeans. PMID:18628960

Sequencing historical specimens: successful preparation of small specimens with low amounts of degraded DNA.

PubMed

Sproul, John S; Maddison, David R

2017-11-01

Despite advances that allow DNA sequencing of old museum specimens, sequencing small-bodied, historical specimens can be challenging and unreliable as many contain only small amounts of fragmented DNA. Dependable methods to sequence such specimens are especially critical if the specimens are unique. We attempt to sequence small-bodied (3-6 mm) historical specimens (including nomenclatural types) of beetles that have been housed, dried, in museums for 58-159 years, and for which few or no suitable replacement specimens exist. To better understand ideal approaches of sample preparation and produce preparation guidelines, we compared different library preparation protocols using low amounts of input DNA (1-10 ng). We also explored low-cost optimizations designed to improve library preparation efficiency and sequencing success of historical specimens with minimal DNA, such as enzymatic repair of DNA. We report successful sample preparation and sequencing for all historical specimens despite our low-input DNA approach. We provide a list of guidelines related to DNA repair, bead handling, reducing adapter dimers and library amplification. We present these guidelines to facilitate more economical use of valuable DNA and enable more consistent results in projects that aim to sequence challenging, irreplaceable historical specimens. © 2017 John Wiley & Sons Ltd.

Broad HPV distribution in the genital region of men from the HPV infection in men (HIM) study.

PubMed

Sichero, Laura; Pierce Campbell, Christine M; Ferreira, Silvaneide; Sobrinho, João S; Luiza Baggio, Maria; Galan, Lenice; Silva, Roberto C; Lazcano-Ponce, Eduardo; Giuliano, Anna R; Villa, Luisa L

2013-09-01

The HPV infection in men (HIM) study examines the natural history of genital HPV infection in men. Genotyping methods used in this study identify 37 α-HPV types; however, the viral type could not be identified in approximately 22% of male genital specimens that were HPV PCR positive. Our aim was to genotype HPV-unclassified specimens by sequencing PGMY09/11, GP5+/6+ or FAP59/64 PCR products. Using this approach we were able to detect 86 unique HPV types among 508 of 931 specimens analyzed. We report for the first time the presence of a broad range of α-, β- and γ-HPV at the male genitals. Copyright © 2013 Elsevier Inc. All rights reserved.

Discovery and mechanistic study of a class of protein arginine methylation inhibitors.

PubMed

Feng, You; Li, Mingyong; Wang, Binghe; Zheng, Yujun George

2010-08-26

Protein arginine methylation regulates multiple biological processes such as chromatin remodeling and RNA splicing. Malfunction of protein arginine methyltransferases (PRMTs) is correlated with many human diseases. Thus, small molecule inhibitors of protein arginine methylation are of great potential for therapeutic development. Herein, we report a type of compound that blocks PRMT1-mediated arginine methylation at micromolar potency through a unique mechanism. Most of the discovered compounds bear naphthalene and sulfonate groups and are structurally different from typical PRMT substrates, for example, histone H4 and glycine- and arginine-rich sequences. To elucidate the molecular basis of inhibition, we conducted a variety of kinetic and biophysical assays. The combined data reveal that this type of naphthyl-sulfo (NS) molecule directly targets the substrates but not PRMTs for the observed inhibition. We also found that suramin effectively inhibited PRMT1 activity. These findings about novel PRMT inhibitors and their unique inhibition mechanism provide a new way for chemical regulation of protein arginine methylation.

Cell type transcriptome atlas for the planarian Schmidtea mediterranea.

PubMed

Fincher, Christopher T; Wurtzel, Omri; de Hoog, Thom; Kravarik, Kellie M; Reddien, Peter W

2018-05-25

The transcriptome of a cell dictates its unique cell type biology. We used single-cell RNA sequencing to determine the transcriptomes for essentially every cell type of a complete animal: the regenerative planarian Schmidtea mediterranea. Planarians contain a diverse array of cell types, possess lineage progenitors for differentiated cells (including pluripotent stem cells), and constitutively express positional information, making them ideal for this undertaking. We generated data for 66,783 cells, defining transcriptomes for known and many previously unknown planarian cell types and for putative transition states between stem and differentiated cells. We also uncovered regionally expressed genes in muscle, which harbors positional information. Identifying the transcriptomes for potentially all cell types for many organisms should be readily attainable and represents a powerful approach to metazoan biology. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Transcriptomic sequencing reveals a set of unique genes activated by butyrate-induced histone modification

USDA-ARS?s Scientific Manuscript database

Butyrate is a nutritional element with strong epigenetic regulatory activity as an inhibitor of histone deacetylases (HDACs). Based on the analysis of differentially expressed genes induced by butyrate in the bovine epithelial cell using deep RNA-sequencing technology (RNA-seq), a set of unique gen...

TagDust2: a generic method to extract reads from sequencing data.

PubMed

Lassmann, Timo

2015-01-28

Arguably the most basic step in the analysis of next generation sequencing data (NGS) involves the extraction of mappable reads from the raw reads produced by sequencing instruments. The presence of barcodes, adaptors and artifacts subject to sequencing errors makes this step non-trivial. Here I present TagDust2, a generic approach utilizing a library of hidden Markov models (HMM) to accurately extract reads from a wide array of possible read architectures. TagDust2 extracts more reads of higher quality compared to other approaches. Processing of multiplexed single, paired end and libraries containing unique molecular identifiers is fully supported. Two additional post processing steps are included to exclude known contaminants and filter out low complexity sequences. Finally, TagDust2 can automatically detect the library type of sequenced data from a predefined selection. Taken together TagDust2 is a feature rich, flexible and adaptive solution to go from raw to mappable NGS reads in a single step. The ability to recognize and record the contents of raw reads will help to automate and demystify the initial, and often poorly documented, steps in NGS data analysis pipelines. TagDust2 is freely available at: http://tagdust.sourceforge.net .

Ultraviolet spectral morphology of the O stars. IV - The OB supergiant sequence

NASA Technical Reports Server (NTRS)

Walborn, Nolan R.; Nichols-Bohlin, Joy

1987-01-01

An atlas of 25 O3-B8 supergiant spectra in the wavelength ranges 1320-1580 A and 1620-1880 A is presented, based on high-resolution data from the IUE archives. The remarkably detailed relationship between the stellar-wind profiles and the optical spectral classifications throughout this sequence is emphasized. For instance, the (Si IV)/(C IV) ratio reverses between O4 and O6.5; and the B0, B0.5, and B0.7 Ia wind characteristics are each qualitatively unique and distinct from one another. The systematic behavior of nine stellar-wind features with ionization potentials ranging from 114 to 19 eV is summarized as a function of advancing spectral type.

A novel monoclonal Perkinsus chesapeaki in vitro isolate from an Australian cockle, Anadara trapezia.

PubMed

Reece, Kimberly S; Scott, Gail P; Dang, Cécile; Dungan, Christopher F

2017-09-01

A monoclonal Perkinsus chesapeaki isolate was established from 1 of 10 infected Australian Anadara trapezia cockles. Morphological features were similar to those of described P. chesapeaki isolates, and also included a unique vermiform schizont cell-type. Perkinsus olseni-specific PCR primers amplified DNAs from all 10 cockles. Perkinsus chesapeaki-specific primers also amplified DNAs from 4/10 cockles, including DNA from the isolate source cockle. Three different sets of DNA sequences from the monoclonal isolate grouped with the homologous, previously deposited, P. chesapeaki sequences in phylogenetic analyses. In situ hybridization assays detected both P. chesapeaki and P. olseni cells in histological sections from the source cockle for monoclonal isolate ATCC PRA-425. Copyright © 2017 Elsevier Inc. All rights reserved.

Defining the healthy "core microbiome" of oral microbial communities

PubMed Central

2009-01-01

Background Most studies examining the commensal human oral microbiome are focused on disease or are limited in methodology. In order to diagnose and treat diseases at an early and reversible stage an in-depth definition of health is indispensible. The aim of this study therefore was to define the healthy oral microbiome using recent advances in sequencing technology (454 pyrosequencing). Results We sampled and sequenced microbiomes from several intraoral niches (dental surfaces, cheek, hard palate, tongue and saliva) in three healthy individuals. Within an individual oral cavity, we found over 3600 unique sequences, over 500 different OTUs or "species-level" phylotypes (sequences that clustered at 3% genetic difference) and 88 - 104 higher taxa (genus or more inclusive taxon). The predominant taxa belonged to Firmicutes (genus Streptococcus, family Veillonellaceae, genus Granulicatella), Proteobacteria (genus Neisseria, Haemophilus), Actinobacteria (genus Corynebacterium, Rothia, Actinomyces), Bacteroidetes (genus Prevotella, Capnocytophaga, Porphyromonas) and Fusobacteria (genus Fusobacterium). Each individual sample harboured on average 266 "species-level" phylotypes (SD 67; range 123 - 326) with cheek samples being the least diverse and the dental samples from approximal surfaces showing the highest diversity. Principal component analysis discriminated the profiles of the samples originating from shedding surfaces (mucosa of tongue, cheek and palate) from the samples that were obtained from solid surfaces (teeth). There was a large overlap in the higher taxa, "species-level" phylotypes and unique sequences among the three microbiomes: 84% of the higher taxa, 75% of the OTUs and 65% of the unique sequences were present in at least two of the three microbiomes. The three individuals shared 1660 of 6315 unique sequences. These 1660 sequences (the "core microbiome") contributed 66% of the reads. The overlapping OTUs contributed to 94% of the reads, while nearly all reads (99.8%) belonged to the shared higher taxa. Conclusions We obtained the first insight into the diversity and uniqueness of individual oral microbiomes at a resolution of next-generation sequencing. We showed that a major proportion of bacterial sequences of unrelated healthy individuals is identical, supporting the concept of a core microbiome at health. PMID:20003481

Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes.

PubMed

Albrechtsen, A; Grarup, N; Li, Y; Sparsø, T; Tian, G; Cao, H; Jiang, T; Kim, S Y; Korneliussen, T; Li, Q; Nie, C; Wu, R; Skotte, L; Morris, A P; Ladenvall, C; Cauchi, S; Stančáková, A; Andersen, G; Astrup, A; Banasik, K; Bennett, A J; Bolund, L; Charpentier, G; Chen, Y; Dekker, J M; Doney, A S F; Dorkhan, M; Forsen, T; Frayling, T M; Groves, C J; Gui, Y; Hallmans, G; Hattersley, A T; He, K; Hitman, G A; Holmkvist, J; Huang, S; Jiang, H; Jin, X; Justesen, J M; Kristiansen, K; Kuusisto, J; Lajer, M; Lantieri, O; Li, W; Liang, H; Liao, Q; Liu, X; Ma, T; Ma, X; Manijak, M P; Marre, M; Mokrosiński, J; Morris, A D; Mu, B; Nielsen, A A; Nijpels, G; Nilsson, P; Palmer, C N A; Rayner, N W; Renström, F; Ribel-Madsen, R; Robertson, N; Rolandsson, O; Rossing, P; Schwartz, T W; Slagboom, P E; Sterner, M; Tang, M; Tarnow, L; Tuomi, T; van't Riet, E; van Leeuwen, N; Varga, T V; Vestmar, M A; Walker, M; Wang, B; Wang, Y; Wu, H; Xi, F; Yengo, L; Yu, C; Zhang, X; Zhang, J; Zhang, Q; Zhang, W; Zheng, H; Zhou, Y; Altshuler, D; 't Hart, L M; Franks, P W; Balkau, B; Froguel, P; McCarthy, M I; Laakso, M; Groop, L; Christensen, C; Brandslund, I; Lauritzen, T; Witte, D R; Linneberg, A; Jørgensen, T; Hansen, T; Wang, J; Nielsen, R; Pedersen, O

2013-02-01

Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m(2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case-control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans. Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10(-14)), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10(-11)) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10(-10)). We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits.

Differentiation of highly virulent strains of Streptococcus suis serotype 2 according to glutamate dehydrogenase electrophoretic and sequence type.

PubMed

Kutz, Russell; Okwumabua, Ogi

2008-10-01

The glutamate dehydrogenase (GDH) enzymes of 19 Streptococcus suis serotype 2 strains, consisting of 18 swine isolates and 1 human clinical isolate from a geographically varied collection, were analyzed by activity staining on a nondenaturing gel. All seven (100%) of the highly virulent strains tested produced an electrophoretic type (ET) distinct from those of moderately virulent and nonvirulent strains. By PCR and nucleotide sequence determination, the gdh genes of the 19 strains and of 2 highly virulent strains involved in recent Chinese outbreaks yielded a 1,820-bp fragment containing an open reading frame of 1,344 nucleotides, which encodes a protein of 448 amino acid residues with a calculated molecular mass of approximately 49 kDa. The nucleotide sequences contained base pair differences, but most were silent. Cluster analysis of the deduced amino acid sequences separated the isolates into three groups. Group I (ETI) consisted of the seven highly virulent isolates and the two Chinese outbreak strains, containing Ala(299)-to-Ser, Glu(305)-to-Lys, and Glu(330)-to-Lys amino acid substitutions compared with groups II and III (ETII). Groups II and III consisted of moderately virulent and nonvirulent strains, which are separated from each other by Tyr(72)-to-Asp and Thr(296)-to-Ala substitutions. Gene exchange studies resulted in the change of ETI to ETII and vice versa. A spectrophotometric activity assay for GDH did not show significant differences between the groups. These results suggest that the GDH ETs and sequence types may serve as useful markers in predicting the pathogenic behavior of strains of this serotype and that the molecular basis for the observed differences in the ETs was amino acid substitutions and not deletion, insertion, or processing uniqueness.

Diversity Analysis of Dairy and Nondairy Lactococcus lactis Isolates, Using a Novel Multilocus Sequence Analysis Scheme and (GTG)5-PCR Fingerprinting▿

PubMed Central

Rademaker, Jan L. W.; Herbet, Hélène; Starrenburg, Marjo J. C.; Naser, Sabri M.; Gevers, Dirk; Kelly, William J.; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E. T.

2007-01-01

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)5-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene. PMID:17890345

Diversity analysis of dairy and nondairy Lactococcus lactis isolates, using a novel multilocus sequence analysis scheme and (GTG)5-PCR fingerprinting.

PubMed

Rademaker, Jan L W; Herbet, Hélène; Starrenburg, Marjo J C; Naser, Sabri M; Gevers, Dirk; Kelly, William J; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E T

2007-11-01

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)(5)-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene.

Structural features of diverse Pin-II proteinase inhibitor genes from Capsicum annuum.

PubMed

Mahajan, Neha S; Dewangan, Veena; Lomate, Purushottam R; Joshi, Rakesh S; Mishra, Manasi; Gupta, Vidya S; Giri, Ashok P

2015-02-01

The proteinase inhibitor (PI) genes from Capsicum annuum were characterized with respect to their UTR, introns and promoter elements. The occurrence of PIs with circularly permuted domain organization was evident. Several potato inhibitor II (Pin-II) type proteinase inhibitor (PI) genes have been analyzed from Capsicum annuum (L.) with respect to their differential expression during plant defense response. However, complete gene characterization of any of these C. annuum PIs (CanPIs) has not been carried out so far. Complete gene architectures of a previously identified CanPI-7 (Beads-on-string, Type A) and a member of newly isolated Bracelet type B, CanPI-69 are reported in this study. The 5' UTR (untranslated region), 3'UTR, and intronic sequences of both the CanPI genes were obtained. The genomic sequence of CanPI-7 exhibited, exon 1 (49 base pair, bp) and exon 2 (740 bp) interrupted by a 294-bp long type I intron. We noted the occurrence of three multi-domain PIs (CanPI-69, 70, 71) with circularly permuted domain organization. CanPI-69 was found to possess exon 1 (49 bp), exon 2 (551 bp) and a 584-bp long type I intron. The upstream sequence analysis of CanPI-7 and CanPI-69 predicted various transcription factor-binding sites including TATA and CAAT boxes, hormone-responsive elements (ABRELATERD1, DOFCOREZM, ERELEE4), and a defense-responsive element (WRKY71OS). Binding of transcription factors such as zinc finger motif MADS-box and MYB to the promoter regions was confirmed using electrophoretic mobility shift assay followed by mass spectrometric identification. The 3' UTR analysis for 25 CanPI genes revealed unique/distinct 3' UTR sequence for each gene. Structures of three domain CanPIs of type A and B were predicted and further analyzed for their attributes. This investigation of CanPI gene architecture will enable the better understanding of the genetic elements present in CanPIs.

The detection and phylogenetic analysis of the alkane 1-monooxygenase gene of members of the genus Rhodococcus.

PubMed

Táncsics, András; Benedek, Tibor; Szoboszlay, Sándor; Veres, Péter G; Farkas, Milán; Máthé, István; Márialigeti, Károly; Kukolya, József; Lányi, Szabolcs; Kriszt, Balázs

2015-02-01

Naturally occurring and anthropogenic petroleum hydrocarbons are potential carbon sources for many bacteria. The AlkB-related alkane hydroxylases, which are integral membrane non-heme iron enzymes, play a key role in the microbial degradation of many of these hydrocarbons. Several members of the genus Rhodococcus are well-known alkane degraders and are known to harbor multiple alkB genes encoding for different alkane 1-monooxygenases. In the present study, 48 Rhodococcus strains, representing 35 species of the genus, were investigated to find out whether there was a dominant type of alkB gene widespread among species of the genus that could be used as a phylogenetic marker. Phylogenetic analysis of rhodococcal alkB gene sequences indicated that a certain type of alkB gene was present in almost every member of the genus Rhodococcus. These alkB genes were common in a unique nucleotide sequence stretch absent from other types of rhodococcal alkB genes that encoded a conserved amino acid motif: WLG(I/V/L)D(G/D)GL. The sequence identity of the targeted alkB gene in Rhodococcus ranged from 78.5 to 99.2% and showed higher nucleotide sequence variation at the inter-species level compared to the 16S rRNA gene (93.9-99.8%). The results indicated that the alkB gene type investigated might be applicable for: (i) differentiating closely related Rhodococcus species, (ii) properly assigning environmental isolates to existing Rhodococcus species, and finally (iii) assessing whether a new Rhodococcus isolate represents a novel species of the genus. Copyright © 2014 Elsevier GmbH. All rights reserved.

Molecular Analysis of the Nitrate-Reducing Community from Unplanted and Maize-Planted Soils

PubMed Central

Philippot, Laurent; Piutti, Séverine; Martin-Laurent, Fabrice; Hallet, Stéphanie; Germon, Jean Claude

2002-01-01

Microorganisms that use nitrate as an alternative terminal electron acceptor play an important role in the global nitrogen cycle. The diversity of the nitrate-reducing community in soil and the influence of the maize roots on the structure of this community were studied. The narG gene encoding the membrane bound nitrate reductase was selected as a functional marker for the nitrate-reducing community. The use of narG is of special interest because the phylogeny of the narG gene closely reflects the 16S ribosomal DNA phylogeny. Therefore, targeting the narG gene provided for the first time a unique insight into the taxonomic composition of the nitrate-reducing community in planted and unplanted soils. The PCR-amplified narG fragments were cloned and analyzed by restriction fragment length polymorphism (RFLP). In all, 60 RFLP types represented by two or more clones were identified in addition to the 58 RFLP types represented by only one clone. At least one clone belonging to each RFLP type was then sequenced. Several of the obtained sequences were not related to the narG genes from cultivated bacteria, suggesting the existence of unidentified nitrate-reducing bacteria in the studied soil. However, environmental sequences were also related to NarG from many bacterial divisions, i.e., Actinobacteria and α, β, and γ Proteobacteria. The presence of the plant roots resulted in a shift in the structure of the nitrate-reducing community between the unplanted and planted soils. Sequencing of RFLP types dominant in the rhizosphere or present only in the rhizosphere revealed that they are related to NarG from the Actinobacteria in an astonishingly high proportion. PMID:12450836

Chemical property based sequence characterization of PpcA and its homolog proteins PpcB-E: A mathematical approach

PubMed Central

Pal Choudhury, Pabitra

2017-01-01

Periplasmic c7 type cytochrome A (PpcA) protein is determined in Geobacter sulfurreducens along with its other four homologs (PpcB-E). From the crystal structure viewpoint the observation emerges that PpcA protein can bind with Deoxycholate (DXCA), while its other homologs do not. But it is yet to be established with certainty the reason behind this from primary protein sequence information. This study is primarily based on primary protein sequence analysis through the chemical basis of embedded amino acids. Firstly, we look for the chemical group specific score of amino acids. Along with this, we have developed a new methodology for the phylogenetic analysis based on chemical group dissimilarities of amino acids. This new methodology is applied to the cytochrome c7 family members and pinpoint how a particular sequence is differing with others. Secondly, we build a graph theoretic model on using amino acid sequences which is also applied to the cytochrome c7 family members and some unique characteristics and their domains are highlighted. Thirdly, we search for unique patterns as subsequences which are common among the group or specific individual member. In all the cases, we are able to show some distinct features of PpcA that emerges PpcA as an outstanding protein compared to its other homologs, resulting towards its binding with deoxycholate. Similarly, some notable features for the structurally dissimilar protein PpcD compared to the other homologs are also brought out. Further, the five members of cytochrome family being homolog proteins, they must have some common significant features which are also enumerated in this study. PMID:28362850

Quantum-Sequencing: Fast electronic single DNA molecule sequencing

NASA Astrophysics Data System (ADS)

Casamada Ribot, Josep; Chatterjee, Anushree; Nagpal, Prashant

2014-03-01

A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free, high-throughput and cost-effective, single-molecule sequencing method. Here, we present the first demonstration of unique ``electronic fingerprint'' of all nucleotides (A, G, T, C), with single-molecule DNA sequencing, using Quantum-tunneling Sequencing (Q-Seq) at room temperature. We show that the electronic state of the nucleobases shift depending on the pH, with most distinct states identified at acidic pH. We also demonstrate identification of single nucleotide modifications (methylation here). Using these unique electronic fingerprints (or tunneling data), we report a partial sequence of beta lactamase (bla) gene, which encodes resistance to beta-lactam antibiotics, with over 95% success rate. These results highlight the potential of Q-Seq as a robust technique for next-generation sequencing.

Dissemination of VIM-2 producing Pseudomonas aeruginosa ST233 at tertiary care hospitals in Egypt.

PubMed

Zafer, Mai Mahmoud; Al-Agamy, Mohamed Hamed; El-Mahallawy, Hadir Ahmed; Amin, Magdy Aly; El Din Ashour, Seif

2015-03-12

Pseudomonas aeruginosa is an important nosocomial pathogen, commonly causing infections in immunocompromised patients. The aim of this study was to examine the genetic relatedness of metallo-beta-lactamase (MBL) producing carbapenem resistant Pseudomonas aeruginosa clinical isolates collected from 2 tertiary hospitals in Cairo, Egypt using Multi Locus sequence typing (MLST). Phenotypic and genotypic detection of metallo-beta-lactamase for forty eight non-duplicate carbapenem resistant P. aeruginosa isolates were carried out. DNA sequencing and MLST were done. The bla VIM-2 gene was highly prevalent (28/33 strains, 85%) among 33 MBL-positive P.aeruginosa isolates. MLST revealed eleven distinct Sequence Types (STs). A unique ST233 clone producing VIM-2 was documented by MLST in P.aeruginosa strains isolated from Cairo university hospitals. The high prevalence of VIM-2 producers was not due to the spread of a single clone. The findings of the present study clearly demonstrate that clones of VIM-2 positive in our hospitals are different from those reported from European studies. Prevalence of VIM-2 producers of the same clone was detected from surgical specimens whereas oncology related specimens were showing diverse clones.

A Unique T-Cell Receptor Amino Acid Sequence Selected by Human T-Cell Lymphotropic Virus Type 1 Tax301-309-Specific Cytotoxic T Cells in HLA-A24:02-Positive Asymptomatic Carriers and Adult T-Cell Leukemia/Lymphoma Patients.

PubMed

Ishihara, Yuko; Tanaka, Yukie; Kobayashi, Seiichiro; Kawamura, Koji; Nakasone, Hideki; Gomyo, Ayumi; Hayakawa, Jin; Tamaki, Masaharu; Akahoshi, Yu; Harada, Naonori; Kusuda, Machiko; Kameda, Kazuaki; Ugai, Tomotaka; Wada, Hidenori; Sakamoto, Kana; Sato, Miki; Terasako-Saito, Kiriko; Kikuchi, Misato; Kimura, Shun-Ichi; Tanihara, Aki; Kako, Shinichi; Uchimaru, Kaoru; Kanda, Yoshinobu

2017-10-01

We previously reported that the T-cell receptor (TCR) repertoire of human T-cell lymphotropic virus type 1 (HTLV-1) Tax 301-309 -specific CD8 + cytotoxic T cells (Tax 301-309 -CTLs) was highly restricted and a particular amino acid sequence motif, the PDR motif, was conserved among HLA-A*24:02-positive (HLA-A*24:02 + ) adult T-cell leukemia/lymphoma (ATL) patients who had undergone allogeneic hematopoietic cell transplantation (allo-HSCT). Furthermore, we found that donor-derived PDR + CTLs selectively expanded in ATL long-term HSCT survivors with strong CTL activity against HTLV-1. On the other hand, the TCR repertoires in Tax 301-309 -CTLs of asymptomatic HTLV-1 carriers (ACs) remain unclear. In this study, we directly identified the DNA sequence of complementarity-determining region 3 (CDR3) of the TCR-β chain of Tax 301-309 -CTLs at the single-cell level and compared not only the TCR repertoires but also the frequencies and phenotypes of Tax 301-309 -CTLs between ACs and ATL patients. We did not observe any essential difference in the frequencies of Tax 301-309 -CTLs between ACs and ATL patients. In the single-cell TCR repertoire analysis of Tax 301-309 -CTLs, 1,458 Tax 301-309 -CTLs and 140 clones were identified in this cohort. Tax 301-309 -CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was exclusively observed in all ACs and ATL patients. However, there was no correlation between PDR + CTL frequencies and HTLV-1 proviral load (PVL). In conclusion, we have identified, for the first time, a unique amino acid sequence, PDR, as a public TCR-CDR3 motif against Tax in HLA-A*24:02 + HTLV-1-infected individuals. Further investigations are warranted to elucidate the role of the PDR + CTL response in the progression from carrier state to ATL. IMPORTANCE ATL is an aggressive T-cell malignancy caused by HTLV-1 infection. The HTLV-1 regulatory protein Tax aggressively promotes the proliferation of HTLV-1-infected lymphocytes and is also a major target antigen for CD8 + CTLs. In our previous evaluation of Tax 301-309 -CTLs, we found that a unique amino acid sequence motif, PDR, in CDR3 of the TCR-β chain of Tax 301-309 -CTLs was conserved among ATL patients after allo-HSCT. Furthermore, the PDR + Tax 301-309 -CTL clones selectively expanded and showed strong cytotoxic activities against HTLV-1. On the other hand, it remains unclear how Tax 301-309 -CTL repertoire exists in ACs. In this study, we comprehensively compared Tax-specific TCR repertoires at the single-cell level between ACs and ATL patients. Tax 301-309 -CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was conserved in all ACs and ATL patients, regardless of clinical subtype in HTLV-1 infection. Copyright © 2017 American Society for Microbiology.

A Unique T-Cell Receptor Amino Acid Sequence Selected by Human T-Cell Lymphotropic Virus Type 1 Tax301-309-Specific Cytotoxic T Cells in HLA-A24:02-Positive Asymptomatic Carriers and Adult T-Cell Leukemia/Lymphoma Patients

PubMed Central

Ishihara, Yuko; Tanaka, Yukie; Kobayashi, Seiichiro; Kawamura, Koji; Nakasone, Hideki; Gomyo, Ayumi; Hayakawa, Jin; Tamaki, Masaharu; Akahoshi, Yu; Harada, Naonori; Kusuda, Machiko; Kameda, Kazuaki; Ugai, Tomotaka; Wada, Hidenori; Sakamoto, Kana; Sato, Miki; Terasako-Saito, Kiriko; Kikuchi, Misato; Kimura, Shun-ichi; Tanihara, Aki; Kako, Shinichi; Uchimaru, Kaoru

2017-01-01

ABSTRACT We previously reported that the T-cell receptor (TCR) repertoire of human T-cell lymphotropic virus type 1 (HTLV-1) Tax301-309-specific CD8+ cytotoxic T cells (Tax301-309-CTLs) was highly restricted and a particular amino acid sequence motif, the PDR motif, was conserved among HLA-A*24:02-positive (HLA-A*24:02+) adult T-cell leukemia/lymphoma (ATL) patients who had undergone allogeneic hematopoietic cell transplantation (allo-HSCT). Furthermore, we found that donor-derived PDR+ CTLs selectively expanded in ATL long-term HSCT survivors with strong CTL activity against HTLV-1. On the other hand, the TCR repertoires in Tax301-309-CTLs of asymptomatic HTLV-1 carriers (ACs) remain unclear. In this study, we directly identified the DNA sequence of complementarity-determining region 3 (CDR3) of the TCR-β chain of Tax301-309-CTLs at the single-cell level and compared not only the TCR repertoires but also the frequencies and phenotypes of Tax301-309-CTLs between ACs and ATL patients. We did not observe any essential difference in the frequencies of Tax301-309-CTLs between ACs and ATL patients. In the single-cell TCR repertoire analysis of Tax301-309-CTLs, 1,458 Tax301-309-CTLs and 140 clones were identified in this cohort. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was exclusively observed in all ACs and ATL patients. However, there was no correlation between PDR+ CTL frequencies and HTLV-1 proviral load (PVL). In conclusion, we have identified, for the first time, a unique amino acid sequence, PDR, as a public TCR-CDR3 motif against Tax in HLA-A*24:02+ HTLV-1-infected individuals. Further investigations are warranted to elucidate the role of the PDR+ CTL response in the progression from carrier state to ATL. IMPORTANCE ATL is an aggressive T-cell malignancy caused by HTLV-1 infection. The HTLV-1 regulatory protein Tax aggressively promotes the proliferation of HTLV-1-infected lymphocytes and is also a major target antigen for CD8+ CTLs. In our previous evaluation of Tax301-309-CTLs, we found that a unique amino acid sequence motif, PDR, in CDR3 of the TCR-β chain of Tax301-309-CTLs was conserved among ATL patients after allo-HSCT. Furthermore, the PDR+ Tax301-309-CTL clones selectively expanded and showed strong cytotoxic activities against HTLV-1. On the other hand, it remains unclear how Tax301-309-CTL repertoire exists in ACs. In this study, we comprehensively compared Tax-specific TCR repertoires at the single-cell level between ACs and ATL patients. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was conserved in all ACs and ATL patients, regardless of clinical subtype in HTLV-1 infection. PMID:28724766

Ongoing outbreak of invasive listeriosis, Germany, 2012 to 2015.

PubMed

Ruppitsch, Werner; Prager, Rita; Halbedel, Sven; Hyden, Patrick; Pietzka, Ariane; Huhulescu, Steliana; Lohr, Dorothee; Schönberger, Katharina; Aichinger, Elisabeth; Hauri, Anja; Stark, Klaus; Vygen, Sabine; Tietze, Erhard; Allerberger, Franz; Wilking, Hendrik

2015-01-01

Listeriosis patient isolates in Germany have shown a new identical pulsed-field gel electrophoresis (PFGE) pattern since 2012 (n = 66). Almost all isolates (Listeria monocytogenes serotype 1/2a) belonged to cases living in southern Germany, indicating an outbreak with a so far unknown source. Case numbers in 2015 are high (n = 28). No outbreak cases outside Germany have been reported. Next generation sequencing revealed the unique cluster type CT1248 and confirmed the outbreak. Investigations into the source are ongoing.

Partial bisulfite conversion for unique template sequencing.

PubMed

Kumar, Vijay; Rosenbaum, Julie; Wang, Zihua; Forcier, Talitha; Ronemus, Michael; Wigler, Michael; Levy, Dan

2018-01-25

We introduce a new protocol, mutational sequencing or muSeq, which uses sodium bisulfite to randomly deaminate unmethylated cytosines at a fixed and tunable rate. The muSeq protocol marks each initial template molecule with a unique mutation signature that is present in every copy of the template, and in every fragmented copy of a copy. In the sequenced read data, this signature is observed as a unique pattern of C-to-T or G-to-A nucleotide conversions. Clustering reads with the same conversion pattern enables accurate count and long-range assembly of initial template molecules from short-read sequence data. We explore count and low-error sequencing by profiling 135 000 restriction fragments in a PstI representation, demonstrating that muSeq improves copy number inference and significantly reduces sporadic sequencer error. We explore long-range assembly in the context of cDNA, generating contiguous transcript clusters greater than 3,000 bp in length. The muSeq assemblies reveal transcriptional diversity not observable from short-read data alone. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Characterization of a species-specific repetitive DNA from a highly endangered wild animal, Rhinoceros unicornis, and assessment of genetic polymorphism by microsatellite associated sequence amplification (MASA).

PubMed

Ali, S; Azfer, M A; Bashamboo, A; Mathur, P K; Malik, P K; Mathur, V B; Raha, A K; Ansari, S

1999-03-04

We have cloned and sequenced a 906bp EcoRI repeat DNA fraction from Rhinoceros unicornis genome. The contig pSS(R)2 is AT rich with 340 A (37.53%), 187 C (20.64%), 173 G (19.09%) and 206 T (22.74%). The sequence contains MALT box, NF-E1, Poly-A signal, lariat consensus sequences, TATA box, translational initiation sequences and several stop codons. Translation of the contig showed seven different types of protein motifs, among which, EGF-like domain cysteine pattern signatures and Bowman-Birk serine protease inhibitor family signatures were prominent. The presence of eukaryotic transcriptional elements, protein signatures and analysis of subset sequences in the 5' region from 1 to 165nt indicating coding potential (test code value=0.97) suggest possible regulatory and/or functional role(s) of these sequences in the rhino genome. Translation of the complementary strand from 906 to 706nt and 190 to 2nt showed proteins of more than 7kDa rich in non-polar residues. This suggests that pSS(R)2 is either a part of, or adjacent to, a functional gene. The contig contains mostly non-consecutive simple repeat units from 2 to 17nt with varying frequencies, of which four base motifs were found to be predominant. Zoo-blot hybridization revealed that pSS(R)2 sequences are unique to R. unicornis genome because they do not cross-hybridize, even with the genomic DNA of South African black rhino Diceros bicornis. Southern blot analysis of R. unicornis genomic DNA with pSS(R)2 and other synthetic oligo probes revealed a high level of genetic homogeneity, which was also substantiated by microsatellite associated sequence amplification (MASA). Owing to its uniqueness, the pSS(R)2 probe has a potential application in the area of conservation biology for unequivocal identification of horn or other body tissues of R. unicornis. The evolutionary aspect of this repeat fraction in the context of comparative genome analysis is discussed.

Unique Trichomonas vaginalis gene sequences identified in multinational regions of Northwest China.

PubMed

Liu, Jun; Feng, Meng; Wang, Xiaolan; Fu, Yongfeng; Ma, Cailing; Cheng, Xunjia

2017-07-24

Trichomonas vaginalis (T. vaginalis) is a flagellated protozoan parasite that infects humans worldwide. This study determined the sequence of the 18S ribosomal RNA gene of T. vaginalis infecting both females and males in Xinjiang, China. Samples from 73 females and 28 males were collected and confirmed for infection with T. vaginalis, a total of 110 sequences were identified when the T. vaginalis 18S ribosomal RNA gene was sequenced. These sequences were used to prepare a phylogenetic network. The rooted network comprised three large clades and several independent branches. Most of the Xinjiang sequences were in one group. Preliminary results suggest that Xinjiang T. vaginalis isolates might be genetically unique, as indicated by the sequence of their 18S ribosomal RNA gene. Low migration rate of local people in this province may contribute to a genetic conservativeness of T. vaginalis. The unique genetic feature of our isolates may suggest a different clinical presentation of trichomoniasis, including metronidazole susceptibility, T. vaginalis virus or Mycoplasma co-infection characteristics. The transmission and evolution of Xinjiang T. vaginalis is of interest and should be studied further. More attention should be given to T. vaginalis infection in both females and males in Xinjiang.

Horizontal gene transfer of chromosomal Type II toxin-antitoxin systems of Escherichia coli.

PubMed

Ramisetty, Bhaskar Chandra Mohan; Santhosh, Ramachandran Sarojini

2016-02-01

Type II toxin-antitoxin systems (TAs) are small autoregulated bicistronic operons that encode a toxin protein with the potential to inhibit metabolic processes and an antitoxin protein to neutralize the toxin. Most of the bacterial genomes encode multiple TAs. However, the diversity and accumulation of TAs on bacterial genomes and its physiological implications are highly debated. Here we provide evidence that Escherichia coli chromosomal TAs (encoding RNase toxins) are 'acquired' DNA likely originated from heterologous DNA and are the smallest known autoregulated operons with the potential for horizontal propagation. Sequence analyses revealed that integration of TAs into the bacterial genome is unique and contributes to variations in the coding and/or regulatory regions of flanking host genome sequences. Plasmids and genomes encoding identical TAs of natural isolates are mutually exclusive. Chromosomal TAs might play significant roles in the evolution and ecology of bacteria by contributing to host genome variation and by moderation of plasmid maintenance. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Pseudomonas aeruginosa Type III Secretory Toxin ExoU and Its Predicted Homologs.

PubMed

Sawa, Teiji; Hamaoka, Saeko; Kinoshita, Mao; Kainuma, Atsushi; Naito, Yoshifumi; Akiyama, Koichi; Kato, Hideya

2016-10-26

Pseudomonas aeruginosa ExoU, a type III secretory toxin and major virulence factor with patatin-like phospholipase activity, is responsible for acute lung injury and sepsis in immunocompromised patients. Through use of a recently updated bacterial genome database, protein sequences predicted to be homologous to Ps. aeruginosa ExoU were identified in 17 other Pseudomonas species ( Ps. fluorescens , Ps. lundensis , Ps. weihenstephanensis , Ps. marginalis, Ps. rhodesiae, Ps. synxantha , Ps. libanensis , Ps. extremaustralis , Ps. veronii , Ps. simiae , Ps. trivialis , Ps. tolaasii , Ps. orientalis , Ps. taetrolens , Ps. syringae , Ps. viridiflava , and Ps. cannabina ) and 8 Gram-negative bacteria from three other genera ( Photorhabdus , Aeromonas , and Paludibacterium ). In the alignment of the predicted primary amino acid sequences used for the phylogenetic analyses, both highly conserved and nonconserved parts of the toxin were discovered among the various species. Further comparative studies of the predicted ExoU homologs should provide us with more detailed information about the unique characteristics of the Ps. aeruginosa ExoU toxin.

Characterization of three types of human alpha s1-casein mRNA transcripts.

PubMed Central

Johnsen, L B; Rasmussen, L K; Petersen, T E; Berglund, L

1995-01-01

Here we report the molecular cloning and sequencing of three types of human alpha s1-casein transcripts and present evidence indicating that exon skipping is responsible for deleted mRNA transcripts. The largest transcript comprised 981 bp encoding a signal peptide of 15 amino acids followed by the mature alpha s1-casein sequence of 170 amino acids. Human alpha s1-casein has been reported to exist naturally as a multimer in complex with kappa-casein in mature human milk, thereby being unique among alpha s1-caseins [Rasmussen, Due and Petersen (1995) Comp. Biochem. Physiol., in the press]. The present demonstration of three cysteines in the mature protein provides a molecular explanation of the interactions in this complex. Tissue-specific expression of human alpha s1-casein was indicated by Northern-blot analysis. In addition, two cryptic exons were localized in the bovine alpha s1-casein gene. Images Figure 3 PMID:7619062

Spatial and temporal plasticity of chromatin during programmed DNA-reorganization in Stylonychia macronuclear development

PubMed Central

Postberg, Jan; Heyse, Katharina; Cremer, Marion; Cremer, Thomas; Lipps, Hans J

2008-01-01

Background: In this study we exploit the unique genome organization of ciliates to characterize the biological function of histone modification patterns and chromatin plasticity for the processing of specific DNA sequences during a nuclear differentiation process. Ciliates are single-cell eukaryotes containing two morphologically and functionally specialized types of nuclei, the somatic macronucleus and the germline micronucleus. In the course of sexual reproduction a new macronucleus develops from a micronuclear derivative. During this process specific DNA sequences are eliminated from the genome, while sequences that will be transcribed in the mature macronucleus are retained. Results: We show by immunofluorescence microscopy, Western analyses and chromatin immunoprecipitation (ChIP) experiments that each nuclear type establishes its specific histone modification signature. Our analyses reveal that the early macronuclear anlage adopts a permissive chromatin state immediately after the fusion of two heterochromatic germline micronuclei. As macronuclear development progresses, repressive histone modifications that specify sequences to be eliminated are introduced de novo. ChIP analyses demonstrate that permissive histone modifications are associated with sequences that will be retained in the new macronucleus. Furthermore, our data support the hypothesis that a PIWI-family protein is involved in a transnuclear cross-talk and in the RNAi-dependent control of developmental chromatin reorganization. Conclusion: Based on these data we present a comprehensive analysis of the spatial and temporal pattern of histone modifications during this nuclear differentiation process. Results obtained in this study may also be relevant for our understanding of chromatin plasticity during metazoan embryogenesis. PMID:19014664

Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB)

PubMed Central

Pamp, Sünje J.; Harrington, Eoghan D.; Quake, Stephen R.; Relman, David A.; Blainey, Paul C.

2012-01-01

Segmented filamentous bacteria (SFB) are host-specific intestinal symbionts that comprise a distinct clade within the Clostridiaceae, designated Candidatus Arthromitus. SFB display a unique life cycle within the host, involving differentiation into multiple cell types. The latter include filaments that attach intimately to intestinal epithelial cells, and from which “holdfasts” and spores develop. SFB induce a multifaceted immune response, leading to host protection from intestinal pathogens. Cultivation resistance has hindered characterization of these enigmatic bacteria. In the present study, we isolated five SFB filaments from a mouse using a microfluidic device equipped with laser tweezers, generated genome sequences from each, and compared these sequences with each other, as well as to recently published SFB genome sequences. Based on the resulting analyses, SFB appear to be dependent on the host for a variety of essential nutrients. SFB have a relatively high abundance of predicted proteins devoted to cell cycle control and to envelope biogenesis, and have a group of SFB-specific autolysins and a dynamin-like protein. Among the five filament genomes, an average of 8.6% of predicted proteins were novel, including a family of secreted SFB-specific proteins. Four ADP-ribosyltransferase (ADPRT) sequence types, and a myosin-cross-reactive antigen (MCRA) protein were discovered; we hypothesize that they are involved in modulation of host responses. The presence of polymorphisms among mouse SFB genomes suggests the evolution of distinct SFB lineages. Overall, our results reveal several aspects of SFB adaptation to the mammalian intestinal tract. PMID:22434425

DNA methylation at hepatitis B viral integrants is associated with methylation at flanking human genomic sequences

PubMed Central

Watanabe, Yoshiyuki; Yamamoto, Hiroyuki; Oikawa, Ritsuko; Toyota, Minoru; Yamamoto, Masakazu; Kokudo, Norihiro; Tanaka, Shinji; Arii, Shigeki; Yotsuyanagi, Hiroshi; Koike, Kazuhiko; Itoh, Fumio

2015-01-01

Integration of DNA viruses into the human genome plays an important role in various types of tumors, including hepatitis B virus (HBV)–related hepatocellular carcinoma. However, the molecular details and clinical impact of HBV integration on either human or HBV epigenomes are unknown. Here, we show that methylation of the integrated HBV DNA is related to the methylation status of the flanking human genome. We developed a next-generation sequencing-based method for structural methylation analysis of integrated viral genomes (denoted G-NaVI). This method is a novel approach that enables enrichment of viral fragments for sequencing using unique baits based on the sequence of the HBV genome. We detected integrated HBV sequences in the genome of the PLC/PRF/5 cell line and found variable levels of methylation within the integrated HBV genomes. Allele-specific methylation analysis revealed that the HBV genome often became significantly methylated when integrated into highly methylated host sites. After integration into unmethylated human genome regions such as promoters, however, the HBV DNA remains unmethylated and may eventually play an important role in tumorigenesis. The observed dynamic changes in DNA methylation of the host and viral genomes may functionally affect the biological behavior of HBV. These findings may impact public health given that millions of people worldwide are carriers of HBV. We also believe our assay will be a powerful tool to increase our understanding of the various types of DNA virus-associated tumorigenesis. PMID:25653310

Identification and verification of hybridoma-derived monoclonal antibody variable region sequences using recombinant DNA technology and mass spectrometry

USDA-ARS?s Scientific Manuscript database

Antibody engineering requires the identification of antigen binding domains or variable regions (VR) unique to each antibody. It is the VR that define the unique antigen binding properties and proper sequence identification is essential for functional evaluation and performance of recombinant antibo...

Genetic diversity among sea otter isolates of Toxoplasma gondii

USGS Publications Warehouse

Sundar, N.; Cole, Rebecca A.; Thomas, N.J.; Majumdar, D.; Dubey, J.P.; Su, C.

2008-01-01

Sea otters (Enhydra lutris) have been reported to become infected with Toxoplasma gondiiand at times succumb to clinical disease. Here, we determined genotypes of 39 T. gondiiisolates from 37 sea otters in two geographically distant locations (25 from California and 12 from Washington). Six genotypes were identified using 10 PCR-RFLP genetic markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico, and by DNA sequencing of loci SAG1 and GRA6 in 13 isolates. Of these 39 isolates, 13 (33%) were clonal Type II which can be further divided into two groups at the locus Apico. Two of the 39 isolates had Type II alleles at all loci except a Type I allele at locus L358. One isolate had Type II alleles at all loci except the Type I alleles at loci L358 and Apico. One isolate had Type III alleles at all loci except Type II alleles at SAG2 and Apico. Two sea otter isolates had a mixed infection. Twenty-one (54%) isolates had an unique allele at SAG1 locus. Further genotyping or DNA sequence analysis for 18 of these 21 isolates at loci SAG1 and GRA6 revealed that there were two different genotypes, including the previously identified Type X (four isolates) and a new genotype named Type A (14 isolates). The results from this study suggest that the sea otter isolates are genetically diverse.

Rhodotorula svalbardensis sp. nov., a novel yeast species isolated from cryoconite holes of Ny-Ålesund, Arctic.

PubMed

Singh, Purnima; Singh, Shiv M; Tsuji, Masaharu; Prasad, Gandham S; Hoshino, Tamotsu

2014-02-01

A psychrophilic yeast species was isolated from glacier cryoconite holes of Svalbard. Nucleotide sequences of the strains were studied using D1/D2 domain, ITS region and partial sequences of mitochondrial cytochrome b gene. The strains belonged to a clade of psychrophilic yeasts, but showed marked differences from related species in the D1/D2 domain and biochemical characters. Effects of temperature, salt and media on growth of the cultures were also studied. Screening of the cultures for amylase, cellulase, protease, lipase, urease and catalase activities was carried out. The strains expressed high amylase and lipase activities. Freeze tolerance ability of the isolates indicated the formation of unique hexagonal ice crystal structures due to presence of 'antifreeze proteins' (AFPs). FAME analysis of cultures showed a unique trend of increase in unsaturated fatty acids with decrease in temperature. The major fatty acids recorded were oleic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid, myristic acid and pentadecanoic acid. Based on sequence data and, physiological and morphological properties of the strains, we propose a novel species, Rhodotorula svalbardensis and designate strains MLB-I (CCP-II) and CRY-YB-1 (CBS 12863, JCM 19699, JCM 19700, MTCC 10952) as its type strains (Etymology: sval.bar.den'sis. N.L. fem. adj. svalbardensis pertaining to Svalbard). Copyright © 2014 Elsevier Inc. All rights reserved.

Whole-Genome-Sequencing characterization of bloodstream infection-causing hypervirulent Klebsiella pneumoniae of capsular serotype K2 and ST374.

PubMed

Wang, Xiaoli; Xie, Yingzhou; Li, Gang; Liu, Jialin; Li, Xiaobin; Tian, Lijun; Sun, Jingyong; Ou, Hong-Yu; Qu, Hongping

2018-01-01

Hypervirulent K. pneumoniae variants (hvKP) have been increasingly reported worldwide, causing metastasis of severe infections such as liver abscesses and bacteremia. The capsular serotype K2 hvKP strains show diverse multi-locus sequence types (MLSTs), but with limited genetics and virulence information. In this study, we report a hypermucoviscous K. pneumoniae strain, RJF293, isolated from a human bloodstream sample in a Chinese hospital. It caused a metastatic infection and fatal septic shock in a critical patient. The microbiological features and genetic background were investigated with multiple approaches. The Strain RJF293 was determined to be multilocis sequence type (ST) 374 and serotype K2, displayed a median lethal dose (LD50) of 1.5 × 10 2 CFU in BALB/c mice and was as virulent as the ST23 K1 serotype hvKP strain NTUH-K2044 in a mouse lethality assay. Whole genome sequencing revealed that the RJF293 genome codes for 32 putative virulence factors and exhibits a unique presence/absence pattern in comparison to the other 105 completely sequenced K. pneumoniae genomes. Whole genome SNP-based phylogenetic analysis revealed that strain RJF293 formed a single clade, distant from those containing either ST66 or ST86 hvKP. Compared to the other sequenced hvKP chromosomes, RJF293 contains several strain-variable regions, including one prophage, one ICEKp1 family integrative and conjugative element and six large genomic islands. The sequencing of the first complete genome of an ST374 K2 hvKP clinical strain should reinforce our understanding of the epidemiology and virulence mechanisms of this bloodstream infection-causing hvKP with clinical significance.

Whole-Genome-Sequencing characterization of bloodstream infection-causing hypervirulent Klebsiella pneumoniae of capsular serotype K2 and ST374

PubMed Central

Wang, Xiaoli; Xie, Yingzhou; Li, Gang; Liu, Jialin; Li, Xiaobin; Tian, Lijun; Sun, Jingyong; Qu, Hongping

2018-01-01

ABSTRACT Hypervirulent K. pneumoniae variants (hvKP) have been increasingly reported worldwide, causing metastasis of severe infections such as liver abscesses and bacteremia. The capsular serotype K2 hvKP strains show diverse multi-locus sequence types (MLSTs), but with limited genetics and virulence information. In this study, we report a hypermucoviscous K. pneumoniae strain, RJF293, isolated from a human bloodstream sample in a Chinese hospital. It caused a metastatic infection and fatal septic shock in a critical patient. The microbiological features and genetic background were investigated with multiple approaches. The Strain RJF293 was determined to be multilocis sequence type (ST) 374 and serotype K2, displayed a median lethal dose (LD50) of 1.5 × 102 CFU in BALB/c mice and was as virulent as the ST23 K1 serotype hvKP strain NTUH-K2044 in a mouse lethality assay. Whole genome sequencing revealed that the RJF293 genome codes for 32 putative virulence factors and exhibits a unique presence/absence pattern in comparison to the other 105 completely sequenced K. pneumoniae genomes. Whole genome SNP-based phylogenetic analysis revealed that strain RJF293 formed a single clade, distant from those containing either ST66 or ST86 hvKP. Compared to the other sequenced hvKP chromosomes, RJF293 contains several strain-variable regions, including one prophage, one ICEKp1 family integrative and conjugative element and six large genomic islands. The sequencing of the first complete genome of an ST374 K2 hvKP clinical strain should reinforce our understanding of the epidemiology and virulence mechanisms of this bloodstream infection-causing hvKP with clinical significance. PMID:29338592

Genetic mutation analysis of human gastric adenocarcinomas using ion torrent sequencing platform.

PubMed

Xu, Zhi; Huo, Xinying; Ye, Hua; Tang, Chuanning; Nandakumar, Vijayalakshmi; Lou, Feng; Zhang, Dandan; Dong, Haichao; Sun, Hong; Jiang, Shouwen; Zhang, Guangchun; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; He, Yan; Yan, Chaowei; Wang, Lu; Su, Ziyi; Li, Yangyang; Gu, Dongying; Zhang, Xiaojing; Wu, Xiaomin; Wei, Xiaowei; Hong, Lingzhi; Zhang, Yangmei; Yang, Jinsong; Gong, Yonglin; Tang, Cuiju; Jones, Lindsey; Huang, Xue F; Chen, Si-Yi; Chen, Jinfei

2014-01-01

Gastric cancer is the one of the major causes of cancer-related death, especially in Asia. Gastric adenocarcinoma, the most common type of gastric cancer, is heterogeneous and its incidence and cause varies widely with geographical regions, gender, ethnicity, and diet. Since unique mutations have been observed in individual human cancer samples, identification and characterization of the molecular alterations underlying individual gastric adenocarcinomas is a critical step for developing more effective, personalized therapies. Until recently, identifying genetic mutations on an individual basis by DNA sequencing remained a daunting task. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent sequencing platform, makes DNA sequencing cheaper, faster, and more reliable. In this study, we aim to identify genetic mutations in the genes which are targeted by drugs in clinical use or are under development in individual human gastric adenocarcinoma samples using Ion Torrent sequencing. We sequenced 737 loci from 45 cancer-related genes in 238 human gastric adenocarcinoma samples using the Ion Torrent Ampliseq Cancer Panel. The sequencing analysis revealed a high occurrence of mutations along the TP53 locus (9.7%) in our sample set. Thus, this study indicates the utility of a cost and time efficient tool such as Ion Torrent sequencing to screen cancer mutations for the development of personalized cancer therapy.

Impact of sequencing depth and read length on single cell RNA sequencing data of T cells.

PubMed

Rizzetto, Simone; Eltahla, Auda A; Lin, Peijie; Bull, Rowena; Lloyd, Andrew R; Ho, Joshua W K; Venturi, Vanessa; Luciani, Fabio

2017-10-06

Single cell RNA sequencing (scRNA-seq) provides great potential in measuring the gene expression profiles of heterogeneous cell populations. In immunology, scRNA-seq allowed the characterisation of transcript sequence diversity of functionally relevant T cell subsets, and the identification of the full length T cell receptor (TCRαβ), which defines the specificity against cognate antigens. Several factors, e.g. RNA library capture, cell quality, and sequencing output affect the quality of scRNA-seq data. We studied the effects of read length and sequencing depth on the quality of gene expression profiles, cell type identification, and TCRαβ reconstruction, utilising 1,305 single cells from 8 publically available scRNA-seq datasets, and simulation-based analyses. Gene expression was characterised by an increased number of unique genes identified with short read lengths (<50 bp), but these featured higher technical variability compared to profiles from longer reads. Successful TCRαβ reconstruction was achieved for 6 datasets (81% - 100%) with at least 0.25 millions (PE) reads of length >50 bp, while it failed for datasets with <30 bp reads. Sufficient read length and sequencing depth can control technical noise to enable accurate identification of TCRαβ and gene expression profiles from scRNA-seq data of T cells.

Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC).

PubMed

Brzuszkiewicz, Elzbieta; Thürmer, Andrea; Schuldes, Jörg; Leimbach, Andreas; Liesegang, Heiko; Meyer, Frauke-Dorothee; Boelter, Jürgen; Petersen, Heiko; Gottschalk, Gerhard; Daniel, Rolf

2011-12-01

The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).

Phylogenetic relationships among Streptococcus agalactiae isolated from piscine, dolphin, bovine and human sources: a dolphin and piscine lineage associated with a fish epidemic in Kuwait is also associated with human neonatal infections in Japan.

PubMed

Evans, Joyce J; Bohnsack, John F; Klesius, Phillip H; Whiting, April A; Garcia, Julio C; Shoemaker, Craig A; Takahashi, Shinji

2008-11-01

Streptococcus agalactiae, commonly known as group B streptococcus (GBS), is a cause of infectious disease in numerous animal species. This study examined the genetic relatedness of piscine, dolphin and human GBS isolates and bovine GBS reference strains from different geographical regions using serological and molecular serotyping and multilocus sequence typing (MLST) techniques. Piscine isolates originating from Kuwait, Brazil, Israel and the USA were capsular serotype Ia, a serotype previously unreported in GBS isolated from fish. Sequence typing of piscine isolates produced six sequence types (ST-7, ST-257, ST-258, ST-259, ST-260 and ST-261), the latter five representing allelic designations and allelic combinations not previously reported in the S. agalactiae MLST database. Genomic diversity existed between dolphin and piscine GBS isolates from Kuwait and other geographical areas. Piscine GBS isolates from Brazil, Israel, Honduras and the USA appeared to represent a distinct genetic population of strains that were largely unrelated to human and bovine GBS. The Kuwait dolphin and piscine lineage (ST-7, Ia) was also associated with human neonatal infections in Japan. Comparative genomics of piscine, human and bovine GBS could help clarify those genes important for host tropism, the emergence of unique pathogenic clones and whether these hosts act as reservoirs of one another's pathogenic lineages.

Mycobacterium pseudoshottsii sp. nov., a slowly growing chromogenic species isolated from Chesapeake Bay striped bass (Morone saxatilis)

USGS Publications Warehouse

Rhodes, M.W.; Kator, H.; McNabb, A.; Deshayes, C.; Reyrat, J.-M.; Brown-Elliott, B. A.; Wallace, R.; Trott, K.A.; Parker, J.M.; Lifland, B.; Osterhout, G.; Kaattari, I.; Reece, K.; Vogelbein, W.; Ottinger, C.A.

2005-01-01

A group of slowly growing photochromogenic mycobacteria was isolated from Chesapeake Bay striped bass (Morone saxatilis) during an epizootic of mycobacteriosis. Growth characteristics, acid-fastness and 16S rRNA gene sequencing results were consistent with those of the genus Mycobacterium. Biochemical reactions, growth characteristics and mycolic acid profiles (HPLC) resembled those of Mycobacterium shottsii, a non-pigmented mycobacterium also isolated during the same epizootic. Sequencing of the 16S rRNA genes, the gene encoding the exported repeated protein (erp) and the gene encoding the 65 kDa heat-shock protein (hsp65) and restriction enzyme analysis of the hsp65 gene demonstrated that this group of isolates is unique. Insertion sequences associated with Mycobacterium ulcerans, IS2404 and IS2606, were detected by PCR. These isolates could be differentiated from other slowly growing pigmented mycobacteria by their inability to grow at 37 ??C, production of niacin and urease, absence of nitrate reductase, negative Tween 80 hydrolysis and resistance to isoniazid (1 ??g ml-1), p-nitrobenzoic acid, thiacetazone and thiophene-2-carboxylic hydrazide. On the basis of this polyphasic study, it is proposed that these isolates represent a novel species, Mycobacterium pseudoshottsii sp. nov. The type strain, L15T, has been deposited in the American Type Culture Collection as ATCC BAA-883T and the National Collection of Type Cultures (UK) as NCTC 13318T. ?? 2005 IUMS.

Triple helix-forming oligonucleotide corresponding to the polypyrimidine sequence in the rat alpha 1(I) collagen promoter specifically inhibits factor binding and transcription.

PubMed

Kovacs, A; Kandala, J C; Weber, K T; Guntaka, R V

1996-01-19

Type I and III fibrillar collagens are the major structural proteins of the extracellular matrix found in various organs including the myocardium. Abnormal and progressive accumulation of fibrillar type I collagen in the interstitial spaces compromises organ function and therefore, the study of transcriptional regulation of this gene and specific targeting of its expression is of major interest. Transient transfection of adult cardiac fibroblasts indicate that the polypurine-polypyrimidine sequence of alpha 1(I) collagen promoter between nucleotides - 200 and -140 represents an overall positive regulatory element. DNase I footprinting and electrophoretic mobility shift assays suggest that multiple factors bind to different elements of this promoter region. We further demonstrate that the unique polypyrimidine sequence between -172 and -138 of the promoter represents a suitable target for a single-stranded polypurine oligonucleotide (TFO) to form a triple helix DNA structure. Modified electrophoretic mobility shift assays show that this TFO specifically inhibits the protein-DNA interaction within the target region. In vitro transcription assays and transient transfection experiments demonstrate that the transcriptional activity of the promoter is inhibited by this oligonucleotide. We propose that TFOs represent a therapeutic potential to specifically influence the expression of alpha 1(I) collagen gene in various disease states where abnormal type I collagen accumulation is known to occur.

Penicillin-Binding Protein Transpeptidase Signatures for Tracking and Predicting β-Lactam Resistance Levels in Streptococcus pneumoniae

PubMed Central

Metcalf, Benjamin J.; Chochua, Sopio; Li, Zhongya; Gertz, Robert E.; Walker, Hollis; Hawkins, Paulina A.; Tran, Theresa; Whitney, Cynthia G.; McGee, Lesley; Beall, Bernard W.

2016-01-01

ABSTRACT β-Lactam antibiotics are the drugs of choice to treat pneumococcal infections. The spread of β-lactam-resistant pneumococci is a major concern in choosing an effective therapy for patients. Systematically tracking β-lactam resistance could benefit disease surveillance. Here we developed a classification system in which a pneumococcal isolate is assigned to a “PBP type” based on sequence signatures in the transpeptidase domains (TPDs) of the three critical penicillin-binding proteins (PBPs), PBP1a, PBP2b, and PBP2x. We identified 307 unique PBP types from 2,528 invasive pneumococcal isolates, which had known MICs to six β-lactams based on broth microdilution. We found that increased β-lactam MICs strongly correlated with PBP types containing divergent TPD sequences. The PBP type explained 94 to 99% of variation in MICs both before and after accounting for genomic backgrounds defined by multilocus sequence typing, indicating that genomic backgrounds made little independent contribution to β-lactam MICs at the population level. We further developed and evaluated predictive models of MICs based on PBP type. Compared to microdilution MICs, MICs predicted by PBP type showed essential agreement (MICs agree within 1 dilution) of >98%, category agreement (interpretive results agree) of >94%, a major discrepancy (sensitive isolate predicted as resistant) rate of <3%, and a very major discrepancy (resistant isolate predicted as sensitive) rate of <2% for all six β-lactams. Thus, the PBP transpeptidase signatures are robust indicators of MICs to different β-lactam antibiotics in clinical pneumococcal isolates and serve as an accurate alternative to phenotypic susceptibility testing. PMID:27302760

Genetic variation and DNA fingerprinting of durian types in Malaysia using simple sequence repeat (SSR) markers.

PubMed

Siew, Ging Yang; Ng, Wei Lun; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Tan, Soon Guan; Yeap, Swee Keong

2018-01-01

Durian ( Durio zibethinus ) is one of the most popular tropical fruits in Asia. To date, 126 durian types have been registered with the Department of Agriculture in Malaysia based on phenotypic characteristics. Classification based on morphology is convenient, easy, and fast but it suffers from phenotypic plasticity as a direct result of environmental factors and age. To overcome the limitation of morphological classification, there is a need to carry out genetic characterization of the various durian types. Such data is important for the evaluation and management of durian genetic resources in producing countries. In this study, simple sequence repeat (SSR) markers were used to study the genetic variation in 27 durian types from the germplasm collection of Universiti Putra Malaysia. Based on DNA sequences deposited in Genbank, seven pairs of primers were successfully designed to amplify SSR regions in the durian DNA samples. High levels of variation among the 27 durian types were observed (expected heterozygosity, H E  = 0.35). The DNA fingerprinting power of SSR markers revealed by the combined probability of identity (PI) of all loci was 2.3×10 -3 . Unique DNA fingerprints were generated for 21 out of 27 durian types using five polymorphic SSR markers (the other two SSR markers were monomorphic). We further tested the utility of these markers by evaluating the clonal status of shared durian types from different germplasm collection sites, and found that some were not clones. The findings in this preliminary study not only shows the feasibility of using SSR markers for DNA fingerprinting of durian types, but also challenges the current classification of durian types, e.g., on whether the different types should be called "clones", "varieties", or "cultivars". Such matters have a direct impact on the regulation and management of durian genetic resources in the region.

Genetic variation and DNA fingerprinting of durian types in Malaysia using simple sequence repeat (SSR) markers

PubMed Central

Siew, Ging Yang; Tan, Sheau Wei; Tan, Soon Guan; Yeap, Swee Keong

2018-01-01

Durian (Durio zibethinus) is one of the most popular tropical fruits in Asia. To date, 126 durian types have been registered with the Department of Agriculture in Malaysia based on phenotypic characteristics. Classification based on morphology is convenient, easy, and fast but it suffers from phenotypic plasticity as a direct result of environmental factors and age. To overcome the limitation of morphological classification, there is a need to carry out genetic characterization of the various durian types. Such data is important for the evaluation and management of durian genetic resources in producing countries. In this study, simple sequence repeat (SSR) markers were used to study the genetic variation in 27 durian types from the germplasm collection of Universiti Putra Malaysia. Based on DNA sequences deposited in Genbank, seven pairs of primers were successfully designed to amplify SSR regions in the durian DNA samples. High levels of variation among the 27 durian types were observed (expected heterozygosity, HE = 0.35). The DNA fingerprinting power of SSR markers revealed by the combined probability of identity (PI) of all loci was 2.3×10−3. Unique DNA fingerprints were generated for 21 out of 27 durian types using five polymorphic SSR markers (the other two SSR markers were monomorphic). We further tested the utility of these markers by evaluating the clonal status of shared durian types from different germplasm collection sites, and found that some were not clones. The findings in this preliminary study not only shows the feasibility of using SSR markers for DNA fingerprinting of durian types, but also challenges the current classification of durian types, e.g., on whether the different types should be called “clones”, “varieties”, or “cultivars”. Such matters have a direct impact on the regulation and management of durian genetic resources in the region. PMID:29511604

Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas.

PubMed

Yabuuchi, E; Yano, I; Oyaizu, H; Hashimoto, Y; Ezaki, T; Yamamoto, H

1990-01-01

Based on the partial nucleotide sequence analysis of 16S ribosomal ribonucleic acid (rRNA), presence of unique sphingoglycolipids in cellular lipid, and the major type of ubiquinone (Q10), we propose Sphingomonas gen. nov. with the type species Sphingomonas paucimobilis (Holmes et al, 1977) comb. nov. From the homology values of deoxyribonucleic acid-deoxyribonucleic acid hybridization and the phenotypic characteristics, three new species, Sphingomonas parapaucimobilis, Sphingomonas yanoikuyae, Sphingomonas adhaesiva, and one new combination, Sphingomonas capsulata, are described. S. parapaucimobilis JCM 7510 (= GIFU 11387), S. yanoikuyae JCM 7371 (= GIFU 9882), and S. adhaesiva JCM 7370 (= GIFU 11458) are designated as the type strains of the three new species. Emended description of the type strain of S. capsulata is presented.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taneja, Bhupesh; Patel, Asmita; Slesarev, Alexei

Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting nomore » conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.« less

Streptococcus mutans in a Wild, Sucrose-Eating Rat Population

PubMed Central

Coykendall, Alan L.; Specht, Patricia A.; Samol, Harry H.

1974-01-01

Streptococcus mutans, an organism implicated in dental caries and not previously found outside of man and certain laboratory animals, was isolated from the mouths of wild rats which ate sugar cane. The strains isolated fermented mannitol and sorbitol, and failed to grow in 6.5% NaCl or at 45 C. They formed in vitro plaques on nichrome wires when grown in sucrose broth. They also stored intracellular polysaccharide which could be catabolized by washed, resting cells. Deoxyribonucleic acid-deoxyribonucleic acid reassociations revealed two genetic types. One type shared extensive deoxyribonucleic acid base sequences with S. mutans strains HS6 and OMZ61, two members of a genetic type found in man and laboratory hamsters. The other type seemed unrelated to any S. mutans genetic type previously encountered. It is concluded that the ecological triad of tooth-sucrose-S. mutans is not a phenomenon unique to man and experimental animals. Images PMID:4601769

Comprehensive analysis of the T-cell receptor beta chain gene in rhesus monkey by high throughput sequencing

PubMed Central

Li, Zhoufang; Liu, Guangjie; Tong, Yin; Zhang, Meng; Xu, Ying; Qin, Li; Wang, Zhanhui; Chen, Xiaoping; He, Jiankui

2015-01-01

Profiling immune repertoires by high throughput sequencing enhances our understanding of immune system complexity and immune-related diseases in humans. Previously, cloning and Sanger sequencing identified limited numbers of T cell receptor (TCR) nucleotide sequences in rhesus monkeys, thus their full immune repertoire is unknown. We applied multiplex PCR and Illumina high throughput sequencing to study the TCRβ of rhesus monkeys. We identified 1.26 million TCRβ sequences corresponding to 643,570 unique TCRβ sequences and 270,557 unique complementarity-determining region 3 (CDR3) gene sequences. Precise measurements of CDR3 length distribution, CDR3 amino acid distribution, length distribution of N nucleotide of junctional region, and TCRV and TCRJ gene usage preferences were performed. A comprehensive profile of rhesus monkey immune repertoire might aid human infectious disease studies using rhesus monkeys. PMID:25961410

Recent progress in the research about Propionibacterium acnes strain diversity and acne: pathogen or bystander?

PubMed

Kwon, Hyuck Hoon; Suh, Dae Hun

2016-11-01

Recent progress has steadily reported the existence of the diverse strains of Propionibacterium acnes, and these studies have contributed to the elucidation of their contradictory roles between normal commensals and pathogens. In this review, the authors aimed to provide an update on the recent understanding of research about P. acnes strain diversity and acne, analyzing the potential implications for clinical applications. Before the era of genomic research, P. acnes was known to be distinguished based on serological agglutination tests, cell wall sugar analysis, or fermentation traits. Since the complete genome sequence of P. acnes was first deciphered, genetic studies based on sequence data have expanded with the introduction of more refined and precise DNA-based typing methods, including multilocus sequence typing and metagenomics. These sophisticated techniques have revealed that P. acnes consists of phylogenetically distinct cluster groups with various pathogenic traits, including elicitation of inflammation, protein secretome profile, and unique distribution patterns in various skin loci. In following large-scale studies from patients' acne samples have revealed that specific sequence types are included within the phylogenetic divisions and further suggested that particular P. acnes strains play an etiologic role in acne while others are associated with health, providing a firm platform for evidential-based research into the exact role of this organism in acne. We strongly believe that future research would provide fruitful results in not only clarifying the apparent controversy with respect to roles of P. acnes but also developing therapeutic drugs by pinpointing specific targets of the pathogenic strain only. © 2016 The International Society of Dermatology.

Genotypes and phylogeographical relationships of infectious hematopoietic necrosis virus in California, USA

USGS Publications Warehouse

Kelley, G.O.; Bendorf, C.M.; Yun, S.C.; Kurath, G.; Hedrick, R.P.

2007-01-01

Infectious hematopoietic necrosis virus (IHNV) contains 3 major genogroups in North America with discreet geographic ranges designated as upper (U), middle (M), and lower (L). A comprehensive genotyping of 237 IHNV isolates from hatchery and wild salmonids in California revealed 25 different sequence types (a to y) all in the L genogroup; specifically, the genogroup contained 14 sequence types that were unique to individual isolates as well as 11 sequence types representing 2 or more identical isolates. The most evident trend was the phylogenetic and geographical division of the L genogroup into 2 distinct subgroups designated as LI and LII. Isolates within Subgroup LI were primarily found within waterways linked to southern Oregon and northern California coastal rivers. Isolates in Subgroup LII were concentrated within inland valley watersheds that included the Sacramento River, San Joaquin River, and their tributaries. The temporal and spatial patterns of virus occurrence suggested that infections among adult Chinook salmon in the hatchery or that spawn in the river are a major source of virus potentially infecting other migrating or resident salmonids in California. Serum neutralization results of the California isolates of IHNV corroborated a temporal trend of sequence divergence; specifically, 2 progressive shifts in which more recent virus isolates represent new serotypes. A comparison of the estimates of divergence rates for Subgroup LI (1 ?? ICT5 mutations per nucleotide site per year) indicated stasis similar to that observed in the U genogroup, while the Subgroup LII rate (1 ?? 10 3 mutations per nucleotide site per year) suggested a more active evolution similar to that of the M genogroup. ?? Inter-Research 2007.

Genomic comparison of multi-drug resistant invasive and colonizing Acinetobacter baumannii isolated from diverse human body sites reveals genomic plasticity.

PubMed

Sahl, Jason W; Johnson, J Kristie; Harris, Anthony D; Phillippy, Adam M; Hsiao, William W; Thom, Kerri A; Rasko, David A

2011-06-04

Acinetobacter baumannii has recently emerged as a significant global pathogen, with a surprisingly rapid acquisition of antibiotic resistance and spread within hospitals and health care institutions. This study examines the genomic content of three A. baumannii strains isolated from distinct body sites. Isolates from blood, peri-anal, and wound sources were examined in an attempt to identify genetic features that could be correlated to each isolation source. Pulsed-field gel electrophoresis, multi-locus sequence typing and antibiotic resistance profiles demonstrated genotypic and phenotypic variation. Each isolate was sequenced to high-quality draft status, which allowed for comparative genomic analyses with existing A. baumannii genomes. A high resolution, whole genome alignment method detailed the phylogenetic relationships of sequenced A. baumannii and found no correlation between phylogeny and body site of isolation. This method identified genomic regions unique to both those isolates found on the surface of the skin or in wounds, termed colonization isolates, and those identified from body fluids, termed invasive isolates; these regions may play a role in the pathogenesis and spread of this important pathogen. A PCR-based screen of 74 A. baumanii isolates demonstrated that these unique genes are not exclusive to either phenotype or isolation source; however, a conserved genomic region exclusive to all sequenced A. baumannii was identified and verified. The results of the comparative genome analysis and PCR assay show that A. baumannii is a diverse and genomically variable pathogen that appears to have the potential to cause a range of human disease regardless of the isolation source.

The Genome of the Netherlands: design, and project goals.

PubMed

Boomsma, Dorret I; Wijmenga, Cisca; Slagboom, Eline P; Swertz, Morris A; Karssen, Lennart C; Abdellaoui, Abdel; Ye, Kai; Guryev, Victor; Vermaat, Martijn; van Dijk, Freerk; Francioli, Laurent C; Hottenga, Jouke Jan; Laros, Jeroen F J; Li, Qibin; Li, Yingrui; Cao, Hongzhi; Chen, Ruoyan; Du, Yuanping; Li, Ning; Cao, Sujie; van Setten, Jessica; Menelaou, Androniki; Pulit, Sara L; Hehir-Kwa, Jayne Y; Beekman, Marian; Elbers, Clara C; Byelas, Heorhiy; de Craen, Anton J M; Deelen, Patrick; Dijkstra, Martijn; den Dunnen, Johan T; de Knijff, Peter; Houwing-Duistermaat, Jeanine; Koval, Vyacheslav; Estrada, Karol; Hofman, Albert; Kanterakis, Alexandros; Enckevort, David van; Mai, Hailiang; Kattenberg, Mathijs; van Leeuwen, Elisabeth M; Neerincx, Pieter B T; Oostra, Ben; Rivadeneira, Fernanodo; Suchiman, Eka H D; Uitterlinden, Andre G; Willemsen, Gonneke; Wolffenbuttel, Bruce H; Wang, Jun; de Bakker, Paul I W; van Ommen, Gert-Jan; van Duijn, Cornelia M

2014-02-01

Within the Netherlands a national network of biobanks has been established (Biobanking and Biomolecular Research Infrastructure-Netherlands (BBMRI-NL)) as a national node of the European BBMRI. One of the aims of BBMRI-NL is to enrich biobanks with different types of molecular and phenotype data. Here, we describe the Genome of the Netherlands (GoNL), one of the projects within BBMRI-NL. GoNL is a whole-genome-sequencing project in a representative sample consisting of 250 trio-families from all provinces in the Netherlands, which aims to characterize DNA sequence variation in the Dutch population. The parent-offspring trios include adult individuals ranging in age from 19 to 87 years (mean=53 years; SD=16 years) from birth cohorts 1910-1994. Sequencing was done on blood-derived DNA from uncultured cells and accomplished coverage was 14-15x. The family-based design represents a unique resource to assess the frequency of regional variants, accurately reconstruct haplotypes by family-based phasing, characterize short indels and complex structural variants, and establish the rate of de novo mutational events. GoNL will also serve as a reference panel for imputation in the available genome-wide association studies in Dutch and other cohorts to refine association signals and uncover population-specific variants. GoNL will create a catalog of human genetic variation in this sample that is uniquely characterized with respect to micro-geographic location and a wide range of phenotypes. The resource will be made available to the research and medical community to guide the interpretation of sequencing projects. The present paper summarizes the global characteristics of the project.

Unique Phylogenetic Lineage Found in the Fusarium-like Clade after Re-examining BCCM/IHEM Fungal Culture Collection Material

PubMed Central

De Cremer, Koen; Piérard, Denis; Hendrickx, Marijke

2016-01-01

Recently, the Fusarium genus has been narrowed based upon phylogenetic analyses and a Fusarium-like clade was adopted. The few species of the Fusarium-like clade were moved to new, re-installed or existing genera or provisionally retained as "Fusarium." Only a limited number of reference strains and DNA marker sequences are available for this clade and not much is known about its actual species diversity. Here, we report six strains, preserved by the Belgian fungal culture collection BCCM/IHEM as a Fusarium species, that belong to the Fusarium-like clade. They showed a slow growth and produced pionnotes, typical morphological characteristics of many Fusarium-like species. Multilocus sequencing with comparative sequence analyses in GenBank and phylogenetic analyses, using reference sequences of type material, confirmed that they were indeed member of the Fusarium-like clade. One strain was identified as "Fusarium" ciliatum whereas another strain was identified as Fusicolla merismoides. The four remaining strains were shown to represent a unique phylogenetic lineage in the Fusarium-like clade and were also found morphologically distinct from other members of the Fusarium-like clade. Based upon phylogenetic considerations, a new genus, Pseudofusicolla gen. nov., and a new species, Pseudofusicolla belgica sp. nov., were installed for this lineage. A formal description is provided in this study. Additional sampling will be required to gather isolates other than the historical strains presented in the present study as well as to further reveal the actual species diversity in the Fusarium-like clade. PMID:27790062

Extensive genetic diversity, unique population structure and evidence of genetic exchange in the sexually transmitted parasite Trichomonas vaginalis.

PubMed

Conrad, Melissa D; Gorman, Andrew W; Schillinger, Julia A; Fiori, Pier Luigi; Arroyo, Rossana; Malla, Nancy; Dubey, Mohan Lal; Gonzalez, Jorge; Blank, Susan; Secor, William E; Carlton, Jane M

2012-01-01

Trichomonas vaginalis is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted infection world-wide. Despite its prevalence, little is known about the genetic diversity and population structure of this haploid parasite due to the lack of appropriate tools. The development of a panel of microsatellite makers and SNPs from mining the parasite's genome sequence has paved the way to a global analysis of the genetic structure of the pathogen and association with clinical phenotypes. Here we utilize a panel of T. vaginalis-specific genetic markers to genotype 235 isolates from Mexico, Chile, India, Australia, Papua New Guinea, Italy, Africa and the United States, including 19 clinical isolates recently collected from 270 women attending New York City sexually transmitted disease clinics. Using population genetic analysis, we show that T. vaginalis is a genetically diverse parasite with a unique population structure consisting of two types present in equal proportions world-wide. Parasites belonging to the two types (type 1 and type 2) differ significantly in the rate at which they harbor the T. vaginalis virus, a dsRNA virus implicated in parasite pathogenesis, and in their sensitivity to the widely-used drug, metronidazole. We also uncover evidence of genetic exchange, indicating a sexual life-cycle of the parasite despite an absence of morphologically-distinct sexual stages. Our study represents the first robust and comprehensive evaluation of global T. vaginalis genetic diversity and population structure. Our identification of a unique two-type structure, and the clinically relevant phenotypes associated with them, provides a new dimension for understanding T. vaginalis pathogenesis. In addition, our demonstration of the possibility of genetic exchange in the parasite has important implications for genetic research and control of the disease.

Multilocus Sequence Analysis of Nectar Pseudomonads Reveals High Genetic Diversity and Contrasting Recombination Patterns

PubMed Central

Álvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M.

2013-01-01

The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas ‘sensu stricto’ isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria. PMID:24116076

Viral Communities Associated with Human Pericardial Fluids in Idiopathic Pericarditis

PubMed Central

Fancello, Laura; Monteil, Sonia; Popgeorgiev, Nikolay; Rivet, Romain; Gouriet, Frédérique; Fournier, Pierre-Edouard; Raoult, Didier; Desnues, Christelle

2014-01-01

Pericarditis is a common human disease defined by inflammation of the pericardium. Currently, 40% to 85% of pericarditis cases have no identified etiology. Most of these cases are thought to be caused by an infection of undetected, unsuspected or unknown viruses. In this work, we used a culture- and sequence-independent approach to investigate the viral DNA communities present in human pericardial fluids. Seven viral metagenomes were generated from the pericardial fluid of patients affected by pericarditis of unknown etiology and one metagenome was generated from the pericardial fluid of a sudden infant death case. As a positive control we generated one metagenome from the pericardial fluid of a patient affected by pericarditis caused by herpesvirus type 3. Furthermore, we used as negative controls a total of 6 pericardial fluids from 6 different individuals affected by pericarditis of non-infectious origin: 5 of them were sequenced as a unique pool and the remaining one was sequenced separately. The results showed a significant presence of torque teno viruses especially in one patient, while herpesviruses and papillomaviruses were present in the positive control. Co-infections by different genotypes of the same viral type (torque teno viruses) or different viruses (herpesviruses and papillomaviruses) were observed. Sequences related to bacteriophages infecting Staphylococcus, Enterobacteria, Streptococcus, Burkholderia and Pseudomonas were also detected in three patients. This study detected torque teno viruses and papillomaviruses, for the first time, in human pericardial fluids. PMID:24690743

Bioprospecting Red Sea Coastal Ecosystems for Culturable Microorganisms and Their Antimicrobial Potential.

PubMed

Al-Amoudi, Soha; Essack, Magbubah; Simões, Marta F; Bougouffa, Salim; Soloviev, Irina; Archer, John A C; Lafi, Feras F; Bajic, Vladimir B

2016-09-10

Microorganisms that inhabit unchartered unique soil such as in the highly saline and hot Red Sea lagoons on the Saudi Arabian coastline, represent untapped sources of potentially new bioactive compounds. In this study, a culture-dependent approach was applied to three types of sediments: mangrove mud (MN), microbial mat (MM), and barren soil (BS), collected from Rabigh harbor lagoon (RHL) and Al-Kharrar lagoon (AKL). The isolated bacteria were evaluated for their potential to produce bioactive compounds. The phylogenetic characterization of 251 bacterial isolates based on the 16S rRNA gene sequencing, supported their assignment to five different phyla: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Planctomycetes. Fifteen putative novel species were identified based on a 16S rRNA gene sequence similarity to other strain sequences in the NCBI database, being ≤98%. We demonstrate that 49 of the 251 isolates exhibit the potential to produce antimicrobial compounds. Additionally, at least one type of biosynthetic gene sequence, responsible for the synthesis of secondary metabolites, was recovered from 25 of the 49 isolates. Moreover, 10 of the isolates had a growth inhibition effect towards Staphylococcus aureus, Salmonella typhimurium and Pseudomonas syringae. We report the previously unknown antimicrobial activity of B. borstelensis, P. dendritiformis and M. salipaludis against all three indicator pathogens. Our study demonstrates the evidence of diverse cultured microbes associated with the Red Sea harbor/lagoon environments and their potential to produce antimicrobial compounds.

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.

The Kolumbo submarine volcano of Santorini island is a large pool of bacterial strains with antimicrobial activity.

PubMed

Bourbouli, Maria; Katsifas, Efstathios A; Papathanassiou, Evangelos; Karagouni, Amalia D

2015-05-01

Microbes in hydrothermal vents with their unique secondary metabolism may represent an untapped potential source of new natural products. In this study, samples were collected from the hydrothermal field of Kolumbo submarine volcano in the Aegean Sea, in order to isolate bacteria with antimicrobial activity. Eight hundred and thirty-two aerobic heterotrophic bacteria were isolated and then differentiated through BOX-PCR analysis at the strain level into 230 genomic fingerprints, which were screened against 13 different type strains (pathogenic and nonpathogenic) of Gram-positive, Gram-negative bacteria and fungi. Forty-two out of 176 bioactive-producing genotypes (76 %) exhibited antimicrobial activity against at least four different type strains and were selected for 16S rDNA sequencing and screening for nonribosomal peptide (NRPS) and polyketide (PKS) synthases genes. The isolates were assigned to genus Bacillus and Proteobacteria, and 20 strains harbored either NRPS, PKS type I or both genes. This is the first report on the diversity of culturable mesophilic bacteria associated with antimicrobial activity from Kolumbo area; the extremely high proportion of antimicrobial-producing strains suggested that this unique environment may represent a potential reservoir of novel bioactive compounds.

Falsirhodobacter sp. alg1 Harbors Single Homologs of Endo and Exo-Type Alginate Lyases Efficient for Alginate Depolymerization

PubMed Central

Takahashi, Mami; Tanaka, Reiji; Miyake, Hideo; Shibata, Toshiyuki; Chow, Seinen; Kuroda, Kouichi; Ueda, Mitsuyoshi; Takeyama, Haruko

2016-01-01

Alginate-degrading bacteria play an important role in alginate degradation by harboring highly efficient and unique alginolytic genes. Although the general mechanism for alginate degradation by these bacteria is fairly understood, much is still required to fully exploit them. Here, we report the isolation of a novel strain, Falsirhodobacter sp. alg1, the first report for an alginate-degrading bacterium from the family Rhodobacteraceae. Genome sequencing reveals that strain alg1 harbors a primary alginate degradation pathway with only single homologs of an endo- and exo-type alginate lyase, AlyFRA and AlyFRB, which is uncommon among such bacteria. Subsequent functional analysis showed that both enzymes were extremely efficient to depolymerize alginate suggesting evolutionary interests in the acquirement of these enzymes. The exo-type alginate lyase, AlyFRB in particular could depolymerize alginate without producing intermediate products making it a highly efficient enzyme for the production of 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Based on our findings, we believe that the discovery of Falsirhodobacter sp. alg1 and its alginolytic genes hints at the potentiality of a more diverse and unique population of alginate-degrading bacteria. PMID:27176711

Geoseq: a tool for dissecting deep-sequencing datasets.

PubMed

Gurtowski, James; Cancio, Anthony; Shah, Hardik; Levovitz, Chaya; George, Ajish; Homann, Robert; Sachidanandam, Ravi

2010-10-12

Datasets generated on deep-sequencing platforms have been deposited in various public repositories such as the Gene Expression Omnibus (GEO), Sequence Read Archive (SRA) hosted by the NCBI, or the DNA Data Bank of Japan (ddbj). Despite being rich data sources, they have not been used much due to the difficulty in locating and analyzing datasets of interest. Geoseq http://geoseq.mssm.edu provides a new method of analyzing short reads from deep sequencing experiments. Instead of mapping the reads to reference genomes or sequences, Geoseq maps a reference sequence against the sequencing data. It is web-based, and holds pre-computed data from public libraries. The analysis reduces the input sequence to tiles and measures the coverage of each tile in a sequence library through the use of suffix arrays. The user can upload custom target sequences or use gene/miRNA names for the search and get back results as plots and spreadsheet files. Geoseq organizes the public sequencing data using a controlled vocabulary, allowing identification of relevant libraries by organism, tissue and type of experiment. Analysis of small sets of sequences against deep-sequencing datasets, as well as identification of public datasets of interest, is simplified by Geoseq. We applied Geoseq to, a) identify differential isoform expression in mRNA-seq datasets, b) identify miRNAs (microRNAs) in libraries, and identify mature and star sequences in miRNAS and c) to identify potentially mis-annotated miRNAs. The ease of using Geoseq for these analyses suggests its utility and uniqueness as an analysis tool.

Novel numerical and graphical representation of DNA sequences and proteins.

PubMed

Randić, M; Novic, M; Vikić-Topić, D; Plavsić, D

2006-12-01

We have introduced novel numerical and graphical representations of DNA, which offer a simple and unique characterization of DNA sequences. The numerical representation of a DNA sequence is given as a sequence of real numbers derived from a unique graphical representation of the standard genetic code. There is no loss of information on the primary structure of a DNA sequence associated with this numerical representation. The novel representations are illustrated with the coding sequences of the first exon of beta-globin gene of half a dozen species in addition to human. The method can be extended to proteins as is exemplified by humanin, a 24-aa peptide that has recently been identified as a specific inhibitor of neuronal cell death induced by familial Alzheimer's disease mutant genes.

Real-Time PCR Assay for a Unique Chromosomal Sequence of Bacillus anthracis

DTIC Science & Technology

2004-12-01

13061 Neisseria lactamica .............................................................. 23970 Bacillus coagulans ...NEG Bacillus coagulane 7050 NEG NEG Bacillus cereus 13472 NEG NEG Bacillus licheniforms 12759 NEG NEG Bacillus cereus 13824 NEG NEG Bacillus ...Assay for a Unique Chromosomal Sequence of Bacillus anthracis Elizabeth Bode,1 William Hurtle,2† and David Norwood1* United States Army Medical

Draft Genome Sequence of the Spore-Forming Probiotic Strain Bacillus coagulans Unique IS-2

PubMed Central

Upadrasta, Aditya; Pitta, Swetha

2016-01-01

Bacillus coagulans Unique IS-2 is a potential spore-forming probiotic that is commercially available on the market. The draft genome sequence presented here provides deep insight into the beneficial features of this strain for its safe use as a probiotic for various human and animal health applications. PMID:27103709

Virulence factors encoded by Legionella longbeachae identified on the basis of the genome sequence analysis of clinical isolate D-4968.

PubMed

Kozak, Natalia A; Buss, Meghan; Lucas, Claressa E; Frace, Michael; Govil, Dhwani; Travis, Tatiana; Olsen-Rasmussen, Melissa; Benson, Robert F; Fields, Barry S

2010-02-01

Legionella longbeachae causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of L. longbeachae-specific diagnostic tests. L. longbeachae displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to Legionella pneumophila, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems, L. longbeachae is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an L. longbeachae clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of L. pneumophila. The results revealed that the D-4968 genome is larger than the L. pneumophila genome and has a gene order that is different from that of the L. pneumophila genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding L. pneumophila Icm/Dot substrates have been found in the D-4968 genome. L. longbeachae encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the L. longbeachae Icm/Dot secretion system. In contrast to the L. pneumophila genome, the L. longbeachae D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of L. longbeachae to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of L. longbeachae may reflect adaptation of this species to life in soil.

Small non-coding RNA profiling in human biofluids and surrogate tissues from healthy individuals: description of the diverse and most represented species.

PubMed

Ferrero, Giulio; Cordero, Francesca; Tarallo, Sonia; Arigoni, Maddalena; Riccardo, Federica; Gallo, Gaetano; Ronco, Guglielmo; Allasia, Marco; Kulkarni, Neha; Matullo, Giuseppe; Vineis, Paolo; Calogero, Raffaele A; Pardini, Barbara; Naccarati, Alessio

2018-01-09

The role of non-coding RNAs in different biological processes and diseases is continuously expanding. Next-generation sequencing together with the parallel improvement of bioinformatics analyses allows the accurate detection and quantification of an increasing number of RNA species. With the aim of exploring new potential biomarkers for disease classification, a clear overview of the expression levels of common/unique small RNA species among different biospecimens is necessary. However, except for miRNAs in plasma, there are no substantial indications about the pattern of expression of various small RNAs in multiple specimens among healthy humans. By analysing small RNA-sequencing data from 243 samples, we have identified and compared the most abundantly and uniformly expressed miRNAs and non-miRNA species of comparable size with the library preparation in four different specimens (plasma exosomes, stool, urine, and cervical scrapes). Eleven miRNAs were commonly detected among all different specimens while 231 miRNAs were globally unique across them. Classification analysis using these miRNAs provided an accuracy of 99.6% to recognize the sample types. piRNAs and tRNAs were the most represented non-miRNA small RNAs detected in all specimen types that were analysed, particularly in urine samples. With the present data, the most uniformly expressed small RNAs in each sample type were also identified. A signature of small RNAs for each specimen could represent a reference gene set in validation studies by RT-qPCR. Overall, the data reported hereby provide an insight of the constitution of the human miRNome and of other small non-coding RNAs in various specimens of healthy individuals.

Bacterial community composition in Brazilian Anthrosols and adjacent soils characterized using culturing and molecular identification.

PubMed

O'Neill, B; Grossman, J; Tsai, M T; Gomes, J E; Lehmann, J; Peterson, J; Neves, E; Thies, J E

2009-07-01

Microbial community composition was examined in two soil types, Anthrosols and adjacent soils, sampled from three locations in the Brazilian Amazon. The Anthrosols, also known as Amazonian dark earths, are highly fertile soils that are a legacy of pre-Columbian settlement. Both Anthrosols and adjacent soils are derived from the same parent material and subject to the same environmental conditions, including rainfall and temperature; however, the Anthrosols contain high levels of charcoal-like black carbon from which they derive their dark color. The Anthrosols typically have higher cation exchange capacity, higher pH, and higher phosphorus and calcium contents. We used culture media prepared from soil extracts to isolate bacteria unique to the two soil types and then sequenced their 16S rRNA genes to determine their phylogenetic placement. Higher numbers of culturable bacteria, by over two orders of magnitude at the deepest sampling depths, were counted in the Anthrosols. Sequences of bacteria isolated on soil extract media yielded five possible new bacterial families. Also, a higher number of families in the bacteria were represented by isolates from the deeper soil depths in the Anthrosols. Higher bacterial populations and a greater diversity of isolates were found in all of the Anthrosols, to a depth of up to 1 m, compared to adjacent soils located within 50-500 m of their associated Anthrosols. Compared to standard culture media, soil extract media revealed diverse soil microbial populations adapted to the unique biochemistry and physiological ecology of these Anthrosols.

Sequence of a new DR12 allele with two silent mutations that affect PCR-SSP typing.

PubMed

Zanone, R; Bettens, F; Tiercy, J-M

2002-02-01

A new HLA-DR12 allele has been identified in a European Caucasoid bone marrow donor. The DRB1*12012 allele differs from DRB1*12011 by two silent substitutions at codons 72 and 78, two polymorphic positions used for DNA subtyping of the DR12 serotype. The co-occurence of the two nucleotide changes is unique to the DR12 group and results in a new PCR-SSP typing pattern. The complete HLA type of the donor is A24, A68; B55, B61; Cw*01, Cw*0304; DRB1*12012, DRB1*1402; DRB3*0101, DRB3*0202; DQB1*0301. HLA-DRB1*12012 is a rare allele as it occurs in < 0.2% of DR12 donors.

Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Ckap4 as a New Modulator of Fibroblasts Activation.

PubMed

Gladka, Monika M; Molenaar, Bas; de Ruiter, Hesther; van der Elst, Stefan; Tsui, Hoyee; Versteeg, Danielle; Lacraz, Grègory P A; Huibers, Manon M H; van Oudenaarden, Alexander; van Rooij, Eva

2018-01-31

Background -Genome-wide transcriptome analysis has greatly advanced our understanding of the regulatory networks underlying basic cardiac biology and mechanisms driving disease. However, so far, the resolution of studying gene expression patterns in the adult heart has been limited to the level of extracts from whole tissues. The use of tissue homogenates inherently causes the loss of any information on cellular origin or cell type-specific changes in gene expression. Recent developments in RNA amplification strategies provide a unique opportunity to use small amounts of input RNA for genome-wide sequencing of single cells. Methods -Here, we present a method to obtain high quality RNA from digested cardiac tissue from adult mice for automated single-cell sequencing of both the healthy and diseased heart. Results -After optimization, we were able to perform single-cell sequencing on adult cardiac tissue under both homeostatic conditions and after ischemic injury. Clustering analysis based on differential gene expression unveiled known and novel markers of all main cardiac cell types. Based on differential gene expression we were also able to identify multiple subpopulations within a certain cell type. Furthermore, applying single-cell sequencing on both the healthy and the injured heart indicated the presence of disease-specific cell subpopulations. As such, we identified cytoskeleton associated protein 4 ( Ckap4 ) as a novel marker for activated fibroblasts that positively correlates with known myofibroblast markers in both mouse and human cardiac tissue. Ckap4 inhibition in activated fibroblasts treated with TGFβ triggered a greater increase in the expression of genes related to activated fibroblasts compared to control, suggesting a role of Ckap4 in modulating fibroblast activation in the injured heart. Conclusions -Single-cell sequencing on both the healthy and diseased adult heart allows us to study transcriptomic differences between cardiac cells, as well as cell type-specific changes in gene expression during cardiac disease. This new approach provides a wealth of novel insights into molecular changes that underlie the cellular processes relevant for cardiac biology and pathophysiology. Applying this technology could lead to the discovery of new therapeutic targets relevant for heart disease.

Population structure and genetic diversity of the parasite Trichomonas vaginalis in Bristol, UK.

PubMed

Hawksworth, Joseph; Levy, Max; Smale, Chloe; Cheung, Dean; Whittle, Alice; Longhurst, Denise; Muir, Peter; Gibson, Wendy

2015-08-01

The protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, an extremely common, but non-life-threatening, sexually-transmitted disease throughout the world. Recent population genetics studies of T. vaginalis have detected high genetic diversity and revealed a two-type population structure, associated with phenotypic differences in sensitivity to metronidazole, the drug commonly used for treatment, and presence of T. vaginalis virus. There is currently a lack of data on UK isolates; most isolates examined to date are from the US. Here we used a recently described system for multilocus sequence typing (MLST) of T. vaginalis to study diversity of clinical isolates from Bristol, UK. We used MLST to characterise 23 clinical isolates of T. vaginalis collected from female patients during 2013. Seven housekeeping genes were PCR-amplified for each isolate and sequenced. The concatenated sequences were then compared with data from other MLST-characterised isolates available from http://tvaginalis.mlst.net/ to analyse the population structure and construct phylogenetic trees. Among the 23 isolates from the Bristol population of T. vaginalis, we found 23 polymorphic nucleotide sites, 25 different alleles and 19 sequence types (genotypes). Most isolates had a unique genotype, in agreement with the high levels of heterogeneity observed elsewhere in the world. A two-type population structure was evident from population genetic analysis and phylogenetic reconstruction split the isolates into two major clades. Tests for recombination in the Bristol population of T. vaginalis gave conflicting results, suggesting overall a clonal pattern of reproduction. We conclude that the Bristol population of T. vaginalis parasites conforms to the two-type population structure found in most other regions of the world. We found the MLST scheme to be an efficient genotyping method. The online MLST database provides a useful repository and resource that will prove invaluable in future studies linking the genetics of T. vaginalis with the clinical manifestation of trichomoniasis. Copyright © 2015 Elsevier B.V. All rights reserved.

Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes

PubMed Central

Shiroguchi, Katsuyuki; Jia, Tony Z.; Sims, Peter A.; Xie, X. Sunney

2012-01-01

RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic to exponential PCR amplification. We developed a simple strategy for mitigating these complications, allowing truly digital RNA-Seq. Following reverse transcription, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends. After PCR, we applied paired-end deep sequencing to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence. We optimized the barcodes to be unambiguously identifiable, even in the presence of multiple sequencing errors. This method allows counting with single-copy resolution despite sequence-dependent bias and PCR-amplification noise, and is analogous to digital PCR but amendable to quantifying a whole transcriptome. We demonstrated transcriptome profiling of Escherichia coli with more accurate and reproducible quantification than conventional RNA-Seq. PMID:22232676

Sequencing Needs for Viral Diagnostics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, S N; Lam, M; Mulakken, N J

2004-01-26

We built a system to guide decisions regarding the amount of genomic sequencing required to develop diagnostic DNA signatures, which are short sequences that are sufficient to uniquely identify a viral species. We used our existing DNA diagnostic signature prediction pipeline, which selects regions of a target species genome that are conserved among strains of the target (for reliability, to prevent false negatives) and unique relative to other species (for specificity, to avoid false positives). We performed simulations, based on existing sequence data, to assess the number of genome sequences of a target species and of close phylogenetic relatives (''nearmore » neighbors'') that are required to predict diagnostic signature regions that are conserved among strains of the target species and unique relative to other bacterial and viral species. For DNA viruses such as variola (smallpox), three target genomes provide sufficient guidance for selecting species-wide signatures. Three near neighbor genomes are critical for species specificity. In contrast, most RNA viruses require four target genomes and no near neighbor genomes, since lack of conservation among strains is more limiting than uniqueness. SARS and Ebola Zaire are exceptional, as additional target genomes currently do not improve predictions, but near neighbor sequences are urgently needed. Our results also indicate that double stranded DNA viruses are more conserved among strains than are RNA viruses, since in most cases there was at least one conserved signature candidate for the DNA viruses and zero conserved signature candidates for the RNA viruses.« less

Search for Variables in the Kepler Field on DASCH Plates

NASA Astrophysics Data System (ADS)

Tang, Sumin; Grindlay, J.; Los, E.; Servillat, M.

2011-01-01

The Digital Access to a Sky Century @ Harvard (DASCH) is a project to digitize the half a million glass photographic plates over the period 1880s-1980s. This 100 year coverage is a unique resource for studying temporal variations in the universe. Here we present our variable search algorithms and variable catalog in the Kepler fields based on 3000 scanned plates. We use the KIC spectral classifications to search for long-term variability of any main sequence stars, particularly M dwarfs. We apply a variability search technique developed for DASCH and set limits on the fraction of main sequence stars, by spectral type, which show detectable (>0.2mag) variability on timescales 10-100y. Such limits are of particular interest for M dwarfs given the recent discoveries of their planet systems.

Circularized Chromosome with a Large Palindromic Structure in Streptomyces griseus Mutants

PubMed Central

Uchida, Tetsuya; Ishihara, Naoto; Zenitani, Hiroyuki; Hiratsu, Keiichiro; Kinashi, Haruyasu

2004-01-01

Streptomyces linear chromosomes display various types of rearrangements after telomere deletion, including circularization, arm replacement, and amplification. We analyzed the new chromosomal deletion mutants Streptomyces griseus 301-22-L and 301-22-M. In these mutants, chromosomal arm replacement resulted in long terminal inverted repeats (TIRs) at both ends; different sizes were deleted again and recombined inside the TIRs, resulting in a circular chromosome with an extremely large palindrome. Short palindromic sequences were found in parent strain 2247, and these sequences might have played a role in the formation of this unique structure. Dynamic structural changes of Streptomyces linear chromosomes shown by this and previous studies revealed extraordinary strategies of members of this genus to keep a functional chromosome, even if it is linear or circular. PMID:15150216

Principles of protein folding--a perspective from simple exact models.

PubMed Central

Dill, K. A.; Bromberg, S.; Yue, K.; Fiebig, K. M.; Yee, D. P.; Thomas, P. D.; Chan, H. S.

1995-01-01

General principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models represent protein chains at a rudimentary level, but they involve few parameters, approximations, or implicit biases, and they allow complete explorations of conformational and sequence spaces. Such simulations have resulted in testable predictions that are sometimes unanticipated: The folding code is mainly binary and delocalized throughout the amino acid sequence. The secondary and tertiary structures of a protein are specified mainly by the sequence of polar and nonpolar monomers. More specific interactions may refine the structure, rather than dominate the folding code. Simple exact models can account for the properties that characterize protein folding: two-state cooperativity, secondary and tertiary structures, and multistage folding kinetics--fast hydrophobic collapse followed by slower annealing. These studies suggest the possibility of creating "foldable" chain molecules other than proteins. The encoding of a unique compact chain conformation may not require amino acids; it may require only the ability to synthesize specific monomer sequences in which at least one monomer type is solvent-averse. PMID:7613459

Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays.

PubMed

Horn, T; Chang, C A; Urdea, M S

1997-12-01

The divergent synthesis of bDNA structures is described. This new type of branched DNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branching network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb molecules were assembled on a solid support using parameters optimized for bDNA synthesis. The chemistry was used to synthesize bDNA comb molecules containing 15 secondary sequences. The bDNA comb molecules were elaborated by enzymatic ligation into branched amplification multimers, large bDNA molecules (a total of 1068 nt) containing an average of 36 repeated DNA oligomer sequences, each capable of hybridizing specifically to an alkaline phosphatase-labeled oligonucleotide. The bDNA comb molecules were characterized by electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The branched amplification multimers have been used as signal amplifiers in nucleic acid quantification assays for detection of viral infection. It is possible to detect as few as 50 molecules with bDNA technology.

Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays.

PubMed Central

Horn, T; Chang, C A; Urdea, M S

1997-01-01

The divergent synthesis of bDNA structures is described. This new type of branched DNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branching network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb molecules were assembled on a solid support using parameters optimized for bDNA synthesis. The chemistry was used to synthesize bDNA comb molecules containing 15 secondary sequences. The bDNA comb molecules were elaborated by enzymatic ligation into branched amplification multimers, large bDNA molecules (a total of 1068 nt) containing an average of 36 repeated DNA oligomer sequences, each capable of hybridizing specifically to an alkaline phosphatase-labeled oligonucleotide. The bDNA comb molecules were characterized by electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The branched amplification multimers have been used as signal amplifiers in nucleic acid quantification assays for detection of viral infection. It is possible to detect as few as 50 molecules with bDNA technology. PMID:9365266

NEBNext Direct: A Novel, Rapid, Hybridization-Based Approach for the Capture and Library Conversion of Genomic Regions of Interest.

PubMed

Emerman, Amy B; Bowman, Sarah K; Barry, Andrew; Henig, Noa; Patel, Kruti M; Gardner, Andrew F; Hendrickson, Cynthia L

2017-07-05

Next-generation sequencing (NGS) is a powerful tool for genomic studies, translational research, and clinical diagnostics that enables the detection of single nucleotide polymorphisms, insertions and deletions, copy number variations, and other genetic variations. Target enrichment technologies improve the efficiency of NGS by only sequencing regions of interest, which reduces sequencing costs while increasing coverage of the selected targets. Here we present NEBNext Direct ® , a hybridization-based, target-enrichment approach that addresses many of the shortcomings of traditional target-enrichment methods. This approach features a simple, 7-hr workflow that uses enzymatic removal of off-target sequences to achieve a high specificity for regions of interest. Additionally, unique molecular identifiers are incorporated for the identification and filtering of PCR duplicates. The same protocol can be used across a wide range of input amounts, input types, and panel sizes, enabling NEBNext Direct to be broadly applicable across a wide variety of research and diagnostic needs. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Community-led comparative genomic and phenotypic analysis of the aquaculture pathogen Pseudomonas baetica a390T sequenced by Ion semiconductor and Nanopore technologies

PubMed Central

Beaton, Ainsley; Lood, Cédric; Cunningham-Oakes, Edward; MacFadyen, Alison; Mullins, Alex J; Bestawy, Walid El; Botelho, João; Chevalier, Sylvie; Dalzell, Chloe; Dolan, Stephen K; Faccenda, Alberto; Ghequire, Maarten G K; Higgins, Steven; Kutschera, Alexander; Murray, Jordan; Redway, Martha; Salih, Talal; Smith, Brian A; Smits, Nathan; Thomson, Ryan; Woodcock, Stuart; Cornelis, Pierre; Lavigne, Rob; van Noort, Vera

2018-01-01

Abstract Pseudomonas baetica strain a390T is the type strain of this recently described species and here we present its high-contiguity draft genome. To celebrate the 16th International Conference on Pseudomonas, the genome of P. baetica strain a390T was sequenced using a unique combination of Ion Torrent semiconductor and Oxford Nanopore methods as part of a collaborative community-led project. The use of high-quality Ion Torrent sequences with long Nanopore reads gave rapid, high-contiguity and -quality, 16-contig genome sequence. Whole genome phylogenetic analysis places P. baetica within the P. koreensis clade of the P. fluorescens group. Comparison of the main genomic features of P. baetica with a variety of other Pseudomonas spp. suggests that it is a highly adaptable organism, typical of the genus. This strain was originally isolated from the liver of a diseased wedge sole fish, and genotypic and phenotypic analyses show that it is tolerant to osmotic stress and to oxytetracycline. PMID:29579234

Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq

PubMed Central

Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

2015-01-01

Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

The Human Microbiome and Understanding the 16S rRNA Gene in Translational Nursing Science.

PubMed

Ames, Nancy J; Ranucci, Alexandra; Moriyama, Brad; Wallen, Gwenyth R

As more is understood regarding the human microbiome, it is increasingly important for nurse scientists and healthcare practitioners to analyze these microbial communities and their role in health and disease. 16S rRNA sequencing is a key methodology in identifying these bacterial populations that has recently transitioned from use primarily in research to having increased utility in clinical settings. The objectives of this review are to (a) describe 16S rRNA sequencing and its role in answering research questions important to nursing science; (b) provide an overview of the oral, lung, and gut microbiomes and relevant research; and (c) identify future implications for microbiome research and 16S sequencing in translational nursing science. Sequencing using the 16S rRNA gene has revolutionized research and allowed scientists to easily and reliably characterize complex bacterial communities. This type of research has recently entered the clinical setting, one of the best examples involving the use of 16S sequencing to identify resistant pathogens, thereby improving the accuracy of bacterial identification in infection control. Clinical microbiota research and related requisite methods are of particular relevance to nurse scientists-individuals uniquely positioned to utilize these techniques in future studies in clinical settings.

Biosynthetic Potential of Phylogenetically Unique Endophytic Actinomycetes from Tropical Plants▿ †

PubMed Central

Janso, Jeffrey E.; Carter, Guy T.

2010-01-01

The culturable diversity of endophytic actinomycetes associated with tropical, native plants is essentially unexplored. In this study, 123 endophytic actinomycetes were isolated from tropical plants collected from several locations in Papua New Guinea and Mborokua Island, Solomon Islands. Isolates were found to be prevalent in roots but uncommon in leaves. Initially, isolates were dereplicated to the strain level by ribotyping. Subsequent characterization of 105 unique strains by 16S rRNA gene sequence analysis revealed that 17 different genera were represented, and rare genera, such as Sphaerisporangium and Planotetraspora, which have never been previously reported to be endophytic, were quite prevalent. Phylogenetic analyses grouped many of the strains into clades distinct from known genera within Thermomonosporaceae and Micromonosporaceae, indicating that they may be unique genera. Bioactivity testing and liquid chromatography-mass spectrometry (LC-MS) profiling of crude fermentation extracts were performed on 91 strains. About 60% of the extracts exhibited bioactivity or displayed LC-MS profiles with spectra indicative of secondary metabolites. The biosynthetic potential of 29 nonproductive strains was further investigated by the detection of putative polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes. Despite their lack of detectable secondary metabolite production in fermentation, most were positive for type I (66%) and type II (79%) PKS genes, and all were positive for NRPS genes. These results suggest that tropical plants from New Guinea and the adjacent archipelago are hosts to unique endophytic actinomycetes that possess significant biosynthetic potential. PMID:20472734

General and inducible hypermutation facilitate parallel adaptation in Pseudomonas aeruginosa despite divergent mutation spectra.

PubMed

Weigand, Michael R; Sundin, George W

2012-08-21

The successful growth of hypermutator strains of bacteria contradicts a clear preference for lower mutation rates observed in the microbial world. Whether by general DNA repair deficiency or the inducible action of low-fidelity DNA polymerases, the evolutionary strategies of bacteria include methods of hypermutation. Although both raise mutation rate, general and inducible hypermutation operate through distinct molecular mechanisms and therefore likely impart unique adaptive consequences. Here we compare the influence of general and inducible hypermutation on adaptation in the model organism Pseudomonas aeruginosa PAO1 through experimental evolution. We observed divergent spectra of single base substitutions derived from general and inducible hypermutation by sequencing rpoB in spontaneous rifampicin-resistant (Rif(R)) mutants. Likewise, the pattern of mutation in a draft genome sequence of a derived inducible hypermutator isolate differed from those of general hypermutators reported in the literature. However, following experimental evolution, populations of both mutator types exhibited comparable improvements in fitness across varied conditions that differed from the highly specific adaptation of nonmutators. Our results suggest that despite their unique mutation spectra, general and inducible hypermutation can analogously influence the ecology and adaptation of bacteria, significantly shaping pathogenic populations where hypermutation has been most widely observed.

Sequencing Adventure Activities: A New Perspective.

ERIC Educational Resources Information Center

Bisson, Christian

Sequencing in adventure education involves putting activities in an order appropriate to the needs of the group. Contrary to the common assumption that each adventure sequence is unique, a review of literature concerning five sequencing models reveals a certain universality. These models present sequences that move through four phases: group…

Molecular Epidemiology and Phylogeny Reveal Complex Spatial Dynamics in Areas Where Canine Parvovirus Is Endemic ▿†

PubMed Central

Clegg, S. R.; Coyne, K. P.; Parker, J.; Dawson, S.; Godsall, S. A.; Pinchbeck, G.; Cripps, P. J.; Gaskell, R. M.; Radford, A. D.

2011-01-01

Canine parvovirus type 2 (CPV-2) is a severe enteric pathogen of dogs, causing high mortality in unvaccinated dogs. After emerging, CPV-2 spread rapidly worldwide. However, there is now some evidence to suggest that international transmission appears to be more restricted. In order to investigate the transmission and evolution of CPV-2 both nationally and in relation to the global situation, we have used a long-range PCR to amplify and sequence the full VP2 gene of 150 canine parvoviruses obtained from a large cross-sectional sample of dogs presenting with severe diarrhea to veterinarians in the United Kingdom, over a 2-year period. Among these 150 strains, 50 different DNA sequence types (S) were identified, and apart from one case, all appeared unique to the United Kingdom. Phylogenetic analysis provided clear evidence for spatial clustering at the international level and for the first time also at the national level, with the geographical range of some sequence types appearing to be highly restricted within the United Kingdom. Evolution of the VP2 gene in this data set was associated with a lack of positive selection. In addition, the majority of predicted amino acid sequences were identical to those found elsewhere in the world, suggesting that CPV VP2 has evolved a highly fit conformation. Based on typing systems using key amino acid mutations, 43% of viruses were CPV-2a, and 57% CPV-2b, with no type 2 or 2c found. However, phylogenetic analysis suggested complex antigenic evolution of this virus, with both type 2a and 2b viruses appearing polyphyletic. As such, typing based on specific amino acid mutations may not reflect the true epidemiology of this virus. The geographical restriction that we observed both within the United Kingdom and between the United Kingdom and other countries, together with the lack of CPV-2c in this population, strongly suggests the spread of CPV within its population may be heterogeneously subject to limiting factors. This cross-sectional study of national and global CPV phylogeographic segregation reveals a substantially more complex epidemic structure than previously described. PMID:21593180

The neXtProt peptide uniqueness checker: a tool for the proteomics community.

PubMed

Schaeffer, Mathieu; Gateau, Alain; Teixeira, Daniel; Michel, Pierre-André; Zahn-Zabal, Monique; Lane, Lydie

2017-11-01

The neXtProt peptide uniqueness checker allows scientists to define which peptides can be used to validate the existence of human proteins, i.e. map uniquely versus multiply to human protein sequences taking into account isobaric substitutions, alternative splicing and single amino acid variants. The pepx program is available at https://github.com/calipho-sib/pepx and can be launched from the command line or through a cgi web interface. Indexing requires a sequence file in FASTA format. The peptide uniqueness checker tool is freely available on the web at https://www.nextprot.org/tools/peptide-uniqueness-checker and from the neXtProt API at https://api.nextprot.org/. lydie.lane@sib.swiss. © The Author(s) 2017. Published by Oxford University Press.

A Unique Sequence of Financial Accounting Courses Featuring Team Teaching, Linked Courses, Challenging Assignments, and Instruments for Evaluation and Assessment

ERIC Educational Resources Information Center

Lundblad, Heidemarie; Wilson, Barbara A.

2008-01-01

The Department of Accounting at California State University Northridge (CSUN) has developed a unique sequence of courses designed to ensure that accounting students are trained not only in technical accounting, but also acquire critical thinking, research and communication skills. The courses have proven effective and have embedded assessment…

Conformation of Tax-response elements in the human T-cell leukemia virus type I promoter.

PubMed

Cox, J M; Sloan, L S; Schepartz, A

1995-12-01

HTLV-I Tax is believed to activate viral gene expression by binding bZIP proteins (such as CREB) and increasing their affinities for proviral TRE target sites. Each 21 bp TRE target site contains an imperfect copy of the intrinsically bent CRE target site (the TRE core) surrounded by highly conserved flanking sequences. These flanking sequences are essential for maximal increases in DNA affinity and transactivation, but they are not, apparently, contacted by protein. Here we employ non-denaturing gel electrophoresis to evaluate TRE conformation in the presence and absence of bZIP proteins, and to explore the role of DNA conformation in viral transactivation. Our results show that the TRE-1 flanking sequences modulate the structure and modestly increase the affinity of a CREB bZIP peptide for the TRE-1 core recognition sequence. These flanking sequences are also essential for a maximal increase in stability of the CREB-DNA complex in the presence of Tax. The CRE-like TRE core and the TRE flanking sequences are both essential for formation of stable CREB-TRE-1 and Tax-CREB-TRE-1 complexes. These two DNA segments may have co-evolved into a unique structure capable of recognizing Tax and a bZIP protein.

Strain-Level Diversity of Secondary Metabolism in Streptomyces albus

PubMed Central

Seipke, Ryan F.

2015-01-01

Streptomyces spp. are robust producers of medicinally-, industrially- and agriculturally-important small molecules. Increased resistance to antibacterial agents and the lack of new antibiotics in the pipeline have led to a renaissance in natural product discovery. This endeavor has benefited from inexpensive high quality DNA sequencing technology, which has generated more than 140 genome sequences for taxonomic type strains and environmental Streptomyces spp. isolates. Many of the sequenced streptomycetes belong to the same species. For instance, Streptomyces albus has been isolated from diverse environmental niches and seven strains have been sequenced, consequently this species has been sequenced more than any other streptomycete, allowing valuable analyses of strain-level diversity in secondary metabolism. Bioinformatics analyses identified a total of 48 unique biosynthetic gene clusters harboured by Streptomyces albus strains. Eighteen of these gene clusters specify the core secondary metabolome of the species. Fourteen of the gene clusters are contained by one or more strain and are considered auxiliary, while 16 of the gene clusters encode the production of putative strain-specific secondary metabolites. Analysis of Streptomyces albus strains suggests that each strain of a Streptomyces species likely harbours at least one strain-specific biosynthetic gene cluster. Importantly, this implies that deep sequencing of a species will not exhaust gene cluster diversity and will continue to yield novelty. PMID:25635820

VISA--Vector Integration Site Analysis server: a web-based server to rapidly identify retroviral integration sites from next-generation sequencing.

PubMed

Hocum, Jonah D; Battrell, Logan R; Maynard, Ryan; Adair, Jennifer E; Beard, Brian C; Rawlings, David J; Kiem, Hans-Peter; Miller, Daniel G; Trobridge, Grant D

2015-07-07

Analyzing the integration profile of retroviral vectors is a vital step in determining their potential genotoxic effects and developing safer vectors for therapeutic use. Identifying retroviral vector integration sites is also important for retroviral mutagenesis screens. We developed VISA, a vector integration site analysis server, to analyze next-generation sequencing data for retroviral vector integration sites. Sequence reads that contain a provirus are mapped to the human genome, sequence reads that cannot be localized to a unique location in the genome are filtered out, and then unique retroviral vector integration sites are determined based on the alignment scores of the remaining sequence reads. VISA offers a simple web interface to upload sequence files and results are returned in a concise tabular format to allow rapid analysis of retroviral vector integration sites.

Ensemble codes involving hippocampal neurons are at risk during delayed performance tests.

PubMed

Hampson, R E; Deadwyler, S A

1996-11-26

Multielectrode recording techniques were used to record ensemble activity from 10 to 16 simultaneously active CA1 and CA3 neurons in the rat hippocampus during performance of a spatial delayed-nonmatch-to-sample task. Extracted sources of variance were used to assess the nature of two different types of errors that accounted for 30% of total trials. The two types of errors included ensemble "miscodes" of sample phase information and errors associated with delay-dependent corruption or disappearance of sample information at the time of the nonmatch response. Statistical assessment of trial sequences and associated "strength" of hippocampal ensemble codes revealed that miscoded error trials always followed delay-dependent error trials in which encoding was "weak," indicating that the two types of errors were "linked." It was determined that the occurrence of weakly encoded, delay-dependent error trials initiated an ensemble encoding "strategy" that increased the chances of being correct on the next trial and avoided the occurrence of further delay-dependent errors. Unexpectedly, the strategy involved "strongly" encoding response position information from the prior (delay-dependent) error trial and carrying it forward to the sample phase of the next trial. This produced a miscode type error on trials in which the "carried over" information obliterated encoding of the sample phase response on the next trial. Application of this strategy, irrespective of outcome, was sufficient to reorient the animal to the proper between trial sequence of response contingencies (nonmatch-to-sample) and boost performance to 73% correct on subsequent trials. The capacity for ensemble analyses of strength of information encoding combined with statistical assessment of trial sequences therefore provided unique insight into the "dynamic" nature of the role hippocampus plays in delay type memory tasks.

VizieR Online Data Catalog: Far-UV spectral atlas of O-type stars (Smith, 2012)

NASA Astrophysics Data System (ADS)

Smith, M. A.

2012-10-01

In this paper, we present a spectral atlas covering the wavelength interval 930-1188Å for O2-O9.5 stars using Far-Ultraviolet Spectroscopic Explorer archival data. The stars selected for the atlas were drawn from three populations: Galactic main-sequence (classes III-V) stars, supergiants, and main-sequence stars in the Magellanic Clouds, which have low metallicities. For several of these stars, we have prepared FITS files comprised of pairs of merged spectra for user access via the Multimission Archive at Space Telescope (MAST). We chose spectra from the first population with spectral types O4, O5, O6, O7, O8, and O9.5 and used them to compile tables and figures with identifications of all possible atmospheric and interstellar medium lines in the region 949-1188Å. Our identified line totals for these six representative spectra are 821 (500), 992 (663), 1077 (749), 1178 (847), 1359 (1001), and 1798 (1392) lines, respectively, where the numbers in parentheses are the totals of lines formed in the atmospheres, according to spectral synthesis models. The total number of unique atmospheric identifications for the six main-sequence O-star template spectra is 1792, whereas the number of atmospheric lines in common to these spectra is 300. The number of identified lines decreases toward earlier types (increasing effective temperature), while the percentages of "missed" features (unknown lines not predicted from our spectral syntheses) drop from a high of 8% at type B0.2, from our recently published B-star far-UV atlas (Cat. J/ApJS/186/175), to 1%-3% for type O spectra. The percentages of overpredicted lines are similar, despite their being much higher for B-star spectra. (4 data files).

Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Acinetobacter baumannii and Interlaboratory Validation of an Optimized Multiple-Locus VNTR Analysis Typing Scheme▿†

PubMed Central

Pourcel, Christine; Minandri, Fabrizia; Hauck, Yolande; D'Arezzo, Silvia; Imperi, Francesco; Vergnaud, Gilles; Visca, Paolo

2011-01-01

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial outbreaks, mostly occurring in intensive care units. Due to the multiplicity of infection sources, reliable molecular fingerprinting techniques are needed to establish epidemiological correlations among A. baumannii isolates. Multiple-locus variable-number tandem-repeat analysis (MLVA) has proven to be a fast, reliable, and cost-effective typing method for several bacterial species. In this study, an MLVA assay compatible with simple PCR- and agarose gel-based electrophoresis steps as well as with high-throughput automated methods was developed for A. baumannii typing. Preliminarily, 10 potential polymorphic variable-number tandem repeats (VNTRs) were identified upon bioinformatic screening of six annotated genome sequences of A. baumannii. A collection of 7 reference strains plus 18 well-characterized isolates, including unique types and representatives of the three international A. baumannii lineages, was then evaluated in a two-center study aimed at validating the MLVA assay and comparing it with other genotyping assays, namely, macrorestriction analysis with pulsed-field gel electrophoresis (PFGE) and PCR-based sequence group (SG) profiling. The results showed that MLVA can discriminate between isolates with identical PFGE types and SG profiles. A panel of eight VNTR markers was selected, all showing the ability to be amplified and good amounts of polymorphism in the majority of strains. Independently generated MLVA profiles, composed of an ordered string of allele numbers corresponding to the number of repeats at each VNTR locus, were concordant between centers. Typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. A database containing information and MLVA profiles for several A. baumannii strains is available from http://mlva.u-psud.fr/. PMID:21147956

Generation and Analysis of a Large-Scale Expressed Sequence Tag Database from a Full-Length Enriched cDNA Library of Developing Leaves of Gossypium hirsutum L

PubMed Central

Pang, Chaoyou; Fan, Shuli; Song, Meizhen; Yu, Shuxun

2013-01-01

Background Cotton (Gossypium hirsutum L.) is one of the world’s most economically-important crops. However, its entire genome has not been sequenced, and limited resources are available in GenBank for understanding the molecular mechanisms underlying leaf development and senescence. Methodology/Principal Findings In this study, 9,874 high-quality ESTs were generated from a normalized, full-length cDNA library derived from pooled RNA isolated from throughout leaf development during the plant blooming stage. After clustering and assembly of these ESTs, 5,191 unique sequences, representative 1,652 contigs and 3,539 singletons, were obtained. The average unique sequence length was 682 bp. Annotation of these unique sequences revealed that 84.4% showed significant homology to sequences in the NCBI non-redundant protein database, and 57.3% had significant hits to known proteins in the Swiss-Prot database. Comparative analysis indicated that our library added 2,400 ESTs and 991 unique sequences to those known for cotton. The unigenes were functionally characterized by gene ontology annotation. We identified 1,339 and 200 unigenes as potential leaf senescence-related genes and transcription factors, respectively. Moreover, nine genes related to leaf senescence and eleven MYB transcription factors were randomly selected for quantitative real-time PCR (qRT-PCR), which revealed that these genes were regulated differentially during senescence. The qRT-PCR for three GhYLSs revealed that these genes express express preferentially in senescent leaves. Conclusions/Significance These EST resources will provide valuable sequence information for gene expression profiling analyses and functional genomics studies to elucidate their roles, as well as for studying the mechanisms of leaf development and senescence in cotton and discovering candidate genes related to important agronomic traits of cotton. These data will also facilitate future whole-genome sequence assembly and annotation in G. hirsutum and comparative genomics among Gossypium species. PMID:24146870

The evolution of neuropeptide signalling: insights from echinoderms.

PubMed

Semmens, Dean C; Elphick, Maurice R

2017-09-01

Neuropeptides are evolutionarily ancient mediators of neuronal signalling that regulate a wide range of physiological processes and behaviours in animals. Neuropeptide signalling has been investigated extensively in vertebrates and protostomian invertebrates, which include the ecdysozoans Drosophila melanogaster (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda). However, until recently, an understanding of evolutionary relationships between neuropeptide signalling systems in vertebrates and protostomes has been impaired by a lack of genome/transcriptome sequence data from non-ecdysozoan invertebrates. The echinoderms-a deuterostomian phylum that includes sea urchins, sea cucumbers and starfish-have been particularly important in providing new insights into neuropeptide evolution. Sequencing of the genome of the sea urchin Strongylocentrotus purpuratus (Class Echinoidea) enabled discovery of (i) the first invertebrate thyrotropin-releasing hormone-type precursor, (ii) the first deuterostomian pedal peptide/orcokinin-type precursors and (iii) NG peptides-the 'missing link' between neuropeptide S in tetrapod vertebrates and crustacean cardioactive peptide in protostomes. More recently, sequencing of the neural transcriptome of the starfish Asterias rubens (Class Asteroidea) enabled identification of 40 neuropeptide precursors, including the first kisspeptin and melanin-concentrating hormone-type precursors to be identified outside of the chordates. Furthermore, the characterization of a corazonin-type neuropeptide signalling system in A. rubens has provided important new insights into the evolution of gonadotropin-releasing hormone-related neuropeptides. Looking forward, the discovery of multiple neuropeptide signalling systems in echinoderms provides opportunities to investigate how these systems are used to regulate physiological and behavioural processes in the unique context of a decentralized, pentaradial bauplan. © The Author 2017. Published by Oxford University Press.

A novel deletion/insertion mutation in the mRNA transcribed from one {alpha}1(I) collagen allele in a family with dominant type III OI and germline mosaicism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, O.; Masters, C.; Lewis, M.B.

1994-09-01

In an 8-year-old girl and her father, both of whom have severe type III OI, we have previously used RNA/RNA hybrid analysis to demonstrate a mismatch in the region of {alpha}1(I) mRNA coding for aa 558-861. We used SSCP to further localize the abnormality to a subregion coding for aa 579-679. This region was subcloned and sequenced. Each patient`s cDNA has a deletion of the sequences coding for the last residue of exon 34, and all of exons 35 and 36 (aa 604-639), followed by an insertion of 156 nt from the 3{prime}-end of intron 36. PCR amplification of leukocytemore » DNA from the patients and the clinically normal paternal grandmother yielded two fragments: a 1007 bp fragment predicted from normal genomic sequences and a 445 bp fragment. Subcloning and sequencing of the shorter genomic PCR product confirmed the presence of a 565 bp genomic deletion from the end of exon 34 to the middle of intron 36. The abnormal protein is apparently synthesized and incorporated into helix. The inserted nucleotides are in frame with the collagenous sequence and contain no stop codons. They encode a 52 aa non-collagenous region. The fibroblast procollagen of the patients has both normal and electrophoretically delayed pro{alpha}(I) bands. The electrophoretically delayed procollagen is very sensitive to pepsin or trypsin digestion, as predicted by its non-collagenous sequence, and cannot be visualized as collagen. This unique OI collagen mutation is an excellent candidate for molecular targeting to {open_quotes}turn off{close_quotes} a dominant mutant allele.« less

Modulation of collagen-induced arthritis by adenovirus-mediated intra-articular expression of modified collagen type II.

PubMed

Tang, Bo; Cullins, David L; Zhou, Jing; Zawaski, Janice A; Park, Hyelee; Brand, David D; Hasty, Karen A; Gaber, M Waleed; Stuart, John M; Kang, Andrew H; Myers, Linda K

2010-01-01

Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA). We engineered an adenoviral vector to contain the CB11 portion of recombinant type II collagen and used PCR to introduce point mutations at three sites within (CII124-402, 260A, 261B, 263D), (rCB11-A9) so that the resulting molecule contained the A9 sequence at the exact site of the wild-type sequence. We used this construct to target intra-articular tissues of mice and utilized the collagen-induced arthritis model to show that this treatment strategy provided a sustained, local therapy for individual arthritic joints, effective whether given to prevent arthritis or as a treatment. We also developed a novel system for in vivo bioimaging, using the firefly luciferase reporter gene to allow serial bioluminescence imaging to show that luciferase can be detected as late as 18 days post injection into the joint. Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells which secrete interleukin (IL)-4. We believe this approach has potential to effectively suppress RA with minimal side effects.

Modulation of collagen-induced arthritis by adenovirus-mediated intra-articular expression of modified collagen type II

PubMed Central

2010-01-01

Introduction Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA). Methods We engineered an adenoviral vector to contain the CB11 portion of recombinant type II collagen and used PCR to introduce point mutations at three sites within (CII124-402, 260A, 261B, 263D), (rCB11-A9) so that the resulting molecule contained the A9 sequence at the exact site of the wild-type sequence. Results We used this construct to target intra-articular tissues of mice and utilized the collagen-induced arthritis model to show that this treatment strategy provided a sustained, local therapy for individual arthritic joints, effective whether given to prevent arthritis or as a treatment. We also developed a novel system for in vivo bioimaging, using the firefly luciferase reporter gene to allow serial bioluminescence imaging to show that luciferase can be detected as late as 18 days post injection into the joint. Conclusions Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells which secrete interleukin (IL)-4. We believe this approach has potential to effectively suppress RA with minimal side effects. PMID:20615221

Comparative evaluation of an automated repetitive-sequence-based PCR instrument versus pulsed-field gel electrophoresis in the setting of a Serratia marcescens nosocomial infection outbreak.

PubMed

Ligozzi, Marco; Fontana, Roberta; Aldegheri, Marco; Scalet, Giovanna; Lo Cascio, Giuliana

2010-05-01

A semiautomated, repetitive-sequence-based PCR (rep-PCR) instrument (DiversiLab system) was evaluated in comparison with pulsed-field gel electrophoresis (PFGE) to investigate an outbreak of Serratia marcescens infections in a neonatal intensive care unit (NICU). A selection of 36 epidemiologically related and 8 epidemiologically unrelated isolates was analyzed. Among the epidemiologically related isolates, PFGE identified five genetically unrelated patterns. Thirty-two isolates from patients and wet nurses showed the same PFGE profile (pattern A). Genetically unrelated PFGE patterns were found in one patient (pattern B), in two wet nurses (patterns C and D), and in an environmental isolate from the NICU (pattern G). Rep-PCR identified seven different patterns, three of which included the 32 isolates of PFGE type A. One or two band differences in isolates of these three types allowed isolates to be categorized as similar and included in a unique cluster. Isolates of different PFGE types were also of unrelated rep-PCR types. All of the epidemiologically unrelated isolates were of different PFGE and rep-PCR types. The level of discrimination exhibited by rep-PCR with the DiversiLab system allowed us to conclude that this method was able to identify genetic similarity in a spatio-temporal cluster of S. marcescens isolates.

Mitochondrial control-region sequence variation in aboriginal Australians.

PubMed Central

van Holst Pellekaan, S; Frommer, M; Sved, J; Boettcher, B

1998-01-01

The mitochondrial D-loop hypervariable segment 1 (mt HVS1) between nucleotides 15997 and 16377 has been examined in aboriginal Australian people from the Darling River region of New South Wales (riverine) and from Yuendumu in central Australia (desert). Forty-seven unique HVS1 types were identified, varying at 49 nucleotide positions. Pairwise analysis by calculation of BEPPI (between population proportion index) reveals statistically significant structure in the populations, although some identical HVS1 types are seen in the two contrasting regions. mt HVS1 types may reflect more-ancient distributions than do linguistic diversity and other culturally distinguishing attributes. Comparison with sequences from five published global studies reveals that these Australians demonstrate greatest divergence from some Africans, least from Papua New Guinea highlanders, and only slightly more from some Pacific groups (Indonesian, Asian, Samoan, and coastal Papua New Guinea), although the HVS1 types vary at different nucleotide sites. Construction of a median network, displaying three main groups, suggests that several hypervariable nucleotide sites within the HVS1 are likely to have undergone mutation independently, making phylogenetic comparison with global samples by conventional methods difficult. Specific nucleotide-site variants are major separators in median networks constructed from Australian HVS1 types alone and for one global selection. The distribution of these, requiring extended study, suggests that they may be signatures of different groups of prehistoric colonizers into Australia, for which the time of colonization remains elusive. PMID:9463317

Hierarchically nested river landform sequences

NASA Astrophysics Data System (ADS)

Pasternack, G. B.; Weber, M. D.; Brown, R. A.; Baig, D.

2017-12-01

River corridors exhibit landforms nested within landforms repeatedly down spatial scales. In this study we developed, tested, and implemented a new way to create river classifications by mapping domains of fluvial processes with respect to the hierarchical organization of topographic complexity that drives fluvial dynamism. We tested this approach on flow convergence routing, a morphodynamic mechanism with different states depending on the structure of nondimensional topographic variability. Five nondimensional landform types with unique functionality (nozzle, wide bar, normal channel, constricted pool, and oversized) represent this process at any flow. When this typology is nested at base flow, bankfull, and floodprone scales it creates a system with up to 125 functional types. This shows how a single mechanism produces complex dynamism via nesting. Given the classification, we answered nine specific scientific questions to investigate the abundance, sequencing, and hierarchical nesting of these new landform types using a 35-km gravel/cobble river segment of the Yuba River in California. The nested structure of flow convergence routing landforms found in this study revealed that bankfull landforms are nested within specific floodprone valley landform types, and these types control bankfull morphodynamics during moderate to large floods. As a result, this study calls into question the prevailing theory that the bankfull channel of a gravel/cobble river is controlled by in-channel, bankfull, and/or small flood flows. Such flows are too small to initiate widespread sediment transport in a gravel/cobble river with topographic complexity.

Making sense of deep sequencing

PubMed Central

Goldman, D.; Domschke, K.

2016-01-01

This review, the first of an occasional series, tries to make sense of the concepts and uses of deep sequencing of polynucleic acids (DNA and RNA). Deep sequencing, synonymous with next-generation sequencing, high-throughput sequencing and massively parallel sequencing, includes whole genome sequencing but is more often and diversely applied to specific parts of the genome captured in different ways, for example the highly expressed portion of the genome known as the exome and portions of the genome that are epigenetically marked either by DNA methylation, the binding of proteins including histones, or that are in different configurations and thus more or less accessible to enzymes that cleave DNA. Deep sequencing of RNA (RNASeq) reverse-transcribed to complementary DNA is invaluable for measuring RNA expression and detecting changes in RNA structure. Important concepts in deep sequencing include the length and depth of sequence reads, mapping and assembly of reads, sequencing error, haplotypes, and the propensity of deep sequencing, as with other types of ‘big data’, to generate large numbers of errors, requiring monitoring for methodologic biases and strategies for replication and validation. Deep sequencing yields a unique genetic fingerprint that can be used to identify a person, and a trove of predictors of genetic medical diseases. Deep sequencing to identify epigenetic events including changes in DNA methylation and RNA expression can reveal the history and impact of environmental exposures. Because of the power of sequencing to identify and deliver biomedically significant information about a person and their blood relatives, it creates ethical dilemmas and practical challenges in research and clinical care, for example the decision and procedures to report incidental findings that will increasingly and frequently be discovered. PMID:24925306

Molecular characterization of canine parvovirus in Vientiane, Laos.

PubMed

Vannamahaxay, Soulasack; Vongkhamchanh, Souliya; Intanon, Montira; Tangtrongsup, Sahatchai; Tiwananthagorn, Saruda; Pringproa, Kidsadagon; Chuammitri, Phongsakorn

2017-05-01

The global emergence of canine parvovirus type 2c (CPV-2c) has been well documented. In the present study, 139 rectal swab samples collected from diarrheic dogs living in Vientiane, Laos, in 2016 were tested for the presence of the canine parvovirus (CPV) VP2 gene by PCR. The results showed that 82.73% (115/139) of dogs were CPV positive by PCR. The partial VP2 gene was sequenced in 94 of the positive samples; 91 samples belonged to CPV-2c (426Glu) subtype, while 3 samples belonged to the CPV-2a (426Asn) subtype. Notably, phylogenetic analysis of amino acid sequences revealed a close relationship between Laotian isolates and novel Chinese CPV-2c isolates. In Laotian CPV isolates, aligned protein sequences indicated a high rate of residue substitutions at positions 305, 324, 345, 370, 375, and 426 in the GH loop. The mutation at residue 370 (Q370R), a single mutation, was characterized as a unique mutant residue specific to the Laotian CPV-2c variant.

Weissella ghanensis sp. nov., isolated from a Ghanaian cocoa fermentation.

PubMed

De Bruyne, Katrien; Camu, Nicholas; Lefebvre, Karen; De Vuyst, Luc; Vandamme, Peter

2008-12-01

During a study on lactic acid bacteria (and their species diversity) in spontaneous heap fermentations of Ghanaian cocoa beans, two strains, designated 215(T) and 194B, were isolated. A phylogenetic analysis based on 16S rRNA gene sequences demonstrated that these strains represented a distinct lineage close to the genus Weissella and showing only 92.1 % 16S rRNA gene sequence similarity with respect to their closest neighbour, Weissella soli LMG 20113(T). Whole-cell protein electrophoresis, fluorescent amplified fragment length polymorphism fingerprinting of whole genomes and physiological and biochemical tests confirmed the unique taxonomic position of the two novel isolates. On the basis of the results of the morphological and biochemical tests and 16S rRNA gene sequence analysis, strains 215(T) and 194B represent the most peripheral lineage of the genus Weissella, for which we propose the name Weissella ghanensis sp. nov. The type strain is 215(T) (=LMG 24286(T)=DSM 19935(T)).

Domain Organization in Clostridium botulinum Neurotoxin Type E is Unique: Its Implication in Faster Translocation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumaran, D.; Eswaramoorthy, S; Furey, W

2009-01-01

Clostridium botulinum produces seven antigenically distinct neurotoxins [C. botulinum neurotoxins (BoNTs) A-G] sharing a significant sequence homology. Based on sequence and functional similarity, it was believed that their three-dimensional structures will also be similar. Indeed, the crystal structures of BoNTs A and B exhibit similar fold and domain association where the translocation domain is flanked on either side by binding and catalytic domains. Here, we report the crystal structure of BoNT E holotoxin and show that the domain association is different and unique, although the individual domains are similar to those of BoNTs A and B. In BoNT E, bothmore » the binding domain and the catalytic domain are on the same side of the translocation domain, and all three have mutual interfaces. This unique association may have an effect on the rate of translocation, with the molecule strategically positioned in the vesicle for quick entry into cytosol. Botulism, the disease caused by BoNT E, sets in faster than any other serotype because of its speedy internalization and translocation, and the present structure offers a credible explanation. We propose that the translocation domain in other BoNTs follows a two-step process to attain translocation-competent conformation as in BoNT E. We also suggest that this translocation-competent conformation in BoNT E is a probable reason for its faster toxic rate compared to BoNT A. However, this needs further experimental elucidation.« less

Unique Features of Human Protein Arginine Methyltransferase 9 (PRMT9) and Its Substrate RNA Splicing Factor SF3B2*

PubMed Central

Hadjikyriacou, Andrea; Yang, Yanzhong; Espejo, Alexsandra; Bedford, Mark T.; Clarke, Steven G.

2015-01-01

Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides. PMID:25979344

A comprehensive characterization of simple sequence repeats in pepper genomes provides valuable resources for marker development in Capsicum.

PubMed

Cheng, Jiaowen; Zhao, Zicheng; Li, Bo; Qin, Cheng; Wu, Zhiming; Trejo-Saavedra, Diana L; Luo, Xirong; Cui, Junjie; Rivera-Bustamante, Rafael F; Li, Shuaicheng; Hu, Kailin

2016-01-07

The sequences of the full set of pepper genomes including nuclear, mitochondrial and chloroplast are now available for use. However, the overall of simple sequence repeats (SSR) distribution in these genomes and their practical implications for molecular marker development in Capsicum have not yet been described. Here, an average of 868,047.50, 45.50 and 30.00 SSR loci were identified in the nuclear, mitochondrial and chloroplast genomes of pepper, respectively. Subsequently, systematic comparisons of various species, genome types, motif lengths, repeat numbers and classified types were executed and discussed. In addition, a local database composed of 113,500 in silico unique SSR primer pairs was built using a homemade bioinformatics workflow. As a pilot study, 65 polymorphic markers were validated among a wide collection of 21 Capsicum genotypes with allele number and polymorphic information content value per marker raging from 2 to 6 and 0.05 to 0.64, respectively. Finally, a comparison of the clustering results with those of a previous study indicated the usability of the newly developed SSR markers. In summary, this first report on the comprehensive characterization of SSR motifs in pepper genomes and the very large set of SSR primer pairs will benefit various genetic studies in Capsicum.

A comprehensive characterization of simple sequence repeats in pepper genomes provides valuable resources for marker development in Capsicum

PubMed Central

Cheng, Jiaowen; Zhao, Zicheng; Li, Bo; Qin, Cheng; Wu, Zhiming; Trejo-Saavedra, Diana L.; Luo, Xirong; Cui, Junjie; Rivera-Bustamante, Rafael F.; Li, Shuaicheng; Hu, Kailin

2016-01-01

The sequences of the full set of pepper genomes including nuclear, mitochondrial and chloroplast are now available for use. However, the overall of simple sequence repeats (SSR) distribution in these genomes and their practical implications for molecular marker development in Capsicum have not yet been described. Here, an average of 868,047.50, 45.50 and 30.00 SSR loci were identified in the nuclear, mitochondrial and chloroplast genomes of pepper, respectively. Subsequently, systematic comparisons of various species, genome types, motif lengths, repeat numbers and classified types were executed and discussed. In addition, a local database composed of 113,500 in silico unique SSR primer pairs was built using a homemade bioinformatics workflow. As a pilot study, 65 polymorphic markers were validated among a wide collection of 21 Capsicum genotypes with allele number and polymorphic information content value per marker raging from 2 to 6 and 0.05 to 0.64, respectively. Finally, a comparison of the clustering results with those of a previous study indicated the usability of the newly developed SSR markers. In summary, this first report on the comprehensive characterization of SSR motifs in pepper genomes and the very large set of SSR primer pairs will benefit various genetic studies in Capsicum. PMID:26739748

Aspergillus Section Fumigati Typing by PCR-Restriction Fragment Polymorphism▿

PubMed Central

Staab, Janet F.; Balajee, S. Arunmozhi; Marr, Kieren A.

2009-01-01

Recent studies have shown that there are multiple clinically important members of the Aspergillus section Fumigati that are difficult to distinguish on the basis of morphological features (e.g., Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae). Identification of these organisms may be clinically important, as some species vary in their susceptibilities to antifungal agents. In a prior study, we utilized multilocus sequence typing to describe A. lentulus as a species distinct from A. fumigatus. The sequence data show that the gene encoding β-tubulin, benA, has high interspecies variability at intronic regions but is conserved among isolates of the same species. These data were used to develop a PCR-restriction fragment length polymorphism (PCR-RFLP) method that rapidly and accurately distinguishes A. fumigatus, A. lentulus, and N. udagawae, three major species within the section Fumigati that have previously been implicated in disease. Digestion of the benA amplicon with BccI generated unique banding patterns; the results were validated by screening a collection of clinical strains and by in silico analysis of the benA sequences of Aspergillus spp. deposited in the GenBank database. PCR-RFLP of benA is a simple method for the identification of clinically important, similar morphotypes of Aspergillus spp. within the section Fumigati. PMID:19403766

Aspergillus section Fumigati typing by PCR-restriction fragment polymorphism.

PubMed

Staab, Janet F; Balajee, S Arunmozhi; Marr, Kieren A

2009-07-01

Recent studies have shown that there are multiple clinically important members of the Aspergillus section Fumigati that are difficult to distinguish on the basis of morphological features (e.g., Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae). Identification of these organisms may be clinically important, as some species vary in their susceptibilities to antifungal agents. In a prior study, we utilized multilocus sequence typing to describe A. lentulus as a species distinct from A. fumigatus. The sequence data show that the gene encoding beta-tubulin, benA, has high interspecies variability at intronic regions but is conserved among isolates of the same species. These data were used to develop a PCR-restriction fragment length polymorphism (PCR-RFLP) method that rapidly and accurately distinguishes A. fumigatus, A. lentulus, and N. udagawae, three major species within the section Fumigati that have previously been implicated in disease. Digestion of the benA amplicon with BccI generated unique banding patterns; the results were validated by screening a collection of clinical strains and by in silico analysis of the benA sequences of Aspergillus spp. deposited in the GenBank database. PCR-RFLP of benA is a simple method for the identification of clinically important, similar morphotypes of Aspergillus spp. within the section Fumigati.

New insights into hydrothermal vent processes in the unique shallow-submarine arc-volcano, Kolumbo (Santorini), Greece

PubMed Central

Kilias, Stephanos P.; Nomikou, Paraskevi; Papanikolaou, Dimitrios; Polymenakou, Paraskevi N.; Godelitsas, Athanasios; Argyraki, Ariadne; Carey, Steven; Gamaletsos, Platon; Mertzimekis, Theo J.; Stathopoulou, Eleni; Goettlicher, Joerg; Steininger, Ralph; Betzelou, Konstantina; Livanos, Isidoros; Christakis, Christos; Bell, Katherine Croff; Scoullos, Michael

2013-01-01

We report on integrated geomorphological, mineralogical, geochemical and biological investigations of the hydrothermal vent field located on the floor of the density-stratified acidic (pH ~ 5) crater of the Kolumbo shallow-submarine arc-volcano, near Santorini. Kolumbo features rare geodynamic setting at convergent boundaries, where arc-volcanism and seafloor hydrothermal activity are occurring in thinned continental crust. Special focus is given to unique enrichments of polymetallic spires in Sb and Tl (±Hg, As, Au, Ag, Zn) indicating a new hybrid seafloor analogue of epithermal-to-volcanic-hosted-massive-sulphide deposits. Iron microbial-mat analyses reveal dominating ferrihydrite-type phases, and high-proportion of microbial sequences akin to "Nitrosopumilus maritimus", a mesophilic Thaumarchaeota strain capable of chemoautotrophic growth on hydrothermal ammonia and CO2. Our findings highlight that acidic shallow-submarine hydrothermal vents nourish marine ecosystems in which nitrifying Archaea are important and suggest ferrihydrite-type Fe3+-(hydrated)-oxyhydroxides in associated low-temperature iron mats are formed by anaerobic Fe2+-oxidation, dependent on microbially produced nitrate. PMID:23939372

Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

PubMed

Clark, Clifford G; Berry, Chrystal; Walker, Matthew; Petkau, Aaron; Barker, Dillon O R; Guan, Cai; Reimer, Aleisha; Taboada, Eduardo N

2016-12-03

Whole genome sequencing (WGS) is useful for determining clusters of human cases, investigating outbreaks, and defining the population genetics of bacteria. It also provides information about other aspects of bacterial biology, including classical typing results, virulence, and adaptive strategies of the organism. Cell culture invasion and protein expression patterns of four related multilocus sequence type 21 (ST21) C. jejuni isolates from a significant Canadian water-borne outbreak were previously associated with the presence of a CJIE1 prophage. Whole genome sequencing was used to examine the genetic diversity among these isolates and confirm that previous observations could be attributed to differential prophage carriage. Moreover, we sought to determine the presence of genome sequences that could be used as surrogate markers to delineate outbreak-associated isolates. Differential carriage of the CJIE1 prophage was identified as the major genetic difference among the four outbreak isolates. High quality single-nucleotide variant (hqSNV) and core genome multilocus sequence typing (cgMLST) clustered these isolates within expanded datasets consisting of additional C. jejuni strains. The number and location of homopolymeric tract regions was identical in all four outbreak isolates but differed from all other C. jejuni examined. Comparative genomics and PCR amplification enabled the identification of large chromosomal inversions of approximately 93 kb and 388 kb within the outbreak isolates associated with transducer-like proteins containing long nucleotide repeat sequences. The 93-kb inversion was characteristic of the outbreak-associated isolates, and the gene content of this inverted region displayed high synteny with the reference strain. The four outbreak isolates were clonally derived and differed mainly in the presence of the CJIE1 prophage, validating earlier findings linking the prophage to phenotypic differences in virulence assays and protein expression. The identification of large, genetically syntenous chromosomal inversions in the genomes of outbreak-associated isolates provided a unique method for discriminating outbreak isolates from the background population. Transducer-like proteins appear to be associated with the chromosomal inversions. CgMLST and hqSNV analysis also effectively delineated the outbreak isolates within the larger C. jejuni population structure.

Points of View: A Survey of Survey Courses--Are They Effective? A Unique Approach? Four Semesters of Biology Core Curriculum

ERIC Educational Resources Information Center

Batzli, Janet M.

2005-01-01

''Why four semesters? How does this track differ from the two-semester course sequence?'' These are the most common questions students have when they learn about the Biology Core Curriculum (Biocore), a unique four-semester honors biology sequence at University of Wisconsin-Madison (UW-Madison). Biocore was first taught at University of Wisconsin…

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.

The Poultry-Associated Microbiome: Network Analysis and Farm-to-Fork Characterizations

PubMed Central

Oakley, Brian B.; Morales, Cesar A.; Line, J.; Berrang, Mark E.; Meinersmann, Richard J.; Tillman, Glenn E.; Wise, Mark G.; Siragusa, Gregory R.; Hiett, Kelli L.; Seal, Bruce S.

2013-01-01

Microbial communities associated with agricultural animals are important for animal health, food safety, and public health. Here we combine high-throughput sequencing (HTS), quantitative-PCR assays, and network analysis to profile the poultry-associated microbiome and important pathogens at various stages of commercial poultry production from the farm to the consumer. Analysis of longitudinal data following two flocks from the farm through processing showed a core microbiome containing multiple sequence types most closely related to genera known to be pathogenic for animals and/or humans, including Campylobacter, Clostridium, and Shigella. After the final stage of commercial poultry processing, taxonomic richness was ca. 2–4 times lower than the richness of fecal samples from the same flocks and Campylobacter abundance was significantly reduced. Interestingly, however, carcasses sampled at 48 hr after processing harboured the greatest proportion of unique taxa (those not encountered in other samples), significantly more than expected by chance. Among these were anaerobes such as Prevotella, Veillonella, Leptrotrichia, and multiple Campylobacter sequence types. Retail products were dominated by Pseudomonas, but also contained 27 other genera, most of which were potentially metabolically active and encountered in on-farm samples. Network analysis was focused on the foodborne pathogen Campylobacter and revealed a majority of sequence types with no significant interactions with other taxa, perhaps explaining the limited efficacy of previous attempts at competitive exclusion of Campylobacter. These data represent the first use of HTS to characterize the poultry microbiome across a series of farm-to-fork samples and demonstrate the utility of HTS in monitoring the food supply chain and identifying sources of potential zoonoses and interactions among taxa in complex communities. PMID:23468931

Single-cell RNA-sequencing reveals a distinct population of proglucagon-expressing cells specific to the mouse upper small intestine.

PubMed

Glass, Leslie L; Calero-Nieto, Fernando J; Jawaid, Wajid; Larraufie, Pierre; Kay, Richard G; Göttgens, Berthold; Reimann, Frank; Gribble, Fiona M

2017-10-01

To identify sub-populations of intestinal preproglucagon-expressing (PPG) cells producing Glucagon-like Peptide-1, and their associated expression profiles of sensory receptors, thereby enabling the discovery of therapeutic strategies that target these cell populations for the treatment of diabetes and obesity. We performed single cell RNA sequencing of PPG-cells purified by flow cytometry from the upper small intestine of 3 GLU-Venus mice. Cells from 2 mice were sequenced at low depth, and from the third mouse at high depth. High quality sequencing data from 234 PPG-cells were used to identify clusters by tSNE analysis. qPCR was performed to compare the longitudinal and crypt/villus locations of cluster-specific genes. Immunofluorescence and mass spectrometry were used to confirm protein expression. PPG-cells formed 3 major clusters: a group with typical characteristics of classical L-cells, including high expression of Gcg and Pyy (comprising 51% of all PPG-cells); a cell type overlapping with Gip-expressing K-cells (14%); and a unique cluster expressing Tph1 and Pzp that was predominantly located in proximal small intestine villi and co-produced 5-HT (35%). Expression of G-protein coupled receptors differed between clusters, suggesting the cell types are differentially regulated and would be differentially targetable. Our findings support the emerging concept that many enteroendocrine cell populations are highly overlapping, with individual cells producing a range of peptides previously assigned to distinct cell types. Different receptor expression profiles across the clusters highlight potential drug targets to increase gut hormone secretion for the treatment of diabetes and obesity. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

High Throughput Sequencing to Detect Differences in Methanotrophic Methylococcaceae and Methylocystaceae in Surface Peat, Forest Soil, and Sphagnum Moss in Cranesville Swamp Preserve, West Virginia, USA

PubMed Central

Lau, Evan; Nolan, Edward J.; Dillard, Zachary W.; Dague, Ryan D.; Semple, Amanda L.; Wentzell, Wendi L.

2015-01-01

Northern temperate forest soils and Sphagnum-dominated peatlands are a major source and sink of methane. In these ecosystems, methane is mainly oxidized by aerobic methanotrophic bacteria, which are typically found in aerated forest soils, surface peat, and Sphagnum moss. We contrasted methanotrophic bacterial diversity and abundances from the (i) organic horizon of forest soil; (ii) surface peat; and (iii) submerged Sphagnum moss from Cranesville Swamp Preserve, West Virginia, using multiplex sequencing of bacterial 16S rRNA (V3 region) gene amplicons. From ~1 million reads, >50,000 unique OTUs (Operational Taxonomic Units), 29 and 34 unique sequences were detected in the Methylococcaceae and Methylocystaceae, respectively, and 24 potential methanotrophs in the Beijerinckiaceae were also identified. Methylacidiphilum-like methanotrophs were not detected. Proteobacterial methanotrophic bacteria constitute <2% of microbiota in these environments, with the Methylocystaceae one to two orders of magnitude more abundant than the Methylococcaceae in all environments sampled. The Methylococcaceae are also less diverse in forest soil compared to the other two habitats. Nonmetric multidimensional scaling analyses indicated that the majority of methanotrophs from the Methylococcaceae and Methylocystaceae tend to occur in one habitat only (peat or Sphagnum moss) or co-occurred in both Sphagnum moss and peat. This study provides insights into the structure of methanotrophic communities in relationship to habitat type, and suggests that peat and Sphagnum moss can influence methanotroph community structure and biogeography. PMID:27682082

The Genome of the Netherlands: design, and project goals

PubMed Central

Boomsma, Dorret I; Wijmenga, Cisca; Slagboom, Eline P; Swertz, Morris A; Karssen, Lennart C; Abdellaoui, Abdel; Ye, Kai; Guryev, Victor; Vermaat, Martijn; van Dijk, Freerk; Francioli, Laurent C; Hottenga, Jouke Jan; Laros, Jeroen F J; Li, Qibin; Li, Yingrui; Cao, Hongzhi; Chen, Ruoyan; Du, Yuanping; Li, Ning; Cao, Sujie; van Setten, Jessica; Menelaou, Androniki; Pulit, Sara L; Hehir-Kwa, Jayne Y; Beekman, Marian; Elbers, Clara C; Byelas, Heorhiy; de Craen, Anton J M; Deelen, Patrick; Dijkstra, Martijn; den Dunnen, Johan T; de Knijff, Peter; Houwing-Duistermaat, Jeanine; Koval, Vyacheslav; Estrada, Karol; Hofman, Albert; Kanterakis, Alexandros; Enckevort, David van; Mai, Hailiang; Kattenberg, Mathijs; van Leeuwen, Elisabeth M; Neerincx, Pieter B T; Oostra, Ben; Rivadeneira, Fernanodo; Suchiman, Eka H D; Uitterlinden, Andre G; Willemsen, Gonneke; Wolffenbuttel, Bruce H; Wang, Jun; de Bakker, Paul I W; van Ommen, Gert-Jan; van Duijn, Cornelia M

2014-01-01

Within the Netherlands a national network of biobanks has been established (Biobanking and Biomolecular Research Infrastructure-Netherlands (BBMRI-NL)) as a national node of the European BBMRI. One of the aims of BBMRI-NL is to enrich biobanks with different types of molecular and phenotype data. Here, we describe the Genome of the Netherlands (GoNL), one of the projects within BBMRI-NL. GoNL is a whole-genome-sequencing project in a representative sample consisting of 250 trio-families from all provinces in the Netherlands, which aims to characterize DNA sequence variation in the Dutch population. The parent–offspring trios include adult individuals ranging in age from 19 to 87 years (mean=53 years; SD=16 years) from birth cohorts 1910–1994. Sequencing was done on blood-derived DNA from uncultured cells and accomplished coverage was 14–15x. The family-based design represents a unique resource to assess the frequency of regional variants, accurately reconstruct haplotypes by family-based phasing, characterize short indels and complex structural variants, and establish the rate of de novo mutational events. GoNL will also serve as a reference panel for imputation in the available genome-wide association studies in Dutch and other cohorts to refine association signals and uncover population-specific variants. GoNL will create a catalog of human genetic variation in this sample that is uniquely characterized with respect to micro-geographic location and a wide range of phenotypes. The resource will be made available to the research and medical community to guide the interpretation of sequencing projects. The present paper summarizes the global characteristics of the project. PMID:23714750

[Identification and phylogenetic application of unique nucleotide sequence of nad7 intron2 in Rhodiola (Crassulaceae) species].

PubMed

Deng, Ke-Jun; Yang, Zu-Jun; Liu, Cheng; Zhao, Wei; Liu, Chang; Feng, Juan; Ren, Zheng-Long

2007-03-01

Genetic characterization of 9 populations of Rhodiola crenulata, R. fastigiata and R. sachalinensis (Crassulaceae) species from Sichuan and Jilin Provinces of China, was investigated using the conserved primer of nad7 intron 2. All PCR products about 800 bp long were shorter than other Crassulaceae plants, which were used as molecular markers to identify the Rhodiola species. The sequence of the products indicated that total exon of 53 bp and intron of 738 bp exhibit only 9 nucleotide variations. Blasting the nad7 sequences to GenBank and the phylogenetic analysis showed that the sequence of Rhodiola species was clusted independently, and the length was smaller than all the registered sequences of higher plants. The result suggests that the Rhiodola species had a unique sequence in this gene region, which might be related to the special growth condition.

Biogeographic and phylogenetic diversity of thermoacidophilic cyanidiales in Yellowstone National Park, Japan, and New Zealand.

PubMed

Toplin, J A; Norris, T B; Lehr, C R; McDermott, T R; Castenholz, R W

2008-05-01

Members of the rhodophytan order Cyanidiales are unique among phototrophs in their ability to live in extreme environments that combine low pH levels ( approximately 0.2 to 4.0) and moderately high temperatures of 40 to 56 degrees C. These unicellular algae occur in far-flung volcanic areas throughout the earth. Three genera (Cyanidium, Galdieria, and Cyanidioschyzon) are recognized. The phylogenetic diversity of culture isolates of the Cyanidiales from habitats throughout Yellowstone National Park (YNP), three areas in Japan, and seven regions in New Zealand was examined by using the chloroplast RuBisCO large subunit gene (rbcL) and the 18S rRNA gene. Based on the nucleotide sequences of both genes, the YNP isolates fall into two groups, one with high identity to Galdieria sulphuraria (type II) and another that is by far the most common and extensively distributed Yellowstone type (type IA). The latter is a spherical, walled cell that reproduces by internal divisions, with a subsequent release of smaller daughter cells. This type, nevertheless, shows a 99 to 100% identity to Cyanidioschyzon merolae (type IB), which lacks a wall, divides by "fission"-like cytokinesis into two daughter cells, and has less than 5% of the cell volume of type IA. The evolutionary and taxonomic ramifications of this disparity are discussed. Although the 18S rRNA and rbcL genes did not reveal diversity among the numerous isolates of type IA, chloroplast short sequence repeats did show some variation by location within YNP. In contrast, Japanese and New Zealand strains showed considerable diversity when we examined only the sequences of 18S and rbcL genes. Most exhibited identities closer to Galdieria maxima than to other strains, but these identities were commonly as low as 91 to 93%. Some of these Japanese and New Zealand strains probably represent undescribed species that diverged after long-term geographic isolation.

DNA Clutch Probes for Circulating Tumor DNA Analysis.

PubMed

Das, Jagotamoy; Ivanov, Ivaylo; Sargent, Edward H; Kelley, Shana O

2016-08-31

Progress toward the development of minimally invasive liquid biopsies of disease is being bolstered by breakthroughs in the analysis of circulating tumor DNA (ctDNA): DNA released from cancer cells into the bloodstream. However, robust, sensitive, and specific methods of detecting this emerging analyte are lacking. ctDNA analysis has unique challenges, since it is imperative to distinguish circulating DNA from normal cells vs mutation-bearing sequences originating from tumors. Here we report the electrochemical detection of mutated ctDNA in samples collected from cancer patients. By developing a strategy relying on the use of DNA clutch probes (DCPs) that render specific sequences of ctDNA accessible, we were able to readout the presence of mutated ctDNA. DCPs prevent reassociation of denatured DNA strands: they make one of the two strands of a dsDNA accessible for hybridization to a probe, and they also deactivate other closely related sequences in solution. DCPs ensure thereby that only mutated sequences associate with chip-based sensors detecting hybridization events. The assay exhibits excellent sensitivity and specificity in the detection of mutated ctDNA: it detects 1 fg/μL of a target mutation in the presence of 100 pg/μL of wild-type DNA, corresponding to detecting mutations at a level of 0.01% relative to wild type. This approach allows accurate analysis of samples collected from lung cancer and melanoma patients. This work represents the first detection of ctDNA without enzymatic amplification.

Analysis of the Legionella longbeachae Genome and Transcriptome Uncovers Unique Strategies to Cause Legionnaires' Disease

PubMed Central

Rusniok, Christophe; Lomma, Mariella; Dervins-Ravault, Delphine; Newton, Hayley J.; Sansom, Fiona M.; Jarraud, Sophie; Zidane, Nora; Ma, Laurence; Bouchier, Christiane; Etienne, Jerôme; Hartland, Elizabeth L.; Buchrieser, Carmen

2010-01-01

Legionella pneumophila and L. longbeachae are two species of a large genus of bacteria that are ubiquitous in nature. L. pneumophila is mainly found in natural and artificial water circuits while L. longbeachae is mainly present in soil. Under the appropriate conditions both species are human pathogens, capable of causing a severe form of pneumonia termed Legionnaires' disease. Here we report the sequencing and analysis of four L. longbeachae genomes, one complete genome sequence of L. longbeachae strain NSW150 serogroup (Sg) 1, and three draft genome sequences another belonging to Sg1 and two to Sg2. The genome organization and gene content of the four L. longbeachae genomes are highly conserved, indicating strong pressure for niche adaptation. Analysis and comparison of L. longbeachae strain NSW150 with L. pneumophila revealed common but also unexpected features specific to this pathogen. The interaction with host cells shows distinct features from L. pneumophila, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system substrates, eukaryotic-like and eukaryotic domain proteins, and encodes additional secretion systems. However, analysis of the ability of a dotA mutant of L. longbeachae NSW150 to replicate in the Acanthamoeba castellanii and in a mouse lung infection model showed that the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens. Furthermore, transcriptome analysis revealed that L. longbeachae has a less pronounced biphasic life cycle as compared to L. pneumophila, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences may account for the different environmental niches and disease epidemiology of these two Legionella species. PMID:20174605

Late-Type Membership of the Open Cluster NGC 2232

NASA Technical Reports Server (NTRS)

Orban, Chris; Patten, Brian

2004-01-01

NGC 2232 is one of the nearest open clusters (approx.360 pc) with an age of approx.25 Myr. This places it in the unique position to study the transition from T Tauri activity to the Zero Age Main Sequence. In order for those studies to begin, late-type members must be identified for the cluster. X-ray observations combined with ground-based photometry and spectroscopy offers the best way to accomplish this goal. We present photometry in the VRI bands, 2MASS near-infrared measurements in the J, H , Ks bands and spectra for the suspected optical counterparts to the X-ray sources in the field of NGC 2232. 46 candidate members were identified through these efforts ranging from F5 to M5.

PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries.

PubMed

Shave, Steven; Mann, Stefan; Koszela, Joanna; Kerr, Alastair; Auer, Manfred

2018-01-01

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.

Bacterial DNA Detected in Japanese Rice Wines and the Fermentation Starters.

PubMed

Terasaki, Momoka; Fukuyama, Akari; Takahashi, Yurika; Yamada, Masato; Nishida, Hiromi

2017-12-01

As Japanese rice wine (sake) brewing is not done aseptically, bacterial contamination is conceivable during the process of sake production. There are two types of the fermentation starter, sokujo-moto and yamahai-moto (kimoto). We identified bacterial DNA found in various sakes, the sokujo-moto and the yamahai-moto making just after sake yeast addition. Each sake has a unique variety of bacterial DNA not observed in other sakes. Although most bacterial DNA sequences detected in the sokujo-moto were found in sakes of different sake breweries, most bacterial DNA sequences detected in the yamahai-moto at the early stage of the starter fermentation were not detected in any sakes. Our findings demonstrate that various bacteria grow and then die during the process of sake brewing, as indicated by the presence of trace levels of bacterial DNA.

Equivalent Indels – Ambiguous Functional Classes and Redundancy in Databases

PubMed Central

Assmus, Jens; Kleffe, Jürgen; Schmitt, Armin O.; Brockmann, Gudrun A.

2013-01-01

There is considerable interest in studying sequenced variations. However, while the positions of substitutions are uniquely identifiable by sequence alignment, the location of insertions and deletions still poses problems. Each insertion and deletion causes a change of sequence. Yet, due to low complexity or repetitive sequence structures, the same indel can sometimes be annotated in different ways. Two indels which differ in allele sequence and position can be one and the same, i.e. the alternative sequence of the whole chromosome is identical in both cases and, therefore, the two deletions are biologically equivalent. In such a case, it is impossible to identify the exact position of an indel merely based on sequence alignment. Thus, variation entries in a mutation database are not necessarily uniquely defined. We prove the existence of a contiguous region around an indel in which all deletions of the same length are biologically identical. Databases often show only one of several possible locations for a given variation. Furthermore, different data base entries can represent equivalent variation events. We identified 1,045,590 such problematic entries of insertions and deletions out of 5,860,408 indel entries in the current human database of Ensembl. Equivalent indels are found in sequence regions of different functions like exons, introns or 5' and 3' UTRs. One and the same variation can be assigned to several different functional classifications of which only one is correct. We implemented an algorithm that determines for each indel database entry its complete set of equivalent indels which is uniquely characterized by the indel itself and a given interval of the reference sequence. PMID:23658777

Mycobacterium intermedium sp. nov.

PubMed

Meier, A; Kirschner, P; Schröder, K H; Wolters, J; Kroppenstedt, R M; Böttger, E C

1993-04-01

Strains of a new type of slowly growing mycobacterium were repeatedly isolated from sputum from a patient with pulmonary disease. This photochromogenic organism grew at 22, 31, 37, and 41 degrees C, possessed catalase, acid phosphatase, esterase, beta-galactosidase, and arylsulfatase activities, and hydrolyzed Tween. It did not produce nicotinic acid or have nitrate reductase, acetamidase, benzamidase, isonicotinamidase, nicotinamidase, pyrazinamidase, succinidamidase, and acid phosphatase activities. Urease activity was variable. The organism is susceptible to ethambutol and resistant to isoniazid and streptomycin. A mycolic acid analysis revealed the presence of alpha-mycolates, alpha'-mycolates, and keto-mycolates. The results of comparative 16S rRNA sequencing placed this organism at an intermediate position between the rapidly and slowly growing mycobacteria. On the basis of the pattern of enzymatic activities and metabolic properties, the results of fatty acid analyses, and the unique 16S rRNA sequence, we propose that this organism represents a new species, for which we propose the name Mycobacterium intermedium. The type strain is strain 1669/91; a culture of this strain has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen as strain DSM 44049.

Adaptive evolution of insect selective excitatory β-type sodium channel neurotoxins from scorpion venom.

PubMed

Wu, Wenlan; Li, Zhongjie; Ma, Yibao

2017-06-01

Insect selective excitatory β-type sodium channel neurotoxins from scorpion venom (β-NaScTxs) are composed of about 70-76 amino acid residues and share a common scaffold stabilized by four unique disulfide bonds. The phylogenetic analysis of these toxins was hindered by limited sequence data. In our recent study, two new insect selective excitatory β-NaScTxs, LmIT and ImIT, were isolated from Lychas mucronatus and Isometrus maculatus, respectively. With the sequences previously reported, we examined the adaptive molecular evolution of insect selective excitatory β-NaScTxs by estimating the nonsynonymous-to-synonymous rate ratio (ω=d N /d S ). The results revealed 12 positively selected sites in the genes of insect selective excitatory β-NaScTxs. Moreover, these positively selected sites match well with the sites important for interacting with sodium channels, as demonstrated in previous mutagenesis study. These results reveal that adaptive evolution after gene duplication is one of the most important genetic mechanisms of scorpion neurotoxin diversification. Copyright © 2017 Elsevier Inc. All rights reserved.

The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation

PubMed Central

Posse, Viktor; Hoberg, Emily; Dierckx, Anke; Shahzad, Saba; Koolmeister, Camilla; Larsson, Nils-Göran; Wilhelmsson, L. Marcus; Hällberg, B. Martin; Gustafsson, Claes M.

2014-01-01

Mammalian mitochondrial transcription is executed by a single subunit mitochondrial RNA polymerase (Polrmt) and its two accessory factors, mitochondrial transcription factors A and B2 (Tfam and Tfb2m). Polrmt is structurally related to single-subunit phage RNA polymerases, but it also contains a unique N-terminal extension (NTE) of unknown function. We here demonstrate that the NTE functions together with Tfam to ensure promoter-specific transcription. When the NTE is deleted, Polrmt can initiate transcription in the absence of Tfam, both from promoters and non-specific DNA sequences. Additionally, when in presence of Tfam and a mitochondrial promoter, the NTE-deleted mutant has an even higher transcription activity than wild-type polymerase, indicating that the NTE functions as an inhibitory domain. Our studies lead to a model according to which Tfam specifically recruits wild-type Polrmt to promoter sequences, relieving the inhibitory effect of the NTE, as a first step in transcription initiation. In the second step, Tfb2m is recruited into the complex and transcription is initiated. PMID:24445803

Orogenic gold deposits: a proposed classification in the context of their crustal distribution and relationship to other gold deposit types

USGS Publications Warehouse

Groves, D.I.; Goldfarb, R.J.; Gebre-Mariam, M.; Hagemann, S.G.; Robert, F.

1998-01-01

The so-called 'mesothermal' gold deposits are associated with reginally metamorphosed terranes of all ages. Ores were formed during compressional to transpressional deformation processes at convergent plate margins in accretionary and collisional orogens. In both types of orogen, hydrated marine sedimentary and volcanic rocks have been added to continental margins during tens to some 100 million years of collision. Subduction-related thermal events, episodically raising geothermal gradients within the hydrated accretionary sequences, initiate and drive long-distance hydrothermal fluid migration. The resulting gold-bearing quartz veins are emplaced over a unique depth range for hydrothermal ore deposits, with gold deposition from 15-20 km to the near surface environment. On the basis of this broad depth range of formation, the term 'mesothermal' is not applicable to this deposit types as a whole. Instead, the unique temporal and spatial association of this deposit type with orogeny means that the vein systems are best termed orogenic gold deposits. Most ores are post-orogenic with respect to to tectonism of their immediate host rocks, but are simultaneously syn-orogenic with respect to ongoing deep-crustal, subduction-related thermal processes and the prefix orogenic satisfies both these conditions. On the basis of their depth of formation, the orogenic deposits are best subdivided into epizonal (12 km) classes.

Genetic homogeneity of Clostridium botulinum type A1 strains with unique toxin gene clusters.

PubMed

Raphael, Brian H; Luquez, Carolina; McCroskey, Loretta M; Joseph, Lavin A; Jacobson, Mark J; Johnson, Eric A; Maslanka, Susan E; Andreadis, Joanne D

2008-07-01

A group of five clonally related Clostridium botulinum type A strains isolated from different sources over a period of nearly 40 years harbored several conserved genetic properties. These strains contained a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502. The strains also had a common toxin gene cluster composition (ha-/orfX+) similar to that associated with bont/A in type A strains containing an unexpressed bont/B [termed A(B) strains]. However, bont/B was not identified in the strains examined. Comparative genomic hybridization demonstrated identical genomic content among the strains relative to C. botulinum strain ATCC 3502. In addition, microarray data demonstrated the absence of several genes flanking the toxin gene cluster among the ha-/orfX+ A1 strains, suggesting the presence of genomic rearrangements with respect to this region compared to the C. botulinum ATCC 3502 strain. All five strains were shown to have identical flaA variable region nucleotide sequences. The pulsed-field gel electrophoresis patterns of the strains were indistinguishable when digested with SmaI, and a shift in the size of at least one band was observed in a single strain when digested with XhoI. These results demonstrate surprising genomic homogeneity among a cluster of unique C. botulinum type A strains of diverse origin.

De novo assembly, characterization and functional annotation of pineapple fruit transcriptome through massively parallel sequencing.

PubMed

Ong, Wen Dee; Voo, Lok-Yung Christopher; Kumar, Vijay Subbiah

2012-01-01

Pineapple (Ananas comosus var. comosus), is an important tropical non-climacteric fruit with high commercial potential. Understanding the mechanism and processes underlying fruit ripening would enable scientists to enhance the improvement of quality traits such as, flavor, texture, appearance and fruit sweetness. Although, the pineapple is an important fruit, there is insufficient transcriptomic or genomic information that is available in public databases. Application of high throughput transcriptome sequencing to profile the pineapple fruit transcripts is therefore needed. To facilitate this, we have performed transcriptome sequencing of ripe yellow pineapple fruit flesh using Illumina technology. About 4.7 millions Illumina paired-end reads were generated and assembled using the Velvet de novo assembler. The assembly produced 28,728 unique transcripts with a mean length of approximately 200 bp. Sequence similarity search against non-redundant NCBI database identified a total of 16,932 unique transcripts (58.93%) with significant hits. Out of these, 15,507 unique transcripts were assigned to gene ontology terms. Functional annotation against Kyoto Encyclopedia of Genes and Genomes pathway database identified 13,598 unique transcripts (47.33%) which were mapped to 126 pathways. The assembly revealed many transcripts that were previously unknown. The unique transcripts derived from this work have rapidly increased of the number of the pineapple fruit mRNA transcripts as it is now available in public databases. This information can be further utilized in gene expression, genomics and other functional genomics studies in pineapple.

De Novo Assembly, Characterization and Functional Annotation of Pineapple Fruit Transcriptome through Massively Parallel Sequencing

PubMed Central

Ong, Wen Dee; Voo, Lok-Yung Christopher; Kumar, Vijay Subbiah

2012-01-01

Background Pineapple (Ananas comosus var. comosus), is an important tropical non-climacteric fruit with high commercial potential. Understanding the mechanism and processes underlying fruit ripening would enable scientists to enhance the improvement of quality traits such as, flavor, texture, appearance and fruit sweetness. Although, the pineapple is an important fruit, there is insufficient transcriptomic or genomic information that is available in public databases. Application of high throughput transcriptome sequencing to profile the pineapple fruit transcripts is therefore needed. Methodology/Principal Findings To facilitate this, we have performed transcriptome sequencing of ripe yellow pineapple fruit flesh using Illumina technology. About 4.7 millions Illumina paired-end reads were generated and assembled using the Velvet de novo assembler. The assembly produced 28,728 unique transcripts with a mean length of approximately 200 bp. Sequence similarity search against non-redundant NCBI database identified a total of 16,932 unique transcripts (58.93%) with significant hits. Out of these, 15,507 unique transcripts were assigned to gene ontology terms. Functional annotation against Kyoto Encyclopedia of Genes and Genomes pathway database identified 13,598 unique transcripts (47.33%) which were mapped to 126 pathways. The assembly revealed many transcripts that were previously unknown. Conclusions The unique transcripts derived from this work have rapidly increased of the number of the pineapple fruit mRNA transcripts as it is now available in public databases. This information can be further utilized in gene expression, genomics and other functional genomics studies in pineapple. PMID:23091603

De novo design of peptide immunogens that mimic the coiled coil region of human T-cell leukemia virus type-1 glycoprotein 21 transmembrane subunit for induction of native protein reactive neutralizing antibodies.

PubMed

Sundaram, Roshni; Lynch, Marcus P; Rawale, Sharad V; Sun, Yiping; Kazanji, Mirdad; Kaumaya, Pravin T P

2004-06-04

Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.

Informational structure of genetic sequences and nature of gene splicing

NASA Astrophysics Data System (ADS)

Trifonov, E. N.

1991-10-01

Only about 1/20 of DNA of higher organisms codes for proteins, by means of classical triplet code. The rest of DNA sequences is largely silent, with unclear functions, if any. The triplet code is not the only code (message) carried by the sequences. There are three levels of molecular communication, where the same sequence ``talks'' to various bimolecules, while having, respectively, three different appearances: DNA, RNA and protein. Since the molecular structures and, hence, sequence specific preferences of these are substantially different, the original DNA sequence has to carry simultaneously three types of sequence patterns (codes, messages), thus, being a composite structure in which one had the same letter (nucleotide) is frequently involved in several overlapping codes of different nature. This multiplicity and overlapping of the codes is a unique feature of the Gnomic, language of genetic sequences. The coexisting codes have to be degenerate in various degrees to allow an optimal and concerted performance of all the encoded functions. There is an obvious conflict between the best possible performance of a given function and necessity to compromise the quality of a given sequence pattern in favor of other patterns. It appears that the major role of various changes in the sequences on their ``ontogenetic'' way from DNA to RNA to protein, like RNA editing and splicing, or protein post-translational modifications is to resolve such conflicts. New data are presented strongly indicating that the gene splicing is such a device to resolve the conflict between the code of DNA folding in chromatin and the triplet code for protein synthesis.

Genetic diversity of the DBLalpha region in Plasmodium falciparum var genes among Asia-Pacific isolates.

PubMed

Fowler, Elizabeth V; Peters, Jennifer M; Gatton, Michelle L; Chen, Nanhua; Cheng, Qin

2002-03-01

In Plasmodium falciparum a highly polymorphic multi-copy gene family, var, encodes the variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1), which has an important role in cytoadherence and immune evasion. Using previously described universal PCR primers for the first Duffy binding-like domain (DBLalpha) of var we analysed the DBLalpha repertoires of Dd2 (originally from Thailand) and eight isolates from the Solomon Islands (n=4), Philippines (n=2), Papua New Guinea (n=1) and Africa (n=1). We found 15-32 unique DBLalpha sequence types among these isolates and estimated detectable DBLalpha repertoire sizes ranging from 33-38 to 52-57 copies per genome. Our data suggest that var gene repertoires generally consist of 40-50 copies per genome. Eighteen DBLalpha sequences appeared in more than one Asia-Pacific isolate with the number of sequences shared between any two isolates ranging from 0 to 6 (mean=2.0 +/-1.6). At the amino acid level DBLalpha sequence similarity within isolates ranged from 45.2 +/- 7.1 to 50.2 +/- 6.9%, and was not significantly different from the DBLalpha amino acid sequence similarity among isolates (P>0.1). Comparisons with published sequences also revealed little overlap among DBLalpha sequences from different regions. High DBLalpha sequence diversity and minimal overlap among these isolates suggest that the global var gene repertoire is immense, and may potentially be selected for by the host's protective immune response to the var gene products, PfEMP1.

Mosaic Graphs and Comparative Genomics in Phage Communities

PubMed Central

Belcaid, Mahdi; Bergeron, Anne

2010-01-01

Abstract Comparing the genomes of two closely related viruses often produces mosaics where nearly identical sequences alternate with sequences that are unique to each genome. When several closely related genomes are compared, the unique sequences are likely to be shared with third genomes, leading to virus mosaic communities. Here we present comparative analysis of sets of Staphylococcus aureus phages that share large identical sequences with up to three other genomes, and with different partners along their genomes. We introduce mosaic graphs to represent these complex recombination events, and use them to illustrate the breath and depth of sequence sharing: some genomes are almost completely made up of shared sequences, while genomes that share very large identical sequences can adopt alternate functional modules. Mosaic graphs also allow us to identify breakpoints that could eventually be used for the construction of recombination networks. These findings have several implications on phage metagenomics assembly, on the horizontal gene transfer paradigm, and more generally on the understanding of the composition and evolutionary dynamics of virus communities. PMID:20874413

Production of Supra-regular Spatial Sequences by Macaque Monkeys.

PubMed

Jiang, Xinjian; Long, Tenghai; Cao, Weicong; Li, Junru; Dehaene, Stanislas; Wang, Liping

2018-06-18

Understanding and producing embedded sequences in language, music, or mathematics, is a central characteristic of our species. These domains are hypothesized to involve a human-specific competence for supra-regular grammars, which can generate embedded sequences that go beyond the regular sequences engendered by finite-state automata. However, is this capacity truly unique to humans? Using a production task, we show that macaque monkeys can be trained to produce time-symmetrical embedded spatial sequences whose formal description requires supra-regular grammars or, equivalently, a push-down stack automaton. Monkeys spontaneously generalized the learned grammar to novel sequences, including longer ones, and could generate hierarchical sequences formed by an embedding of two levels of abstract rules. Compared to monkeys, however, preschool children learned the grammars much faster using a chunking strategy. While supra-regular grammars are accessible to nonhuman primates through extensive training, human uniqueness may lie in the speed and learning strategy with which they are acquired. Copyright © 2018 Elsevier Ltd. All rights reserved.

Unique core genomes of the bacterial family vibrionaceae: insights into niche adaptation and speciation.

PubMed

Kahlke, Tim; Goesmann, Alexander; Hjerde, Erik; Willassen, Nils Peder; Haugen, Peik

2012-05-10

The criteria for defining bacterial species and even the concept of bacterial species itself are under debate, and the discussion is apparently intensifying as more genome sequence data is becoming available. However, it is still unclear how the new advances in genomics should be used most efficiently to address this question. In this study we identify genes that are common to any group of genomes in our dataset, to determine whether genes specific to a particular taxon exist and to investigate their potential role in adaptation of bacteria to their specific niche. These genes were named unique core genes. Additionally, we investigate the existence and importance of unique core genes that are found in isolates of phylogenetically non-coherent groups. These groups of isolates, that share a genetic feature without sharing a closest common ancestor, are termed genophyletic groups. The bacterial family Vibrionaceae was used as the model, and we compiled and compared genome sequences of 64 different isolates. Using the software orthoMCL we determined clusters of homologous genes among the investigated genome sequences. We used multilocus sequence analysis to build a host phylogeny and mapped the numbers of unique core genes of all distinct groups of isolates onto the tree. The results show that unique core genes are more likely to be found in monophyletic groups of isolates. Genophyletic groups of isolates, in contrast, are less common especially for large groups of isolate. The subsequent annotation of unique core genes that are present in genophyletic groups indicate a high degree of horizontally transferred genes. Finally, the annotation of the unique core genes of Vibrio cholerae revealed genes involved in aerotaxis and biosynthesis of the iron-chelator vibriobactin. The presented work indicates that genes specific for any taxon inside the bacterial family Vibrionaceae exist. These unique core genes encode conserved metabolic functions that can shed light on the adaptation of a species to its ecological niche. Additionally, our study suggests that unique core genes can be used to aid classification of bacteria and contribute to a bacterial species definition on a genomic level. Furthermore, these genes may be of importance in clinical diagnostics and drug development.

Comparative Genomics of Non-TNL Disease Resistance Genes from Six Plant Species.

PubMed

Nepal, Madhav P; Andersen, Ethan J; Neupane, Surendra; Benson, Benjamin V

2017-09-30

Disease resistance genes (R genes), as part of the plant defense system, have coevolved with corresponding pathogen molecules. The main objectives of this project were to identify non-Toll interleukin receptor, nucleotide-binding site, leucine-rich repeat (nTNL) genes and elucidate their evolutionary divergence across six plant genomes. Using reference sequences from Arabidopsis , we investigated nTNL orthologs in the genomes of common bean, Medicago , soybean, poplar, and rice. We used Hidden Markov Models for sequence identification, performed model-based phylogenetic analyses, visualized chromosomal positioning, inferred gene clustering, and assessed gene expression profiles. We analyzed 908 nTNL R genes in the genomes of the six plant species, and classified them into 12 subgroups based on the presence of coiled-coil (CC), nucleotide binding site (NBS), leucine rich repeat (LRR), resistance to Powdery mildew 8 (RPW8), and BED type zinc finger domains. Traditionally classified CC-NBS-LRR (CNL) genes were nested into four clades (CNL A-D) often with abundant, well-supported homogeneous subclades of Type-II R genes. CNL-D members were absent in rice, indicating a unique R gene retention pattern in the rice genome. Genomes from Arabidopsis , common bean, poplar and soybean had one chromosome without any CNL R genes. Medicago and Arabidopsis had the highest and lowest number of gene clusters, respectively. Gene expression analyses suggested unique patterns of expression for each of the CNL clades. Differential gene expression patterns of the nTNL genes were often found to correlate with number of introns and GC content, suggesting structural and functional divergence.

Comparative Genomics of Non-TNL Disease Resistance Genes from Six Plant Species

PubMed Central

Andersen, Ethan J.; Neupane, Surendra; Benson, Benjamin V.

2017-01-01

Disease resistance genes (R genes), as part of the plant defense system, have coevolved with corresponding pathogen molecules. The main objectives of this project were to identify non-Toll interleukin receptor, nucleotide-binding site, leucine-rich repeat (nTNL) genes and elucidate their evolutionary divergence across six plant genomes. Using reference sequences from Arabidopsis, we investigated nTNL orthologs in the genomes of common bean, Medicago, soybean, poplar, and rice. We used Hidden Markov Models for sequence identification, performed model-based phylogenetic analyses, visualized chromosomal positioning, inferred gene clustering, and assessed gene expression profiles. We analyzed 908 nTNL R genes in the genomes of the six plant species, and classified them into 12 subgroups based on the presence of coiled-coil (CC), nucleotide binding site (NBS), leucine rich repeat (LRR), resistance to Powdery mildew 8 (RPW8), and BED type zinc finger domains. Traditionally classified CC-NBS-LRR (CNL) genes were nested into four clades (CNL A-D) often with abundant, well-supported homogeneous subclades of Type-II R genes. CNL-D members were absent in rice, indicating a unique R gene retention pattern in the rice genome. Genomes from Arabidopsis, common bean, poplar and soybean had one chromosome without any CNL R genes. Medicago and Arabidopsis had the highest and lowest number of gene clusters, respectively. Gene expression analyses suggested unique patterns of expression for each of the CNL clades. Differential gene expression patterns of the nTNL genes were often found to correlate with number of introns and GC content, suggesting structural and functional divergence. PMID:28973974

Unique Variants in OPN1LW Cause Both Syndromic and Nonsyndromic X-Linked High Myopia Mapped to MYP1.

PubMed

Li, Jiali; Gao, Bei; Guan, Liping; Xiao, Xueshan; Zhang, Jianguo; Li, Shiqiang; Jiang, Hui; Jia, Xiaoyun; Yang, Jianhua; Guo, Xiangming; Yin, Ye; Wang, Jun; Zhang, Qingjiong

2015-06-01

MYP1 is a locus for X-linked syndromic and nonsyndromic high myopia. Recently, unique haplotypes in OPN1LW were found to be responsible for X-linked syndromic high myopia mapped to MYP1. The current study is to test if such variants in OPN1LW are also responsible for X-linked nonsyndromic high myopia mapped to MYP1. The proband of the family previously mapped to MYP1 was initially analyzed using whole-exome sequencing and whole-genome sequencing. Additional probands with early-onset high myopia were analyzed using whole-exome sequencing. Variants in OPN1LW were selected and confirmed by Sanger sequencing. Long-range and second PCR were used to determine the haplotype and the first gene of the red-green gene array. Candidate variants were further validated in family members and controls. The unique LVAVA haplotype in OPN1LW was detected in the family with X-linked nonsyndromic high myopia mapped to MYP1. In addition, this haplotype and a novel frameshift mutation (c.617_620dup, p.Phe208Argfs*51) in OPN1LW were detected in two other families with X-linked high myopia. The unique haplotype cosegregated with high myopia in the two families, with a maximum LOD score of 3.34 and 2.31 at θ = 0. OPN1LW with the variants in these families was the first gene in the red-green gene array and was not present in 247 male controls. Reevaluation of the clinical data in both families with the unique haplotype suggested nonsyndromic high myopia. Our study confirms the findings that unique variants in OPN1LW are responsible for both syndromic and nonsyndromic X-linked high myopia mapped to MYP1.

Cortical Spreading Depression Causes Unique Dysregulation of Inflammatory Pathways in a Transgenic Mouse Model of Migraine.

PubMed

Eising, Else; Shyti, Reinald; 't Hoen, Peter A C; Vijfhuizen, Lisanne S; Huisman, Sjoerd M H; Broos, Ludo A M; Mahfouz, Ahmed; Reinders, Marcel J T; Ferrari, Michel D; Tolner, Else A; de Vries, Boukje; van den Maagdenberg, Arn M J M

2017-05-01

Familial hemiplegic migraine type 1 (FHM1) is a rare monogenic subtype of migraine with aura caused by mutations in CACNA1A that encodes the α 1A subunit of voltage-gated Ca V 2.1 calcium channels. Transgenic knock-in mice that carry the human FHM1 R192Q missense mutation ('FHM1 R192Q mice') exhibit an increased susceptibility to cortical spreading depression (CSD), the mechanism underlying migraine aura. Here, we analysed gene expression profiles from isolated cortical tissue of FHM1 R192Q mice 24 h after experimentally induced CSD in order to identify molecular pathways affected by CSD. Gene expression profiles were generated using deep serial analysis of gene expression sequencing. Our data reveal a signature of inflammatory signalling upon CSD in the cortex of both mutant and wild-type mice. However, only in the brains of FHM1 R192Q mice specific genes are up-regulated in response to CSD that are implicated in interferon-related inflammatory signalling. Our findings show that CSD modulates inflammatory processes in both wild-type and mutant brains, but that an additional unique inflammatory signature becomes expressed after CSD in a relevant mouse model of migraine.

Penicillium araracuarense sp. nov., Penicillium elleniae sp. nov., Penicillium penarojense sp. nov., Penicillium vanderhammenii sp. nov. and Penicillium wotroi sp. nov., isolated from leaf litter.

PubMed

Houbraken, Jos; López-Quintero, Carlos A; Frisvad, Jens C; Boekhout, Teun; Theelen, Bart; Franco-Molano, Ana Esperanza; Samson, Robert A

2011-06-01

Several species of the genus Penicillium were isolated during a survey of the mycobiota of leaf litter and soil in Colombian Amazon forest. Five species, Penicillium penarojense sp. nov. (type strain CBS 113178(T) = IBT 23262(T)), Penicillium wotroi sp. nov. (type strain CBS 118171(T) = IBT 23253(T)), Penicillium araracuarense sp. nov. (type strain CBS 113149(T) = IBT 23247(T)), Penicillium elleniae sp. nov. (type strain CBS 118135(T) = IBT 23229(T)) and Penicillium vanderhammenii sp. nov. (type strain CBS 126216(T) = IBT 23203(T)) are described here as novel species. Their taxonomic novelty was determined using a polyphasic approach, combining phenotypic, molecular (ITS and partial β-tubulin sequences) and extrolite data. Phylogenetic analyses showed that each novel species formed a unique clade for both loci analysed and that they were most closely related to Penicillium simplicissimum, Penicillium janthinellum, Penicillium daleae and Penicillium brasilianum. An overview of the phylogeny of this taxonomically difficult group is presented, and 33 species are accepted. Each of the five novel species had a unique extrolite profile of known and uncharacterized metabolites and various compounds, such as penicillic acid, andrastin A, pulvilloric acid, paxillin, paspaline and janthitrem, were commonly produced by these phylogenetically related species. The novel species had a high growth rate on agar media, but could be distinguished from each other by several macro- and microscopical characteristics.

Global profiling of alternative RNA splicing events provides insights into molecular differences between various types of hepatocellular carcinoma.

PubMed

Tremblay, Marie-Pier; Armero, Victoria E S; Allaire, Andréa; Boudreault, Simon; Martenon-Brodeur, Camille; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S; Bisaillon, Martin

2016-08-26

Dysregulations in alternative splicing (AS) patterns have been associated with many human diseases including cancer. In the present study, alterations to the global RNA splicing landscape of cellular genes were investigated in a large-scale screen from 377 liver tissue samples using high-throughput RNA sequencing data. Our study identifies modifications in the AS patterns of transcripts encoded by more than 2500 genes such as tumor suppressor genes, transcription factors, and kinases. These findings provide insights into the molecular differences between various types of hepatocellular carcinoma (HCC). Our analysis allowed the identification of 761 unique transcripts for which AS is misregulated in HBV-associated HCC, while 68 are unique to HCV-associated HCC, 54 to HBV&HCV-associated HCC, and 299 to virus-free HCC. Moreover, we demonstrate that the expression pattern of the RNA splicing factor hnRNPC in HCC tissues significantly correlates with patient survival. We also show that the expression of the HBx protein from HBV leads to modifications in the AS profiles of cellular genes. Finally, using RNA interference and a reverse transcription-PCR screening platform, we examined the implications of cellular proteins involved in the splicing of transcripts involved in apoptosis and demonstrate the potential contribution of these proteins in AS control. This study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in hepatocellular carcinoma. Moreover, these data allowed us to identify unique signatures of genes for which AS is misregulated in the different types of HCC.

A full genomic characterization of the development of a stable Small Colony Variant cell-type by a clinical Staphylococcus aureus strain.

PubMed

Bui, Long M G; Kidd, Stephen P

2015-12-01

A key to persistent and recurrent Staphylococcus aureus infections is its ability to adapt to diverse and toxic conditions. This ability includes a switch into a biofilm or to the quasi-dormant Small Colony Variant (SCV). The development and molecular attributes of SCVs have been difficult to study due to their rapid reversion to their parental cell-type. We recently described the unique induction of a matrix-embedded and stable SCV cell-type in a clinical S. aureus strain (WCH-SK2) by growing the cells with limiting conditions for a prolonged timeframe. Here we further study their characteristics. They possessed an increased viability in the presence of antibiotics compared to their non-SCV form. Their stability implied that there had been genetic changes; we therefore determined both the genome sequence of WCH-SK2 and its stable SCV form at a single base resolution, employing Single Molecular Real-Time (SMRT) sequencing that enabled the methylome to also be determined. The genetic features of WCH-SK2 have been identified; the SCCmec type, the pathogenicity and genetic islands and virulence factors. The genetic changes that had occurred in the stable SCV form were identified; most notably being in MgrA, a global regulator, and RsbU, a phosphoserine phosphatase within the regulatory pathway of the sigma factor SigB. There was a shift in the methylomes of the non-SCV and stable SCV forms. We have also shown a similar induction of this cell-type in other S. aureus strains and performed a genetic comparison to these and other S. aureus genomes. We additionally map RNAseq data to the WCH-SK2 genome in a transcriptomic analysis of the parental, SCV and stable SCV cells. The results from this study represent the unique identification of a suite of epigenetic, genetic and transcriptional factors that are implicated in the switch in S. aureus to its persistent SCV form. Copyright © 2015 Elsevier B.V. All rights reserved.

Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers

PubMed Central

2011-01-01

Background Panax notoginseng (Burk) F.H. Chen is important medicinal plant of the Araliacease family. Triterpene saponins are the bioactive constituents in P. notoginseng. However, available genomic information regarding this plant is limited. Moreover, details of triterpene saponin biosynthesis in the Panax species are largely unknown. Results Using the 454 pyrosequencing technology, a one-quarter GS FLX titanium run resulted in 188,185 reads with an average length of 410 bases for P. notoginseng root. These reads were processed and assembled by 454 GS De Novo Assembler software into 30,852 unique sequences. A total of 70.2% of unique sequences were annotated by Basic Local Alignment Search Tool (BLAST) similarity searches against public sequence databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG) assignment discovered 41 unique sequences representing 11 genes involved in triterpene saponin backbone biosynthesis in the 454-EST dataset. In particular, the transcript encoding dammarenediol synthase (DS), which is the first committed enzyme in the biosynthetic pathway of major triterpene saponins, is highly expressed in the root of four-year-old P. notoginseng. It is worth emphasizing that the candidate cytochrome P450 (Pn02132 and Pn00158) and UDP-glycosyltransferase (Pn00082) gene most likely to be involved in hydroxylation or glycosylation of aglycones for triterpene saponin biosynthesis were discovered from 174 cytochrome P450s and 242 glycosyltransferases by phylogenetic analysis, respectively. Putative transcription factors were detected in 906 unique sequences, including Myb, homeobox, WRKY, basic helix-loop-helix (bHLH), and other family proteins. Additionally, a total of 2,772 simple sequence repeat (SSR) were identified from 2,361 unique sequences, of which, di-nucleotide motifs were the most abundant motif. Conclusion This study is the first to present a large-scale EST dataset for P. notoginseng root acquired by next-generation sequencing (NGS) technology. The candidate genes involved in triterpene saponin biosynthesis, including the putative CYP450s and UGTs, were obtained in this study. Additionally, the identification of SSRs provided plenty of genetic makers for molecular breeding and genetics applications in this species. These data will provide information on gene discovery, transcriptional regulation and marker-assisted selection for P. notoginseng. The dataset establishes an important foundation for the study with the purpose of ensuring adequate drug resources for this species. PMID:22369100

The Human Microbiome and Understanding the 16S rRNA Gene in Translational Nursing Science

PubMed Central

Ames, Nancy J.; Ranucci, Alexandra; Moriyama, Brad; Wallen, Gwenyth R.

2017-01-01

Background As more is understood regarding the human microbiome, it is increasingly important for nurse scientists and health care practitioners to analyze these microbial communities and their role in health and disease.16S rRNA sequencing is a key methodology in identifying these bacterial populations that has recently transitioned from use primarily in research to having increased utility in clinical settings. Objectives The objectives of this review are to: (a) describe 16S rRNA sequencing and its role in answering research questions important to nursing science; (b) provide an overview of the oral, lung and gut microbiomes and relevant research; and (c) identify future implications for microbiome research and 16S sequencing in translational nursing science. Discussion Sequencing using the 16S rRNA gene has revolutionized research and allowed scientists to easily and reliably characterize complex bacterial communities. This type of research has recently entered the clinical setting, one of the best examples involving the use of 16S sequencing to identify resistant pathogens, thereby improving the accuracy of bacterial identification in infection control. Clinical microbiota research and related requisite methods are of particular relevance to nurse scientists—individuals uniquely positioned to utilize these techniques in future studies in clinical settings. PMID:28252578

Barcode Identifiers as a Practical Tool for Reliable Species Assignment of Medically Important Black Yeast Species

PubMed Central

Heinrichs, Guido; de Hoog, G. Sybren

2012-01-01

Herpotrichiellaceous black yeasts and relatives comprise severe pathogens flanked by nonpathogenic environmental siblings. Reliable identification by conventional methods is notoriously difficult. Molecular identification is hampered by the sequence variability in the internal transcribed spacer (ITS) domain caused by difficult-to-sequence homopolymeric regions and by poor taxonomic attribution of sequences deposited in GenBank. Here, we present a potential solution using short barcode identifiers (27 to 50 bp) based on ITS2 ribosomal DNA (rDNA), which allows unambiguous definition of species-specific fragments. Starting from proven sequences of ex-type and authentic strains, we were able to describe 103 identifiers. Multiple BLAST searches of these proposed barcode identifiers in GenBank revealed uniqueness for 100 taxonomic entities, whereas the three remaining identifiers each matched with two entities, but the species of these identifiers could easily be discriminated by differences in the remaining ITS regions. Using the proposed barcode identifiers, a 4.1-fold increase of 100% matches in GenBank was achieved in comparison to the classical approach using the complete ITS sequences. The proposed barcode identifiers will be made accessible for the diagnostic laboratory in a permanently updated online database, thereby providing a highly practical, reliable, and cost-effective tool for identification of clinically important black yeasts and relatives. PMID:22785187

De Novo Assembly of Human Herpes Virus Type 1 (HHV-1) Genome, Mining of Non-Canonical Structures and Detection of Novel Drug-Resistance Mutations Using Short- and Long-Read Next Generation Sequencing Technologies

PubMed Central

Karamitros, Timokratis; Piorkowska, Renata; Katzourakis, Aris; Magiorkinis, Gkikas; Mbisa, Jean Lutamyo

2016-01-01

Human herpesvirus type 1 (HHV-1) has a large double-stranded DNA genome of approximately 152 kbp that is structurally complex and GC-rich. This makes the assembly of HHV-1 whole genomes from short-read sequencing data technically challenging. To improve the assembly of HHV-1 genomes we have employed a hybrid genome assembly protocol using data from two sequencing technologies: the short-read Roche 454 and the long-read Oxford Nanopore MinION sequencers. We sequenced 18 HHV-1 cell culture-isolated clinical specimens collected from immunocompromised patients undergoing antiviral therapy. The susceptibility of the samples to several antivirals was determined by plaque reduction assay. Hybrid genome assembly resulted in a decrease in the number of contigs in 6 out of 7 samples and an increase in N(G)50 and N(G)75 of all 7 samples sequenced by both technologies. The approach also enhanced the detection of non-canonical contigs including a rearrangement between the unique (UL) and repeat (T/IRL) sequence regions of one sample that was not detectable by assembly of 454 reads alone. We detected several known and novel resistance-associated mutations in UL23 and UL30 genes. Genome-wide genetic variability ranged from <1% to 53% of amino acids in each gene exhibiting at least one substitution within the pool of samples. The UL23 gene had one of the highest genetic variabilities at 35.2% in keeping with its role in development of drug resistance. The assembly of accurate, full-length HHV-1 genomes will be useful in determining genetic determinants of drug resistance, virulence, pathogenesis and viral evolution. The numerous, complex repeat regions of the HHV-1 genome currently remain a barrier towards this goal. PMID:27309375

De Novo Assembly of Human Herpes Virus Type 1 (HHV-1) Genome, Mining of Non-Canonical Structures and Detection of Novel Drug-Resistance Mutations Using Short- and Long-Read Next Generation Sequencing Technologies.

PubMed

Karamitros, Timokratis; Harrison, Ian; Piorkowska, Renata; Katzourakis, Aris; Magiorkinis, Gkikas; Mbisa, Jean Lutamyo

2016-01-01

Human herpesvirus type 1 (HHV-1) has a large double-stranded DNA genome of approximately 152 kbp that is structurally complex and GC-rich. This makes the assembly of HHV-1 whole genomes from short-read sequencing data technically challenging. To improve the assembly of HHV-1 genomes we have employed a hybrid genome assembly protocol using data from two sequencing technologies: the short-read Roche 454 and the long-read Oxford Nanopore MinION sequencers. We sequenced 18 HHV-1 cell culture-isolated clinical specimens collected from immunocompromised patients undergoing antiviral therapy. The susceptibility of the samples to several antivirals was determined by plaque reduction assay. Hybrid genome assembly resulted in a decrease in the number of contigs in 6 out of 7 samples and an increase in N(G)50 and N(G)75 of all 7 samples sequenced by both technologies. The approach also enhanced the detection of non-canonical contigs including a rearrangement between the unique (UL) and repeat (T/IRL) sequence regions of one sample that was not detectable by assembly of 454 reads alone. We detected several known and novel resistance-associated mutations in UL23 and UL30 genes. Genome-wide genetic variability ranged from <1% to 53% of amino acids in each gene exhibiting at least one substitution within the pool of samples. The UL23 gene had one of the highest genetic variabilities at 35.2% in keeping with its role in development of drug resistance. The assembly of accurate, full-length HHV-1 genomes will be useful in determining genetic determinants of drug resistance, virulence, pathogenesis and viral evolution. The numerous, complex repeat regions of the HHV-1 genome currently remain a barrier towards this goal.

Cell cycle, differentiation and tissue-independent expression of ribosomal protein L37.

PubMed

Su, S; Bird, R C

1995-09-15

A unique human cDNA (hG1.16) that encodes a mRNA of 450 nucleotides was isolated from a subtractive library derived from HeLa cells. The relative expression level of hG1.16 during different cell-cycle phases was determined by Northern-blot analysis of cells synchronized by double-thymidine block and serum deprivation/refeeding. hG1.16 was constitutively expressed during all phases of the cell cycle, including the quiescent phase when even most constitutively expressed genes experience some suppression of expression. The expression level of hG1.16 did not change during terminal differentiation of myoblasts to myotubes, during which cells become permanently post-mitotic. Examination of other tissues revealed that the relative expression level of hG1.16 was constitutive in all embryonic mouse tissues examined, including brain, eye, heart, kidney, liver, lung and skeletal muscle. This was unusual in that expression was not down-modulated during differentiation and did not vary appreciably between tissue types. Analysis by inter-species Northern-blot analysis revealed that hG1.16 was highly conserved among all vertebrates studied (from fish to humans but not in insects). DNA sequence analysis of hG1.16 revealed a high level of similarity to rat ribosomal protein L37, identifying hG1.16 as a new member of this multigene family. The deduced amino acid sequence of hG1.16 was identical to rat ribosomal protein L37 that contained 97 amino acids, many of which are highly positively charged (15 arginine and 14 lysine residues with a predicted M(r) of 11,065). hG1.16 protein has a single C2-C2 zinc-finger-like motif which is also present in rat ribosomal protein L37. Using primers designed from the sequence of hG1.16, unique bovine and rat cDNAs were also isolated by 5'-rapid-amplification of cDNA ends. DNA sequences of bovine and rat G1.16, clones were 92.8% and 92.2% similar to human G1.16 while the deduced amino acid sequences derived from bovine and rat cDNAs each differed by a single amino acid from the sequence of hG1.16 and the published rat L37 sequence. Southern-blot analysis revealed that hG1.16 exists in multiple copies in human, rat and mouse genomes. These G1.16 clones encode unique human, rat and bovine members of the ribosomal protein L37 gene family, which are constitutively expressed even during transitions from quiescence to active cell proliferation or terminal differentiation, in all tissues and all vertebrates investigated.

Animal selection for whole genome sequencing by quantifying the unique contribution of homozygous haplotypes sequenced

USDA-ARS?s Scientific Manuscript database

Major whole genome sequencing projects promise to identify rare and causal variants within livestock species; however, the efficient selection of animals for sequencing remains a major problem within these surveys. The goal of this project was to develop a library of high accuracy genetic variants f...

Insights to Genetic Characterization Tools for Epidemiological Tracking of Francisella tularensis in Sweden

PubMed Central

Wahab, Tara; Birdsell, Dawn N.; Hjertqvist, Marika; Mitchell, Cedar L.; Wagner, David M.; Keim, Paul S.; Hedenström, Ingela; Löfdahl, Sven

2014-01-01

Tularaemia, caused by the bacterium Francisella tularensis, is endemic in Sweden and is poorly understood. The aim of this study was to evaluate the effectiveness of three different genetic typing systems to link a genetic type to the source and place of tularemia infection in Sweden. Canonical single nucleotide polymorphisms (canSNPs), MLVA including five variable number of tandem repeat loci and PmeI-PFGE were tested on 127 F. tularensis positive specimens collected from Swedish case-patients. All three typing methods identified two major genetic groups with near-perfect agreement. Higher genetic resolution was obtained with canSNP and MLVA compared to PFGE; F. tularensis samples were first assigned into ten phylogroups based on canSNPs followed by 33 unique MLVA types. Phylogroups were geographically analysed to reveal complex phylogeographic patterns in Sweden. The extensive phylogenetic diversity found within individual counties posed a challenge to linking specific genetic types with specific geographic locations. Despite this, a single phylogroup (B.22), defined by a SNP marker specific to a lone Swedish sequenced strain, did link genetic type with a likely geographic place. This result suggests that SNP markers, highly specific to a particular reference genome, may be found most frequently among samples recovered from the same location where the reference genome originated. This insight compels us to consider whole-genome sequencing (WGS) as the appropriate tool for effectively linking specific genetic type to geography. Comparing the WGS of an unknown sample to WGS databases of archived Swedish strains maximizes the likelihood of revealing those rare geographically informative SNPs. PMID:25401326

The respective roles of polar/nonpolar binary patterns and amino acid composition in protein regular secondary structures explored exhaustively using hydrophobic cluster analysis.

PubMed

Rebehmed, Joseph; Quintus, Flavien; Mornon, Jean-Paul; Callebaut, Isabelle

2016-05-01

Several studies have highlighted the leading role of the sequence periodicity of polar and nonpolar amino acids (binary patterns) in the formation of regular secondary structures (RSS). However, these were based on the analysis of only a few simple cases, with no direct mean to correlate binary patterns with the limits of RSS. Here, HCA-derived hydrophobic clusters (HC) which are conditioned binary patterns whose positions fit well those of RSS, were considered. All the HC types, defined by unique binary patterns, which were commonly observed in three-dimensional (3D) structures of globular domains, were analyzed. The 180 HC types with preferences for either α-helices or β-strands distinctly contain basic binary units typical of these RSS. Therefore a general trend supporting the "binary pattern preference" assumption was observed. HC for which observed RSS are in disagreement with their expected behavior (discordant HC) were also examined. They were separated in HC types with moderate preferences for RSS, having "weak" binary patterns and versatile RSS and HC types with high preferences for RSS, having "strong" binary patterns and then displaying nonpolar amino acids at the protein surface. It was shown that in both cases, discordant HC could be distinguished from concordant ones by well-differentiated amino acid compositions. The obtained results could, thus, help to complement the currently available methods for the accurate prediction of secondary structures in proteins from the only information of a single amino acid sequence. This can be especially useful for characterizing orphan sequences and for assisting protein engineering and design. © 2016 Wiley Periodicals, Inc.

Lateral Transfer of a Lectin-Like Antifreeze Protein Gene in Fishes

PubMed Central

Graham, Laurie A.; Lougheed, Stephen C.; Ewart, K. Vanya; Davies, Peter L.

2008-01-01

Fishes living in icy seawater are usually protected from freezing by endogenous antifreeze proteins (AFPs) that bind to ice crystals and stop them from growing. The scattered distribution of five highly diverse AFP types across phylogenetically disparate fish species is puzzling. The appearance of radically different AFPs in closely related species has been attributed to the rapid, independent evolution of these proteins in response to natural selection caused by sea level glaciations within the last 20 million years. In at least one instance the same type of simple repetitive AFP has independently originated in two distant species by convergent evolution. But, the isolated occurrence of three very similar type II AFPs in three distantly related species (herring, smelt and sea raven) cannot be explained by this mechanism. These globular, lectin-like AFPs have a unique disulfide-bonding pattern, and share up to 85% identity in their amino acid sequences, with regions of even higher identity in their genes. A thorough search of current databases failed to find a homolog in any other species with greater than 40% amino acid sequence identity. Consistent with this result, genomic Southern blots showed the lectin-like AFP gene was absent from all other fish species tested. The remarkable conservation of both intron and exon sequences, the lack of correlation between evolutionary distance and mutation rate, and the pattern of silent vs non-silent codon changes make it unlikely that the gene for this AFP pre-existed but was lost from most branches of the teleost radiation. We propose instead that lateral gene transfer has resulted in the occurrence of the type II AFPs in herring, smelt and sea raven and allowed these species to survive in an otherwise lethal niche. PMID:18612417

Identification, characterization and description of Arcobacter faecis sp. nov., isolated from a human waste septic tank.

PubMed

Whiteduck-Léveillée, Kerri; Whiteduck-Léveillée, Jenni; Cloutier, Michel; Tambong, James T; Xu, Renlin; Topp, Edward; Arts, Michael T; Chao, Jerry; Adam, Zaky; Lévesque, C André; Lapen, David R; Villemur, Richard; Khan, Izhar U H

2016-03-01

A study on the taxonomic classification of Arcobacter species was performed on the cultures isolated from various fecal sources where an Arcobacter strain AF1078(T) from human waste septic tank near Ottawa, Ontario, Canada was characterized using a polyphasic approach. Genetic investigations including 16S rRNA, atpA, cpn60, gyrA, gyrB and rpoB gene sequences of strain AF1078(T) are unique in comparison with other arcobacters. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain is most closely related to Arcobacter lanthieri and Arcobacter cibarius. Analyses of atpA, cpn60, gyrA, gyrB and rpoB gene sequences suggested that strain AF1078(T) formed a phylogenetic lineage independent of other species in the genus. Whole-genome sequence, DNA-DNA hybridization, fatty acid profile and phenotypic analysis further supported the conclusion that strain AF1078(T) represents a novel Arcobacter species, for which the name Arcobacter faecis sp. nov. is proposed, with type strain AF1078(T) (=LMG 28519(T); CCUG 66484(T)). Crown Copyright © 2015. Published by Elsevier GmbH. All rights reserved.

Whole-Genome Sequence Analysis of Bombella intestini LMG 28161T, a Novel Acetic Acid Bacterium Isolated from the Crop of a Red-Tailed Bumble Bee, Bombus lapidarius.

PubMed

Li, Leilei; Illeghems, Koen; Van Kerrebroeck, Simon; Borremans, Wim; Cleenwerck, Ilse; Smagghe, Guy; De Vuyst, Luc; Vandamme, Peter

2016-01-01

The whole-genome sequence of Bombella intestini LMG 28161T, an endosymbiotic acetic acid bacterium (AAB) occurring in bumble bees, was determined to investigate the molecular mechanisms underlying its metabolic capabilities. The draft genome sequence of B. intestini LMG 28161T was 2.02 Mb. Metabolic carbohydrate pathways were in agreement with the metabolite analyses of fermentation experiments and revealed its oxidative capacity towards sucrose, D-glucose, D-fructose and D-mannitol, but not ethanol and glycerol. The results of the fermentation experiments also demonstrated that the lack of effective aeration in small-scale carbohydrate consumption experiments may be responsible for the lack of reproducibility of such results in taxonomic studies of AAB. Finally, compared to the genome sequences of its nearest phylogenetic neighbor and of three other insect associated AAB strains, the B. intestini LMG 28161T genome lost 69 orthologs and included 89 unique genes. Although many of the latter were hypothetical they also included several type IV secretion system proteins, amino acid transporter/permeases and membrane proteins which might play a role in the interaction with the bumble bee host.

An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex

PubMed Central

Chen, Kenian; Sloan, Steven A.; Bennett, Mariko L.; Scholze, Anja R.; O'Keeffe, Sean; Phatnani, Hemali P.; Guarnieri, Paolo; Caneda, Christine; Ruderisch, Nadine; Deng, Shuyun; Liddelow, Shane A.; Zhang, Chaolin; Daneman, Richard; Maniatis, Tom; Barres, Ben A.

2014-01-01

The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain. PMID:25186741

Improved traceability of Shiga-toxin-producing Escherichia coli using CRISPRs for detection and typing.

PubMed

Delannoy, Sabine; Beutin, Lothar; Fach, Patrick

2016-05-01

Among strains of Shiga-toxin-producing Escherichia coli (STEC), seven serogroups (O26, O45, O103, O111, O121, O145, and O157) are frequently associated with severe clinical illness in humans. The development of methods for their reliable detection from complex samples such as food has been challenging thus far, and is currently based on the PCR detection of the major virulence genes stx1, stx2, and eae, and O-serogroup-specific genes. However, this approach lacks resolution. Moreover, new STEC serotypes are continuously emerging worldwide. For example, in May 2011, strains belonging to the hitherto rarely detected STEC serotype O104:H4 were identified as causative agents of one of the world's largest outbreak of disease with a high incidence of hemorrhagic colitis and hemolytic uremic syndrome in the infected patients. Discriminant typing of pathogens is crucial for epidemiological surveillance and investigations of outbreaks, and especially for tracking and tracing in case of accidental and deliberate contamination of food and water samples. Clustered regularly interspaced short palindromic repeats (CRISPRs) are composed of short, highly conserved DNA repeats separated by unique sequences of similar length. This distinctive sequence signature of CRISPRs can be used for strain typing in several bacterial species including STEC. This review discusses how CRISPRs have recently been used for STEC identification and typing.

Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

PubMed Central

Mizuno, Takako; Sridharan, Anusha; Du, Yina; Guo, Minzhe; Wikenheiser-Brokamp, Kathryn A.; Perl, Anne-Karina T.; Funari, Vincent A.; Gokey, Jason J.; Stripp, Barry R.; Whitsett, Jeffrey A.

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease. PMID:27942595

Photoautotrophic symbiont and geography are major factors affecting highly structured and diverse bacterial communities in the lichen microbiome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hodkinson, Brendan P; Gottel, Neil R; Schadt, Christopher Warren

2011-01-01

Although common knowledge dictates that the lichen thallus is formed solely by a fungus (mycobiont) that develops a symbiotic relationship with an alga and/or cyanobacterium (photobiont), the non-photoautotrophic bacteria found in lichen microbiomes are increasingly regarded as integral components of lichen thalli. For this study, comparative analyses were conducted on lichen-associated bacterial communities to test for effects of photobiont-types (i.e. green algal vs. cyanobacterial), mycobiont-types and large-scale spatial distances (from tropical to arctic latitudes). Amplicons of the 16S (SSU) rRNA gene were examined using both Sanger sequencing of cloned fragments and barcoded pyrosequencing. Rhizobiales is typically the most abundant andmore » taxonomically diverse order in lichen microbiomes; however, overall bacterial diversity in lichens is shown to be much higher than previously reported. Members of Acidobacteriaceae, Acetobacteraceae, Brucellaceae and sequence group LAR1 are the most commonly found groups across the phylogenetically and geographically broad array of lichens examined here. Major bacterial community trends are significantly correlated with differences in large-scale geography, photobiont-type and mycobiont-type. The lichen as a microcosm represents a structured, unique microbial habitat with greater ecological complexity and bacterial diversity than previously appreciated and can serve as a model system for studying larger ecological and evolutionary principles.« less

High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs.

PubMed

Panda, Amaresh C; De, Supriyo; Grammatikakis, Ioannis; Munk, Rachel; Yang, Xiaoling; Piao, Yulan; Dudekula, Dawood B; Abdelmohsen, Kotb; Gorospe, Myriam

2017-07-07

High-throughput RNA sequencing methods coupled with specialized bioinformatic analyses have recently uncovered tens of thousands of unique circular (circ)RNAs, but their complete sequences, genes of origin and functions are largely unknown. Given that circRNAs lack free ends and are thus relatively stable, their association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression programs. While exoribonuclease treatment is widely used to degrade linear RNAs and enrich circRNAs in RNA samples, it does not efficiently eliminate all linear RNAs. Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

Regulation of the Human Endogenous Retrovirus K (HML-2) Transcriptome by the HIV-1 Tat Protein

PubMed Central

Gonzalez-Hernandez, Marta J.; Cavalcoli, James D.; Sartor, Maureen A.; Contreras-Galindo, Rafael; Meng, Fan; Dai, Manhong; Dube, Derek; Saha, Anjan K.; Gitlin, Scott D.; Omenn, Gilbert S.; Kaplan, Mark H.

2014-01-01

ABSTRACT Approximately 8% of the human genome is made up of endogenous retroviral sequences. As the HIV-1 Tat protein activates the overall expression of the human endogenous retrovirus type K (HERV-K) (HML-2), we used next-generation sequencing to determine which of the 91 currently annotated HERV-K (HML-2) proviruses are regulated by Tat. Transcriptome sequencing of total RNA isolated from Tat- and vehicle-treated peripheral blood lymphocytes from a healthy donor showed that Tat significantly activates expression of 26 unique HERV-K (HML-2) proviruses, silences 12, and does not significantly alter the expression of the remaining proviruses. Quantitative reverse transcription-PCR validation of the sequencing data was performed on Tat-treated PBLs of seven donors using provirus-specific primers and corroborated the results with a substantial degree of quantitative similarity. IMPORTANCE The expression of HERV-K (HML-2) is tightly regulated but becomes markedly increased following infection with HIV-1, in part due to the HIV-1 Tat protein. The findings reported here demonstrate the complexity of the genome-wide regulation of HERV-K (HML-2) expression by Tat. This work also demonstrates that although HERV-K (HML-2) proviruses in the human genome are highly similar in terms of DNA sequence, modulation of the expression of specific proviruses in a given biological situation can be ascertained using next-generation sequencing and bioinformatics analysis. PMID:24872592

High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs

PubMed Central

De, Supriyo; Grammatikakis, Ioannis; Munk, Rachel; Yang, Xiaoling; Piao, Yulan; Dudekula, Dawood B.; Gorospe, Myriam

2017-01-01

Abstract High-throughput RNA sequencing methods coupled with specialized bioinformatic analyses have recently uncovered tens of thousands of unique circular (circ)RNAs, but their complete sequences, genes of origin and functions are largely unknown. Given that circRNAs lack free ends and are thus relatively stable, their association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression programs. While exoribonuclease treatment is widely used to degrade linear RNAs and enrich circRNAs in RNA samples, it does not efficiently eliminate all linear RNAs. Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions. PMID:28444238

Two new miniature inverted-repeat transposable elements in the genome of the clam Donax trunculus.

PubMed

Šatović, Eva; Plohl, Miroslav

2017-10-01

Repetitive sequences are important components of eukaryotic genomes that drive their evolution. Among them are different types of mobile elements that share the ability to spread throughout the genome and form interspersed repeats. To broaden the generally scarce knowledge on bivalves at the genome level, in the clam Donax trunculus we described two new non-autonomous DNA transposons, miniature inverted-repeat transposable elements (MITEs), named DTC M1 and DTC M2. Like other MITEs, they are characterized by their small size, their A + T richness, and the presence of terminal inverted repeats (TIRs). DTC M1 and DTC M2 are 261 and 286 bp long, respectively, and in addition to TIRs, both of them contain a long imperfect palindrome sequence in their central parts. These elements are present in complete and truncated versions within the genome of the clam D. trunculus. The two new MITEs share only structural similarity, but lack any nucleotide sequence similarity to each other. In a search for related elements in databases, blast search revealed within the Crassostrea gigas genome a larger element sharing sequence similarity only to DTC M1 in its TIR sequences. The lack of sequence similarity with any previously published mobile elements indicates that DTC M1 and DTC M2 elements may be unique to D. trunculus.

Construction, database integration, and application of an Oenothera EST library.

PubMed

Mrácek, Jaroslav; Greiner, Stephan; Cho, Won Kyong; Rauwolf, Uwe; Braun, Martha; Umate, Pavan; Altstätter, Johannes; Stoppel, Rhea; Mlcochová, Lada; Silber, Martina V; Volz, Stefanie M; White, Sarah; Selmeier, Renate; Rudd, Stephen; Herrmann, Reinhold G; Meurer, Jörg

2006-09-01

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.

A Review of Living Collections with Special Emphasis on Sustainability and Its Impact on Research Across Multiple Disciplines

PubMed Central

2017-01-01

Formal living collections have unique characteristics that distinguish them from other types of biorepositories. Comprising diverse resources, microbe culture collections, crop and biodiversity plant germplasm collections, and animal germplasm repositories are commonly allied with specific research communities or stakeholder groups. Among living collections, microbial culture collections have very long and unique life histories, with some being older than 100 years. Regulatory, financial, and technical developments have impacted living collections in many ways. International treaty obligations and restrictions on release of genetically modified organisms complicate the activities of living collections. Funding for living collections is a continuing challenge and threatens to create a two-tier system where medically relevant collections are well funded and all other collections are underfunded and hence understaffed. Molecular, genetic, and whole genome sequence analysis of contents of microbes and other living resource collections bring additional value to living collections. PMID:27869477

A Review of Living Collections with Special Emphasis on Sustainability and Its Impact on Research Across Multiple Disciplines.

PubMed

McCluskey, Kevin

2017-02-01

Formal living collections have unique characteristics that distinguish them from other types of biorepositories. Comprising diverse resources, microbe culture collections, crop and biodiversity plant germplasm collections, and animal germplasm repositories are commonly allied with specific research communities or stakeholder groups. Among living collections, microbial culture collections have very long and unique life histories, with some being older than 100 years. Regulatory, financial, and technical developments have impacted living collections in many ways. International treaty obligations and restrictions on release of genetically modified organisms complicate the activities of living collections. Funding for living collections is a continuing challenge and threatens to create a two-tier system where medically relevant collections are well funded and all other collections are underfunded and hence understaffed. Molecular, genetic, and whole genome sequence analysis of contents of microbes and other living resource collections bring additional value to living collections.

Unique Crystallization of Fullerenes: Fullerene Flowers

PubMed Central

Kim, Jungah; Park, Chibeom; Song, Intek; Lee, Minkyung; Kim, Hyungki; Choi, Hee Cheul

2016-01-01

Solution-phase crystallization of fullerene molecules strongly depends on the types of solvent and their ratios because solvent molecules are easily included in the crystal lattice and distort its structure. The C70 (solute)–mesitylene (solvent) system yields crystals with various morphologies and structures, such as cubes, tubes, and imperfect rods. Herein, using C60 and C70 dissolved in mesitylene, we present a novel way to grow unique flower-shaped crystals with six symmetric petals. The different solubility of C60 and C70 in mesitylene promotes nucleation of C70 with sixfold symmetry in the early stage, which is followed by co-crystallization of both C60 and C70 molecules, leading to lateral petal growth. Based on the growth mechanism, we obtained more complex fullerene crystals, such as multi-deck flowers and tube-flower complexes, by changing the sequence and parameters of crystallization. PMID:27561446

Using the self-select paradigm to delineate the nature of speech motor programming.

PubMed

Wright, David L; Robin, Don A; Rhee, Jooyhun; Vaculin, Amber; Jacks, Adam; Guenther, Frank H; Fox, Peter T

2009-06-01

The authors examined the involvement of 2 speech motor programming processes identified by S. T. Klapp (1995, 2003) during the articulation of utterances differing in syllable and sequence complexity. According to S. T. Klapp, 1 process, INT, resolves the demands of the programmed unit, whereas a second process, SEQ, oversees the serial order demands of longer sequences. A modified reaction time paradigm was used to assess INT and SEQ demands. Specifically, syllable complexity was dependent on syllable structure, whereas sequence complexity involved either repeated or unique syllabi within an utterance. INT execution was slowed when articulating single syllables in the form CCCV compared to simpler CV syllables. Planning unique syllables within a multisyllabic utterance rather than repetitions of the same syllable slowed INT but not SEQ. The INT speech motor programming process, important for mental syllabary access, is sensitive to changes in both syllable structure and the number of unique syllables in an utterance.

Whole-Genome Sequencing and Variant Analysis of Human Papillomavirus 16 Infections.

PubMed

van der Weele, Pascal; Meijer, Chris J L M; King, Audrey J

2017-10-01

Human papillomavirus (HPV) is a strongly conserved DNA virus, high-risk types of which can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A to D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole-genome level. We analyzed HPV16 major variant diversity and conservation in persistent infections and performed a single nucleotide polymorphism (SNP) comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to 3 years. Our analysis shows a remarkably large variant diversity in the population. Whole-genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified where the initial and follow-up samples clustered differently. Non-A1/A2 variants seemed to clear preferentially ( P = 0.02). Our analysis shows that population-wide HPV16 sequence diversity is very large. In persistent infections, the HPV16 sequence was fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strongly acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to 3 years of follow-up. These findings suggest the progression of an early HPV16 infection could be host related. IMPORTANCE Human papillomavirus 16 (HPV16) is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge of variants circulating in European populations, especially with longitudinal follow-up, is limited. By sequencing a number of infections with known follow-up for up to 3 years, we gained initial insights into the genetic diversity of HPV16 and the effects of the viral genome on the persistence of infections. A SNP comparison between sequences obtained from clearing and persistent infections did not identify strongly acting DNA variations responsible for these infection outcomes. In addition, we identified an HPV16 reinfection event where sequencing of initial and follow-up samples showed different HPV16 variants. Based on conventional genotyping, this infection would incorrectly be considered a persistent HPV16 infection. In the context of vaccine efficacy and monitoring studies, such infections could potentially cause reduced reported efficacy or efficiency. Copyright © 2017 van der Weele et al.

Fast fMRI provides high statistical power in the analysis of epileptic networks.

PubMed

Jacobs, Julia; Stich, Julia; Zahneisen, Benjamin; Assländer, Jakob; Ramantani, Georgia; Schulze-Bonhage, Andreas; Korinthenberg, Rudolph; Hennig, Jürgen; LeVan, Pierre

2014-03-01

EEG-fMRI is a unique method to combine the high temporal resolution of EEG with the high spatial resolution of MRI to study generators of intrinsic brain signals such as sleep grapho-elements or epileptic spikes. While the standard EPI sequence in fMRI experiments has a temporal resolution of around 2.5-3s a newly established fast fMRI sequence called MREG (Magnetic-Resonance-Encephalography) provides a temporal resolution of around 100ms. This technical novelty promises to improve statistics, facilitate correction of physiological artifacts and improve the understanding of epileptic networks in fMRI. The present study compares simultaneous EEG-EPI and EEG-MREG analyzing epileptic spikes to determine the yield of fast MRI in the analysis of intrinsic brain signals. Patients with frequent interictal spikes (>3/20min) underwent EEG-MREG and EEG-EPI (3T, 20min each, voxel size 3×3×3mm, EPI TR=2.61s, MREG TR=0.1s). Timings of the spikes were used in an event-related analysis to generate activation maps of t-statistics. (FMRISTAT, |t|>3.5, cluster size: 7 voxels, p<0.05 corrected). For both sequences, the amplitude and location of significant BOLD activations were compared with the spike topography. 13 patients were recorded and 33 different spike types could be analyzed. Peak T-values were significantly higher in MREG than in EPI (p<0.0001). Positive BOLD effects correlating with the spike topography were found in 8/29 spike types using the EPI and in 22/33 spikes types using the MREG sequence. Negative BOLD responses in the default mode network could be observed in 3/29 spike types with the EPI and in 19/33 with the MREG sequence. With the latter method, BOLD changes were observed even when few spikes occurred during the investigation. Simultaneous EEG-MREG thus is possible with good EEG quality and shows higher sensitivity in regard to the localization of spike-related BOLD responses than EEG-EPI. The development of new methods of analysis for this sequence such as modeling of physiological noise, temporal analysis of the BOLD signal and defining appropriate thresholds is required to fully profit from its high temporal resolution. © 2013.

Next-Generation DNA Sequencing of VH/VL Repertoires: A Primer and Guide to Applications in Single-Domain Antibody Discovery.

PubMed

Henry, Kevin A

2018-01-01

Immunogenetic analyses of expressed antibody repertoires are becoming increasingly common experimental investigations and are critical to furthering our understanding of autoimmunity, infectious disease, and cancer. Next-generation DNA sequencing (NGS) technologies have now made it possible to interrogate antibody repertoires to unprecedented depths, typically by sequencing of cDNAs encoding immunoglobulin variable domains. In this chapter, we describe simple, fast, and reliable methods for producing and sequencing multiplex PCR amplicons derived from the variable regions (V H , V H H or V L ) of rearranged immunoglobulin heavy and light chain genes using the Illumina MiSeq platform. We include complete protocols and primer sets for amplicon sequencing of V H /V H H/V L repertoires directly from human, mouse, and llama lymphocytes as well as from phage-displayed V H /V H H/V L libraries; these can be easily be adapted to other types of amplicons with little modification. The resulting amplicons are diverse and representative, even using as few as 10 3 input B cells, and their generation is relatively inexpensive, requiring no special equipment and only a limited set of primers. In the absence of heavy-light chain pairing, single-domain antibodies are uniquely amenable to NGS analyses. We present a number of applications of NGS technology useful in discovery of single-domain antibodies from phage display libraries, including: (i) assessment of library functionality; (ii) confirmation of desired library randomization; (iii) estimation of library diversity; and (iv) monitoring the progress of panning experiments. While the case studies presented here are of phage-displayed single-domain antibody libraries, the principles extend to other types of in vitro display libraries.

Exobasidium maculosum, a new species causing leaf and fruit spots on blueberry in the southeastern USA and its relationship with other Exobasidium spp. parasitic to blueberry and cranberry.

PubMed

Brewer, Marin Talbot; Turner, Ashley N; Brannen, Phillip M; Cline, William O; Richardson, Elizabeth A

2014-01-01

Exobasidium leaf and fruit spot of blueberry (Vaccinium section Cyanococcus) is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA. To determine whether this disease is caused by a new species of Exobasidium, we studied the morphology and phylogenetic relationship of the causal fungus compared with other members of the genus, including the type species E. vaccinii and other species that parasitize blueberry and cranberry (V. macrocarpon). Both scanning electron microscopy and light microscopy were used for morphological characterization. For phylogenetic analyses, we sequenced the large subunit of the rDNA (LSU) from 10 isolates collected from leaf or fruit spots of rabbiteye blueberry (V. virgatum), highbush blueberry (V. corymbosum) and southern highbush blueberry (Vaccinium interspecific hybrid) from Georgia and North Carolina and six isolates from leaf spots of lowbush blueberry (V. angustifolium) from Maine and Nova Scotia, Canada. LSU was sequenced from isolates causing red leaf disease of lowbush blueberry and red leaf spot (E. rostrupii) and red shoot (E. perenne) of cranberry. In addition, LSU sequences from GenBank, including sequences with high similarity to the emerging parasite and from Exobasidium spp. parasitizing other Vaccinium spp. and related hosts, were obtained. All sequences were aligned and subjected to phylogenetic analyses. Results indicated that the emerging parasite in the southeastern USA differs morphologically and phylogenetically from other described species and is described herein as Exobasidium maculosum. Within the southeastern USA, clustering based on host species, host tissue type (leaf or fruit) or geographic region was not detected; however, leaf spot isolates from lowbush blueberry were genetically different and likely represent a unique species. © 2014 by The Mycological Society of America.

Genome Sequence of Saccharomyces carlsbergensis, the World’s First Pure Culture Lager Yeast

PubMed Central

Walther, Andrea; Hesselbart, Ana; Wendland, Jürgen

2014-01-01

Lager yeast beer production was revolutionized by the introduction of pure culture strains. The first established lager yeast strain is known as the bottom fermenting Saccharomyces carlsbergensis, which was originally termed Unterhefe No. 1 by Emil Chr. Hansen and has been used in production in since 1883. S. carlsbergensis belongs to group I/Saaz-type lager yeast strains and is better adapted to cold growth conditions than group II/Frohberg-type lager yeasts, e.g., the Weihenstephan strain WS34/70. Here, we sequenced S. carlsbergensis using next generation sequencing technologies. Lager yeasts are descendants from hybrids formed between a S. cerevisiae parent and a parent similar to S. eubayanus. Accordingly, the S. carlsbergensis 19.5-Mb genome is substantially larger than the 12-Mb S. cerevisiae genome. Based on the sequence scaffolds, synteny to the S. cerevisae genome, and by using directed polymerase chain reaction for gap closure, we generated a chromosomal map of S. carlsbergensis consisting of 29 unique chromosomes. We present evidence for genome and chromosome evolution within S. carlsbergensis via chromosome loss and loss of heterozygosity specifically of parts derived from the S. cerevisiae parent. Based on our sequence data and via fluorescence-activated cell-sorting analysis, we determined the ploidy of S. carlsbergensis. This inferred that this strain is basically triploid with a diploid S. eubayanus and haploid S. cerevisiae genome content. In contrast the Weihenstephan strain, which we resequenced, is essentially tetraploid composed of two diploid S. cerevisiae and S. eubayanus genomes. Based on conserved translocations between the parental genomes in S. carlsbergensis and the Weihenstephan strain we propose a joint evolutionary ancestry for lager yeast strains. PMID:24578374

Multilocus sequence analysis of Thermoanaerobacter isolates reveals recombining, but differentiated, populations from geothermal springs of the Uzon Caldera, Kamchatka, Russia

PubMed Central

Wagner, Isaac D.; Varghese, Litty B.; Hemme, Christopher L.; Wiegel, Juergen

2013-01-01

Thermal environments have island-like characteristics and provide a unique opportunity to study population structure and diversity patterns of microbial taxa inhabiting these sites. Strains having ≥98% 16S rRNA gene sequence similarity to the obligately anaerobic Firmicutes Thermoanaerobacter uzonensis were isolated from seven geothermal springs, separated by up to 1600 m, within the Uzon Caldera (Kamchatka, Russian Far East). The intraspecies variation and spatial patterns of diversity for this taxon were assessed by multilocus sequence analysis (MLSA) of 106 strains. Analysis of eight protein-coding loci (gyrB, lepA, leuS, pyrG, recA, recG, rplB, and rpoB) revealed that all loci were polymorphic and that nucleotide substitutions were mostly synonymous. There were 148 variable nucleotide sites across 8003 bp concatenates of the protein-coding loci. While pairwise FST values indicated a small but significant level of genetic differentiation between most subpopulations, there was a negligible relationship between genetic divergence and spatial separation. Strains with the same allelic profile were only isolated from the same hot spring, occasionally from consecutive years, and single locus variant (SLV) sequence types were usually derived from the same spring. While recombination occurred, there was an “epidemic” population structure in which a particular T. uzonensis sequence type rose in frequency relative to the rest of the population. These results demonstrate spatial diversity patterns for an anaerobic bacterial species in a relative small geographic location and reinforce the view that terrestrial geothermal springs are excellent places to look for biogeographic diversity patterns regardless of the involved distances. PMID:23801987

Deciphering the Resistome of the Widespread Pseudomonas aeruginosa Sequence Type 175 International High-Risk Clone through Whole-Genome Sequencing

PubMed Central

López-Causapé, Carla; Ocampo-Sosa, Alain A.; Sommer, Lea M.; Domínguez, María Ángeles; Zamorano, Laura; Juan, Carlos; Tubau, Fe; Rodríguez, Cristina; Moyà, Bartolomé; Martínez-Martínez, Luis; Plesiat, Patrick

2016-01-01

Whole-genome sequencing (WGS) was used for the characterization of the frequently extensively drug resistant (XDR) Pseudomonas aeruginosa sequence type 175 (ST175) high-risk clone. A total of 18 ST175 isolates recovered from 8 different Spanish hospitals were analyzed; 4 isolates from 4 different French hospitals were included for comparison. The typical resistance profile of ST175 included penicillins, cephalosporins, monobactams, carbapenems, aminoglycosides, and fluoroquinolones. In the phylogenetic analysis, the four French isolates clustered together with two isolates from one of the Spanish regions. Sequence variation was analyzed for 146 chromosomal genes related to antimicrobial resistance, and horizontally acquired genes were explored using online databases. The resistome of ST175 was determined mainly by mutational events; resistance traits common to all or nearly all of the strains included specific ampR mutations leading to ampC overexpression, specific mutations in oprD conferring carbapenem resistance, or a mexZ mutation leading to MexXY overexpression. All isolates additionally harbored an aadB gene conferring gentamicin and tobramycin resistance. Several other resistance traits were specific to certain geographic areas, such as a streptomycin resistance gene, aadA13, detected in all four isolates from France and in the two isolates from the Cantabria region and a glpT mutation conferring fosfomycin resistance, detected in all but these six isolates. Finally, several unique resistance mutations were detected in single isolates; particularly interesting were those in genes encoding penicillin-binding proteins (PBP1A, PBP3, and PBP4). Thus, these results provide information valuable for understanding the genetic basis of resistance and the dynamics of the dissemination and evolution of high-risk clones. PMID:27736752

Deciphering the Resistome of the Widespread Pseudomonas aeruginosa Sequence Type 175 International High-Risk Clone through Whole-Genome Sequencing.

PubMed

Cabot, Gabriel; López-Causapé, Carla; Ocampo-Sosa, Alain A; Sommer, Lea M; Domínguez, María Ángeles; Zamorano, Laura; Juan, Carlos; Tubau, Fe; Rodríguez, Cristina; Moyà, Bartolomé; Peña, Carmen; Martínez-Martínez, Luis; Plesiat, Patrick; Oliver, Antonio

2016-12-01

Whole-genome sequencing (WGS) was used for the characterization of the frequently extensively drug resistant (XDR) Pseudomonas aeruginosa sequence type 175 (ST175) high-risk clone. A total of 18 ST175 isolates recovered from 8 different Spanish hospitals were analyzed; 4 isolates from 4 different French hospitals were included for comparison. The typical resistance profile of ST175 included penicillins, cephalosporins, monobactams, carbapenems, aminoglycosides, and fluoroquinolones. In the phylogenetic analysis, the four French isolates clustered together with two isolates from one of the Spanish regions. Sequence variation was analyzed for 146 chromosomal genes related to antimicrobial resistance, and horizontally acquired genes were explored using online databases. The resistome of ST175 was determined mainly by mutational events; resistance traits common to all or nearly all of the strains included specific ampR mutations leading to ampC overexpression, specific mutations in oprD conferring carbapenem resistance, or a mexZ mutation leading to MexXY overexpression. All isolates additionally harbored an aadB gene conferring gentamicin and tobramycin resistance. Several other resistance traits were specific to certain geographic areas, such as a streptomycin resistance gene, aadA13, detected in all four isolates from France and in the two isolates from the Cantabria region and a glpT mutation conferring fosfomycin resistance, detected in all but these six isolates. Finally, several unique resistance mutations were detected in single isolates; particularly interesting were those in genes encoding penicillin-binding proteins (PBP1A, PBP3, and PBP4). Thus, these results provide information valuable for understanding the genetic basis of resistance and the dynamics of the dissemination and evolution of high-risk clones. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

'Candidatus Phytoplasma palmicola', associated with a lethal yellowing-type disease of coconut (Cocos nucifera L.) in Mozambique.

PubMed

Harrison, Nigel A; Davis, Robert E; Oropeza, Carlos; Helmick, Ericka E; Narváez, María; Eden-Green, Simon; Dollet, Michel; Dickinson, Matthew

2014-06-01

In this study, the taxonomic position and group classification of the phytoplasma associated with a lethal yellowing-type disease (LYD) of coconut (Cocos nucifera L.) in Mozambique were addressed. Pairwise similarity values based on alignment of nearly full-length 16S rRNA gene sequences (1530 bp) revealed that the Mozambique coconut phytoplasma (LYDM) shared 100% identity with a comparable sequence derived from a phytoplasma strain (LDN) responsible for Awka wilt disease of coconut in Nigeria, and shared 99.0-99.6% identity with 16S rRNA gene sequences from strains associated with Cape St Paul wilt (CSPW) disease of coconut in Ghana and Côte d'Ivoire. Similarity scores further determined that the 16S rRNA gene of the LYDM phytoplasma shared <97.5% sequence identity with all previously described members of 'Candidatus Phytoplasma'. The presence of unique regions in the 16S rRNA gene sequence distinguished the LYDM phytoplasma from all currently described members of 'Candidatus Phytoplasma', justifying its recognition as the reference strain of a novel taxon, 'Candidatus Phytoplasma palmicola'. Virtual RFLP profiles of the F2n/R2 portion (1251 bp) of the 16S rRNA gene and pattern similarity coefficients delineated coconut LYDM phytoplasma strains from Mozambique as novel members of established group 16SrXXII, subgroup A (16SrXXII-A). Similarity coefficients of 0.97 were obtained for comparisons between subgroup 16SrXXII-A strains and CSPW phytoplasmas from Ghana and Côte d'Ivoire. On this basis, the CSPW phytoplasma strains were designated members of a novel subgroup, 16SrXXII-B.

Quantitative statistical analysis of cis-regulatory sequences in ABA/VP1- and CBF/DREB1-regulated genes of Arabidopsis.

PubMed

Suzuki, Masaharu; Ketterling, Matthew G; McCarty, Donald R

2005-09-01

We have developed a simple quantitative computational approach for objective analysis of cis-regulatory sequences in promoters of coregulated genes. The program, designated MotifFinder, identifies oligo sequences that are overrepresented in promoters of coregulated genes. We used this approach to analyze promoter sequences of Viviparous1 (VP1)/abscisic acid (ABA)-regulated genes and cold-regulated genes, respectively, of Arabidopsis (Arabidopsis thaliana). We detected significantly enriched sequences in up-regulated genes but not in down-regulated genes. This result suggests that gene activation but not repression is mediated by specific and common sequence elements in promoters. The enriched motifs include several known cis-regulatory sequences as well as previously unidentified motifs. With respect to known cis-elements, we dissected the flanking nucleotides of the core sequences of Sph element, ABA response elements (ABREs), and the C repeat/dehydration-responsive element. This analysis identified the motif variants that may correlate with qualitative and quantitative differences in gene expression. While both VP1 and cold responses are mediated in part by ABA signaling via ABREs, these responses correlate with unique ABRE variants distinguished by nucleotides flanking the ACGT core. ABRE and Sph motifs are tightly associated uniquely in the coregulated set of genes showing a strict dependence on VP1 and ABA signaling. Finally, analysis of distribution of the enriched sequences revealed a striking concentration of enriched motifs in a proximal 200-base region of VP1/ABA and cold-regulated promoters. Overall, each class of coregulated genes possesses a discrete set of the enriched motifs with unique distributions in their promoters that may account for the specificity of gene regulation.

Hide and vanish: data sets where the most parsimonious tree is known but hard to find, and their implications for tree search methods.

PubMed

Goloboff, Pablo A

2014-10-01

Three different types of data sets, for which the uniquely most parsimonious tree can be known exactly but is hard to find with heuristic tree search methods, are studied. Tree searches are complicated more by the shape of the tree landscape (i.e. the distribution of homoplasy on different trees) than by the sheer abundance of homoplasy or character conflict. Data sets of Type 1 are those constructed by Radel et al. (2013). Data sets of Type 2 present a very rugged landscape, with narrow peaks and valleys, but relatively low amounts of homoplasy. For such a tree landscape, subjecting the trees to TBR and saving suboptimal trees produces much better results when the sequence of clipping for the tree branches is randomized instead of fixed. An unexpected finding for data sets of Types 1 and 2 is that starting a search from a random tree instead of a random addition sequence Wagner tree may increase the probability that the search finds the most parsimonious tree; a small artificial example where these probabilities can be calculated exactly is presented. Data sets of Type 3, the most difficult data sets studied here, comprise only congruent characters, and a single island with only one most parsimonious tree. Even if there is a single island, missing entries create a very flat landscape which is difficult to traverse with tree search algorithms because the number of equally parsimonious trees that need to be saved and swapped to effectively move around the plateaus is too large. Minor modifications of the parameters of tree drifting, ratchet, and sectorial searches allow travelling around these plateaus much more efficiently than saving and swapping large numbers of equally parsimonious trees with TBR. For these data sets, two new related criteria for selecting taxon addition sequences in Wagner trees (the "selected" and "informative" addition sequences) produce much better results than the standard random or closest addition sequences. These new methods for Wagner trees and for moving around plateaus can be useful when analyzing phylogenomic data sets formed by concatenation of genes with uneven taxon representation ("sparse" supermatrices), which are likely to present a tree landscape with extensive plateaus. Copyright © 2014 Elsevier Inc. All rights reserved.

Extensive Genetic Diversity, Unique Population Structure and Evidence of Genetic Exchange in the Sexually Transmitted Parasite Trichomonas vaginalis

PubMed Central

Conrad, Melissa D.; Gorman, Andrew W.; Schillinger, Julia A.; Fiori, Pier Luigi; Arroyo, Rossana; Malla, Nancy; Dubey, Mohan Lal; Gonzalez, Jorge; Blank, Susan; Secor, William E.; Carlton, Jane M.

2012-01-01

Background Trichomonas vaginalis is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted infection world-wide. Despite its prevalence, little is known about the genetic diversity and population structure of this haploid parasite due to the lack of appropriate tools. The development of a panel of microsatellite makers and SNPs from mining the parasite's genome sequence has paved the way to a global analysis of the genetic structure of the pathogen and association with clinical phenotypes. Methodology/Principal Findings Here we utilize a panel of T. vaginalis-specific genetic markers to genotype 235 isolates from Mexico, Chile, India, Australia, Papua New Guinea, Italy, Africa and the United States, including 19 clinical isolates recently collected from 270 women attending New York City sexually transmitted disease clinics. Using population genetic analysis, we show that T. vaginalis is a genetically diverse parasite with a unique population structure consisting of two types present in equal proportions world-wide. Parasites belonging to the two types (type 1 and type 2) differ significantly in the rate at which they harbor the T. vaginalis virus, a dsRNA virus implicated in parasite pathogenesis, and in their sensitivity to the widely-used drug, metronidazole. We also uncover evidence of genetic exchange, indicating a sexual life-cycle of the parasite despite an absence of morphologically-distinct sexual stages. Conclusions/Significance Our study represents the first robust and comprehensive evaluation of global T. vaginalis genetic diversity and population structure. Our identification of a unique two-type structure, and the clinically relevant phenotypes associated with them, provides a new dimension for understanding T. vaginalis pathogenesis. In addition, our demonstration of the possibility of genetic exchange in the parasite has important implications for genetic research and control of the disease. PMID:22479659

Analysis of dissimilatory sulfite reductase and 16S rRNA gene fragments from deep-sea hydrothermal sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific.

PubMed

Nakagawa, Tatsunori; Ishibashi, Jun-Ichiro; Maruyama, Akihiko; Yamanaka, Toshiro; Morimoto, Yusuke; Kimura, Hiroyuki; Urabe, Tetsuro; Fukui, Manabu

2004-01-01

This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5 degrees C and natural vent fluids at 7 degrees C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with the Aquificae and gamma- and epsilon-Proteobacteria as well as the occurrence of archaeal phylotypes affiliated with the Thermococcales and of a unique archaeon sequence that clustered with "Nanoarchaeota." The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300 degrees C were affiliated with the delta-Proteobacteria, genus Thiomicrospira, and Pelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4 degrees C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments.

Linguistic Analysis of the Human Heartbeat Using Frequency and Rank Order Statistics

NASA Astrophysics Data System (ADS)

Yang, Albert C.-C.; Hseu, Shu-Shya; Yien, Huey-Wen; Goldberger, Ary L.; Peng, C.-K.

2003-03-01

Complex physiologic signals may carry unique dynamical signatures that are related to their underlying mechanisms. We present a method based on rank order statistics of symbolic sequences to investigate the profile of different types of physiologic dynamics. We apply this method to heart rate fluctuations, the output of a central physiologic control system. The method robustly discriminates patterns generated from healthy and pathologic states, as well as aging. Furthermore, we observe increased randomness in the heartbeat time series with physiologic aging and pathologic states and also uncover nonrandom patterns in the ventricular response to atrial fibrillation.

Packaging of Human Chromosome 19-Specific Adeno-Associated Virus (AAV) Integration Sites in AAV Virions during AAV Wild-Type and Recombinant AAV Vector Production

PubMed Central

Hüser, Daniela; Weger, Stefan; Heilbronn, Regine

2003-01-01

Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought. PMID:12663794

The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific

PubMed Central

Rusch, Douglas B; Halpern, Aaron L; Sutton, Granger; Heidelberg, Karla B; Williamson, Shannon; Yooseph, Shibu; Wu, Dongying; Eisen, Jonathan A; Hoffman, Jeff M; Remington, Karin; Beeson, Karen; Tran, Bao; Smith, Hamilton; Baden-Tillson, Holly; Stewart, Clare; Thorpe, Joyce; Freeman, Jason; Andrews-Pfannkoch, Cynthia; Venter, Joseph E; Li, Kelvin; Kravitz, Saul; Heidelberg, John F; Utterback, Terry; Rogers, Yu-Hui; Falcón, Luisa I; Souza, Valeria; Bonilla-Rosso, Germán; Eguiarte, Luis E; Karl, David M; Sathyendranath, Shubha; Platt, Trevor; Bermingham, Eldredge; Gallardo, Victor; Tamayo-Castillo, Giselle; Ferrari, Michael R; Strausberg, Robert L; Nealson, Kenneth; Friedman, Robert; Frazier, Marvin; Venter, J. Craig

2007-01-01

The world's oceans contain a complex mixture of micro-organisms that are for the most part, uncharacterized both genetically and biochemically. We report here a metagenomic study of the marine planktonic microbiota in which surface (mostly marine) water samples were analyzed as part of the Sorcerer II Global Ocean Sampling expedition. These samples, collected across a several-thousand km transect from the North Atlantic through the Panama Canal and ending in the South Pacific yielded an extensive dataset consisting of 7.7 million sequencing reads (6.3 billion bp). Though a few major microbial clades dominate the planktonic marine niche, the dataset contains great diversity with 85% of the assembled sequence and 57% of the unassembled data being unique at a 98% sequence identity cutoff. Using the metadata associated with each sample and sequencing library, we developed new comparative genomic and assembly methods. One comparative genomic method, termed “fragment recruitment,” addressed questions of genome structure, evolution, and taxonomic or phylogenetic diversity, as well as the biochemical diversity of genes and gene families. A second method, termed “extreme assembly,” made possible the assembly and reconstruction of large segments of abundant but clearly nonclonal organisms. Within all abundant populations analyzed, we found extensive intra-ribotype diversity in several forms: (1) extensive sequence variation within orthologous regions throughout a given genome; despite coverage of individual ribotypes approaching 500-fold, most individual sequencing reads are unique; (2) numerous changes in gene content some with direct adaptive implications; and (3) hypervariable genomic islands that are too variable to assemble. The intra-ribotype diversity is organized into genetically isolated populations that have overlapping but independent distributions, implying distinct environmental preference. We present novel methods for measuring the genomic similarity between metagenomic samples and show how they may be grouped into several community types. Specific functional adaptations can be identified both within individual ribotypes and across the entire community, including proteorhodopsin spectral tuning and the presence or absence of the phosphate-binding gene PstS. PMID:17355176

A Detailed Far-ultraviolet Spectral Atlas of O-type Stars

NASA Astrophysics Data System (ADS)

Smith, Myron A.

2012-10-01

In this paper, we present a spectral atlas covering the wavelength interval 930-1188 Å for O2-O9.5 stars using Far-Ultraviolet Spectroscopic Explorer archival data. The stars selected for the atlas were drawn from three populations: Galactic main-sequence (classes III-V) stars, supergiants, and main-sequence stars in the Magellanic Clouds, which have low metallicities. For several of these stars, we have prepared FITS files comprised of pairs of merged spectra for user access via the Multimission Archive at Space Telescope (MAST). We chose spectra from the first population with spectral types O4, O5, O6, O7, O8, and O9.5 and used them to compile tables and figures with identifications of all possible atmospheric and interstellar medium lines in the region 949-1188 Å. Our identified line totals for these six representative spectra are 821 (500), 992 (663), 1077 (749), 1178 (847), 1359 (1001), and 1798 (1392) lines, respectively, where the numbers in parentheses are the totals of lines formed in the atmospheres, according to spectral synthesis models. The total number of unique atmospheric identifications for the six main-sequence O-star template spectra is 1792, whereas the number of atmospheric lines in common to these spectra is 300. The number of identified lines decreases toward earlier types (increasing effective temperature), while the percentages of "missed" features (unknown lines not predicted from our spectral syntheses) drop from a high of 8% at type B0.2, from our recently published B-star far-UV atlas, to 1%-3% for type O spectra. The percentages of overpredicted lines are similar, despite their being much higher for B-star spectra. We discuss the statistics of line populations among the various elemental ionization states. Also, as an aid to users we list those isolated lines that can be used to determine stellar temperatures and the presence of possible chemical anomalies. Finally, we have prepared FITS files that give pairs of merged spectra for stars in our population sequences, for access via MAST.

Technical Considerations for Reduced Representation Bisulfite Sequencing with Multiplexed Libraries

PubMed Central

Chatterjee, Aniruddha; Rodger, Euan J.; Stockwell, Peter A.; Weeks, Robert J.; Morison, Ian M.

2012-01-01

Reduced representation bisulfite sequencing (RRBS), which couples bisulfite conversion and next generation sequencing, is an innovative method that specifically enriches genomic regions with a high density of potential methylation sites and enables investigation of DNA methylation at single-nucleotide resolution. Recent advances in the Illumina DNA sample preparation protocol and sequencing technology have vastly improved sequencing throughput capacity. Although the new Illumina technology is now widely used, the unique challenges associated with multiplexed RRBS libraries on this platform have not been previously described. We have made modifications to the RRBS library preparation protocol to sequence multiplexed libraries on a single flow cell lane of the Illumina HiSeq 2000. Furthermore, our analysis incorporates a bioinformatics pipeline specifically designed to process bisulfite-converted sequencing reads and evaluate the output and quality of the sequencing data generated from the multiplexed libraries. We obtained an average of 42 million paired-end reads per sample for each flow-cell lane, with a high unique mapping efficiency to the reference human genome. Here we provide a roadmap of modifications, strategies, and trouble shooting approaches we implemented to optimize sequencing of multiplexed libraries on an a RRBS background. PMID:23193365

Novel application of the MSSCP method in biodiversity studies.

PubMed

Tomczyk-Żak, Karolina; Kaczanowski, Szymon; Górecka, Magdalena; Zielenkiewicz, Urszula

2012-02-01

Analysis of 16S rRNA sequence diversity is widely performed for characterizing the biodiversity of microbial samples. The number of determined sequences has a considerable impact on complete results. Although the cost of mass sequencing is decreasing, it is often still too high for individual projects. We applied the multi-temperature single-strand conformational polymorphism (MSSCP) method to decrease the number of analysed sequences. This was a novel application of this method. As a control, the same sample was analysed using random sequencing. In this paper, we adapted the MSSCP technique for screening of unique sequences of the 16S rRNA gene library and bacterial strains isolated from biofilms growing on the walls of an ancient gold mine in Poland and determined whether the results obtained by both methods differed and whether random sequencing could be replaced by MSSCP. Although it was biased towards the detection of rare sequences in the samples, the qualitative results of MSSCP were not different than those of random sequencing. Unambiguous discrimination of unique clones and strains creates an opportunity to effectively estimate the biodiversity of natural communities, especially in populations which are numerous but species poor. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Whole genome sequencing of Oryza sativa L. cv. Seeragasamba identifies a new fragrance allele in rice

PubMed Central

Bindusree, Ganigara; Natarajan, Purushothaman; Kalva, Sukesh

2017-01-01

Fragrance of rice is an important trait that confers a large economic benefit to the farmers who cultivate aromatic rice varieties. Several aromatic rice varieties have limited geographic distribution, and are endowed with variety-specific unique fragrances. BADH2 was identified as a fragrance gene in 2005, and it is essential to identify the fragrance alleles from diverse geographical locations and genetic backgrounds. Seeragasamba is a short-grain aromatic rice variety of the indica type, which is cultivated in a limited area in India. Whole genome sequencing of this variety identified a new badh2 allele (badh2-p) with an 8 bp insertion in the promoter region of the BADH2 gene. When the whole genome sequences of 76 aromatic varieties in the 3000 rice genome project were analyzed, the badh2-p allele was present in 13 varieties (approximately 17%) of both indica and japonica types. In addition, the badh2-p allele was present in 17 varieties that already had the loss-of-function allele, badh2-E7. Taken together, the frequency of badh2-p allele (approximately 40%) was found to be greater than that of the badh2-E7 allele (approximately 34%) among the aromatic rice varieties. Therefore, it is suggested to include badh2-p as a predominant allele when screening for fragrance alleles in aromatic rice varieties. PMID:29190814

Comparative genomic analysis of Acinetobacter strains isolated from murine colonic crypts.

PubMed

Saffarian, Azadeh; Touchon, Marie; Mulet, Céline; Tournebize, Régis; Passet, Virginie; Brisse, Sylvain; Rocha, Eduardo P C; Sansonetti, Philippe J; Pédron, Thierry

2017-07-11

A restricted set of aerobic bacteria dominated by the Acinetobacter genus was identified in murine intestinal colonic crypts. The vicinity of such bacteria with intestinal stem cells could indicate that they protect the crypt against cytotoxic and genotoxic signals. Genome analyses of these bacteria were performed to better appreciate their biodegradative capacities. Two taxonomically different clusters of Acinetobacter were isolated from murine proximal colonic crypts, one was identified as A. modestus and the other as A. radioresistens. Their identification was performed through biochemical parameters and housekeeping gene sequencing. After selection of one strain of each cluster (A. modestus CM11G and A. radioresistens CM38.2), comparative genomic analysis was performed on whole-genome sequencing data. The antibiotic resistance pattern of these two strains is different, in line with the many genes involved in resistance to heavy metals identified in both genomes. Moreover whereas the operon benABCDE involved in benzoate metabolism is encoded by the two genomes, the operon antABC encoding the anthranilate dioxygenase, and the phenol hydroxylase gene cluster are absent in the A. modestus genomic sequence, indicating that the two strains have different capacities to metabolize xenobiotics. A common feature of the two strains is the presence of a type IV pili system, and the presence of genes encoding proteins pertaining to secretion systems such as Type I and Type II secretion systems. Our comparative genomic analysis revealed that different Acinetobacter isolated from the same biological niche, even if they share a large majority of genes, possess unique features that could play a specific role in the protection of the intestinal crypt.

Genomic analysis of the symbiotic marine crenarchaeon, Cenarchaeumsymbiosum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hallam, Steven J.; Konstantinidis, Konstantinos T.; Brochier,Celine

2006-06-24

Crenarchaea are ubiquitous and abundant microbial constituents of soils, sediments, lakes and ocean waters, yet relatively little is known about their fundamental evolutionary, ecological, and physiological properties. To better describe the ubiquitous nonthermophilic Crenarchaea, we analyzed the genome sequence of one representative, the uncultivated sponge symbiont, Cenarchaeum symbiosum. C. symbiosum genotypes coinhabiting the same host partitioned into two dominant populations, corresponding to previously described a- and b-type ribosomal RNA variants. Although synthetic, overlapping a- and b-type ribotypes harbored significant genetic variability. A single tiling path comprising the dominant a-type genotype was assembled, and used to explore the biological properties ofmore » C. symbiosum and its planktonic relatives. Out of a total of 2,066 predicted open reading frames, 36% were more highly conserved with other Archaea. The remainder partitioned between bacteria (18%), eukaryotes (1.5%) and viruses (0.1%). A total of 525 open reading frames were more highly conserved with sequences derived from marine environmental genomic surveys, most probably representing orthologous genes found in free-living planktonic Crenarchaea. The remaining genes partitioned between functional RNAs (2.4%), and hypotheticals (42%) with limited homology to known functional genes. The latter category likely contains genes specifically involved in mediated archaeal-sponge symbiosis. Phylogenetic analyses placed C. symbiosum as a basal crenarchaeon, sharing specific genomic features in common with either Crenarchaea, Euryarchaea, or both. The genome sequence of C. symbiosum reflect a unique and unusual evolutionary, physiological, and ecological history, one remarkably distinct from that of any other previously known microbial lineage.« less

Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation Sequencing

PubMed Central

Yoon, Song-Ro; Arnheim, Norman; Calabrese, Peter

2016-01-01

We used targeted next generation deep-sequencing (Safe Sequencing System) to measure ultra-rare de novo mutation frequencies in the human male germline by attaching a unique identifier code to each target DNA molecule. Segments from three different human genes (FGFR3, MECP2 and PTPN11) were studied. Regardless of the gene segment, the particular testis donor or the 73 different testis pieces used, the frequencies for any one of the six different mutation types were consistent. Averaging over the C>T/G>A and G>T/C>A mutation types the background mutation frequency was 2.6x10-5 per base pair, while for the four other mutation types the average background frequency was lower at 1.5x10-6 per base pair. These rates far exceed the well documented human genome average frequency per base pair (~10−8) suggesting a non-biological explanation for our data. By computational modeling and a new experimental procedure to distinguish between pre-mutagenic lesion base mismatches and a fully mutated base pair in the original DNA molecule, we argue that most of the base-dependent variation in background frequency is due to a mixture of deamination and oxidation during the first two PCR cycles. Finally, we looked at a previously studied disease mutation in the PTPN11 gene and could easily distinguish true mutations from the SSS background. We also discuss the limits and possibilities of this and other methods to measure exceptionally rare mutation frequencies, and we present calculations for other scientists seeking to design their own such experiments. PMID:27341568

Wickerhamomyces mori sp. nov., an anamorphic yeast species found in the guts of wood-boring insect larvae.

PubMed

Hui, Feng-Li; Chen, Liang; Chu, Xue-Ying; Niu, Qiu-Hong; Ke, Tao

2013-03-01

A novel anamorphic yeast species is described to accommodate three isolates recovered from the guts of three different wood-boring insect larvae collected in Henan, central China. On the basis of sequence analyses of the D1/D2 domains of the large-subunit rRNA gene and the internal transcribed spacer regions, the three strains are assigned to a novel species of the genus Wickerhamomyces, although the formation of ascospores was not observed. These strains also exhibited a number of distinct morphological and physiological characteristics that clearly differentiated them from Wickerhamomyces mucosus, Candida odintsovae and Wickerhamomyces rabaulensis, the most closely related species. In view of the phenotypic differences and unique rRNA gene sequences, we consider that these three isolates represent a novel species of the genus Wickerhamomyces, Wickerhamomyces mori sp. nov. The type strain is NYNU 1216(T) ( = CICC 1983(T)  = CBS 12678(T)).

Outbreak of betanodavirus infection in tilapia, Oreochromis niloticus (L.), in fresh water.

PubMed

Bigarré, L; Cabon, J; Baud, M; Heimann, M; Body, A; Lieffrig, F; Castric, J

2009-08-01

A betanodavirus associated with a massive mortality was isolated from larvae of tilapia, Oreochromis niloticus, maintained in fresh water at 30 degrees C. Histopathology revealed vacuolation of the nervous system, suggesting an infection by a betanodavirus. The virus was identified by indirect fluorescent antibody test in the SSN1 cell line and further characterized by sequencing of a PCR product. Sequencing of the T4 region of the coat protein gene indicated a phylogenetic clustering of this isolate within the red-spotted grouper nervous necrosis virus type. However, the tilapia isolate formed a unique branch distinct from other betanodavirus isolates. The disease was experimentally reproduced by bath infection of young tilapia at 30 degrees C. The reservoir of virus at the origin of the outbreak remains unidentified. To our knowledge, this is the first report of natural nodavirus infection in tilapia reared in fresh water.

Identification of Novel Betaherpesviruses in Iberian Bats Reveals Parallel Evolution

PubMed Central

Vázquez-Morón, Sonia; Aznar-López, Carolina; Ibáñez, Carlos; Garin, Inazio; Aihartza, Joxerra; Casas, Inmaculada; Tenorio, Antonio; Echevarría, Juan Emilio

2016-01-01

A thorough search for bat herpesviruses was carried out in oropharyngeal samples taken from most of the bat species present in the Iberian Peninsula from the Vespertilionidae, Miniopteridae, Molossidae and Rhinolophidae families, in addition to a colony of captive fruit bats from the Pteropodidae family. By using two degenerate consensus PCR methods targeting two conserved genes, distinct and previously unrecognized bat-hosted herpesviruses were identified for the most of the tested species. All together a total of 42 potentially novel bat herpesviruses were partially characterized. Thirty-two of them were tentatively assigned to the Betaherpesvirinae subfamily while the remaining 10 were allocated into the Gammaherpesvirinae subfamily. Significant diversity was observed among the novel sequences when compared with type herpesvirus species of the ICTV-approved genera. The inferred phylogenetic relationships showed that most of the betaherpesviruses sequences fell into a well-supported unique monophyletic clade and support the recognition of a new betaherpesvirus genus. This clade is subdivided into three major clades, corresponding to the families of bats studied. This supports the hypothesis of a species-specific parallel evolution process between the potentially new betaherpesviruses and their bat hosts. Interestingly, two of the betaherpesviruses’ sequences detected in rhinolophid bats clustered together apart from the rest, closely related to viruses that belong to the Roseolovirus genus. This suggests a putative third roseolo lineage. On the contrary, no phylogenetic structure was detected among several potentially novel bat-hosted gammaherpesviruses found in the study. Remarkably, all of the possible novel bat herpesviruses described in this study are linked to a unique bat species. PMID:28036408

High sequence variations in the region containing genes encoding a cellular morphogenesis protein and the repressor of sexual development help to reveal origins of Aspergillus oryzae.

PubMed

Chang, Perng-Kuang; Scharfenstein, Leslie L; Solorzano, Cesar D; Abbas, Hamed K; Hua, Sui-Sheng T; Jones, Walker A; Zablotowicz, Robert M

2015-05-04

Aspergillus oryzae and Aspergillus flavus are closely related fungal species. The A. flavus morphotype that produces numerous small sclerotia (S strain) and aflatoxin has a unique 1.5 kb deletion in the norB-cypA region of the aflatoxin gene cluster (i.e. the S genotype). Phylogenetic studies have indicated that an isolate of the nonaflatoxigenic A. flavus with the S genotype is the ancestor of A. oryzae. Genome sequence comparison between A. flavus NRRL3357, which produces large sclerotia (L strain), and S-strain A. flavus 70S identified a region (samA-rosA) that was highly variable in the two morphotypes. A third type of samA-rosA region was found in A. oryzae RIB40. The three samA-rosA types were later revealed to be commonly present in A. flavus L-strain populations. Of the 182 L-strain A. flavus field isolates examined, 46%, 15% and 39% had the samA-rosA type of NRRL3357, 70S and RIB40, respectively. The three types also were found in 18 S-strain A. flavus isolates with different proportions. For A. oryzae, however, the majority (80%) of the 16 strains examined had the RIB40 type and none had the NRRL3357 type. The results suggested that A. oryzae strains in the current culture collections were mostly derived from the samA-rosA/RIB40 lineage of the nonaflatoxigenic A. flavus with the S genotype. Published by Elsevier B.V.

Developmentally distinct MYB genes encode functionally equivalent proteins in Arabidopsis.

PubMed

Lee, M M; Schiefelbein, J

2001-05-01

The duplication and divergence of developmental control genes is thought to have driven morphological diversification during the evolution of multicellular organisms. To examine the molecular basis of this process, we analyzed the functional relationship between two paralogous MYB transcription factor genes, WEREWOLF (WER) and GLABROUS1 (GL1), in Arabidopsis. The WER and GL1 genes specify distinct cell types and exhibit non-overlapping expression patterns during Arabidopsis development. Nevertheless, reciprocal complementation experiments with a series of gene fusions showed that WER and GL1 encode functionally equivalent proteins, and their unique roles in plant development are entirely due to differences in their cis-regulatory sequences. Similar experiments with a distantly related MYB gene (MYB2) showed that its product cannot functionally substitute for WER or GL1. Furthermore, an analysis of the WER and GL1 proteins shows that conserved sequences correspond to specific functional domains. These results provide new insights into the evolution of the MYB gene family in Arabidopsis, and, more generally, they demonstrate that novel developmental gene function may arise solely by the modification of cis-regulatory sequences.

Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.

PubMed

Truong, Andrew S; Lochbaum, Christian A; Boyce, Matthew W; Lockett, Matthew R

2015-11-17

Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

Validation of use of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for typing strains belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex and application of the method to the investigation of a hospital outbreak.

PubMed Central

Snelling, A M; Gerner-Smidt, P; Hawkey, P M; Heritage, J; Parnell, P; Porter, C; Bodenham, A R; Inglis, T

1996-01-01

Acinetobacter spp. are being reported with increasing frequency as causes of nosocomial infection. In order to identify reservoirs of infection as quickly as possible, a rapid typing method that can differentiate epidemic strains from environmental and nonepidemic strains is needed. In 1993, a cluster of Acinetobacter baumannii isolates from five patients in the adult intensive therapy unit of our tertiary-care teaching hospital led us to develop and optimize a rapid repetitive extragenic palindromic sequence-based PCR (REP-PCR) typing protocol for members of the Acinetobacter calcoaceticus-A. baumannii complex that uses boiled colonies and consensus primers aimed at repetitive extragenic palindromic sequences. Four of the five patient isolates gave the same REP-PCR typing pattern as isolates of A. baumannii obtained from the temperature probe of a Bennett humidifier; the fifth isolate had a unique profile. Disinfection of the probe with 70% ethanol, as recommended by the manufacturer, proved ineffective, as A. baumannii with the same REP-PCR pattern was isolated from it 10 days after cleaning, necessitating a change in our decontamination procedure. Results obtained with REP-PCR were subsequently confirmed by ribotyping. To evaluate the discriminatory power (D) of REP-PCR for typing members of the A. calcoaceticus-A. baumannii complex, compared with that of ribotyping, we have applied both methods to a collection of 85 strains that included representatives of six DNA groups within the complex. Ribotyping using EcoRI digests yielded 53 patterns (D = 0.98), whereas 68 different REP-PCR patterns were observed (D = 0.99). By computer-assisted analysis of gel images, 74 patterns were observed with REP-PCR (D = 1.0). Overall, REP-PCR typing proved to be slightly more discriminatory than ribotyping. Our results indicate that REP-PCR typing used boiled colonies is a simple, rapid, and effective means of typing members of the A. calcoaceticus-A. baumannii complex. PMID:8727902

Unique LCR variations among lineages of HPV16, 18 and 45 isolates from women with normal cervical cytology in Ghana.

PubMed

Awua, Adolf K; Adanu, Richard M K; Wiredu, Edwin K; Afari, Edwin A; Zubuch, Vanessa A; Asmah, Richard H; Severini, Alberto

2017-04-21

In addition to being useful for classification, sequence variations of human Papillomavirus (HPV) genotypes have been implicated in differential oncogenic potential and a differential association with the different histological forms of invasive cervical cancer. These associations have also been indicated for HPV genotype lineages and sub-lineages. In order to better understand the potential implications of lineage variation in the occurrence of cervical cancers in Ghana, we studied the lineages of the three most prevalent HPV genotypes among women with normal cytology as baseline to further studies. Of previously collected self- and health personnel-collected cervical specimen, 54, which were positive for HPV16, 18 and 45, were selected and the long control region (LCR) of each HPV genotype was separately amplified by a nested PCR. DNA sequences of 41 isolates obtained with the forward and reverse primers by Sanger sequencing were analysed. Nucleotide sequence variations of the HPV16 genotypes were observed at 30 positions within the LCR (7460 - 7840). Of these, 19 were the known variations for the lineages B and C (African lineages), while the other 11 positions had variations unique to the HPV16 isolates of this study. For the HPV18 isolates, the variations were at 35 positions, 22 of which were known variations of Africa lineages and the other 13 were unique variations observed for the isolates obtained in this study (at positions 7799 and 7813). HPV45 isolates had variations at 35 positions and 2 (positions 7114 and 97) were unique to the isolates of this study. This study provides the first data on the lineages of HPV 16, 18 and 45 isolates from Ghana. Although the study did not obtain full genome sequence data for a comprehensive comparison with known lineages, these genotypes were predominately of the Africa lineages and had some unique sequence variations at positions that suggest potential oncogenic implications. These data will be useful for comparison with lineages of these genotypes from women with cervical lesion and all the forms of invasive cervical cancers.

Differential 3’ processing of specific transcripts expands regulatory and protein diversity across neuronal cell types

PubMed Central

Jereb, Saša; Hwang, Hun-Way; Van Otterloo, Eric; Govek, Eve-Ellen; Fak, John J; Yuan, Yuan; Hatten, Mary E

2018-01-01

Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3’UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3’UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3’UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies. PMID:29578408

DSAP: deep-sequencing small RNA analysis pipeline.

PubMed

Huang, Po-Jung; Liu, Yi-Chung; Lee, Chi-Ching; Lin, Wei-Chen; Gan, Richie Ruei-Chi; Lyu, Ping-Chiang; Tang, Petrus

2010-07-01

DSAP is an automated multiple-task web service designed to provide a total solution to analyzing deep-sequencing small RNA datasets generated by next-generation sequencing technology. DSAP uses a tab-delimited file as an input format, which holds the unique sequence reads (tags) and their corresponding number of copies generated by the Solexa sequencing platform. The input data will go through four analysis steps in DSAP: (i) cleanup: removal of adaptors and poly-A/T/C/G/N nucleotides; (ii) clustering: grouping of cleaned sequence tags into unique sequence clusters; (iii) non-coding RNA (ncRNA) matching: sequence homology mapping against a transcribed sequence library from the ncRNA database Rfam (http://rfam.sanger.ac.uk/); and (iv) known miRNA matching: detection of known miRNAs in miRBase (http://www.mirbase.org/) based on sequence homology. The expression levels corresponding to matched ncRNAs and miRNAs are summarized in multi-color clickable bar charts linked to external databases. DSAP is also capable of displaying miRNA expression levels from different jobs using a log(2)-scaled color matrix. Furthermore, a cross-species comparative function is also provided to show the distribution of identified miRNAs in different species as deposited in miRBase. DSAP is available at http://dsap.cgu.edu.tw.

Abamectin, pymetrozine and azadirachtin sequence as a unique solution to control the leafminer Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) infesting garden beans (Phaseolus vulgaris L.) in Egypt.

PubMed

Saad, A S A; Massoud, M A; Abdel-Megeed, A A M; Hamid, N A; Mourad, A K K; Barakat, A S T

2007-01-01

Field trails were conducted to determine the performance of three different sequences as a unique solution for the control of the leaf miner Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) infesting garden beans (Phaseolus vulgaris L.) during the two successive seasons of 2004 and 2005. Furthermore, during the evaluation period, the side effect against the ectoparasite Diglyphus isaea (Walker) (Hymenoptera: Eulophidae) was put into consideration. Meanwhile, the comparative evaluation of the pesticides alone showed that abamectin and azadirachtin were highly effective against Liriomyza trifolii, while carbosulfan, pymetrozine and thiamethoxam provided to be of a moderate effect. Moreover, carbosulfan showed harmful effect to the larvae of the ectoparasite Diglyphus isaea (Walker), while abamectin and azadirachtin gave a moderate effect. Thiamethoxam and the the detergent (Masrol 410) had slight effect in this respect. The highly effective sequence among the sequences was abamectin, pymetrozine and azadirachtin, against Liriomyza trifolii (Burgess), with slight harmful effect on Diglyphus isaea (Walker). However the sequence of azadirachtin, pymetrozine and abamectin had a moderate effect on Liriomyza trifolii (Burgess) and exhibited a slight toxic effect on Diglyphus isaea (Walker). In contrast, the sequence of carbosulfan, thiamethoxam and pymetrozine was the least effective and represented a slight effect on Diglyphus isaea (Walker). From this study, it was concluded that abamectin, pymetrozine and azadirachtin sequence has proved to be a unique solution for the control of the leaf miner Liriomyza trifolii (Burgess) infesting garden beans (Phaseolus vulgaris L.) in Egypt.

Image Encryption Algorithm Based on Hyperchaotic Maps and Nucleotide Sequences Database

PubMed Central

2017-01-01

Image encryption technology is one of the main means to ensure the safety of image information. Using the characteristics of chaos, such as randomness, regularity, ergodicity, and initial value sensitiveness, combined with the unique space conformation of DNA molecules and their unique information storage and processing ability, an efficient method for image encryption based on the chaos theory and a DNA sequence database is proposed. In this paper, digital image encryption employs a process of transforming the image pixel gray value by using chaotic sequence scrambling image pixel location and establishing superchaotic mapping, which maps quaternary sequences and DNA sequences, and by combining with the logic of the transformation between DNA sequences. The bases are replaced under the displaced rules by using DNA coding in a certain number of iterations that are based on the enhanced quaternary hyperchaotic sequence; the sequence is generated by Chen chaos. The cipher feedback mode and chaos iteration are employed in the encryption process to enhance the confusion and diffusion properties of the algorithm. Theoretical analysis and experimental results show that the proposed scheme not only demonstrates excellent encryption but also effectively resists chosen-plaintext attack, statistical attack, and differential attack. PMID:28392799

Molecular identification and genetic diversity of open reading frame 7 field isolated porcine reproductive and respiratory syndrome in North Sumatera, Indonesia, in the period of 2008-2014.

PubMed

Faisal, Faisal; Widayanti, Rini; Haryanto, Aris; Tabu, Charles Rangga

2015-07-01

Molecular identification and genetic diversity of open reading frame 7 (ORF7) of field isolated porcine reproductive and respiratory syndrome virus (PRRSV) in North Sumatera, Indonesia, in the period of 2008-2014. A total of 47 PRRSV samples were collected from the death case of pigs. The samples were collected from different districts in the period of 2008-2014 from North Sumatera province. Two pairs of primer were designed to amplify ORF7 of Type 1 and 2 PRRSV based on the sequence of reference viruses VR2332 and Lelystad. Viral RNAs were extracted from samples using PureLink™ micro-to-Midi total RNA purification system (Invitrogen). To amplify the ORF7 of PRRSV, the synthesis cDNA and DNA amplification were performed by reverse transcription polymerase chain reaction (RT-PCR) and nested PCR method. Then the DNA sequencing of PCR products and phylogenetic analysis were accomplished by molecular evolutionary genetics analysis version 6.0 software program. RT-: PCR and nested PCR used in this study had successfully detected of 18 samples positive PRRS virus with the amplification products at 703bp and 508bp, respectively. Sequencing of the ORF7 shows that 18 PRRS viruses isolated from North Sumatera belonged to North American (NA). JXA1 Like and classic NA type viruses. Several mutations were detected, particularly in the area of nuclear localization signal (NLS1) and in NLS2. In the local viruses, which were related closed to JXA1 virus; there are two differences in amino acids in position 12 and 43 of ORF7. Our tested viruses showed that the amino acid positions 12 and 43 are Asparagine and Arginine, while the reference virus (VR2332, Lelystad, and JXA1) occupied both by Lysine. Based on differences in two amino acids at position 12 and 43 showed that viruses from North Sumatera has its own uniqueness and related closed to highly pathogenic PRRS (HP-PRRS) virus (JXA1). The results demonstrated that North Sumatera type PRRS virus has caused PRRS outbreaks in pig in North Sumatera between 2008 and 2014. The JAX1 like viruses had unique amino acid residue in position 12 and 43 of asparagine and lysine, and these were genetic determinants of North Sumatera viruses compared to other PRRS viruses.

comK Prophage Junction Fragments as Markers for Listeria monocytogenes Genotypes Unique to Individual Meat and Poultry Processing Plants and a Model for Rapid Niche-Specific Adaptation, Biofilm Formation, and Persistence ▿ †

PubMed Central

Verghese, Bindhu; Lok, Mei; Wen, Jia; Alessandria, Valentina; Chen, Yi; Kathariou, Sophia; Knabel, Stephen

2011-01-01

Different strains of Listeria monocytogenes are well known to persist in individual food processing plants and to contaminate foods for many years; however, the specific genotypic and phenotypic mechanisms responsible for persistence of these unique strains remain largely unknown. Based on sequences in comK prophage junction fragments, different strains of epidemic clones (ECs), which included ECII, ECIII, and ECV, were identified and shown to be specific to individual meat and poultry processing plants. The comK prophage-containing strains showed significantly higher cell densities after incubation at 30°C for 48 h on meat and poultry food-conditioning films than did strains lacking the comK prophage (P < 0.05). Overall, the type of strain, the type of conditioning film, and the interaction between the two were all highly significant (P < 0.001). Recombination analysis indicated that the comK prophage junction fragments in these strains had evolved due to extensive recombination. Based on the results of the present study, we propose a novel model in which the concept of defective comK prophage was replaced with the rapid adaptation island (RAI). Genes within the RAI were recharacterized as “adaptons,” as these genes may allow L. monocytogenes to rapidly adapt to different food processing facilities and foods. If confirmed, the model presented would help explain Listeria's rapid niche adaptation, biofilm formation, persistence, and subsequent transmission to foods. Also, comK prophage junction fragment sequences may permit accurate tracking of persistent strains back to and within individual food processing operations and thus allow the design of more effective intervention strategies to reduce contamination and enhance food safety. PMID:21441318

Population Genetics of Lactobacillus sakei Reveals Three Lineages with Distinct Evolutionary Histories

PubMed Central

Chaillou, Stéphane; Lucquin, Isabelle; Najjari, Afef; Zagorec, Monique; Champomier-Vergès, Marie-Christine

2013-01-01

Lactobacillus sakei plays a major role in meat fermentation and in the preservation of fresh meat. The large diversity of L. sakei strains represents a valuable and exploitable asset in the development of a variety of industrial applications; however, an efficient method to identify and classify these strains has yet to be developed. In this study, we used multilocus sequence typing (MLST) to analyze the polymorphism and allelic distribution of eight loci within an L. sakei population of 232 strains collected worldwide. Within this population, we identified 116 unique sequence types with an average pairwise nucleotide diversity per site (π) of 0.13%. Results from Structure, goeBurst, and ClonalFrame software analyses demonstrated that the L. sakei population analyzed here is derived from three ancestral lineages, each of which shows evidence of a unique evolutionary history influenced by independent selection scenarios. However, the signature of selective events in the contemporary population of isolates was somewhat masked by the pervasive phenomenon of homologous recombination. Our results demonstrate that lineage 1 is a completely panmictic subpopulation in which alleles have been continually redistributed through the process of intra-lineage recombination. In contrast, lineage 2 was characterized by a high degree of clonality. Lineage 3, the earliest-diverging branch in the genealogy, showed evidence of both clonality and recombination. These evolutionary histories strongly indicate that the three lineages may correspond to distinct ecotypes, likely linked or specialized to different environmental reservoirs. The MLST scheme developed in this study represents an easy and straightforward tool that can be used to further analyze the population dynamics of L. sakei strains in food products. PMID:24069179

Population genetics of Lactobacillus sakei reveals three lineages with distinct evolutionary histories.

PubMed

Chaillou, Stéphane; Lucquin, Isabelle; Najjari, Afef; Zagorec, Monique; Champomier-Vergès, Marie-Christine

2013-01-01

Lactobacillus sakei plays a major role in meat fermentation and in the preservation of fresh meat. The large diversity of L. sakei strains represents a valuable and exploitable asset in the development of a variety of industrial applications; however, an efficient method to identify and classify these strains has yet to be developed. In this study, we used multilocus sequence typing (MLST) to analyze the polymorphism and allelic distribution of eight loci within an L. sakei population of 232 strains collected worldwide. Within this population, we identified 116 unique sequence types with an average pairwise nucleotide diversity per site (π) of 0.13%. Results from Structure, goeBurst, and ClonalFrame software analyses demonstrated that the L. sakei population analyzed here is derived from three ancestral lineages, each of which shows evidence of a unique evolutionary history influenced by independent selection scenarios. However, the signature of selective events in the contemporary population of isolates was somewhat masked by the pervasive phenomenon of homologous recombination. Our results demonstrate that lineage 1 is a completely panmictic subpopulation in which alleles have been continually redistributed through the process of intra-lineage recombination. In contrast, lineage 2 was characterized by a high degree of clonality. Lineage 3, the earliest-diverging branch in the genealogy, showed evidence of both clonality and recombination. These evolutionary histories strongly indicate that the three lineages may correspond to distinct ecotypes, likely linked or specialized to different environmental reservoirs. The MLST scheme developed in this study represents an easy and straightforward tool that can be used to further analyze the population dynamics of L. sakei strains in food products.

comK prophage junction fragments as markers for Listeria monocytogenes genotypes unique to individual meat and poultry processing plants and a model for rapid niche-specific adaptation, biofilm formation, and persistence.

PubMed

Verghese, Bindhu; Lok, Mei; Wen, Jia; Alessandria, Valentina; Chen, Yi; Kathariou, Sophia; Knabel, Stephen

2011-05-01

Different strains of Listeria monocytogenes are well known to persist in individual food processing plants and to contaminate foods for many years; however, the specific genotypic and phenotypic mechanisms responsible for persistence of these unique strains remain largely unknown. Based on sequences in comK prophage junction fragments, different strains of epidemic clones (ECs), which included ECII, ECIII, and ECV, were identified and shown to be specific to individual meat and poultry processing plants. The comK prophage-containing strains showed significantly higher cell densities after incubation at 30°C for 48 h on meat and poultry food-conditioning films than did strains lacking the comK prophage (P < 0.05). Overall, the type of strain, the type of conditioning film, and the interaction between the two were all highly significant (P < 0.001). Recombination analysis indicated that the comK prophage junction fragments in these strains had evolved due to extensive recombination. Based on the results of the present study, we propose a novel model in which the concept of defective comK prophage was replaced with the rapid adaptation island (RAI). Genes within the RAI were recharacterized as "adaptons," as these genes may allow L. monocytogenes to rapidly adapt to different food processing facilities and foods. If confirmed, the model presented would help explain Listeria's rapid niche adaptation, biofilm formation, persistence, and subsequent transmission to foods. Also, comK prophage junction fragment sequences may permit accurate tracking of persistent strains back to and within individual food processing operations and thus allow the design of more effective intervention strategies to reduce contamination and enhance food safety.

Genetic diversity of the merozoite surface protein-3 gene in Plasmodium falciparum populations in Thailand.

PubMed

Pattaradilokrat, Sittiporn; Sawaswong, Vorthon; Simpalipan, Phumin; Kaewthamasorn, Morakot; Siripoon, Napaporn; Harnyuttanakorn, Pongchai

2016-10-21

An effective malaria vaccine is an urgently needed tool to fight against human malaria, the most deadly parasitic disease of humans. One promising candidate is the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum. This antigenic protein, encoded by the merozoite surface protein (msp-3) gene, is polymorphic and classified according to size into the two allelic types of K1 and 3D7. A recent study revealed that both the K1 and 3D7 alleles co-circulated within P. falciparum populations in Thailand, but the extent of the sequence diversity and variation within each allelic type remains largely unknown. The msp-3 gene was sequenced from 59 P. falciparum samples collected from five endemic areas (Mae Hong Son, Kanchanaburi, Ranong, Trat and Ubon Ratchathani) in Thailand and analysed for nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity. The gene was also subject to population genetic analysis (F st ) and neutrality tests (Tajima's D, Fu and Li D* and Fu and Li' F* tests) to determine any signature of selection. The sequence analyses revealed eight unique DNA haplotypes and seven amino acid sequence variants, with a haplotype and nucleotide diversity of 0.828 and 0.049, respectively. Neutrality tests indicated that the polymorphism detected in the alanine heptad repeat region of MSP-3 was maintained by positive diversifying selection, suggesting its role as a potential target of protective immune responses and supporting its role as a vaccine candidate. Comparison of MSP-3 variants among parasite populations in Thailand, India and Nigeria also inferred a close genetic relationship between P. falciparum populations in Asia. This study revealed the extent of the msp-3 gene diversity in P. falciparum in Thailand, providing the fundamental basis for the better design of future blood stage malaria vaccines against P. falciparum.

Genetic structure of Plasmodium vivax using the merozoite surface protein 1 icb5-6 fragment reveals new hybrid haplotypes in southern Mexico

PubMed Central

2014-01-01

Background Plasmodium vivax is a protozoan parasite with an extensive worldwide distribution, being highly prevalent in Asia as well as in Mesoamerica and South America. In southern Mexico, P. vivax transmission has been endemic and recent studies suggest that these parasites have unique biological and genetic features. The msp1 gene has shown high rate of nucleotide substitutions, deletions, insertions, and its mosaic structure reveals frequent events of recombination, maybe between highly divergent parasite isolates. Methods The nucleotide sequence variation in the polymorphic icb5-6 fragment of the msp1 gene of Mexican and worldwide isolates was analysed. To understand how genotype diversity arises, disperses and persists in Mexico, the genetic structure and genealogical relationships of local isolates were examined. To identify new sequence hybrids and their evolutionary relationships with other P. vivax isolates circulating worldwide two haplotype networks were constructed questioning that two portions of the icb5-6 have different evolutionary history. Results Twelve new msp1 icb5-6 haplotypes of P. vivax from Mexico were identified. These nucleotide sequences show mosaic structure comprising three partially conserved and two variable subfragments and resulted into five different sequence types. The variable subfragment sV1 has undergone recombination events and resulted in hybrid sequences and the haplotype network allocated the Mexican haplotypes to three lineages, corresponding to the Sal I and Belem types, and other more divergent group. In contrast, the network from icb5-6 fragment but not sV1 revealed that the Mexican haplotypes belong to two separate lineages, none of which are closely related to Sal I or Belem sequences. Conclusions These results suggest that the new hybrid haplotypes from southern Mexico were the result of at least three different recombination events. These rearrangements likely resulted from the recombination between haplotypes of highly divergent lineages that are frequently distributed in South America and Asia and diversified rapidly. PMID:24472213

Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome.

PubMed

Nicholson, Matthew J; Theodorou, Michael K; Brookman, Jayne L

2005-01-01

The anaerobic gut fungi occupy a unique niche in the intestinal tract of large herbivorous animals and are thought to act as primary colonizers of plant material during digestion. They are the only known obligately anaerobic fungi but molecular analysis of this group has been hampered by difficulties in their culture and manipulation, and by their extremely high A+T nucleotide content. This study begins to answer some of the fundamental questions about the structure and organization of the anaerobic gut fungal genome. Directed plasmid libraries using genomic DNA digested with highly or moderately rich AT-specific restriction enzymes (VspI and EcoRI) were prepared from a polycentric Orpinomyces isolate. Clones were sequenced from these libraries and the breadth of genomic inserts, both genic and intergenic, was characterized. Genes encoding numerous functions not previously characterized for these fungi were identified, including cytoskeletal, secretory pathway and transporter genes. A peptidase gene with no introns and having sequence similarity to a gene encoding a bacterial peptidase was also identified, extending the range of metabolic enzymes resulting from apparent trans-kingdom transfer from bacteria to fungi, as previously characterized largely for genes encoding plant-degrading enzymes. This paper presents the first thorough analysis of the genic, intergenic and rDNA regions of a variety of genomic segments from an anaerobic gut fungus and provides observations on rules governing intron boundaries, the codon biases observed with different types of genes, and the sequence of only the second anaerobic gut fungal promoter reported. Large numbers of retrotransposon sequences of different types were found and the authors speculate on the possible consequences of any such transposon activity in the genome. The coding sequences identified included several orphan gene sequences, including one with regions strongly suggestive of structural proteins such as collagens and lampirin. This gene was present as a single copy in Orpinomyces, was expressed during vegetative growth and was also detected in genomes from another gut fungal genus, Neocallimastix.

The Unique Morgue Ubiquitination Protein Is Conserved in a Diverse but Restricted Set of Invertebrates

PubMed Central

Zhou, Ying; Carpenter, Zachary W.; Brennan, Gregory

2009-01-01

Drosophila Morgue is a unique ubiquitination protein that facilitates programmed cell death and associates with DIAP1, a critical cell death inhibitor with E3 ubiquitin ligase activity. Morgue possesses a unique combination of functional domains typically associated with distinct types of ubiquitination enzymes. This includes an F box characteristic of the substrate-binding subunit in Skp, Cullin, and F box (SCF)-type ubiquitin E3 ligase complexes and a variant ubiquitin E2 conjugase domain where the active site cysteine is replaced by a glycine. Morgue also contains a single C4-type zinc finger motif. This architecture suggests potentially novel ubiquitination activities for Morgue. In this study, we address the evolutionary origins of this distinctive protein utilizing a combination of bioinformatics and molecular biology approaches. We find that Morgue exhibits widespread but restricted phylogenetic distribution among metazoans. Morgue proteins were identified in a wide range of Protostome phyla, including Arthropoda, Annelida, Mollusca, Nematoda, and Platyhelminthes. However, with one potential exception, Morgue was not detected in Deuterostomes, including Chordates, Hemichordates, or Echinoderms. Morgue was also not found in Ctenophora, Cnidaria, Placozoa, or Porifera. Characterization of Morgue sequences within specific animal lineages suggests that gene deletion or acquisition has occurred during divergence of nematodes and that at least one arachnid expresses an atypical form of Morgue consisting only of the variant E2 conjugase domain. Analysis of the organization of several morgue genes suggests that exon-shuffling events have contributed to the evolution of the Morgue protein. These results suggest that Morgue mediates conserved and distinctive ubiquitination functions in specific cell death pathways. PMID:19602541

Je, a versatile suite to handle multiplexed NGS libraries with unique molecular identifiers.

PubMed

Girardot, Charles; Scholtalbers, Jelle; Sauer, Sajoscha; Su, Shu-Yi; Furlong, Eileen E M

2016-10-08

The yield obtained from next generation sequencers has increased almost exponentially in recent years, making sample multiplexing common practice. While barcodes (known sequences of fixed length) primarily encode the sample identity of sequenced DNA fragments, barcodes made of random sequences (Unique Molecular Identifier or UMIs) are often used to distinguish between PCR duplicates and transcript abundance in, for example, single-cell RNA sequencing (scRNA-seq). In paired-end sequencing, different barcodes can be inserted at each fragment end to either increase the number of multiplexed samples in the library or to use one of the barcodes as UMI. Alternatively, UMIs can be combined with the sample barcodes into composite barcodes, or with standard Illumina® indexing. Subsequent analysis must take read duplicates and sample identity into account, by identifying UMIs. Existing tools do not support these complex barcoding configurations and custom code development is frequently required. Here, we present Je, a suite of tools that accommodates complex barcoding strategies, extracts UMIs and filters read duplicates taking UMIs into account. Using Je on publicly available scRNA-seq and iCLIP data containing UMIs, the number of unique reads increased by up to 36 %, compared to when UMIs are ignored. Je is implemented in JAVA and uses the Picard API. Code, executables and documentation are freely available at http://gbcs.embl.de/Je . Je can also be easily installed in Galaxy through the Galaxy toolshed.

An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis.

PubMed

Tang, Qi; Ma, Xiaojun; Mo, Changming; Wilson, Iain W; Song, Cai; Zhao, Huan; Yang, Yanfang; Fu, Wei; Qiu, Deyou

2011-07-05

Siraitia grosvenorii (Luohanguo) is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in S. grosvenorii, especially the late steps of the pathway. In this study, a cDNA library generated from of equal amount of RNA taken from S. grosvenorii fruit at 50 days after flowering (DAF) and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9%) unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450) and ninety UDP-glucosyltransferase (UDPG) unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE) analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven CYP450s and five UDPGs were selected as the candidates most likely to be involved in mogrosides biosynthesis. A combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying candidate genes encoding enzymes responsible for the biosynthesis of novel secondary metabolites in a non-model plant. Seven CYP450s and five UDPGs were selected as potential candidates involved in mogrosides biosynthesis. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the fruit extract from S. grosvenorii.

Extremely low Helicobacter pylori prevalence in North Sulawesi, Indonesia and identification of a Maori-tribe type strain: a cross sectional study.

PubMed

Miftahussurur, Muhammad; Tuda, Josef; Suzuki, Rumiko; Kido, Yasutoshi; Kawamoto, Fumihiko; Matsuda, Miyuki; Tantular, Indah S; Pusarawati, Suhintam; Nasronudin; Harijanto, Paul N; Yamaoka, Yoshio

2014-01-01

Sulawesi in Indonesia has a unique geographical profile with assumed separation from Sundaland. Studies of Helicobacter pylori in this region are rare due to the region's rural location and lack of endoscopy equipment. Indirect methods are, therefore, the most appropriate for measuring H. pylori infection in these areas; with the disposable gastric brush test, we can obtain gastric juice as well as small gastric tissue samples for H. pylori culture. We investigated the prevalence of H. pylori infection and evaluated human migration patterns in the remote areas of North Sulawesi. We recruited a total of 251 consecutive adult volunteers and 131 elementary school children. H. pylori infection was determined by urine antibody test. A gastric brush test was used to culture H. pylori. We used next-generation and polymerase chain reaction based sequencing to determine virulence factors and multi-locus sequence typing (MLST). The overall H. pylori prevalence was only 14.3% for adults and 3.8% for children, and 13.6% and 16.7% in Minahasanese and Mongondownese participants, respectively. We isolated a single H. pylori strain, termed -Manado-1. Manado-1 was East Asian type cagA (ABD type), vacA s1c-m1b, iceA1 positive/iceA2 negative, jhp0562-positive/β-(1,3) galT-negative, oipA "on", and dupA-negative. Phylogenetic analyses showed the strain to be hspMaori type, a major type observed in native Taiwanese and Maori tribes. Our data support that very low H. pylori infection prevalence in Indonesia. Identification of hspMaori type H. pylori in North Sulawesi may support the hypothesis that North Sulawesi people migrated from north.

Proteins without unique 3D structures: biotechnological applications of intrinsically unstable/disordered proteins.

PubMed

Uversky, Vladimir N

2015-03-01

Intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) are functional proteins or regions that do not have unique 3D structures under functional conditions. Therefore, from the viewpoint of their lack of stable 3D structure, IDPs/IDPRs are inherently unstable. As much as structure and function of normal ordered globular proteins are determined by their amino acid sequences, the lack of unique 3D structure in IDPs/IDPRs and their disorder-based functionality are also encoded in the amino acid sequences. Because of their specific sequence features and distinctive conformational behavior, these intrinsically unstable proteins or regions have several applications in biotechnology. This review introduces some of the most characteristic features of IDPs/IDPRs (such as peculiarities of amino acid sequences of these proteins and regions, their major structural features, and peculiar responses to changes in their environment) and describes how these features can be used in the biotechnology, for example for the proteome-wide analysis of the abundance of extended IDPs, for recombinant protein isolation and purification, as polypeptide nanoparticles for drug delivery, as solubilization tools, and as thermally sensitive carriers of active peptides and proteins. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rhnull syndrome: identification of a novel mutation in RHce.

PubMed

Rosa, K A; Reid, M E; Lomas-Francis, C; Powell, V I; Costa, F F; Stinghen, S T; Watanabe, A M; Carboni, E K; Baldon, J P; Jucksch, M M F; Castilho, L

2005-11-01

The deficiency of Rh proteins on red blood cells (RBCs) from individuals of the Rh(null) amorph type are the result of homozygosity for a silent RHCE in cis with a deleted RHD. A novel mutation in RHce was identified in two Caucasian Brazilian girls with the amorph type of Rh(null) who were born to parents who were first cousins. RBCs from the Rh(null) sisters and from family members were analyzed by serology and flow cytometry with specific antibodies. Genomic DNA and transcripts were tested by polymerase chain reaction and sequence analysis. Rh(null) RBCs were nonreactive with anti-Rh and anti-LW. Molecular analyses showed a deletion of RHD and of one nucleotide (960/963; GGGG-->GGG) in exon 7 of the RHce. This deletion introduced a frameshift after Gly321, a new C-terminal sequence, and a premature stop codon, resulting in a shorter predicted protein with 357 amino acids. The detection of a unique RHce transcript indicated that the two sisters were homozygous, whereas the other family members were heterozygous for the mutation. A novel mutation resulting in the amorph Rh(null) with loss of Rh antigen expression is described.

Genetic structure of the mating-type locus of Chlamydomonas reinhardtii.

PubMed Central

Ferris, Patrick J; Armbrust, E Virginia; Goodenough, Ursula W

2002-01-01

Portions of the cloned mating-type (MT) loci (mt(+) and mt(-)) of Chlamydomonas reinhardtii, defined as the approximately 1-Mb domains of linkage group VI that are under recombinational suppression, were subjected to Northern analysis to elucidate their coding capacity. The four central rearranged segments of the loci were found to contain both housekeeping genes (expressed during several life-cycle stages) and mating-related genes, while the sequences unique to mt(+) or mt(-) carried genes expressed only in the gametic or zygotic phases of the life cycle. One of these genes, Mtd1, is a candidate participant in gametic cell fusion; two others, Mta1 and Ezy2, are candidate participants in the uniparental inheritance of chloroplast DNA. The identified housekeeping genes include Pdk, encoding pyruvate dehydrogenase kinase, and GdcH, encoding glycine decarboxylase complex subunit H. Unusual genetic configurations include three genes whose sequences overlap, one gene that has inserted into the coding region of another, several genes that have been inactivated by rearrangements in the region, and genes that have undergone tandem duplication. This report extends our original conclusion that the MT locus has incurred high levels of mutational change. PMID:11805055

Fine-scale phylogenetic architecture of a complex bacterial community.

PubMed

Acinas, Silvia G; Klepac-Ceraj, Vanja; Hunt, Dana E; Pharino, Chanathip; Ceraj, Ivica; Distel, Daniel L; Polz, Martin F

2004-07-29

Although molecular data have revealed the vast scope of microbial diversity, two fundamental questions remain unanswered even for well-defined natural microbial communities: how many bacterial types co-exist, and are such types naturally organized into phylogenetically discrete units of potential ecological significance? It has been argued that without such information, the environmental function, population biology and biogeography of microorganisms cannot be rigorously explored. Here we address these questions by comprehensive sampling of two large 16S ribosomal RNA clone libraries from a coastal bacterioplankton community. We show that compensation for artefacts generated by common library construction techniques reveals fine-scale patterns of community composition. At least 516 ribotypes (unique rRNA sequences) were detected in the sample and, by statistical extrapolation, at least 1,633 co-existing ribotypes in the sampled population. More than 50% of the ribotypes fall into discrete clusters containing less than 1% sequence divergence. This pattern cannot be accounted for by interoperon variation, indicating a large predominance of closely related taxa in this community. We propose that such microdiverse clusters arise by selective sweeps and persist because competitive mechanisms are too weak to purge diversity from within them.

Gut microbiome analysis of type 2 diabetic patients from the Chinese minority ethnic groups the Uygurs and Kazaks.

PubMed

Wang, Ye; Luo, Xin; Mao, Xinmin; Tao, Yicun; Ran, Xinjian; Zhao, Haixia; Xiong, Jianhui; Li, Linlin

2017-01-01

The gut microbiome may have an important influence on the development of diabetes mellitus type 2 (DM2). To better understand the DM2 pandemic in ethnic minority groups in China, we investigated and compared the composition and richness of the gut microbiota of healthy, normal glucose tolerant (NGT) individuals and DM2 patients from two ethnic minority groups in Xinjiang, northwest China, the Uygurs and Kazaks. The conserved V6 region of the 16S rRNA gene was amplified by PCR from the isolated DNA. The amplified DNA was sequenced and analyzed. An average of 4047 high quality reads of unique tag sequences were obtained from the 40 Uygurs and Kazaks. The 3 most dominant bacterial families among all participants, both healthy and DM2 patients, were the Ruminococcaceae, Lachnospiraceae, and Enterobacteriaceae. Significant differences in intestinal microbiota were found between the NGT individuals and DM2 patients, as well as between the two ethnic groups. Our findings shed new light on the gut microbiome in relation to DM2. The differentiated microbiota data may be used for potential biomarkers for DM2 diagnosis and prevention.

Using the Self-Select Paradigm to Delineate the Nature of Speech Motor Programming

PubMed Central

Wright, David L.; Robin, Don A.; Rhee, Jooyhun; Vaculin, Amber; Jacks, Adam; Guenther, Frank H.; Fox, Peter T.

2015-01-01

Purpose The authors examined the involvement of 2 speech motor programming processes identified by S. T. Klapp (1995, 2003) during the articulation of utterances differing in syllable and sequence complexity. According to S. T. Klapp, 1 process, INT, resolves the demands of the programmed unit, whereas a second process, SEQ, oversees the serial order demands of longer sequences. Method A modified reaction time paradigm was used to assess INT and SEQ demands. Specifically, syllable complexity was dependent on syllable structure, whereas sequence complexity involved either repeated or unique syllabi within an utterance. Results INT execution was slowed when articulating single syllables in the form CCCV compared to simpler CV syllables. Planning unique syllables within a multisyllabic utterance rather than repetitions of the same syllable slowed INT but not SEQ. Conclusions The INT speech motor programming process, important for mental syllabary access, is sensitive to changes in both syllable structure and the number of unique syllables in an utterance. PMID:19474396

Enriching Genomic Resources and Marker Development from Transcript Sequences of Jatropha curcas for Microgravity Studies

PubMed Central

Tian, Wenlan; Paudel, Dev

2017-01-01

Jatropha (Jatropha curcas L.) is an economically important species with a great potential for biodiesel production. To enrich the jatropha genomic databases and resources for microgravity studies, we sequenced and annotated the transcriptome of jatropha and developed SSR and SNP markers from the transcriptome sequences. In total 1,714,433 raw reads with an average length of 441.2 nucleotides were generated. De novo assembling and clustering resulted in 115,611 uniquely assembled sequences (UASs) including 21,418 full-length cDNAs and 23,264 new jatropha transcript sequences. The whole set of UASs were fully annotated, out of which 59,903 (51.81%) were assigned with gene ontology (GO) term, 12,584 (10.88%) had orthologs in Eukaryotic Orthologous Groups (KOG), and 8,822 (7.63%) were mapped to 317 pathways in six different categories in Kyoto Encyclopedia of Genes and Genome (KEGG) database, and it contained 3,588 putative transcription factors. From the UASs, 9,798 SSRs were discovered with AG/CT as the most frequent (45.8%) SSR motif type. Further 38,693 SNPs were detected and 7,584 remained after filtering. This UAS set has enriched the current jatropha genomic databases and provided a large number of genetic markers, which can facilitate jatropha genetic improvement and many other genetic and biological studies. PMID:28154822

Temporal variation of aftershocks by means of multifractal characterization of their inter-event time and cluster analysis

NASA Astrophysics Data System (ADS)

Figueroa-Soto, A.; Zuñiga, R.; Marquez-Ramirez, V.; Monterrubio-Velasco, M.

2017-12-01

. The inter-event time characteristics of seismic aftershock sequences can provide important information to discern stages in the aftershock generation process. In order to investigate whether separate dynamic stages can be identified, (1) aftershock series after selected earthquake mainshocks, which took place at similar tectonic regimes were analyzed. To this end we selected two well-defined aftershock sequences from New Zealand and one aftershock sequence for Mexico, we (2) analyzed the fractal behavior of the logarithm of inter-event times (also called waiting times) of aftershocks by means of Holdeŕs exponent, and (3) their magnitude and spatial location based on a methodology proposed by Zaliapin and Ben Zion [2011] which accounts for the clustering properties of the sequence. In general, more than two coherent process stages can be identified following the main rupture, evidencing a type of "cascade" process which precludes implying a single generalized power law even though the temporal rate and average fractal character appear to be unique (as in a single Omorís p value). We found that aftershock processes indeed show multi-fractal characteristics, which may be related to different stages in the process of diffusion, as seen in the temporary-spatial distribution of aftershocks. Our method provides a way of defining the onset of the return to seismic background activity and the end of the main aftershock sequence.

RUCS: rapid identification of PCR primers for unique core sequences.

PubMed

Thomsen, Martin Christen Frølund; Hasman, Henrik; Westh, Henrik; Kaya, Hülya; Lund, Ole

2017-12-15

Designing PCR primers to target a specific selection of whole genome sequenced strains can be a long, arduous and sometimes impractical task. Such tasks would benefit greatly from an automated tool to both identify unique targets, and to validate the vast number of potential primer pairs for the targets in silico. Here we present RUCS, a program that will find PCR primer pairs and probes for the unique core sequences of a positive genome dataset complement to a negative genome dataset. The resulting primer pairs and probes are in addition to simple selection also validated through a complex in silico PCR simulation. We compared our method, which identifies the unique core sequences, against an existing tool called ssGeneFinder, and found that our method was 6.5-20 times more sensitive. We used RUCS to design primer pairs that would target a set of genomes known to contain the mcr-1 colistin resistance gene. Three of the predicted pairs were chosen for experimental validation using PCR and gel electrophoresis. All three pairs successfully produced an amplicon with the target length for the samples containing mcr-1 and no amplification products were produced for the negative samples. The novel methods presented in this manuscript can reduce the time needed to identify target sequences, and provide a quick virtual PCR validation to eliminate time wasted on ambiguously binding primers. Source code is freely available on https://bitbucket.org/genomicepidemiology/rucs. Web service is freely available on https://cge.cbs.dtu.dk/services/RUCS. mcft@cbs.dtu.dk. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Giraffe genome sequence reveals clues to its unique morphology and physiology

PubMed Central

Agaba, Morris; Ishengoma, Edson; Miller, Webb C.; McGrath, Barbara C.; Hudson, Chelsea N.; Bedoya Reina, Oscar C.; Ratan, Aakrosh; Burhans, Rico; Chikhi, Rayan; Medvedev, Paul; Praul, Craig A.; Wu-Cavener, Lan; Wood, Brendan; Robertson, Heather; Penfold, Linda; Cavener, Douglas R.

2016-01-01

The origins of giraffe's imposing stature and associated cardiovascular adaptations are unknown. Okapi, which lacks these unique features, is giraffe's closest relative and provides a useful comparison, to identify genetic variation underlying giraffe's long neck and cardiovascular system. The genomes of giraffe and okapi were sequenced, and through comparative analyses genes and pathways were identified that exhibit unique genetic changes and likely contribute to giraffe's unique features. Some of these genes are in the HOX, NOTCH and FGF signalling pathways, which regulate both skeletal and cardiovascular development, suggesting that giraffe's stature and cardiovascular adaptations evolved in parallel through changes in a small number of genes. Mitochondrial metabolism and volatile fatty acids transport genes are also evolutionarily diverged in giraffe and may be related to its unusual diet that includes toxic plants. Unexpectedly, substantial evolutionary changes have occurred in giraffe and okapi in double-strand break repair and centrosome functions. PMID:27187213

Genomic analyses of Clostridium perfringens isolates from five toxinotypes.

PubMed

Hassan, Karl A; Elbourne, Liam D H; Tetu, Sasha G; Melville, Stephen B; Rood, Julian I; Paulsen, Ian T

2015-05-01

Clostridium perfringens can be isolated from a range of environments, including soil, marine and fresh water sediments, and the gastrointestinal tracts of animals and humans. Some C. perfringens strains have attractive industrial applications, e.g., in the degradation of waste products or the production of useful chemicals. However, C. perfringens has been most studied as the causative agent of a range of enteric and soft tissue infections of varying severities in humans and animals. Host preference and disease type in C. perfringens are intimately linked to the production of key extracellular toxins and on this basis toxigenic C. perfringens strains have been classified into five toxinotypes (A-E). To date, twelve genome sequences have been generated for a diverse collection of C. perfringens isolates, including strains associated with human and animal infections, a human commensal strain, and a strain with potential industrial utility. Most of the sequenced strains are classified as toxinotype A. However, genome sequences of representative strains from each of the other four toxinotypes have also been determined. Analysis of this collection of sequences has highlighted a lack of features differentiating toxinotype A strains from the other isolates, indicating that the primary defining characteristic of toxinotype A strains is their lack of key plasmid-encoded extracellular toxin genes associated with toxinotype B to E strains. The representative B-E strains sequenced to date each harbour many unique genes. Additional genome sequences are needed to determine if these genes are characteristic of their respective toxinotypes. Copyright © 2014. Published by Elsevier Masson SAS.

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens

PubMed Central

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-01-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue® mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents. PMID:22735701

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens.

PubMed

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-08-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.

Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

NASA Technical Reports Server (NTRS)

Sheridan, Peter P.; Miteva, Vanya I.; Brenchley, Jean E.

2003-01-01

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.

How to design a single-cell RNA-sequencing experiment: pitfalls, challenges and perspectives.

PubMed

Dal Molin, Alessandra; Di Camillo, Barbara

2018-01-31

The sequencing of the transcriptome of single cells, or single-cell RNA-sequencing, has now become the dominant technology for the identification of novel cell types in heterogeneous cell populations or for the study of stochastic gene expression. In recent years, various experimental methods and computational tools for analysing single-cell RNA-sequencing data have been proposed. However, most of them are tailored to different experimental designs or biological questions, and in many cases, their performance has not been benchmarked yet, thus increasing the difficulty for a researcher to choose the optimal single-cell transcriptome sequencing (scRNA-seq) experiment and analysis workflow. In this review, we aim to provide an overview of the current available experimental and computational methods developed to handle single-cell RNA-sequencing data and, based on their peculiarities, we suggest possible analysis frameworks depending on specific experimental designs. Together, we propose an evaluation of challenges and open questions and future perspectives in the field. In particular, we go through the different steps of scRNA-seq experimental protocols such as cell isolation, messenger RNA capture, reverse transcription, amplification and use of quantitative standards such as spike-ins and Unique Molecular Identifiers (UMIs). We then analyse the current methodological challenges related to preprocessing, alignment, quantification, normalization, batch effect correction and methods to control for confounding effects. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core

PubMed Central

Sheridan, Peter P.; Miteva, Vanya I.; Brenchley, Jean E.

2003-01-01

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at −9°C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 × 107 cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at −2°C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years. PMID:12676695

DAMe: a toolkit for the initial processing of datasets with PCR replicates of double-tagged amplicons for DNA metabarcoding analyses.

PubMed

Zepeda-Mendoza, Marie Lisandra; Bohmann, Kristine; Carmona Baez, Aldo; Gilbert, M Thomas P

2016-05-03

DNA metabarcoding is an approach for identifying multiple taxa in an environmental sample using specific genetic loci and taxa-specific primers. When combined with high-throughput sequencing it enables the taxonomic characterization of large numbers of samples in a relatively time- and cost-efficient manner. One recent laboratory development is the addition of 5'-nucleotide tags to both primers producing double-tagged amplicons and the use of multiple PCR replicates to filter erroneous sequences. However, there is currently no available toolkit for the straightforward analysis of datasets produced in this way. We present DAMe, a toolkit for the processing of datasets generated by double-tagged amplicons from multiple PCR replicates derived from an unlimited number of samples. Specifically, DAMe can be used to (i) sort amplicons by tag combination, (ii) evaluate PCR replicates dissimilarity, and (iii) filter sequences derived from sequencing/PCR errors, chimeras, and contamination. This is attained by calculating the following parameters: (i) sequence content similarity between the PCR replicates from each sample, (ii) reproducibility of each unique sequence across the PCR replicates, and (iii) copy number of the unique sequences in each PCR replicate. We showcase the insights that can be obtained using DAMe prior to taxonomic assignment, by applying it to two real datasets that vary in their complexity regarding number of samples, sequencing libraries, PCR replicates, and used tag combinations. Finally, we use a third mock dataset to demonstrate the impact and importance of filtering the sequences with DAMe. DAMe allows the user-friendly manipulation of amplicons derived from multiple samples with PCR replicates built in a single or multiple sequencing libraries. It allows the user to: (i) collapse amplicons into unique sequences and sort them by tag combination while retaining the sample identifier and copy number information, (ii) identify sequences carrying unused tag combinations, (iii) evaluate the comparability of PCR replicates of the same sample, and (iv) filter tagged amplicons from a number of PCR replicates using parameters of minimum length, copy number, and reproducibility across the PCR replicates. This enables an efficient analysis of complex datasets, and ultimately increases the ease of handling datasets from large-scale studies.

Molecular Typing of Staphylococcus aureus Isolated from Patients with Autosomal Dominant Hyper IgE Syndrome

PubMed Central

Sastalla, Inka; Williams, Kelli W.; Anderson, Erik D.; Myles, Ian A.; Reckhow, Jensen D.; Espinoza-Moraga, Marlene; Freeman, Alexandra F.; Datta, Sandip K.

2017-01-01

Autosomal dominant hyper IgE syndrome (AD-HIES) is a primary immunodeficiency caused by a loss-of-function mutation in the Signal Transducer and Activator of Transcription 3 (STAT3). This immune disorder is clinically characterized by increased susceptibility to cutaneous and sinopulmonary infections, in particular with Candida and Staphylococcus aureus. It has recently been recognized that the skin microbiome of patients with AD-HIES is altered with an overrepresentation of certain Gram-negative bacteria and Gram-positive staphylococci. However, these alterations have not been characterized at the species- and strain-level. Since S. aureus infections are influenced by strain-specific expression of virulence factors, information on colonizing strain characteristics may provide insights into host-pathogen interactions and help guide management strategies for treatment and prophylaxis. The aim of this study was to determine whether the immunodeficiency of AD-HIES selects for unique strains of colonizing S. aureus. Using multi-locus sequence typing (MLST), protein A (spa) typing, and PCR-based detection of toxin genes, we performed a detailed analysis of the S. aureus isolates (n = 13) found on the skin of twenty-one patients with AD-HIES. We found a low diversity of sequence types, and an abundance of strains that expressed methicillin resistance, Panton-Valentine leukocidin (PVL), and staphylococcal enterotoxins K and Q (SEK, SEQ). Our results indicate that patients with AD-HIES may often carry antibiotic-resistant strains that harbor key virulence factors. PMID:28587312

Position-based scanning for comparative genomics and identification of genetic islands in Haemophilus influenzae type b.

PubMed

Bergman, Nicholas H; Akerley, Brian J

2003-03-01

Bacteria exhibit extensive genetic heterogeneity within species. In many cases, these differences account for virulence properties unique to specific strains. Several such loci have been discovered in the genome of the type b serotype of Haemophilus influenzae, a human pathogen able to cause meningitis, pneumonia, and septicemia. Here we report application of a PCR-based scanning procedure to compare the genome of a virulent type b (Hib) strain with that of the laboratory-passaged Rd KW20 strain for which a complete genome sequence is available. We have identified seven DNA segments or H. influenzae genetic islands (HiGIs) present in the type b genome and absent from the Rd genome. These segments vary in size and content and show signs of horizontal gene transfer in that their percent G+C content differs from that of the rest of the H. influenzae genome, they contain genes similar to those found on phages or other mobile elements, or they are flanked by DNA repeats. Several of these loci represent potential pathogenicity islands, because they contain genes likely to mediate interactions with the host. These newly identified genetic islands provide areas of investigation into both the evolution and pathogenesis of H. influenzae. In addition, the genome scanning approach developed to identify these islands provides a rapid means to compare the genomes of phenotypically diverse bacterial strains once the genome sequence of one representative strain has been determined.

Complete Genome Sequences of Bacillus Phages Janet and OTooleKemple52

PubMed Central

2018-01-01

ABSTRACT We report here the genome sequences of two novel Bacillus cereus group-infecting bacteriophages, Janet and OTooleKemple52. These bacteriophages are double-stranded DNA-containing Myoviridae isolated from soil samples. While their genomes share a high degree of sequence identity with one another, their host preferences are unique. PMID:29748396

Novel Insights into Tree Biology and Genome Evolution as Revealed Through Genomics.

PubMed

Neale, David B; Martínez-García, Pedro J; De La Torre, Amanda R; Montanari, Sara; Wei, Xiao-Xin

2017-04-28

Reference genome sequences are the key to the discovery of genes and gene families that determine traits of interest. Recent progress in sequencing technologies has enabled a rapid increase in genome sequencing of tree species, allowing the dissection of complex characters of economic importance, such as fruit and wood quality and resistance to biotic and abiotic stresses. Although the number of reference genome sequences for trees lags behind those for other plant species, it is not too early to gain insight into the unique features that distinguish trees from nontree plants. Our review of the published data suggests that, although many gene families are conserved among herbaceous and tree species, some gene families, such as those involved in resistance to biotic and abiotic stresses and in the synthesis and transport of sugars, are often expanded in tree genomes. As the genomes of more tree species are sequenced, comparative genomics will further elucidate the complexity of tree genomes and how this relates to traits unique to trees.

Generation of a total of 6483 expressed sequence tags from 60 day-old bovine whole fetus and fetal placenta.

PubMed

Oishi, M; Gohma, H; Lejukole, H Y; Taniguchi, Y; Yamada, T; Suzuki, K; Shinkai, H; Uenishi, H; Yasue, H; Sasaki, Y

2004-05-01

Expressed sequence tags (ESTs) generated based on characterization of clones isolated randomly from cDNA libraries are used to study gene expression profiles in specific tissues and to provide useful information for characterizing tissue physiology. In this study, two directionally cloned cDNA libraries were constructed from 60 day-old bovine whole fetus and fetal placenta. We have characterized 5357 and 1126 clones, and then identified 3464 and 795 unique sequences for the fetus and placenta cDNA libraries: 1851 and 504 showed homology to already identified genes, and 1613 and 291 showed no significant matches to any of the sequences in DNA databases, respectively. Further, we found 94 unique sequences overlapping in both the fetus and the placenta, leading to a catalog of 4165 genes expressed in 60 day-old fetus and placenta. The catalog is used to examine expression profile of genes in 60 day-old bovine fetus and placenta.

UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy

PubMed Central

2017-01-01

Unique Molecular Identifiers (UMIs) are random oligonucleotide barcodes that are increasingly used in high-throughput sequencing experiments. Through a UMI, identical copies arising from distinct molecules can be distinguished from those arising through PCR amplification of the same molecule. However, bioinformatic methods to leverage the information from UMIs have yet to be formalized. In particular, sequencing errors in the UMI sequence are often ignored or else resolved in an ad hoc manner. We show that errors in the UMI sequence are common and introduce network-based methods to account for these errors when identifying PCR duplicates. Using these methods, we demonstrate improved quantification accuracy both under simulated conditions and real iCLIP and single-cell RNA-seq data sets. Reproducibility between iCLIP replicates and single-cell RNA-seq clustering are both improved using our proposed network-based method, demonstrating the value of properly accounting for errors in UMIs. These methods are implemented in the open source UMI-tools software package. PMID:28100584

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in a urease-positive thermophilic Campylobacter sp. (UPTC).

PubMed

Tasaki, E; Hirayama, J; Tazumi, A; Hayashi, K; Hara, Y; Ueno, H; Moore, J E; Millar, B C; Matsuda, M

2012-02-01

Novel clustered regularly-interspaced short palindromic repeats (CRISPRs) locus [7,500 base pairs (bp) in length] occurred in the urease-positive thermophilic Campylobacter (UPTC) Japanese isolate, CF89-12. The 7,500 bp gene loci consisted of the 5'-methylaminomethyl-2-thiouridylate methyltransferase gene, putative (P) CRISPR associated (p-Cas), putative open reading frames, Cas1 and Cas2, leader sequence region (146 bp), 12 CRISPRs consensus sequence repeats (each 36 bp) separated by a non-repetitive unique spacer region of similar length (26-31 bp) and the phosphatidyl glycerophosphatase A gene. When the CRISPRs loci in the UPTC CF89-12 and five C. jejuni isolates were compared with one another, these six isolates contained p-Cas, Cas1 and Cas2 within the loci. Four to 12 CRISPRs consensus sequence repeats separated by a non-repetitive unique spacer region occurred in six isolates and the nucleotide sequences of those repeats gave approximately 92-100% similarity with each other. However, no sequence similarity occurred in the unique spacer regions among these isolates. The putative σ(70) transcriptional promoter and the hypothetical ρ-independent terminator structures for the CRISPRs and Cas were detected. No in vivo transcription of p-Cas, Cas1 and Cas2 was confirmed in the UPTC cells.

Structural analysis of a set of proteins resulting from a bacterial genomics project.

PubMed

Badger, J; Sauder, J M; Adams, J M; Antonysamy, S; Bain, K; Bergseid, M G; Buchanan, S G; Buchanan, M D; Batiyenko, Y; Christopher, J A; Emtage, S; Eroshkina, A; Feil, I; Furlong, E B; Gajiwala, K S; Gao, X; He, D; Hendle, J; Huber, A; Hoda, K; Kearins, P; Kissinger, C; Laubert, B; Lewis, H A; Lin, J; Loomis, K; Lorimer, D; Louie, G; Maletic, M; Marsh, C D; Miller, I; Molinari, J; Muller-Dieckmann, H J; Newman, J M; Noland, B W; Pagarigan, B; Park, F; Peat, T S; Post, K W; Radojicic, S; Ramos, A; Romero, R; Rutter, M E; Sanderson, W E; Schwinn, K D; Tresser, J; Winhoven, J; Wright, T A; Wu, L; Xu, J; Harris, T J R

2005-09-01

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB. Copyright 2005 Wiley-Liss, Inc.

Conservation of the Human Integrin-Type Beta-Propeller Domain in Bacteria

PubMed Central

Chouhan, Bhanupratap; Denesyuk, Alexander; Heino, Jyrki; Johnson, Mark S.; Denessiouk, Konstantin

2011-01-01

Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca2+-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca2+-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem copies are found. PMID:22022374

Complete sequences of a novel blaNDM-1-harbouring plasmid from Providencia rettgeri and an FII-type plasmid from Klebsiella pneumoniae identified in Canada.

PubMed

Mataseje, L F; Boyd, D A; Lefebvre, B; Bryce, E; Embree, J; Gravel, D; Katz, K; Kibsey, P; Kuhn, M; Langley, J; Mitchell, R; Roscoe, D; Simor, A; Taylor, G; Thomas, E; Turgeon, N; Mulvey, M R

2014-03-01

Emergence of plasmids harbouring bla(NDM-1) is a major public health concern due to their association with multidrug resistance and their potential mobility. PCR was used to detect bla(NDM-1) from clinical isolates of Providencia rettgeri (PR) and Klebsiella pneumoniae (KP). Antimicrobial susceptibilities were determined using Vitek 2. The complete DNA sequence of two bla(NDM-1) plasmids (pPrY2001 and pKp11-42) was obtained using a 454-Genome Sequencer FLX. Contig assembly and gap closures were confirmed by PCR-based sequencing. Comparative analysis was done using BLASTn and BLASTp algorithms. Both clinical isolates were resistant to all β-lactams, carbapenems, aminoglycosides, ciprofloxacin and trimethoprim/sulfamethoxazole, and susceptible to tigecycline. Plasmid pPrY2001 (113 295 bp) was isolated from PR. It did not show significant homology to any known plasmid backbone and contained a truncated repA and novel repB. Two bla(NDM-1)-harbouring plasmids from Acinetobacter lwoffii (JQ001791 and JQ060896) shared 100% similarity to a 15 kb region that contained bla(NDM-1). pPrY2001 also contained a type II toxin/antitoxin system. pKp11-42 (146 695 bp) was isolated from KP. It contained multiple repA genes. The plasmid backbone had the highest homology to the IncFIIk plasmid type (51% coverage, 100% nucleotide identity). The bla(NDM-1) region was unique in that it was flanked upstream by IS3000 and downstream by a novel transposon designated Tn6229. pKp11-42 also contained a number of mutagenesis and plasmid stability proteins. pPrY2001 differed from all known plasmids due to its novel backbone and repB. pKp11-42 was similar to IncFIIk plasmids and contained a number of genes that aid in plasmid persistence.

Method for identifying and quantifying nucleic acid sequence aberrations

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

1998-01-01

A method for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe.

Method for identifying and quantifying nucleic acid sequence aberrations

DOEpatents

Lucas, J.N.; Straume, T.; Bogen, K.T.

1998-07-21

A method is disclosed for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe. 11 figs.

[Soil propagule bank of ectomycorrhizal fungi in natural forest of Pinus bungeana].

PubMed

Zhao, Nan Xing; Han, Qi Sheng; Huang, Jian

2017-12-01

To conserve and restore the forest of Pinu bungeana, we investigated the soil propagule bank of ectomycorrhizal (ECM) fungi in a severely disturbed natural forest of P. bungeana in Shaanxi Province, China. We used a seedling-bioassay method to bait the ECM fungal propagules in the soils collected from the forest site. ECM was identified by combining morph typing with ITS-PCR-sequencing. We obtained 73 unique sequences from the ECM associated with P. bungeana seedlings, and assigned them into 12 ECM fungal OTUs at the threshold of 97% based on the sequence similarity. Rarefaction curve displayed almost all ECM fungi in the propagule bank were detected. The most frequent OTU (80%) showed poor similarity (75%) with existing sequences in the online database, which suggested it might be a new species. Cenococcum geophilum, Tomentella sp., Tuber sp. were common species in the propagule bank. Although C. geophilum and Tomentella sp. were frequently detected in other soil propagule banks of pine forest, the most frequent OTU was not assigned to known genus or family, which indicated the host-specif of ECM propagule banks associa-ted with P. bungeana. This result confirmed the importance of the special ECM propagule banks associated with P. bungeana for natural forest restoration.

Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding

PubMed Central

Best, Katharine; Oakes, Theres; Heather, James M.; Shawe-Taylor, John; Chain, Benny

2015-01-01

The polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology. In combination with High Throughput Sequencing (HTS), PCR is widely used to quantify transcript abundance for RNA-seq, and in the context of analysis of T and B cell receptor repertoires. In this study, we combine DNA barcoding with HTS to quantify PCR output from individual target molecules. We develop computational tools that simulate both the PCR branching process itself, and the subsequent subsampling which typically occurs during HTS sequencing. We explore the influence of different types of heterogeneity on sequencing output, and compare them to experimental results where the efficiency of amplification is measured by barcodes uniquely identifying each molecule of starting template. Our results demonstrate that the PCR process introduces substantial amplification heterogeneity, independent of primer sequence and bulk experimental conditions. This heterogeneity can be attributed both to inherited differences between different template DNA molecules, and the inherent stochasticity of the PCR process. The results demonstrate that PCR heterogeneity arises even when reaction and substrate conditions are kept as constant as possible, and therefore single molecule barcoding is essential in order to derive reproducible quantitative results from any protocol combining PCR with HTS. PMID:26459131

Non-contiguous genome sequence of Mycobacterium simiae strain DSM 44165(T.).

PubMed

Sassi, Mohamed; Robert, Catherine; Raoult, Didier; Drancourt, Michel

2013-01-01

Mycobacterium simiae is a non-tuberculosis mycobacterium causing pulmonary infections in both immunocompetent and imunocompromized patients. We announce the draft genome sequence of M. simiae DSM 44165(T). The 5,782,968-bp long genome with 65.15% GC content (one chromosome, no plasmid) contains 5,727 open reading frames (33% with unknown function and 11 ORFs sizing more than 5000 -bp), three rRNA operons, 52 tRNA, one 66-bp tmRNA matching with tmRNA tags from Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium marinum, and Mycobacterium africanum and 389 DNA repetitive sequences. Comparing ORFs and size distribution between M. simiae and five other Mycobacterium species M. simiae clustered with M. abscessus and M. smegmatis. A 40-kb prophage was predicted in addition to two prophage-like elements, 7-kb and 18-kb in size, but no mycobacteriophage was seen after the observation of 10(6) M. simiae cells. Fifteen putative CRISPRs were found. Three genes were predicted to encode resistance to aminoglycosides, betalactams and macrolide-lincosamide-streptogramin B. A total of 163 CAZYmes were annotated. M. simiae contains ESX-1 to ESX-5 genes encoding for a type-VII secretion system. Availability of the genome sequence may help depict the unique properties of this environmental, opportunistic pathogen.

Exploitation of the diverse insertion sequence element content of dairy Lactobacillus helveticus starters as a rapid method to identify different strains.

PubMed

Kaleta, Pawel; Callanan, Michael J; O'Callaghan, John; Fitzgerald, Gerald F; Beresford, Thomas P; Ross, R Paul

2009-10-01

The species Lactobacillus helveticus is a commonly used thermophilic starter and/or adjunct culture for Swiss and Cheddar cheese manufacture. Its use is normally associated with flavour improvement which is known to be associated with culture traits such as rapid autolysis and high proteolytic activity. The genome of the commercial strain, DPC4571, was recently sequenced and found to have an abundance of IS sequences in terms of both abundance (213 intact) and diversity (21 types). Given this unique diversity for a lactic acid bacterium, we investigated whether PCR-based IS fingerprinting could be used as a discriminatory tool to distinguish between different strains of Lb. helveticus. A set of ten primers targeting five of the most numerous groups (ISL1201, ISLhe65, ISLhe2, ISLhe15 and ISL2) of IS elements was designed. Multiplex-PCR with all primers resulted in 1-12 discreet amplicons for each strain tested. The resultant fingerprints (in the 0.5 kb-3 kb range) were found to be strain specific and reproducible. This approach thus provides a valuable method to distinguish between Lb. helveticus strains while giving some indication of the relative abundance of IS sequences in each strain.

Molecular epidemiology of early and acute HIV type 1 infections in the United States Navy and Marine Corps, 2005-2010.

PubMed

Heipertz, Richard A; Sanders-Buell, Eric; Kijak, Gustavo; Howell, Shana; Lazzaro, Michelle; Jagodzinski, Linda L; Eggleston, John; Peel, Sheila; Malia, Jennifer; Armstrong, Adam; Michael, Nelson L; Kim, Jerome H; O'Connell, Robert J; Scott, Paul T; Brett-Major, David M; Tovanabutra, Sodsai

2013-10-01

The U.S. military represents a unique population within the human immunodeficiency virus 1 (HIV-1) pandemic. The last comprehensive study of HIV-1 in members of the U.S. Navy and Marine Corps (Sea Services) was completed in 2000, before large-scale combat operations were taking place. Here, we present molecular characterization of HIV-1 from 40 Sea Services personnel who were identified during their seroconversion window and initially classified as HIV-1 negative during screening. Protease/reverse transcriptase (pro/rt) and envelope (env) sequences were obtained from each member of the cohort. Phylogenetic analyses were carried out on these regions to determine relatedness within the cohort and calculate the most recent common ancestor for the related sequences. We identified 39 individuals infected with subtype B and one infected with CRF01_AE. Comparison of the pairwise genetic distance of Sea Service sequences and reference sequences in the env and pro/rt regions showed that five samples were part of molecular clusters, a group of two and a group of three, confirmed by single genome amplification. Real-time molecular monitoring of new HIV-1 acquisitions in the Sea Services may have a role in facilitating public health interventions at sites where related HIV-1 infections are identified.

Detecting very low allele fraction variants using targeted DNA sequencing and a novel molecular barcode-aware variant caller.

PubMed

Xu, Chang; Nezami Ranjbar, Mohammad R; Wu, Zhong; DiCarlo, John; Wang, Yexun

2017-01-03

Detection of DNA mutations at very low allele fractions with high accuracy will significantly improve the effectiveness of precision medicine for cancer patients. To achieve this goal through next generation sequencing, researchers need a detection method that 1) captures rare mutation-containing DNA fragments efficiently in the mix of abundant wild-type DNA; 2) sequences the DNA library extensively to deep coverage; and 3) distinguishes low level true variants from amplification and sequencing errors with high accuracy. Targeted enrichment using PCR primers provides researchers with a convenient way to achieve deep sequencing for a small, yet most relevant region using benchtop sequencers. Molecular barcoding (or indexing) provides a unique solution for reducing sequencing artifacts analytically. Although different molecular barcoding schemes have been reported in recent literature, most variant calling has been done on limited targets, using simple custom scripts. The analytical performance of barcode-aware variant calling can be significantly improved by incorporating advanced statistical models. We present here a highly efficient, simple and scalable enrichment protocol that integrates molecular barcodes in multiplex PCR amplification. In addition, we developed smCounter, an open source, generic, barcode-aware variant caller based on a Bayesian probabilistic model. smCounter was optimized and benchmarked on two independent read sets with SNVs and indels at 5 and 1% allele fractions. Variants were called with very good sensitivity and specificity within coding regions. We demonstrated that we can accurately detect somatic mutations with allele fractions as low as 1% in coding regions using our enrichment protocol and variant caller.

High-throughput analysis of the protein sequence-stability landscape using a quantitative "yeast surface two-hybrid" system and fragment reconstitution

PubMed Central

Dutta, Sanjib; Koide, Akiko; Koide, Shohei

2008-01-01

Stability evaluation of many mutants can lead to a better understanding of the sequence determinants of a structural motif and of factors governing protein stability and protein evolution. The traditional biophysical analysis of protein stability is low throughput, limiting our ability to widely explore the sequence space in a quantitative manner. In this study, we have developed a high-throughput library screening method for quantifying stability changes, which is based on protein fragment reconstitution and yeast surface display. Our method exploits the thermodynamic linkage between protein stability and fragment reconstitution and the ability of the yeast surface display technique to quantitatively evaluate protein-protein interactions. The method was applied to a fibronectin type III (FN3) domain. Characterization of fragment reconstitution was facilitated by the co-expression of two FN3 fragments, thus establishing a "yeast surface two-hybrid" method. Importantly, our method does not rely on competition between clones and thus eliminates a common limitation of high-throughput selection methods in which the most stable variants are predominantly recovered. Thus, it allows for the isolation of sequences that exhibits a desired level of stability. We identified over one hundred unique sequences for a β-bulge motif, which was significantly more informative than natural sequences of the FN3 family in revealing the sequence determinants for the β-bulge. Our method provides a powerful means to rapidly assess stability of many variants, to systematically assess contribution of different factors to protein stability and to enhance protein stability. PMID:18674545

Centromere location in Arabidopsis is unaltered by extreme divergence in CENH3 protein sequence

PubMed Central

2017-01-01

During cell division, spindle fibers attach to chromosomes at centromeres. The DNA sequence at regional centromeres is fast evolving with no conserved genetic signature for centromere identity. Instead CENH3, a centromere-specific histone H3 variant, is the epigenetic signature that specifies centromere location across both plant and animal kingdoms. Paradoxically, CENH3 is also adaptively evolving. An ongoing question is whether CENH3 evolution is driven by a functional relationship with the underlying DNA sequence. Here, we demonstrate that despite extensive protein sequence divergence, CENH3 histones from distant species assemble centromeres on the same underlying DNA sequence. We first characterized the organization and diversity of centromere repeats in wild-type Arabidopsis thaliana. We show that A. thaliana CENH3-containing nucleosomes exhibit a strong preference for a unique subset of centromeric repeats. These sequences are largely missing from the genome assemblies and represent the youngest and most homogeneous class of repeats. Next, we tested the evolutionary specificity of this interaction in a background in which the native A. thaliana CENH3 is replaced with CENH3s from distant species. Strikingly, we find that CENH3 from Lepidium oleraceum and Zea mays, although specifying epigenetically weaker centromeres that result in genome elimination upon outcrossing, show a binding pattern on A. thaliana centromere repeats that is indistinguishable from the native CENH3. Our results demonstrate positional stability of a highly diverged CENH3 on independently evolved repeats, suggesting that the sequence specificity of centromeres is determined by a mechanism independent of CENH3. PMID:28223399

Mining on scorpion venom biodiversity.

PubMed

Rodríguez de la Vega, Ricardo C; Schwartz, Elisabeth F; Possani, Lourival D

2010-12-15

Scorpion venoms are complex mixtures of dozens or even hundreds of distinct proteins, many of which are inter-genome active elements. Fifty years after the first scorpion toxin sequences were determined, chromatography-assisted purification followed by automated protein sequencing or gene cloning, on a case-by-case basis, accumulated nearly 250 amino acid sequences of scorpion venom components. A vast majority of the available sequences correspond to proteins adopting a common three-dimensional fold, whose ion channel modulating functions have been firmly established or could be confidently inferred. However, the actual molecular diversity contained in scorpion venoms -as revealed by bioassay-driven purification, some unexpected activities of "canonical" neurotoxins and even serendipitous discoveries- is much larger than those "canonical" toxin types. In the last few years mining into the molecular diversity contained in scorpion has been assisted by high-throughput Mass Spectrometry techniques and large-scale DNA sequencing, collectively accounting for the more than twofold increase in the number of known sequences of scorpion venom components (now reaching 500 unique sequences). This review, from a comparative perspective, deals with recent data obtained by proteomic and transcriptomic studies on scorpion venoms and venom glands. Altogether, these studies reveal a large contribution of non canonical venom components, which would account for more than half of the total protein diversity of any scorpion venom. On top of aiding at the better understanding of scorpion venom biology, whether in the context of venom function or within the venom gland itself, these "novel" venom components certainly are an interesting source of bioactive proteins, whose characterization is worth pursuing. Copyright © 2009 Elsevier Ltd. All rights reserved.

Genetic variation and dynamics of infections of equid herpesvirus 5 in individual horses.

PubMed

Back, Helena; Ullman, Karin; Leijon, Mikael; Söderlund, Robert; Penell, Johanna; Ståhl, Karl; Pringle, John; Valarcher, Jean-François

2016-01-01

Equid herpesvirus 5 (EHV-5) is related to the human Epstein-Barr virus (human herpesvirus 4) and has frequently been observed in equine populations worldwide. EHV-5 was previously assumed to be low to non-pathogenic; however, studies have also related the virus to the severe lung disease equine multinodular pulmonary fibrosis (EMPF). Genetic information of EHV-5 is scanty: the whole genome was recently described and only limited nucleotide sequences are available. In this study, samples were taken twice 1 year apart from eight healthy horses at the same professional training yard and samples from a ninth horse that was diagnosed with EMPF with samples taken pre- and post-mortem to analyse partial glycoprotein B (gB) gene of EHV-5 by using next-generation sequencing. The analysis resulted in 27 partial gB gene sequences, 11 unique sequence types and five amino acid sequences. These sequences could be classified within four genotypes (I-IV) of the EHV-5 gB gene based on the degree of similarity of the nucleotide and amino acid sequences, and in this work horses were shown to be identified with up to three different genotypes simultaneously. The observations showed a range of interactions between EHV-5 and the host over time, where the same virus persists in some horses, whereas others have a more dynamic infection pattern including strains from different genotypes. This study provides insight into the genetic variation and dynamics of EHV-5, and highlights that further work is needed to understand the EHV-5 interaction with its host.

The Caenorhabditis elegans gene unc-89, required fpr muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains

PubMed Central

1996-01-01

Mutations in the Caenorhabditis elegans gene unc-89 result in nematodes having disorganized muscle structure in which thick filaments are not organized into A-bands, and there are no M-lines. Beginning with a partial cDNA from the C. elegans sequencing project, we have cloned and sequenced the unc-89 gene. An unc-89 allele, st515, was found to contain an 84-bp deletion and a 10-bp duplication, resulting in an in- frame stop codon within predicted unc-89 coding sequence. Analysis of the complete coding sequence for unc-89 predicts a novel 6,632 amino acid polypeptide consisting of sequence motifs which have been implicated in protein-protein interactions. UNC-89 begins with 67 residues of unique sequences, SH3, dbl/CDC24, and PH domains, 7 immunoglobulins (Ig) domains, a putative KSP-containing multiphosphorylation domain, and ends with 46 Ig domains. A polyclonal antiserum raised to a portion of unc-89 encoded sequence reacts to a twitchin-sized polypeptide from wild type, but truncated polypeptides from st515 and from the amber allele e2338. By immunofluorescent microscopy, this antiserum localizes to the middle of A-bands, consistent with UNC-89 being a structural component of the M-line. Previous studies indicate that myofilament lattice assembly begins with positional cues laid down in the basement membrane and muscle cell membrane. We propose that the intracellular protein UNC-89 responds to these signals, localizes, and then participates in assembling an M-line. PMID:8603916

Analysis of Dissimilatory Sulfite Reductase and 16S rRNA Gene Fragments from Deep-Sea Hydrothermal Sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific

PubMed Central

Nakagawa, Tatsunori; Ishibashi, Jun-Ichiro; Maruyama, Akihiko; Yamanaka, Toshiro; Morimoto, Yusuke; Kimura, Hiroyuki; Urabe, Tetsuro; Fukui, Manabu

2004-01-01

This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5°C and natural vent fluids at 7°C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with the Aquificae and γ- and ɛ-Proteobacteria as well as the occurrence of archaeal phylotypes affiliated with the Thermococcales and of a unique archaeon sequence that clustered with “Nanoarchaeota.” The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300°C were affiliated with the δ-Proteobacteria, genus Thiomicrospira, and Pelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4°C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments. PMID:14711668

Erwinia amylovora CRISPR Elements Provide New Tools for Evaluating Strain Diversity and for Microbial Source Tracking

PubMed Central

McGhee, Gayle C.; Sundin, George W.

2012-01-01

Clustered regularly interspaced short palindromic repeats (CRISPRs) comprise a family of short DNA repeat sequences that are separated by non repetitive spacer sequences and, in combination with a suite of Cas proteins, are thought to function as an adaptive immune system against invading DNA. The number of CRISPR arrays in a bacterial chromosome is variable, and the content of each array can differ in both repeat number and in the presence or absence of specific spacers. We utilized a comparative sequence analysis of CRISPR arrays of the plant pathogen Erwinia amylovora to uncover previously unknown genetic diversity in this species. A total of 85 E. amylovora strains varying in geographic isolation (North America, Europe, New Zealand, and the Middle East), host range, plasmid content, and streptomycin sensitivity/resistance were evaluated for CRISPR array number and spacer variability. From these strains, 588 unique spacers were identified in the three CRISPR arrays present in E. amylovora, and these arrays could be categorized into 20, 17, and 2 patterns types, respectively. Analysis of the relatedness of spacer content differentiated most apple and pear strains isolated in the eastern U.S. from western U.S. strains. In addition, we identified North American strains that shared CRISPR genotypes with strains isolated on other continents. E. amylovora strains from Rubus and Indian hawthorn contained mostly unique spacers compared to apple and pear strains, while strains from loquat shared 79% of spacers with apple and pear strains. Approximately 23% of the spacers matched known sequences, with 16% targeting plasmids and 5% targeting bacteriophage. The plasmid pEU30, isolated in E. amylovora strains from the western U.S., was targeted by 55 spacers. Lastly, we used spacer patterns and content to determine that streptomycin-resistant strains of E. amylovora from Michigan were low in diversity and matched corresponding streptomycin-sensitive strains from the background population. PMID:22860008

Erwinia amylovora CRISPR elements provide new tools for evaluating strain diversity and for microbial source tracking.

PubMed

McGhee, Gayle C; Sundin, George W

2012-01-01

Clustered regularly interspaced short palindromic repeats (CRISPRs) comprise a family of short DNA repeat sequences that are separated by non repetitive spacer sequences and, in combination with a suite of Cas proteins, are thought to function as an adaptive immune system against invading DNA. The number of CRISPR arrays in a bacterial chromosome is variable, and the content of each array can differ in both repeat number and in the presence or absence of specific spacers. We utilized a comparative sequence analysis of CRISPR arrays of the plant pathogen Erwinia amylovora to uncover previously unknown genetic diversity in this species. A total of 85 E. amylovora strains varying in geographic isolation (North America, Europe, New Zealand, and the Middle East), host range, plasmid content, and streptomycin sensitivity/resistance were evaluated for CRISPR array number and spacer variability. From these strains, 588 unique spacers were identified in the three CRISPR arrays present in E. amylovora, and these arrays could be categorized into 20, 17, and 2 patterns types, respectively. Analysis of the relatedness of spacer content differentiated most apple and pear strains isolated in the eastern U.S. from western U.S. strains. In addition, we identified North American strains that shared CRISPR genotypes with strains isolated on other continents. E. amylovora strains from Rubus and Indian hawthorn contained mostly unique spacers compared to apple and pear strains, while strains from loquat shared 79% of spacers with apple and pear strains. Approximately 23% of the spacers matched known sequences, with 16% targeting plasmids and 5% targeting bacteriophage. The plasmid pEU30, isolated in E. amylovora strains from the western U.S., was targeted by 55 spacers. Lastly, we used spacer patterns and content to determine that streptomycin-resistant strains of E. amylovora from Michigan were low in diversity and matched corresponding streptomycin-sensitive strains from the background population.

Diversity of human immunodeficiency virus type 1 subtypes in Kagera and Kilimanjaro regions, Tanzania.

PubMed

Nyombi, Balthazar M; Kristiansen, Knut I; Bjune, Gunnar; Müller, Fredrik; Holm-Hansen, Carol

2008-06-01

A strategy to prevent the spread of HIV-1 worldwide is complicated by the high genetic diversity of the virus. To gain a better understanding of the HIV-1 genetic diversity in Tanzania, a molecular epidemiological investigation was conducted in Kagera and Kilimanjaro regions. While several studies have addressed HIV-1 subtypes in Tanzania, this is the first study to describe the virus subtypes circulating in Kagera. The Kagera region is the epicenter of the HIV-1 epidemic in Africa, and it was therefore of interest to compare the prevalence of HIV subtypes in this region and Kilimanjaro. Blood samples were obtained from 246 HIV-1-infected pregnant women attending antenatal clinics. Plasma HIV-1 RNA was extracted, amplified, and sequenced in the env C2V3 and/or pol regions from 209 samples. Based on the analysis of env C2V3 and pol sequences, 47.4% had concordant subtypes, 19.1% were discordant indicating recombination, and for 33.5% sequences were obtained for only one region. The distribution HIV-1 subtypes based on the phylogenetic analysis of paired env C2V3/ pol sequences in Kagera region was A/A (27.8%), C/C (29.6%), D/D (16.7%), and unique recombinant forms (25.9%), and in Kilimanjaro region was A/A (32.9%), C/C (25.9%), D/D (10.6%), CRF10_CD (1.2%), and unique recombinant forms (29.4%). The env C2V3 subsubtype A2 and env C2V3/pol CRF10_CD were also observed indicating that these recombinants are circulating in Tanzania. The high diversity of HIV-1 subtypes and the high prevalence of recombinants demonstrated in this study necessitate expanded and continuous monitoring of the epidemic in Tanzania. The trend may have implications for current national control strategies against the HIV-1 epidemic.

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

Kilo-sequencing: an ordered strategy for rapid DNA sequence data acquisition.

PubMed Central

Barnes, W M; Bevan, M

1983-01-01

A strategy for rapid DNA sequence acquisition in an ordered, nonrandom manner, while retaining all of the conveniences of the dideoxy method with M13 transducing phage DNA template, is described. Target DNA 3 to 14 kb in size can be stably carried by our M13 vectors. Suitable targets are stretches of DNA which lack an enzyme recognition site which is unique on our cloning vectors and adjacent to the sequencing primer; current sites that are so useful when lacking are Pst, Xba, HindIII, BglII, EcoRI. By an in vitro procedure, we cut RF DNA once randomly and once specifically, to create thousands of deletions which start at the unique restriction site adjacent to the dideoxy sequencing primer and extend various distances across the target DNA. Phage carrying a desired size of deletions, whose DNA as template will give rise to DNA sequence data in a desired location along the target DNA, may be purified by electrophoresis alive on agarose gels. Phage running in the same location on the agarose gel thus conveniently give rise to nucleotide sequence data from the same kilobase of target DNA. Images PMID:6298723

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

PubMed Central

2013-01-01

Background Microsatellites are widely used for many genetic studies. In contrast to single nucleotide polymorphism (SNP) and genotyping-by-sequencing methods, they are readily typed in samples of low DNA quality/concentration (e.g. museum/non-invasive samples), and enable the quick, cheap identification of species, hybrids, clones and ploidy. Microsatellites also have the highest cross-species utility of all types of markers used for genotyping, but, despite this, when isolated from a single species, only a relatively small proportion will be of utility. Marker development of any type requires skill and time. The availability of sufficient “off-the-shelf” markers that are suitable for genotyping a wide range of species would not only save resources but also uniquely enable new comparisons of diversity among taxa at the same set of loci. No other marker types are capable of enabling this. We therefore developed a set of avian microsatellite markers with enhanced cross-species utility. Results We selected highly-conserved sequences with a high number of repeat units in both of two genetically distant species. Twenty-four primer sets were designed from homologous sequences that possessed at least eight repeat units in both the zebra finch (Taeniopygia guttata) and chicken (Gallus gallus). Each primer sequence was a complete match to zebra finch and, after accounting for degenerate bases, at least 86% similar to chicken. We assessed primer-set utility by genotyping individuals belonging to eight passerine and four non-passerine species. The majority of the new Conserved Avian Microsatellite (CAM) markers amplified in all 12 species tested (on average, 94% in passerines and 95% in non-passerines). This new marker set is of especially high utility in passerines, with a mean 68% of loci polymorphic per species, compared with 42% in non-passerine species. Conclusions When combined with previously described conserved loci, this new set of conserved markers will not only reduce the necessity and expense of microsatellite isolation for a wide range of genetic studies, including avian parentage and population analyses, but will also now enable comparisons of genetic diversity among different species (and populations) at the same set of loci, with no or reduced bias. Finally, the approach used here can be applied to other taxa in which appropriate genome sequences are available. PMID:23497230

Application of combinatorial biocatalysis for a unique ring expansion of dihydroxymethylzearalenone

USDA-ARS?s Scientific Manuscript database

Combinatorial biocatalysis was applied to generate a diverse set of dihydroxymethylzearalenone derivatives with modified ring structure. In one chemoenzymatic reaction sequence, dihydroxymethylzearalenone was first subjected to a unique enzyme-catalyzed oxidative ring opening reaction that creates ...

Annotating Mutational Effects on Proteins and Protein Interactions: Designing Novel and Revisiting Existing Protocols.

PubMed

Li, Minghui; Goncearenco, Alexander; Panchenko, Anna R

2017-01-01

In this review we describe a protocol to annotate the effects of missense mutations on proteins, their functions, stability, and binding. For this purpose we present a collection of the most comprehensive databases which store different types of sequencing data on missense mutations, we discuss their relationships, possible intersections, and unique features. Next, we suggest an annotation workflow using the state-of-the art methods and highlight their usability, advantages, and limitations for different cases. Finally, we address a particularly difficult problem of deciphering the molecular mechanisms of mutations on proteins and protein complexes to understand the origins and mechanisms of diseases.

Genomic analysis of a Raoultella ornithinolytica strain causing prosthetic joint infection in an immunocompetent patient.

PubMed

Beye, Mamadou; Hasni, Issam; Seng, Piseth; Michelle, Caroline; La Scola, Bernard; Raoult, Didier; Fournier, Pierre-Edouard

2018-06-21

We sequenced the genome of Raoultella ornithinolytica strain Marseille-P1025 that caused a rare case of prosthetic joint infection in a 67-year-old immunocompetent male. The 6.7-Mb genome exhibited a genomic island (RoGI) that was unique among R. ornithinolytica strains. RoGI was likely acquired by lateral gene transfer from a member of the Pectobacterium genus and coded for a type IVa secretion system found in other pathogenic bacteria and that may have conferred strain Marseille-P1025 an increased virulence. Strain Marseille-P1025 was also able to infect, multiply within, and kill Acanthamoaeba castellanii amoebae.

A cricket Gene Index: a genomic resource for studying neurobiology, speciation, and molecular evolution

PubMed Central

Danley, Patrick D; Mullen, Sean P; Liu, Fenglong; Nene, Vishvanath; Quackenbush, John; Shaw, Kerry L

2007-01-01

Background As the developmental costs of genomic tools decline, genomic approaches to non-model systems are becoming more feasible. Many of these systems may lack advanced genetic tools but are extremely valuable models in other biological fields. Here we report the development of expressed sequence tags (EST's) in an orthopteroid insect, a model for the study of neurobiology, speciation, and evolution. Results We report the sequencing of 14,502 EST's from clones derived from a nerve cord cDNA library, and the subsequent construction of a Gene Index from these sequences, from the Hawaiian trigonidiine cricket Laupala kohalensis. The Gene Index contains 8607 unique sequences comprised of 2575 tentative consensus (TC) sequences and 6032 singletons. For each of the unique sequences, an attempt was made to assign a provisional annotation and to categorize its function using a Gene Ontology-based classification through a sequence-based comparison to known proteins. In addition, a set of unique 70 base pair oligomers that can be used for DNA microarrays was developed. All Gene Index information is posted at the DFCI Gene Indices web page Conclusion Orthopterans are models used to understand the neurophysiological basis of complex motor patterns such as flight and stridulation. The sequences presented in the cricket Gene Index will provide neurophysiologists with many genetic tools that have been largely absent in this field. The cricket Gene Index is one of only two gene indices to be developed in an evolutionary model system. Species within the genus Laupala have speciated recently, rapidly, and extensively. Therefore, the genes identified in the cricket Gene Index can be used to study the genomics of speciation. Furthermore, this gene index represents a significant EST resources for basal insects. As such, this resource is a valuable comparative tool for the understanding of invertebrate molecular evolution. The sequences presented here will provide much needed genomic resources for three distinct but overlapping fields of inquiry: neurobiology, speciation, and molecular evolution. PMID:17459168

Population analysis of Vibrio parahaemolyticus originating from different geographical regions demonstrates a high genetic diversity.

PubMed

Urmersbach, Sara; Alter, Thomas; Koralage, Madura Sanjeevani Gonsal; Sperling, Lisa; Gerdts, Gunnar; Messelhäusser, Ute; Huehn, Stephan

2014-03-08

Vibrio parahaemolyticus is frequently isolated from environmental and seafood samples and associated with gastroenteritis outbreakes in American, European, Asian and African countries. To distinguish between different lineages of V. parahaemolyticus various genotyping techniques have been used, incl. multilocus sequence typing (MLST). Even though some studies have already applied MLST analysis to characterize V. parahaemolyticus strain sets, these studies have been restricted to specific geographical areas (e.g. U.S. coast, Thailand and Peru), have focused exclusively on pandemic or non-pandemic pathogenic isolates or have been based on a limited strain number. To generate a global picture of V. parahaemolyticus genotype distribution, a collection of 130 environmental and seafood related V. parahaemolyticus isolates of different geographical origins (Sri Lanka, Ecuador, North Sea and Baltic Sea as well as German retail) was subjected to MLST analysis after modification of gyrB and recA PCRs. The V. parahaemolyticus population was composed of 82 unique Sequence Types (STs), of which 68 (82.9%) were new to the pubMLST database. After translating the in-frame nucleotide sequences into amino acid sequences, less diversity was detectable: a total of 31 different peptide Sequence Types (pSTs) with 19 (61.3%) new pSTs were generated from the analyzed isolates. Most STs did not show a global dissemination, but some were supra-regionally distributed and clusters of STs were dependent on geographical origin. On peptide level no general clustering of strains from specific geographical regions was observed, thereby the most common pSTs were found on all continents (Asia, South America and Europe) and rare pSTs were restricted to distinct countries or even geographical regions. One lineage of pSTs associated only with strains from North and Baltic Sea strains was identified. Our study reveals a high genetic diversity in the analyzed V. parahaemolyticus strain set as well as for geographical strain subsets, with a high proportion of newly discovered alleles and STs. Differences between the subsets were identified. Our data support the postulated population structure of V. parahaemolyticus which follows the 'epidemic' model of clonal expansion. Application of peptide based AA-MLST allowed the identification of reliable relationships between strains.

Genetic differentiation of methicillin-resistant Staphylococcus aureus strains from Korea and Japan.

PubMed

Soo Ko, Kwan; Peck, Kyong Ran; Sup Oh, Won; Lee, Nam Yong; Hiramatsu, Keiichi; Song, Jae-Hoon

2005-01-01

In this study, we evaluated genetic differentiation between methicillin-resistant Staphylococcus aureus (MRSA) strains from Korea and Japan. Seventy-five MRSA strains, including 25 h VISA strains, were analyzed by molecular typing methods, including multilocus sequence typing (MLST), SCC mec typing, and spa typing. The most prevalent genotype of MRSA strains, in both Korea and Japan, was ST 5-MRSA-II with the DMGMK spa motif, characteristic of the New York/Japan MRSA clone. In spite of these common features in MRSA strains from Korea and Japan, we also observed some genotypic divergence in MRSA from the two countries. Several spa types might be differentiated from a prevalent prototype (TJMBMDMGMK) that is shared by the two countries, revealing a unique geographic distribution. SCC mec type II lacking pUB110, designated type IIA, was found more frequently in Korea than in Japan. The rate of gentamicin resistance was also dramatically different between the two countries: 87.2% (Korea) vs. 28.6% (Japan). These preliminary findings suggested that MRSA strains from Korea and Japan might have originated from a common ancestor, but then clearly differentiated according to locality. A further comprehensive study should be performed to document the hypotheses from this study.

Hydraulic fracturing and the Crooked Lake Sequences: Insights gleaned from regional seismic networks

NASA Astrophysics Data System (ADS)

Schultz, Ryan; Stern, Virginia; Novakovic, Mark; Atkinson, Gail; Gu, Yu Jeffrey

2015-04-01

Within central Alberta, Canada, a new sequence of earthquakes has been recognized as of 1 December 2013 in a region of previous seismic quiescence near Crooked Lake, ~30 km west of the town of Fox Creek. We utilize a cross-correlation detection algorithm to detect more than 160 events to the end of 2014, which is temporally distinguished into five subsequences. This observation is corroborated by the uniqueness of waveforms clustered by subsequence. The Crooked Lake Sequences have come under scrutiny due to its strong temporal correlation (>99.99%) to the timing of hydraulic fracturing operations in the Duvernay Formation. We assert that individual subsequences are related to fracturing stimulation and, despite adverse initial station geometry, double-difference techniques allow us to spatially relate each cluster back to a unique horizontal well. Overall, we find that seismicity in the Crooked Lake Sequences is consistent with first-order observations of hydraulic fracturing induced seismicity.

Cross-Species Analyses Identify the BNIP-2 and Cdc42GAP Homology (BCH) Domain as a Distinct Functional Subclass of the CRAL_TRIO/Sec14 Superfamily

PubMed Central

Gupta, Anjali Bansal; Wee, Liang En; Zhou, Yi Ting; Hortsch, Michael; Low, Boon Chuan

2012-01-01

The CRAL_TRIO protein domain, which is unique to the Sec14 protein superfamily, binds to a diverse set of small lipophilic ligands. Similar domains are found in a range of different proteins including neurofibromatosis type-1, a Ras GTPase-activating Protein (RasGAP) and Rho guanine nucleotide exchange factors (RhoGEFs). Proteins containing this structural protein domain exhibit a low sequence similarity and ligand specificity while maintaining an overall characteristic three-dimensional structure. We have previously demonstrated that the BNIP-2 and Cdc42GAP Homology (BCH) protein domain, which shares a low sequence homology with the CRAL_TRIO domain, can serve as a regulatory scaffold that binds to Rho, RhoGEFs and RhoGAPs to control various cell signalling processes. In this work, we investigate 175 BCH domain-containing proteins from a wide range of different organisms. A phylogenetic analysis with ∼100 CRAL_TRIO and similar domains from eight representative species indicates a clear distinction of BCH-containing proteins as a novel subclass within the CRAL_TRIO/Sec14 superfamily. BCH-containing proteins contain a hallmark sequence motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs (‘h’ is large and hydrophobic residue and ‘s’ is small and weekly polar residue) and can be further subdivided into three unique subtypes associated with BNIP-2-N, macro- and RhoGAP-type protein domains. A previously unknown group of genes encoding ‘BCH-only’ domains is also identified in plants and arthropod species. Based on an analysis of their gene-structure and their protein domain context we hypothesize that BCH domain-containing genes evolved through gene duplication, intron insertions and domain swapping events. Furthermore, we explore the point of divergence between BCH and CRAL-TRIO proteins in relation to their ability to bind small GTPases, GAPs and GEFs and lipid ligands. Our study suggests a need for a more extensive analysis of previously uncharacterized BCH, ‘BCH-like’ and CRAL_TRIO-containing proteins and their significance in regulating signaling events involving small GTPases. PMID:22479462

Diversity and Variation of Bacterial Community Revealed by MiSeq Sequencing in Chinese Dark Teas

PubMed Central

Fu, Jianyu; Lv, Haipeng; Chen, Feng

2016-01-01

Chinese dark teas (CDTs) are now among the popular tea beverages worldwide due to their unique health benefits. Because the production of CDTs involves fermentation that is characterized by the effect of microbes, microorganisms are believed to play critical roles in the determination of the chemical characteristics of CDTs. Some dominant fungi have been identified from CDTs. In contrast, little, if anything, is known about the composition of bacterial community in CDTs. This study was set to investigate the diversity and variation of bacterial community in four major types of CDTs from China. First, the composition of the bacterial community of CDTs was determined using MiSeq sequencing. From the four typical CDTs, a total of 238 genera that belong to 128 families of bacteria were detected, including most of the families of beneficial bacteria known to be associated with fermented food. While different types of CDTs had generally distinct bacterial structures, the two types of brick teas produced from adjacent regions displayed strong similarity in bacterial composition, suggesting that the producing environment and processing condition perhaps together influence bacterial succession in CDTs. The global characterization of bacterial communities in CDTs is an essential first step for us to understand their function in fermentation and their potential impact on human health. Such knowledge will be important guidance for improving the production of CDTs with higher quality and elevated health benefits. PMID:27690376

The Genetics of Symbiotic Nitrogen Fixation: Comparative Genomics of 14 Rhizobia Strains by Resolution of Protein Clusters

PubMed Central

Black, Michael; Moolhuijzen, Paula; Chapman, Brett; Barrero, Roberto; Howieson, John; Hungria, Mariangela; Bellgard, Matthew

2012-01-01

The symbiotic relationship between legumes and nitrogen fixing bacteria is critical for agriculture, as it may have profound impacts on lowering costs for farmers, on land sustainability, on soil quality, and on mitigation of greenhouse gas emissions. However, despite the importance of the symbioses to the global nitrogen cycling balance, very few rhizobial genomes have been sequenced so far, although there are some ongoing efforts in sequencing elite strains. In this study, the genomes of fourteen selected strains of the order Rhizobiales, all previously fully sequenced and annotated, were compared to assess differences between the strains and to investigate the feasibility of defining a core ‘symbiome’—the essential genes required by all rhizobia for nodulation and nitrogen fixation. Comparison of these whole genomes has revealed valuable information, such as several events of lateral gene transfer, particularly in the symbiotic plasmids and genomic islands that have contributed to a better understanding of the evolution of contrasting symbioses. Unique genes were also identified, as well as omissions of symbiotic genes that were expected to be found. Protein comparisons have also allowed the identification of a variety of similarities and differences in several groups of genes, including those involved in nodulation, nitrogen fixation, production of exopolysaccharides, Type I to Type VI secretion systems, among others, and identifying some key genes that could be related to host specificity and/or a better saprophytic ability. However, while several significant differences in the type and number of proteins were observed, the evidence presented suggests no simple core symbiome exists. A more abstract systems biology concept of nitrogen fixing symbiosis may be required. The results have also highlighted that comparative genomics represents a valuable tool for capturing specificities and generalities of each genome. PMID:24704847

Multiple cis-acting elements involved in up-regulation of a cytochrome P450 gene conferring resistance to deltamethrin in smal brown planthopper, Laodelphax striatellus (Fallén).

PubMed

Pu, Jian; Sun, Haina; Wang, Jinda; Wu, Min; Wang, Kangxu; Denholm, Ian; Han, Zhaojun

2016-11-01

As well as arising from single point mutations in binding sites or detoxifying enzymes, it is likely that insecticide resistance mechanisms are frequently controlled by multiple genetic factors, resulting in resistance being inherited as a quantitative trait. However, empirical evidence for this is still rare. Here we analyse the causes of up-regulation of CYP6FU1, a monoxygenase implicated in resistance to deltamethrin in the rice pest Laodelphax striatellus. The 5'-flanking region of this gene was cloned and sequenced from individuals of a susceptible and a resistant strain. A luminescent reporter assay was used to evaluate different 5'-flanking regions and their fragments for promoter activity. Mutations enhancing promoter activity in various fragments were characterized, singly and in combination, by site mutation recovery. Nucleotide diversity in flanking sequences was greatly reduced in deltamethrin-resistant insects compared to susceptible ones. Phylogenetic sequence analysis found that CYP6FU1 had five different types of 5'-flanking region. All five types were present in a susceptible strain but only a single type showing the highest promoter activity was present in a resistant strain. Four cis-acting elements were identified whose influence on up-regulation was much more pronounced in combination than when present singly. Of these, two were new transcription factor (TF) binding sites produced by mutations, another one was also a new TF binding site alternated from an existing one, and the fourth was a unique transcription start site. These results demonstrate that multiple cis-acting elements are involved in up-regulating CYP6FU1 to generate a resistance phenotype. Copyright © 2016 Elsevier Ltd. All rights reserved.

Truncating variants in the majority of the cytoplasmic domain of PCDH15 are unlikely to cause Usher syndrome 1F.

PubMed

Perreault-Micale, Cynthia; Frieden, Alexander; Kennedy, Caleb J; Neitzel, Dana; Sullivan, Jessica; Faulkner, Nicole; Hallam, Stephanie; Greger, Valerie

2014-11-01

Loss of function variants in the PCDH15 gene can cause Usher syndrome type 1F, an autosomal recessive disease associated with profound congenital hearing loss, vestibular dysfunction, and retinitis pigmentosa. The Ashkenazi Jewish population has an increased incidence of Usher syndrome type 1F (founder variant p.Arg245X accounts for 75% of alleles), yet the variant spectrum in a panethnic population remains undetermined. We sequenced the coding region and intron-exon borders of PCDH15 using next-generation DNA sequencing technology in approximately 14,000 patients from fertility clinics. More than 600 unique PCDH15 variants (single nucleotide changes and small indels) were identified, including previously described pathogenic variants p.Arg3X, p.Arg245X (five patients), p.Arg643X, p.Arg929X, and p.Arg1106X. Novel truncating variants were also found, including one in the N-terminal extracellular domain (p.Leu877X), but all other novel truncating variants clustered in the exon 33 encoded C-terminal cytoplasmic domain (52 patients, 14 variants). One variant was observed predominantly in African Americans (carrier frequency of 2.3%). The high incidence of truncating exon 33 variants indicates that they are unlikely to cause Usher syndrome type 1F even though many remove a large portion of the gene. They may be tolerated because PCDH15 has several alternate cytoplasmic domain exons and differentially spliced isoforms may function redundantly. Effects of some PCDH15 truncating variants were addressed by deep sequencing of a panethnic population. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Further characterization of a new recombinant group of Plum pox virus isolates, PPV-T, found in orchards in the Ankara province of Turkey.

PubMed

Serçe, Ciğdem Ulubaş; Candresse, Thierry; Svanella-Dumas, Laurence; Krizbai, Laszlo; Gazel, Mona; Cağlayan, Kadriye

2009-06-01

Sixteen Plum pox virus (PPV) isolates collected in the Ankara region of Turkey were analyzed using available serological and molecular typing assays. Surprisingly, despite the fact that all isolates except one, which was a mix infection, were typed as belonging to the PPV-M strain in four independent molecular assays, nine of them (60%) reacted with both PPV-M specific and PPV-D specific monoclonal antibodies. Partial 5' and 3' genomic sequence analysis on four isolates demonstrated that irrespective of their reactivity towards the PPV-D specific monoclonal antibody, they were all closely related to a recombinant PPV isolate from Turkey, Ab-Tk. All three isolates for which the relevant genomic sequence was obtained showed the same recombination event as Ab-Tk in the HC-Pro gene, around position 1566 of the genome. Complete genomic sequencing of Ab-Tk did not provide evidence for additional recombination events in its evolutionary history. Taken together, these results indicate that a group of closely related PPV isolates characterized by a unique recombination in the HC-Pro gene is prevalent under field conditions in the Ankara region of Turkey. Similar to the situation with the PPV-Rec strain, we propose that these isolates represent a novel strain of PPV, for which the name PPV-T (Turkey) is proposed. Given that PPV-T isolates cannot be identified by currently available typing techniques, it is possible that their presence has been overlooked in other situations. Further efforts should allow a precise description of their prevalence and of their geographical distribution in Turkey and, possibly, in other countries.

The Conformational Stability and Biophysical Properties of the Eukaryotic Thioredoxins of Pisum Sativum Are Not Family-Conserved

PubMed Central

Aguado-Llera, David; Martínez-Gómez, Ana Isabel; Prieto, Jesús; Marenchino, Marco; Traverso, José Angel; Gómez, Javier; Chueca, Ana; Neira, José L.

2011-01-01

Thioredoxins (TRXs) are ubiquitous proteins involved in redox processes. About forty genes encode TRX or TRX-related proteins in plants, grouped in different families according to their subcellular localization. For instance, the h-type TRXs are located in cytoplasm or mitochondria, whereas f-type TRXs have a plastidial origin, although both types of proteins have an eukaryotic origin as opposed to other TRXs. Herein, we study the conformational and the biophysical features of TRXh1, TRXh2 and TRXf from Pisum sativum. The modelled structures of the three proteins show the well-known TRX fold. While sharing similar pH-denaturations features, the chemical and thermal stabilities are different, being PsTRXh1 (Pisum sativum thioredoxin h1) the most stable isoform; moreover, the three proteins follow a three-state denaturation model, during the chemical-denaturations. These differences in the thermal- and chemical-denaturations result from changes, in a broad sense, of the several ASAs (accessible surface areas) of the proteins. Thus, although a strong relationship can be found between the primary amino acid sequence and the structure among TRXs, that between the residue sequence and the conformational stability and biophysical properties is not. We discuss how these differences in the biophysical properties of TRXs determine their unique functions in pea, and we show how residues involved in the biophysical features described (pH-titrations, dimerizations and chemical-denaturations) belong to regions involved in interaction with other proteins. Our results suggest that the sequence demands of protein-protein function are relatively rigid, with different protein-binding pockets (some in common) for each of the three proteins, but the demands of structure and conformational stability per se (as long as there is a maintained core), are less so. PMID:21364950

Multilocus Sequence Typing of Pathogenic Treponemes Isolated from Cloven-Hoofed Animals and Comparison to Treponemes Isolated from Humans

PubMed Central

Carter, Stuart D.; Birtles, Richard J.; Brown, Jennifer M.; Hart, C. Anthony; Evans, Nicholas J.

2016-01-01

ABSTRACT Treponema species are implicated in many diseases of humans and animals. Digital dermatitis (DD) treponemes are reported to cause severe lesions in cattle, sheep, pigs, goats, and wild elk, causing substantial global animal welfare issues and economic losses. The fastidiousness of these spirochetes has previously precluded studies investigating within-phylogroup genetic diversity. An archive of treponemes that we isolated enabled multilocus sequence typing to quantify the diversity and population structure of DD treponemes. Isolates (n = 121) were obtained from different animal hosts in nine countries on three continents. The analyses herein of currently isolated DD treponemes at seven housekeeping gene loci confirm the classification of the three previously designated phylogroups: the Treponema medium, Treponema phagedenis, and Treponema pedis phylogroups. Sequence analysis of seven DD treponeme housekeeping genes revealed a generally low level of diversity among the strains within each phylogroup, removing the need for the previously used “-like” suffix. Surprisingly, all isolates within each phylogroup clustered together, regardless of host or geographic origin, suggesting that the same sequence types (STs) can infect different animals. Some STs were derived from multiple animals from the same farm, highlighting probable within-farm transmissions. Several STs infected multiple hosts from similar geographic regions, identifying probable frequent between-host transmissions. Interestingly, T. pedis appears to be evolving more quickly than the T. medium or T. phagedenis DD treponeme phylogroup, by forming two unique ST complexes. The lack of phylogenetic discrimination between treponemes isolated from different hosts or geographic regions substantially contrasts with the data for other clinically relevant spirochetes. IMPORTANCE The recent expansion of the host range of digital dermatitis (DD) treponemes from cattle to sheep, goats, pigs, and wild elk, coupled with the high level of 16S rRNA gene sequence similarity across hosts and with human treponemes, suggests that the same bacterial species can cause disease in multiple different hosts. This multilocus sequence typing (MLST) study further demonstrates that these bacteria isolated from different hosts are indeed very similar, raising the potential for cross-species transmission. The study also shows that infection spread occurs frequently, both locally and globally, suggesting transmission by routes other than animal-animal transmission alone. These results indicate that on-farm biosecurity is important for controlling disease spread in domesticated species. Continued surveillance and vigilance are important for ascertaining the evolution and tracking any further host range expansion of these important pathogens. PMID:27208135

Multilocus Sequence Typing of Pathogenic Treponemes Isolated from Cloven-Hoofed Animals and Comparison to Treponemes Isolated from Humans.

PubMed

Clegg, Simon R; Carter, Stuart D; Birtles, Richard J; Brown, Jennifer M; Hart, C Anthony; Evans, Nicholas J

2016-08-01

Treponema species are implicated in many diseases of humans and animals. Digital dermatitis (DD) treponemes are reported to cause severe lesions in cattle, sheep, pigs, goats, and wild elk, causing substantial global animal welfare issues and economic losses. The fastidiousness of these spirochetes has previously precluded studies investigating within-phylogroup genetic diversity. An archive of treponemes that we isolated enabled multilocus sequence typing to quantify the diversity and population structure of DD treponemes. Isolates (n = 121) were obtained from different animal hosts in nine countries on three continents. The analyses herein of currently isolated DD treponemes at seven housekeeping gene loci confirm the classification of the three previously designated phylogroups: the Treponema medium, Treponema phagedenis, and Treponema pedis phylogroups. Sequence analysis of seven DD treponeme housekeeping genes revealed a generally low level of diversity among the strains within each phylogroup, removing the need for the previously used "-like" suffix. Surprisingly, all isolates within each phylogroup clustered together, regardless of host or geographic origin, suggesting that the same sequence types (STs) can infect different animals. Some STs were derived from multiple animals from the same farm, highlighting probable within-farm transmissions. Several STs infected multiple hosts from similar geographic regions, identifying probable frequent between-host transmissions. Interestingly, T. pedis appears to be evolving more quickly than the T. medium or T. phagedenis DD treponeme phylogroup, by forming two unique ST complexes. The lack of phylogenetic discrimination between treponemes isolated from different hosts or geographic regions substantially contrasts with the data for other clinically relevant spirochetes. The recent expansion of the host range of digital dermatitis (DD) treponemes from cattle to sheep, goats, pigs, and wild elk, coupled with the high level of 16S rRNA gene sequence similarity across hosts and with human treponemes, suggests that the same bacterial species can cause disease in multiple different hosts. This multilocus sequence typing (MLST) study further demonstrates that these bacteria isolated from different hosts are indeed very similar, raising the potential for cross-species transmission. The study also shows that infection spread occurs frequently, both locally and globally, suggesting transmission by routes other than animal-animal transmission alone. These results indicate that on-farm biosecurity is important for controlling disease spread in domesticated species. Continued surveillance and vigilance are important for ascertaining the evolution and tracking any further host range expansion of these important pathogens. Copyright © 2016 Clegg et al.

High-throughput T-cell receptor sequencing across chronic liver diseases reveals distinct disease-associated repertoires.

PubMed

Liaskou, Evaggelia; Klemsdal Henriksen, Eva Kristine; Holm, Kristian; Kaveh, Fatemeh; Hamm, David; Fear, Janine; Viken, Marte K; Hov, Johannes Roksund; Melum, Espen; Robins, Harlan; Olweus, Johanna; Karlsen, Tom H; Hirschfield, Gideon M

2016-05-01

Hepatic T-cell infiltrates and a strong genetic human leukocyte antigen association represent characteristic features of various immune-mediated liver diseases. Conceptually the presence of disease-associated antigens is predicted to be reflected in T-cell receptor (TCR) repertoires. Here, we aimed to determine if disease-associated TCRs could be identified in the nonviral chronic liver diseases primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and alcoholic liver disease (ALD). We performed high-throughput sequencing of the TCRβ chain complementarity-determining region 3 of liver-infiltrating T cells from PSC (n = 20), PBC (n = 10), and ALD (n = 10) patients, alongside genomic human leukocyte antigen typing. The frequency of TCRβ nucleotide sequences was significantly higher in PSC samples (2.53 ± 0.80, mean ± standard error of the mean) compared to PBC samples (1.13 ± 0.17, P < 0.0001) and ALD samples (0.62 ± 0.10, P < 0.0001). An average clonotype overlap of 0.85% was detected among PSC samples, significantly higher compared to the average overlap of 0.77% seen within the PBC (P = 0.024) and ALD groups (0.40%, P < 0.0001). From eight to 42 clonotypes were uniquely detected in each of the three disease groups (≥30% of the respective patient samples). Multiple, unique sequences using different variable family genes encoded the same amino acid clonotypes, providing additional support for antigen-driven selection. In PSC and PBC, disease-associated clonotypes were detected among patients with human leukocyte antigen susceptibility alleles. We demonstrate liver-infiltrating disease-associated clonotypes in all three diseases evaluated, and evidence for antigen-driven clonal expansions. Our findings indicate that differential TCR signatures, as determined by high-throughput sequencing, may represent an imprint of distinctive antigenic repertoires present in the different chronic liver diseases; this thereby opens up the prospect of studying disease-relevant T cells in order to better understand and treat liver disease. © 2015 by the American Association for the Study of Liver Diseases.

Brucella papionis sp. nov., isolated from baboons (Papio spp.)

PubMed Central

Davison, Nicholas; Cloeckaert, Axel; Al Dahouk, Sascha; Zygmunt, Michel S.; Brew, Simon D.; Perrett, Lorraine L.; Koylass, Mark S.; Vergnaud, Gilles; Quance, Christine; Scholz, Holger C.; Dick, Edward J.; Hubbard, Gene; Schlabritz-Loutsevitch, Natalia E.

2014-01-01

Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60T and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60T and F8/08-61 could be distinguished clearly from all known species of the genus Brucellaand their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucellasuggested by the ICSP Subcommittee on the Taxonomy of Brucella, they represent a novel species within the genus Brucella, for which the name Brucella papionis sp. nov. is proposed, with the type strain F8/08-60T ( = NCTC 13660T = CIRMBP 0958T). PMID:25242540

Genomic Diversification in Strains of Rickettsia felis Isolated from Different Arthropods

PubMed Central

Gillespie, Joseph J.; Driscoll, Timothy P.; Verhoeve, Victoria I.; Utsuki, Tadanobu; Husseneder, Claudia; Chouljenko, Vladimir N.; Azad, Abdu F.; Macaluso, Kevin R.

2015-01-01

Rickettsia felis (Alphaproteobacteria: Rickettsiales) is the causative agent of an emerging flea-borne rickettsiosis with worldwide occurrence. Originally described from the cat flea, Ctenocephalides felis, recent reports have identified R. felis from other flea species, as well as other insects and ticks. This diverse host range for R. felis may indicate an underlying genetic variability associated with host-specific strains. Accordingly, to determine a potential genetic basis for host specialization, we sequenced the genome of R. felis str. LSU-Lb, which is an obligate mutualist of the parthenogenic booklouse Liposcelis bostrychophila (Insecta: Psocoptera). We also sequenced the genome of R. felis str. LSU, the second genome sequence for cat flea-associated strains (cf. R. felis str. URRWXCal2), which are presumably facultative parasites of fleas. Phylogenomics analysis revealed R. felis str. LSU-Lb diverged from the flea-associated strains. Unexpectedly, R. felis str. LSU was found to be divergent from R. felis str. URRWXCal2, despite sharing similar hosts. Although all three R. felis genomes contain the pRF plasmid, R. felis str. LSU-Lb carries an additional unique plasmid, pLbaR (plasmid of L. bostrychophila associated Rickettsia), nearly half of which encodes a unique 23-gene integrative conjugative element. Remarkably, pLbaR also encodes a repeats-in-toxin-like type I secretion system and associated toxin, heretofore unknown from other Rickettsiales genomes, which likely originated from lateral gene transfer with another obligate intracellular parasite of arthropods, Cardinium (Bacteroidetes). Collectively, our study reveals unexpected genomic diversity across three R. felis strains and identifies several diversifying factors that differentiate facultative parasites of fleas from obligate mutualists of booklice. PMID:25477419

Molecular Analysis of an Outbreak of Lethal Postpartum Sepsis Caused by Streptococcus pyogenes

PubMed Central

Turner, Claire E.; Dryden, Matthew; Holden, Matthew T. G.; Davies, Frances J.; Lawrenson, Richard A.; Farzaneh, Leili; Bentley, Stephen D.; Efstratiou, Androulla

2013-01-01

Sepsis is now the leading direct cause of maternal death in the United Kingdom, and Streptococcus pyogenes is the leading pathogen. We combined conventional and genomic analyses to define the duration and scale of a lethal outbreak. Two postpartum deaths caused by S. pyogenes occurred within 24 h; one was characterized by bacteremia and shock and the other by hemorrhagic pneumonia. The women gave birth within minutes of each other in the same maternity unit 2 days earlier. Seven additional infections in health care and household contacts were subsequently detected and treated. All cluster-associated S. pyogenes isolates were genotype emm1 and were initially indistinguishable from other United Kingdom emm1 isolates. Sequencing of the virulence gene sic revealed that all outbreak isolates had the same unique sic type. Genome sequencing confirmed that the cluster was caused by a unique S. pyogenes clone. Transmission between patients occurred on a single day and was associated with casual contact only. A single isolate from one patient demonstrated a sequence change in sic consistent with longer infection duration. Transmission to health care workers was traced to single clinical contacts with index cases. The last case was detected 18 days after the first case. Following enhanced surveillance, the outbreak isolate was not detected again. Mutations in bacterial regulatory genes played no detectable role in this outbreak, illustrating the intrinsic ability of emm1 S. pyogenes to spread while retaining virulence. This fast-moving outbreak highlights the potential of S. pyogenes to cause a range of diseases in the puerperium with rapid transmission, underlining the importance of immediate recognition and response by clinical infection and occupational health teams. PMID:23616448

De novo assembly and characterization of Muscovy duck liver transcriptome and analysis of differentially regulated genes in response to heat stress.

PubMed

Zeng, Tao; Zhang, Liping; Li, Jinjun; Wang, Deqian; Tian, Yong; Lu, Lizhi

2015-05-01

High temperature is a major abiotic stress limiting animal growth and productivity worldwide. The Muscovy duck (Cairina moschata), sometimes called the Barbary drake, is a type of duck with a fairly unusual domestication history. In Southeast Asia, duck meat is one of the top meats consumed, and as such, the production of the meat is an important topic of research. The transcriptomic and genomic data presently available are insufficient to understanding the molecular mechanism underlying the heat tolerance of Muscovy ducks. Thus, transcriptome and expression profiling data for this species are required as important resource for identifying genes and developing molecular marker. In this study, de novo transcriptome assembly and gene expression analysis using Illumina sequencing technology were performed. More than 225 million clean reads were generated and assembled into 36,903 unique transcripts with an average length of 1,135 bp. A total of 21,221 (57.50 %) unigenes were annotated. Gene Ontology (GO) analysis of the annotated unigenes revealed that the majority of sequenced genes were associated with transcription, signal transduction, and apoptosis. We also performed gene expression profiling analysis upon heat treatment in Muscovy ducks and identified 470 heat-response unique transcripts. GO term enrichment showed that protein folding and chaperone binding were significant enrichment, whereas KEGG pathway analyses showed that Ras and MAPKs were activated after heat stress in Muscovy ducks. Our research enriched sequences information of Muscovy duck, provided novel insights into responses to heat stress in these ducks, and serve as candidate genes or markers that can be used to guide future efforts to breed heat-tolerant duck strains.

Generation and analysis of expressed sequence tags from a cDNA library of the fruiting body of Ganoderma lucidum

PubMed Central

2010-01-01

Background Little genomic or trancriptomic information on Ganoderma lucidum (Lingzhi) is known. This study aims to discover the transcripts involved in secondary metabolite biosynthesis and developmental regulation of G. lucidum using an expressed sequence tag (EST) library. Methods A cDNA library was constructed from the G. lucidum fruiting body. Its high-quality ESTs were assembled into unique sequences with contigs and singletons. The unique sequences were annotated according to sequence similarities to genes or proteins available in public databases. The detection of simple sequence repeats (SSRs) was preformed by online analysis. Results A total of 1,023 clones were randomly selected from the G. lucidum library and sequenced, yielding 879 high-quality ESTs. These ESTs showed similarities to a diverse range of genes. The sequences encoding squalene epoxidase (SE) and farnesyl-diphosphate synthase (FPS) were identified in this EST collection. Several candidate genes, such as hydrophobin, MOB2, profilin and PHO84 were detected for the first time in G. lucidum. Thirteen (13) potential SSR-motif microsatellite loci were also identified. Conclusion The present study demonstrates a successful application of EST analysis in the discovery of transcripts involved in the secondary metabolite biosynthesis and the developmental regulation of G. lucidum. PMID:20230644

Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations.

PubMed

Lathe, R

1985-05-05

Synthetic probes deduced from amino acid sequence data are widely used to detect cognate coding sequences in libraries of cloned DNA segments. The redundancy of the genetic code dictates that a choice must be made between (1) a mixture of probes reflecting all codon combinations, and (2) a single longer "optimal" probe. The second strategy is examined in detail. The frequency of sequences matching a given probe by chance alone can be determined and also the frequency of sequences closely resembling the probe and contributing to the hybridization background. Gene banks cannot be treated as random associations of the four nucleotides, and probe sequences deduced from amino acid sequence data occur more often than predicted by chance alone. Probe lengths must be increased to confer the necessary specificity. Examination of hybrids formed between unique homologous probes and their cognate targets reveals that short stretches of perfect homology occurring by chance make a significant contribution to the hybridization background. Statistical methods for improving homology are examined, taking human coding sequences as an example, and considerations of codon utilization and dinucleotide frequencies yield an overall homology of greater than 82%. Recommendations for probe design and hybridization are presented, and the choice between using multiple probes reflecting all codon possibilities and a unique optimal probe is discussed.

Complete Genome Sequence of Sporisorium scitamineum and Biotrophic Interaction Transcriptome with Sugarcane

PubMed Central

Benevenuto, Juliana; Peters, Leila P.; Carvalho, Giselle; Palhares, Alessandra; Quecine, Maria C.; Nunes, Filipe R. S.; Kmit, Maria C. P.; Wai, Alvan; Hausner, Georg; Aitken, Karen S.; Berkman, Paul J.; Fraser, James A.; Moolhuijzen, Paula M.; Coutinho, Luiz L.; Creste, Silvana; Vieira, Maria L. C.; Kitajima, João P.; Monteiro-Vitorello, Claudia B.

2015-01-01

Sporisorium scitamineum is a biotrophic fungus responsible for the sugarcane smut, a worldwide spread disease. This study provides the complete sequence of individual chromosomes of S. scitamineum from telomere to telomere achieved by a combination of PacBio long reads and Illumina short reads sequence data, as well as a draft sequence of a second fungal strain. Comparative analysis to previous available sequences of another strain detected few polymorphisms among the three genomes. The novel complete sequence described herein allowed us to identify and annotate extended subtelomeric regions, repetitive elements and the mitochondrial DNA sequence. The genome comprises 19,979,571 bases, 6,677 genes encoding proteins, 111 tRNAs and 3 assembled copies of rDNA, out of our estimated number of copies as 130. Chromosomal reorganizations were detected when comparing to sequences of S. reilianum, the closest smut relative, potentially influenced by repeats of transposable elements. Repetitive elements may have also directed the linkage of the two mating-type loci. The fungal transcriptome profiling from in vitro and from interaction with sugarcane at two time points (early infection and whip emergence) revealed that 13.5% of the genes were differentially expressed in planta and particular to each developmental stage. Among them are plant cell wall degrading enzymes, proteases, lipases, chitin modification and lignin degradation enzymes, sugar transporters and transcriptional factors. The fungus also modulates transcription of genes related to surviving against reactive oxygen species and other toxic metabolites produced by the plant. Previously described effectors in smut/plant interactions were detected but some new candidates are proposed. Ten genomic islands harboring some of the candidate genes unique to S. scitamineum were expressed only in planta. RNAseq data was also used to reassure gene predictions. PMID:26065709

Discovery of Escherichia coli CRISPR sequences in an undergraduate laboratory.

PubMed

Militello, Kevin T; Lazatin, Justine C

2017-05-01

Clustered regularly interspaced short palindromic repeats (CRISPRs) represent a novel type of adaptive immune system found in eubacteria and archaebacteria. CRISPRs have recently generated a lot of attention due to their unique ability to catalog foreign nucleic acids, their ability to destroy foreign nucleic acids in a mechanism that shares some similarity to RNA interference, and the ability to utilize reconstituted CRISPR systems for genome editing in numerous organisms. In order to introduce CRISPR biology into an undergraduate upper-level laboratory, a five-week set of exercises was designed to allow students to examine the CRISPR status of uncharacterized Escherichia coli strains and to allow the discovery of new repeats and spacers. Students started the project by isolating genomic DNA from E. coli and amplifying the iap CRISPR locus using the polymerase chain reaction (PCR). The PCR products were analyzed by Sanger DNA sequencing, and the sequences were examined for the presence of CRISPR repeat sequences. The regions between the repeats, the spacers, were extracted and analyzed with BLASTN searches. Overall, CRISPR loci were sequenced from several previously uncharacterized E. coli strains and one E. coli K-12 strain. Sanger DNA sequencing resulted in the discovery of 36 spacer sequences and their corresponding surrounding repeat sequences. Five of the spacers were homologous to foreign (non-E. coli) DNA. Assessment of the laboratory indicates that improvements were made in the ability of students to answer questions relating to the structure and function of CRISPRs. Future directions of the laboratory are presented and discussed. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(3):262-269, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.

Rotary pin-in-maze discriminator

DOEpatents

Benavides, Gilbert L.

1997-01-01

A discriminator apparatus and method that discriminates between a unique signal and any other (incorrect) signal. The unique signal is a sequence of events; each event can assume one of two possible event states. Given the unique signal, a maze wheel is allowed to rotate fully in one direction. Given an incorrect signal, both the maze wheel and a pin wheel lock in position.

Direct typing of Canine parvovirus (CPV) from infected dog faeces by rapid mini sequencing technique.

PubMed

V, Pavana Jyothi; S, Akila; Selvan, Malini K; Naidu, Hariprasad; Raghunathan, Shwethaa; Kota, Sathish; Sundaram, R C Raja; Rana, Samir Kumar; Raj, G Dhinakar; Srinivasan, V A; Mohana Subramanian, B

2016-12-01

Canine parvovirus (CPV) is a non-enveloped single stranded DNA virus with an icosahedral capsid. Mini-sequencing based CPV typing was developed earlier to detect and differentiate all the CPV types and FPV in a single reaction. This technique was further evaluated in the present study by performing the mini-sequencing directly from fecal samples which avoided tedious virus isolation steps by cell culture system. Fecal swab samples were collected from 84 dogs with enteritis symptoms, suggestive of parvoviral infection from different locations across India. Seventy six of these samples were positive by PCR; the subsequent mini-sequencing reaction typed 74 of them as type 2a virus, and 2 samples as type 2b. Additionally, 25 of the positive samples were typed by cycle sequencing of PCR products. Direct CPV typing from fecal samples using mini-sequencing showed 100% correlation with CPV typing by cycle sequencing. Moreover, CPV typing was achieved by mini-sequencing even with faintly positive PCR amplicons which was not possible by cycle sequencing. Therefore, the mini-sequencing technique is recommended for regular epidemiological follow up of CPV types, since the technique is rapid, highly sensitive and high capacity method for CPV typing. Copyright © 2016. Published by Elsevier B.V.