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Sample records for esox lucius full-length

  1. Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome

    PubMed Central

    2010-01-01

    Background Salmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. This duplication greatly impacts species diversification, functional specialization, and adaptation. Extensive new genomic resources have recently become available for Atlantic salmon (Salmo salar), but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution. Results From existing expressed sequence tag (EST) resources and three new full-length cDNA libraries, 9,057 reference quality full-length gene insert clones were identified for Atlantic salmon. A further 1,365 reference full-length clones were annotated from 29,221 northern pike (Esox lucius) ESTs. Pairwise dN/dS comparisons within each of 408 sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates. Conclusions 9,057 full-length reference genes were characterized in S. salar and can be used to identify alleles and gene family members. Comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. In addition, there is evidence that evolution has acted asymmetrically on paralogs, allowing one of the pair to diverge at a faster rate. PMID:20433749

  2. Early life migration patterns of Baltic Sea pike Esox lucius.

    PubMed

    Rohtla, M; Vetemaa, M; Urtson, K; Soesoo, A

    2012-04-01

    This study investigated the movement patterns of Baltic Sea pike Esox lucius in Matsalu Bay, Estonia, using otolith microchemistry. Migration patterns of E. lucius were remarkably diverse, but distinct groups were evident. Of the E. lucius analysed (n = 28), 82% hatched in fresh water and 74% of them left this biotope during the first growth season.

  3. Prey capture of pike Esox lucius larvae in turbid water.

    PubMed

    Salonen, M; Engström-Ost, J

    2010-06-01

    Pike Esox lucius larvae captured fewer calanoid and cyclopoid copepods in turbid than in clear water, whereas no differences were detected in feeding rates on Daphnia longispina. Decreased capture of copepods may lead to lower growth and survival of E. lucius larvae in turbid areas, in particular, if cladocerans are scarce.

  4. Trophic flexibility and opportunism in pike Esox lucius.

    PubMed

    Pedreschi, D; Mariani, S; Coughlan, J; Voigt, C C; O'Grady, M; Caffrey, J; Kelly-Quinn, M

    2015-10-01

    The first comprehensive investigation of pike Esox lucius trophic ecology in a region (Ireland) where they have long been thought to be a non-native species is presented. Diet was investigated across habitat types (lake, river and canal) through the combined methods of stable-isotope and stomach content analyses. Variations in niche size, specialization and the timing of the ontogenetic dietary switch were examined, revealing pronounced opportunism and feeding plasticity in E. lucius, along with a high occurrence of invertivory (up to 60 cm fork length, LF ) and a concomitant delayed switch to piscivory. Furthermore, E. lucius were found to primarily prey upon the highly available non-native roach Rutilus rutilus, which may alleviate predation pressure on brown trout Salmo trutta, highlighting the complexity of dynamic systems and the essential role of research in informing effective management.

  5. Morphological study of the northern pike (Esox lucius) tongue.

    PubMed

    Sadeghinezhad, Javad; Rahmati-holasoo, Hooman; Fayyaz, Sahel; Zargar, Ashkan

    2015-09-01

    The northern pike (Esox lucius) is a fresh water species belonging to the Esocidae family. It is a carnivorous fish feeding mostly on invertebrates and fishes. Due to the scantiness of relevant literature regarding the morphology of the tongue in fish we carried out this study with the aim of providing information on the dorsal surface morphology and histological structures of the tongue in E. lucius. The tongues of five E. lucius were examined using light- and scanning electron- microscopy (SEM) techniques. The SEM studies revealed the presence of numerous teeth, longitudinal mucosal strands and scattered taste buds spread on the tongue surface. Histological studies using hematoxylin and eosin and Masson's trichrome staining showed that the musculature was not visible in the tongue of E. lucius. The tongue is composed of mucosa, and submucosa supported by osteocartilagionous skeleton. The mucosa consists of several layers of unicellular mucous cells interrupted by numerous teeth. The derivation of teeth from the underlying bronchial skeleton was visible in longitudinal section. The scattered taste buds with a typical onion shape were also present. Overall, the morphological features of the E. lucius tongue together suggested its mechanical and sensory roles. The findings of this study together with morphological and physiological data from other fishes contribute to the knowledge of the nutrition and feeding behavior in aquaculture species.

  6. Northern pike (Esox lucius) collagen: Extraction, characterization and potential application.

    PubMed

    Kozlowska, J; Sionkowska, A; Skopinska-Wisniewska, J; Piechowicz, K

    2015-11-01

    Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the scales of northern pike (Esox lucius) were extracted and characterized. It was the first time that this species was used as sources of collagen. FT-IR and amino acid analysis results revealed the presence of collagen. Glycine accounts for one-third of its amino acid residues and specific for collagen amino acid - hydroxyproline - is present in isolated protein. The content of imino acid: proline and hydroxyproline in ASC and PSC was similar (12.5% Pro and 6.5% Hyp). Both ASC and PSC were type I collagen. The denaturation temperature of ASC and PSC were 28.5 and 27°C, respectively. Thin collagen films were obtained by casting of collagen solution onto glass plates. The surface properties of ASC and PSC films were different - the surface of ASC collagen film was more polar and less rough than PSC and we can observe the formation of collagen fibrils after solvent evaporation. ASC films showed much higher tensile properties than PSC. The obtained results suggest that northern pike scales have potential as an alternative source of collagen for use in various fields.

  7. Ovarian alterations in wild northern pike Esox lucius females.

    PubMed

    Zarski, Daniel; Rechulicz, Jacek; Krejszeff, Sławomir; Czarkowski, Tomasz K; Stańczak, Katarzyna; Palińska, Katarzyna; Gryzińska, Magdalena; Targońska, Katarzyna; Kozłowski, Krzysztof; Mamcarz, Andrzej; Hliwa, Piotr

    2013-09-24

    The aim of the present study was to analyse the occurrence of macroscopically visible ovary alterations in 2 populations of northern pike Esox lucius L. originating from lakes in the Mazurian Lake District (NE Poland). The alterations were characterised by ovary tissue that was morphologically malformed, in part or in whole, and contained immature oocytes, i.e. trophoplastic or previtellogenic oocytes instead of vitellogenic oocytes. These alterations were found only in the ovaries, and no morphological alterations of the testes were noted. Macroscopic and histological analyses were carried out in order to classify the observed alterations in the ovaries. Three types of alterations were identified in which morphological malformations as well as histological investigation of the ovaries were considered. An analysis of the size and age of the fish in relation to the occurrence of alterations as well as of the macroscopic and histological nature of the alteration types was made. The data obtained revealed no lake or age dependency of the observed alterations. Based on the results obtained, we suggest that the presence of endocrine disruptors in the environment or/and genetic factors could be responsible for these kinds of gonad anomalies. However, our results did not allow us to determine the aetiology of the alterations.

  8. Mercury elimination by a top predator, Esox lucius.

    PubMed

    Van Walleghem, Jillian L A; Blanchfield, Paul J; Hrenchuk, Lee E; Hintelmann, Holger

    2013-05-07

    Top-level piscivores are highly sought after for consumption in freshwater fisheries, yet these species contain the highest levels of the neurotoxin monomethylmercury (MMHg) and therefore present the greatest concern for MMHg exposure to humans. The slow elimination of MMHg is one factor that contributes to high levels of this contaminant in fish; however, little quantitative information exists on elimination rates by top predators in nature. We determined rates of MMHg elimination in northern pike (Esox lucius) by transferring fish that had naturally accumulated isotope-enriched MMHg (spike MMHg) through a whole-lake Hg loading study to a different lake. Over a period of ~7 y, pike were periodically recaptured and a small amount of muscle tissue was extracted using a nonlethal biopsy. Spike total mercury (THg) persisted in muscle tissue throughout the entire study despite discontinuing exposure upon transfer to the new lake. Spike THg burdens increased for the first ~460 d, followed by a decline to 65% of original burden levels over the next 200 d, and subsequently reached a plateau near original burden levels for the remainder of the study. We estimated the half-life of muscle THg to be 3.3 y (1193 d), roughly 1.2- to 2.7-fold slower than predicted by current elimination models. We advocate for further long-term field studies that examine kinetics of MMHg in fish to better inform predictive models estimating the recovery of MMHg-contaminated fisheries.

  9. Foraging success of juvenile pike Esox lucius depends on visual conditions and prey pigmentation.

    PubMed

    Jönsson, M; Hylander, S; Ranåker, L; Nilsson, P A; Brönmark, C

    2011-07-01

    Young-of-the-year pike Esox lucius foraging on copepods experienced different foraging success depending on prey pigmentation in water visually degraded by brown colouration or algae. Both attack rate and prey consumption rate were higher for E. lucius foraging on transparent prey in brown water, whereas the opposite was true in algal turbid water. Pigments in copepod prey may have a cryptic function in brown water instead of a photo-protective function even if prey-size selectivity was stronger than selection based on pigmentation in juvenile E. lucius.

  10. Mixed mesodermal and mesenchymal origin of myotomal muscles in pike (Esox lucius: Teleostei).

    PubMed

    Kacperczyk, A; Daczewska, M

    2006-02-01

    During the myotomal myogenesis in pike (Esox lucius) two phases of muscle differentiation can be distinguished. In the first phase, the somite cells-derived stock, the primary myoblasts (of mesodermal origin), fuse to form multinucleate myotubes. Participation of myotomal cells of mesodermal origin is insufficient for further muscle development. In the second stage mesenchymal cells migrate, via myosepts, into the myotome between myotubes. Immunocytochemical detection of proliferating cell nuclear antigen (marker of S phase of cell cycle) showed their mitotic activity. Transmission electron microscope analysis revealed that the differentiation of these cells depends on their position. Cells remaining in the myosepts develop into fibroblasts and produce collagen fibres, while those that have migrated into the myotomes transform into secondary myoblasts. Mesenchymal cells in the studied species are believed to participate in hypertrophy and hyperplasy of muscle fibres. Thus the muscle fibres in pike (E. lucius) are of mesodermal-mesenchymal origin.

  11. Rhipidocotyle fennica (Digenea: Bucephalidae) from Anodonta anatina and pike Esox lucius in Lithuania.

    PubMed

    Stunžėnas, Virmantas; Petkevičiūtė, Romualda; Stanevičiūtė, Gražina; Binkienė, Rasa

    2014-10-01

    Ribosomal DNA sequences of Rhipidocotyle sp. adults from Esox lucius were shown to be identical to sequences of larval Rhipidocotyle fennica, occurring in Anodonta anatina in Lake Vilkokšnis, Lithuania. Morphological features and host specificity of this adult worm correspond with that, determinate in the first description of R. fennica in Finland. These data give the first evidence that a viable population of R. fennica exists in east central Europe. Bucephalus polymorphus which was reported in unionids in all previous publications is probably R. fennica.

  12. Pike Esox lucius as an emerging model organism for studies in ecology and evolutionary biology: a review.

    PubMed

    Forsman, A; Tibblin, P; Berggren, H; Nordahl, O; Koch-Schmidt, P; Larsson, P

    2015-08-01

    The pike Esox lucius is a large, long-lived, iteroparous, top- predator fish species with a circumpolar distribution that occupies a broad range of aquatic environments. This study reports on a literature search and demonstrates that the publication rate of E. lucius research increases both in absolute terms and relative to total scientific output, and that the focus of investigation has changed over time from being dominated by studies on physiology and disease to being gradually replaced by studies on ecology and evolution. Esox lucius can be exploited as a model in future research for identifying causes and consequences of phenotypic and genetic variation at the levels of individuals, populations and species as well as for investigating community processes.

  13. Mechanical suppression of northern pike (Esox lucius) populations in small Arizona reservoirs

    USGS Publications Warehouse

    Kuzmenko, Yuliya; Spesiviy, Timofy; Bonar, Scott A.

    2010-01-01

    Introduced populations of northern pike Esox lucius have provided angling opportunities in the western United States (McMahon and Bennett 1996). However, the northern pike is a voracious piscivore and its large size, high fecundity, and broad physiological tolerance make it capable of drastically altering ecosystems it invades (Marchetti et al. 2004). Indeed, predation by northern pike has been shown to significantly alter fish community structure and put native fishes at a higher extinction risk (He and Kitchell 1990, Findlay et al. 2000). Predation by northern pike is viewed as a significant threat to native stocks of salmonids in Washington, British Columbia, and California (McMahon and Bennett 1996, California Department of Fish and Game [CDFG] 2003).

  14. Mercury elimination rates for adult northern pike Esox lucius: evidence for a sex effect

    USGS Publications Warehouse

    Madenjian, Charles P.; Blanchfield, Paul J.; Hrenchuk, Lee E.; Van Walleghem, Jillian L. A.

    2014-01-01

    We examined the effect of sex on mercury elimination in fish by monitoring isotope-enriched mercury concentrations in the muscle tissue of three adult female and three adult male northern pike Esox lucius, which had accumulated the isotope-enriched mercury via a whole-lake manipulation and were subsequently moved to a clean lake. Mercury elimination rates for female and male northern pike were estimated to be 0.00034 and 0.00073 day−1, respectively. Thus, males were capable of eliminating mercury at more than double the rate than that of females. To the best of our knowledge, our study represents the first documentation of mercury elimination rates varying between the sexes of fish. This sex difference in elimination rates should be taken into account when comparing mercury accumulation between the sexes of fish from the same population. Further, our findings should eventually lead to an improved understanding of mechanisms responsible for mercury elimination in vertebrates.

  15. 'Soft' harness for external attachment of large radio transmitters to northern pike (Esox lucius)

    USGS Publications Warehouse

    Herke, S.W.; Moring, J.R.

    1999-01-01

    We developed a 'soft' harness for dorsally attaching large, external radio transmitters to northern pike (Esox lucius). The key harness component was a soft, flexible, thick-walled tubing that prevented tissue abrasion by the attachment lines which passed through the tubing. Six field-tagged fish (1.5-7.5 kg) were monitored for 45-115 days before tracking was terminated. Tracking patterns of fish indicated no apparent effect of these large, external transmitters on movement behavior; further, the transmitters did not appear to entangle the fish in vegetation. One fish with its transmitter still secure was recaptured after 54 days, and there was minimal tissue erosion under the transmitter. With minor improvements for the attachment lines and the transmitter saddle, the method is suitable for externally attaching large telemetry transmitters to fish.

  16. Altered energetics and parasitism in juvenile northern pike (Esox lucius) inhabiting metal-mining contaminated lakes.

    PubMed

    Kelly, Jocelyn M; Janz, David M

    2008-07-01

    The objective of this study was to evaluate possible factors that could be contributing to altered bioenergetics of juvenile northern pike (Esox lucius) living in lakes receiving effluent from the Key Lake uranium mill in northern Saskatchewan, Canada. Although glycogen and triglycerides stores in liver and muscle were significantly greater in pike from exposure lakes compared to the reference, triglycerides stores of aquatic insects and spottail shiners that are prey items of juvenile pike showed no overall differences among lakes. Measures of parasitism, on the other hand, were negatively correlated with pike bioenergetics thereby reflecting a possible energetic cost of parasitism on reference lake fishes. The degree of infection, as measured by the abundance and biomass of intestinal parasites and the abundance of monogeneans on pike gills, was greatest in reference fishes and intermediate in low-exposure pike, whereas high-exposure fishes harbored no parasites.

  17. Spatial genetic structure of northern pike (Esox lucius) in the Baltic Sea.

    PubMed

    Laikre, Linda; Miller, Loren M; Palmé, Anna; Palm, Stefan; Kapuscinski, Anne R; Thoresson, Gunnar; Ryman, Nils

    2005-06-01

    The genetic relationships among 337 northern pike (Esox lucius) collected from the coastal zone of the central Baltic region and the Finnish islands of Aland were analysed using five microsatellite loci. Spatial structure was delineated using both traditional F-statistics and individually based approaches including spatial autocorrelation analysis. Our results indicate that the observed genotypic distribution is incompatible with that of a single, panmictic population. Isolation by distance appears important for shaping the genetic structure of pike in this region resulting in a largely continuous genetic change over the study area. Spatial autocorrelation analysis (Moran's I) of individual pairwise genotypic data show significant positive genetic correlation among pike collected within geographical distances of less than c. 100-150 km (genetic patch size). We suggest that the genetic patch size may be used as a preliminary basis for identifying management units for pike in the Baltic Sea.

  18. Age-dependent accumulation of (137)Cs by pike Esox lucius in the Yenisei River.

    PubMed

    Zotina, T A; Trofimova, E A; Dementyev, D V; Bolsunovsky, A Ya

    2016-05-01

    Age-dependent accumulation of (137)Cs in the muscles and bodies of the pike Esox lucius (aged two to seven years) inhabiting a section of the Yenisei River polluted with artificial radionuclides has been studied. The content of (137)Cs in muscles varied from 0.5 to 7.0 Bq/kg of fresh weight. The maximum content of the radionuclide has been found in juveniles. The content of (137)Cs in pike muscles and body decreased considerably with age. The high content of (137)Cs in the muscles of juveniles is probably a consequence of their higher intensity of feeding as compared to older individuals, which is due to the intense growth of juveniles.

  19. Mercury elimination rates for adult northern pike Esox lucius: evidence for a sex effect.

    PubMed

    Madenjian, Charles P; Blanchfield, Paul J; Hrenchuk, Lee E; Van Walleghem, Jillian L A

    2014-08-01

    We examined the effect of sex on mercury elimination in fish by monitoring isotope-enriched mercury concentrations in the muscle tissue of three adult female and three adult male northern pike Esox lucius, which had accumulated the isotope-enriched mercury via a whole-lake manipulation and were subsequently moved to a clean lake. Mercury elimination rates for female and male northern pike were estimated to be 0.00034 and 0.00073 day(-1), respectively. Thus, males were capable of eliminating mercury at more than double the rate than that of females. To the best of our knowledge, our study represents the first documentation of mercury elimination rates varying between the sexes of fish. This sex difference in elimination rates should be taken into account when comparing mercury accumulation between the sexes of fish from the same population. Further, our findings should eventually lead to an improved understanding of mechanisms responsible for mercury elimination in vertebrates.

  20. Biogenic amines formation in high-pressure processed pike flesh (Esox lucius) during storage.

    PubMed

    Křížek, Martin; Matějková, Kateřina; Vácha, František; Dadáková, Eva

    2014-05-15

    The effects of vacuum packaging followed by high pressure processing on the shelf-life of fillets of pike (Esox lucius) were examined. Samples were pressure-treated at 300 and 500 MPa and stored at 3.5 and 12 °C for up to 70 days. The content of eight biogenic amines (putrescine, cadaverine, spermidine, spermine, histamine, tyramine, tryptamine and phenylethylamine) were determined. Putrescine showed very good correspondence with the level of applied pressure and organoleptic properties. Polyamines spermidine and spermine did not show statistically significant changes with the level of applied pressure and the time of storage. Increased cadaverine and tyramine contents were found in samples with good sensory signs, stored for longer time and/or kept at 12 °C, thus indicating the loss of freshness. Tryptamine and phenylethylamine were not detected in pressure-treated samples kept at 3.5 °C. Histamine was not detected in samples of good quality.

  1. Age and growth of pike (Esox lucius) in Chivyrkui Bay, Lake Baikal

    USGS Publications Warehouse

    Owens, Randall W.; Pronin, Nikolai M.

    2000-01-01

    The purpose of this study was to describe age and growth of pike (Esox lucius) in Lake Baikal. Pike were collected with gill nets and by angling in Chivyrkui Bay in late July-early August 1993 and by gill nets in June 1995. Total length (mm), weight (g), and sex were recorded and scales and cleithra were collected for aging. In 1993, pike, ages 1 to 3, ranged in length from 331 to 810 mm and in 1995 , pike, ages 2 to 10, ranged in length from 365 to 1,111 mm but only three percent were age 7 or older. Most growth in length occurred during the first two years of life. The length-weight relation for pike from Chivyrkui Bay was similar to that of pike from the St. Lawrence River. Calculated total length of pike from Lake Baikal equalled or exceeded the lengths of pike from lakes Erie or Ontario. Good agreement was found between ages from cleithra and from scales. Lengths at age in June 1995 (N=108) varied widely among pike. Females were generally larger than males at a given age among fish age-3 and older. When compared with the circumpolar growth standard, based on the von Bertalanffy growth curve, growth of Lake Baikal pike exceeded all other Asian populations, and equalled or exceeded many other northern hemisphere populations.

  2. Composition of seminal plasma and ovarian fluid in Ide Leuciscus idus and Northern pike Esox lucius.

    PubMed

    Siddique, Mam; Linhart, O; Kujawa, R; Krejszeff, S; Butts, Iae

    2016-12-01

    Seminal plasma (SP) and ovarian fluid (OF) plays an important role as storage media to prevent the activation of gametes both in vivo and under artificial conditions. The objectives of this study were to quantify gamete biochemistry and explore correlations among quantitative characteristics of SP, OF and sperm performance traits of Ide Leuciscus idus and Northern pike Esox lucius. Generally, Na(+) , K(+) and Cl(-) were found to be the most dominating ions, although concentrations of K(+) were higher in SP, while Na(+) and Cl(-) concentrations were higher in OF for both species. Several significant correlations among the biochemical properties such as total protein, glucose, osmolality, cholesterol, K(+) , Ca(2+) , Cl(-) and Mg(2+) were observed for SP and OF. Total protein content of Ide SP was positively correlated with sperm activity traits (r ≥ .89, p ≤ .05), while K(+) concentration was negatively correlated with sperm traits (r ≥ -.89, p ≤ .05). Moreover, Ca(2+) concentration in Northern pike SP was positively correlated with the percentage of sperm motility (r = . 98, p < .01). In conclusion, these results can be used to better understand the biochemistry of SP and OF, improve methods for short- and long-term storage of gametes and standardize fertilization protocols. © 2016 Blackwell Verlag GmbH.

  3. Selective exploitation of large pike Esox lucius--effects on mercury concentrations in fish populations.

    PubMed

    Sharma, Chhatra Mani; Borgstrøm, Reidar; Huitfeldt, Jørgen Sinkaberg; Rosseland, Bjørn Olav

    2008-07-25

    The present study outlines two main trends of mercury transfer patterns through the fish community: 1) the Hg concentrations increase with increase in the trophic level, with top predators having the highest concentrations, and 2) a fast growth rate may dilute the concentrations of Hg in fish muscle tissue (growth biodilution). In 2004, an extensive reduction in number of large pike (Esox lucius L.), was initiated by selective gillnet fishing in Lake Arungen, Norway, in order to increase the pike recruitment due to an expected reduction in cannibalism. In this connection, total mercury (THg) concentrations in the fish community were studied both before (2003) and after (2005) the removal of large pike. The delta(15)N signatures and stomach content analyses indicated that pike and perch (Perca fluviatilis L.) occupied the highest trophic position, while roach (Rutilus rutilus (L.)) was at the lower level, and rudd (Scardinius erythrophthalmus L.) at the lowest. The piscivores, pike and perch, had the highest concentrations of THg. The biomagnification rate of THg through the food web in the fish community was 0.163 (per thousand delta(15)N), with the highest uptake rate (0.232) in perch. A significant decrease in THg concentrations was found in all fish species in 2005 compared to 2003. Removal of the top predators in an Hg contaminated lake might thus be an important management tool for reducing Hg levels in fish, thereby reducing health risk to humans.

  4. Susceptibility of pike Esox lucius to a panel of Ranavirus isolates.

    PubMed

    Jensen, Britt Bang; Ersbøll, Annette Kjaer; Ariel, Ellen

    2009-02-25

    In order to study the pathogenicity of ranaviruses to a wild European freshwater fish species, pike Esox lucius fry were challenged with the following Ranavirus isolates: epizootic haematopoietic necrosis virus (EHNV), European sheatfish virus (ESV), European catfish virus (ECV), pike-perch iridovirus (PPIV), New Zealand eel virus (NZeelV) and frog virus 3 (FV3). The fry were infected using bath challenge at 12 and 22 degrees C. Significant mortalities were observed at 12 degrees C for EHNV, ESV, PPIV and NZeelV. Background mortality was too high in the experiments performed at 22 degrees C for any conclusions about viral pathogenicity at this temperature to be drawn. Viruses could be re-isolated from samples from all challenged groups, and their presence in infected tissue was demonstrated using immunohistochemistry. The findings suggest that pike fry are susceptible to EHNV, ESV, PPIV and NZeelV and can be a vector for ECV and FV3. Statistical analysis of the factors associated with positive virus re-isolation showed that the number of fish in the sample influenced the outcome of virus re-isolation. Moreover, the likelihood of positive virus re-isolation significantly differed among the 6 viral isolates. The temperature from where the sample was taken and the number of days after infection were not associated with the probability of a positive virus re-isolation.

  5. Temporal change estimation of mercury concentrations in northern pike (Esox lucius L.) in Swedish lakes.

    PubMed

    Åkerblom, Staffan; Nilsson, Mats; Yu, Jun; Ranneby, Bo; Johansson, Kjell

    2012-02-01

    Adequate temporal trend analysis of mercury (Hg) in freshwater ecosystems is critical to evaluate if actions from the human society have affected Hg concentrations ([Hg]) in fresh water biota. This study examined temporal change in [Hg] in Northern pike (Esox lucius L.) in Swedish freshwater lakes between 1994 and 2006. To achieve this were lake-specific, multiple-linear-regression models used to estimate pike [Hg], including indicator variables representing time and fish weight and their interactions. This approach permitted estimation of the direction and magnitude of temporal changes in 25 lakes selected from the Swedish national database on Hg in freshwater biota. A significant increase was found in 36% of the studied lakes with an average increase in pike [Hg] of 3.7±6.7% per year that was found to be positively correlated with total organic carbon. For lakes with a significant temporal change the dataset was based on a mean of 30 fish, while for lakes with no temporal change it was based on a mean of 13 fish. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Visibility conditions and diel period affect small-scale spatio-temporal behaviour of pike Esox lucius in the absence of prey and conspecifics.

    PubMed

    Nilsson, P A; Baktoft, H; Boel, M; Meier, K; Jacobsen, L; Rokkjaer, E M; Clausen, T; Skov, C

    2012-05-01

    Pike Esox lucius in the absence of prey and conspecifics were shown to have the highest habitat-change activity during dusk and to decrease preference for complex habitats in turbid water. As the behaviours indicate routine responses in the absence of behavioural interactions, E. lucius spatio-temporal distributions should be directly affected and thereby more easily assessed and avoided by prey, with potential consequences for encounter rates.

  7. DDT in northern pike (Esox lucius) from the Richelieu River, Québec, Canada, 1974-75.

    PubMed

    Boileau, S; Baril, M; Alary, J G

    1979-12-01

    Concentrations of DDT, TDE, DDE, and sigma DDT were determined in homogenized whole fish samples of 129 northern pike (Esox lucius). These fish were netted between June 1974 and June 1975 in the first 10 km of the Richelieu River flowing in Canadian territory. Two years after the banning of DDT, sigma DDT levels ranged from 0.2 ppm fresh body weight in two-year-old specimens to 1.5 ppm in a six-year-old pike. Residues increased greatly with age, and significant seasonal variations in the sigma DDT levels were found in five- and six-year-old pike.

  8. Genetic structure of pike (Esox lucius) reveals a complex and previously unrecognized colonization history of Ireland

    PubMed Central

    Pedreschi, Debbi; Kelly-Quinn, Mary; Caffrey, Joe; O’Grady, Martin; Mariani, Stefano; Phillimore, Albert

    2014-01-01

    Aim We investigated genetic variation of Irish pike populations and their relationship with European outgroups, in order to elucidate the origin of this species to the island, which is largely assumed to have occurred as a human-mediated introduction over the past few hundred years. We aimed thereby to provide new insights into population structure to improve fisheries and biodiversity management in Irish freshwaters. Location Ireland, Britain and continental Europe. Methods A total of 752 pike (Esox lucius) were sampled from 15 locations around Ireland, and 9 continental European sites, and genotyped at six polymorphic microsatellite loci. Patterns and mechanisms of population genetic structure were assessed through a diverse array of methods, including Bayesian clustering, hierarchical analysis of molecular variance, and approximate Bayesian computation. Results Varying levels of genetic diversity and a high degree of population genetic differentiation were detected. Clear substructure within Ireland was identified, with two main groups being evident. One of the Irish populations showed high similarity with British populations. The other, more widespread, Irish strain did not group with any European population examined. Approximate Bayesian computation suggested that this widespread Irish strain is older, and may have colonized Ireland independently of humans. Main conclusions Population genetic substructure in Irish pike is high and comparable to the levels observed elsewhere in Europe. A comparison of evolutionary scenarios upholds the possibility that pike may have colonized Ireland in two ‘waves’, the first of which, being independent of human colonization, would represent the first evidence for natural colonization of a non-anadromous freshwater fish to the island of Ireland. Although further investigations using comprehensive genomic techniques will be necessary to confirm this, the present results warrant a reappraisal of current management strategies

  9. Mercury toxicity in livers of northern pike (Esox lucius) from Isle Royale, USA.

    PubMed

    Drevnick, Paul E; Roberts, Aaron P; Otter, Ryan R; Hammerschmidt, Chad R; Klaper, Rebecca; Oris, James T

    2008-04-01

    Many laboratory studies have documented that mercury can be toxic to fish, but it is largely unknown if mercury is toxic to fish in their natural environments. The objective of our study was to investigate the toxic effects of mercury on northern pike (Esox lucius) at Isle Royale, Michigan. In 124 northern pike from eight inland lakes, concentrations of total mercury in skin-on fillets ranged from 0.069 to 0.622 microg/g wet mass (wet wt). Concentrations of total mercury in livers increased exponentially compared with concentrations in fillets, to a maximum of 3.1 microg/g wet wt. Methylmercury constituted a majority of the mercury in livers with total mercury concentrations <0.5 microg/g wet wt, but declined to 28-51% of the mercury in livers with total mercury concentrations >0.5 microg/g wet wt. Liver color (absorbance at 400 nm) varied among northern pike and was positively related to liver total mercury concentration. The pigment causing variation in liver color was identified as lipofuscin, which results from lipid peroxidation of membranous organelles. An analysis of covariance revealed lipofuscin accumulation was primarily associated with mercury exposure, and this association obscured any normal accumulation from aging. We also documented decreased lipid reserves in livers and poor condition factors of northern pike with high liver total mercury concentrations. Our results suggest (i) northern pike at Isle Royale are experiencing toxicity at concentrations of total mercury common for northern pike and other piscivorous fish elsewhere in North America and (ii) liver color may be useful for indicating mercury exposure and effects in northern pike at Isle Royale and possibly other aquatic ecosystems and other fish species.

  10. Larval deformities associated with selenium accumulation in northern pike (Esox lucius) exposed to metal mining effluent.

    PubMed

    Muscatello, Jorgelina R; Bennett, Pamela M; Himbeault, Kevin T; Belknap, Andrew M; Janz, David M

    2006-10-15

    The objective of this study was to investigate selenium toxicosis in larval northern pike (Esox lucius) originating from reproductively mature pike collected downstream of a uranium milling operation in northern Saskatchewan, Canada. Eggs were obtained from female pike collected from a reference site and three sites representing an exposure gradient (approximately 2, 10, and 15 km downstream of effluent discharge). Embryos were incubated following a two-way (crossover) analysis of variance experimental design that allowed discrimination between effects due to maternal transfer to eggs and effects due to site water exposure in the developing embryos. The major finding of this study was a significant increase in the frequencies of individual deformities (skeletal curvatures, craniofacial deformities, and fin deformities) and edema in fry originating from high and medium exposure site females (mean selenium concentrations of 48.23 and 31.28 microg/g egg dry weight and 38.27 and 16.58 microg/g muscle dry weight, respectively) compared to reference site females. Selenium concentrations resulting in a 20% increase in total deformities above background levels (EC20S) were 33.55 and 21.54 micro/g dry weight in eggs and muscle, respectively. Mathematical conversion of the egg- and muscle-derived relationships to whole body selenium levels resulted in similar EC20S of 15.56 and 17.72 microg/g dry weight, respectively. These relationships between tissue selenium levels and larval deformities suggest that northern pike are within the same range of sensitivity to selenium as the majority of warm water (e.g., centrarchids and cyprinids) and cold water (e.g., salmonids) fish species studied to date.

  11. Effects of osmolality on sperm morphology, motility and flagellar wave parameters in Northern pike (Esox lucius L.).

    PubMed

    Alavi, S M Hadi; Rodina, Marek; Viveiros, Ana T M; Cosson, Jacky; Gela, David; Boryshpolets, Sergei; Linhart, Otomar

    2009-07-01

    Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 microm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 microm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg(-1) (average: 283.88+/-33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P<0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg(-1). Osmolality above 375 mOsmol kg(-1) inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile

  12. Biomarkers of contaminant exposure in northern pike (Esox lucius) from the Yukon River Basin, Alaska

    USGS Publications Warehouse

    Hinck, J.E.; Blazer, V.S.; Denslow, N.D.; Myers, M.S.; Gross, T.S.; Tillitt, D.E.

    2007-01-01

    As part of a larger investigation, northern pike (n = 158; Esox lucius) were collected from ten sites in the Yukon River Basin (YRB), Alaska, to document biomarkers and their correlations with organochlorine pesticide (total p,p'-DDT, total chlordane, dieldrin, and toxaphene), total polychlorinated biphenyls (PCBs), and elemental contaminant (arsenic, cadmium, copper, lead, total mercury, selenium, and zinc) concentrations. A suite of biomarkers including somatic indices, hepatic 7-ethoxyresorufin O-deethylase (EROD) activity, vitellogenin concentrations, steroid hormone (17B- ustradiol and 16-kebtestosteront) concentrations, splenic macrophage aggregates (MAs), oocyte atresia, and other microscopic anomalies in various tissues were documented in YRB pike. Mean condition factor (0.50 to 0.68), hepatosomatic index (1.00% to 3.56%), and splenosomatic index (0.09% to 0.18%) were not anomalous at any site nor correlated with any contaminant concentration. Mean EROD activity (0.71 to 17.51 pmol/min/mg protein) was similar to basal activity levels previously measured in pike and was positively correlated with selenium concentrations (r = 0.88, P < 0.01). Vitellogenin concentrations in female (0.09 to 5.32 mg/mL) and male (0.01 mg/mL in male pike from multiple sites indicated exposure to estrogenic compounds. Mean steroid hormone concentrations and percent oocyte atresia were not anomalous in pike from any YRB site. Few site differences were significant for mean MA density (1.86 to 6.42 MA/mm2), size (812 to 1481 ??m2), and tissue occupied (MA-%; 0.24% to 0.75%). A linear regression between MA-% and total PCBs was significant, although PCB concentrations were generally low in YRB pike (???63 ng/g), and MA-% values in female pike (0.24% to 0.54%) were lower than in male pike (0.32% to 0.75%) at similar PCB concentrations. Greater numbers of MAs were found as zinc concentrations increased in YRB female pike, but it is unlikely that this is a causative relationship

  13. Biomarkers of contaminant exposure in Northern Pike (Esox lucius) from the Yukon River Basin, Alaska.

    PubMed

    Hinck, J E; Blazer, V S; Denslow, N D; Myers, M S; Gross, T S; Tillitt, D E

    2007-05-01

    As part of a larger investigation, northern pike (n = 158; Esox lucius) were collected from ten sites in the Yukon River Basin (YRB), Alaska, to document biomarkers and their correlations with organochlorine pesticide (total p,p'-DDT, total chlordane, dieldrin, and toxaphene), total polychlorinated biphenyls (PCBs), and elemental contaminant (arsenic, cadmium, copper, lead, total mercury, selenium, and zinc) concentrations. A suite of biomarkers including somatic indices, hepatic 7-ethoxyresorufin O-deethylase (EROD) activity, vitellogenin concentrations, steroid hormone (17B- ustradiol and 16-kebtestosteront) concentrations, splenic macrophage aggregates (MAs), oocyte atresia, and other microscopic anomalies in various tissues were documented in YRB pike. Mean condition factor (0.50 to 0.68), hepatosomatic index (1.00% to 3.56%), and splenosomatic index (0.09% to 0.18%) were not anomalous at any site nor correlated with any contaminant concentration. Mean EROD activity (0.71 to 17.51 pmol/min/mg protein) was similar to basal activity levels previously measured in pike and was positively correlated with selenium concentrations (r = 0.88, P < 0.01). Vitellogenin concentrations in female (0.09 to 5.32 mg/mL) and male (<0.0005 to 0.097 mg/mL) pike were not correlated with any contaminant, but vitellogenin concentrations >0.01 mg/mL in male pike from multiple sites indicated exposure to estrogenic compounds. Mean steroid hormone concentrations and percent oocyte atresia were not anomalous in pike from any YRB site. Few site differences were significant for mean MA density (1.86 to 6.42 MA/mm(2)), size (812 to 1481 microm(2)), and tissue occupied (MA-%; 0.24% to 0.75%). A linear regression between MA-% and total PCBs was significant, although PCB concentrations were generally low in YRB pike (< or =63 ng/g), and MA-% values in female pike (0.24% to 0.54%) were lower than in male pike (0.32% to 0.75%) at similar PCB concentrations. Greater numbers of MAs were found as

  14. [Taxonomic characteristics and physiological properties of microorganisms from the gut of pike (Esox lucius)].

    PubMed

    Izveskova, G I; Nemtseva, N V; Plotnikov, A O

    2008-01-01

    The taxonomic composition and distribution of microorganisms differing in the degree of association with the intestinal mucosa of the pike (Lucius lucius) has been studied. Microorgansism of the families Enterobacteriaceae, Aeromonadaceae, and Vibrionaceae dominate in the gut microflora. Numerically prevailing bacterial species are characterized by high proteolytic and amylolytic enzyme activities as well as by high persistence accounted for by antilysozyme and antihistone activities. The results of this study show that Hafnia alvei, Yersinia ruckeri, Vibrio vulnificus, V. furnissii, Aeromonas salmonicida, and Shewanella putrefaciens may be regarded as normal components of the pike gut microflora.

  15. Post-glacial dispersal patterns of Northern pike inferred from an 8800 year old pike (Esox cf. lucius) skull from interior Alaska

    NASA Astrophysics Data System (ADS)

    Wooller, Matthew J.; Gaglioti, Benjamin; Fulton, Tara L.; Lopez, Andres; Shapiro, Beth

    2015-07-01

    The biogeography of freshwater fish species during and after late-Pleistocene glaciations relate to how these species are genetically organized today, and the management of these often disjunct populations. Debate exists concerning the biogeography and routes of dispersal for Northern pike (Esox lucius) after the last glaciation. A hypothesis to account for the relatively low modern genetic diversity for E. lucius is post-glacial radiation from refugia, including lakes from within the un-glaciated portions of eastern Beringia. We report the remains of a Northern pike (E. cf. lucius) skull, including bones, teeth, bone collagen and ancient DNA. The remains were preserved at a depth of between 440 and 446 cm in a 670 cm long core of sediment from Quartz Lake, which initiated at ˜11,200 cal yr BP in interior Alaska. A calibrated accelerator mass spectrometer (AMS) radiocarbon age of the collagen extracted from the preserved bones indicated that the organism was dated to 8820 cal yr BP and is bracketed by AMS values from analyses of terrestrial plant macrofossils, avoiding any potential aquatic reservoir effect that could have influenced the radiocarbon age of the bones. Scanning electron microscope images of the specimen show the hinged tooth anatomy typically of E. lucius. Molar C:N (3.5, 1σ = 0.1) value of the collagen from the specimen indicated well-preserved collagen and its mean stable nitrogen isotope value is consistent with the known predatory feeding ecology of E. lucius. Ancient DNA in the bones showed that the specimen was identical to modern E. lucius. Our record of E. lucius from interior Alaska is consistent with a biogeographic scenario involving rapid dispersal of this species from glacial refugia in the northern hemisphere after the last glaciation.

  16. Fish Community Responses to the Establishment of a Piscivore, Northern Pike (Esox lucius), in a Nebraska Sandhill Lake

    USGS Publications Warehouse

    DeBates, T.J.; Paukert, C.P.; Willis, D.W.

    2003-01-01

    Northern pike (Esox lucius) was first documented in West Long Lake, Nebraska, in 1998 when two pike <380 mm were collected. In 2002, a Peterson mark-recapture population estimate on northern pike revealed density and standing stock (i.e., biomass) estimates of 35.8 fish/ha (95% CI= ?? 8.8) and 22.0 kg/ha (95% CI= ?? 5.4), respectively. Consequently, West Long Lake was sampled in 2002 to compare relative abundance, size structure, and growth of bluegill (Lepomis macrochirus), largemouth bass (Micropterus salmoides), and yellow perch (Perca flavescens) prior to and after the establishment of a high-density northern pike population. Bluegill, largemouth bass, and yellow perch relative abundances were significantly lower in 2002 than 1998. Similarly, size structures of all three species were significantly different between years. Size structure declined for both bluegill and yellow perch, and increased for largemouth bass. Growth was significantly higher for bluegill, largemouth bass, and yellow perch in 2002 than 1998. While the fish community changes were expected with the establishment of northern pike, they occurred in a relatively short time period (i.e., four years).

  17. Quantifying selection differentials caused by recreational fishing: development of modeling framework and application to reproductive investment in pike (Esox lucius)

    PubMed Central

    Arlinghaus, Robert; Matsumura, Shuichi; Dieckmann, Ulf

    2009-01-01

    Methods for quantifying selection pressures on adaptive traits affected by size-selective fishing are still scarce, and none have as yet been developed for recreational fishing. We present an ecologically realistic age-structured model specifically tailored to recreational fishing that allows estimating selection differentials on adaptive life-history traits. The model accounts for multiple ecological feedbacks, which result in density-dependent and frequency-dependent selection. We study selection differentials on annual reproductive investment under size-selective exploitation in a highly demanded freshwater recreational fish species, northern pike (Esox lucius L.). We find that recreational angling mortality exerts positive selection differentials on annual reproductive investment, in agreement with predictions from life-history theory. The strength of selection increases with the intensity of harvesting. We also find that selection on reproductive investment can be reduced by implementing simple harvest regulations such as minimum-size limits. The general, yet computationally simple, methods introduced here allow evaluating and comparing selection pressures on adaptive traits in other fish populations and species, and thus have the potential to become a tool for evolutionary impact assessment of harvesting. PMID:25567885

  18. Endocrine (sexual) disruption is not a prominent feature in the pike (Esox lucius), a top predator, living in English waters.

    PubMed

    Vine, Emma; Shears, Jan; van Aerle, Ronny; Tyler, Charles R; Sumpter, John P

    2005-06-01

    The high incidence of intersex roach (Rutilus rutilus) in some United Kingdom rivers that has been associated with exposure to sewage treatment works (STWs) effluent led us to hypothesize that top predator fish also may be affected by estrogenic chemicals, because they are likely to bioaccumulate lipophilic compounds through a predator-prey relationship. To investigate this possibility, pike (Esox lucius) were sampled both upstream and downstream of STWs and then examined for total estrogenic activity of their bile, as measured using a yeast-based estrogen assay to determine the degree of recent exposure of the pike to estrogens and vitellogenin induction, and for possible disruption of sexual development, as measured using histological analysis of the gonads. No evidence of severe disruption was found in the sampled fish, which came from 16 sampling sites that were representative of English rivers. However, 14% of pike were intersex, of which 15 of 16 showed patches of male germ cells among predominantly female gonadal tissue. The incidence of masculinization was independent of whether the pike had been sampled upstream or downstream of STWs. Although pike are gonochoristic, it is not known if this masculinization of presumptive female pike is normal or, instead, indicative of endocrine disruption. Vitellogenin concentrations were not elevated in male pike at sites either upstream or downstream of STWs. The results suggest that sexual disruption is not common in pike, a fish at the top of the food chain in the freshwaters of England.

  19. Between-population similarity in intestinal parasite community structure of pike (Esox lucius)--effects of distance and historical connections.

    PubMed

    Karvonen, Anssi; Valtonen, E Tellervo

    2009-06-01

    The effect of geographical distance on similarity in parasite communities of freshwater fish has received considerable attention in recent years, and it has become evident that these apparently simple relationships are influenced by, among other things, colonization ability of parasites and degree of connectivity between the populations. In the present paper, we explored qualitative and quantitative similarity in the intestinal parasite communities of pike (Esox lucius) in a particular system where previously interconnected groups of lakes became isolated ca. 8,400 yr ago. Contrary to our expectations, we did not find differences in similarity between the lake groups or a negative effect of distance among the populations. This supports the role of common ancestral colonization events and shows that no significant loss of species has occurred during the past 8,000 yr. However, the communities were dominated by a single parasite species, the cestode Triaenophorus nodulosus. The exclusion of this species from the data had a significant negative impact on the community similarities and also revealed a negative relationship between distance and quantitative similarity. This suggests that patterns of community organization may be obscured by a single dominant species. We also highlight the need for further studies in different systems and host species, as well as detailed reanalysis of existing data sets, to unravel the controversy in the relationship between distance and similarity in parasite communities.

  20. {sup 32}P-postlabeling analysis of DNA adducts in wild perch (Perca fluviatilis) and northern pike (Esox lucius)

    SciTech Connect

    Ericson, G.; Liewenborg, B.; Balk, L.

    1995-12-31

    Several previous studies have demonstrated a correlation between high concentrations of sediment-associated contaminants and elevated levels of aromatic/hydrophobic DNA adduct levels in the liver of benthic fish species. In the present study DNA adducts was analyzed in coastal populations of perch (Perca fluviatilis) and northern pike (Esox lucius). Fish were sampled from four different sites in a gradient from a heavily industrialized area at the Swedish Baltic coast. For comparison, fish were also caught in a reference area with no main industries and comparatively low levels of contaminants of anthropogenic origin. DNA was extracted from liver and several extrahepatic tissues and DNA adducts were analyzed by the nuclease PI version of the {sup 32}P-postlabeling assay. The autoradiograms derived from DNA of fish from the contaminated sites showed several adduct spots not visible on the autoradiograms derived from fish from the reference area. Total adduct levels were significantly elevated in several tissues in fish from contaminated sites compared to the reference area. Species and tissue-specific differences in adduct levels and the use of {sup 32}P-postlabeling analysis of DNA adducts as a biomarker to monitor the presence and effects of genotoxic chemicals in the aquatic environment are discussed.

  1. Behaviour in a standardized assay, but not metabolic or growth rate, predicts behavioural variation in an adult aquatic top predator Esox lucius in the wild.

    PubMed

    Laskowski, K L; Monk, C T; Polverino, G; Alós, J; Nakayama, S; Staaks, G; Mehner, T; Arlinghaus, R

    2016-04-01

    This study tested for links among behaviour, state and life-history variables as predicted by the pace-of-life hypothesis in adult pike Esox lucius. First, a standardized open-field behavioural assay was developed to assess individual behaviour of wild-captured adult E. lucius. Behaviour within the standardized assay predicted swimming behaviour in the lake, providing an ecological validation of the assay. There was no relationship between standardized behaviour and any of the life-history and state variables, including metabolism, body condition, juvenile growth rate and adult growth rate in contrast to predictions from the pace-of-life hypothesis. This study demonstrates that it is possible to assess ecologically relevant behavioural variation in a large-bodied top predator using a standard open-field assay, but it is noteworthy that this standardized behaviour is not systematically related to standard metabolism or growth.

  2. Assessment of oxidative stress and histopathology in juvenile northern pike (Esox lucius) inhabiting lakes downstream of a uranium mill.

    PubMed

    Kelly, Jocelyn M; Janz, David M

    2009-05-17

    Lakes receiving effluent from the Key Lake uranium mill in northern Saskatchewan contain elevated trace metals, some of which are associated with increased reactive oxygen species (ROS) in cells and tissues causing oxidative stress. The potential for oxidative stress was assessed in juvenile (age 1+) northern pike (Esox lucius) collected from two exposure (high and low) and one reference lake near the Key Lake operation. The concentrations of total, reduced and oxidized glutathione and the ratio of oxidized to reduced glutathione in liver and kidney did not differ significantly among pike collected from exposure and reference lakes, with the exception of low exposure pike kidney that had significantly greater oxidized glutathione and ratio of oxidized to reduced glutathione. The concentrations of by-products of lipid peroxidation (malondialdehyde and 4-hydroxyalkenal) were significantly greater in kidney of pike collected from the reference lake compared to both exposure lakes. The activity of the antioxidant enzyme glutathione peroxidase in liver was greater in pike collected from the high exposure lake compared to the reference lake. Histopathological evaluations revealed greater pathology in reference lake pike as indicated by a greater number of pyknotic and fragmented nuclei and dilated tubules as well as a thickening of Bowman's capsule in kidney, and as a thickening of the primary filament epithelial padding in gills. In liver, hepatocyte morphology, including transsectional area and degree of vacuolation, differed among lakes without any clear signs of pathology. Trace metal analyses of muscle showed that eight elements (arsenic, cobalt, copper, iron, molybdenum, selenium, thallium, and uranium) were significantly elevated in pike collected from both exposure lakes compared to reference. These results provide only limited evidence of oxidative stress in exposure pike tissues and no evidence of histopathology despite indications that trace metals, most

  3. Determination of polychlorinated biphenyls and total mercury in two fish species (Esox lucius and Carassius auratus) in Anzali Wetland, Iran.

    PubMed

    Sakizadeh, Mohammad; Esmaeili Sari, Abas; Abdoli, Asghar; Bahramifar, Nader; Hashemi, Seyed Hossein

    2012-05-01

    The Anzali Wetland is one of the most important ecosystems in the north of Iran, and parts of it were registered as a Ramsar site in 1975. However, even though, due to many problems, including eutrophication produced by inflow of excess nutrients and organic materials, the wetland was also listed on the Montreux Record indicating the need to take urgent remedial action. This study was conducted to study the levels of polychlorinated biphenyls (PCBs) and total mercury (THg) in two fish species (Esox lucius and Carassius auratus) as bio-indicators of the ecosystem condition in eastern part of Anzali Wetland. The sampling was carried out in six different periods between years 2009 and 2010. The results showed that the amounts of PCBs in the muscle of northern pike were below the detection limit of gas chromatography, whereas the average concentration in goldfish was 0.449 mg/kg wet weight. Some possible reasons for the higher levels of PCBs in goldfish in comparison with pike have been discussed. No significant (p < 0.05) correlation was observed between PCBs and biological factors (weight, length, lipid content) for both species. On the other hand, the mean concentration of THg in the muscle of pike and goldfish were 182.22 and 75.27 ng/g dry weight, respectively. Although these concentrations were below US-EPA criterion for human consumption (0.3 mg/kg), it pointed up a significant deterioration of the ecosystem condition during the past years. Finally, statistical analysis revealed a significant correlation between THg with weight and an insignificant correlation with length for pike specimens.

  4. Spatial analysis of Cd and Pb in the Pike (Esox lucius) from Western Anzali wetlands of Iran.

    PubMed

    Zamani-Ahmadmahmoodi, R; Esmaili-Sari, A; Mohammadi, J; Riyahi Bakhtiari, A; Savabieasfahani, M

    2013-04-01

    Geostatistical studies are used to estimate pollution burden in aquatic ecosystems and to plan large-scale control programs to protect these environments. Geostatistical studies allow us to predicted pollutant concentrations for areas that have not been sampled. This is done by taking into account the spatial correlations between estimated and sampled points and by minimizing the variance of estimation error. The use of geostatistical techniques in biomonitoring of fish species can illuminate extent and source of pollution, thereby providing an effective tool for developing intervention strategies to protect such environments. This study investigates the spatial distribution patterns of cadmium and lead in the Pike (Esox lucius). Fish were captured in the western parts of the Anzali wetlands located on the Caspian Sea in Iran. The muscle tissue of Anzali Pike had 5 ± 0.25 and 168 ± 18.4 (ng/g dw) cadmium and lead, respectively. Positive relationships were detected between Pike's length and weight (r = 0.85, p < 0.05), length and age (r = 0.35, p < 0.05), and muscle cadmium and lead (r = 0.45, p < 0.05). By contrast, there was a negative relationship between lead levels and weight in Pike (r = -0.36, p < 0.05). For both metals, the resulting metal concentration maps indicated higher pollutant concentrations in the southeast parts of the study area. Considerable boat traffic activity and agricultural activity contribute to the pollution in these areas, undermining the integrity of local habitat for fish survival and reproduction.

  5. Landscape variability explains spatial pattern of population structure of northern pike (Esox lucius) in a large fluvial system

    PubMed Central

    Ouellet-Cauchon, Geneviève; Mingelbier, Marc; Lecomte, Frédéric; Bernatchez, Louis

    2014-01-01

    A growing number of studies have been investigating the influence of contemporary environmental factors on population genetic structure, but few have addressed the issue of spatial patterns in the variable intensity of factors influencing the extent of population structure, and particularly so in aquatic ecosystems. In this study, we document the landscape genetics of northern pike (Esox lucius), based on the analysis of nearly 3000 individuals from 40 sampling sites using 22 microsatellites along the Lake Ontario – St. Lawrence River system (750 km) that locally presents diverse degrees of interannual water level variation. Genetic structure was globally very weak (FST = 0.0208) but spatially variable with mean level of differentiation in the upstream section of the studied area being threefold higher (FST = 0.0297) than observed in the downstream sector (FST = 0.0100). Beside interannual water level fluctuation, 19 additional variables were considered and a multiple regression on distance matrices model (R2 = 0.6397, P < 0.001) revealed that water masses (b = 0.3617, P < 0.001) and man-made dams (b = 0.4852, P < 0.005) reduced genetic connectivity. Local level of interannual water level stability was positively associated to the extent of genetic differentiation (b = 0.3499, P < 0.05). As water level variation impacts on yearly quality and localization of spawning habitats, our study illustrates how temporal variation in local habitat availability, caused by interannual water level fluctuations, may locally decrease population genetic structure by forcing fish to move over longer distances to find suitable habitat. This study thus represents one of the rare examples of how environmental fluctuations may influence spatial variation in the extent of population genetic structure within a given species. PMID:25614787

  6. Landscape variability explains spatial pattern of population structure of northern pike (Esox lucius) in a large fluvial system.

    PubMed

    Ouellet-Cauchon, Geneviève; Mingelbier, Marc; Lecomte, Frédéric; Bernatchez, Louis

    2014-10-01

    A growing number of studies have been investigating the influence of contemporary environmental factors on population genetic structure, but few have addressed the issue of spatial patterns in the variable intensity of factors influencing the extent of population structure, and particularly so in aquatic ecosystems. In this study, we document the landscape genetics of northern pike (Esox lucius), based on the analysis of nearly 3000 individuals from 40 sampling sites using 22 microsatellites along the Lake Ontario - St. Lawrence River system (750 km) that locally presents diverse degrees of interannual water level variation. Genetic structure was globally very weak (F ST = 0.0208) but spatially variable with mean level of differentiation in the upstream section of the studied area being threefold higher (F ST = 0.0297) than observed in the downstream sector (F ST = 0.0100). Beside interannual water level fluctuation, 19 additional variables were considered and a multiple regression on distance matrices model (R (2)  = 0.6397, P < 0.001) revealed that water masses (b = 0.3617, P < 0.001) and man-made dams (b = 0.4852, P < 0.005) reduced genetic connectivity. Local level of interannual water level stability was positively associated to the extent of genetic differentiation (b = 0.3499, P < 0.05). As water level variation impacts on yearly quality and localization of spawning habitats, our study illustrates how temporal variation in local habitat availability, caused by interannual water level fluctuations, may locally decrease population genetic structure by forcing fish to move over longer distances to find suitable habitat. This study thus represents one of the rare examples of how environmental fluctuations may influence spatial variation in the extent of population genetic structure within a given species.

  7. Potential of Environmental DNA to Evaluate Northern Pike (Esox lucius) Eradication Efforts: An Experimental Test and Case Study.

    PubMed

    Dunker, Kristine J; Sepulveda, Adam J; Massengill, Robert L; Olsen, Jeffrey B; Russ, Ora L; Wenburg, John K; Antonovich, Anton

    2016-01-01

    Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling

  8. Potential of Environmental DNA to Evaluate Northern Pike (Esox lucius) Eradication Efforts: An Experimental Test and Case Study

    PubMed Central

    Dunker, Kristine J.; Sepulveda, Adam J.; Massengill, Robert L.; Olsen, Jeffrey B.; Russ, Ora L.; Wenburg, John K.; Antonovich, Anton

    2016-01-01

    Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling

  9. Potential of environmental DNA to evaluate Northern pike (Esox lucius) eradication efforts: An experimental test and case study

    USGS Publications Warehouse

    Dunker, Kristine J.; Sepulveda, Adam; Massengill, Robert L.; Olsen, Jeffrey B.; Russ, Ora L.; Wenburg, John K.; Antonovich, Anton

    2016-01-01

    Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling

  10. Investigation of first year biotic and abiotic influences on the recruitment of pike Esox lucius over 48 years in Windermere, UK.

    PubMed

    Paxton, C G M; Winfield, I J; Fletcher, J M; George, D G; Hewitt, D P

    2009-07-01

    Estimated pike Esox lucius recruitment varied by a factor of 16 for females from 1944 to 1991 and by a factor of 27 for males from 1943 to 1990 in Windermere, a temperate, mesotrophic U.K. lake. No significant stock-recruitment relationships were found, but analysis with general additive models (GAMs) revealed that early autumnal water temperature, strength and direction of the North Atlantic Oscillation displacement (corresponding to different climatic conditions in winter) and zooplankton abundance but above all, late summer water temperature were important explanatory variables over the entire time series. Female recruitment was also influenced by young-of-the-year winter temperature. There was no evidence that perch Perca fluviatilis year-class strength, lake level or the summer position of the Gulf Stream influenced recruitment. The fitted models explained up to c. 65% of the overall observed variation between years.

  11. Environmental factors regulate the effects of roach Rutilus rutilus and pike Esox lucius on perch Perca fluviatilis populations in small boreal forest lakes.

    PubMed

    Olin, M; Vinni, M; Lehtonen, H; Rask, M; Ruuhijärvi, J; Saulamo, K; Ala-Opas, P

    2010-04-01

    In this study of 18 small boreal forest lakes, the effects of abiotic and biotic factors (roach Rutilus rutilus and pike Esox lucius) on various population variables of perch Perca fluviatilis were examined. As a single variable, the gillnet catch per unit effort (CPUE) of R. rutilus was negatively related to the mean mass of small (< 200 mm) and the growth rate of young (1-2 years) P. fluviatilis. The mean mass of large (> or = 200 mm) P. fluviatilis was the highest at intermediate CPUE of R. rutilus. Redundancy analysis including environmental factors and P. fluviatilis population variables suggested that 'predation-productivity-humus' gradient affected P. fluviatilis populations by decreasing the CPUE and mean mass of small individuals but increasing these variables of large individuals. The CPUE of R. rutilus and lake area had a negative effect on small and a positive effect on large P. fluviatilis growth rate. In small boreal forest lakes, P. fluviatilis populations are affected by the partially opposite forces of competition by R. rutilus and predation by E. lucius, and the intensity of these interactions is regulated by several environmental factors.

  12. Uptake and distribution of (/sup 3/H)benzo(a)pyrene in the Northern pike (Esox lucius). Examination by whole-body autoradiography and scintillation counting

    SciTech Connect

    Balk, L.; Meijer, J.; DePierre, J.W.; Appelgren, L.E.

    1984-07-01

    The uptake and distribution of the polyaromatic hydrocarbon benzo(a)pyrene in Northern pike (Esox lucius) were investigated by whole body autoradiography and scintillation counting. (/sup 3/H)Benzo(a)pyrene was administered either in the diet or in the water. The uptake and distribution of this compound and its metabolites were followed from 10 hr to 21 days after the initial exposure. The autoradiography patterns observed here with both routes of administration suggest, as expected, that benzo(a)pyrene is taken up through the gastrointestinal system and the gills, metabolized in the liver, and excreted in the urine and bile. Other findings indicate that the gills may not be a major route of excretion for benzo(a)pyrene and its metabolites in the Northern pike; that benzo(a)pyrene may be taken up from the water directly into the skin of this fish; that benzo(a)pyrene and its metabolites are heterogeneously distributed in the kidney of the Northern pike; and that very little radioactivity accumulates in the adipose tissue. With scintillation counting, uptake of radioactivity from the water was found to occur rapidly in all organs, reaching a plateau in most cases after about 0.8 days. The concentrations of radioactivity in different organs ranged between 50 (many organs) and 80,000 (gallbladder + bile) times that found in the surrounding water.

  13. Perfluoroalkylphosphinic Acids in Northern Pike (Esox lucius), Double-Crested Cormorants (Phalacrocorax auritus), and Bottlenose Dolphins (Tursiops truncatus) in Relation to Other Perfluoroalkyl Acids.

    PubMed

    De Silva, Amila O; Spencer, Christine; Ho, Ki Chung D; Al Tarhuni, Mohammed; Go, Christopher; Houde, Magali; de Solla, Shane R; Lavoie, Raphael A; King, Laura E; Muir, Derek C G; Fair, Patricia A; Wells, Randall S; Bossart, Gregory D

    2016-10-18

    Perfluoroalkyl phosphinic acids (PFPIAs) are perfluoroalkyl acids (PFAAs) that are used for their surfactant properties in a variety of applications, resulting in their presence in environmental waters; however, they have not been widely studied in biota. A survey of PFPIAs was conducted in fish, dolphins, and birds from various locations in North America. Northern pike (Esox lucius) were collected at two locations in 2011 near Montréal Island in the St. Lawrence River, Canada, double-crested cormorants (Phalacrocorax auritus) were collected from bird colonies in the Great Lakes in 2010-2012, and bottlenose dolphins (Tursiops truncatus) from Sarasota Bay, FL and Charleston Harbor, SC were sampled in 2004-2009. PFPIAs had a detection frequency of 100% in all animals. This is the first report of PFPIAs in fish, dolphin, and bird plasma. Total PFPIA levels (mean ± standard deviation, 1.87 ± 2.17 ng/g wet weight (ww), range of 0.112-15.3 ng/g ww) were 1-2 orders of magnitude lower than those of perfluoroalkyl carboxylates (PFCA) and perfluoroalkanesulfonates (PFSA) in the same samples. The predominant congeners were 6:8 PFPIA (cormorants and pike) and 6:6 PFPIA (dolphins). Total PFPIAs in cormorants from Hamilton Harbour (5.02 ± 2.80 ng/g ww) were statistically higher than in other areas and taxonomic groups. The ubiquity of PFPIAs warrants further research on sources and effects of these unique compounds.

  14. Bioenergetics and growth of young-of the-year northern pike (Esox lucius) and burbot (Lota lota) exposed to metal mining effluent.

    PubMed

    Bennett, Pamela M; Janz, David M

    2007-09-01

    We hypothesized that exposure to metal mining effluent would reduce the ability of young-of-the-year fishes to accumulate energy reserves to survive the overwinter period (known as "winter stress syndrome") in a Canadian boreal forest watershed. Northern pike (Esox lucius) and burbot (Lota lota) were collected immediately before and after winter from a reference lake and two lakes receiving effluent. Unexpectedly, total body lipid and triglyceride, and liver triglyceride levels were greater in effluent-exposed pike and burbot in both fall and spring. However, there were no lake or season differences in growth indices of length, weight, muscle RNA/DNA ratio, or muscle protein levels in pike. In addition, total lipids and triglycerides in burbot were greater in spring compared to fall, while no seasonal differences were observed in pike, suggesting that burbot continued to feed during winter. Findings do not support the winter stress syndrome hypothesis and suggest possible direct and indirect effects of metal mining effluent on lipid dynamics of juvenile fishes.

  15. Metabolic enzymes activity and histomorphology in the liver of whitefish (Coregonus lavaretus L.) and pike (Esox lucius L.) inhabiting a mineral contaminated lake.

    PubMed

    Churova, Maria V; Murzina, Svetlana A; Meshcheryakova, Olga V; Nemova, Nina N

    2014-12-01

    The effects of wastewater from a mining and ore-dressing mill on fish in Lake Kostomukshskoe, which is used as a cesspool of circulating water and for storage of industrial wastes produced by the Kostomuksha mining and ore-dressing mill in northwest Russia, were studied. The lake is characterized by heavy mineralization, high pH, elevated levels of K(+), Li(+), SO4 (2-), NO(2-), Cl(-), Li, Mn, and Ni, and the presence of a fine-dispersed mechanical suspension. To assess the impact of contamination on fish and determine the mechanisms of their adaptation, we investigated the biochemical indices and histology of the liver of whitefish (Coregonus lavaretus L.) and pike (Esox lucius L.) inhabiting Lake Kostomukshskoe, downstream Lake Koyvas (64° 47' 30° 59'), and Lake Kamennoe, which is located in a nature preserve and has not been affected by anthropogenic activity (64° 28' 30° 13'). Changes were detected in the activity of metabolic enzymes (cytochrome c oxidase (COX), lactate dehydrogenase, and glucose-6-phosphate dehydrogenase) in the liver. Specifically, the COX activity in the liver of both fish species from the contaminated lake decreased, indicating a low level of aerobic metabolism. Lipid infiltration was the most visible and widespread change observed in the liver of both fish species; therefore, it can be considered a marker of such long-term contamination. Lesions in pike liver demonstrated a wider range of severity than in those of whitefish. In summary, metabolic enzyme activity and histomorphology of the liver of whitefish and pike differed among lakes in a species-specific manner. The changes in enzyme activity and histomorphological alterations in fish that were observed can be applied for evaluation of freshwater systems that may be subjected to mineral pollution.

  16. Assessment of larval deformities and selenium accumulation in northern pike (Esox lucius) and white sucker (Catostomus commersoni) exposed to metal mining effluent.

    PubMed

    Muscatello, Jorgelina R; Janz, David M

    2009-03-01

    Uranium mining and milling operations in northern Saskatchewan (Canada) release effluents with elevated levels of certain trace metals and metalloids, including selenium. The goal of the present study was to evaluate the presence of selenium-induced deformities in northern pike (Esox lucius) and white sucker (Catostomus commersoni) larvae originating from adults collected downstream of a uranium mine. Eggs were fertilized in the field and incubated in the laboratory following a two-way (crossover) analysis-of-variance experimental design to discriminate effects from maternal transfer versus those from exposure to site water in the developing embryos. Selenium concentrations in northern pike and white sucker eggs (8.02 and 4.89 microg/g dry wt, respectively; mean +/- standard error throughout) from the exposure site were approximately two- to threefold higher than reference (2.35 +/- 0.20 and 1.94 +/- 0.25 microg/g dry wt, respectively). Among all evaluated deformities (skeletal curvatures, craniofacial deformities, fin deformities, and edema), only edema in white sucker fry from the exposure site was slightly elevated ( approximately 3%) compared to reference. The occurrence of edema, however, can be associated with factors other than selenium (e.g., other metals and organic compounds). Both fish species displayed strong linear relationships between the selenium concentrations in eggs and other tissues (muscle, liver, kidney, and bone), suggesting that selenium concentrations in eggs could be predicted from selenium concentrations in adult tissues. The lack of a clear, toxic response in the present study is in agreement with selenium thresholds for early life-stage deformities reported in other studies, with egg selenium concentrations in northern pike and white sucker collected at the exposure site being less than the 10 microg/g (dry wt) threshold associated with the presence of deformities.

  17. The effect of hatching time on the bioenergetics of northern pike (Esox lucius) larvae from a single egg batch during the endogenous feeding period.

    PubMed

    Trabelsi, Awatef; Jaworski, Andrzej; Kamler, Ewa; Gardeur, Jean-Noël; Teletchea, Fabrice; Ayadi, Habib; Fontaine, Pascal

    2016-04-01

    Size, caloric value and chemical composition were measured separately in the progeny of two northern pike (Esox lucius) females at 3-day intervals during the endogenous feeding period from hatching to final yolk resorption. Tissue, yolk and entire larvae were analysed separately in three groups of larvae that hatched at different times (between 88 and 106 degree-days post-fertilization). An integrated approach with the Gompertz model was used to compute the yolk conversion efficiency and time to maximum tissue size in early, mid and late hatched larvae. At hatching, unresorbed yolk of early hatched larvae contained more energy (39.20 J) and more protein (0.99 mg) compared to the yolk of larvae that hatched later (38.13 J and 0.92 mg protein for late hatched larvae, p < 0.05). In contrast, a significant reduction in tissue weight (-0.7 mg DW) and protein content (-0.5 mg) was found in early hatched larvae compared to those which hatched later (p < 0.05). Between days 9 and 12 post-hatching (108 and 144 degree-days post-hatching), close to the final yolk resorption, late hatched larvae stopped growing and their tissue began to be resorbed. This tissue resorption time was delayed in early hatched larvae which presented at the end of the experiment a greater tissue weight than late hatched ones. Yolk conversion efficiency in term of energy from hatching to complete yolk resorption stage was significantly higher for early and mid hatched larvae (51%) compared to late hatched ones (44%) (p = 0.004). Furthermore, the time to maximum tissue size was found to be negatively related to hatching time which implies that early hatched larvae take longer time to switch from one developmental stage to the next. The maximum tissue dry weight and energy content were found to be reached at approximately the same age post-fertilization for both early hatched and late hatched larvae, suggesting that the principal steps in a fish's lifespan are better correlated with time of

  18. Gill reaction to pollutants from the Tamiš River in three freshwater fish species, Esox lucius L. 1758, Sander lucioperca (L. 1758) and Silurus glanis L. 1758: a comparative study.

    PubMed

    Lujić, J; Matavulj, M; Poleksić, V; Rašković, B; Marinović, Z; Kostić, D; Miljanović, B

    2015-04-01

    The study evaluated the effects of waterborne pollutants from the Tamiš River on gill histology and possible differences in gill reaction patterns between three freshwater fish species, pike Esox lucius L. 1758, pike-perch Sander lucioperca (L. 1758) and wels catfish Silurus glanis L. 1758 from the Tamiš River. Gills from analysed fish species showed moderate to intense histopathological alterations. The most frequent progressive alteration was hyperplasia of epithelium, whereas the most frequent regressive alteration was epithelial lifting. Circulatory disturbances were most often manifested in the form of hyperaemia. During comparative analysis, differences in gill indices, reaction and alteration indices, as well as in gill and filament prevalence between analysed species, were observed. Although all analysed fish species did show both progressive and regressive alterations, there was a significant difference in the level of expression of these reaction patterns. Gill index obtained for pike clearly stands out as the lowest. Wels catfish showed the highest progressive reaction index, significantly higher in comparison with the other two species (P < 0.05), while pike-perch showed the highest regressive reaction index, also significantly higher in comparison with the other species (P < 0.001). These results may implicate species-specific gill reactions and thus present a useful tool for better understanding toxic mechanisms of various pollutants.

  19. Development of new microsatellite loci and multiplex reactions for muskellunge (Esox masquinongy)

    USGS Publications Warehouse

    Sloss, Brian L.; Franckowiak, R.P.; Murphy, E.L.

    2008-01-01

    The muskellunge (Esox masquinongy) is a valued fisheries species throughout its native range. Numerous studies have documented performance and phenotypic differences among muskellunge populations, but genetic markers for assessment have been lacking. We characterized 14 microsatellite loci and developed five multiplex polymerase chain reactions. Successful amplification of northern pike (Esox lucius) was observed for seven loci. These microsatellites will be useful for analysing population structure, performance characteristics of propagated strains, and helping to develop and monitor hatchery management guidelines for muskellunge. ?? 2008 Blackwell Publishing Ltd.

  20. Mitochondrial genome of Esox flaviae (Southern pike): announcement and comparison with other Esocidae.

    PubMed

    Gandolfi, Andrea; Fontaneto, Diego; Natali, Mauro; Lucentini, Livia

    2016-07-01

    Pikes are fish species belonging to order Esociformes, family Esocidae, genus Esox. Species of the genus Esox are common, large, and economically important for food and fishing activities. Recently, a new species, southern pike E. flaviae, was described for a well-studied area such as Italy, using also two mtDNA markers: cox1 and cytb. A scant number of remnant populations of the species persist in Italy, threatened by habitat loss and degradation and by competition and possible hybridization with E. lucius, massively and recurrently stocked to sustain angling pressure. The availability of new mtDNA markers will possibly contribute to the conservation of the species. Currently, whole mitogenome information for the genus is available only for E. lucius and for E. reichertii. The aim of the present paper is to report novel mitogenomic information for southern pike.

  1. Full-length fuel rod behavior under severe accident conditions

    SciTech Connect

    Lombardo, N J; Lanning, D D; Panisko, F E

    1992-12-01

    This document presents an assessment of the severe accident phenomena observed from four Full-Length High-Temperature (FLHT) tests that were performed by the Pacific Northwest Laboratory (PNL) in the National Research Universal (NRU) reactor at Chalk River, Ontario, Canada. These tests were conducted for the US Nuclear Regulatory Commission (NRC) as part of the Severe Accident Research Program. The objectives of the test were to simulate conditions and provide information on the behavior of full-length fuel rods during hypothetical, small-break, loss-of-coolant severe accidents, in commercial light water reactors.

  2. Recovering full-length viral genomes from metagenomes

    PubMed Central

    Smits, Saskia L.; Bodewes, Rogier; Ruiz-González, Aritz; Baumgärtner, Wolfgang; Koopmans, Marion P.; Osterhaus, Albert D. M. E.; Schürch, Anita C.

    2015-01-01

    Infectious disease metagenomics is driven by the question: “what is causing the disease?” in contrast to classical metagenome studies which are guided by “what is out there?” In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral genome from a metagenomic sample. However, retrieval of a full-length genome of a divergent virus is technically challenging and can be time-consuming and costly. Here we discuss different assembly and fragment linkage strategies such as iterative assembly, motif searches, k-mer frequency profiling, coverage profile binning, and other strategies used to recover genomes of potential viral pathogens in a timely and cost-effective manner. PMID:26483782

  3. Irradiation performance of full-length metallic IFR fuels

    SciTech Connect

    Tsai, H.; Neimark, L.A.

    1992-07-01

    An assembly irradiation of 169 full-length U-Pu-Zr metallic fuel pins was successfully completed in FFTF to a goal burnup of 10 at.%. All test fuel pins maintained their cladding integrity during the irradiation. Postirradiation examination showed minimal fuel/cladding mechanical interaction and excellent stability of the fuel column. Fission-gas release was normal and consistent with the existing data base from irradiation testing of shorter metallic fuel pins in EBR-II.

  4. Molecular and Phenotypic Evidence of a New Species of Genus Esox (Esocidae, Esociformes, Actinopterygii): The Southern Pike, Esox flaviae

    PubMed Central

    Lucentini, Livia; Puletti, Maria Elena; Ricciolini, Claudia; Gigliarelli, Lilia; Fontaneto, Diego; Lanfaloni, Luisa; Bilò, Fabiana; Natali, Mauro; Panara, Fausto

    2011-01-01

    We address the taxonomic position of the southern European individuals of pike, performing a series of tests and comparisons from morphology, DNA taxonomy and population genetics parameters, in order to support the hypothesis that two species of pike, and not only one, exist in Europe. A strong relationship emerged between a northern genotype supported by COI, Cytb, AFLP and specific fragments, and a phenotype with round spot skin colour pattern and a large number of scales in the lateral line, clearly separated from a southern genotype with other skin colour pattern and a low number of scales in the lateral line. DNA taxonomy, based on a coalescent approach (GMYC) from phylogenetic reconstructions on COI and Cytb together with AFLP admixture analysis, supported the existence of two independently evolving entities. Such differences are not simply due to geographic distances, as northern European samples are more similar to Canadian and Chinese samples than the southern Europe ones. Thus, given that the differences between the two groups of European pike are significant at the phenotypic, genotypic and geographical levels, we propose the identification of two pike species: the already known northern pike (Esox lucius) and the southern pike (E. flaviae n.sp.). The correct identification of these two lineages as independent species should give rise to a ban on the introduction of northern pikes in southern Europe for recreational fishing, due to potential problems of hybridisation. PMID:22164201

  5. Molecular and phenotypic evidence of a new species of genus Esox (Esocidae, Esociformes, Actinopterygii): the southern pike, Esox flaviae.

    PubMed

    Lucentini, Livia; Puletti, Maria Elena; Ricciolini, Claudia; Gigliarelli, Lilia; Fontaneto, Diego; Lanfaloni, Luisa; Bilò, Fabiana; Natali, Mauro; Panara, Fausto

    2011-01-01

    We address the taxonomic position of the southern European individuals of pike, performing a series of tests and comparisons from morphology, DNA taxonomy and population genetics parameters, in order to support the hypothesis that two species of pike, and not only one, exist in Europe. A strong relationship emerged between a northern genotype supported by COI, Cytb, AFLP and specific fragments, and a phenotype with round spot skin colour pattern and a large number of scales in the lateral line, clearly separated from a southern genotype with other skin colour pattern and a low number of scales in the lateral line. DNA taxonomy, based on a coalescent approach (GMYC) from phylogenetic reconstructions on COI and Cytb together with AFLP admixture analysis, supported the existence of two independently evolving entities. Such differences are not simply due to geographic distances, as northern European samples are more similar to Canadian and Chinese samples than the southern Europe ones. Thus, given that the differences between the two groups of European pike are significant at the phenotypic, genotypic and geographical levels, we propose the identification of two pike species: the already known northern pike (Esox lucius) and the southern pike (E. flaviae n.sp.). The correct identification of these two lineages as independent species should give rise to a ban on the introduction of northern pikes in southern Europe for recreational fishing, due to potential problems of hybridisation.

  6. Technology development for gene discovery and full-length sequencing

    SciTech Connect

    Marcelo Bento Soares

    2004-07-19

    In previous years, with support from the U.S. Department of Energy, we developed methods for construction of normalized and subtracted cDNA libraries, and constructed hundreds of high-quality libraries for production of Expressed Sequence Tags (ESTs). Our clones were made widely available to the scientific community through the IMAGE Consortium, and millions of ESTs were produced from our libraries either by collaborators or by our own sequencing laboratory at the University of Iowa. During this grant period, we focused on (1) the development of a method for preferential cloning of tissue-specific and/or rare transcripts, (2) its utilization to expedite EST-based gene discovery for the NIH Mouse Brain Molecular Anatomy Project, (3) further development and optimization of a method for construction of full-length-enriched cDNA libraries, and (4) modification of a plasmid vector to maximize efficiency of full-length cDNA sequencing by the transposon-mediated approach. It is noteworthy that the technology developed for preferential cloning of rare mRNAs enabled identification of over 2,000 mouse transcripts differentially expressed in the hippocampus. In addition, the method that we optimized for construction of full-length-enriched cDNA libraries was successfully utilized for the production of approximately fifty libraries from the developing mouse nervous system, from which over 2,500 full-ORF-containing cDNAs have been identified and accurately sequenced in their entirety either by our group or by the NIH-Mammalian Gene Collection Program Sequencing Team.

  7. Conformational states of the full-length glucagon receptor

    PubMed Central

    Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

    2015-01-01

    Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism. PMID:26227798

  8. Development of a full-length human protein production pipeline

    PubMed Central

    Saul, Justin; Petritis, Brianne; Sau, Sujay; Rauf, Femina; Gaskin, Michael; Ober-Reynolds, Benjamin; Mineyev, Irina; Magee, Mitch; Chaput, John; Qiu, Ji; LaBaer, Joshua

    2014-01-01

    There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high-throughput (HT) methods, we transferred the genes of 31 full-length proteins into three expression vectors, and expressed the collection as N-terminal HaloTag fusion proteins in Escherichia coli and two commercial cell-free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip® GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full-length human proteins in these three expression systems. PMID:24806540

  9. Full-Length Minor Ampullate Spidroin Gene Sequence

    PubMed Central

    Chen, Gefei; Liu, Xiangqin; Zhang, Yunlong; Lin, Senzhu; Yang, Zijiang; Johansson, Jan; Rising, Anna; Meng, Qing

    2012-01-01

    Spider silk includes seven protein based fibers and glue-like substances produced by glands in the spider's abdomen. Minor ampullate silk is used to make the auxiliary spiral of the orb-web and also for wrapping prey, has a high tensile strength and does not supercontract in water. So far, only partial cDNA sequences have been obtained for minor ampullate spidroins (MiSps). Here we describe the first MiSp full-length gene sequence from the spider species Araneus ventricosus, using a multidimensional PCR approach. Comparative analysis of the sequence reveals regulatory elements, as well as unique spidroin gene and protein architecture including the presence of an unusually large intron. The spliced full-length transcript of MiSp gene is 5440 bp in size and encodes 1766 amino acid residues organized into conserved nonrepetitive N- and C-terminal domains and a central predominantly repetitive region composed of four units that are iterated in a non regular manner. The repeats are more conserved within A. ventricosus MiSp than compared to repeats from homologous proteins, and are interrupted by two nonrepetitive spacer regions, which have 100% identity even at the nucleotide level. PMID:23251707

  10. A drosophila full-length cDNA resource

    SciTech Connect

    Stapleton, Mark; Carlson, Joseph; Brokstein, Peter; Yu, Charles; Champe, Mark; George, Reed; Guarin, Hannibal; Kronmiller, Brent; Pacleb, Joanne; Park, Soo; Rubin, Gerald M.; Celniker, Susan E.

    2003-05-09

    Background: A collection of sequenced full-length cDNAs is an important resource both for functional genomics studies and for the determination of the intron-exon structure of genes. Providing this resource to the Drosophila melanogaster research community has been a long-term goal of the Berkeley Drosophila Genome Project. We have previously described the Drosophila Gene Collection (DGC), a set of putative full-length cDNAs that was produced by generating and analyzing over 250,000 expressed sequence tags (ESTs) derived from a variety of tissues and developmental stages. Results: We have generated high-quality full-insert sequence for 8,921 clones in the DGC. We compared the sequence of these clones to the annotated Release 3 genomic sequence, and identified more than 5,300 cDNAs that contain a complete and accurate protein-coding sequence. This corresponds to at least one splice form for 40 percent of the predicted D. melanogaster genes. We also identified potential new cases of RNA editing. Conclusions: We show that comparison of cDNA sequences to a high-quality annotated genomic sequence is an effective approach to identifying and eliminating defective clones from a cDNA collection and ensure its utility for experimentation. Clones were eliminated either because they carry single nucleotide discrepancies, which most probably result from reverse transcriptase errors, or because they are truncated and contain only part of the protein-coding sequence.

  11. Conformational states of the full-length glucagon receptor

    NASA Astrophysics Data System (ADS)

    Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

    2015-07-01

    Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism.

  12. Structural photoactivation of a full-length bacterial phytochrome

    PubMed Central

    Björling, Alexander; Berntsson, Oskar; Lehtivuori, Heli; Takala, Heikki; Hughes, Ashley J.; Panman, Matthijs; Hoernke, Maria; Niebling, Stephan; Henry, Léocadie; Henning, Robert; Kosheleva, Irina; Chukharev, Vladimir; Tkachenko, Nikolai V.; Menzel, Andreas; Newby, Gemma; Khakhulin, Dmitry; Wulff, Michael; Ihalainen, Janne A.; Westenhoff, Sebastian

    2016-01-01

    Phytochromes are light sensor proteins found in plants, bacteria, and fungi. They function by converting a photon absorption event into a conformational signal that propagates from the chromophore through the entire protein. However, the structure of the photoactivated state and the conformational changes that lead to it are not known. We report time-resolved x-ray scattering of the full-length phytochrome from Deinococcus radiodurans on micro- and millisecond time scales. We identify a twist of the histidine kinase output domains with respect to the chromophore-binding domains as the dominant change between the photoactivated and resting states. The time-resolved data further show that the structural changes up to the microsecond time scales are small and localized in the chromophore-binding domains. The global structural change occurs within a few milliseconds, coinciding with the formation of the spectroscopic meta-Rc state. Our findings establish key elements of the signaling mechanism of full-length bacterial phytochromes. PMID:27536728

  13. Simulations of The Dalles Dam Proposed Full Length Spillwall

    SciTech Connect

    Rakowski, Cynthia L.; Perkins, William A.; Richmond, Marshall C.; Serkowski, John A.

    2008-02-25

    This report presents results of a computational fluid dynamics (CFD) modeling study to evaluatethe impacts of a full-length spillwall at The Dalles Dam. The full-length spillwall is being designed and evaluated as a structural means to improve tailrace egress and thus survival of juvenile fish passing through the spillway. During the course of this study, a full-length spillwall at Bays 6/7 and 8/9 were considered. The U.S. Army Corps of Engineers (USACE) has proposed extending the spillwall constructed in the stilling basin between spillway Bays 6 and 7 about 590 ft farther downstream. It is believed that the extension of the spillwall will improve egress conditions for downstream juvenile salmonids by moving them more rapidly into the thalweg of the river hence reducing their exposure to predators. A numerical model was created, validated, and applied the The Dalles Dam tailrace. The models were designed to assess impacts to flow, tailrace egress, navigation, and adult salmon passage of a proposed spill wall extension. The more extensive model validation undertaken in this study greatly improved our confidence in the numerical model to represent the flow conditions in The Dalles tailrace. This study used these validated CFD models to simulate the potential impacts of a spillwall extension for The Dalles Dam tailrace for two locations. We determined the following: (1)The construction of an extended wall (between Bays 6/7) will not adversely impact entering or exiting the navigation lock. Impact should be less if a wall were constructed between Bays 8/9. (2)The construction of a wall between Bays 6/7 will increase the water surface elevation between the wall and the Washington shore. Although the increased water surface elevation would be beneficial to adult upstream migrants in that it decreases velocities on the approach to the adult ladder, the increased flow depth would enhance dissolved gas production, impacting potential operations of the project because of

  14. Analysis and Optimization of "Full-Length" Diodes

    SciTech Connect

    Schock, Alfred

    2012-01-19

    A method of analyzing the axial variation of the heat generation rate, temperature, voltage, current density and emitter heat flux in a thermionic converter is described. The method is particularly useful for the case of "long" diodes, each extending over the full length of the reactor core. For a given diode geometry and fuel distribution, the analysis combines a nuclear solution of the axial fission density profile with the iterative solution of four differential equations representing the thermal, electrical, and thermionic interactions within the diode. The digital computer program developed to solve these equations can also perform a design optimization with respect to lead resistance, load voltage, and emitter thickness, for a specified maximum emitter temperature. Typical results are presented, and the use of this analysis for predicting the diode operating characteristics is illustrated.

  15. Universal full-length nucleosome mapping sequence probe.

    PubMed

    Tripathi, Vijay; Salih, Bilal; Trifonov, Edward N

    2015-01-01

    For the computational sequence-directed mapping of the nucleosomes, the knowledge of the nucleosome positioning motifs - 10-11 base long sequences - and respective matrices of bendability, is not sufficient, since there is no justified way to fuse these motifs in one continuous nucleosome DNA sequence. Discovery of the strong nucleosome (SN) DNA sequences, with visible sequence periodicity allows derivation of the full-length nucleosome DNA bendability pattern as matrix or consensus sequence. The SN sequences of three species (A. thaliana, C. elegans, and H. sapiens) are aligned (512 sequences for each species), and long (115 dinucleotides) matrices of bendability derived for the species. The matrices have strong common property - alternation of runs of purine-purine (RR) and pyrimidine-pyrimidine (YY) dinucleotides, with average period 10.4 bases. On this basis the universal [R,Y] consensus of the nucleosome DNA sequence is derived, with exactly defined positions of respective penta- and hexamers RRRRR, RRRRRR, YYYYY, and YYYYYY.

  16. Crystal Structure of a Full-Length [beta]-Catenin

    SciTech Connect

    Xing, Yi; Takemaru, Ken-Ichi; Liu, Jing; Berndt, Jason D.; Zheng, Jie J.; Moon, Randall T.; Xu, Wenqing

    2008-08-19

    {beta}-catenin plays essential roles in cell adhesion and Wnt signaling, while deregulation of {beta}-catenin is associated with multiple diseases including cancers. Here, we report the crystal structures of full-length zebrafish {beta}-catenin and a human {beta}-catenin fragment that contains both the armadillo repeat and the C-terminal domains. Our structures reveal that the N-terminal region of the C-terminal domain, a key component of the C-terminal transactivation domain, forms a long {alpha} helix that packs on the C-terminal end of the armadillo repeat domain, and thus forms part of the {beta}-catenin superhelical core. The existence of this helix redefines our view of interactions of {beta}-catenin with some of its critical partners, including ICAT and Chibby, which may form extensive interactions with this C-terminal domain {alpha} helix. Our crystallographic and NMR studies also suggest that the unstructured N-terminal and C-terminal tails interact with the ordered armadillo repeat domain in a dynamic and variable manner.

  17. Full-length genome analysis of canine coronavirus type I.

    PubMed

    Decaro, Nicola; Mari, Viviana; Elia, Gabriella; Lanave, Gianvito; Dowgier, Giulia; Colaianni, Maria Loredana; Martella, Vito; Buonavoglia, Canio

    2015-12-02

    Canine coronavirus types I (CCoV-I) and II (CCoV-II) are usually responsible for mild enteritis in dogs. While the CCoV-II genome has been completely sequenced, to date there are no complete genomic sequence data available publicly for CCoV-I. Thus, the aim of the present study was to analyze the full-length genome of a CCoV-I prototype strain that had been recovered from a dog with diarrhea in Italy. CCoV-I strain 23/03 has a genome of 30,000 nucleotides, excluding the 3' poly(A) tail, displaying the typical Alphacoronavirus-1 organization and the highest genetic relatedness to CCoV-II. However, two distinct features were observed in the CCoV-I genome: (i) the presence of an additional ORF between the spike (S) protein gene and ORF3a; (ii) the diversity of the S protein, which is more closely related to that of feline coronavirus type I and presents a furin cleavage site. The present study may contribute to a better understanding of the Alphacoronavirus-1 evolutionary pattern and may be paradigmatic of how coronaviruses evolve through gene losses, acquisition and exchanges among different members. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Full-Length Genomic Analysis of Korean Porcine Sapelovirus Strains

    PubMed Central

    Kwon, Joseph; Choi, Jong-Soon; Kang, Mun-Il; Belsham, Graham J.; Cho, Kyoung-Oh

    2014-01-01

    Porcine sapelovirus (PSV), a species of the genus Sapelovirus within the family Picornaviridae, is associated with diarrhea, pneumonia, severe neurological disorders, and reproductive failure in pigs. However, the structural features of the complete PSV genome remain largely unknown. To analyze the structural features of PSV genomes, the full-length nucleotide sequences of three Korean PSV strains were determined and analyzed using bioinformatic techniques in comparison with other known PSV strains. The Korean PSV genomes ranged from 7,542 to 7,566 nucleotides excluding the 3′ poly(A) tail, and showed the typical picornavirus genome organization; 5′untranslated region (UTR)-L-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3′UTR. Three distinct cis-active RNA elements, the internal ribosome entry site (IRES) in the 5′UTR, a cis-replication element (CRE) in the 2C coding region and 3′UTR were identified and their structures were predicted. Interestingly, the structural features of the CRE and 3′UTR were different between PSV strains. The availability of these first complete genome sequences for PSV strains will facilitate future investigations of the molecular pathogenesis and evolutionary characteristics of PSV. PMID:25229940

  19. Crystal Structure of a Full-Length Autotransporter

    SciTech Connect

    van den Berg, B.

    2010-01-01

    The autotransporter (AT) secretion mechanism is the most common mechanism for the secretion of virulence factors across the outer membrane (OM) from pathogenic Gram-negative bacteria. In addition, ATs have attracted biotechnological and biomedical interest for protein display on bacterial cell surfaces. Despite their importance, the mechanism by which passenger domains of ATs pass the OM is still unclear. The classical view is that the {beta}-barrel domain provides the conduit through which the unfolded passenger moves, with the energy provided by vectorial folding of the {beta}-strand-rich passenger on the extracellular side of the OM. We present here the first structure of a full-length AT, the esterase EstA from Pseudomonas aeruginosa, at a resolution of 2.5 {angstrom}. EstA has a relatively narrow, 12-stranded {beta}-barrel that is covalently attached to the passenger domain via a long, curved helix that occupies the lumen of the {beta}-barrel. The passenger has a structure that is dramatically different from that of other known passengers, with a globular fold that is dominated by {alpha}-helices and loops. The arrangement of secondary-structure elements suggests that the passenger can fold sequentially, providing the driving force for passenger translocation. The esterase active-site residues are located at the apical surface of the passenger, at the entrance of a large hydrophobic pocket that contains a bound detergent molecule that likely mimics substrate. The EstA structure provides insight into AT mechanism and will facilitate the design of fusion proteins for cell surface display.

  20. Molecular Architecture of Full-Length KcsA

    PubMed Central

    Cortes, D. Marien; Cuello, Luis G.; Perozo, Eduardo

    2001-01-01

    The molecular architecture of the NH2 and COOH termini of the prokaryotic potassium channel KcsA has been determined using site-directed spin-labeling methods and paramagnetic resonance EPR spectroscopy. Cysteine mutants were generated (residues 5–24 and 121–160) and spin labeled, and the X-band CW EPR spectra were obtained from liposome-reconstituted channels at room temperature. Data on probe mobility (ΔHo−1), accessibility parameters (ΠO2 and ΠNiEdda), and inter-subunit spin-spin interaction (Ω) were used as structural constraints to build a three-dimensional folding model of these cytoplasmic domains from a set of simulated annealing and restrained molecular dynamics runs. 32 backbone structures were generated and averaged using fourfold symmetry, and a final mean structure was obtained from the eight lowest energy runs. Based on the present data, together with information from the KcsA crystal structure, a model for the three-dimensional fold of full-length KcsA was constructed. In this model, the NH2 terminus of KcsA forms an α-helix anchored at the membrane–water interface, while the COOH terminus forms a right-handed four-helix bundle that extend some 40–50 Å towards the cytoplasm. Functional analysis of COOH-terminal deletion constructs suggest that, while the COOH terminus does not play a substantial role in determining ion permeation properties, it exerts a modulatory role in the pH-dependent gating mechanism. PMID:11158168

  1. Pike Esox Lucius Distribution and Feeding Comparisons in Natural and Historically Channelized River Sections

    NASA Astrophysics Data System (ADS)

    Ivanovs, Kaspars

    2016-12-01

    During the last century a large portion of small and medium-sized rivers in Latvia were channelized, hydroelectric power stations were also built, which led to changes in the hydrodynamic conditions, geomorphological structure, as well as a change in the fish fauna. Fish are an integral part of any community in natural or man-made bodies of water. They actively participate in maintaining the system, balancing/equilibrium, energy, substance transformation and biomass production. They are able to influence other organisms in the ecosystem in which they live. The aim of the paper "Pike distribution and feeding comparisons in natural and historically channelized river sections" is to find out what pike feed on in different environments in Latvian rivers, such as natural and straightened river sections, as well as what main factors determine the composition of their food. Several points were assessed during the course of the study: the impact of environmental conditions on the feeding habits and the distribution of pike; the general feeding habits of predators in Latvian rivers; the feeding differences of predators in natural and straightened river sections; and lastly, rhithral and pothamal habitats were compared. The study was based on data from 2014 and 2015 on fish fauna monitoring. During the study, 347 pike were collected from 136 plots using electrofishing method.

  2. Length-weight relationship of northern pike, Esox lucius, from East Harbor, Ohio

    USGS Publications Warehouse

    Brown, Edward H.; Clark, Clarence F.

    1965-01-01

    The northern pike is one of Ohio's largest game fish but is well known to comparatively few anglers. Large numbers of the big fish spawn in the Ohio marshes adjacent to Lake Erie. Movements related to spawning reach a peak in late March or early April. Later the spawning population disperses and is seldom represented in catches by experimental gear or by anglers. The short period of availability was used to obtain life history information in March of 1951 through 1953. No comprehensive length-weight data for this species have previously been published from this area. East Harbor is a sandspit pond separated from Lake Erie by a large sand bar. Waters and fish populations of the harbor and lake can mix freely through a permanent connecting channel. The larger part of the 850 surface acres of the harbor is normally less than 8 feet deep. The male northern pike averaged 20.5 inches in length and ranged from 13.5 to 28.5 inches. The conspicuously larger females averaged 26.0 inches and ranged from 15.5 to 37.5 inches.

  3. Fluence rate or cumulative dose? Vulnerability of larval northern pike (Esox lucius) to ultraviolet radiation.

    PubMed

    Vehniäinen, E-R; Häkkinen, Jani M; Oikari, Aimo O J

    2007-01-01

    Newly hatched larvae of northern pike were exposed in the laboratory to four fluence rates of ultraviolet radiation (UVR; 290-400 nm) over three different time periods, resulting in total doses ranging from 3.0 +/- 0.2 to 63.0 +/- 4.4 kJ.m(-2). Mortality and behavior of the larvae were followed for 8-12 days, and growth measured at the end of the experiment. Also, the principle of reciprocity-that the UVR-induced mortality depends on the cumulative dose, independent of fluence rate-was tested. Fluence rates higher than 1480 +/- 150 mW.m(-2) caused mortality and growth retardation. The highest fluence rate (3040 +/- 210 mW.m(-2)) caused 100% mortality in 5 days. All fluence rates caused behavioral disorders, which led to death at fluence rates higher than 1480 mW.m(-2). Reciprocity failure occurred with the lowest and highest dose (550 +/- 45 and 3040 +/- 210 mW.m(-2), respectively). The results show that fluence rate is of primary importance when assessing the UVR-related risk.

  4. Fine-scale oscillatory banding in otoliths from arctic charr (Salveninus alpinus) and pike (Esox lucius)

    SciTech Connect

    Meldrum, A.; Halden, N.M.

    1997-12-31

    Transmission electron microscopy of otoliths from the inner ear of arctic charr and pike has revealed the presence of fine banding on the scale of several nanometers. The thickness of the bands was observed to vary in different portions of the sample, and some areas were not banded. EDS analysis could not detect chemical differences within the bands, but electron diffraction showed that the crystallographic orientation of the bands is related by a lattice mismatch. Previously, banding on the scale of 50 to 100 microns was observed by SEM in otoliths from arctic charr and was attributed to seasonal variations in growth. The fine-scale banding observed in this study, however, is unlikely to represent a daily variation. Electron diffraction from the pike samples shows that the material is composed of CaCO{sub 3} having the both the vaterite and aragonite structure, and hydrous CaCO{sub 3} was also observed. The large-scale banding previously identified by SEM was not observed in the TEM despite attempts to intersect the boundaries of the micron-sized layers. The interaction of the electron beam with the sample material was investigated by conducting several electron-irradiation experiments. The electron beam was observed to interact strongly with the sample and caused the precipitation of cubic CaO from the calcium carbonate matrix. Bright-field imaging showed the development of fine grained ({approximately} 5 nm) randomly oriented crystallites which accumulated with increasing electron dose. These initial results suggest that the precipitation of CaO is not driven by electron-beam beating. Previously, a similar phase-change phenomenon has been observed in hydroxyapatite from dental enamel. Other Ca-bearing biominerals may therefore also be expected to be sensitive to electron irradiation.

  5. Higher-order organisation of extremely amplified, potentially functional and massively methylated 5S rDNA in European pikes (Esox sp.).

    PubMed

    Symonová, Radka; Ocalewicz, Konrad; Kirtiklis, Lech; Delmastro, Giovanni Battista; Pelikánová, Šárka; Garcia, Sonia; Kovařík, Aleš

    2017-05-18

    Pikes represent an important genus (Esox) harbouring a pre-duplication karyotype (2n = 2x = 50) of economically important salmonid pseudopolyploids. Here, we have characterized the 5S ribosomal RNA genes (rDNA) in Esox lucius and its closely related E. cisalpinus using cytogenetic, molecular and genomic approaches. Intragenomic homogeneity and copy number estimation was carried out using Illumina reads. The higher-order structure of rDNA arrays was investigated by the analysis of long PacBio reads. Position of loci on chromosomes was determined by FISH. DNA methylation was analysed by methylation-sensitive restriction enzymes. The 5S rDNA loci occupy exclusively (peri)centromeric regions on 30-38 acrocentric chromosomes in both E. lucius and E. cisalpinus. The large number of loci is accompanied by extreme amplification of genes (>20,000 copies), which is to the best of our knowledge one of the highest copy number of rRNA genes in animals ever reported. Conserved secondary structures of predicted 5S rRNAs indicate that most of the amplified genes are potentially functional. Only few SNPs were found in genic regions indicating their high homogeneity while intergenic spacers were more heterogeneous and several families were identified. Analysis of 10-30 kb-long molecules sequenced by the PacBio technology (containing about 40% of total 5S rDNA) revealed that the vast majority (96%) of genes are organised in large several kilobase-long blocks. Dispersed genes or short tandems were less common (4%). The adjacent 5S blocks were directly linked, separated by intervening DNA and even inverted. The 5S units differing in the intergenic spacers formed both homogeneous and heterogeneous (mixed) blocks indicating variable degree of homogenisation between the loci. Both E. lucius and E. cisalpinus 5S rDNA was heavily methylated at CG dinucleotides. Extreme amplification of 5S rRNA genes in the Esox genome occurred in the absence of significant pseudogenisation

  6. Retrotransposon mdg3 of Drosophila: General structure and functional domains of the full-length copy

    SciTech Connect

    Avedisov, S.N.; Ilyin, Yu.V.

    1995-09-01

    A full-length copy of the transposable element mdg3 from the plasmid clone Dm38 of Drosophila melanogaster was obtained by screening the DNA library of the cell culture 67J25D. Previous work demonstrated that only full-length copies of mdg3 (5.5 kb) are amplified in this culture, whereas the number of deleted copies probably has not changed since the cell line was established. We sequenced the full-length copy of mdg3 from cDm38 by the method described by Sanger. 10 refs., 2 figs., 2 tabs.

  7. Fabrication and Testing of Full-Length Single-Cell Externally Fueled Converters for Thermionic Reactors

    SciTech Connect

    Schock, Alfred

    1995-08-01

    Paper presented at the 29th IECEC in Monterey, CA in August 1994. The present paper describes the fabrication and testing of full-length prototypcial converters, both unfueled and fueled, and presents parametric results of electrically heated tests.

  8. In vitro translation of the full-length RNA transcript of figwort mosaic virus (Caulimovirus).

    PubMed

    Ranu, R S; Gowda, S; Scholthof, H; Wu, F C; Shepherd, R J

    1996-01-01

    The circular DNA genome of FMV consists of seven tandemly arranged genes placed successively on a full-length RNA transcript that spans the entire circular viral genome. This transcript is a tentative mRNA for at least five of the six major conserved genes of this virus (genes I-V) that are positioned on this transcript. The sixth major gene (gene VI) is expressed as a separate monocistronic transcript. A long 5'-nontranslated leader (598 nucleotides), a small nonconserved gene (VII), and a short intergenic region (57 nucleotides) precede the five major conserved genes (I through V) on the full-length transcript. A reporter gene (CAT), as a separate cistron or fused in-frame, to viral cistrons in various downstream positions in cloned versions of the viral genome was used in a transcription vector to generate artificial full-length transcripts of FMV. When these mRNAs were translated in vitro (rabbit reticulocyte lysate system), the reporter gene was translated efficiently in all positions. Translation of internal native viral gene positioned on the full-length transcript of FMV was also determined (the gene VI product). These observations suggest that the full-length FMV transcript functions as a polycistronic mRNA in plants. Results are best explained on the basis of translational coupling/relay race model.

  9. A simple method for generating full length cDNA from low abundance partial genomic clones

    PubMed Central

    Wang, Yongxin; Fugaro, Joseph M; Siddiq, Fauzia; Goparaju, Chandra Mouli V; Lonardo, Fulvio; Wali, Anil; Lechner, John F; Pass, Harvey I

    2000-01-01

    Background PCR amplification of target molecules involves sequence specific primers that flank the region to be amplified. While this technique is generally routine, its applicability may not be sufficient to generate a desired target molecule from two separate regions involving intron /exon boundaries. For these situations, the generation of full-length complementary DNAs from two partial genomic clones becomes necessary for the family of low abundance genes. Results The first approach we used for the isolation of full-length cDNA from two known genomic clones of Hox genes was based on fusion PCR. Here we describe a simple and efficient method of amplification for homeobox D13 (HOXD13) full length cDNA from two partial genomic clones. Specific 5' and 3' untranslated region (UTR) primer pairs and website program (primer3_www.cgv0.2) were key steps involved in this process. Conclusions We have devised a simple, rapid and easy method for generating cDNA clone from genomic sequences. The full length HOXD13 clone (1.1 kb) generated with this technique was confirmed by sequence analysis. This simple approach can be utilized to generate full-length cDNA clones from available partial genomic sequences. PMID:11114844

  10. The Full-length Unprocessed Hedgehog Protein Is an Active Signaling Molecule*

    PubMed Central

    Tokhunts, Robert; Singh, Samer; Chu, Tehyen; D'Angelo, Gisela; Baubet, Valerie; Goetz, John A.; Huang, Zhen; Yuan, Ziqiang; Ascano, Manuel; Zavros, Yana; Thérond, Pascal P.; Kunes, Sam; Dahmane, Nadia; Robbins, David J.

    2010-01-01

    The hedgehog (HH) family of ligands plays an important instructional role in metazoan development. HH proteins are initially produced as ∼45-kDa full-length proteins, which undergo an intramolecular cleavage to generate an amino-terminal product that subsequently becomes cholesterol-modified (HH-Np). It is well accepted that this cholesterol-modified amino-terminal cleavage product is responsible for all HH-dependent signaling events. Contrary to this model we show here that full-length forms of HH proteins are able to traffic to the plasma membrane and participate directly in cell-cell signaling, both in vitro and in vivo. We were also able to rescue a Drosophila eye-specific hh loss of function phenotype by expressing a full-length form of hh that cannot be processed into HH-Np. These results suggest that in some physiological contexts full-length HH proteins may participate directly in HH signaling and that this novel activity of full-length HH may be evolutionarily conserved. PMID:19920144

  11. Chinese hamster ovary cells contain transcriptionally active full-length type C proviruses.

    PubMed

    Lie, Y S; Penuel, E M; Low, M A; Nguyen, T P; Mangahas, J O; Anderson, K P; Petropoulos, C J

    1994-12-01

    We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons.

  12. Secretion of full-length tau or tau fragments in a cell culture model.

    PubMed

    Pérez, Mar; Cuadros, Raquel; Hernández, Félix; Avila, Jesús

    2016-11-10

    Tau is a microtubule-associated protein that plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease. Several studies have suggested that tau may be secreted to extracellular medium and may be responsible of spreading of neurodegeneration. The overexpression of tau in cultured non-neuronal cells leads to the secretion of this protein. The proline-rich region of tau may serve as a membrane-binding site during the secretion of the full-length tau molecule. Tau fragments lacking this proline-region are either not secreted or are secreted in a distinct manner to the full-length molecule.

  13. Lentiviral vectors can be used for full-length dystrophin gene therapy.

    PubMed

    Counsell, John R; Asgarian, Zeinab; Meng, Jinhong; Ferrer, Veronica; Vink, Conrad A; Howe, Steven J; Waddington, Simon N; Thrasher, Adrian J; Muntoni, Francesco; Morgan, Jennifer E; Danos, Olivier

    2017-12-01

    Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

  14. Structure of full-length PPARgamma-RXRalpha: a snapshot of a functional complex?

    PubMed

    Moras, Dino

    2009-01-07

    The first crystal structure of a full-length nuclear receptor complex on DNA reveals an interdomain contact between the ligand- and DNA-binding domains of subunits and illuminates the role of the 5' extension of the binding site in polarity of positioning. These findings have possible functional implications.

  15. Lentiviral vectors can be used for full-length dystrophin gene therapy

    PubMed Central

    Counsell, John R.; Asgarian, Zeinab; Meng, Jinhong; Ferrer, Veronica; Vink, Conrad A.; Howe, Steven J.; Waddington, Simon N.; Thrasher, Adrian J.; Muntoni, Francesco; Morgan, Jennifer E.; Danos, Olivier

    2017-01-01

    Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a ‘template-switching’ lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD. PMID:28303972

  16. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts

    PubMed Central

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  17. Near full-length genome analysis of HCV genotype 5 strains from South Africa.

    PubMed

    Gededzha, Maemu P; Selabe, Selokela G; Blackard, Jason T; Kyaw, Thanda; Mphahlele, M Jeffrey

    2014-01-01

    Hepatitis C virus (HCV) genotype 5 is the predominant genotype in South Africa. However, to date, only 2 full-length genotype 5 genomes have been sequenced and only one is from South Africa. This study characterized HCV genotype 5 sequences from South Africa, including six near full-length genomes, as well as the E1 region from an additional 12 genotype 5 samples. Phylogenetic analysis of these near full-length genome sequences revealed that all genotype 5 sequences formed a close cluster with high bootstrap support. Bayesian analysis of the E1 region was used to estimate the time of the most recent common ancestor (tMRCA). The tMRCA for HCV genotype 5a was estimated at 114-134 years before the last sampling date. In conclusion, this study provides six near full-length genotype 5 nucleotide sequences for use as references to design efficient vaccines and for the development of new antiviral agents, and provides further insight into the diversity of HCV genotypes circulating in South Africa. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Synthesis of full length and truncated microcin B17 analogues as DNA gyrase poisons.

    PubMed

    Thompson, Robert E; Collin, Frédéric; Maxwell, Anthony; Jolliffe, Katrina A; Payne, Richard J

    2014-03-14

    Microcin B17 (MccB17) is a post-translationally modified peptide containing thiazole and oxazole heterocycles that interrupt the peptide backbone. MccB17 is capable of poisoning DNA gyrase through stabilization of the gyrase-DNA cleavage complex and has therefore attracted significant attention. Using a combination of Fmoc-strategy solid-phase peptide synthesis and solution-phase fragment assembly we have prepared a library of full-length and truncated MccB17 analogues to investigate key structural requirements for gyrase-poisoning activity. Synthetic peptides lacking the glycine-rich N-terminal portion of the full-length sequence showed strong stabilization of the gyrase-DNA cleavage complex with increased potency relative to the full-length sequences. This truncation, however, led to a decrease in antibacterial activity of these analogues relative to their full-length counterparts indicating a potential role of the N-terminal region of the natural product for cellular uptake.

  19. Native mutant huntingtin in human brain: evidence for prevalence of full-length monomer.

    PubMed

    Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B; Vonsattel, Jean-Paul; Young, Anne B; Wexler, Nancy; DiFiglia, Marian

    2012-04-13

    Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575-850 kDa in control brain and at 650-885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1-17)) and increased when lysates were treated with denaturants (SDS, 8 M urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670-880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43-50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 M urea + DTT. Native full-length mutant htt in embryonic HD(140Q/140Q) mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer.

  20. Four decades of opposing natural and human-induced artificial selection acting on Windermere pike (Esox lucius).

    PubMed

    Carlson, Stephanie M; Edeline, Eric; Asbjørn Vøllestad, L; Haugen, Thrond O; Winfield, Ian J; Fletcher, Janice M; Ben James, J; Stenseth, Nils Chr

    2007-06-01

    The ability of natural selection to drive local adaptation has been appreciated ever since Darwin. Whether human impacts can impede the adaptive process has received less attention. We tested this hypothesis by quantifying natural selection and harvest selection acting on a freshwater fish (pike) over four decades. Across the time series, directional natural selection tended to favour large individuals whereas the fishery targeted large individuals. Moreover, non-linear natural selection tended to favour intermediate sized fish whereas the fishery targeted intermediate sized fish because the smallest and largest individuals were often not captured. Thus, our results unequivocally demonstrate that natural selection and fishery selection often acted in opposite directions within this natural system. Moreover, the two selective factors combined to produce reduced fitness overall and stronger stabilizing selection relative to natural selection acting alone. The long-term ramifications of such human-induced modifications to adaptive landscapes are currently unknown and certainly warrant further investigation.

  1. UV-B exposure causes DNA damage and changes in protein expression in northern pike (Esox lucius) posthatched embryos.

    PubMed

    Vehniäinen, Eeva-Riikka; Vähäkangas, Kirsi; Oikari, Aimo

    2012-01-01

    The ongoing anthropogenically caused ozone depletion and climate change has increased the amount of biologically harmful UV-B radiation, which is detrimental to fish in embryonal stages. The effects of UV-B radiation on the levels and locations of DNA damage manifested as cyclobutane pyrimidine dimers (CPDs), heat shock protein 70 (HSP70) and p53 protein in newly hatched embryos of pike were examined. Pike larvae were exposed in the laboratory to current and enhanced doses of UV-B radiation. UV-B exposure caused the formation of CPDs in a fluence rate-dependent manner, and the CPDs were found deeper in the tissues with increasing fluence rates. UV-B radiation induced HSP70 in epidermis, and caused plausible p53 activation in the brain and epidermis of some individuals. Also at a fluence rate occurring in nature, the DNA damage in the brain and eyes of pike and changes in protein expression were followed by severe behavioral disorders, suggesting that neural molecular changes were associated with functional consequences.

  2. Historical analysis of genetic variation reveals low effective population size in a northern pike (Esox lucius) population.

    PubMed

    Miller, L M; Kapuscinski, A R

    1997-11-01

    Effective population size (Ne) of a natural fish population was estimated from temporal changes in allele frequencies at seven microsatellite loci. Use of a historical collection of fish scales made it possible to increase the precision of estimates by increasing the time interval between samples and to use an equation developed for discrete generations without correcting for demographic parameters. Estimates of Ne for the time intervals 1961-1977 and 1977-1993 were 35 and 72, respectively. For the entire interval, 1961-1993, the estimate of Ne was 48 when based on a weighted mean derived from the above two estimates or 125 when calculated from 1961 and 1993 samples only. Corresponding ratios of effective size to adult census size ranged from 0.03 to 0.14. An Ne of 48 over a 32-year period would imply that this population lost as much as 8% of its heterozygosity in that time. Results suggest the potential for using genetic methods based on microsatellite loci data to compare historical trends in Ne with population dynamic parameters. Such comparisons will help to evaluate the relationship between genetic diversity and long-term persistence of natural populations.

  3. Historical Analysis of Genetic Variation Reveals Low Effective Population Size in a Northern Pike (Esox Lucius) Population

    PubMed Central

    Miller, L. M.; Kapuscinski, A. R.

    1997-01-01

    Effective population size (N(e)) of a natural fish population was estimated from temporal changes in allele frequencies at seven microsatellite loci. Use of a historical collection of fish scales made it possible to increase the precision of estimates by increasing the time interval between samples and to use an equation developed for discrete generations without correcting for demographic parameters. Estimates of N(e) for the time intervals 1961-1977 and 1977-1993 were 35 and 72, respectively. For the entire interval, 1961-1993, the estimate of N(e) was 48 when based on a weighted mean derived from the above two estimates or 125 when calculated from 1961 and 1993 samples only. Corresponding ratios of effective size to adult census size ranged from 0.03 to 0.14. An N(e) of 48 over a 32-year period would imply that this population lost as much as 8% of its heterozygosity in that time. Results suggest the potential for using genetic methods based on microsatellite loci data to compare historical trends in N(e) with population dynamic parameters. Such comparisons will help to evaluate the relationship between genetic diversity and long-term persistence of natural populations. PMID:9383067

  4. Investigation of parasites of pikes (Esox lucius Linnaeus, 1785) from Chamkhale River, Anzali and Amirkelayeh wetlands, Iran.

    PubMed

    Sadrinejad, Ali; Khara, Hossein; Gudarzi, Majid

    2016-09-01

    To investigate the health status, the parasite diversity and abundance were studied in three pike populations. For this purpose, totally 90 fish were caught from three natural habitats of pike including Chamkhale River, Anzali and Amirkelayeh wetlands, Iran. After biometry and age determination, fish were dissected and parasites were investigated in intestine, gill and eye. According to results, five parasite species were identified in whole body of pikes including Eustrongylides exises, Raphidascaris acus and Triaeophorus crassus in intestine, Tetraonchus monenteron in gill and Diplostomum spathaceum in eye. The highest values of quantitative indices of parasite population [i.e. incidence percent (86.66 %), mean intensity (60.83 ± 39.92), mean abundance (58.14 ± 34.6) and abundance range (1-232)] were recorded for T. monenteron in all three sampling regions. The lowest values of these quantitative indices were found for D. spathaceum, E. exises and Tetraonchus crassus in Anzali wetland, Chamkhale River and Amirkelayeh wetland respectively. In 2, 3 and 4 years old pikes, T. monenteron had the highest values of quantitative indices of parasite population. In conclusion, our results showed that Anzali wetland have more parasite pollution compared to other sampling regions. This may be due to the more rapid eutrophication in this wetland compared to Amirkelayeh wetland and Chamkhale River.

  5. Pathogenicity of the gill artery worm Philometra obturans (Nematoda) in northern pike (Esox lucius) in southwest Finland.

    PubMed

    Kåll, S; Fagerholm, H P; Sarvala, J

    2004-02-01

    The pathogenicity of female Philometra obturans (Prenant, 1886) was investigated. Fish samples were taken in May and June 2001 from Lake Littoistenjärvi, in southwest Finland. The intensity of infection varied between 1 and 4 worms, these being found in the afferent artery of the 2 outermost gill arches. The prevalence was 33.9% (n = 59). Host condition and tissue reactions were investigated through dissection and analysis of histopathology. The parasites obstructed the gill arteries, and the arterial wall was stretched around intact worms. Hypertrophy of the arterial wall was observed, especially when only disintegrating remains of the parasites were present. Infected pike were in a poorer condition after catch, transportation, and holding in aquaria and thus less tolerant to stress than uninfected pike. This was correlated with the number of infected gill arteries (P < 0.05). The results show that infection with this nematode has a detrimental effect on the host when combined with environmental or physiological stress and may result in host mortality.

  6. Construction of an infectious full-length cDNA clone of potato virus M.

    PubMed

    Flatken, S; Ungewickell, V; Menzel, W; Maiss, E

    2008-01-01

    An infectious full-length cDNA clone of potato virus M (PVM) was produced. Total RNA was extracted from PVM-infected Nicotiana hesperis plants and used for cDNA synthesis. Subsequent RT-PCR produced two DNA fragments of about 5.5 and 3.2 kbp, which were ligated downstream of an enhanced 35S cauliflower mosaic virus promoter. After cloning of the enhanced 35S promoter with the PVM sequence into a modified pBIN19 plasmid and electroporation of Agrobacterium tumefaciens, the agroinoculated PVM full-length clone (pPVM-flc) led to systemic PVM infections in different host plants, causing symptoms indistinguishable from those caused by wild-type PVM.

  7. Structure of the full-length TRPV2 channel by cryo-EM

    NASA Astrophysics Data System (ADS)

    Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

    2016-03-01

    Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a `minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ~5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

  8. Genetic characterization of near full length SIVdrl genomes from four captive drills (Mandrillus leucophaeus).

    PubMed

    Dietrich, Ursula; Landersz, Margot; Stahl-Hennig, Christiane; Geiger, Christina; Foley, Brian T

    2015-03-01

    We sequenced near full length SIVdrl genomes from four captive drills (Mandrillus leucophaeus). All four animals were born in captivity in German zoos. Although serologically SIV negative before acquisition in zoo A in 2008 and 2009, during a routine analysis all four animals were determined to be SIV antibody positive in 2011. Comparisons of the four new SIVdrl sequences showed high identity among each other (90.7-97.7% in env) and to the only published full length sequence SIVdrl FAO (90.5-92.8% in env), which is also derived from a captive drill. SIVdrl infections seem to be highly prevalent in captive drills, probably resulting from frequent animal transfers between the zoos in an effort to maintain this highly endangered species and its genetic diversity. This should be kept in mind as SIVdrl may be transmitted to uninfected animals in open groups and potentially also to animal keepers having contact with these nonhuman primates.

  9. 3G vector-primer plasmid for constructing full-length-enriched cDNA libraries.

    PubMed

    Zheng, Dong; Zhou, Yanna; Zhang, Zidong; Li, Zaiyu; Liu, Xuedong

    2008-09-01

    We designed a 3G vector-primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector-primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5' end of RNA transcript) approach.

  10. Full-length RNA-seq from single cells using Smart-seq2.

    PubMed

    Picelli, Simone; Faridani, Omid R; Björklund, Asa K; Winberg, Gösta; Sagasser, Sven; Sandberg, Rickard

    2014-01-01

    Emerging methods for the accurate quantification of gene expression in individual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multiplexing ability. We recently introduced Smart-seq for transcriptome analysis from single cells, and we subsequently optimized the method for improved sensitivity, accuracy and full-length coverage across transcripts. Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. The entire protocol takes ∼2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1-3 d depending on the strategy and sequencer. The current limitations are the lack of strand specificity and the inability to detect nonpolyadenylated (polyA(-)) RNA.

  11. Calcium-dependent regulation of the motor activity of recombinant full-length Physarum myosin.

    PubMed

    Zhang, Ying; Kawamichi, Hozumi; Tanaka, Hideyuki; Yoshiyama, Shinji; Kohama, Kazuhiro; Nakamura, Akio

    2012-08-01

    We successfully synthesized full-length and the mutant Physarum myosin and heavy meromyosin (HMM) constructs associated with Physarum regulatory light chain and essential light chain (PhELC) using Physarum myosin heavy chain in Sf-9 cells, and examined their Ca(2+)-mediated regulation. Ca(2+) inhibited the motility and ATPase activities of Physarum myosin and HMM. The Ca(2+) effect is also reversible at the in vitro motility of Physarum myosin. We demonstrated that full-length myosin increases the Ca(2+) inhibition more effectively than HMM. Furthermore, Ca(2+) did not affect the motility and ATPase activities of the mutant Physarum myosin with PhELC that lost Ca(2+)-binding ability. Therefore, we conclude that PhELC plays a critical role in Ca(2+)-dependent regulation of Physarum myosin.

  12. Full-length high-temperature severe fuel damage test No. 1

    SciTech Connect

    Rausch, W.N.; Hesson, G.M.; Pilger, J.P.; King, L.L.; Goodman, R.L.; Panisko, F.E.

    1993-08-01

    This report describes the first full-length high-temperature test (FLHT-1) performed by Pacific Northwest Laboratory (PNL) in the National Research Universal (NRU) reactor at Chalk River, Ontario, Canada. The test is part of a series of experiments being performed for the NRC as a part of their Severe Fuel Damage Program and is one of several planned for PNL`s Coolant Boilaway and Damage Progression Program. The report summarizes the test design and test plan. it also provides a summary and discussion of the data collected during the test and of the photos taken during the post-test examination. All objectives for the test were met. The key objective was to demonstrate that severe fuel damage tests on full-length fuel bundles can be safely conducted in the NRU reactor.

  13. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

    PubMed

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K

    2016-01-29

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Results of simulated abnormal heating events for full-length nuclear fuel rods

    SciTech Connect

    Guenther, R.J.

    1983-01-01

    Full-length nuclear fuel rods were tested in a furnace to simulate the slow heating rates postulated for commercial pressurized water reactor fuel rods exposed to an overheating event in a storage cask. Fuel rod temperatures and internal gas pressures were monitored during the test and are presented along with mensural data for cladding. Metallography of the cladding provided data on grain growth, hydriding, oxidation, cladding stresses, and the general nature of the failures.

  15. A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling

    PubMed Central

    Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

    2017-01-01

    Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein–EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here. PMID:28099529

  16. A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling.

    PubMed

    Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

    2017-01-01

    Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein-EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here.

  17. Scalable Production in Human Cells and Biochemical Characterization of Full-Length Normal and Mutant Huntingtin

    PubMed Central

    Huang, Bin; Lucas, Tanja; Kueppers, Claudia; Dong, Xiaomin; Krause, Maike; Bepperling, Alexander; Buchner, Johannes; Voshol, Hans; Weiss, Andreas; Gerrits, Bertran; Kochanek, Stefan

    2015-01-01

    Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington’s disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on “gutless” adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different. PMID:25799558

  18. Synaptonemal complex extension from clustered telomeres mediates full-length chromosome pairing in Schmidtea mediterranea

    PubMed Central

    Xiang, Youbin; Miller, Danny E.; Ross, Eric J.; Sánchez Alvarado, Alejandro; Hawley, R. Scott

    2014-01-01

    In the 1920s, József Gelei proposed that chromosome pairing in flatworms resulted from the formation of a telomere bouquet followed by the extension of synapsis from telomeres at the base of the bouquet, thus facilitating homolog pairing in a processive manner. A modern interpretation of Gelei’s model postulates that the synaptonemal complex (SC) is nucleated close to the telomeres and then extends progressively along the full length of chromosome arms. We used the easily visible meiotic chromosomes, a well-characterized genome, and RNAi in the sexual biotype of the planarian Schmidtea mediterranea to test that hypothesis. By identifying and characterizing S. mediterranea homologs of genes encoding synaptonemal complex protein 1 (SYCP1), the topoisomerase-like protein SPO11, and RAD51, a key player in homologous recombination, we confirmed that SC formation begins near the telomeres and progresses along chromosome arms during zygotene. Although distal regions pair at the time of bouquet formation, pairing of a unique interstitial locus is not observed until the formation of full-length SC at pachytene. Moreover, neither full extension of the SC nor homologous pairing is dependent on the formation of double-strand breaks. These findings validate Gelei’s speculation that full-length pairing of homologous chromosomes is mediated by the extension of the SC formed near the telomeres. S. mediterranea thus becomes the first organism described (to our knowledge) that forms a canonical telomere bouquet but does not require double-strand breaks for synapsis between homologous chromosomes. However, the initiation of SC formation at the base of the telomere bouquet, which then is followed by full-length homologous pairing in planarian spermatocytes, is not observed in other species and may not be conserved. PMID:25404302

  19. Full-length high-temperature severe fuel damage test No. 2. Final safety analysis

    SciTech Connect

    Hesson, G.M.; Lombardo, N.J.; Pilger, J.P.; Rausch, W.N.; King, L.L.; Hurley, D.E.; Parchen, L.J.; Panisko, F.E.

    1993-09-01

    Hazardous conditions associated with performing the Full-Length High- Temperature (FLHT). Severe Fuel Damage Test No. 2 experiment have been analyzed. Major hazards that could cause harm or damage are (1) radioactive fission products, (2) radiation fields, (3) reactivity changes, (4) hydrogen generation, (5) materials at high temperature, (6) steam explosion, and (7) steam pressure pulse. As a result of this analysis, it is concluded that with proper precautions the FLHT- 2 test can be safely conducted.

  20. 78 FR 13071 - Guidance for Industry: Implementation of an Acceptable Full-Length and Abbreviated Donor History...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-26

    ...- Length and Abbreviated Donor History Questionnaires and Accompanying Materials for Use in Screening... ``Guidance for Industry: Implementation of an Acceptable Full-Length and Abbreviated Donor History... guidance document recognizes the standardized full-length and abbreviated donor history questionnaires...

  1. Computational analysis of full-length mouse cDNAs compared with human genome sequences.

    PubMed

    Kondo, S; Shinagawa, A; Saito, T; Kiyosawa, H; Yamanaka, I; Aizawa, K; Fukuda, S; Hara, A; Itoh, M; Kawai, J; Shibata, K; Hayashizaki, Y

    2001-09-01

    Although the sequencing of the human genome is complete, identification of encoded genes and determination of their structures remain a major challenge. In this report, we introduce a method that effectively uses full-length mouse cDNAs to complement efforts in carrying out these difficult tasks. A total of 61,227 RIKEN mouse cDNAs (21,076 full-length and 40,151 EST sequences containing certain redundancies) were aligned with the draft human sequences. We found 35,141 non-redundant genomic regions that showed a significant alignment with the mouse cDNAs. We analyzed the structures and compositional properties of the regions detected by the full-length cDNAs, including cross-species comparisons, and noted a systematic bias of GENSCAN against exons of small size and/or low GC-content. Of the cDNAs locating the 35,141 genomic regions, 3,217 did not match any sequences of the known human genes or ESTs. Among those 3,217 cDNAs, 1,141 did not show any significant similarity to any protein sequence in the GenBank non-redundant protein database and thus are candidates for novel genes.

  2. Construction of a full-length infectious cDNA clone of Cowpea mild mottle virus.

    PubMed

    Carvalho, Silvia L; Nagata, Tatsuya; Junqueira, Bruna R; Zanardo, Larissa G; Paiva, Ana C S; Carvalho, Claudine M

    2017-02-01

    Infectious cDNA clones are an important tool to study the molecular and cellular process of RNA virus infection. In vitro and in vivo transcription systems are the two main strategies used in the generation of infectious cDNA clones for RNA viruses. This study describes the first generation of a full-length infectious cDNA clone of Cowpea mild mottle virus (CPMMV), a Carlavirus. The full-length genome was synthesized by Overlap Extension PCR of two overlapping fragments and cloned in a pUC-based vector under control of the SP6 RNA polymerase promoter. After in vitro run-off transcription, the produced RNA was mechanically inoculated into soybean plants cv. CD206. The systemic infection was confirmed by RT-PCR and further sequencing of amplified cDNA fragments. To simplify the transfection process, the complete genome was subcloned into a binary vector under control of the 35S promoter of cauliflower mosaic virus by the Gibson Assembly protocol. The resulting clones were inoculated by particle bombardment onto soybean seedlings and the recovery of the virus was confirmed 2 weeks later by RT-PCR. Our results indicate the constructs of the full-length cDNA of CPMMV are fully infectious in both in vitro and in vivo transcription strategies.

  3. Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction.

    PubMed

    Zhu, Y Y; Machleder, E M; Chenchik, A; Li, R; Siebert, P D

    2001-04-01

    Here, we describe a fast, simple method for constructing full-length cDNA libraries using SMART technology. This novel procedure uses the template-switching activity of Moloney murine leukemia virus (MMLV) reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, three cycles of PCR are performed using a modified oligo(dT) primer and an anchor primer to enrich the cDNA population for full-length sequences. Starting with 1 microgram human skeletal muscle poly(A)+ RNA, a cDNA library was constructed that contained 3 x 10(6) independent clones with an average insert size of 2 kb. Sequence analysis of 172 randomly selected clones showed that 77% of cDNA clones corresponding to known genes contained intact open reading frames. The average length of complete open reading frames was 2.4 kb. Furthermore, 86% of the full-length clones retained longer 5' UTR sequences than the longest 5' end deposited in the GenBank database. cDNA libraries generated using this method will be useful for accelerating the collection of mRNA 5' end sequence information, which is currently very limited in GenBank.

  4. An improved and validated RNA HLA class I SBT approach for obtaining full length coding sequences.

    PubMed

    Gerritsen, K E H; Olieslagers, T I; Groeneweg, M; Voorter, C E M; Tilanus, M G J

    2014-11-01

    The functional relevance of human leukocyte antigen (HLA) class I allele polymorphism beyond exons 2 and 3 is difficult to address because more than 70% of the HLA class I alleles are defined by exons 2 and 3 sequences only. For routine application on clinical samples we improved and validated the HLA sequence-based typing (SBT) approach based on RNA templates, using either a single locus-specific or two overlapping group-specific polymerase chain reaction (PCR) amplifications, with three forward and three reverse sequencing reactions for full length sequencing. Locus-specific HLA typing with RNA SBT of a reference panel, representing the major antigen groups, showed identical results compared to DNA SBT typing. Alleles encountered with unknown exons in the IMGT/HLA database and three samples, two with Null and one with a Low expressed allele, have been addressed by the group-specific RNA SBT approach to obtain full length coding sequences. This RNA SBT approach has proven its value in our routine full length definition of alleles.

  5. Pleiotrophin Gene Therapy for Peripheral Ischemia: Evaluation of Full-Length and Truncated Gene Variants

    PubMed Central

    Fang, Qizhi; Mok, Pamela Y.; Thomas, Anila E.; Haddad, Daniel J.; Saini, Shereen A.; Clifford, Brian T.; Kapasi, Neel K.; Danforth, Olivia M.; Usui, Minako; Ye, Weisheng; Luu, Emmy; Sharma, Rikki; Bartel, Maya J.; Pathmanabhan, Jeremy A.; Ang, Andrew A. S.; Sievers, Richard E.; Lee, Randall J.; Springer, Matthew L.

    2013-01-01

    Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model. PMID:23630585

  6. Crystallization and X-ray structure of full-length recombinant human butyrylcholinesterase

    SciTech Connect

    Ngamelue, Michelle N.; Homma, Kohei; Lockridge, Oksana; Asojo, Oluwatoyin A.

    2007-09-01

    The first crystals and the 2.8 Å X-ray structure of full-length recombinant human butyrylcholinesterase are reported. Human butyrylcholinesterase (BChE) has been shown to function as an endogenous scavenger of diverse poisons. BChE is a 340 kDa tetrameric glycoprotein that is present in human serum at a concentration of 5 mg l{sup −1}. The well documented therapeutic effects of BChE on cocaine toxicity and organophosphorus agent poisoning has increased the need for effective methods of producing recombinant therapeutic BChE. In order to be therapeutically useful, BChE must have a long circulatory residence time or associate as tetramers. Full-length recombinant BChE produced in Chinese hamster ovary (CHO) cells or human embryonic kidney cells has been shown to associate as monomers, with a shorter circulatory residence time than the naturally occurring tetrameric serum protein. Based on the preceding observation as well as the need to develop novel methodologies to facilitate the mass production of therapeutic recombinant BChE, studies have been initiated to determine the structural basis of tetramer formation. Towards these ends, full-length monomeric recombinant BChE has been crystallized for the first time. A 2.8 Å X-ray structure was solved in space group P42{sub 1}2, with unit-cell parameters a = b = 156, c = 146 Å.

  7. cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing

    PubMed Central

    Hartwig, Benjamin; Reinhardt, Richard; Schneeberger, Korbinian

    2016-01-01

    The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci. PMID:27327613

  8. Defining 'full-length' recombinant factor VIII: a comparative structural analysis.

    PubMed

    Jankowski, M A; Patel, H; Rouse, J C; Marzilli, L A; Weston, S B; Sharpe, P J

    2007-01-01

    Coagulation factor VIII (FVIII) is an important glycoprotein co-factor involved in haemostasis, functioning to accelerate activation of factor X by activated factor IX. Insertion of expression vectors containing the full-length cDNA sequence of human FVIII into mammalian cell lines results in the production of recombinant factor VIII (rFVIII), typically referred to as 'full-length' rFVIII (FLrFVIII). Both FLrFVIII and plasma-derived FVIII exist primarily as heterodimeric proteins, consisting of a heterogenous light and heavy chain. The objectives of this study were to compare the structural heterogeneity of high-purity FVIII preparations and further define the term 'full length' as it refers to rFVIII protein structure. Five commercially available FVIII concentrates were characterized based on SDS-PAGE, N-terminal sequencing, and peptide and domain mapping coupled to mass spectrometry. The major heavy chain species identified in FLrFVIII included various B-domain-truncated forms of FVIII, with the predominant species terminating at Arg(1313). This study demonstrates that the use of full-sequence FVIII cDNA for the production of rFVIII does not result in a homogeneous FLrFVIII protein product. Rather, commercially available FLrFVIII represents a heterogenous mixture of various B-domain-truncated forms of the molecule, with no evidence of a contiguous, intact B-domain.

  9. Pulp regeneration in a full-length human tooth root using a hierarchical nanofibrous microsphere system.

    PubMed

    Li, Xiangwei; Ma, Chi; Xie, Xiaohua; Sun, Hongchen; Liu, Xiaohua

    2016-04-15

    While pulp regeneration using tissue engineering strategy has been explored for over a decade, successful regeneration of pulp tissues in a full-length human root with a one-end seal that truly simulates clinical endodontic treatment has not been achieved. To address this challenge, we designed and synthesized a unique hierarchical growth factor-loaded nanofibrous microsphere scaffolding system. In this system, vascular endothelial growth factor (VEGF) binds with heparin and is encapsulated in heparin-conjugated gelatin nanospheres, which are further immobilized in the nanofibers of an injectable poly(l-lactic acid) (PLLA) microsphere. This hierarchical microsphere system not only protects the VEGF from denaturation and degradation, but also provides excellent control of its sustained release. In addition, the nanofibrous PLLA microsphere integrates the extracellular matrix-mimicking architecture with a highly porous injectable form, efficiently accommodating dental pulp stem cells (DPSCs) and supporting their proliferation and pulp tissue formation. Our in vivo study showed the successful regeneration of pulp-like tissues that fulfilled the entire apical and middle thirds and reached the coronal third of the full-length root canal. In addition, a large number of blood vessels were regenerated throughout the canal. For the first time, our work demonstrates the success of pulp tissue regeneration in a full-length root canal, making it a significant step toward regenerative endodontics. The regeneration of pulp tissues in a full-length tooth root canal has been one of the greatest challenges in the field of regenerative endodontics, and one of the biggest barriers for its clinical application. In this study, we developed a unique approach to tackle this challenge, and for the first time, we successfully regenerated living pulp tissues in a full-length root canal, making it a significant step toward regenerative endodontics. This study will make positive scientific

  10. Transition from meeting abstract to full-length journal article for randomized controlled trials.

    PubMed

    Toma, Mustafa; McAlister, Finlay A; Bialy, Liza; Adams, Denise; Vandermeer, Ben; Armstrong, Paul W

    2006-03-15

    Not all research presented at scientific meetings is subsequently published and, even when it is, there may be inconsistencies between these results and what is ultimately printed. Although late-breaking trials sessions are now integrated into several major scientific meetings and the results are often promptly and prominently communicated, no studies have examined the publication fate and degree of consistency between meeting abstracts or presentations and subsequent full-length article publications for randomized controlled trials (RCTs) presented at these sessions. To compare RCT abstracts presented in the late-breaking trials session vs other sessions at a major scientific meeting and subsequent full-length publications. RCTs were identified by hand searching abstract proceedings booklets and related Web sites for the American College of Cardiology scientific meetings (1999-2002). Subsequent full-length articles were identified via electronic databases. Publication fate and degree of consistency between meeting abstract results and subsequent full-length publication results. The 86 late-breaking RCTs were significantly larger (median, 2737 patients vs 896; P<.001), were more likely to be preceded by a published design paper (27 [31%] vs 13 [13%]; P = .002), had higher quality scores when eventually published (mean Jadad score 2.69 vs 2.19; P = .01), and were less likely to report favorable results for the intervention than the 100 randomly chosen comparison RCTs presented in other sessions (50 [58%] vs 75 [75%]; P = .01; odds ratio 0.46; 95% confidence interval, 0.24-0.90). RCTs presented at the late-breaking trials sessions were significantly more likely to be published (79 [92%] vs 69 [69%]; P<.001) and appeared earlier after presentation (median 11.5 months vs 22.0 months; P<.001) than RCTs presented in other sessions, an association that persisted even after adjusting for sample size, conclusion of study, and RCT design: adjusted hazard ratio, 1.80 (95

  11. Regulation of tumor growth by circulating full-length chromogranin A

    PubMed Central

    Gasparri, Anna; Sacchi, Angelina; Colombo, Barbara; Fiocchi, Martina; Perani, Laura; Venturini, Massimo; Tacchetti, Carlo; Sen, Suvajit; Borges, Ricardo; Dondossola, Eleonora; Esposito, Antonio; Mahata, Sushil K.; Corti, Angelo

    2016-01-01

    Chromogranin A (CgA), a neuroendocrine secretory protein, and its fragments are present in variable amounts in the blood of normal subjects and cancer patients. We investigated whether circulating CgA has a regulatory function in tumor biology and progression. Systemic administration of full-length CgA, but not of fragments lacking the C-terminal region, could reduce tumor growth in murine models of fibrosarcoma, mammary adenocarcinoma, Lewis lung carcinoma, and primary and metastatic melanoma, with U-shaped dose-response curves. Tumor growth inhibition was associated with reduction of microvessel density and blood flow in neoplastic tissues. Neutralization of endogenous CgA with antibodies against its C-terminal region (residues 410-439) promoted tumor growth. Structure-function studies showed that the C-terminal region of CgA contains a bioactive site and that cleavage of this region causes a marked loss of anti-angiogenic and anti-tumor potency. Mechanistic studies showed that full-length CgA could induce, with a U-shaped dose-response curve, the production of protease nexin-1 in endothelial cells, a serine protease inhibitor endowed of anti-angiogenic activity. Gene silencing or neutralization of protease nexin-1 with specific antibodies abolished both anti-angiogenic and anti-tumor effects of CgA. These results suggest that circulating full-length CgA is an important inhibitor of angiogenesis and tumor growth, and that cleavage of its C-terminal region markedly reduces its activity. Pathophysiological changes in CgA blood levels and/or its fragmentation might regulate disease progression in cancer patients. PMID:27683038

  12. BioRAT: extracting biological information from full-length papers.

    PubMed

    Corney, David P A; Buxton, Bernard F; Langdon, William B; Jones, David T

    2004-11-22

    Converting the vast quantity of free-format text found in journals into a concise, structured format makes the researcher's quest for information easier. Recently, several information extraction systems have been developed that attempt to simplify the retrieval and analysis of biological and medical data. Most of this work has used the abstract alone, owing to the convenience of access and the quality of data. Abstracts are generally available through central collections with easy direct access (e.g. PubMed). The full-text papers contain more information, but are distributed across many locations (e.g. publishers' web sites, journal web sites and local repositories), making access more difficult. In this paper, we present BioRAT, a new information extraction (IE) tool, specifically designed to perform biomedical IE, and which is able to locate and analyse both abstracts and full-length papers. BioRAT is a Biological Research Assistant for Text mining, and incorporates a document search ability with domain-specific IE. We show first, that BioRAT performs as well as existing systems, when applied to abstracts; and second, that significantly more information is available to BioRAT through the full-length papers than via the abstracts alone. Typically, less than half of the available information is extracted from the abstract, with the majority coming from the body of each paper. Overall, BioRAT recalled 20.31% of the target facts from the abstracts with 55.07% precision, and achieved 43.6% recall with 51.25% precision on full-length papers.

  13. A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification

    PubMed Central

    2013-01-01

    Background Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. Results In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3′-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. Conclusions The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are

  14. TargetIdentifier: a webserver for identifying full-length cDNAs from EST sequences

    PubMed Central

    Min, Xiang Jia; Butler, Gregory; Storms, Reginald; Tsang, Adrian

    2005-01-01

    TargetIdentifier is a webserver that identifies full-length cDNA sequences from the expressed sequence tag (EST)-derived contig and singleton data. To accomplish this TargetIdentifier uses BLASTX alignments as a guide to locate protein coding regions and potential start and stop codons. This information is then used to determine whether the EST-derived sequences include their translation start codons. The algorithm also uses the BLASTX output to assign putative functions to the query sequences. The server is available at . PMID:15980559

  15. Full-Length High-Temperature Severe Fuel Damage Test No. 5: Final safety analysis

    SciTech Connect

    Lanning, D.D.; Lombardo, N.J.; Panisko, F.E.

    1993-09-01

    This report presents the final safety analysis for the preparation, conduct, and post-test discharge operation for the Full-Length High Temperature Experiment-5 (FLHT-5) to be conducted in the L-24 position of the National Research Universal (NRU) Reactor at Chalk River Nuclear Laboratories (CRNL), Ontario, Canada. The test is sponsored by an international group organized by the US Nuclear Regulatory Commission. The test is designed and conducted by staff from Pacific Northwest Laboratory with CRNL staff support. The test will study the consequences of loss-of-coolant and the progression of severe fuel damage.

  16. Full-length high-temperature severe fuel damage test No. 5

    SciTech Connect

    Lanning, D.D.; Lombardo, N.J.; Hensley, W.K.; Fitzsimmons, D.E.; Panisko, F.E.; Hartwell, J.K.

    1993-09-01

    This report describes and presents data from a severe fuel damage test that was conducted in the National Research Universal (NRU) reactor at Chalk River Nuclear Laboratories (CRNL), Ontario, Canada. The test, designated FLHT-5, was the fourth in a series of full-length high-temperature (FLHT) tests on light-water reactor fuel. The tests were designed and performed by staff from the US Department of Energy`s Pacific Northwest Laboratory (PNL), operated by Battelle Memorial Institute. The test operation and test results are described in this report. The fuel bundle in the FLHT-5 experiment included 10 unirradiated full-length pressurized-water reactor (PWR) rods, 1 irradiated PWR rod and 1 dummy gamma thermometer. The fuel rods were subjected to a very low coolant flow while operating at low fission power. This caused coolant boilaway, rod dryout and overheating to temperatures above 2600 K, severe fuel rod damage, hydrogen generation, and fission product release. The test assembly and its effluent path were extensively instrumented to record temperatures, pressures, flow rates, hydrogen evolution, and fission product release during the boilaway/heatup transient. Post-test gamma scanning of the upper plenum indicated significant iodine and cesium release and deposition. Both stack gas activity and on-line gamma spectrometer data indicated significant ({approximately}50%) release of noble fission gases. Post-test visual examination of one side of the fuel bundle revealed no massive relocation and flow blockage; however, rundown of molten cladding was evident.

  17. Full-length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells.

    PubMed

    Meregalli, Mirella; Navarro, Claire; Sitzia, Clementina; Farini, Andrea; Montani, Erica; Wein, Nicolas; Razini, Paola; Beley, Cyriaque; Cassinelli, Letizia; Parolini, Daniele; Belicchi, Marzia; Parazzoli, Dario; Garcia, Luis; Torrente, Yvan

    2013-12-01

    The protein dysferlin is abundantly expressed in skeletal and cardiac muscles, where its main function is membrane repair. Mutations in the dysferlin gene are involved in two autosomal recessive muscular dystrophies: Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. Development of effective therapies remains a great challenge. Strategies to repair the dysferlin gene by skipping mutated exons, using antisense oligonucleotides (AONs), may be suitable only for a subset of mutations, while cell and gene therapy can be extended to all mutations. AON-treated blood-derived CD133+ stem cells isolated from patients with Miyoshi myopathy led to partial dysferlin reconstitution in vitro but failed to express dysferlin after intramuscular transplantation into scid/blAJ dysferlin null mice. We thus extended these experiments producing the full-length dysferlin mediated by a lentiviral vector in blood-derived CD133+ stem cells isolated from the same patients. Transplantation of engineered blood-derived CD133+ stem cells into scid/blAJ mice resulted in sufficient dysferlin expression to correct functional deficits in skeletal muscle membrane repair. Our data suggest for the first time that lentivirus-mediated delivery of full-length dysferlin in stem cells isolated from Miyoshi myopathy patients could represent an alternative therapeutic approach for treatment of dysferlinopathies.

  18. Structural Organization of a Full-Length Gp130/LIF-R Cytokine Receptor Transmembrane Complex

    SciTech Connect

    Skiniotis, G.; Lupardus, P.J.; Martick, M.; Walz, T.; Garcia, K.C.

    2009-05-26

    gp130 is a shared receptor for at least nine cytokines, and can signal either as a homodimer, or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-R{alpha}). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6R{alpha} hexameric complex, CNTF/CNTF-R{alpha} heterodimerizes gp130 and LIF-R via non-cooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic (EM) analysis of the full-length gp130/LIF-R/CNTF-R{alpha}/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the 'tall' class of gp130-family cytokine receptor complexes including LIF, IL-27, IL-12, and others.

  19. Full length parathyroid hormone (1–84) in the treatment of osteoporosis in postmenopausal women

    PubMed Central

    Jódar-Gimeno, Esteban

    2007-01-01

    Objective: To review the pharmacological properties and the available clinical data of full length parathyroid hormone (PTH) in post-menopausal osteoporosis. Sources: A MEDLINE search was completed, together with a review of information obtained from the manufacturer and from the medicine regulatory agencies. Study and data selection: Studies were selected according to relevance and availability. Relevant information (design, objectives, patients’ characteristics, outcomes, adverse events, dosing, etc) was analyzed. Results: Different studies have shown that, when administered intermittently as a subcutaneous injection in the abdomen, PTH increases bone mineral density (BMD) and prevents vertebral fractures. On completion of PTH therapy (up to 24 months), there is evidence that sequential treatment with alendronate is associated with a therapeutic benefit in terms of increase in BMD. Further trials are necessary to determine long-term safety and the role of PTH in combination with other treatments for osteoporosis and the effect of repeated cycles of PTH followed by an anti-catabolic agent. There are currently no completed comparative trials with other osteoporosis treatments. Conclusions: Full length PTH, given intermittently as an abdominal subcutaneous injection, appears to be a safe and efficacious treatment option for high risk osteoporosis. More data are needed to determine its specific role in osteoporosis treatment. PMID:18044089

  20. Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle

    PubMed Central

    Escobar, Helena; Schöwel, Verena; Spuler, Simone; Marg, Andreas; Izsvák, Zsuzsanna

    2016-01-01

    Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysfprmd Prkdcscid/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy. PMID:26784637

  1. Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle.

    PubMed

    Escobar, Helena; Schöwel, Verena; Spuler, Simone; Marg, Andreas; Izsvák, Zsuzsanna

    2016-01-19

    Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysf(prmd) Prkdc(scid)/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy.

  2. Full-length hdmX transcripts decrease following genotoxic stress

    PubMed Central

    Markey, M; Berberich, SJ

    2008-01-01

    Previous studies have suggested that the mdmX gene is constitutively transcribed, and that MdmX protein activity is instead controlled by cellular localization and DNA damage induced Mdm2-mediated ubiquitination leading to proteasomal degradation. In these studies, we report that the human mdmX (hdmX) mRNA is reproducibly decreased in various human cell lines following treatment with various DNA-damaging agents. Repression of hdmX transcripts is observed in DNAdamaged HCT116 colon cancer cells and in isogenic p53−/− cells, suggesting that this effect is p53-independent. Reduction in the amount of hdmX transcript occurs in both human tumor cell lines and primary human diploid fibroblasts, and results in a significant reduction of HdmX protein. Examination of hdmX promoter activity suggests that damage-induced repression of hdmX mRNA is not significantly impacted by transcription initiation. In contrast, changes in hdmX mRNA splicing appear to partly explain the reduction in full-length hdmX mRNA levels in tumor cell lines with the destabilization of full-length hdmX transcripts, potentially through microRNA miR-34a regulation, also impacting transcript levels. Taken together, this study uncovers previously unrecognized cellular mechanisms by which hdmX mRNA levels are kept low following genotoxic stress. PMID:18711402

  3. Design, fabrication, and testing of an external fuel (UO2), full-length thermionic converter

    NASA Technical Reports Server (NTRS)

    Schock, A.; Raab, B.

    1971-01-01

    The development of a full-length external-fuel thermionic converter for in-pile testing is described. The development program includes out-of-pile performance testing of the fully fueled-converter, using RF-induction heating, before its installation in the in-pile test capsule. The external-fuel converter is cylindrical in shape, and consists of an inner, centrally cooled collector, and an outer emitter surrounded by nuclear fuel. The term full-length denotes that the converter is long enough to extend over the full height of the reactor core. Thus, the converter is not a scaled-down test device, but a full-scale fuel element of the thermionic reactor. The external-fuel converter concept permits a number of different design options, particularly with respect to the fuel composition and shape, and the collector cooling arrangement. The converter described was developed for the Jet Propulsion Laboratory, and is based on their concept for a thermionic reactor with uninsulated collector cooling as previously described. The converter is double-ended, with through-flow cooling, and with ceramic seals and emitter and collector power take-offs at both ends. The design uses a revolver-shaped tungsten emitter body, with the central emitter hole surrounded by six peripheral fuel holes loaded with cylindrical UO2 pellets.

  4. High-efficiency cloning of full-length cDNA.

    PubMed Central

    Okayama, H; Berg, P

    1982-01-01

    A widely recognized difficulty of presently used methods for cDNA cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 10(5) plasmid-cDNA recombinants obtained per microgram of rabbit reticulocyte mRNA, about 10% contained a complete alpha- of beta-globin mRNA sequence, and at least 30 to 50%, but very likely more, contained the entire globin coding regions. We attribute the high efficiency of cloning full- or nearly full-length cDNA to (i) the fact that the plasmid DNA vector itself serves as the primer for first- and second-strand cDNA synthesis, (ii) the lack of any nuclease treatment of the products, and (iii) the fact that one of the steps in the procedure results in preferential cloning of recombinants with full-length cDNA's over those with truncated cDNA's. Images PMID:6287227

  5. Shear-Induced Unfolding and Enzymatic Cleavage of Full-Length VWF Multimers

    PubMed Central

    Lippok, Svenja; Radtke, Matthias; Obser, Tobias; Kleemeier, Lars; Schneppenheim, Reinhard; Budde, Ulrich; Netz, Roland R.; Rädler, Joachim O.

    2016-01-01

    Proteolysis of the multimeric blood coagulation protein von Willebrand Factor (VWF) by ADAMTS13 is crucial for prevention of microvascular thrombosis. ADAMTS13 cleaves VWF within the mechanosensitive A2 domain, which is believed to open under shear flow. In this study, we combine fluorescence correlation spectroscopy (FCS) and a microfluidic shear cell to monitor real-time kinetics of full-length VWF proteolysis as a function of shear stress. For comparison, we also measure the Michaelis-Menten kinetics of ADAMTS13 cleavage of wild-type VWF in the absence of shear but partially denaturing conditions. Under shear, ADAMTS13 activity on full-length VWF arises without denaturing agent as evidenced by FCS and gel-based multimer analysis. In agreement with Brownian hydrodynamics simulations, we find a sigmoidal increase of the enzymatic rate as a function of shear at a threshold shear rate γ˙1/2 = 5522/s. The same flow-rate dependence of ADAMTS13 activity we also observe in blood plasma, which is relevant to predict hemostatic dysfunction. PMID:26840720

  6. Synthesis of full length cDNAs from four partially purified oviduct mRNAs.

    PubMed

    Buell, G N; Wickens, M P; Payvar, F; Schimke, R T

    1978-04-10

    Total poly(A)-containing RNA prepared from hen oviduct and centrifuged on an isokinetic sucrose gradient displays four peaks of optical absorbance. These have been identified by translation in vitro as lysozyme, ovomucoid, ovalbumin, and conalbumin mRNAs. Isolation and recentrifugation of the peaks results in partial purification of each mRNA. Molecular weights have been determined for the mRNAs on agarose gels containing 20 mM methylmercury hydroxide. Each mRNA possesses a number of apparently untranslated nucleotides ranging from approximately 900 bases for ovalbumin and conalbumin mRNAs to 200 bases for ovomucoid and lysozyme mRNAs. The mRNAs have been copied with avian myeloblastosis virus reverse transcriptase. Each mRNA with the exception of conalbumin gives rise to a high proportion of full length cDNA. Several parameters previously reported to influence the size distribution of cDNA had no effect on the length of cDNA made from any mRNA fraction. The proportion of full length copy does depend on the reverse transcriptase lot.

  7. Crystallization and X-ray structure of full-length recombinant human butyrylcholinesterase.

    PubMed

    Ngamelue, Michelle N; Homma, Kohei; Lockridge, Oksana; Asojo, Oluwatoyin A

    2007-09-01

    Human butyrylcholinesterase (BChE) has been shown to function as an endogenous scavenger of diverse poisons. BChE is a 340 kDa tetrameric glycoprotein that is present in human serum at a concentration of 5 mg l(-1). The well documented therapeutic effects of BChE on cocaine toxicity and organophosphorus agent poisoning has increased the need for effective methods of producing recombinant therapeutic BChE. In order to be therapeutically useful, BChE must have a long circulatory residence time or associate as tetramers. Full-length recombinant BChE produced in Chinese hamster ovary (CHO) cells or human embryonic kidney cells has been shown to associate as monomers, with a shorter circulatory residence time than the naturally occurring tetrameric serum protein. Based on the preceding observation as well as the need to develop novel methodologies to facilitate the mass production of therapeutic recombinant BChE, studies have been initiated to determine the structural basis of tetramer formation. Towards these ends, full-length monomeric recombinant BChE has been crystallized for the first time. A 2.8 A X-ray structure was solved in space group P42(1)2, with unit-cell parameters a = b = 156, c = 146 A.

  8. Crystallization and X-ray structure of full-length recombinant human butyrylcholinesterase

    PubMed Central

    Ngamelue, Michelle N.; Homma, Kohei; Lockridge, Oksana; Asojo, Oluwatoyin A.

    2007-01-01

    Human butyrylcholinesterase (BChE) has been shown to function as an endogenous scavenger of diverse poisons. BChE is a 340 kDa tetrameric glycoprotein that is present in human serum at a concentration of 5 mg l−1. The well documented therapeutic effects of BChE on cocaine toxicity and organophosphorus agent poisoning has increased the need for effective methods of producing recombinant therapeutic BChE. In order to be therapeutically useful, BChE must have a long circulatory residence time or associate as tetramers. Full-length recombinant BChE produced in Chinese hamster ovary (CHO) cells or human embryonic kidney cells has been shown to associate as monomers, with a shorter circulatory residence time than the naturally occurring tetrameric serum protein. Based on the preceding observation as well as the need to develop novel methodologies to facilitate the mass production of therapeutic recombinant BChE, studies have been initiated to determine the structural basis of tetramer formation. Towards these ends, full-length monomeric recombinant BChE has been crystallized for the first time. A 2.8 Å X-ray structure was solved in space group P4212, with unit-cell parameters a = b = 156, c = 146 Å. PMID:17768338

  9. Dental Pulp Tissue Engineering in Full-length Human Root Canals

    PubMed Central

    Rosa, V.; Zhang, Z.; Grande, R.H.M.; Nör, J.E.

    2013-01-01

    The clinical translation of stem-cell-based dental pulp regeneration will require the use of injectable scaffolds. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) can generate a functional dental pulp when injected into full-length root canals. SHED survived and began to express putative markers of odontoblastic differentiation after 7 days when mixed with Puramatrix™ (peptide hydrogel), or after 14 days when mixed with recombinant human Collagen (rhCollagen) type I, and injected into the root canals of human premolars in vitro. Roots of human premolars injected with scaffolds (Puramatrix™ or rhCollagen) containing SHED were implanted subcutaneously into immunodeficient mice (CB-17 SCID). We observed pulp-like tissues with odontoblasts capable of generating new tubular dentin throughout the root canals. Notably, the pulp tissue engineered with SHED injected with either Puramatrix™ or rhCollagen type I presented similar cellularity and vascularization when compared with control human dental pulps. Analysis of these data, collectively, demonstrates that SHED injected into full-length human root canals differentiate into functional odontoblasts, and suggests that such a strategy might facilitate the completion of root formation in necrotic immature permanent teeth. PMID:24056227

  10. Dental pulp tissue engineering in full-length human root canals.

    PubMed

    Rosa, V; Zhang, Z; Grande, R H M; Nör, J E

    2013-11-01

    The clinical translation of stem-cell-based dental pulp regeneration will require the use of injectable scaffolds. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) can generate a functional dental pulp when injected into full-length root canals. SHED survived and began to express putative markers of odontoblastic differentiation after 7 days when mixed with Puramatrix™ (peptide hydrogel), or after 14 days when mixed with recombinant human Collagen (rhCollagen) type I, and injected into the root canals of human premolars in vitro. Roots of human premolars injected with scaffolds (Puramatrix™ or rhCollagen) containing SHED were implanted subcutaneously into immunodeficient mice (CB-17 SCID). We observed pulp-like tissues with odontoblasts capable of generating new tubular dentin throughout the root canals. Notably, the pulp tissue engineered with SHED injected with either Puramatrix™ or rhCollagen type I presented similar cellularity and vascularization when compared with control human dental pulps. Analysis of these data, collectively, demonstrates that SHED injected into full-length human root canals differentiate into functional odontoblasts, and suggests that such a strategy might facilitate the completion of root formation in necrotic immature permanent teeth.

  11. Diversity, distribution and dynamics of full-length Copia and Gypsy LTR retroelements in Solanum lycopersicum.

    PubMed

    Paz, Rosalía Cristina; Kozaczek, Melisa Eliana; Rosli, Hernán Guillermo; Andino, Natalia Pilar; Sanchez-Puerta, Maria Virginia

    2017-08-03

    Transposable elements are the most abundant components of plant genomes and can dramatically induce genetic changes and impact genome evolution. In the recently sequenced genome of tomato (Solanum lycopersicum), the estimated fraction of elements corresponding to retrotransposons is nearly 62%. Given that tomato is one of the most important vegetable crop cultivated and consumed worldwide, understanding retrotransposon dynamics can provide insight into its evolution and domestication processes. In this study, we performed a genome-wide in silico search of full-length LTR retroelements in the tomato nuclear genome and annotated 736 full-length Gypsy and Copia retroelements. The dispersion level across the 12 chromosomes, the diversity and tissue-specific expression of those elements were estimated. Phylogenetic analysis based on the retrotranscriptase region revealed the presence of 12 major lineages of LTR retroelements in the tomato genome. We identified 97 families, of which 77 and 20 belong to the superfamilies Copia and Gypsy, respectively. Each retroelement family was characterized according to their element size, relative frequencies and insertion time. These analyses represent a valuable resource for comparative genomics within the Solanaceae, transposon-tagging and for the design of cultivar-specific molecular markers in tomato.

  12. Structural organization of a full-length gp130/LIF-R cytokine receptor transmembrane complex

    PubMed Central

    Skiniotis, Georgios; Lupardus, Patrick; Martick, Monika; Walz, Thomas; Garcia, K. Christopher

    2008-01-01

    Summary gp130 is a shared receptor for at least nine cytokines, and can signal either as a homodimer, or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-Rα). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6Rα hexameric complex, CNTF/CNTF-Rα heterodimerizes gp130 and LIF-R via non-cooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic (EM) analysis of the full-length gp130/LIF-R/CNTF-Rα/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the ‘tall’ class of gp130-family cytokine receptor complexes including LIF, IL-27, IL-12, and others. PMID:18775332

  13. Full-length sequence analysis of four IBDV strains with different pathogenicities.

    PubMed

    Petkov, Daniel; Linnemann, Erich; Kapczynski, Darrell R; Sellers, Holly S

    2007-06-01

    Characterization of field isolate 9109, Lukert, Edgar cell culture-adapted (CCA), and Edgar chicken embryo-adapted (CEA) serotype 1 IBDV strains using full-length genomic sequences is reported. IBDV genomic segments A and B were sequenced and the nucleotide and deduced amino acid (aa) sequences were compared with previously reported full-length sequenced IBDV strains. We found that the viral protein VPX and amino acid sequences between aa 202-451 and 210-473 of VP2 but not the entire VP2 protein are the best representatives of the entire IBDV genome. The greatest variability was found in the VP2 and 5' non-coding region of segment B among IBDV strains. The deduced amino acid sequences of the VP1 protein varies in length among the strains analyzed. The RNA-dependent, RNA-polymerase motifs within VP1 and the VP5 protein were highly conserved among isolates. Although within the VP2 processing site, amino acid sequence of Lukert was similar to the classical while the Edgar CCA, and CEA were more similar to the very virulent strains, it was determined that these strains have sequence characteristics of the classical strains. In addition, close relatedness between Lukert, Edgar CCA and CEA was observed. Although phylogenetic analysis of the VP1, VP3, and VP4 proteins indicated that 9109 is a classical type virus, this isolate shares unique amino acid changes with very virulent strains within the same proteins. Phylogenetic analysis of the 3' and 5' non-coding regions of segment A revealed that 9109 is more similar to the very virulent strains compared to the classical strains. In the VP2 protein, several amino acids were conserved between variant E and 9109 strains. Thus, it appears that 9109 isolate has characteristics of classical, very virulent, and variant strains. Our analysis indicates that although VPX amino acid comparison may be initially useful for molecular typing, full-length genomic sequence analysis is essential for thorough molecular characterization as

  14. Characterization of full-length sequenced cDNA inserts (FLIcs) from Atlantic salmon (Salmo salar)

    PubMed Central

    Andreassen, Rune; Lunner, Sigbjørn; Høyheim, Bjørn

    2009-01-01

    Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs) are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP), the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91%) of the transcripts were annotated using Gene Ontology (GO) terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS). The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS). This suggests that the remaining c

  15. Characterisation of full-length cDNA sequences provides insights into the Eimeria tenellatranscriptome

    PubMed Central

    2012-01-01

    Background Eimeria tenella is an apicomplexan parasite that causes coccidiosis in the domestic fowl. Infection with this parasite is diagnosed frequently in intensively reared poultry and its control is usually accorded a high priority, especially in chickens raised for meat. Prophylactic chemotherapy has been the primary method used for the control of coccidiosis. However, drug efficacy can be compromised by drug-resistant parasites and the lack of new drugs highlights demands for alternative control strategies including vaccination. In the long term, sustainable control of coccidiosis will most likely be achieved through integrated drug and vaccination programmes. Characterisation of the E. tenella transcriptome may provide a better understanding of the biology of the parasite and aid in the development of a more effective control for coccidiosis. Results More than 15,000 partial sequences were generated from the 5' and 3' ends of clones randomly selected from an E. tenella second generation merozoite full-length cDNA library. Clustering of these sequences produced 1,529 unique transcripts (UTs). Based on the transcript assembly and subsequently primer walking, 433 full-length cDNA sequences were successfully generated. These sequences varied in length, ranging from 441 bp to 3,083 bp, with an average size of 1,647 bp. Simple sequence repeat (SSR) analysis identified CAG as the most abundant trinucleotide motif, while codon usage analysis revealed that the ten most infrequently used codons in E. tenella are UAU, UGU, GUA, CAU, AUA, CGA, UUA, CUA, CGU and AGU. Subsequent analysis of the E. tenella complete coding sequences identified 25 putative secretory and 60 putative surface proteins, all of which are now rational candidates for development as recombinant vaccines or drug targets in the effort to control avian coccidiosis. Conclusions This paper describes the generation and characterisation of full-length cDNA sequences from E. tenella second generation

  16. Structure and function of the Zika virus full-length NS5 protein

    PubMed Central

    Zhao, Baoyu; Yi, Guanghui; Du, Fenglei; Chuang, Yin-Chih; Vaughan, Robert C.; Sankaran, Banumathi; Kao, C. Cheng; Li, Pingwei

    2017-01-01

    The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions to those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Overall, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication. PMID:28345656

  17. Structure of the Full-length VEGFR-1 Extracellular Domain in Complex with VEGF-A.

    PubMed

    Markovic-Mueller, Sandra; Stuttfeld, Edward; Asthana, Mayanka; Weinert, Tobias; Bliven, Spencer; Goldie, Kenneth N; Kisko, Kaisa; Capitani, Guido; Ballmer-Hofer, Kurt

    2017-02-07

    Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases: VEGFR-1, -2, and -3. Partial structures of VEGFR/VEGF complexes based on single-particle electron microscopy, small-angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and domain arrangement of individual receptor subdomains. Here, we describe the structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A at 4 Å resolution. We combined X-ray crystallography, single-particle electron microscopy, and molecular modeling for structure determination and validation. The structure reveals the molecular details of ligand-induced receptor dimerization, in particular of homotypic receptor interactions in immunoglobulin homology domains 4, 5, and 7. Functional analyses of ligand binding and receptor activation confirm the relevance of these homotypic contacts and identify them as potential therapeutic sites to allosterically inhibit VEGFR-1 activity.

  18. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria.

    PubMed

    Robinson, Michael-Paul; Ke, Na; Lobstein, Julie; Peterson, Cristen; Szkodny, Alana; Mansell, Thomas J; Tuckey, Corinna; Riggs, Paul D; Colussi, Paul A; Noren, Christopher J; Taron, Christopher H; DeLisa, Matthew P; Berkmen, Mehmet

    2015-08-27

    Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named 'cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation.

  19. Mechanism of activation gating in the full-length KcsA K[superscript +] channel

    SciTech Connect

    Uysal, Serdar; Cuello, Luis G.; Cortes, D. Marien; Koide, Shohei; Kossiakoff, Anthony A.; Perozo, Eduardo

    2012-10-25

    Using a constitutively active channel mutant, we solved the structure of full-length KcsA in the open conformation at 3.9 {angstrom}. The structure reveals that the activation gate expands about 20 {angstrom}, exerting a strain on the bulge helices in the C-terminal domain and generating side windows large enough to accommodate hydrated K{sup +} ions. Functional and spectroscopic analysis of the gating transition provides direct insight into the allosteric coupling between the activation gate and the selectivity filter. We show that the movement of the inner gate helix is transmitted to the C-terminus as a straightforward expansion, leading to an upward movement and the insertion of the top third of the bulge helix into the membrane. We suggest that by limiting the extent to which the inner gate can open, the cytoplasmic domain also modulates the level of inactivation occurring at the selectivity filter.

  20. Full-length apolipoprotein E protects against the neurotoxicity of an apoE-related peptide

    PubMed Central

    Crutcher, K.A.; Lilley, H.N.; Anthony, S. R.; Zhou, W.; Narayanaswami, V.

    2009-01-01

    Apolipoprotein E was found to protect against the neurotoxic effects of a dimeric peptide derived from the receptor-binding region of this protein (residues 141–149). Both apoE3 and apoE4 conferred protection but the major N-terminal fragment of each isoform did not. Nor was significant protection provided by bovine serum albumin or apoA-I. Full-length apoE3 and apoE4 also inhibited the uptake of a fluorescent-labeled derivative of the peptide, suggesting that the mechanism of inhibition might involve competition for cell surface receptors/proteoglycans that mediate endocytosis and/or signaling pathways. These results might bear on the question of the role of apoE in neuronal degeneration, such as occurs in Alzheimer’s disease where apoE4 confers a significantly greater risk of pathology. PMID:19836363

  1. Cocrystallization studies of full-length recombinant butyrylcholinesterase (BChE) with cocaine

    SciTech Connect

    Asojo, Oluwatoyin Ajibola; Asojo, Oluyomi Adebola; Ngamelue, Michelle N.; Homma, Kohei; Lockridge, Oksana

    2011-09-16

    Human butyrylcholinesterase (BChE; EC 3.1.1.8) is a 340 kDa tetrameric glycoprotein that is present in human serum at about 5 mg l{sup -1} and has well documented therapeutic effects on cocaine toxicity. BChE holds promise as a therapeutic that reduces and finally eliminates the rewarding effects of cocaine, thus weaning an addict from the drug. There have been extensive computational studies of cocaine hydrolysis by BChE. Since there are no reported structures of BChE with cocaine or any of the hydrolysis products, full-length monomeric recombinant wild-type BChE was cocrystallized with cocaine. The refined 3 {angstrom} resolution structure appears to retain the hydrolysis product benzoic acid in sufficient proximity to form a hydrogen bond to the active-site Ser198.

  2. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

    PubMed Central

    Robinson, Michael-Paul; Ke, Na; Lobstein, Julie; Peterson, Cristen; Szkodny, Alana; Mansell, Thomas J.; Tuckey, Corinna; Riggs, Paul D.; Colussi, Paul A.; Noren, Christopher J.; Taron, Christopher H.; DeLisa, Matthew P.; Berkmen, Mehmet

    2015-01-01

    Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named ‘cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. PMID:26311203

  3. The preparation of an infectious full-length cDNA clone of Saffold virus.

    PubMed

    Himeda, Toshiki; Hosomi, Takushi; Asif, Naeem; Shimizu, Hiroyuki; Okuwa, Takako; Muraki, Yasushi; Ohara, Yoshiro

    2011-03-09

    The pathogenicity of Saffold virus (SAFV) among humans still remains unclear, although it was identified as a novel human cardiovirus in 2007. In order to encourage the molecular pathogenetic studies of SAFV, we generated an infectious cDNA clone of SAFV type 3 (SAFV-3). The present study demonstrated that the synthesis of the full-length infectious RNA by T7 RNA polymerase was terminated by a homologous sequence motif with the human preproparathyroid hormone (PTH) signal in the SAFV-3 genome. To obtain the infectious RNA using T7 promoter, a variant of T7 RNA polymerase, which fails to recognize the PTH signal, was useful. This study will provide a valuable technical insight into the reverse genetics of SAFV.

  4. The preparation of an infectious full-length cDNA clone of Saffold virus

    PubMed Central

    2011-01-01

    The pathogenicity of Saffold virus (SAFV) among humans still remains unclear, although it was identified as a novel human cardiovirus in 2007. In order to encourage the molecular pathogenetic studies of SAFV, we generated an infectious cDNA clone of SAFV type 3 (SAFV-3). The present study demonstrated that the synthesis of the full-length infectious RNA by T7 RNA polymerase was terminated by a homologous sequence motif with the human preproparathyroid hormone (PTH) signal in the SAFV-3 genome. To obtain the infectious RNA using T7 promoter, a variant of T7 RNA polymerase, which fails to recognize the PTH signal, was useful. This study will provide a valuable technical insight into the reverse genetics of SAFV. PMID:21385468

  5. Structure and function of the Zika virus full-length NS5 protein.

    PubMed

    Zhao, Baoyu; Yi, Guanghui; Du, Fenglei; Chuang, Yin-Chih; Vaughan, Robert C; Sankaran, Banumathi; Kao, C Cheng; Li, Pingwei

    2017-03-27

    The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions to those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Overall, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.

  6. MELCOR benchmarking: The NRU full-length high-temperature-4 test

    SciTech Connect

    Madni, I.K.; Guo, X.D. )

    1993-01-01

    The purpose of this paper is to describe a MELCOR simulation of the national research universal (NRU) full-length high-temperature-4 (FLHT-4) test and to compare results with test data and predictions from the SCDAP mechanistic code. This study provides code-to-code as well as code-to-data comparisons and will help to identify input refinements and model improvements for MELCOR. It will also help in assessing MELCOR's ability and limitations in predicting events and conditions associated with the early phase of severe core damage. This benchmarking analysis was performed as part of an overall MELCOR assessment effort being carried out for the U.S. Nuclear Regulatory Commission by Brookhaven National Laboratory. The results are considered applicable equally to the recently released MELCOR 1.8.2 as to the earlier version 1.8.1.

  7. Cocrystallization studies of full-length recombinant butyrylcholinesterase (BChE) with cocaine

    PubMed Central

    Asojo, Oluwatoyin Ajibola; Asojo, Oluyomi Adebola; Ngamelue, Michelle N.; Homma, Kohei; Lockridge, Oksana

    2011-01-01

    Human butyrylcholinesterase (BChE; EC 3.1.1.8) is a 340 kDa tetrameric glycoprotein that is present in human serum at about 5 mg l−1 and has well documented therapeutic effects on cocaine toxicity. BChE holds promise as a therapeutic that reduces and finally eliminates the rewarding effects of cocaine, thus weaning an addict from the drug. There have been extensive computational studies of cocaine hydrolysis by BChE. Since there are no reported structures of BChE with cocaine or any of the hydrolysis products, full-length monomeric recombinant wild-type BChE was cocrystallized with cocaine. The refined 3 Å resolution structure appears to retain the hydrolysis product benzoic acid in sufficient proximity to form a hydrogen bond to the active-site Ser198. PMID:21505234

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

    PubMed

    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.

  9. Aggregation Behavior of Chemically Synthesized, Full-Length Huntingtin Exon1

    PubMed Central

    2015-01-01

    Repeat length disease thresholds vary among the 10 expanded polyglutamine (polyQ) repeat diseases, from about 20 to about 50 glutamine residues. The unique amino acid sequences flanking the polyQ segment are thought to contribute to these repeat length thresholds. The specific portions of the flanking sequences that modulate polyQ properties are not always clear, however. This ambiguity may be important in Huntington’s disease (HD), for example, where in vitro studies of aggregation mechanisms have led to distinctly different mechanistic models. Most in vitro studies of the aggregation of the huntingtin (HTT) exon1 fragment implicated in the HD mechanism have been conducted on inexact molecules that are imprecise either on the N-terminus (recombinantly produced peptides) or on the C-terminus (chemically synthesized peptides). In this paper, we investigate the aggregation properties of chemically synthesized HTT exon1 peptides that are full-length and complete, containing both normal and expanded polyQ repeat lengths, and compare the results directly to previously investigated molecules containing truncated C-termini. The results on the full-length peptides are consistent with a two-step aggregation mechanism originally developed based on studies of the C-terminally truncated analogues. Thus, we observe relatively rapid formation of spherical oligomers containing from 100 to 600 HTT exon1 molecules and intermediate formation of short protofibril-like structures containing from 500 to 2600 molecules. In contrast to this relatively rapid assembly, mature HTT exon1 amyloid requires about one month to dissociate in vitro, which is similar to the time required for neuronal HTT exon1 aggregates to disappear in vivo after HTT production is discontinued. PMID:24921664

  10. Targeting a Complex Transcriptome: The Construction of the Mouse Full-Length cDNA Encyclopedia

    PubMed Central

    Carninci, Piero; Waki, Kazunori; Shiraki, Toshiyuki; Konno, Hideaki; Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Arakawa, Takahiro; Ishii, Yoshiyuki; Sasaki, Daisuke; Bono, Hidemasa; Kondo, Shinji; Sugahara, Yuichi; Saito, Rintaro; Osato, Naoki; Fukuda, Shiro; Sato, Kenjiro; Watahiki, Akira; Hirozane-Kishikawa, Tomoko; Nakamura, Mari; Shibata, Yuko; Yasunishi, Ayako; Kikuchi, Noriko; Yoshiki, Atsushi; Kusakabe, Moriaki; Gustincich, Stefano; Beisel, Kirk; Pavan, William; Aidinis, Vassilis; Nakagawara, Akira; Held, William A.; Iwata, Hiroo; Kono, Tomohiro; Nakauchi, Hiromitsu; Lyons, Paul; Wells, Christine; Hume, David A.; Fagiolini, Michela; Hensch, Takao K.; Brinkmeier, Michelle; Camper, Sally; Hirota, Junji; Mombaerts, Peter; Muramatsu, Masami; Okazaki, Yasushi; Kawai, Jun; Hayashizaki, Yoshihide

    2003-01-01

    We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3′-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5′ end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5′-end clusters identify regions that are potential promoters for 8637 known genes and 5′-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete. PMID:12819125

  11. Fabrication and Testing of Full-Length Single-Cell Externally Fueled Converters for Thermionic Reactors

    SciTech Connect

    Schock, Alfred

    1994-06-01

    The preceding paper described designs and analyses of thermionic reactors employing full-core-length single-cell converters with their heated emitters located on the outside of their internally cooled collectors, and it presented results of detailed parametric analyses which illustrate the benefits of this unconventional design. The present paper describes the fabrication and testing of full-length prototypical converters, both unfueled and fueled, and presents parametric results of electrically heated tests. The unfueled converter tests demonstrated the practicality of operating such long converters without shorting across a 0.3-mm interelectrode gap. They produced a measured peak output of 751 watts(e) from a single diode and a peak efficiency of 15.4%. The fueled converter tests measured the parametric performance of prototypic UO(subscript 2)-fueled converters designed for subsequent in-pile testing. They employed revolver-shaped tungsten elements with a central emitter hole surrounded by six fuel chambers. The full-length converters were heated by a water-cooled RF-induction coil inside an ion-pumped vacuum chamber. This required development of high-vacuum coaxial RF feedthroughs. In-pile test rules required multiple containment of the UO (subscript 2)-fuel, which complicated the fabrication of the test article and required successful development of techniques for welding tungsten and other refractory components. The test measured a peak power output of 530 watts(e) or 7.1 watts/cm (superscript 2) at an efficiency of 11.5%. There are three copies in the file. Cross-Reference a copy FSC-ESD-217-94-529 in the ESD files with a CID #8574.

  12. Characterization of full-length recombinant human Proteoglycan 4 as an ocular surface boundary lubricant.

    PubMed

    Samsom, Michael L; Morrison, Sheila; Masala, Nemanja; Sullivan, Benjamin D; Sullivan, David A; Sheardown, Heather; Schmidt, Tannin A

    2014-10-01

    Proteoglycan 4 (PRG4, or lubricin) is a lubricating mucin-like glycoprotein recently discovered at the ocular surface, where it functions as a boundary lubricant and appears to play a protective role. Recent technological advances have enabled abundant expression of full-length recombinant human PRG4 (rhPRG4). The objectives of this study were to 1) biochemically characterize the gross structure and glycosylations of full-length rhPRG4, and 2) assess the ocular surface boundary lubricating ability of rhPRG4 at both human cornea-eyelid and human cornea-polydimethylsiloxane (PDMS) biointerfaces. rhPRG4 expressed by a Chinese hamster ovary cell line was characterized and compared to native bovine PRG4 by SDS-PAGE western blotting, and protein identity was assessed by tandem mass spectrometry (MS/MS). Human corneas were articulated against PDMS or human eyelids, at effective sliding velocities of 0.3-30 mm/s under physiological loads of ∼15 kPa, to assess and compare the ocular lubricating ability of rhPRG4 to PRG4. Samples were tested serially in PRG4, rhPRG4 (both 300 μg/ml), then saline. Western blotting indicated that rhPRG4 had immunoreactivity at the appropriate apparent molecular weight, and possessed O-linked glycosylation consistent with that of PRG4. rhPRG4 protein identity was confirmed by MS/MS. Both PRG4 and rhPRG4 significantly, and similarly, reduced friction compared to saline at both human cornea - PDMS and human cornea-eyelid biointerfaces. In conclusion, the rhPRG4 studied here demonstrated appropriate higher order structure, O-linked glycosylations, and ocular surface boundary lubricating. Purified rhPRG4 may have clinical utility as a topical treatment of dry eye disease or contact lens biomaterial coating to promote more comfortable wear. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Features of Arabidopsis genes and genome discovered using full-length cDNAs.

    PubMed

    Alexandrov, Nickolai N; Troukhan, Maxim E; Brover, Vyacheslav V; Tatarinova, Tatiana; Flavell, Richard B; Feldmann, Kenneth A

    2006-01-01

    Arabidopsis is currently the reference genome for higher plants. A new, more detailed statistical analysis of Arabidopsis gene structure is presented including intron and exon lengths, intergenic distances, features of promoters, and variant 5'-ends of mRNAs transcribed from the same transcription unit. We also provide a statistical characterization of Arabidopsis transcripts in terms of their size, UTR lengths, 3'-end cleavage sites, splicing variants, and coding potential. These analyses were facilitated by scrutiny of our collection of sequenced full-length cDNAs and much larger collection of 5'-ESTs, together with another set of full-length cDNAs from Salk/Stanford/Plant Gene Expression Center/RIKEN. Examples of alternative splicing are observed for transcripts from 7% of the genes and many of these genes display multiple spliced isoforms. Most splicing variants lie in non-coding regions of the transcripts. Non-canonical splice sites constitute less than 1% of all splice sites. Genes with fewer than four introns display reduced average mRNA levels. Putative alternative transcription start sites were observed in 30% of highly expressed genes and in more than 50% of the genes with low expression. Transcription start sites correlate remarkably well with a CG skew peak in the DNA sequences. The intergenic distances vary considerably, those where genes are transcribed towards one another being significantly shorter. New transcripts, missing in the current TIGR genome annotation and ESTs that are non-coding, including those antisense to known genes, are derived and cataloged in the Supplementary Material. They identify 148 new loci in the Arabidopsis genome. The conclusions drawn provide a better understanding of the Arabidopsis genome and how the gene transcripts are processed. The results also allow better predictions to be made for, as yet, poorly defined genes and provide a reference for comparisons with other plant genomes whose complete sequences are currently

  14. Targeting a complex transcriptome: the construction of the mouse full-length cDNA encyclopedia.

    PubMed

    Carninci, Piero; Waki, Kazunori; Shiraki, Toshiyuki; Konno, Hideaki; Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Arakawa, Takahiro; Ishii, Yoshiyuki; Sasaki, Daisuke; Bono, Hidemasa; Kondo, Shinji; Sugahara, Yuichi; Saito, Rintaro; Osato, Naoki; Fukuda, Shiro; Sato, Kenjiro; Watahiki, Akira; Hirozane-Kishikawa, Tomoko; Nakamura, Mari; Shibata, Yuko; Yasunishi, Ayako; Kikuchi, Noriko; Yoshiki, Atsushi; Kusakabe, Moriaki; Gustincich, Stefano; Beisel, Kirk; Pavan, William; Aidinis, Vassilis; Nakagawara, Akira; Held, William A; Iwata, Hiroo; Kono, Tomohiro; Nakauchi, Hiromitsu; Lyons, Paul; Wells, Christine; Hume, David A; Fagiolini, Michela; Hensch, Takao K; Brinkmeier, Michelle; Camper, Sally; Hirota, Junji; Mombaerts, Peter; Muramatsu, Masami; Okazaki, Yasushi; Kawai, Jun; Hayashizaki, Yoshihide

    2003-06-01

    We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5'-end clusters identify regions that are potential promoters for 8637 known genes and 5'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

  15. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.

    PubMed

    Arturo, Emilia C; Gupta, Kushol; Héroux, Annie; Stith, Linda; Cross, Penelope J; Parker, Emily J; Loll, Patrick J; Jaffe, Eileen K

    2016-03-01

    Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.

  16. Dual redundant sequencing strategy: Full-length gene characterisation of 1056 novel and confirmatory HLA alleles.

    PubMed

    Albrecht, V; Zweiniger, C; Surendranath, V; Lang, K; Schöfl, G; Dahl, A; Winkler, S; Lange, V; Böhme, I; Schmidt, A H

    2017-08-01

    The high-throughput department of DKMS Life Science Lab encounters novel human leukocyte antigen (HLA) alleles on a daily basis. To characterise these alleles, we have developed a system to sequence the whole gene from 5'- to 3'-UTR for the HLA loci A, B, C, DQB1 and DPB1 for submission to the European Molecular Biology Laboratory - European Nucleotide Archive (EMBL-ENA) and the IPD-IMGT/HLA Database. Our workflow is based on a dual redundant sequencing strategy. Using shotgun sequencing on an Illumina MiSeq instrument and single molecule real-time (SMRT) sequencing on a PacBio RS II instrument, we are able to achieve highly accurate HLA full-length consensus sequences. Remaining conflicts are resolved using the R package DR2S (Dual Redundant Reference Sequencing). Given the relatively high throughput of this strategy, we have developed the semi-automated web service TypeLoader, to aid in the submission of sequences to the EMBL-ENA and the IPD-IMGT/HLA Database. In the IPD-IMGT/HLA Database release 3.24.0 (April 2016; prior to the submission of the sequences described here), only 5.2% of all known HLA alleles have been fully characterised together with intronic and UTR sequences. So far, we have applied our strategy to characterise and submit 1056 HLA alleles, thereby more than doubling the number of fully characterised alleles. Given the increasing application of next generation sequencing (NGS) for full gene characterisation in clinical practice, extending the HLA database concomitantly is highly desirable. Therefore, we propose this dual redundant sequencing strategy as a workflow for submission of novel full-length alleles and characterisation of sequences that are as yet incomplete. This would help to mitigate the predominance of partially known alleles in the database. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. New insights in HLA-E polymorphism by refined analysis of the full-length gene.

    PubMed

    Olieslagers, T I; Voorter, C E M; Groeneweg, M; Xu, Y; Wieten, L; Tilanus, M G J

    2017-03-01

    Human leukocyte antigen (HLA)-E is a non-classical HLA class I molecule that plays a role in both the innate and the adaptive immune response through interaction with receptors on natural killer- and T-cells. The HLA-E gene is characterized by limited polymorphism compared with the classical HLA loci on chromosome 6. At the start of this study, only 13 variable sites had been identified (IPD-IMGT/HLA Database v3.18.0). While most previous studies focused on polymorphism in exons 2 and 3 or specific gene regions, polymorphism in the other exons and introns could influence protein expression and function as well. Studies that investigate extended HLA-E polymorphism are therefore needed to better understand the functional relevance of HLA-E in health and disease. The aim of this study was to examine the variability of the full-length HLA-E gene region in individuals originating from different populations. A total of 7 new HLA-E alleles were identified using full-length HLA-E sequencing of 123 individuals from Asian, Dutch or Hunan Han origin. Furthermore, genome variation analysis of the third phase of the 1000 genomes database showed 107 new variable sites in 2504 individuals originating from 26 different populations. Our study demonstrates that the nucleotide variability of the HLA-E gene is much higher than previously known, albeit in only a limited number of individuals. Overall only 2 variants, HLA-E*01:01 and *01:03, are frequently present worldwide, suggesting that balancing selection is acting on HLA-E. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli

    PubMed Central

    Gonçalves, Andrezza N.; Meschiari, Cesar A.; Stetler-Stevenson, William G.; Nonato, M. Cristina; Alves, Cleidson P.; Espreafico, Enilza M.; Gerlach, Raquel F.

    2012-01-01

    Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18 °C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12 mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase. PMID:22001844

  19. Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

    NASA Astrophysics Data System (ADS)

    Hamid, Nur Athirah Abd; Ismail, Ismanizan

    2013-11-01

    Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

  20. Structure of full-length Toxascaris leonina galectin with two carbohydrate-recognition domains.

    PubMed

    Jeong, Mi Suk; Hwang, Hyun Gi; Yu, Hak Sun; Jang, Se Bok

    2013-02-01

    The full-length crystal structure of Toxascaris leonine galectin (Tl-gal), a galectin-9 homologue protein, was determined at a resolution of 2.0 Å. Galectin-9 exhibits a variety of biological functions, including cell aggregation, eosinophil chemoattraction, activation and apoptosis of murine thymocytes, T cells and human melanoma cells. Similar to this galectin, Tl-gal may function as a regulatory molecule in the host immune system; however, no molecular or structural information has been reported for Tl-gal. Moreover, until now, there have been no reports of a full-length galectin structure. There are two molecules of Tl-gal per asymmetric unit in space group P2(1)2(1)2(1), and the N-terminal and C-terminal carbohydrate-recognition domains (NCRD and CCRD) of Tl-gal are composed of six-stranded β-sheets and five-stranded β-sheets with a short α-helix. The NCRD of Tl-gal resembles that of human galectin-7 and its CCRD resembles human galectin-9, but the residues in the interface and loop regions of the NCRD and CCRD are flexible and are related to interaction. Engagement of the T-cell immunoglobulin mucin-3 (Tim-3) immunoglobulin variable (IgV) domain by a galectin-9 ligand is known to be important for appropriate termination of T-helper 1 immune responses. To investigate the binding site of Tl-gal, the interaction between Tl-gal and Tim-3 was modelled. Tim-3 is docked into a major groove of the Tl-gal structure, which is larger and deeper than the minor groove. The structural information presented here will provide insight into the development of novel anti-inflammatory agents or selective modulators of immune response.

  1. [Cloning, expression and activity analysis of full-length gene encoding thioredoxin peroxidase from Oncomelania hupensis].

    PubMed

    Ma, Xian-Liang; Liu, Qin; Zhang, Yi

    2012-08-30

    To clone and express full-length thioredoxin peroxidase (TPx) gene of Oncomelania hupensis and study on the peroxidase activity of the recombinant protein. Total RNA was obtained from the cultivated O. hupensis and a cDNA sequence of the TPx gene was cloned by RT-PCR. The TPx cDNA ends were amplified by the SMARTer RACE cDNA Amplification Kit. After sequencing, blasting and matching, the full-length cDNA of the TPx gene was obtained. The TPx cDNA was ligated with the pGEM-Teasy and transformed into E. coli DH5alpha. After sequencing and blasting, the characteristics of biological information of the TPx gene was analyzed. The positive recombinants with pGEM-Teasy/TPx and expression vector pET-28a were digested by the double restriction enzymes, ligated each other, transformed into E. coli BL21 (DE3), and induced by IPTG for expression. The recombinant TPx was expressed as a histidine fusion protein and was purified with Ni chromatography and NTA cation exchange chromatography. The expressed and purified TPx was analyzed by SDS-PAGE. The different concentrations of TPx recombinant protein (10, 20, 30, 40, and 50 microg/ml) were added into hydrogen peroxide (H2O2) reduction test in vitro to calculate the clearance rate of H2O2, each concentration with parallel control of dithiothreitol sugar alcohol (DTT). In the protection test of super-coiled DNA, the TPx protein was added with a concentration of 2.5, 5.0, and 10 microg/ml, respectively, to observe the protective effect of super-coiled DNA in metal-catalyzed oxidation (MCO). The complete cDNA encoding TPx was 992 bp. ORF was 747 bp with GenBank accession number of JN831437. The ORF encoded 249 amino acids, and the relative molecular weight (M(r)) of predicted protein was 27 000. The recombinant plasmid pET28a/TPx was built, and the soluble recombinant protein was obtained by induction and purification. The results of SDS-PAGE showed that the M(r) was 27 000. H2O2 reduction test in vitro showed that the H2O2

  2. Structure and dynamics of full-length HIV-1 capsid protein in solution.

    PubMed

    Deshmukh, Lalit; Schwieters, Charles D; Grishaev, Alexander; Ghirlando, Rodolfo; Baber, James L; Clore, G Marius

    2013-10-30

    The HIV-1 capsid protein plays a crucial role in viral infectivity, assembling into a cone that encloses the viral RNA. In the mature virion, the N-terminal domain of the capsid protein forms hexameric and pentameric rings, while C-terminal domain homodimers connect adjacent N-terminal domain rings to one another. Structures of disulfide-linked hexamer and pentamer assemblies, as well as structures of the isolated domains, have been solved previously. The dimer configuration in C-terminal domain constructs differs in solution (residues 144-231) and crystal (residues 146-231) structures by ∼30°, and it has been postulated that the former connects the hexamers while the latter links pentamers to hexamers. Here we study the structure and dynamics of full-length capsid protein in solution, comprising a mixture of monomeric and dimeric forms in dynamic equilibrium, using ensemble simulated annealing driven by experimental NMR residual dipolar couplings and X-ray scattering data. The complexity of the system necessitated the development of a novel computational framework that should be generally applicable to many other challenging systems that currently escape structural characterization by standard application of mainstream techniques of structural biology. We show that the orientation of the C-terminal domains in dimeric full-length capsid and isolated C-terminal domain constructs is the same in solution, and we obtain a quantitative description of the conformational space sampled by the N-terminal domain relative to the C-terminal domain on the nano- to millisecond time scale. The positional distribution of the N-terminal domain relative to the C-terminal domain is large and modulated by the oligomerization state of the C-terminal domain. We also show that a model of the hexamer/pentamer assembly can be readily generated with a single configuration of the C-terminal domain dimer, and that capsid assembly likely proceeds via conformational selection of sparsely

  3. Construction, characterization and expression of full length cDNA clone of sheep YAP1 gene.

    PubMed

    Sun, Wei; Li, Da; Su, Rui; Musa, Hassan H; Chen, Ling; Zhou, Hong

    2014-02-01

    RT-PCR, 5'RACE, 3'RACE were used to clone sheep full length cDNA sequence of YAP1 (Yes-associated protein 1), eukaryotic expression plasmid and a mutant that cannot be phosphorylated at Ser42 was successfully constructed. The amino acid sequence analysis revealed that sheep YAP1 gene encoded water-soluble protein and its relative molecular weight and isoelectric point was 44,079.0 Da and 4.91, respectively. Sub-cellular localization of YAP1 was in the nucleus, it is hydrophilic non-transmembrane and non-secreted protein. YAP1 protein contained 33 phosphorylation sites, seven glycosylation sites and two WW domains. The secondary structure of YAP1 was mainly composed of random coil, while the tertiary structure of domain area showed a forniciform helix structure. YAP1 gene was expressed in different tissues, the highest expression was in kidney and the lowest was in hypothalamus. The CDS of sheep YAP1was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, cloned into pMD19-T simple vector by T/A ligation. YAP1 coding region was further sub-cloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR, restriction enzyme and sequencing were used to confirm the recombinant plasmid. The sheep full-length YAP1 cDNA sequence is 1712 in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. The result showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, this will help for further studies on the YAP1 protein expression and its biological activities.

  4. Structural and functional characterization of full-length heparin-binding growth associated molecule.

    PubMed Central

    Hampton, B S; Marshak, D R; Burgess, W H

    1992-01-01

    Heparin-binding growth-associated molecule (HB-GAM) was purified from adult bovine brain and chicken heart. The yield of HB-GAM is increased by 5- to 10-fold when 250 mM NaCl is added to the homogenization buffer, indicating that HB-GAM may exist as a complex with an insoluble component of the tissue. The complete amino acid sequence of the brain-derived HB-GAM was established by automated Edman degradation of the intact protein and chemically or enzymatically derived fragments. The mass of bovine HB-GAM as determined by plasma desorption time-of-flight mass spectrometry is 15,291 mass units, which compares favorably with the calculated mass of 15,289 based on the amino acid sequence. Therefore, HB-GAM has not undergone any major post-translational modifications other than cleavage of the signal peptide. These results indicate that previous amino acid sequence analysis of this protein was carried out using truncated HB-GAM. Full-length HB-GAM is not a mitogen for Balb/3T3 clone A31, Balb MK, NRK, or human umbilical vein endothelial cells. HB-GAM does, however, have adhesive properties and neurite extension activity for chick embryo cerebral cortical derived neurons when presented to these cells as a substrate. HB-GAM had little neurite extension activity when presented as a soluble factor. Images PMID:1550956

  5. Use of Dried Blood Spots to Elucidate Full-Length Transmitted/Founder HIV-1 Genomes

    PubMed Central

    Salazar-Gonzalez, Jesus F.; Salazar, Maria G.; Tully, Damien C.; Ogilvie, Colin B.; Learn, Gerald H.; Allen, Todd M.; Heath, Sonya L.; Goepfert, Paul; Bar, Katharine J.

    2016-01-01

    Background Identification of HIV-1 genomes responsible for establishing clinical infection in newly infected individuals is fundamental to prevention and pathogenesis research. Processing, storage, and transportation of the clinical samples required to perform these virologic assays in resource-limited settings requires challenging venipuncture and cold chain logistics. Here, we validate the use of dried-blood spots (DBS) as a simple and convenient alternative to collecting and storing frozen plasma. Methods We performed parallel nucleic acid extraction, single genome amplification (SGA), next generation sequencing (NGS), and phylogenetic analyses on plasma and DBS. Results We demonstrated the capacity to extract viral RNA from DBS and perform SGA to infer the complete nucleotide sequence of the transmitted/founder (TF) HIV-1 envelope gene and full-length genome in two acutely infected individuals. Using both SGA and NGS methodologies, we showed that sequences generated from DBS and plasma display comparable phylogenetic patterns in both acute and chronic infection. SGA was successful on samples with a range of plasma viremia, including samples as low as 1,700 copies/ml and an estimated ∼50 viral copies per blood spot. Further, we demonstrated reproducible efficiency in gp160 env sequencing in DBS stored at ambient temperature for up to three weeks or at -20°C for up to five months. Conclusions These findings support the use of DBS as a practical and cost-effective alternative to frozen plasma for clinical trials and translational research conducted in resource-limited settings. PMID:27819061

  6. Identification of Putative Noncoding RNAs Among the RIKEN Mouse Full-Length cDNA Collection

    PubMed Central

    Numata, Koji; Kanai, Akio; Saito, Rintaro; Kondo, Shinji; Adachi, Jun; Wilming, Laurens G.; Hume, David A.; Hayashizaki, Yoshihide; Tomita, Masaru

    2003-01-01

    With the sequencing and annotation of genomes and transcriptomes of several eukaryotes, the importance of noncoding RNA (ncRNA)—RNA molecules that are not translated to protein products—has become more evident. A subclass of ncRNA transcripts are encoded by highly regulated, multi-exon, transcriptional units, are processed like typical protein-coding mRNAs and are increasingly implicated in regulation of many cellular functions in eukaryotes. This study describes the identification of candidate functional ncRNAs from among the RIKEN mouse full-length cDNA collection, which contains 60,770 sequences, by using a systematic computational filtering approach. We initially searched for previously reported ncRNAs and found nine murine ncRNAs and homologs of several previously described nonmouse ncRNAs. Through our computational approach to filter artifact-free clones that lack protein coding potential, we extracted 4280 transcripts as the largest-candidate set. Many clones in the set had EST hits, potential CpG islands surrounding the transcription start sites, and homologies with the human genome. This implies that many candidates are indeed transcribed in a regulated manner. Our results demonstrate that ncRNAs are a major functional subclass of processed transcripts in mammals. PMID:12819127

  7. Screening of novel malaria DNA vaccine candidates using full-length cDNA library.

    PubMed

    Shibui, Akiko; Nakae, Susumu; Watanabe, Junichi; Sato, Yoshitaka; Tolba, Mohammed E M; Doi, Junko; Shiibashi, Takashi; Nogami, Sadao; Sugano, Sumio; Hozumi, Nobumichi

    2013-11-01

    No licensed malaria vaccine exists, in spite of intensive development efforts. We have been investigating development of a DNA vaccine to prevent malaria infection. To date, we have established a full-length cDNA expression library from the erythrocytic-stage murine malaria parasite, Plasmodium berghei. We found that immunization of mice with combined 2000 clones significantly prolonged survival after challenge infection and that splenocytes from the immunized mice showed parasite-specific cytokine production. We determined the 5'-end one-pass sequence of these clones and mapped a draft genomic sequence for P. berghei for use in screening vaccine candidates for efficacy. In this study, we annotated these cDNA clones by comparing them with the genomic sequence of Plasmodium falciparum. We then divided them into several subsets based on their characteristics and examined their protective effects against malaria infection. Consequently, we selected 104 clones that strongly induced specific IgG production and decreased the mortality rate in the early phase. Most of these 104 clones coded for unknown proteins. The results suggest that these clones represent potential novel malaria vaccine candidates.

  8. Improving the diffraction of full-length human selenomethionyl metavinculin crystals by streak-seeding

    SciTech Connect

    Rangarajan, Erumbi S.; Izard, Tina

    2012-06-28

    Metavinculin is an alternatively spliced isoform of vinculin that has a 68-residue insert in its tail domain (1134 total residues) and is exclusively expressed in cardiac and smooth muscle tissue, where it plays important roles in myocyte adhesion complexes. Mutations in the metavinculin-specific insert are associated with dilated cardiomyopathy (DCM) in man. Crystals of a DCM-associated mutant of full-length selenomethionine-labeled metavinculin grown by hanging-drop vapor diffusion diffracted poorly and were highly sensitive to radiation, preventing the collection of a complete X-ray diffraction data set at the highest possible resolution. Streak-seeding markedly improved the stability, crystal-growth rate and diffraction quality of DCM-associated mutant metavinculin crystals, allowing complete data collection to 3.9 {angstrom} resolution. These crystals belonged to space group P4{sub 3}2{sub 1}2, with two molecules in the asymmetric unit and unit-cell parameters a = b = 170, c = 211 {angstrom}, {alpha} = {beta} = {gamma} = 90{sup o}.

  9. Pulse-field electrophoresis indicates full-length Mycoplasma chromosomes range widely in size.

    PubMed Central

    Neimark, H C; Lange, C S

    1990-01-01

    Full-size linear chromosomes were prepared from mycoplasmas by using gamma-irradiation to introduce one (on average) double-strand break in their circular chromosomes. Chromosome sizes were estimated by pulsed-field gel electrophoresis (PFGE) from the mobilities of these full-length molecules relative to DNA size references. Sizes estimated for Ureaplasma urealyticum T960 and 16 Mycoplasma species ranged from 684 kbp (M. hominis) to 1315 kbp (M. iowae). Using this sample, we found no correlation between the mobility of the full-size linear chromosomes and their G + C content. Sizes for A. laidlawii and A. hippikon were within the range expected from renaturation kinetics. PFGE size estimates are in good agreement with sizes determined by other methods, including electron microscopy, an ordered clone library, and summation of restriction fragments. Our estimates also agree with those from renaturation kinetics for both the largest and some of the smallest chromosomes, but in the intermediate size range, renaturation kinetics consistently provides lower values than PFGE or electron microscopy. Our PFGE estimates show that mycoplasma chromosomes span a continual range of sizes, with several intermediate values falling between the previously recognized large and small chromosome size clusters. Images PMID:2216718

  10. Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation

    SciTech Connect

    Rivera-Cancel, Giomar; Ko, Wen-huang; Tomchick, Diana R.; Correa, Fernando; Gardner, Kevin H.

    2014-12-02

    Although histidine kinases (HKs) are critical sensors of external stimuli in prokaryotes, the mechanisms by which their sensor domains control enzymatic activity remain unclear. In this paper, we report the full-length structure of a blue light-activated HK from Erythrobacter litoralis HTCC2594 (EL346) and the results of biochemical and biophysical studies that explain how it is activated by light. Contrary to the standard view that signaling occurs within HK dimers, EL346 functions as a monomer. Its structure reveals that the light–oxygen–voltage (LOV) sensor domain both controls kinase activity and prevents dimerization by binding one side of a dimerization/histidine phosphotransfer-like (DHpL) domain. The DHpL domain also contacts the catalytic/ATP-binding (CA) domain, keeping EL346 in an inhibited conformation in the dark. Upon light stimulation, interdomain interactions weaken to facilitate activation. Our data suggest that the LOV domain controls kinase activity by affecting the stability of the DHpL/CA interface, releasing the CA domain from an inhibited conformation upon photoactivation. Finally, we suggest parallels between EL346 and dimeric HKs, with sensor-induced movements in the DHp similarly remodeling the DHp/CA interface as part of activation.

  11. Accumulation of Stable Full-Length Circular Group I Intron RNAs during Heat-Shock.

    PubMed

    Andersen, Kasper L; Beckert, Bertrand; Masquida, Benoit; Johansen, Steinar D; Nielsen, Henrik

    2016-10-31

    Group I introns in nuclear ribosomal RNA of eukaryotic microorganisms are processed by splicing or circularization. The latter results in formation of full-length circular introns without ligation of the exons and has been proposed to be active in intron mobility. We applied qRT-PCR to estimate the copy number of circular intron RNA from the myxomycete Didymium iridis. In exponentially growing amoebae, the circular introns are nuclear and found in 70 copies per cell. During heat-shock, the circular form is up-regulated to more than 500 copies per cell. The intron harbours two ribozymes that have the potential to linearize the circle. To understand the structural features that maintain circle integrity, we performed chemical and enzymatic probing of the splicing ribozyme combined with molecular modeling to arrive at models of the inactive circular form and its active linear counterpart. We show that the two forms have the same overall structure but differ in key parts, including the catalytic core element P7 and the junctions at which reactions take place. These differences explain the relative stability of the circular species, demonstrate how it is prone to react with a target molecule for circle integration and thus supports the notion that the circular form is a biologically significant molecule possibly with a role in intron mobility.

  12. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain

    PubMed Central

    Sükösd, Zsuzsanna; Andersen, Ebbe S.; Seemann, Stefan E.; Jensen, Mads Krogh; Hansen, Mathias; Gorodkin, Jan; Kjems, Jørgen

    2015-01-01

    A distance constrained secondary structural model of the ≈10 kb RNA genome of the HIV-1 has been predicted but higher-order structures, involving long distance interactions, are currently unknown. We present the first global RNA secondary structure model for the HIV-1 genome, which integrates both comparative structure analysis and information from experimental data in a full-length prediction without distance constraints. Besides recovering known structural elements, we predict several novel structural elements that are conserved in HIV-1 evolution. Our results also indicate that the structure of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping protein-coding regions the COS is supported by a particular high frequency of compensatory base changes, suggesting functional importance for this element. This new structural element potentially organizes the whole genome into three major domains protruding from a conserved core structure with potential roles in replication and evolution for the virus. PMID:26476446

  13. Comprehensive Analysis of the Green-to-Blue Photoconversion of Full-Length Cyanobacteriochrome Tlr0924

    PubMed Central

    Hardman, Samantha J.O.; Hauck, Anna F.E.; Clark, Ian P.; Heyes, Derren J.; Scrutton, Nigel S.

    2014-01-01

    Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of central importance in biological light-activated signaling mechanisms. These photoreceptors are known to reversibly convert between two states in a photoinitiated process that involves a basic E/Z isomerization of the bilin chromophore and, in certain cases, the breakage of a thioether linkage to a conserved cysteine residue in the bulk protein structure. The exact details and timescales of the reactions involved in these photoconversions have not been conclusively shown. The cyanobacteriochrome Tlr0924 contains phycocyanobilin and phycoviolobilin chromophores, both of which photoconvert between two species: blue-absorbing and green-absorbing, and blue-absorbing and red-absorbing, respectively. Here, we followed the complete green-to-blue photoconversion process of the phycoviolobilin chromophore in the full-length form of Tlr0924 over timescales ranging from femtoseconds to seconds. Using a combination of time-resolved visible and mid-infrared transient absorption spectroscopy and cryotrapping techniques, we showed that after photoisomerization, which occurs with a lifetime of 3.6 ps, the phycoviolobilin twists or distorts slightly with a lifetime of 5.3 μs. The final step, the formation of the thioether linkage with the protein, occurs with a lifetime of 23.6 ms. PMID:25418104

  14. Efficient plant gene identification based on interspecies mapping of full-length cDNAs.

    PubMed

    Amano, Naoki; Tanaka, Tsuyoshi; Numa, Hisataka; Sakai, Hiroaki; Itoh, Takeshi

    2010-10-01

    We present an annotation pipeline that accurately predicts exon-intron structures and protein-coding sequences (CDSs) on the basis of full-length cDNAs (FLcDNAs). This annotation pipeline was used to identify genes in 10 plant genomes. In particular, we show that interspecies mapping of FLcDNAs to genomes is of great value in fully utilizing FLcDNA resources whose availability is limited to several species. Because low sequence conservation at 5'- and 3'-ends of FLcDNAs between different species tends to result in truncated CDSs, we developed an improved algorithm to identify complete CDSs by the extension of both ends of truncated CDSs. Interspecies mapping of 71 801 monocot FLcDNAs to the Oryza sativa genome led to the detection of 22 142 protein-coding regions. Moreover, in comparing two mapping programs and three ab initio prediction programs, we found that our pipeline was more capable of identifying complete CDSs. As demonstrated by monocot interspecies mapping, in which nucleotide identity between FLcDNAs and the genome was ∼80%, the resultant inferred CDSs were sufficiently accurate. Finally, we applied both inter- and intraspecies mapping to 10 monocot and dicot genomes and identified genes in 210 551 loci. Interspecies mapping of FLcDNAs is expected to effectively predict genes and CDSs in newly sequenced genomes.

  15. Near Full-Length Sequence Analysis of HIV Type 1 BF Recombinants from Italy

    PubMed Central

    Foley, Brian T.; Rosi, Andrea; Vicenti, Ilaria; Nannetti, Giulio; Meini, Genny; Razzolini, Francesca; Zazzi, Maurizio

    2012-01-01

    Abstract Recombination between HIV-1 subtypes B and F has generated several circulating and unique recombinant forms, particularly in Latin American areas. In Italy, subtype B is highly prevalent while subtype F is the most common pure non-B subtype. To investigate the recombination pattern in Italian BF recombinant viruses, we characterized full-length sequences derived from 15 adult patients, mostly Italian and infected by the heterosexual route. One of the BF mosaics was a CRF29, three sequences clustered with low bootstrap values with CRF39, CRF40, and CRF42. With the exception of the CRF29-like sequence, the other recombination patterns were unique, but two possible clusters were identified. Analysis of the gp120 V3 domain suggested a possible link with subtype F from Eastern Europe rather than from Latin America, favoring the hypothesis of local recombination between clade B and F viruses over that of import of BF recombinants from Latin America. HIV-1 subtypes B and F appear prone to generation of unique recombinants in Italy, warranting epidemiological surveillance and investigation of a possible clinical significance. PMID:21740272

  16. Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes

    PubMed Central

    Ayoub, Nadia A.; Garb, Jessica E.; Tinghitella, Robin M.; Collin, Matthew A.; Hayashi, Cheryl Y.

    2007-01-01

    Spider dragline (major ampullate) silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons), recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs) that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers. PMID:17565367

  17. Molecular cloning and biological characterization of full-length HIV-1 subtype C from Botswana.

    PubMed

    Ndung'u, T; Renjifo, B; Novitsky, V A; McLane, M F; Gaolekwe, S; Essex, M

    2000-12-20

    Human immunodeficiency virus type 1 (HIV-1) subtype C is now responsible for more than half of all HIV-1 infections in the global epidemic and for the high levels of HIV-1 prevalence in southern Africa. To facilitate studies of the biological nature and the underlying molecular determinants of this virus, we constructed eight full-length proviral clones from two asymptomatic and three AIDS patients infected with HIV-1 subtype C from Botswana. Analysis of viral lysates showed that Gag, Pol, and Env structural proteins were present in the virions. In four clones, the analysis suggested inefficient envelope glycoprotein processing. Nucleotide sequence analysis of the eight clones did not reveal frameshifts, deletions, premature truncations, or translational stop codons in any structural, regulatory, or accessory genes. None of the subtype C clones were replication competent in donor peripheral blood mononuclear cells (PBMCs), macrophages, Jurkat(tat) cells, or U87. CD4.CCR5 cells. However, infection by two clones could be rescued by complementation with a functional subtype C envelope clone, resulting in a productive infection of PBMCs, macrophages, and U87. CD4.CCR5 cells.

  18. Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation.

    PubMed

    Rivera-Cancel, Giomar; Ko, Wen-huang; Tomchick, Diana R; Correa, Fernando; Gardner, Kevin H

    2014-12-16

    Although histidine kinases (HKs) are critical sensors of external stimuli in prokaryotes, the mechanisms by which their sensor domains control enzymatic activity remain unclear. Here, we report the full-length structure of a blue light-activated HK from Erythrobacter litoralis HTCC2594 (EL346) and the results of biochemical and biophysical studies that explain how it is activated by light. Contrary to the standard view that signaling occurs within HK dimers, EL346 functions as a monomer. Its structure reveals that the light-oxygen-voltage (LOV) sensor domain both controls kinase activity and prevents dimerization by binding one side of a dimerization/histidine phosphotransfer-like (DHpL) domain. The DHpL domain also contacts the catalytic/ATP-binding (CA) domain, keeping EL346 in an inhibited conformation in the dark. Upon light stimulation, interdomain interactions weaken to facilitate activation. Our data suggest that the LOV domain controls kinase activity by affecting the stability of the DHpL/CA interface, releasing the CA domain from an inhibited conformation upon photoactivation. We suggest parallels between EL346 and dimeric HKs, with sensor-induced movements in the DHp similarly remodeling the DHp/CA interface as part of activation.

  19. BAY 81-8973, a full-length recombinant factor VIII: manufacturing processes and product characteristics.

    PubMed

    Garger, S; Severs, J; Regan, L; Hesslein, A; Ignowski, J; Wu, P; Long, E; Gupta, S; Liu, S; Wang, W

    2017-03-01

    BAY 81-8973 (Kovaltry(®) , Bayer, Berkeley, CA, USA) is an unmodified, full-length recombinant human factor VIII (FVIII) approved for prophylaxis and on-demand treatment of bleeding episodes in patients with haemophilia A. The BAY 81-8973 manufacturing process is based on the process used for sucrose-formulated recombinant FVIII (rFVIII-FS), with changes and enhancements made to improve production efficiency, further augment pathogen safety, and eliminate animal- and human-derived raw materials from the production processes. The baby hamster kidney cell line used for BAY 81-8973 was developed by introducing the gene for human heat shock protein 70 into the rFVIII-FS cell line, a change that improved cell line robustness and productivity. Pathogen safety was enhanced by including a 20-nm filtration step, which can remove viruses, transmissible spongiform encephalopathy agents and potential protein aggregates. No human- or animal-derived proteins are added to the cell culture process, purification or final formulation. The BAY 81-8973 manufacturing process results in a product of enhanced purity with a consistently high degree of sialylation of N-linked glycans on the molecular surface. The innovative manufacturing techniques used for BAY 81-8973 yield an effective rFVIII product with a favourable safety profile for treatment of haemophilia A. © 2016 Bayer. Haemophilia Published by John Wiley & Sons Ltd.

  20. Quench performance of Fermilab/General Dynamics built full length SSC collider dipole magnets

    SciTech Connect

    Strait, J.; Orris, D.; Mazur, P.O.; Bleadon, M.; Bossert, R.; Carson, J.; Delchamps, S.W.; Gourlay, S.; Hanft, R.; Koska, W.; Kuchnir, M.; Lamm, M.J.; Ozelis, J.; Wake, M.; Devred, A.; DiMarco, J.; Kuzminski, J.; Nah, W.; Ogitsu, T.; Puglisi, M.; Tompkins, J.C.; Yu, Y.; Zhao, Y.; Zheng, H.

    1992-04-01

    In this paper we present results of quench testing of full length SSC dipole magnets at Fermilab. The data are from the first six of a series of thirteen 15 m long, 50 mm aperture SSC dipole magnets which are being built and tested at Fermilab. These magnets were designed jointly by Fermilab, Brookhaven Laboratory, Lawrence Berkeley Laboratory and the SSC laboratory. Among the major goals for this series of magnets are to transfer magnet production technology to the lead vendor for the Collider Dipole Magnet, the General Dynamics Corporation, and to demonstrate industrial production by the vendor. The first magnet in the series, DCA311, was built by Fermilab technicians to establish assembly procedures. The second magnet, DCA312, was the ``technology transfer magnet`` and was built jointly by Fermilab and General Dynamics technicians. The next seven, DCA313- 319 are being built by General Dynamics personnel using Fermilab facilities and procedures. However, Fermilab personnel still operate the major tooling, provide the welders, perform assembly of items that would not be part of production magnets (e.g. voltage taps), and oversee the QA program. Five of these 7 GD-built magnets will be used in the Accelerator Systems String Test (ASST) to be carried out in Dallas later this year. The last four magnets, DCA320-323, are being built by Fermilab alone.

  1. Quench performance of Fermilab/General Dynamics built full length SSC collider dipole magnets

    SciTech Connect

    Strait, J.; Orris, D.; Mazur, P.O.; Bleadon, M.; Bossert, R.; Carson, J.; Delchamps, S.W.; Gourlay, S.; Hanft, R.; Koska, W.; Kuchnir, M.; Lamm, M.J.; Ozelis, J.; Wake, M. ); Devred, A.; DiMarco, J.; Kuzminski, J.; Nah, W.; Ogitsu, T.; Puglisi, M.; Tompkins, J.C.; Yu, Y.; Zhao, Y.; Zheng, H. )

    1992-04-01

    In this paper we present results of quench testing of full length SSC dipole magnets at Fermilab. The data are from the first six of a series of thirteen 15 m long, 50 mm aperture SSC dipole magnets which are being built and tested at Fermilab. These magnets were designed jointly by Fermilab, Brookhaven Laboratory, Lawrence Berkeley Laboratory and the SSC laboratory. Among the major goals for this series of magnets are to transfer magnet production technology to the lead vendor for the Collider Dipole Magnet, the General Dynamics Corporation, and to demonstrate industrial production by the vendor. The first magnet in the series, DCA311, was built by Fermilab technicians to establish assembly procedures. The second magnet, DCA312, was the ''technology transfer magnet'' and was built jointly by Fermilab and General Dynamics technicians. The next seven, DCA313- 319 are being built by General Dynamics personnel using Fermilab facilities and procedures. However, Fermilab personnel still operate the major tooling, provide the welders, perform assembly of items that would not be part of production magnets (e.g. voltage taps), and oversee the QA program. Five of these 7 GD-built magnets will be used in the Accelerator Systems String Test (ASST) to be carried out in Dallas later this year. The last four magnets, DCA320-323, are being built by Fermilab alone.

  2. Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies.

    PubMed

    Njengele, Zikhona; Kleynhans, Ronel; Sayed, Yasien; Mosebi, Salerwe

    2016-12-01

    Vpu is one of four accessory proteins encoded by human immunodeficiency virus type I (HIV-1). Vpu modulates the expression of several cellular restriction factors within the HIV-1 infected cell including CD4, CD74, the bone marrow stromal antigen 2 (BST-2) and NK-T-and-B antigen. The interaction of HIV-1 Vpu with these proteins interferes with the innate immune response directed against HIV-1; thereby promoting viral persistence. The involvement of HIV-1 Vpu in manipulating the cellular environment in ways that favor viral replication makes it an attractive target for anti-HIV drug intervention. This paper describes the over-expression and purification of a soluble HIV-1 Vpu from inclusion bodies by ion-exchange chromatography, allowing production of 6 mg of highly purified protein (>95% purity) per 10 mg of pelleted cells obtained from 1 L of bacterial culture. Far-UV circular dichroism showed that the recombinant protein is folded and retained its secondary structure. Moreover, using ELISA, known HIV-1 Vpu binding partners, BST-2 and CD74, showed that the refolded purified protein is functional or at least assumes a conformation that is capable of binding these putative binding partners. To our knowledge, this is the first report of the purification and successful solubilization of full-length, wild-type HIV-1 Vpu from inclusion bodies in Escherichia coli. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

    PubMed Central

    Bezem, Maria T.; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I.

    2016-01-01

    Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

  4. Membrane Yeast Two-Hybrid (MYTH) Mapping of Full-Length Membrane Protein Interactions.

    PubMed

    Snider, Jamie; Stagljar, Igor

    2016-01-04

    Mapping of protein interaction networks is a major strategy for obtaining a global understanding of protein function in cells and represents one of the primary goals of proteomics research. Membrane proteins, which play key roles in human disease and as drug targets, are of considerable interest; however, because of their hydrophobic nature, mapping their interactions presents significant technical challenges and requires the use of special methodological approaches. One powerful approach is the membrane yeast two-hybrid (MYTH) assay, a split-ubiquitin-based system specifically suited to the study of full-length membrane protein interactions in vivo using the yeast Saccharomyces cerevisiae as a host. The system can be used in both low- and high-throughput formats to study proteins from a wide range of different organisms. There are two primary variants of MYTH: integrated (iMYTH), which involves endogenous expression and tagging of baits and is suitable for studying native yeast membrane proteins, and traditional (tMYTH), which involves ectopic plasmid-based expression of tagged baits and is suitable for studying membrane proteins from other organisms. Here we provide an introduction to the MYTH assay, including both the iMYTH and tMYTH variants. MYTH can be set up in almost any laboratory environment, with results typically obtainable within 4 to 6 wk. © 2016 Cold Spring Harbor Laboratory Press.

  5. Expression of full-length utrophin prevents muscular dystrophy in mdx mice.

    PubMed

    Tinsley, J; Deconinck, N; Fisher, R; Kahn, D; Phelps, S; Gillis, J M; Davies, K

    1998-12-01

    Duchenne muscular dystrophy (DMD) is a lethal, progressive muscle wasting disease caused by a loss of sarcolemmal bound dystrophin, which results in the death of the muscle fiber leading to the gradual depletion of skeletal muscle. The molecular structure of dystrophin is very similar to that of the related protein utrophin. Utrophin is found in all tissues and is confined to the neuromuscular and myotendinous junctions in mature muscle. Sarcolemmal localization of a truncated utrophin transgene in the dystrophin-deficient mdx mouse significantly improves the dystrophic muscle phenotype. Therefore, up-regulation of utrophin by drug therapy is a plausible therapeutic approach in the treatment of DMD. Here we demonstrate that expression of full-length utrophin in mdx mice prevents the development of muscular dystrophy. We assessed muscle morphology, fiber regeneration and mechanical properties (force development and resistance to stretch) of mdx and transgenic mdx skeletal and diaphragm muscle. The utrophin levels required in muscle are significantly less than the normal endogenous utrophin levels seen in lung and kidney, and we provide evidence that the pathology depends on the amount of utrophin expression. These results also have important implications for DMD therapies in which utrophin replacement is achieved by delivery using exogenous vectors.

  6. Alternative interdomain configurations of the full-length MMP-2 enzyme explored by molecular dynamics simulations.

    PubMed

    Díaz, Natalia; Suárez, Dimas

    2012-03-08

    Conformational freedom between the different domains of the matrix metalloproteinase family of enzymes has been repeatedly invoked to explain the mechanism of hydrolysis of some of their most complex macromolecular substrates. This proposed interdomain motion has been experimentally confirmed to occur in solution for matrix metalloproteinases MMP-1, MMP-9, and MMP-12. In this work, we computationally assess the likely conformational freedom in aqueous solution of the full-length form of the MMP-2 enzyme in the absence of its pro-peptide domain. To this end, we perform molecular dynamics (MD) simulations and approximate free energy analyses in four different arrangements of the protein domains that correspond to (a) the compact conformation observed in the X-ray structure; (b) an initially elongated structure in which the hemopexin (HPX) domain is separated from the catalytic (CAT) and fibronectin domains; and (c-d) two alternative conformations suggested by protein-protein docking calculations. Overall, our results indicate that the interdomain flexibility is very likely a general property of the MMP-2 enzyme in solution.

  7. Efficient Plant Gene Identification Based on Interspecies Mapping of Full-Length cDNAs

    PubMed Central

    Amano, Naoki; Tanaka, Tsuyoshi; Numa, Hisataka; Sakai, Hiroaki; Itoh, Takeshi

    2010-01-01

    We present an annotation pipeline that accurately predicts exon–intron structures and protein-coding sequences (CDSs) on the basis of full-length cDNAs (FLcDNAs). This annotation pipeline was used to identify genes in 10 plant genomes. In particular, we show that interspecies mapping of FLcDNAs to genomes is of great value in fully utilizing FLcDNA resources whose availability is limited to several species. Because low sequence conservation at 5′- and 3′-ends of FLcDNAs between different species tends to result in truncated CDSs, we developed an improved algorithm to identify complete CDSs by the extension of both ends of truncated CDSs. Interspecies mapping of 71 801 monocot FLcDNAs to the Oryza sativa genome led to the detection of 22 142 protein-coding regions. Moreover, in comparing two mapping programs and three ab initio prediction programs, we found that our pipeline was more capable of identifying complete CDSs. As demonstrated by monocot interspecies mapping, in which nucleotide identity between FLcDNAs and the genome was ∼80%, the resultant inferred CDSs were sufficiently accurate. Finally, we applied both inter- and intraspecies mapping to 10 monocot and dicot genomes and identified genes in 210 551 loci. Interspecies mapping of FLcDNAs is expected to effectively predict genes and CDSs in newly sequenced genomes. PMID:20668003

  8. Full-length infectious clone of an Iranian isolate of chicken anemia virus.

    PubMed

    Kaffashi, Amir; Eshratabadi, Fatemeh; Shoushtari, Abdelhamed

    2017-04-01

    An Iranian field strain of chicken anemia virus (CAV), designated IR CAV, was isolated in the Marek's disease virus-transformed lymphoblastoid cell line MDCC-MSB1 (MSB1) culture for the first time. The full-length CAV DNA of this strain was cloned in the bacterial plasmid pTZ57R/T to create the molecular clone pTZ-CAV. The nucleotide and deduced amino acid sequences of viral proteins of IR CAV were compared with those of representative CAV sequences including reference and commercial vaccine strains. IR CAV was not related to vaccine strains and also found to have glutamine at positions 139 and 144 confirming previous studies in which such mutations were associated with a slow rate of virus spread in cell culture. pTZ-CAV was digested with PstI to release IR CAV DNA and then transfected into MSB1 cell by electroporation. The transfected cells showed cytopathic effect similar to virion-initiated infection. One-day old specific pathogen-free chicks were inoculated with the regenerated virus, which had been obtained from transfected MSB1 cells, and compared with the chicks inoculated with IR CAV. Gross lesions in the birds inoculated with the regenerated virus illustrated the infectious nature of the regenerated virus from the cloned IR CAV DNA.

  9. Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation

    DOE PAGES

    Rivera-Cancel, Giomar; Ko, Wen-huang; Tomchick, Diana R.; ...

    2014-12-02

    Although histidine kinases (HKs) are critical sensors of external stimuli in prokaryotes, the mechanisms by which their sensor domains control enzymatic activity remain unclear. In this paper, we report the full-length structure of a blue light-activated HK from Erythrobacter litoralis HTCC2594 (EL346) and the results of biochemical and biophysical studies that explain how it is activated by light. Contrary to the standard view that signaling occurs within HK dimers, EL346 functions as a monomer. Its structure reveals that the light–oxygen–voltage (LOV) sensor domain both controls kinase activity and prevents dimerization by binding one side of a dimerization/histidine phosphotransfer-like (DHpL) domain.more » The DHpL domain also contacts the catalytic/ATP-binding (CA) domain, keeping EL346 in an inhibited conformation in the dark. Upon light stimulation, interdomain interactions weaken to facilitate activation. Our data suggest that the LOV domain controls kinase activity by affecting the stability of the DHpL/CA interface, releasing the CA domain from an inhibited conformation upon photoactivation. Finally, we suggest parallels between EL346 and dimeric HKs, with sensor-induced movements in the DHp similarly remodeling the DHp/CA interface as part of activation.« less

  10. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain.

    PubMed

    Sükösd, Zsuzsanna; Andersen, Ebbe S; Seemann, Stefan E; Jensen, Mads Krogh; Hansen, Mathias; Gorodkin, Jan; Kjems, Jørgen

    2015-12-02

    A distance constrained secondary structural model of the ≈10 kb RNA genome of the HIV-1 has been predicted but higher-order structures, involving long distance interactions, are currently unknown. We present the first global RNA secondary structure model for the HIV-1 genome, which integrates both comparative structure analysis and information from experimental data in a full-length prediction without distance constraints. Besides recovering known structural elements, we predict several novel structural elements that are conserved in HIV-1 evolution. Our results also indicate that the structure of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping protein-coding regions the COS is supported by a particular high frequency of compensatory base changes, suggesting functional importance for this element. This new structural element potentially organizes the whole genome into three major domains protruding from a conserved core structure with potential roles in replication and evolution for the virus.

  11. Systematic assembly of a full-length infectious clone of human coronavirus NL63.

    PubMed

    Donaldson, Eric F; Yount, Boyd; Sims, Amy C; Burkett, Susan; Pickles, Raymond J; Baric, Ralph S

    2008-12-01

    Historically, coronaviruses were predominantly associated with mild upper respiratory disease in humans. More recently, three novel coronaviruses associated with severe human respiratory disease were found, including (i) the severe acute respiratory syndrome coronavirus, associated with a significant atypical pneumonia and 10% mortality; (ii) HKU-1, associated with chronic pulmonary disease; and (iii) NL63, associated with both upper and lower respiratory tract disease in children and adults worldwide. These discoveries establish coronaviruses as important human pathogens and underscore the need for continued research toward the development of platforms that will enable genetic manipulation of the viral genome, allowing for rapid and rational development and testing of candidate vaccines, vaccine vectors, and therapeutics. In this report, we describe a reverse genetics system for NL63, whereby five contiguous cDNAs that span the entire genome were used to generate a full-length cDNA. Recombinant NL63 viruses which contained the expected marker mutations replicated as efficiently as the wild-type NL63 virus. In addition, we engineered the heterologous green fluorescent protein gene in place of open reading frame 3 (ORF3) of the NL63 clone, simultaneously creating a unique marker for NL63 infection and demonstrating that the ORF3 protein product is nonessential for the replication of NL63 in cell culture. The availability of the NL63 and NL63gfp clones and recombinant viruses provides powerful tools that will help advance our understanding of this important human pathogen.

  12. Molecular cloning and properties of a full-length putative thyroid hormone receptor coactivator.

    PubMed

    Takeshita, A; Yen, P M; Misiti, S; Cardona, G R; Liu, Y; Chin, W W

    1996-08-01

    Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. The conserved carboxy-terminal region of the ligand-binding domain (AF-2) has been thought to play a critical role in mediating ligand-dependent transactivation by the interaction with coactivator(s). Using bacterially-expressed TR as a probe, far-Western-based expression cDNA library screening identified cDNAs that encode, in part, the recently reported partial steroid receptor coactivator-1 (SRC-1) sequence. Additional work, including 5' RACE, has characterized a full-length cDNA that encodes a approximately 160 kD protein as a putative thyroid hormone receptor coactivator (F-SRC-1). In vitro binding studies show that F-SRC-1 binds to a variety of nuclear hormone receptors in a ligand-dependent manner, along with TBP and TFIIB, suggesting that F-SRC-1 may play a role as a bridging molecule between nuclear hormone receptors and general transcription factors. Interestingly, AF-2 mutants also retain ligand-dependent interaction with F-SRC-1. Although F-SRC-1 recognizes the ligand-induced conformational changes of nuclear hormone receptors, our observations suggest that F-SRC-1 may bind directly with subregion(s) in nuclear hormone receptors other than the AF-2 region.

  13. Purification and Activity Testing of the Full-Length YycFGHI Proteins of Staphylococcus aureus

    PubMed Central

    Türck, Michael; Bierbaum, Gabriele

    2012-01-01

    Background The YycFG two-component regulatory system (TCS) of Staphylococcus aureus represents the only essential TCS that is almost ubiquitously distributed in Gram-positive bacteria with a low G+C-content. YycG (WalK/VicK) is a sensor histidine-kinase and YycF (WalR/VicR) is the cognate response regulator. Both proteins play an important role in the biosynthesis of the cell envelope and mutations in these proteins have been involved in development of vancomycin and daptomycin resistance. Methodology/Principal Findings Here we present high yield expression and purification of the full-length YycG and YycF proteins as well as of the auxiliary proteins YycH and YycI of Staphylococcus aureus. Activity tests of the YycG kinase and a mutated version, that harbours an Y306N exchange in its cytoplasmic PAS domain, in a detergent-micelle-model and a phosholipid-liposome-model showed kinase activity (autophosphorylation and phosphoryl group transfer to YycF) only in the presence of elevated concentrations of alkali salts. A direct comparison of the activity of the kinases in the liposome-model indicated a higher activity of the mutated YycG kinase. Further experiments indicated that YycG responds to fluidity changes in its microenvironment. Conclusions/Significance The combination of high yield expression, purification and activity testing of membrane and membrane-associated proteins provides an excellent experimental basis for further protein-protein interaction studies and for identification of all signals received by the YycFGHI system. PMID:22276191

  14. Designing a soluble near full-length HIV-1 gp41 trimer.

    PubMed

    Gao, Guofen; Wieczorek, Lindsay; Peachman, Kristina K; Polonis, Victoria R; Alving, Carl R; Rao, Mangala; Rao, Venigalla B

    2013-01-04

    The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates virus entry by binding to host receptors, whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has been extensively studied, the detailed mechanism is still poorly understood. Design of gp41 recombinants that mimic key intermediates is essential to elucidate the mechanism as well as to develop potent therapeutics and vaccines. Here, using molecular genetics and biochemical approaches, a series of hypotheses was tested to overcome the extreme hydrophobicity of HIV-1 gp41 and design a soluble near full-length gp41 trimer. The two long heptad repeat helices HR1 and HR2 of gp41 ectodomain were mutated to disrupt intramolecular HR1-HR2 interactions but not intermolecular HR1-HR1 interactions. This resulted in reduced aggregation and improved solubility. Attachment of a 27-amino acid foldon at the C terminus and slow refolding channeled gp41 into trimers. The trimers appear to be stabilized in a prehairpin-like structure, as evident from binding of a HR2 peptide to exposed HR1 grooves, lack of binding to hexa-helical bundle-specific NC-1 mAb, and inhibition of virus neutralization by broadly neutralizing antibodies 2F5 and 4E10. Fusion to T4 small outer capsid protein, Soc, allowed display of gp41 trimers on the phage nanoparticle. These approaches for the first time led to the design of a soluble gp41 trimer containing both the fusion peptide and the cytoplasmic domain, providing insights into the mechanism of entry and development of gp41-based HIV-1 vaccines.

  15. Characterization of Full-Length and Truncated Recombinant κ-Carrageenase Expressed in Pichia pastoris

    PubMed Central

    Yu, Yuan; Liu, Zhemin; Yang, Min; Chen, Meng; Wei, Zhihan; Shi, Lixia; Li, Li; Mou, Haijin

    2017-01-01

    κ-Carrageenase belongs to glycoside hydrolase family 16 and cleaves the β-(1→4) linkages of κ-carrageenan. In this study, genes encoding the full-length (cgkZ), Por secretion tail-truncated (cgkZΔPst) and carbohydrate binding domain-truncated (cgkZΔCBM) κ-carrageenase proteins were expressed in Pichia pastoris. The copy numbers of gene cgkZ, cgkZΔPst and cgkZΔCBM were 7, 7 and 6, respectively. The enzymatic activities of recombinant enzymes cgkZ, cgkZΔPst and cgkZΔCBM reached 4.68, 5.70, and 3.02 U/mL, respectively, after 120 h of shake flask fermentation at 22°C and pH 6 in the presence of 1 % (v/v) methanol. The molecular weights of recombinant cgkZ, cgkZΔPst, and cgkZΔCBM were approximately 65, 45, and 40 kDa; their Km values were 2.07, 1.85, and 1.04 mg/mL; and they exhibited optimal activity at 45–50°C and pH 6–7. All the recombinant enzymes were stimulated by Na+, Mg2+, Ca2+, and dithiothreitol. The end-products of enzymatic hydrolysis were mainly composed of κ-carrageenan tetrasaccharide and hexasaccharide. The removal of the Por secretion tail of κ-carrageenase promoted the transcription of κ-carrageenase gene, enhancing the specific activity of κ-carrageenase without significantly changing its catalytic properties. Although the transcription level of κ-carrageenase gene after the removal of the carbohydrate binding domain was relatively high, the specific activity of the recombinant enzyme significantly decreased. The comprehensive application of the P. pastoris expression system combined with the rational modification of genes may provide a novel approach for the heterologous expression of various marine enzymes with high activities. PMID:28861059

  16. Globular adiponectin but not full-length adiponectin induces increased procoagulability in human endothelial cells.

    PubMed

    Bobbert, Peter; Antoniak, Silvio; Schultheiss, Heinz-Peter; Rauch, Ursula

    2008-02-01

    Adiponectin (APN), a recently discovered adipocytokine, is present in human serum in a full length (fAPN) and a globular form (gAPN). gAPN is a proteolytic cleavage product of fAPN and seems to show independent biological activities compared to the properties of fAPN. The influence of gAPN and fAPN on procoagulability of cells is still unknown. This study examined the effect of gAPN and fAPN on the expression of tissue factor (TF), the initiator of the extrinsic coagulation system, in human umbilical vein endothelial cells (HUVECs). TF activity was measured by a chromogenic assay, TF mRNA by real-time PCR and TF protein by western blot. We found TF activity to be increased after activation by gAPN (3 microg/mL) compared to a non-stimulated control (169.0+/-19.23 U versus 501.9+/-38.95 U, p<0.001). Furthermore, TF mRNA and TF protein was increased dose-dependently after gAPN stimulation. The gAPN-induced rise of TF activity and TF mRNA was significantly reduced by inhibition of the MAP kinases ERK1/2, p38 and JNK. Contrary to gAPN, stimulation with fAPN did not lead to these procoagulant effects. In conclusion, gAPN increased TF transcription, expression and activity in HUVECs. Therefore, our data support the theory that gAPN but not fAPN supports the cellular procoagulability via TF upregulation.

  17. Pharmacokinetic properties of BAY 81-8973, a full-length recombinant factor VIII.

    PubMed

    Shah, A; Delesen, H; Garger, S; Lalezari, S

    2015-11-01

    BAY 81-8973 is a full-length recombinant factor VIII (FVIII) with the same primary amino acid sequence as sucrose-formulated recombinant FVIII (rFVIII-FS) but is produced with advanced manufacturing technologies. To analyse the pharmacokinetics (PK) of BAY 81-8973 after single and multiple dosing across different age and ethnic groups in the LEOPOLD clinical trial programme. The LEOPOLD trials enrolled patients with severe haemophilia A aged 12-65 years (LEOPOLD I and II) or ≤12 years (LEOPOLD Kids) with ≥150 (LEOPOLD I and II) or ≥50 (LEOPOLD Kids) exposure days to any FVIII product and no history of FVIII inhibitors. PK were assessed using chromogenic and one-stage assays (only chromogenic assay for LEOPOLD Kids) after a single 50-IU kg(-1) dose of BAY 81-8973 and, in a subset of patients in LEOPOLD I, after repeated dosing. Pharmacokinetic analyses were also performed based on age (18 to 65, 12 to <18, 6 to <12 and <6 years) and ethnicity (Asian and non-Asian). Pharmacokinetic assessments in the LEOPOLD I trial showed non-inferiority of BAY 81-8973 vs. rFVIII-FS. The PK of BAY 81-8973 were comparable after single and multiple dosing. Age-based analysis in the three trials showed that plasma concentrations were slightly lower for children, but similar for adolescents compared with adults. Pharmacokinetic results were similar in the different ethnic groups. Results of the LEOPOLD trials show that the BAY 81-8973 pharmacokinetic profile is non-inferior to rFVIII-FS. Similar BAY 81-8973 pharmacokinetic values were observed following single and repeated dosing and across ethnic groups. © 2015 John Wiley & Sons Ltd.

  18. Superacid-Surfactant Exchange: Enabling Nondestructive Dispersion of Full-Length Carbon Nanotubes in Water.

    PubMed

    Wang, Peng; Kim, Mijin; Peng, Zhiwei; Sun, Chuan-Fu; Mok, Jasper; Lieberman, Anna; Wang, YuHuang

    2017-09-26

    Attaining aqueous solutions of individual, long single-walled carbon nanotubes is a critical first step for harnessing the extraordinary properties of these materials. However, the widely used ultrasonication-ultracentrifugation approach and its variants inadvertently cut the nanotubes into short pieces. The process is also time-consuming and difficult to scale. Here we present an unexpectedly simple solution to this decade-old challenge by directly neutralizing a nanotube-chlorosulfonic acid solution in the presence of sodium deoxycholate. This straightforward superacid-surfactant exchange eliminates the need for both ultrasonication and ultracentrifugation altogether, allowing aqueous solutions of individual nanotubes to be prepared within minutes and preserving the full length of the nanotubes. We found that the average length of the processed nanotubes is more than 350% longer than sonicated controls, with a significant fraction approaching ∼9 μm, a length that is limited by only the raw material. The nondestructive nature is manifested by an extremely low density of defects, bright and homogeneous photoluminescence in the near-infrared, and ultrahigh electrical conductivity in transparent thin films (130 Ω/sq at 83% transmittance), which well exceeds that of indium tin oxide. Furthermore, we demonstrate that our method is fully compatible with established techniques for sorting nanotubes by their electronic structures and can also be readily applied to graphene. This surprisingly simple method thus enables nondestructive aqueous solution processing of high-quality carbon nanomaterials at large-scale and low-cost with the potential for a wide range of fundamental studies and applications, including, for example, transparent conductors, near-infrared imaging, and high-performance electronics.

  19. Identification and Nearly Full-Length Genome Characterization of Novel Porcine Bocaviruses

    PubMed Central

    Huang, Can-ping; Yao, Dong-ping; Liu, Na; Cui, Shu-xian; Jin, Yu; Duan, Zhao-jun

    2010-01-01

    The genus bocavirus includes bovine parvovirus (BPV), minute virus of canines (MVC), and a group of human bocaviruses (HBoV1-4). Using sequence-independent single primer amplification (SISPA), a novel bocavirus group was discovered with high prevalence (12.59%) in piglet stool samples. Two nearly full-length genome sequences were obtained, which were approximately 5,100 nucleotides in length. Multiple alignments revealed that they share 28.7–56.8% DNA sequence identity with other members of Parvovirinae. Phylogenetic analyses indicated their closest neighbors were members of the genus bocavirus. The new viruses had a putative non-structural NP1 protein, which was unique to bocaviruses. They were provisionally named porcine bocavirus 1 and 2 (PBoV1, PBoV2). PBoV1 and PBoV2 shared 94.2% nucleotide identity in NS1 gene sequence, suggesting that they represented two different bocavirus species. Two additional samples (6V, 7V) were amplified for 2,407 bp and 2,434 bp products, respectively, including a partial NP1 gene and the complete VP1 gene; Phylogenetic analysis indicated that 6Vand 7V grouped with PBoV1 and PBoV2 in the genus of bocavirus, but were in the separate clusters. Like other parvoviruses, PBoV1, PBoV2, 6Vand 7V also contained a putative secretory phospholipase A2 (sPLA2) motif in the VP1 unique region, with a conserved HDXXY motif in the catalytic center. The conserved motif YXGXF of the Ca2+-binding loop of sPLA2 identified in human bocavirus was also found in porcine bocavirus, which differs from the YXGXG motif carried by most other parvoviruses. The observation of PBoV and potentially other new bocavirus genus members may aid in molecular and functional characterization of the genus bocavirus. PMID:21049037

  20. Biomimetic Precipitation of Uniaxially Grown Calcium Phosphate Crystals from Full-Length Human Amelogenin Sols.

    PubMed

    Uskoković, Vuk; Li, Wu; Habelitz, Stefan

    2011-06-10

    Human dental enamel forms over a period of 2 - 4 years by substituting the enamel matrix, a protein gel mostly composed of a single protein, amelogenin with fibrous apatite nanocrystals. Self-assembly of a dense amelogenin matrix is presumed to direct the growth of apatite fibers and their organization into bundles that eventually comprise the mature enamel, the hardest tissue in the mammalian body. This work aims to establish the physicochemical and biochemical conditions for the synthesis of fibrous apatite crystals under the control of a recombinant full-length human amelogenin matrix in combination with a programmable titration system. The growth of apatite substrates was initiated from supersaturated calcium phosphate solutions in the presence of dispersed amelogenin assemblies. It was shown earlier and confirmed in this study that binding of amelogenin onto apatite surfaces presents the first step that leads to substrate-specific crystal growth. In this work, we report enhanced nucleation and growth under conditions at which amelogenin and apatite carry opposite charges and adsorption of the protein onto the apatite seeds is even more favored. Experiments at pH below the isoelectric point of amelogenin showed increased protein binding to apatite and at low Ca/P molar ratios resulted in a change in crystal morphology from plate-like to fibrous and rod-shaped. Concentrations of calcium and phosphate ions in the supernatant did not show drastic decreases throughout the titration period, indicating controlled precipitation from the protein suspension metastable with respect to calcium phosphate. It is argued that ameloblasts in the developing enamel may vary the density of the protein matrix at the nano scale by varying local pH, and thus control the interaction between the mineral and protein phases. The biomimetic experimental setting applied in this study has thus proven as convenient for gaining insight into the fundamental nature of the process of

  1. Structural characterization suggests models for monomeric and dimeric forms of full-length ezrin.

    PubMed

    Phang, Juanita M; Harrop, Stephen J; Duff, Anthony P; Sokolova, Anna V; Crossett, Ben; Walsh, James C; Beckham, Simone A; Nguyen, Cuong D; Davies, Roberta B; Glöckner, Carina; Bromley, Elizabeth H C; Wilk, Krystyna E; Curmi, Paul M G

    2016-09-15

    Ezrin is a member of the ERM (ezrin-radixin-moesin) family of proteins that have been conserved through metazoan evolution. These proteins have dormant and active forms, where the latter links the actin cytoskeleton to membranes. ERM proteins have three domains: an N-terminal FERM [band Four-point-one (4.1) ERM] domain comprising three subdomains (F1, F2, and F3); a helical domain; and a C-terminal actin-binding domain. In the dormant form, FERM and C-terminal domains form a stable complex. We have determined crystal structures of the active FERM domain and the dormant FERM:C-terminal domain complex of human ezrin. We observe a bistable array of phenylalanine residues in the core of subdomain F3 that is mobile in the active form and locked in the dormant form. As subdomain F3 is pivotal in binding membrane proteins and phospholipids, these transitions may facilitate activation and signaling. Full-length ezrin forms stable monomers and dimers. We used small-angle X-ray scattering to determine the solution structures of these species. As expected, the monomer shows a globular domain with a protruding helical coiled coil. The dimer shows an elongated dumbbell structure that is twice as long as the monomer. By aligning ERM sequences spanning metazoan evolution, we show that the central helical region is conserved, preserving the heptad repeat. Using this, we have built a dimer model where each monomer forms half of an elongated antiparallel coiled coil with domain-swapped FERM:C-terminal domain complexes at each end. The model suggests that ERM dimers may bind to actin in a parallel fashion.

  2. Complete mitochondrial genome of Esox reichertii (Amur pike).

    PubMed

    Liu, Yu; Yang, Jun

    2015-01-01

    The whole mitochondrial genome of Esox reichertii (fish) was first sequenced and characterized. It was determined to be 16,909 bp long, which contains the control region (CR), the origin of light-strand replication (OL), 22 transfer RNA genes, 2 ribosomal genes and 13 protein-coding genes. Overall base composition of the complete mitochondrial DNA was 28.65% A, 28.67% T, 27.28% C and 15.41% G, with 57.32%AT.

  3. A novel copper(II) coordination at His186 in full-length murine prion protein

    SciTech Connect

    Watanabe, Yasuko; Hiraoka, Wakako; Igarashi, Manabu; Ito, Kimihito; Shimoyama, Yuhei; Horiuchi, Motohiro; Yamamori, Tohru; Yasui, Hironobu; Kuwabara, Mikinori; Inagaki, Fuyuhiko; Inanami, Osamu

    2010-04-09

    To explore Cu(II) ion coordination by His{sup 186} in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP{sup C}.

  4. Soluble expression, purification and characterization of the full length IS2 Transposase

    PubMed Central

    2011-01-01

    Background The two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein. Results A soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common

  5. [Rapid construction of full-length MnSOD cDNA of chickens by one-step 3'RACE].

    PubMed

    Bu, You-Quan; Luo, Xu-Gang; Liu, Bin; Li, Su-Fen

    2004-07-01

    RACE (rapid amplification of cDNA ends) is a popular technique to rapidly obtain the full-length cDNA. After obtaining the 3' cDNA and 5' cDNA fragments with a overlapped region by 3' RACE and 5' RACE, the full-length cDNA could be generated by end-to-end PCR or subcloning. In this study, 3' RACE combined with touch-down PCR was successfully used for the rapid construction of full-length MnSOD cDNA of chickens. Compared with the conventional end-to-end PCR or subcloning, this method, called one-step 3' RACE, is fast, economical and highly specific. It especially fits the rapid construction of full-length cDNA by RACE method.

  6. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones

    PubMed Central

    Wellenreuther, Ruth; Schupp, Ingo; Poustka, Annemarie; Wiemann, Stefan

    2004-01-01

    Background cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library. Results We describe a method to construct SMART full-length enriched cDNA libraries with large insert sizes. Sub-libraries were generated from size-fractionated cDNA with an average insert size of up to seven kb. The percentage of full-length clones was calculated for different size ranges from BLAST results of over 12,000 5'ESTs. Conclusions The presented technique is suitable to generate full-length enriched cDNA libraries with large average insert sizes in a straightforward and robust way. The representation of full-coding clones is high also for large cDNAs (70%, 4–10 kb), when high-quality starting mRNA is used. PMID:15198809

  7. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones.

    PubMed

    Wellenreuther, Ruth; Schupp, Ingo; Poustka, Annemarie; Wiemann, Stefan

    2004-06-15

    cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library. We describe a method to construct SMART full-length enriched cDNA libraries with large insert sizes. Sub-libraries were generated from size-fractionated cDNA with an average insert size of up to seven kb. The percentage of full-length clones was calculated for different size ranges from BLAST results of over 12,000 5'ESTs. The presented technique is suitable to generate full-length enriched cDNA libraries with large average insert sizes in a straightforward and robust way. The representation of full-coding clones is high also for large cDNAs (70%, 4-10 kb), when high-quality starting mRNA is used.

  8. Analysis of 4,664 high-quality sequence-finished poplar full-length

    SciTech Connect

    Ralph, S.; Gunter, Lee E; Tuskan, Gerald A; Douglas, Carl; Holt, Robert A.; Jones, Steven; Marra, Marco; Bohlmann, J.

    2008-01-01

    The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL)-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa x P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones for genes that were differentially expressed in

  9. Full-length novel MHC class I allele discovery by next-generation sequencing: two platforms are better than one

    PubMed Central

    Dudley, Dawn M.; Karl, Julie A.; Creager, Hannah M.; Bohn, Patrick S.; Wiseman, Roger W.; O'Connor, David H.

    2013-01-01

    Deep sequencing has revolutionized major histocompatibility complex (MHC) class I analysis of nonhuman primates by enabling high-throughput, economical, and comprehensive genotyping. Full-length MHC class I cDNA sequences, which are required to generate reagents such as MHC:peptide tetramers, cannot be directly obtained by short read deep sequencing. We combined data from two next-generation sequencing platforms to discover novel full-length MHC class I mRNA/cDNA transcripts in Chinese rhesus macaques. We first genotyped macaques by Roche/454 pyrosequencing using a 530 bp amplicon spanning the densely polymorphic exons 2 through 4 of the MHC class I loci that encode the peptide-binding region. We then mapped short paired-end 250 bp Illumina sequence reads spanning the full-length transcript to each 530 bp amplicon at high stringency and used paired-end information to reconstruct full-length allele sequences. We characterized 65 full-length sequences from 6 Chinese rhesus macaques. Overall, approximately 70% of the alleles distinguished in these 6 animals contained new sequence information, including 29 novel transcripts. The flexibility of this approach should make full-length MHC class I allele genotyping accessible for any nonhuman primate population of interest. We are currently optimizing this method for full-length characterization of other highly polymorphic, duplicated loci such as the MHC class II DRB and killer immunoglobulin-like receptors. We anticipate that this method will facilitate rapid expansion and near completion of sequence libraries of polymorphic loci, such as MHC class I, within a few years. PMID:24241691

  10. Full-length novel MHC class I allele discovery by next-generation sequencing: two platforms are better than one.

    PubMed

    Dudley, Dawn M; Karl, Julie A; Creager, Hannah M; Bohn, Patrick S; Wiseman, Roger W; O'Connor, David H

    2014-01-01

    Deep sequencing has revolutionized major histocompatibility complex (MHC) class I analysis of nonhuman primates by enabling high-throughput, economical, and comprehensive genotyping. Full-length MHC class I cDNA sequences, which are required to generate reagents such as MHC-peptide tetramers, cannot be directly obtained by short read deep sequencing. We combined data from two next-generation sequencing platforms to discover novel full-length MHC class I mRNA/cDNA transcripts in Chinese rhesus macaques. We first genotyped macaques by Roche/454 pyrosequencing using a 530-bp amplicon spanning the densely polymorphic exons 2 through 4 of the MHC class I loci that encode the peptide-binding region. We then mapped short paired-end 250 bp Illumina sequence reads spanning the full-length transcript to each 530-bp amplicon at high stringency and used paired-end information to reconstruct full-length allele sequences. We characterized 65 full-length sequences from six Chinese rhesus macaques. Overall, approximately 70 % of the alleles distinguished in these six animals contained new sequence information, including 29 novel transcripts. The flexibility of this approach should make full-length MHC class I allele genotyping accessible for any nonhuman primate population of interest. We are currently optimizing this method for full-length characterization of other highly polymorphic, duplicated loci such as the MHC class II DRB and killer immunoglobulin-like receptors. We anticipate that this method will facilitate rapid expansion and near completion of sequence libraries of polymorphic loci, such as MHC class I, within a few years.

  11. Full-length HLA-DRB1 coding sequences generated by a hemizygous RNA-SBT approach.

    PubMed

    Gerritsen, K E H; Groeneweg, M; Meertens, C M H; Voorter, C E M; Tilanus, M G J

    2015-11-01

    Currently 1582 HLA-DRB1 alleles have been identified in the IMGT/HLA database (v3.18). Among those alleles, more than 90% have incomplete allele sequences, which complicates the analysis of the functional relevance of polymorphism beyond exon 2. The polymorphic index of each individual exon of the currently known allele sequences, shows that polymorphism is present in all exons, albeit not equally abundant. Full-length HLA-DRB1 RNA sequencing identifies polymorphism of the complete coding region. Here we describe a hemizygous full-length RNA sequence-based typing (SBT) approach based on group-specific HLA-DRB1 amplification and subsequent sequencing. RNA full-length sequences can easily be accessed because of the short amplicon length (801 bp). The RNA-SBT approach was successfully validated on a panel of DRB1 alleles having fully known coding sequences according to the IMGT/HLA database, and cover all serological equivalents. Subsequently, the approach was applied on a panel of 54 alleles with incomplete allele sequences, resulting in full-length coding sequences and the identification of one new and one corrected allele. This study shows the universal applicability of the RNA-based sequencing approach to identify full-length coding sequences and to define the polymorphic content of HLA-DRB1 alleles.

  12. Recovery of duck hepatitis A virus 3 from a stable full-length infectious cDNA clone.

    PubMed

    Pan, Meng; Yang, Xiaorong; Du, Jige; Zhou, Lei; Ge, Xinna; Guo, Xin; Liu, Jinhua; Zhang, Dabing; Yang, Hanchun

    2011-09-01

    Recently, duck hepatitis A virus 3 (DHAV-3) with genetically distinct characteristics from DHAV-1 and DHAV-2 was recognized in South Korea and China. In this short communication, we successfully constructed a stable full-length infectious cDNA clone derived from DHAV-3 by solving instability of cloned full-length cDNA in Escherichia coli (E. coli). The cDNA fragments amplified from the genome of DHAV-3 were assembled and inserted into a low-copy-number plasmid. Finally, a full-length cDNA clone containing an engineered SacII site that served as a genetic marker was obtained. The cDNA clone showed stable by serial passages in E. coli when propagated at 25°C under low level of antibiotic selection. BHK-21 cells were transfected with transcribed RNA from the full-length cDNA clone; infectious viral particles were rescued, showing its fatality to 10-day-old duck embryos. The results indicated that the constructed full-length cDNA clone of DHAV-3 is infectious. By various virological assays, our results indicated that the rescued virus exhibited similar biological properties with the parental virus. Animal experiments revealed that the rescued virus retained the high pathogenicity to 1-day-old ducklings and could induce a fatal hepatitis indistinguishable from its parental virus. Our present studies provide a useful tool for future research on genomic functions and molecular pathogenesis of DHAV-3.

  13. Detection of full-length and truncated neurokinin-1 receptor mRNA expression in human brain regions.

    PubMed

    Lai, Jian-Ping; Cnaan, Avital; Zhao, Huaqing; Douglas, Steven D

    2008-02-15

    We have applied a newly developed SYBR green-based real-time RT-PCR assay for quantification of full-length and truncated neurokinin-1 receptor (NK1R) mRNA expression in nine regions of human brain tissues obtained from 23 subjects who died with no evidence of neurological or neurodegenerative disease. The following brain regions were examined: cingulate cortex, cerebellum, nucleus accumbens, caudate nucleus, putamen, pons, hippocampus, locus coeruleus, and basal ganglia. The SYBR green-based real-time PCR was more sensitive than TaqMan probe-based real-time PCR in amplifying both full-length and truncated NK1R mRNA. The real-time RT-PCR assay had excellent specificity and sensitivity, with a dynamic range of detection between 100 and 1,000,000 copies of the NK1R cDNA per reaction. The truncated NK1R mRNA levels were more abundant than those of the full-length NK1R in most of the regions examined and there was no significant difference in the truncated NK1R mRNA levels among the nine regions studied. There was, however, a significant difference in the expression of full-length NK1R mRNA levels among the nine regions (P=0.0024), and the putamen region expressed the highest full-length NK1R mRNA. Further studies are needed in order to examine the differences between full-length and truncated NK1R in signal transduction and functional consequences in order to delineate the significance of the co-presence of the two forms of NK1R in the human brain.

  14. Detection of Full-Length and Truncated Neurokinin-1 Receptor mRNA Expression in Human Brain Regions

    PubMed Central

    Lai, Jian-Ping; Cnaan, Avital; Zhao, Huaqing; Douglas, Steven D.

    2008-01-01

    We have applied a newly developed SYBR green based real-time RT-PCR assay for quantification of full-length and truncated neurokinin-1 receptor (NK1R) mRNA expression in 9 regions of human brain tissues obtained from 23 subjects who died with no evidence of neurological or neurodegenerative disease. The following brain regions were examined: cingulate cortex, cerebellum, nucleus accumbens, caudate nucleus, putamen, pons, hippocampus, locus coeruleus, and basal ganglia. The SYBR green based-real-time PCR was more sensitive than TaqMan probe based real-time PCR in amplifying both full-length and truncated NK1R mRNA. The real-time RT-PCR assay had excellent specificity and sensitivity, with a dynamic range of detection between 100 and 1000,000 copies of the NK1R cDNA per reaction. The truncated NK1R mRNA levels were more abundant than those of the full-length NK1R in most of the regions examined and there was no significant difference in the truncated NK1R mRNA levels among the nine regions studied. There was, however, a significant difference in the expression of full-length NK1R mRNA levels among the nine regions (P=0.0024), and the putamen region expressed the highest full-length NK1R mRNA. Further studies are needed in order to examine the differences between full-length and truncated NK1R in signal transduction and functional consequences in order to delineate the significance of the copresence of the two forms of NK1R in the human brain. PMID:18035424

  15. In vitro read-through polysome/ribosome display of full-length protein ORF and it's applications.

    PubMed

    Ogawa, Atsushi; Sando, Shinsuke; Aoyama, Yasuhiro

    2005-01-01

    Combination of nonsense suppression and protein-ribosome-mRNA (PRM) complexation techniques leads to a new strategy "read-through polysome/ribosome display", which is designed to display full-length open reading frame (ORF) domain of the protein on the natural mRNA templates. The optimised conditions are to use the anticodon-adjusted tRNA for Leu as a nonsense suppressor in a reconstituted translation system containing diminished amounts of release factors (RFs). When applied to pseudo-natural mRNAs of Escherichia coli dihydrofolate reductase (E. coli DHFR), the input mRNA was recovered as a polysome complex displaying full-length DHFR.

  16. Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification.

    PubMed

    Wagner, Josef; Coupland, Paul; Browne, Hilary P; Lawley, Trevor D; Francis, Suzanna C; Parkhill, Julian

    2016-11-14

    Currently, bacterial 16S rRNA gene analyses are based on sequencing of individual variable regions of the 16S rRNA gene (Kozich, et al Appl Environ Microbiol 79:5112-5120, 2013).This short read approach can introduce biases. Thus, full-length bacterial 16S rRNA gene sequencing is needed to reduced biases. A new alternative for full-length bacterial 16S rRNA gene sequencing is offered by PacBio single molecule, real-time (SMRT) technology. The aim of our study was to validate PacBio P6 sequencing chemistry using three approaches: 1) sequencing the full-length bacterial 16S rRNA gene from a single bacterial species Staphylococcus aureus to analyze error modes and to optimize the bioinformatics pipeline; 2) sequencing the full-length bacterial 16S rRNA gene from a pool of 50 different bacterial colonies from human stool samples to compare with full-length bacterial 16S rRNA capillary sequence; and 3) sequencing the full-length bacterial 16S rRNA genes from 11 vaginal microbiome samples and compare with in silico selected bacterial 16S rRNA V1V2 gene region and with bacterial 16S rRNA V1V2 gene regions sequenced using the Illumina MiSeq. Our optimized bioinformatics pipeline for PacBio sequence analysis was able to achieve an error rate of 0.007% on the Staphylococcus aureus full-length 16S rRNA gene. Capillary sequencing of the full-length bacterial 16S rRNA gene from the pool of 50 colonies from stool identified 40 bacterial species of which up to 80% could be identified by PacBio full-length bacterial 16S rRNA gene sequencing. Analysis of the human vaginal microbiome using the bacterial 16S rRNA V1V2 gene region on MiSeq generated 129 operational taxonomic units (OTUs) from which 70 species could be identified. For the PacBio, 36,000 sequences from over 58,000 raw reads could be assigned to a barcode, and the in silico selected bacterial 16S rRNA V1V2 gene region generated 154 OTUs grouped into 63 species, of which 62% were shared with the MiSeq dataset. The Pac

  17. Giardia canis: ultrastructural analysis of G. canis trophozoites transfected with full length G. canis virus cDNA transcripts

    USDA-ARS?s Scientific Manuscript database

    Giardia canis virus (GCV) is a double-stranded RNA (dsRNA) virus of the family Totiviridae. In this study, the full-length cDNA of the G. canis virus was constructed in pPoly2/sfinot vector and RNA was transcribed in vitro. Virus-free G. canis trophozoites were transfected with in vitro transcribed ...

  18. Assessment and optimization of theileria parva sporozoite full-length p67 antigen expression in mammalian cells

    USDA-ARS?s Scientific Manuscript database

    Delivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant vers...

  19. Generation of Arabidopsis mutants by heterologous expression of a full length cDNA library from tomato fruits

    USDA-ARS?s Scientific Manuscript database

    Heterologous expression of cDNA libraries in Arabidopsis and other plants has been used for gene identifications. To identify functions of tomato genes, we expressed a tomato full-length cDNA library in Arabidopsis thaliana and generated over 7,000 mutants. We constructed a tomato cDNA library with ...

  20. Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon.

    PubMed

    Clepet, Christian; Joobeur, Tarek; Zheng, Yi; Jublot, Delphine; Huang, Mingyun; Truniger, Veronica; Boualem, Adnane; Hernandez-Gonzalez, Maria Elena; Dolcet-Sanjuan, Ramon; Portnoy, Vitaly; Mascarell-Creus, Albert; Caño-Delgado, Ana I; Katzir, Nurit; Bendahmane, Abdelhafid; Giovannoni, James J; Aranda, Miguel A; Garcia-Mas, Jordi; Fei, Zhangjun

    2011-05-20

    Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot plants. Codon

  1. Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

    PubMed Central

    2011-01-01

    Background Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. Result We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot

  2. Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation.

    PubMed

    Wang, Jin; Gines, Silvia; MacDonald, Marcy E; Gusella, James F

    2005-01-13

    Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1-171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 < 15 microM, including gossypol, gambogic acid, juglone, celastrol, sanguinarine and anthralin. Of these, both juglone and celastrol were effective in reversing the abnormal cellular localization of full-length mutant huntingtin observed in mutant HdhQ111/Q111 striatal cells. At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused by full-length mutant

  3. Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation

    PubMed Central

    Wang, Jin; Gines, Silvia; MacDonald, Marcy E; Gusella, James F

    2005-01-01

    Background Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. Results We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1–171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 < 15 μM, including gossypol, gambogic acid, juglone, celastrol, sanguinarine and anthralin. Of these, both juglone and celastrol were effective in reversing the abnormal cellular localization of full-length mutant huntingtin observed in mutant HdhQ111/Q111 striatal cells. Conclusions At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused

  4. Integrating de novo transcriptome assembly and cloning to obtain chicken Ovocleidin-17 full-length cDNA.

    PubMed

    Zhang, Quan; Liu, Long; Zhu, Feng; Ning, ZhongHua; Hincke, Maxwell T; Yang, Ning; Hou, ZhuoCheng

    2014-01-01

    Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not 'finished'. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length

  5. [Construction of dengue virus-specific full-length fully human antibody libraries by mammalian display technology].

    PubMed

    Wen, Yangming; Lan, Kaijian; Wang, Junjie; Yu, Jingyi; Qu, Yarong; Zhao, Wei; Zhang, Fuchun; Tan, Wanlong; Cao, Hong; Zhou, Chen

    2013-06-01

    To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry. Using 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)]. Using 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).

  6. [Full-length cDNA cloning of flavonol synthase genes of Carthamus tinctorius and construction plant expression vector].

    PubMed

    Yang, Wen-ting; Liu, Xiu-ming; Wan, Qiu; Yao, Na; Wang, Nan; Zhang, Xue-meng; Jiao, Zhong-da; Li, Hai-yan; Li, Xiao-kun

    2015-02-01

    Flavonol synthase (FLS) is one of the key enzymes in flavonoids metabolic pathways. In this study, middle sequence was obtained from Carthamus tinctorius transcriptome sequencing results. Full-length cDNAs of FLS was cloned from petals of C. tinctorius to FLS by using RT-PCR and RACE technology. Its full-length cDNA was 1,201 bp, with an open reading frame of 1,101 bp and 336 encoded amino acids. The phylogenetic analysis showed that, FLS gene encoded amino acids in C. tinctorius were highly homologous with amino acids in congeneric Compositae species, especially Rudbeckia laciniata. The pBASTA-FLS plant expression vector was successfully built by the molecular biology method, which lays a foundation for further studying biology functions of the gene and biosynthesis mechanism of flavonoids.

  7. Caspase 3 inactivates biologically active full length interleukin-33 as a classical cytokine but does not prohibit nuclear translocation

    SciTech Connect

    Ali, Shafaqat; Nguyen, Dang Quan; Falk, Werner; Martin, Michael Uwe

    2010-01-15

    IL-33 is a member of the IL-1 family of cytokines with dual function which either activates cells via the IL-33 receptor in a paracrine fashion or translocates to the nucleus to regulate gene transcription in an intracrine manner. We show that full length murine IL-33 is active as a cytokine and that it is not processed by caspase 1 to mature IL-33 but instead cleaved by caspase 3 at aa175 to yield two products which are both unable to bind to the IL-33 receptor. Full length IL-33 and its N-terminal caspase 3 breakdown product, however, translocate to the nucleus. Finally, bioactive IL-33 is not released by cells constitutively or after activation. This suggests that IL-33 is not a classical cytokine but exerts its function in the nucleus of intact cells and only activates others cells via its receptor as an alarm mediator after destruction of the producing cell.

  8. Two novel lncRNAs discovered in human mitochondrial DNA using PacBio full-length transcriptome data.

    PubMed

    Gao, Shan; Tian, Xiaoxuan; Chang, Hong; Sun, Yu; Wu, Zhenfeng; Cheng, Zhi; Dong, Pengzhi; Zhao, Qiang; Ruan, Jishou; Bu, Wenjun

    2017-08-09

    In this study, we established a general framework to use PacBio full-length transcriptome sequencing for the investigation of mitochondrial RNAs. As a result, we produced the first full-length human mitochondrial transcriptome using public PacBio data and characterized the human mitochondrial genome with more comprehensive and accurate information. Other results included determination of the H-strand primary transcript, identification of the ND5/ND6AS/tRNA(Glu)AS transcript, discovery of palindrome small RNAs (psRNAs) and construction of the "mitochondrial cleavage" model, etc. These results reported for the first time in this study fundamentally changed annotations of human mitochondrial genome and enriched knowledge in the field of animal mitochondrial studies. The most important finding was two novel long non-coding RNAs (lncRNAs) of MDL1 and MDL1AS exist ubiquitously in animal mitochondrial genomes. Copyright © 2017. Published by Elsevier B.V.

  9. Platelet full length TFPI-α in healthy volunteers is not affected by sex or hormonal use

    PubMed Central

    Winckers, Kristien; Thomassen, Stella; ten Cate, Hugo; Hackeng, Tilman M.

    2017-01-01

    Background Only 10% of plasma TFPIα (TFPI) exists in the full length form, the rest circulates as a C-terminally truncated form. However, blood platelets exclusively contain full length TFPI, which is released at the site of injury upon platelet activation, and which could play an important local regulatory role in thrombin generation and prevention of thrombosis. Methods The anticoagulant activities of full length and truncated TFPI were investigated using thrombin generation assays. Blood samples were obtained from 30 healthy volunteers (10 male subjects, 10 female subjects, and 10 females using oral contraceptives). Platelet TFPI was released in platelet rich plasma and in platelet isolates using convulxin or thrombin, and measured by free TFPI ELISA and thrombin generation assays. Results Full length TFPI and platelet TFPI were much more potent inhibitors of thrombin generation than truncated TFPI, which was virtually inactive. Although mean plasma TFPI antigen levels decreased from men (0.30 nM) to women (0.20 nM) to women using oral contraceptives (0.11 nM), no relevant differences were found in platelet TFPI among those subgroups. Conclusions Platelets release similar amounts of TFPI regardless of plasma TFPI concentrations and is unaffected by sex or oral contraceptive use. We speculate that platelet TFPI is important to prevent systemic coagulation and thrombosis and restrict thrombus formation to the site of the growing platelet plug. The stable contribution of platelet TFPI to the anticoagulant potential in plasma is likely to become particularly relevant under conditions of low plasma TFPI levels in combination of oral contraceptives use. PMID:28158181

  10. Massive Collection of Full-Length Complementary DNA Clones and Microarray Analyses:. Keys to Rice Transcriptome Analysis

    NASA Astrophysics Data System (ADS)

    Kikuchi, Shoshi

    2009-02-01

    Completion of the high-precision genome sequence analysis of rice led to the collection of about 35,000 full-length cDNA clones and the determination of their complete sequences. Mapping of these full-length cDNA sequences has given us information on (1) the number of genes expressed in the rice genome; (2) the start and end positions and exon-intron structures of rice genes; (3) alternative transcripts; (4) possible encoded proteins; (5) non-protein-coding (np) RNAs; (6) the density of gene localization on the chromosome; (7) setting the parameters of gene prediction programs; and (8) the construction of a microarray system that monitors global gene expression. Manual curation for rice gene annotation by using mapping information on full-length cDNA and EST assemblies has revealed about 32,000 expressed genes in the rice genome. Analysis of major gene families, such as those encoding membrane transport proteins (pumps, ion channels, and secondary transporters), along with the evolution from bacteria to higher animals and plants, reveals how gene numbers have increased through adaptation to circumstances. Family-based gene annotation also gives us a new way of comparing organisms. Massive amounts of data on gene expression under many kinds of physiological conditions are being accumulated in rice oligoarrays (22K and 44K) based on full-length cDNA sequences. Cluster analyses of genes that have the same promoter cis-elements, that have similar expression profiles, or that encode enzymes in the same metabolic pathways or signal transduction cascades give us clues to understanding the networks of gene expression in rice. As a tool for that purpose, we recently developed "RiCES", a tool for searching for cis-elements in the promoter regions of clustered genes.

  11. Expression and purification of full-length anti-apoptotic Bcl-2 using cell-free protein synthesis.

    PubMed

    Pedersen, Anders; Wallgren, Marcus; Karlsson, B Göran; Gröbner, Gerhard

    2011-06-01

    The anti-apoptotic B cell CLL/lymphoma-2 (Bcl-2) protein is a key player in the regulation of programmed cell death and is linked to various types of cancer and their resistance to drug treatment. Biophysical and structural studies of the full-length intact Bcl-2 have been hampered due to difficulties in expression and severe solubility problems, precluding isolation of this hydrophobic membrane protein. Therefore, previous work has so far mainly been carried out using structurally modified Bcl-2 variants, lacking the transmembrane region. Thus, biophysical information regarding the full-length protein is still missing. Here, a protocol is presented for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)). A batch-based cell-free expression system, using extract isolated from Escherichia coli (E. coli) was employed to produce recombinant protein encoded by an optimized gene sequence. Presence of polyoxyethylene-(20)-cetyl-ether (Brij-58) in the reaction mixture and subsequently in the immobilized metal-affinity purification steps was crucial to keep Bcl-2(2) soluble. The obtained yield was 0.25-0.3mg per ml of cell-free reaction. Far-UV circular dichroism (CD) spectroscopy confirmed the α-helical structure of the purified protein, characteristic for members of the Bcl-2 protein family. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule.

    PubMed

    Majava, Viivi; Wang, Chaozhan; Myllykoski, Matti; Kangas, Salla M; Kang, Sung Ung; Hayashi, Nobuhiro; Baumgärtel, Peter; Heape, Anthony M; Lubec, Gert; Kursula, Petri

    2010-06-01

    Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP-CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein-protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP-CaM interaction, including a 3D model of the complex between full-length proteins.

  13. Near Full-Length Identification of a Novel HIV-1 CRF01_AE/B/C Recombinant in Northern Myanmar.

    PubMed

    Zhou, Yan-Heng; Chen, Xin; Liang, Yue-Bo; Pang, Wei; Qin, Wei-Hong; Zhang, Chiyu; Zheng, Yong-Tang

    2015-08-01

    The Myanmar-China border appears to be the "hot spot" region for the occurrence of HIV-1 recombination. The majority of the previous analyses of HIV-1 recombination were based on partial genomic sequences, which obviously cannot reflect the reality of the genetic diversity of HIV-1 in this area well. Here, we present a near full-length characterization of a novel HIV-1 CRF01_AE/B/C recombinant isolated from a long-distance truck driver in Northern Myanmar. It is the first description of a near full-length genomic sequence in Myanmar since 2003, and might be one of the most complicated HIV-1 chimeras ever detected in Myanmar, containing four CRF01_AE, six B segments, and five C segments separated by 14 breakpoints throughout its genome. The discovery and characterization of this new CRF01_AE/B/C recombinant indicate that intersubtype recombination is ongoing in Myanmar, continuously generating new forms of HIV-1. More work based on near full-length sequence analyses is urgently needed to better understand the genetic diversity of HIV-1 in these regions.

  14. RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

    PubMed

    Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José

    2016-02-01

    During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks.

  15. Database Independent Protein Sequencing (DiPS) enables full-length de-novo protein and antibody sequence determination.

    PubMed

    Savidor, Alon; Barzilay, Rotem; Elinger, Dalia; Yarden, Yosef; Lindzen, Moshit; Gabashvili, Alexandra; Adiv Tal, Ophir; Levin, Yishai

    2017-03-27

    Traditional 'bottom-up' proteomics approaches use proteolytic digestion, LC-MS/MS and database searching to elucidate peptide identities and their parent proteins. Protein sequences absent from the database cannot be identified, and even if present in the database, complete sequence coverage is rarely achieved even for the most abundant proteins in the sample. Thus, sequencing of unknown proteins such as antibodies or constituents of metaproteomes remains a challenging problem. To date, there is no available method for full-length protein sequencing, independent of a reference database, in high throughput. Here we present Database Independent Protein Sequencing (DiPS), a method for unambiguous, rapid, database independent, full-length protein sequencing. The method is a novel combination of non-enzymatic, semi-random cleavage of the protein, LC-MS/MS analysis, peptide de novo sequencing, extraction of peptide tags, and their assembly into a consensus sequence using an algorithm named "Peptide Tag Assembler" (pTA). As proof-of-concept, the method was applied to samples of three known proteins representing three size classes and to a previously un-sequenced, clinically relevant, monoclonal antibody. Excluding leucine/isoleucine and glutamic-acid/deamidated glutamine ambiguities, end-to-end, full-length de novo sequencing was achieved with 99-100% accuracy for all benchmarking proteins and the antibody light chain. Accuracy of the sequenced antibody heavy chain, including the entire variable region, was also 100% but there was a 23 residue gap in the constant region sequence.

  16. Identification of full-length proviral DNA of porcine endogenous retrovirus from Chinese Wuzhishan miniature pigs inbred.

    PubMed

    Ma, Yuyuan; Lv, Maomin; Xu, Shu; Wu, Jianmin; Tian, Kegong; Zhang, Jingang

    2010-07-01

    Existence of porcine endogenous retrovirus (PERV) hinders pigs to be used in clinical xenotransplantation to alleviate the shortage of human transplants. Chinese miniature pigs are potential organ donors for xenotransplantation in China. However, so far, an adequate level of information on the molecular characteristics of PERV from Chinese miniature pigs has not been available. We described here the cloning and characterization of full-length proviral DNA of PERV from Chinese Wuzhishan miniature pigs inbred (WZSP). Full-length nucleotide sequences of PERV-WZSP and other PERVs were aligned and phylogenetic tree was constructed from deduced amino-acid sequences of env. The results demonstrated that the full-length proviral DNA of PERV-WZSP belongs to gammaretrovirus and shares high similarity with other PERVs. Sequence analysis also suggested that different patterns of LTR existed in the same porcine germ line and partial PERV-C sequence may recombine with PERV-A sequence in LTR. (c) 2008 Elsevier Ltd. All rights reserved.

  17. Genetic suppression of neurodegeneration and neurotransmitter release abnormalities caused by expanded full-length huntingtin accumulating in the cytoplasm.

    PubMed Central

    Romero, Eliana; Cha, Guang-Ho; Verstreken, Patrik; Ly, Cindy V.; Hughes, Robert; Bellen, Hugo J.; Botas, Juan

    2008-01-01

    Summary Huntington's Disease (HD) is a dominantly inherited neurodegenerative disorder caused by expansion of a translated CAG repeat in the N-terminus of the huntingtin protein. Here we describe the generation and characterization of a novel full-length HD Drosophila model to reveal a previously unknown disease mechanism that occurs early in the course of pathogenesis, before expanded huntingtin is cleaved and imported into the nucleus in detectable amounts. We find that expanded full-length huntingtin (128QhttFL) leads to behavioral, neurodegenerative, and electrophysiological phenotypes. These phenotypes are caused by a Ca2+-dependent increase in neurotransmitter release efficiency in 128QhttFL animals. Partial loss of function in synaptic transmission (Syntaxin, Snap, Rop) and voltage-gated Ca2+ channel genes suppresses both the electrophysiological and the neurodegenerative phenotypes. Thus, our data indicate that increased neurotransmission is at the root of neuronal degeneration caused by expanded full-length huntingtin during early stages of pathogenesis. PMID:18184562

  18. Identification and Functional Analyses of 11 769 Full-length Human cDNAs Focused on Alternative Splicing

    PubMed Central

    Wakamatsu, Ai; Kimura, Kouichi; Yamamoto, Jun-ichi; Nishikawa, Tetsuo; Nomura, Nobuo; Sugano, Sumio; Isogai, Takao

    2009-01-01

    We analyzed diversity of mRNA produced as a result of alternative splicing in order to evaluate gene function. First, we predicted the number of human genes transcribed into protein-coding mRNAs by using the sequence information of full-length cDNAs and 5′-ESTs and obtained 23 241 of such human genes. Next, using these genes, we analyzed the mRNA diversity and consequently sequenced and identified 11 769 human full-length cDNAs whose predicted open reading frames were different from other known full-length cDNAs. Especially, 30% of the cDNAs we identified contained variation in the transcription start site (TSS). Our analysis, which particularly focused on multiple variable first exons (FEVs) formed due to the alternative utilization of TSSs, led to the identification of 261 FEVs expressed in the tissue-specific manner. Quantification of the expression profiles of 13 genes by real-time PCR analysis further confirmed the tissue-specific expression of FEVs, e.g. OXR1 had specific TSS in brain and tumor tissues, and so on. Finally, based on the results of our mRNA diversity analysis, we have created the FLJ Human cDNA Database. From our result, it has been understood mechanisms that one gene produces suitable protein-coding transcripts responding to the situation and the environment. PMID:19880432

  19. Significance of the variant and full-length forms of the very low density lipoprotein receptor in brain.

    PubMed

    Nakamura, Y; Yamamoto, M; Kumamaru, E

    2001-12-20

    The very low density lipoprotein receptor (VLDLR) is a newly described receptor which binds to apolipoprotein E (apoE) specifically. The authors designed a synthetic peptide of 17 amino acids representing the N-terminus of the putative first ligand binding domain of human VLDLR, this being a unique domain for VLDLR. When the synthetic peptide was used as the antigen, two different monoclonal antibodies were obtained (anti-VLDLR1 and anti-VLDLR2). Expressional cloning revealed that anti-VLDLR1 recognized the variant form of VLDLR which lacks 84 bp of O-linked sugar domain and anti-VLDLR2 recognized the full length form of VLDLR. The variant VLDLR was expressed in neuroblasts as well as matrix cells and Cajal-Retzius cells in the early stages of the developing human brain; later its expression was sequentially found in glioblasts, astrocytes, oligodendrocytes and finally in myelin. The expression of a full length form of VLDLR was detected in senile plaques and some neurons and satellite glia in aged and Alzheimer brains. This suggests that the variant VLDLR is important for the developing human brain and the full length VLDLR has modified functions in aged and Alzheimer brains.

  20. The full-length Saccharomyces cerevisiae Sgs1 protein is a vigorous DNA helicase that preferentially unwinds holliday junctions.

    PubMed

    Cejka, Petr; Kowalczykowski, Stephen C

    2010-03-12

    The highly conserved RecQ family of DNA helicases has multiple roles in the maintenance of genome stability. Sgs1, the single RecQ homologue in Saccharomyces cerevisiae, acts both early and late during homologous recombination. Here we present the expression, purification, and biochemical analysis of full-length Sgs1. Unlike the truncated form of Sgs1 characterized previously, full-length Sgs1 binds diverse single-stranded and double-stranded DNA substrates, including DNA duplexes with 5'- and 3'-single-stranded DNA overhangs. Similarly, Sgs1 unwinds a variety of DNA substrates, including blunt-ended duplex DNA. Significantly, a substrate containing a Holliday junction is unwound most efficiently. DNA unwinding is catalytic, requires ATP, and is stimulated by replication protein A. Unlike RecQ homologues from multicellular organisms, Sgs1 is remarkably active at picomolar concentrations and can efficiently unwind duplex DNA molecules as long as 23,000 base pairs. Our analysis shows that Sgs1 resembles Escherichia coli RecQ protein more than any of the human RecQ homologues with regard to its helicase activity. The full-length recombinant protein will be invaluable for further investigation of Sgs1 biochemistry.

  1. Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

    PubMed

    Gao, Ruimin; Niu, Shengniao; Dai, Weifang; Kitajima, Elliot; Wong, Sek-Man

    2016-10-01

    A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone.

  2. Type III secretion as a generalizable strategy for the production of full-length biopolymer-forming proteins.

    PubMed

    Azam, Anum; Li, Cheng; Metcalf, Kevin J; Tullman-Ercek, Danielle

    2016-11-01

    Biopolymer-forming proteins are integral in the development of customizable biomaterials, but recombinant expression of these proteins is challenging. In particular, biopolymer-forming proteins have repetitive, glycine-rich domains and, like many heterologously expressed proteins, are prone to incomplete translation, aggregation, and proteolytic degradation in the production host. This necessitates tailored purification processes to isolate each full-length protein of interest from the truncated forms as well as other contaminating proteins; owing to the repetitive nature of these proteins, the truncated polypeptides can have very similar chemistry to the full-length form and are difficult to separate from the full-length protein. We hypothesized that bacterial expression and secretion would be a promising alternative option for biomaterials-forming proteins, simplifying isolation of the full-length target protein. By using a selective secretion system, truncated forms of the protein are not secreted and thus are not found in the culture harvest. We show that a synthetically upregulated type III secretion system leads to a general increase in secretion titer for each protein that we tested. Moreover, we observe a substantial enhancement in the homogeneity of full-length forms of pro-resilin, tropo-elastin crosslinking domains, and silk proteins produced in this manner, as compared with proteins purified from the cytosol. Secretion via the type III apparatus limits co-purification of truncated forms of the target protein and increases protein purity without extensive purification steps. Demonstrating the utility of such a system, we introduce several modifications to resilin-based peptides and use an un-optimized, single-column process to purify these proteins. The resulting materials are of sufficiently high quantity and yield for the production of antimicrobial hydrogels with highly reproducible rheological properties. The ease of this process and its

  3. 20(S)-Protopanaxadiol-aglycone Downregulation of the Full-length and Splice Variants of Androgen Receptor

    PubMed Central

    Cao, Bo; Liu, Xichun; Li, Jing; Liu, Shuang; Qi, Yanfeng; Xiong, Zhenggang; Zhang, Allen; Wiese, Thomas; Fu, Xueqi; Gu, Jingkai; Rennie, Paul S.; Sartor, Oliver; Lee, Benjamin R.; Ip, Clement; Zhao, Lijuan; Zhang, Haitao; Dong, Yan

    2012-01-01

    As a public health problem, prostate cancer engenders huge economic and life-quality burden. Developing effective chemopreventive regimens to alleviate the burden remains a major challenge. Androgen signaling is vital to the development and progression of prostate cancer. Targeting androgen signaling via blocking the production of the potent ligand dihydrotestosterone has been shown to decrease prostate cancer incidence. However, the potential of increasing the incidence of high-grade prostate cancers has been a concern. Mechanisms of disease progression after the intervention may include increased expression of androgen receptor (AR) in prostate tissue and expression of the constitutively-active AR splice variants (AR-Vs) lacking the ligand-binding domain. Thus, novel agents targeting the receptor, preferentially both the full-length and AR-Vs, are urgently needed. In the present study, we show that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) effectively downregulates the expression and activity of both the full-length AR and AR-Vs. The effects of PPD on AR and AR-Vs are manifested by an immediate drop in proteins followed by a reduction in transcripts, attributed to PPD induction of proteasome-mediated degradation and inhibition of the transcription of the AR gene. We further show that although PPD inhibits the growth as well as AR expression and activity in LNCaP xenograft tumors, the morphology and AR expression in normal prostates are not affected. This study is the first to show that PPD suppresses androgen signaling through downregulating both the full-length AR and AR-Vs, and provides strong rationale for further developing PPD as a promising agent for the prevention and/or treatment of prostate cancer. PMID:22907191

  4. Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili

    PubMed Central

    Futamura, Norihiro; Totoki, Yasushi; Toyoda, Atsushi; Igasaki, Tomohiro; Nanjo, Tokihiko; Seki, Motoaki; Sakaki, Yoshiyuki; Mari, Adriano; Shinozaki, Kazuo; Shinohara, Kenji

    2008-01-01

    Background Cryptomeria japonica D. Don is one of the most commercially important conifers in Japan. However, the allergic disease caused by its pollen is a severe public health problem in Japan. Since large-scale analysis of expressed sequence tags (ESTs) in the male strobili of C. japonica should help us to clarify the overall expression of genes during the process of pollen development, we constructed a full-length enriched cDNA library that was derived from male strobili at various developmental stages. Results We obtained 36,011 expressed sequence tags (ESTs) from either one or both ends of 19,437 clones derived from the cDNA library of C. japonica male strobili at various developmental stages. The 19,437 cDNA clones corresponded to 10,463 transcripts. Approximately 80% of the transcripts resembled ESTs from Pinus and Picea, while approximately 75% had homologs in Arabidopsis. An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology. Approximately 2% of the transcripts appeared to encode transcription factors. We identified twelve genes for MADS-box proteins among these transcription factors. The twelve MADS-box genes were classified as DEF/GLO/GGM13-, AG-, AGL6-, TM3- and TM8-like MIKCC genes and type I MADS-box genes. Conclusion Our full-length enriched cDNA library derived from C. japonica male strobili provides information on expression of genes during the development of male reproductive organs. We provided potential allergens in C. japonica. We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica. Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species. PMID:18691438

  5. Structural investigation of disordered stress proteins. Comparison of full-length dehydrins with isolated peptides of their conserved segments.

    PubMed

    Mouillon, Jean-Marie; Gustafsson, Petter; Harryson, Pia

    2006-06-01

    Dehydrins constitute a class of intrinsically disordered proteins that are expressed under conditions of water-related stress. Characteristic of the dehydrins are some highly conserved stretches of seven to 17 residues that are repetitively scattered in their sequences, the K-, S-, Y-, and Lys-rich segments. In this study, we investigate the putative role of these segments in promoting structure. The analysis is based on comparative analysis of four full-length dehydrins from Arabidopsis (Arabidopsis thaliana; Cor47, Lti29, Lti30, and Rab18) and isolated peptide mimics of the K-, Y-, and Lys-rich segments. In physiological buffer, the circular dichroism spectra of the full-length dehydrins reveal overall disordered structures with a variable content of poly-Pro helices, a type of elongated secondary structure relying on bridging water molecules. Similar disordered structures are observed for the isolated peptides of the conserved segments. Interestingly, neither the full-length dehydrins nor their conserved segments are able to adopt specific structure in response to altered temperature, one of the factors that regulate their expression in vivo. There is also no structural response to the addition of metal ions, increased protein concentration, or the protein-stabilizing salt Na(2)SO(4). Taken together, these observations indicate that the dehydrins are not in equilibrium with high-energy folded structures. The result suggests that the dehydrins are highly evolved proteins, selected to maintain high configurational flexibility and to resist unspecific collapse and aggregation. The role of the conserved segments is thus not to promote tertiary structure, but to exert their biological function more locally upon interaction with specific biological targets, for example, by acting as beads on a string for specific recognition, interaction with membranes, or intermolecular scaffolding. In this perspective, it is notable that the Lys-rich segment in Cor47 and Lti29 shows

  6. Structural Investigation of Disordered Stress Proteins. Comparison of Full-Length Dehydrins with Isolated Peptides of Their Conserved Segments1

    PubMed Central

    Mouillon, Jean-Marie; Gustafsson, Petter; Harryson, Pia

    2006-01-01

    Dehydrins constitute a class of intrinsically disordered proteins that are expressed under conditions of water-related stress. Characteristic of the dehydrins are some highly conserved stretches of seven to 17 residues that are repetitively scattered in their sequences, the K-, S-, Y-, and Lys-rich segments. In this study, we investigate the putative role of these segments in promoting structure. The analysis is based on comparative analysis of four full-length dehydrins from Arabidopsis (Arabidopsis thaliana; Cor47, Lti29, Lti30, and Rab18) and isolated peptide mimics of the K-, Y-, and Lys-rich segments. In physiological buffer, the circular dichroism spectra of the full-length dehydrins reveal overall disordered structures with a variable content of poly-Pro helices, a type of elongated secondary structure relying on bridging water molecules. Similar disordered structures are observed for the isolated peptides of the conserved segments. Interestingly, neither the full-length dehydrins nor their conserved segments are able to adopt specific structure in response to altered temperature, one of the factors that regulate their expression in vivo. There is also no structural response to the addition of metal ions, increased protein concentration, or the protein-stabilizing salt Na2SO4. Taken together, these observations indicate that the dehydrins are not in equilibrium with high-energy folded structures. The result suggests that the dehydrins are highly evolved proteins, selected to maintain high configurational flexibility and to resist unspecific collapse and aggregation. The role of the conserved segments is thus not to promote tertiary structure, but to exert their biological function more locally upon interaction with specific biological targets, for example, by acting as beads on a string for specific recognition, interaction with membranes, or intermolecular scaffolding. In this perspective, it is notable that the Lys-rich segment in Cor47 and Lti29 shows

  7. An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library

    PubMed Central

    Lu, Chaofu; Wallis, James G; Browse, John

    2007-01-01

    Background Castor seeds are a major source for ricinoleate, an important industrial raw material. Genomics studies of castor plant will provide critical information for understanding seed metabolism, for effectively engineering ricinoleate production in transgenic oilseeds, or for genetically improving castor plants by eliminating toxic and allergic proteins in seeds. Results Full-length cDNAs are useful resources in annotating genes and in providing functional analysis of genes and their products. We constructed a full-length cDNA library from developing castor endosperm, and obtained 4,720 ESTs from 5'-ends of the cDNA clones representing 1,908 unique sequences. The most abundant transcripts are genes encoding storage proteins, ricin, agglutinin and oleosins. Several other sequences are also very numerous, including two acidic triacylglycerol lipases, and the oleate hydroxylase (FAH12) gene that is responsible for ricinoleate biosynthesis. The role(s) of the lipases in developing castor seeds are not clear, and co-expressing of a lipase and the FAH12 did not result in significant changes in hydroxy fatty acid accumulation in transgenic Arabidopsis seeds. Only one oleate desaturase (FAD2) gene was identified in our cDNA sequences. Sequence and functional analyses of the castor FAD2 were carried out since it had not been characterized previously. Overexpression of castor FAD2 in a FAH12-expressing Arabidopsis line resulted in decreased accumulation of hydroxy fatty acids in transgenic seeds. Conclusion Our results suggest that transcriptional regulation of FAD2 and FAH12 genes maybe one of the mechanisms that contribute to a high level of ricinoleate accumulation in castor endosperm. The full-length cDNA library will be used to search for additional genes that affect ricinoleate accumulation in seed oils. Our EST sequences will also be useful to annotate the castor genome, which whole sequence is being generated by shotgun sequencing at the Institute for Genome

  8. FOX-superroots of Lotus corniculatus, overexpressing Arabidopsis full-length cDNA, show stable variations in morphological traits.

    PubMed

    Himuro, Yasuyo; Tanaka, Hidenori; Hashiguchi, Masatsugu; Ichikawa, Takanari; Nakazawa, Miki; Seki, Motoaki; Fujita, Miki; Shinozaki, Kazuo; Matsui, Minami; Akashi, Ryo; Hoffmann, Franz

    2011-01-15

    Using the full-length cDNA overexpressor (FOX) gene-hunting system, we have generated 130 Arabidopsis FOX-superroot lines in bird's-foot trefoil (Lotus corniculatus) for the systematic functional analysis of genes expressed in roots and for the selection of induced mutants with interesting root growth characteristics. We used the Arabidopsis-FOX Agrobacterium library (constructed by ligating pBIG2113SF) for the Agrobacterium-mediated transformation of superroots (SR) and the subsequent selection of gain-of-function mutants with ectopically expressed Arabidopsis genes. The original superroot culture of L. corniculatus is a unique host system displaying fast root growth in vitro, allowing continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Several of the Arabidopsis FOX-superroot lines show interesting deviations from normal growth and morphology of roots from SR-plants, such as differences in pigmentation, growth rate, length or diameter. Some of these mutations are of potential agricultural interest. Genomic PCR analysis revealed that 100 (76.9%) out of the 130 transgenic lines showed the amplification of single fragments. Sequence analysis of the PCR fragments from these 100 lines identified full-length cDNA in 74 of them. Forty-three out of 74 full-length cDNA carried known genes. The Arabidopsis FOX-superroot lines of L. corniculatus, produced in this study, expand the FOX hunting system and provide a new tool for the genetic analysis and control of root growth in a leguminous forage plant.

  9. The Juxtamembrane Linker of Full-length Synaptotagmin 1 Controls Oligomerization and Calcium-dependent Membrane Binding*

    PubMed Central

    Lu, Bin; Kiessling, Volker; Tamm, Lukas K.; Cafiso, David S.

    2014-01-01

    Synaptotagmin 1 (Syt1) is the calcium sensor for synchronous neurotransmitter release. The two C2 domains of Syt1, which may mediate fusion by bridging the vesicle and plasma membranes, are connected to the vesicle membrane by a 60-residue linker. Here, we use site-directed spin labeling and a novel total internal reflection fluorescence vesicle binding assay to characterize the juxtamembrane linker and to test the ability of reconstituted full-length Syt1 to interact with opposing membrane surfaces. EPR spectroscopy demonstrates that the majority of the linker interacts with the membrane interface, thereby limiting the extension of the C2A and C2B domains into the cytoplasm. Pulse dipolar EPR spectroscopy provides evidence that purified full-length Syt1 is oligomerized in the membrane, and mutagenesis indicates that a glycine zipper/GXXXG motif within the linker helps mediate oligomerization. The total internal reflection fluorescence-based vesicle binding assay demonstrates that full-length Syt1 that is reconstituted into supported lipid bilayers will capture vesicles containing negatively charged lipid in a Ca2+-dependent manner. Moreover, the rate of vesicle capture increases with Syt1 density, and mutations in the GXXXG motif that inhibit oligomerization of Syt1 reduce the rate of vesicle capture. This work demonstrates that modifications within the 60-residue linker modulate both the oligomerization of Syt1 and its ability to interact with opposing bilayers. In addition to controlling its activity, the oligomerization of Syt1 may play a role in organizing proteins within the active zone of membrane fusion. PMID:24973220

  10. Identification of 48 full-length MHC-DAB functional alleles in miiuy croaker and evidence for positive selection.

    PubMed

    Liu, Jiang; Sun, Yueyan; Xu, Tianjun

    2016-07-01

    Major histocompatibility complex (MHC) molecules play a vital role in the immune response and are a highly polymorphic gene superfamily in vertebrates. As the molecular marker associated with polymorphism and disease susceptibility/resistance, the polymorphism of MHC genes has been investigated in many tetrapods and teleosts. Most studies were focused on the polymorphism of the second exon, which encodes the peptide-binding region (PBR) in the α1- or β1-domain, but few studies have examined the full-length coding region. To comprehensive investigate the polymorphism of MHC gene, we identified 48 full-length miiuy croaker (Miichthys miiuy) MHC class IIB (Mimi-DAB) functional alleles from 26 miiuy croaker individuals. All of the alleles encode 34 amino acid sequences, and a high level of polymorphism was detected in Mimi-DAB alleles. The rate of non-synonymous substitutions (dN) occurred at a significantly higher frequency than that of synonymous substitutions (dS) in the PBR, and this result suggests that balancing selection maintains polymorphisms at the Mimi-DAB locus. Phylogenetic analysis based on the full-length and exon 2 sequences of Mimi-DAB alleles both showed that the Mimi-DAB alleles were clustered into two major groups. A total of 19 positive selected sites were identified on the Mimi-DAB alleles after testing for positive selection, and 14 sites were predicted to be associated with antigen-binding sites, which suggests that most of selected sites are significant for disease resistance. The polymorphism of Mimi-DAB alleles provides an important resource for analyzing the association between the polymorphism of MHC gene and disease susceptibility/resistance, and for researching the molecular selective breeding of miiuy croaker with enhanced disease resistance. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Synthesis of full length PB1-F2 influenza A virus proteins from 'Spanish flu' and 'bird flu'.

    PubMed

    Röder, René; Bruns, Karsten; Sharma, Alok; Eissmann, André; Hahn, Friedrich; Studtrucker, Nicole; Fossen, Torgils; Wray, Victor; Henklein, Peter; Schubert, Ulrich

    2008-08-01

    The proapoptotic influenza A virus PB1-F2 protein contributes to viral pathogenicity and is present in most human and avian isolates. Previous synthetic protocols have been improved to provide a synthetic full length H1N1 type PB1-F2 protein that is encoded by the 'Spanish flu' isolate and an equivalent protein from an avian host that is representative of a highly pathogenic H5N1 'bird flu' isolate, termed SF2 and BF2, respectively. Full length SF2, different mutants of BF2 and a number of fragments of these peptides have been synthesized by either the standard solid-phase peptide synthesis method or by native chemical ligation of unprotected N- and C-terminal peptide fragments. For SF2 chemical ligation made use of the histidine and the cysteine residues located in positions 41 and 42 of the native sequence, respectively, to afford a highly efficient synthesis of SF2 compared to the standard SPPS elongation method. By-product formation at the aspartic acid residue in position 23 was prevented by specific modifications of the SPPS protocol. As the native sequence of BF2 does not contain a cysteine residue two different mutants of BF2 (Y42C) and BF2 (S47C) with appropriate cysteine exchanges were produced. In addition to the full length molecules, fragments of the native sequences were synthesized for comparison of their physical characteristics with those from the H1N1 human isolate A/Puerto Rico/8/34 (H1N1). All peptides were analyzed by mass spectrometry, (1)H NMR spectroscopy, and SDS-PAGE. The protocols allow the synthesis of significant amounts of PB1-F2 and its related peptides. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.

  12. The Drosophila gene collection: Identification of putative full-length cDNAs for 70 percent of D. melanogaster genes

    SciTech Connect

    Stapleton, Mark; Liao, Guochun; Brokstein, Peter; Hong, Ling; Carninci, Piero; Shiraki, Toshiyuki; Hayashizaki, Yoshihide; Champe, Mark; Pacleb, Joanne; Wan, Ken; Yu, Charles; Carlson, Joe; George, Reed; Celniker, Susan; Rubin, Gerald M.

    2002-08-12

    Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5prime expressed sequence tags (EST) from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to {approx}40 percent of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remaining genes, we have generated an additional 157,835 5prime ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22hr embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70 percent of the predicted genes in Drosophila.

  13. Analysis of a shortened form of human carbonic anhydrase VII expressed in vitro compared to the full-length enzyme.

    PubMed

    Bootorabi, Fatemeh; Jänis, Janne; Smith, Elona; Waheed, Abdul; Kukkurainen, Sampo; Hytönen, Vesa; Valjakka, Jarkko; Supuran, Claudiu T; Vullo, Daniela; Sly, William S; Parkkila, Seppo

    2010-08-01

    Carbonic anhydrase (CA) enzymes are expressed in all organs of the mammalian body where they participate in important physiological functions. CA VII is a cytosolic isozyme which may be expressed as two forms according to the recent GenBank data. We designed a present study to express and characterize the human CA VII forms: full-length CA VII and short form (predicted to lack 56 residues from the N-terminus). Reverse transcriptase PCR analysis revealed mRNAs for both CA VII forms in the human brain. These different forms were expressed as recombinant proteins to investigate their biochemical properties. The full-length CA VII was used to raise a polyclonal antiserum in a rabbit, and the antiserum was then employed in western blot analyses and immunohistochemistry of mouse tissues. Data from mass spectrometry and comparative modeling showed that CA VII protein contains a single intramolecular disulfide bridge (Cys-56 to Cys-180) which is lacking in the short form. The computer model suggested distinctly different folding for the different forms. The more exposed structure and the absence of the disulfide bridge in the short form could make this protein more susceptible to degradation. In fact, this was realized in several protein purification efforts in which the short form readily degraded during the experimental procedures. From these results, we conclude that the full-length CA VII is a predominant active form in human brain and also in other tissues. In addition to the brain, CA VII is expressed in several other organs including the stomach, duodenum, colon, liver, and skeletal muscle. The distribution pattern suggests multiple functions for CA VII in different organs.

  14. The juxtamembrane linker of full-length synaptotagmin 1 controls oligomerization and calcium-dependent membrane binding.

    PubMed

    Lu, Bin; Kiessling, Volker; Tamm, Lukas K; Cafiso, David S

    2014-08-08

    Synaptotagmin 1 (Syt1) is the calcium sensor for synchronous neurotransmitter release. The two C2 domains of Syt1, which may mediate fusion by bridging the vesicle and plasma membranes, are connected to the vesicle membrane by a 60-residue linker. Here, we use site-directed spin labeling and a novel total internal reflection fluorescence vesicle binding assay to characterize the juxtamembrane linker and to test the ability of reconstituted full-length Syt1 to interact with opposing membrane surfaces. EPR spectroscopy demonstrates that the majority of the linker interacts with the membrane interface, thereby limiting the extension of the C2A and C2B domains into the cytoplasm. Pulse dipolar EPR spectroscopy provides evidence that purified full-length Syt1 is oligomerized in the membrane, and mutagenesis indicates that a glycine zipper/GXXXG motif within the linker helps mediate oligomerization. The total internal reflection fluorescence-based vesicle binding assay demonstrates that full-length Syt1 that is reconstituted into supported lipid bilayers will capture vesicles containing negatively charged lipid in a Ca(2+)-dependent manner. Moreover, the rate of vesicle capture increases with Syt1 density, and mutations in the GXXXG motif that inhibit oligomerization of Syt1 reduce the rate of vesicle capture. This work demonstrates that modifications within the 60-residue linker modulate both the oligomerization of Syt1 and its ability to interact with opposing bilayers. In addition to controlling its activity, the oligomerization of Syt1 may play a role in organizing proteins within the active zone of membrane fusion. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Activated full-length myosin-X moves processively on filopodia with large steps toward diverse two-dimensional directions

    PubMed Central

    Sato, Osamu; Jung, Hyun Suk; Komatsu, Satoshi; Tsukasaki, Yoshikazu; Watanabe, Tomonobu M.; Homma, Kazuaki; Ikebe, Mitsuo

    2017-01-01

    Myosin-X, (Myo 10), is an unconventional myosin that transports the specific cargos to filopodial tips, and is associated with the mechanism underlying filopodia formation and extension. To clarify the innate motor characteristic, we studied the single molecule movement of a full-length myosin-X construct with leucine zipper at the C-terminal end of the tail (M10FullLZ) and the tail-truncated myosin-X without artificial dimerization motif (BAP-M101–979HMM). M10FullLZ localizes at the tip of filopodia like myosin-X full-length (M10Full). M10FullLZ moves on actin filaments in the presence of PI(3,4,5)P3, an activator of myosin-X. Single molecule motility analysis revealed that the step sizes of both M10FullLZ and BAP-M101–979HMM are widely distributed on single actin filaments that is consistent with electron microscopy observation. M10FullLZ moves on filopodial actin bundles of cells with a mean step size (~36 nm), similar to the step size on single actin filaments (~38 nm). Cartesian plot analysis revealed that M10FullLZ meandered on filopodial actin bundles to both x- and y- directions. These results suggest that the lever-arm of full-length myosin-X is flexible enough to processively steps on different actin filaments within the actin bundles of filopodia. This characteristic of myosin-X may facilitate actin filament convergence for filopodia production. PMID:28287133

  16. [Construction of full-length complementary DNA of hepatitis C virus genome from an HCV infected patient].

    PubMed

    Mao, Hong-xia; Hu, Yun-wen; Wu, Ying; Lan, Shui-yun; Yuan, Zheng-hong

    2004-06-01

    To construct the full-length complementary DNA of HCV genome from an HCV infected patient. Four HCV gene fragments (1.6, 3.5, 2.4 kb and 2.6 kb) were amplified by RT-PCR from serum of a Chinese patient and fused and connected together to produce a 9.2 kb subgenomic fragment, which was further cloned into a cassette vector with fixed 5-prime and 3-prime termini of HCV to make the full-length cDNA. The cDNA heterogeneity was analyzed by comparing the sequences of 4 isolated HVR1 regions. The prokaryotic expressed Core, NS3 protease, NS3 helicase were detected for their specific reactivities with patient serum by Western blot analysis. And the protease activity of NS3 was evaluated in a cell-based NS3/4A-SEAP expression system. The cDNA covered the near full-length of HCV genome from the patient's serum. The difference among HVR1 regions indicated no selection of HCV variants during RT-PCR and the quasi-species characteristic of the amplified cDNA. The prokaryotic expressed viral proteins could be identified by patient serum. In the NS3/4A-SEAP system, NS3 could cleave the 4A-4B site between NS4A and SEAP proteins and resulted in the secretion of SEAP in culture media. These results suggest that the cloned HCV cDNA encodes a complete and functional open reading frame and will be useful for further construction of infectious cDNA clone.

  17. Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.)

    PubMed Central

    2011-01-01

    Background Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean. Results Two full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes. Conclusions The present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the

  18. Expression of functional full-length hSRC-1 in eukaryotic cells using modified vaccinia virus Ankara and baculovirus.

    PubMed

    Osz, Judit; Pradeau-Aubreton, Karine; Drillien, Robert; Troffer-Charlier, Nathalie; Kolb-Cheynel, Isabelle; Poterszman, Arnaud; Ruff, Marc; Moras, Dino; Rochel, Natacha

    2012-07-15

    Purified protein expression level and quality are contingent upon specific host expression systems. This differential production is particularly observed for proteins of high molecular weight, hampering further structural studies. We developed an expression method aimed at producing proteins in Escherichia coli, insect, and mammalian systems. Our novel protocol was used to produce in large scale the full-length 160-kDa steroid receptor coactivator 1 (SRC-1), a coregulator of nuclear receptors. The results indicate that we can produce biologically active human SRC-1 in mammalian and insect cells in large scale.

  19. Recognition of intermolecular G-quadruplexes by full length nucleophosmin. Effect of a leukaemia-associated mutation.

    PubMed

    Bañuelos, Sonia; Lectez, Benoît; Taneva, Stefka G; Ormaza, Georgina; Alonso-Mariño, Marián; Calle, Xabier; Urbaneja, María A

    2013-07-11

    Nucleophosmin (NPM) is a nucleolar protein involved in ribosome biogenesis. NPM1 gene is frequently mutated in acute myeloid leukaemia (AML), correlating with aberrant cytoplasmic localization of the protein. NPM attachment to the nucleolus in physiological conditions probably depends on binding to nucleic acids, and this recognition could be altered in AML. NPM associates to guanine-rich DNA sequences, able to fold as "G-quadruplexes". We have analyzed the interaction of pentameric, full length NPM with G-rich oligonucleotides, finding that the protein binds preferentially high-order G-quadruplexes. AML-associated mutation significantly hampers DNA binding, pointing to a possible mechanism contributing to pathological mislocalization of NPM.

  20. Construction of a normalized full-length cDNA library of cephalopod Amphioctopus fangsiao and development of microsatellite markers

    NASA Astrophysics Data System (ADS)

    Feng, Yanwei; Liu, Wenfen; Xu, Xin; Yang, Jianmin; Wang, Weijun; Wei, Xiumei; Liu, Xiangquan; Sun, Guohua

    2017-10-01

    Amphioctopus fangsiao is one of the most economically important species and has been considered to be a candidate for aquaculture. In order to facilitate its fine-scale genetic analyses, we constructed a normalized full-length library successfully and developed a set of microsatellite markers in this study. The normalized full-length library had a storage capacity of 6.9×105 independent clones. The recombination efficiency was 95% and the average size of inserted fragments was longer than 1000 bp. A total of 3440 high quality ESTs were obtained, which were assembled into 1803 unigenes. Of these unigenes, 450 (25%) were assigned into 33 Gene Ontology terms, 576 (31.9%) into 153 Kyoto Encyclopedia of Genes and Genomes pathways, and 275 (15.3%) into 22 Clusters of Orthologous Groups. Seventy-six polymorphic microsatellite markers were identified. The number of alleles per locus ranged from 4 to 17, and the observed and expected heterozygosities varied between 0.167 and 0.967 and between 0.326 and 0.944, respectively. Twelve loci were significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no linkage disequilibrium was found between different loci. This study provided not only a useful resource for the isolation of the functional genes, but also a set of informative microsatellites for the assessment of population structure and conservation genetics of A. fangsiao.

  1. Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

    SciTech Connect

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J. )

    1988-10-01

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 {times} 10{sup 6} recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria.

  2. An efficient full-length cDNA amplification strategy based on bioinformatics technology and multiplexed PCR methods.

    PubMed

    Chen, Nan; Wang, Wei-Min; Wang, Huan-Ling

    2016-01-13

    A novel strategy for amplification full-length cDNA and promoter sequences has been developed using bioinformatics technology and multiplexed PCR methods in this study. The amplification of 3' ends of cDNA is performed according to the modified classic 3' RACE techniques, therein the more efficient and effective oligo(dT)-anchor primer with hairpin structure is specially designed. For the amplification of 5' ends of cDNA, two or three-round TAIL-PCR or touch-down PCR using arbitrary degenerate (AD) and sequence-specific reverse (SPR) primers is performed until the 5' sequence of multi-assembled fragment reaches the exon1 region identified by aligning this fragment to reference genome database. Then another TAIL-PCR or touch-down PCR using genomic DNA as template is conducted to obtain the remaining 5' and promoter sequences. The 5' end sites of cDNA are predicted by aligning finally assembled fragment to homologous reference genes of other species, and screening the relative locations of common characteristic cis-elements in silico on promoter. The putative 5' ends are further validated by primers corresponding to these predicted sites in cDNAs. This method is suitable for researchers to isolate limited full-length cDNA sequences due to its operability, inexpensiveness, efficiency and speediness.

  3. No assembly required: Full-length MHC class I allele discovery by PacBio circular consensus sequencing.

    PubMed

    Westbrook, Catherine J; Karl, Julie A; Wiseman, Roger W; Mate, Suzanne; Koroleva, Galina; Garcia, Karla; Sanchez-Lockhart, Mariano; O'Connor, David H; Palacios, Gustavo

    2015-12-01

    Single-molecule real-time (SMRT) sequencing technology with the Pacific Biosciences (PacBio) RS II platform offers the potential to obtain full-length coding regions (∼1100-bp) from MHC class I cDNAs. Despite the relatively high error rate associated with SMRT technology, high quality sequences can be obtained by circular consensus sequencing (CCS) due to the random nature of the error profile. In the present study we first validated the ability of SMRT-CCS to accurately identify class I transcripts in Mauritian-origin cynomolgus macaques (Macaca fascicularis) that have been characterized previously by cloning and Sanger-based sequencing as well as pyrosequencing approaches. We then applied this SMRT-CCS method to characterize 60 novel full-length class I transcript sequences expressed by a cohort of cynomolgus macaques from China. The SMRT-CCS method described here provides a straightforward protocol for characterization of unfragmented single-molecule cDNA transcripts that will potentially revolutionize MHC class I allele discovery in nonhuman primates and other species. Published by Elsevier Inc.

  4. Identification of an IRF-1 splicing transcript in APL cells sharing similar transactivation activity of the full length one.

    PubMed

    Lou, Yejiang; Xia, Di; Yu, Mengxia; Tong, Jianhua; Jin, Jie

    2017-03-20

    Interferon regulatory factor-1 (IRF-1) is a member of the interferon regulatory factor family. It acts as a transcriptional activator and plays a critical role in antiviral defense, immune response, cell growth regulation, apoptosis and cell differentiation. Deletions, mutations or aberrant splicing of IRF-1 would result in its functional inactivation, and closely related to the tumorigenesis. In this work, we identified an IRF-1 splicing transcript (IRF-1-s) in all-trans retinoic acid (ATRA)-treated acute promyelocytic leukemia (APL) cell line NB4 cells. It lost the exon 8 and 9 of the full length IRF-1, expressed in numerous cell types and could be induced to expression by ATRA in NB4 cells. It turned out similar biological activity as full length IRF-1 to enhance the transcription of interferon stimulated response element (ISRE)-containing target genes. Identification of IRF-1-s in NB4 cells would be benefit for our further exploring the signaling pathway of ATRA and interferons, as well as the mechanisms of differentiation of APL cells.

  5. Construction and biological activity of a full-length molecular clone of human Torque teno virus (TTV) genotype 6.

    PubMed

    Kakkola, Laura; Tommiska, Johanna; Boele, Linda C L; Miettinen, Simo; Blom, Tea; Kekarainen, Tuija; Qiu, Jianming; Pintel, David; Hoeben, Rob C; Hedman, Klaus; Söderlund-Venermo, Maria

    2007-09-01

    Torque teno virus (TTV) is a non-enveloped human virus with a circular negative-sense (approximately 3800 nucleotides) ssDNA genome. TTV resembles in genome organization the chicken anemia virus, the animal pathogen of the Circoviridae family, and is currently classified as a member of a new, floating genus, Anellovirus. Molecular and cell biological research on TTV has been restricted by the lack of permissive cell lines and functional, replication-competent plasmid clones. In order to examine the key biological activities (i.e. RNA transcription and DNA replication) of this still poorly characterized ssDNA virus, we cloned the full-length genome of TTV genotype 6 and transfected it into cells of several types. TTV mRNA transcription was detected by RT-PCR in all the cell types: KU812Ep6, Cos-1, 293, 293T, Chang liver, Huh7 and UT7/Epo-S1. Replicating TTV DNA was detected in the latter five cell types by a DpnI-based restriction enzyme method coupled with Southern analysis, a novel approach to assess TTV DNA replication. The replicating full-length clone, the cell lines found to support TTV replication, and the methods presented here will facilitate the elucidation of the molecular biology and the life cycle of this recently identified human virus.

  6. An expanded taxonomy of hepatitis C virus genotype 6: Characterization of 22 new full-length viral genomes.

    PubMed

    Li, Chunhua; Barnes, Eleanor; Newton, Paul N; Fu, Yongshui; Vongsouvath, Manivanh; Klenerman, Paul; Okamoto, Hiroaki; Abe, Kenji; Pybus, Oliver G; Lu, Ling

    2015-02-01

    We characterized the full-length genomes of 22 hepatitis C virus genotype 6 (HCV-6) isolates: 10 from Vietnam (classified into subtypes 6e, 6h, 6p, 6r, 6s, and 6u), one from China (confirmed as a new subtype 6xd), and 11 from the Lao PDR (representing a new subtype 6xe plus eight novel variants). With these 22 new genomes, HCV-6 now has a diverse and extended taxonomic structure, comprised of 28 assigned subtypes (denoted 6a-6xe) and 27 unassigned lineages, all of which have been represented by full-length genomes. Our phylogenetic analyses also included many partially-sequenced novel variants of HCV-6 from Lao PDR. This revealed that Lao HCV isolates are genetically very diverse and are phylogenetically distributed in multiple lineages within genotype 6. Our results suggest that HCV-6 has been maintained in Laos, a landlocked country, since the common ancestor of genotype 6 and indicates historical dispersal of HCV-6 across Southeast Asia.

  7. Full-length sequence analysis of chloroquine resistance transporter gene in Plasmodium falciparum isolates from Sabah, Malaysia.

    PubMed

    Tan, Lii Lian; Lau, Tiek Ying; Timothy, William; Prabakaran, Dhanaraj

    2014-01-01

    Chloroquine resistance (CQR) in falciparum malaria was identified to be associated with several mutations in the chloroquine resistance transporter gene (pfcrt) that encodes the transmembrane transporter in digestive vacuole membrane of the parasite. This study aimed to investigate the point mutations across the full-length pfcrt in Plasmodium falciparum isolates in Sabah, Malaysia. A total of 31 P. falciparum positive samples collected from Keningau, Kota Kinabalu, and Kudat, Sabah, were analyzed. pfcrt was PCR amplified and cloned prior to sequence analysis. This study showed that all the previously described 10 point mutations associated with CQR at codons 72, 74, 75, 76, 97, 220, 271, 326, 356, and 371 were found with different prevalence. Besides, two novel point mutations, I166V and H273N, were identified with 22.5% and 19.3%, respectively. Three haplotypes, namely, CVMNK (29%), CVIET (3.2%), and SVMNT (67.7%), were identified. High prevalence of SVMNT among P. falciparum isolates from Sabah showed that these isolates are closer to the P. falciparum isolates from Papua New Guinea rather than to the more proximal Southeast Asian CVIET haplotype. Full-length analysis of pfcrt showed that chloroquine resistant P. falciparum in Sabah is still prevalent despite the withdrawal of chloroquine usage since 1979.

  8. Full-Length Sequence Analysis of Chloroquine Resistance Transporter Gene in Plasmodium falciparum Isolates from Sabah, Malaysia

    PubMed Central

    Tan, Lii Lian; Lau, Tiek Ying; Timothy, William; Prabakaran, Dhanaraj

    2014-01-01

    Chloroquine resistance (CQR) in falciparum malaria was identified to be associated with several mutations in the chloroquine resistance transporter gene (pfcrt) that encodes the transmembrane transporter in digestive vacuole membrane of the parasite. This study aimed to investigate the point mutations across the full-length pfcrt in Plasmodium falciparum isolates in Sabah, Malaysia. A total of 31 P. falciparum positive samples collected from Keningau, Kota Kinabalu, and Kudat, Sabah, were analyzed. pfcrt was PCR amplified and cloned prior to sequence analysis. This study showed that all the previously described 10 point mutations associated with CQR at codons 72, 74, 75, 76, 97, 220, 271, 326, 356, and 371 were found with different prevalence. Besides, two novel point mutations, I166V and H273N, were identified with 22.5% and 19.3%, respectively. Three haplotypes, namely, CVMNK (29%), CVIET (3.2%), and SVMNT (67.7%), were identified. High prevalence of SVMNT among P. falciparum isolates from Sabah showed that these isolates are closer to the P. falciparum isolates from Papua New Guinea rather than to the more proximal Southeast Asian CVIET haplotype. Full-length analysis of pfcrt showed that chloroquine resistant P. falciparum in Sabah is still prevalent despite the withdrawal of chloroquine usage since 1979. PMID:25574497

  9. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting.

    PubMed

    Fu, Jun; Bian, Xiaoying; Hu, Shengbaio; Wang, Hailong; Huang, Fan; Seibert, Philipp M; Plaza, Alberto; Xia, Liqiu; Müller, Rolf; Stewart, A Francis; Zhang, Youming

    2012-05-01

    Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.

  10. Expression and characterization of full-length Ampullaria crossean endoglucanase EG65s and their two functional modules.

    PubMed

    Yin, Qiuyu; Teng, Yigang; Li, Yanhong; Ding, Ming; Zhao, Fukun

    2011-01-01

    Three endoglucanase cDNAs, eg65a, eg65b, and eg65c, were cloned from the mollusk Ampullaria crossean in previous work. To characterize the full-length enzymes as well as their individual functional modules via heterologous expression analysis, the three full-length putative endoglucanases (rEG65a, rEG65b, and rEG65c) and the corresponding catalytic modules (EG65a-CM, EG65b-CM, and EG65c-CM) were expressed in Pichia pastoris GS115, and the three corresponding carbohydrate-binding modules (EG65a-CBM, EG65b-CBM, and EG65c-CBM) were expressed in Escherichia coli BL21 (DE3). The properties of recombinant rEG65b, EG65a-CM, EG65b-CM, and EG65c-CM were characterized. Binding assays of CBMs with insoluble polysaccharides indicated that both EG65b-CBM and EG65c-CBM bound to phosphoric-acid swollen cellulose (PASC), Avicel, and oat-spelt xylan, while EG65a-CBM did not. The relative equilibrium constants (K(r)) of EG65b-CBM and EG65c-CBM were determined by absorption isotherm measurements. In this study, the CBMs of animal cellulases were expressed and characterized for the first time.

  11. A comparison of the psychometric properties of the psychopathic personality inventory full-length and short-form versions.

    PubMed

    Kastner, Rebecca M; Sellbom, Martin; Lilienfeld, Scott O

    2012-03-01

    The Psychopathic Personality Inventory (PPI) has shown promising construct validity as a measure of psychopathy. Because of its relative efficiency, a short-form version of the PPI (PPI-SF) was developed and has proven useful in many psychopathy studies. The validity of the PPI-SF, however, has not been thoroughly examined, and no studies have directly compared the validity of the short form with that of the full-length version. The current study was designed to compare the psychometric properties of both PPI versions, with an emphasis on convergent and discriminant validity in predicting external criteria conceptually relevant to psychopathy. We used both prison (n = 558) and college samples (n = 322) for this investigation. PPI scale scores were more reliable and more strongly correlated with the conceptually relevant criterion measures compared with the PPI-SF, particularly in the prison sample. There were no differences in relative discriminant validity. Thus, overall, the PPI full-length version showed more evidence of construct validity than did the short form, and the consequences of this psychometric difference should be considered when evaluating the clinical utility of each measure.

  12. Purification, crystallization and preliminary crystallographic analysis of the full-length cystathionine β-synthase from Apis mellifera.

    PubMed

    Oyenarte, Iker; Majtan, Tomas; Ereño, June; Corral-Rodríguez, María Angeles; Klaudiny, Jaroslav; Majtan, Juraj; Kraus, Jan P; Martínez-Cruz, Luis Alfonso

    2012-11-01

    Cystathionine β-synthase (CBS) is a pyridoxal-5'-phosphate-dependent enzyme that catalyzes the first step of the transsulfuration pathway, namely the condensation of serine with homocysteine to form cystathionine. Mutations in the CBS gene are the single most common cause of hereditary homocystinuria, a multisystemic disease affecting to various extents the vasculature, connective tissues and central nervous system. At present, the crystal structure of CBS from Drosophila melanogaster is the only available structure of the full-length enzyme. Here we describe a cloning, overexpression, purification and preliminary crystallographic analysis of a full-length CBS from Apis mellifera (AmCBS) which maintains 51 and 46% sequence identity with its Drosophila and human homologs, respectively. The AmCBS yielded crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a=85.90, b=95.87, c=180.33 Å. Diffraction data were collected to a resolution of 3.0 Å. The crystal structure contained two molecules in the asymmetric unit which presumably correspond to the dimeric species observed in solution.

  13. Predicting the strength of UP-elements and full-length E. coli σE promoters

    PubMed Central

    Rhodius, Virgil A.; Mutalik, Vivek K.; Gross, Carol A.

    2012-01-01

    Predicting the location and strength of promoters from genomic sequence requires accurate sequenced-based promoter models. We present the first model of a full-length bacterial promoter, encompassing both upstream sequences (UP-elements) and core promoter modules, based on a set of 60 promoters dependent on σE, an alternative ECF-type σ factor. UP-element contribution, best described by the length and frequency of A- and T-tracts, in combination with a PWM-based core promoter model, accurately predicted promoter strength both in vivo and in vitro. This model also distinguished active from weak/inactive promoters. Systematic examination of promoter strength as a function of RNA polymerase (RNAP) concentration revealed that UP-element contribution varied with RNAP availability and that the σE regulon is comprised of two promoter types, one of which is active only at high concentrations of RNAP. Distinct promoter types may be a general mechanism for increasing the regulatory capacity of the ECF group of alternative σ's. Our findings provide important insights into the sequence requirements for the strength and function of full-length promoters and establish guidelines for promoter prediction and for forward engineering promoters of specific strengths. PMID:22156164

  14. Characterization of 40 full-length MHC class IIA functional alleles in miiuy croaker: Polymorphism and positive selection.

    PubMed

    Xu, Tianjun; Liu, Jiang; Sun, Yueyan; Zhu, Zhihuang; Liu, Tianxing

    2016-02-01

    The major histocompatibility complex is a highly polymorphic gene superfamily in vertebrates that plays an important role in adaptive immune response. In the present study, we identified 40 full-length miiuy croaker MHC class IIA (Mimi-DAA) functional alleles from 26 miiuy croaker individuals and found that the alleles encode 30 amino acid sequences. A high level of polymorphism in Mimi-DAA was detected in miiuy croaker. The rate of non-synonymous substitutions (d(N)) occurred at a significantly higher frequency than that of synonymous substitutions (d(S)) in the peptide-binding region (PBR) and non-PBR. This result suggests that balancing selection maintains polymorphisms at the Mimi-DAA locus. Phylogenetic analysis based on the full-length sequences showed that the Mimi-DAA alleles clustered into three groups. However, the phylogenetic tree constructed using the exon 2 sequences indicated that the Mimi-DAA alleles clustered into two groups. A total of 22 positively selected sites were identified on the Mimi-DAA alleles after testing for positive selection, and five sites were predicted to be associated with the binding of peptide antigen, suggesting that a few selected residues may play a significant role in immune function. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Cloning, characterisation and bacterial expression of full length cDNA for the mouse liver microsomal glutathione S-transferase.

    PubMed

    Raza, H; Mullick, J; John, A; Bhagwat, S V; Avadhani, N G

    2000-01-01

    We have isolated a cDNA encoding full length microsomal glutathione S-transferase (MGST) from mouse liver. The cDNA was isolated by RT-PCR using primers designed from published cDNA sequence of rat MGST with the addition of 5' Nde-1 and 3' HindIII sites, and cloned into bacterial expression vector pSP19T7LT. Deduced amino acid sequence (155 amino acids, calculated mol.mass 17512 Dalton) confirmed the identity of microsomal GST from mouse liver which has sequence homology with that of rat and human liver MGST1. Recombinant GST cDNA (Genbank accession # 159050) was expressed in BL21(DE3) in the presence of 1 mM IPTG at 30 degrees C. The expressed GST protein was found to be localised in the bacterial membrane as determined by measuring catalytic activity using CDNB and cumene hydroperoxide substrates, SDS-PAGE and Western blot analysis. We have demonstrated the cloning and expression of full length cDNA for MGST from mouse liver and have characterised the functionally active product as MGST protein. These results should facilitate studies on the role of MGST in the regulation of chemical carcinogenesis and in the prevention of oxidative stress caused by endogenous and exogenous chemicals.

  16. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing

    PubMed Central

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  17. Functional and expression analyses of transcripts based on full-length cDNAs of Sorghum bicolor.

    PubMed

    Shimada, Setsuko; Makita, Yuko; Kuriyama-Kondou, Tomoko; Kawashima, Mika; Mochizuki, Yoshiki; Hirakawa, Hideki; Sato, Shusei; Toyoda, Tetsuro; Matsui, Minami

    2015-12-01

    Sorghum bicolor is one of the most important crops for food and bioethanol production. Its small diploid genome and resistance to environmental stress make sorghum an attractive model for studying the functional genomics of the Saccharinae and other C4 grasses. We analyzed the domain-based functional annotation of the cDNAs using the gene ontology (GO) categories for molecular function to characterize all the genes cloned in the full-length cDNA library of sorghum. The sorghum cDNA library successfully captured a wide range of cDNA-encoded proteins with various functions. To characterize the protein function of newly identified cDNAs, a search of their deduced domains and comparative analyses in the Oryza sativa and Zea mays genomes were carried out. Furthermore, genes on the sense strand corresponding to antisense transcripts were classified based on the GO of molecular function. To add more information about these genes, we have analyzed the expression profiles using RNA-Seq of three tissues (spikelet, seed and stem) during the starch-filling phase. We performed functional analysis of tissue-specific genes and expression analysis of genes of starch biosynthesis enzymes. This functional analysis of sorghum full-length cDNAs and the transcriptome information will facilitate further analysis of the Saccharinae and grass families.

  18. Functional and expression analyses of transcripts based on full-length cDNAs of Sorghum bicolor

    PubMed Central

    Shimada, Setsuko; Makita, Yuko; Kuriyama-Kondou, Tomoko; Kawashima, Mika; Mochizuki, Yoshiki; Hirakawa, Hideki; Sato, Shusei; Toyoda, Tetsuro; Matsui, Minami

    2015-01-01

    Sorghum bicolor is one of the most important crops for food and bioethanol production. Its small diploid genome and resistance to environmental stress make sorghum an attractive model for studying the functional genomics of the Saccharinae and other C4 grasses. We analyzed the domain-based functional annotation of the cDNAs using the gene ontology (GO) categories for molecular function to characterize all the genes cloned in the full-length cDNA library of sorghum. The sorghum cDNA library successfully captured a wide range of cDNA-encoded proteins with various functions. To characterize the protein function of newly identified cDNAs, a search of their deduced domains and comparative analyses in the Oryza sativa and Zea mays genomes were carried out. Furthermore, genes on the sense strand corresponding to antisense transcripts were classified based on the GO of molecular function. To add more information about these genes, we have analyzed the expression profiles using RNA-Seq of three tissues (spikelet, seed and stem) during the starch-filling phase. We performed functional analysis of tissue-specific genes and expression analysis of genes of starch biosynthesis enzymes. This functional analysis of sorghum full-length cDNAs and the transcriptome information will facilitate further analysis of the Saccharinae and grass families. PMID:26546227

  19. Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2)

    PubMed Central

    Derewenda, Urszula; Artamonov, Mykhaylo V.; Somlyo, Avril V.; Derewenda, Zygmunt S.

    2016-01-01

    Ribosomal S6 kinases (RSK) play important roles in cell signaling through the mitogen-activated protein kinase (MAPK) pathway. Each of the four RSK isoforms (RSK1-4) is a single polypeptide chain containing two kinase domains connected by a linker sequence with regulatory phosphorylation sites. Here, we demonstrate that full-length RSK2—which is implicated in several types of cancer, and which is linked to the genetic Coffin-Lowry syndrome—can be overexpressed with high yields in Escherichia coli as a fusion with maltose binding protein (MBP), and can be purified to homogeneity after proteolytic removal of MBP by affinity and size-exclusion chromatography. The purified protein can be fully activated in vitro by phosphorylation with protein kinases ERK2 and PDK1. Compared to full-length RSK2 purified from insect host cells, the bacterially expressed and phosphorylated murine RSK2 shows the same levels of catalytic activity after phosphorylation, and sensitivity to inhibition by RSK-specific inhibitor SL0101. Interestingly, we detect low levels of phosphorylation in the nascent RSK2 on Ser386, owing to autocatalysis by the C-terminal domain, independent of ERK. This observation has implications for in vivo signaling, as it suggests that full activation of RSK2 by PDK1 alone is possible, circumventing at least in some cases the requirement for ERK. PMID:27732676

  20. Near full-length genomic characterization of a HIV type 1 BC recombinant strain from Manipur, India.

    PubMed

    Sarkar, Roni; Sarkar, Kamalesh; Singh, N Brajachand; Singh, Y Manihar; Chakrabarti, Sekhar

    2012-10-01

    Genetic complexity of HIV-1 is brought about by recombination between HIV-1 subtypes which leads to the development of epidemiologically significant founder strains. In the present study, the near full-length genome sequence of an HIV-1 isolate from an injecting drug user of Manipur (India) was determined, which evidenced the presence of a novel HIV-1 BC recombinant strain. Near full-length genome was amplified by polymerase chain reaction using primer walking approach. The recombination break points were detected using bootscan and simplot analyses. This isolate exhibited a mosaic structure consisting of subtype C backbone with subtype B insertions at the upstream of pol gene (3026-3259) and the downstream of env gene which spanned till the nef gene (8183-8961). Phylogenetic relationships determined with neighbor-joining trees, revealed that the subtype C sequences clustered with sequences from Indian subtype C HIV-1 strains, and the subtype B sequences clustered with HIV-1 subtype B strains from Thailand. This finding may create a complex scenario of HIV-1 epidemic among the injecting drug users of Manipur in near future.

  1. Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

    PubMed Central

    Anbouhi, Mahdi Habibi; Baraz, Aida Feiz; Bouzari, Saeid; Abolhassani, Mohsen; Khanahmad, Hossein; Golkar, Majid; Aghasadeghi, Mohammad Reza; Behdani, Mahdi; Najafabadi, Ali Jahanian; Shokrgozar, Mohammad Ali

    2012-01-01

    Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. Methods: In this study, we expressed the extra membrane loop of hCD20 (exCD20) consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a(+) expression vector. The desired protein was expressed in fusion with thioredoxin and 6× His tag in E. coli Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. Results: We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin (exCD20) can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies. PMID:23023212

  2. Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs.

    PubMed

    Okazaki, Y; Furuno, M; Kasukawa, T; Adachi, J; Bono, H; Kondo, S; Nikaido, I; Osato, N; Saito, R; Suzuki, H; Yamanaka, I; Kiyosawa, H; Yagi, K; Tomaru, Y; Hasegawa, Y; Nogami, A; Schönbach, C; Gojobori, T; Baldarelli, R; Hill, D P; Bult, C; Hume, D A; Quackenbush, J; Schriml, L M; Kanapin, A; Matsuda, H; Batalov, S; Beisel, K W; Blake, J A; Bradt, D; Brusic, V; Chothia, C; Corbani, L E; Cousins, S; Dalla, E; Dragani, T A; Fletcher, C F; Forrest, A; Frazer, K S; Gaasterland, T; Gariboldi, M; Gissi, C; Godzik, A; Gough, J; Grimmond, S; Gustincich, S; Hirokawa, N; Jackson, I J; Jarvis, E D; Kanai, A; Kawaji, H; Kawasawa, Y; Kedzierski, R M; King, B L; Konagaya, A; Kurochkin, I V; Lee, Y; Lenhard, B; Lyons, P A; Maglott, D R; Maltais, L; Marchionni, L; McKenzie, L; Miki, H; Nagashima, T; Numata, K; Okido, T; Pavan, W J; Pertea, G; Pesole, G; Petrovsky, N; Pillai, R; Pontius, J U; Qi, D; Ramachandran, S; Ravasi, T; Reed, J C; Reed, D J; Reid, J; Ring, B Z; Ringwald, M; Sandelin, A; Schneider, C; Semple, C A M; Setou, M; Shimada, K; Sultana, R; Takenaka, Y; Taylor, M S; Teasdale, R D; Tomita, M; Verardo, R; Wagner, L; Wahlestedt, C; Wang, Y; Watanabe, Y; Wells, C; Wilming, L G; Wynshaw-Boris, A; Yanagisawa, M; Yang, I; Yang, L; Yuan, Z; Zavolan, M; Zhu, Y; Zimmer, A; Carninci, P; Hayatsu, N; Hirozane-Kishikawa, T; Konno, H; Nakamura, M; Sakazume, N; Sato, K; Shiraki, T; Waki, K; Kawai, J; Aizawa, K; Arakawa, T; Fukuda, S; Hara, A; Hashizume, W; Imotani, K; Ishii, Y; Itoh, M; Kagawa, I; Miyazaki, A; Sakai, K; Sasaki, D; Shibata, K; Shinagawa, A; Yasunishi, A; Yoshino, M; Waterston, R; Lander, E S; Rogers, J; Birney, E; Hayashizaki, Y

    2002-12-05

    Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

  3. Purification, crystallization and preliminary crystallographic analysis of the full-length cystathionine β-synthase from Apis mellifera

    PubMed Central

    Oyenarte, Iker; Majtan, Tomas; Ereño, June; Corral-Rodríguez, María Angeles; Klaudiny, Jaroslav; Majtan, Juraj; Kraus, Jan P.; Martínez-Cruz, Luis Alfonso

    2012-01-01

    Cystathionine β-synthase (CBS) is a pyridoxal-5′-phosphate-dependent enzyme that catalyzes the first step of the transsulfuration pathway, namely the condensation of serine with homocysteine to form cystathionine. Mutations in the CBS gene are the single most common cause of hereditary homocystinuria, a multisystemic disease affecting to various extents the vasculature, connective tissues and central nervous system. At present, the crystal structure of CBS from Drosophila melanogaster is the only available structure of the full-length enzyme. Here we describe a cloning, overexpression, purification and preliminary crystallographic analysis of a full-length CBS from Apis mellifera (AmCBS) which maintains 51 and 46% sequence identity with its Drosophila and human homologs, respectively. The AmCBS yielded crystals belonging to space group P212121, with unit-cell parameters a = 85.90, b = 95.87, c = 180.33 Å. Diffraction data were collected to a resolution of 3.0 Å. The crystal structure contained two molecules in the asymmetric unit which presumably correspond to the dimeric species observed in solution. PMID:23143241

  4. An efficient full-length cDNA amplification strategy based on bioinformatics technology and multiplexed PCR methods

    PubMed Central

    Chen, Nan; Wang, Wei-Min; Wang, Huan-Ling

    2016-01-01

    A novel strategy for amplification full-length cDNA and promoter sequences has been developed using bioinformatics technology and multiplexed PCR methods in this study. The amplification of 3′ ends of cDNA is performed according to the modified classic 3′ RACE techniques, therein the more efficient and effective oligo(dT)-anchor primer with hairpin structure is specially designed. For the amplification of 5′ ends of cDNA, two or three-round TAIL-PCR or touch-down PCR using arbitrary degenerate (AD) and sequence-specific reverse (SPR) primers is performed until the 5′ sequence of multi-assembled fragment reaches the exon1 region identified by aligning this fragment to reference genome database. Then another TAIL-PCR or touch-down PCR using genomic DNA as template is conducted to obtain the remaining 5′ and promoter sequences. The 5′ end sites of cDNA are predicted by aligning finally assembled fragment to homologous reference genes of other species, and screening the relative locations of common characteristic cis-elements in silico on promoter. The putative 5′ ends are further validated by primers corresponding to these predicted sites in cDNAs. This method is suitable for researchers to isolate limited full-length cDNA sequences due to its operability, inexpensiveness, efficiency and speediness. PMID:26758040

  5. Update on Acanthamoeba jacobsi genotype T15, including full-length 18S rDNA molecular phylogeny.

    PubMed

    Corsaro, Daniele; Köhsler, Martina; Montalbano Di Filippo, Margherita; Venditti, Danielle; Monno, Rosa; Di Cave, David; Berrilli, Federica; Walochnik, Julia

    2017-04-01

    Free-living amoebae of the genus Acanthamoeba are worldwide present in natural and artificial environments, and are also clinically important, as causative agents of diseases in humans and other animals. Acanthamoeba comprises several species, historically assigned to one of the three groups based on their cyst morphology, but presently recognized as at least 20 genotypes (T1-T20) on the basis of their nuclear 18S ribosomal RNA (rRNA) gene (18S rDNA) sequences. While strain identification may usually be achieved targeting short (<500 bp) 18S ribosomal DNA (rDNA) fragments, the use of full-length gene sequences (>2200 bp) is necessary for correct genotype description and reliable molecular phylogenetic inference. The genotype T15, corresponding to Acanthamoeba jacobsi, is the only genotype described on the basis of partial sequences (~1500 bp). While this feature does not prevent the correct identification of the strains, having only partial sequences renders the genotype T15 not completely defined and may furthermore affect its position in the Acanthamoeba molecular tree. Here, we complete this gap, by obtaining full-length 18S rDNA sequences from eight A. jacobsi strains, genotype T15. Morphologies and physiological features of isolated strains are reported. Molecular phylogeny based on full 18S rDNA confirms some previous suggestions for a genetic link between T15 and T13, T16, and T19, with T19 as sister-group to T15.

  6. Genetic manipulation of porcine epidemic diarrhoea virus recovered from a full-length infectious cDNA clone.

    PubMed

    Jengarn, Juggragarn; Wongthida, Phonphimon; Wanasen, Nanchaya; Frantz, Phanramphoei Namprachan; Wanitchang, Asawin; Jongkaewwattana, Anan

    2015-08-01

    Porcine epidemic diarrhoea virus (PEDV) causes acute diarrhoea and dehydration in swine of all ages, with significant mortality in neonatal pigs. The recent rise of PEDV outbreaks in Asia and North America warrants an urgent search for effective vaccines. However, PEDV vaccine research has been hampered by difficulties in isolating and propagating the virus in mammalian cells, thereby complicating the recovery of infectious PEDV using a full-length infectious clone. Here, we engineered VeroE6 cells to stably express porcine aminopeptidase N (pAPN) and used them as a platform to obtain a high-growth variant of PEDV, termed PEDVAVCT12. Subsequently, the full-length cDNA clone was constructed by assembling contiguous cDNA fragments encompassing the complete genome of PEDVAVCT12 in a bacterial artificial chromosome. Infectious PEDV could be recovered, and the rescued virus displayed phenotypic properties identical to the parental virus. Interestingly, we found that PEDVAVCT12 contained a C-terminal deletion of the spike gene, resulting in disruption of the ORF3 start codon. When a functional ORF3 gene was restored, the recombinant virus could not be rescued, suggesting that ORF3 could suppress PEDV replication in vitro. In addition, a high-growth and genetically stable recombinant PEDV expressing a foreign protein could be rescued by replacing the ORF3 gene with the mCherry gene. Together, the results of this study provide a means to generate genetically defined PEDV as a promising vaccine candidate.

  7. Clusterin: full-length protein and one of its chains show opposing effects on cellular lipid accumulation

    PubMed Central

    Matukumalli, Suvarsha Rao; Tangirala, Ramakrishna; Rao, C. M.

    2017-01-01

    Proteins, made up of either single or multiple chains, are designed to carry out specific biological functions. We found an interesting example of a two-chain protein where administration of one of its chains leads to a diametrically opposite outcome than that reported for the full-length protein. Clusterin is a highly glycosylated protein consisting of two chains, α- and β-clusterin. We have investigated the conformational features, cellular localization, lipid accumulation, in vivo effects and histological changes upon administration of recombinant individual chains of clusterin. We demonstrate that recombinant α- and β-chains exhibit structural and functional differences and differ in their sub-cellular localization. Full-length clusterin is known to lower lipid levels. In contrast, we find that β-chain-treated cells accumulate 2-fold more lipid than controls. Interestingly, α-chain-treated cells do not show such increase. Rabbits injected with β-chain, but not α-chain, show ~40% increase in weight, with adipocyte hypertrophy, liver and kidney steatosis. Many, sometimes contrasting, roles are ascribed to clusterin in obesity, metabolic syndrome and related conditions. Our findings of differential localization and activities of individual chains of clusterin should help in understanding better the roles of clusterin in metabolism. PMID:28120874

  8. Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg.

    PubMed

    Bordat, Amandine; Houvenaghel, Marie-Christine; German-Retana, Sylvie

    2015-06-14

    Approaches to simplify and accelerate the construction of full-length infectious cDNA clones for plant potyviruses have been described, based on cloning strategies involving in vitro ligation or homologous recombination in yeast. In the present study, we developed a faster and more efficient in vitro recombination system using Gibson assembly (GA), to engineer a Lettuce mosaic virus (LMV) infectious clone expressing an ectopic mcherry-tagged VPg (Viral protein genome-linked) for in planta subcellular localization of the viral protein in an infection context. Three overlapping long distance PCR fragments were amplified and assembled in a single-step process based on in vitro recombination (Gibson assembly). The resulting 17.5 kbp recombinant plasmids (LMVmchVPg_Ec) were inoculated by biolistic on lettuce plants and then propagated mechanically on Nicotiana benthamiana. Confocal microscopy was used to analyze the subcellular localization of the ectopically expressed mcherry-VPg fusion protein. The Gibson assembly allowed the cloning of the expected plasmids without any deletion. All the inoculated plants displayed symptoms characteristic of LMV infection. The majority of the mcherry fluorescent signal observed using confocal microscopy was located in the nucleus and nucleolus as expected for a potyviral VPg. This is the first report of the use of the Gibson assembly method to construct full-length infectious cDNA clones of a potyvirus genome. This is also the first description of the ectopic expression of a tagged version of a potyviral VPg without affecting the viability of the recombinant potyvirus.

  9. Global Identification of the Full-Length Transcripts and Alternative Splicing Related to Phenolic Acid Biosynthetic Genes in Salvia miltiorrhiza

    PubMed Central

    Xu, Zhichao; Luo, Hongmei; Ji, Aijia; Zhang, Xin; Song, Jingyuan; Chen, Shilin

    2016-01-01

    Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing) of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and four alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that six candidate cytochrome P450s and five candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza. PMID:26904067

  10. Full length articles published in BJOMS during 2010-11--an analysis by sub-specialty and study type.

    PubMed

    Arakeri, Gururaj; Colbert, Serryth; Rosenbaum, Gavin; Brennan, Peter A

    2012-12-01

    Full length articles such as prospective and retrospective studies, case series, laboratory-based research and reviews form the majority of papers published in the British Journal of Oral and Maxillofacial Surgery (BJOMS). We were interested to evaluate the breakdown of these types of articles both by sub-specialty and the type of study as well as the proportion that are written by UK colleagues compared to overseas authors over a 2 year period (2010-11). A total of 191 full length articles across all sub-specialties of our discipline were published, with 107 papers (56%) coming from UK authors. There were proportionately more oncology papers arising from the UK than overseas (60 and 30% of total respectively) while the opposite was found for cleft/deformity studies (10% and 22%). There was only one laboratory-based study published from the UK compared with 27 papers from overseas. The number of quality papers being submitted to the Journal continues to increase, and the type of article being published between UK and overseas probably reflects different practices and case-loads amongst colleagues. The relatively few UK laboratory based studies published in BJOMS compared to overseas authors are most likely due to authors seeking the most prestigious journals possible for their work. Copyright © 2012 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  11. A Novel Strategy to Engineer Pre-Vascularized Full-Length Dental Pulp-like Tissue Constructs.

    PubMed

    Athirasala, Avathamsa; Lins, Fernanda; Tahayeri, Anthony; Hinds, Monica; Smith, Anthony J; Sedgley, Christine; Ferracane, Jack; Bertassoni, Luiz E

    2017-06-12

    The requirement for immediate vascularization of engineered dental pulp poses a major hurdle towards successful implementation of pulp regeneration as an effective therapeutic strategy for root canal therapy, especially in adult teeth. Here, we demonstrate a novel strategy to engineer pre-vascularized, cell-laden hydrogel pulp-like tissue constructs in full-length root canals for dental pulp regeneration. We utilized gelatin methacryloyl (GelMA) hydrogels with tunable physical and mechanical properties to determine the microenvironmental conditions (microstructure, degradation, swelling and elastic modulus) that enhanced viability, spreading and proliferation of encapsulated odontoblast-like cells (OD21), and the formation of endothelial monolayers by endothelial colony forming cells (ECFCs). GelMA hydrogels with higher polymer concentration (15% w/v) and stiffness enhanced OD21 cell viability, spreading and proliferation, as well as endothelial cell spreading and monolayer formation. We then fabricated pre-vascularized, full-length, dental pulp-like tissue constructs by dispensing OD21 cell-laden GelMA hydrogel prepolymer in root canals of extracted teeth and fabricating 500 µm channels throughout the root canals. ECFCs seeded into the microchannels successfully formed monolayers and underwent angiogenic sprouting within 7 days in culture. In summary, the proposed approach is a simple and effective strategy for engineering of pre-vascularized dental pulp constructs offering potentially beneficial translational outcomes.

  12. Human anti-EGFL7 recombinant full-length antibodies selected from a mammalian cell-based antibody display library.

    PubMed

    Li, Feng; Liu, Yan-Hong; Li, Yan-Wen; Ju, Qian; Chen, Lin; Xie, Ping-Li; Li, Yue-Hui; Li, Guan-Cheng

    2012-06-01

    Epidermal growth factor-like domain 7 (EGFL7) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. The advent of antibody display technology (phage, bacteria, and yeast) led to an enormous revival in the use of antibodies as diagnostic and therapeutic tools for fighting cancer. However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. We describe here the isolation of an EGFL7-specific antibody from a mammalian cell-based full-length antibody display library generated from peripheral blood mononuclear cells of patients with hepatocellular carcinoma. Using a novel vector, contained glycosylphosphatidylinositol anchor and restriction enzyme sites NheI and ClaI, antibody libraries are displayed as whole IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and measured by cell ELISA. Anti-EGFL7 antibody was successfully isolated from the library. The mammalian cell-based full-length antibody display library is a great potential application for rapid identification and cloning of human mAbs of targeting hepatocellular carcinoma.

  13. The effects of season on fatty acid composition and ω3/ω6 ratios of northern pike ( Esox lucius L., 1758) muscle lipids

    NASA Astrophysics Data System (ADS)

    Mert, Ramazan; Bulut, Sait; Konuk, Muhsin

    2015-01-01

    In the present study, the effects of season on fatty acid composition, total lipids, and ω3/ω6 ratios of northern pike muscle lipids in Kizilirmak River (Kirikkale, Turkey) were investigated. A total of 35 different fatty acids were determined in gas chromatography. Among these, palmitic, oleic, and palmitoleic acids had the highest proportion. The main polyunsaturated fatty acids (PUFAs) were found to be docosahexaenoic acid, eicosapentaenoic acid, and arachidonic acid. There were more PUFAs than monounsaturated fatty acids (MUFA) in all seasons. Similarly, the percentages of ω3 fatty acids were higher than those of total ω6 fatty acids in the fatty acid composition. ω3/ω6 ratios were calculated as 1.53, 1.32, 1.97, and 1.71 in spring, summer, autumn and winter, respectively. Overall, we found that the fatty acid composition and ω3/ω6 fatty acid ratio in the muscle of northern pike were significantly influenced by season.

  14. The Genome and Linkage Map of the Northern Pike (Esox lucius): Conserved Synteny Revealed between the Salmonid Sister Group and the Neoteleostei

    PubMed Central

    Rondeau, Eric B.; Minkley, David R.; Leong, Jong S.; Messmer, Amber M.; Jantzen, Johanna R.; von Schalburg, Kristian R.; Lemon, Craig; Bird, Nathan H.; Koop, Ben F.

    2014-01-01

    The northern pike is the most frequently studied member of the Esociformes, the closest order to the diverse and economically important Salmoniformes. The ancestor of all salmonids purportedly experienced a whole-genome duplication (WGD) event, making salmonid species ideal for studying the early impacts of genome duplication while complicating their use in wider analyses of teleost evolution. Studies suggest that the Esociformes diverged from the salmonid lineage prior to the WGD, supporting the use of northern pike as a pre-duplication outgroup. Here we present the first genome assembly, reference transcriptome and linkage map for northern pike, and evaluate the suitability of this species to provide a representative pre-duplication genome for future studies of salmonid and teleost evolution. The northern pike genome sequence is composed of 94,267 contigs (N50 = 16,909 bp) contained in 5,688 scaffolds (N50 = 700,535 bp); the total scaffolded genome size is 878 million bases. Multiple lines of evidence suggest that over 96% of the protein-coding genome is present in the genome assembly. The reference transcriptome was constructed from 13 tissues and contains 38,696 transcripts, which are accompanied by normalized expression data in all tissues. Gene-prediction analysis produced a total of 19,601 northern pike-specific gene models. The first-generation linkage map identifies 25 linkage groups, in agreement with northern pike's diploid karyotype of 2N = 50, and facilitates the placement of 46% of assembled bases onto linkage groups. Analyses reveal a high degree of conserved synteny between northern pike and other model teleost genomes. While conservation of gene order is limited to smaller syntenic blocks, the wider conservation of genome organization implies the northern pike exhibits a suitable approximation of a non-duplicated Protacanthopterygiian genome. This dataset will facilitate future studies of esocid biology and empower ongoing examinations of the Atlantic salmon and rainbow trout genomes by facilitating their comparison with other major teleost groups. PMID:25069045

  15. Reproductive status and lipid content as factors in PCB, DDT and HCH contamination of a population of pike (Esox lucius L. )

    SciTech Connect

    Larsson, P.; Okla, L.; Collvin, L. )

    1993-05-01

    Levels of persistent pollutants, including PCBs, [Sigma]DDT, and [gamma]-hexachlorocyclohexane, were examined in a pike population inhabiting a eutrophic lake in southern Scandinavia. For females, levels of persistent pollutants decreased linearly with age, weight, or length. This decline was ascribed to the seasonal elimination of the lipophilic pollutants in roe, which contained up to 10 times higher fat levels compared to muscle and over 10 times the amounts of pollutants. Male pike contained higher levels of pollutants than females, probably due to the lower elimination via gonadal products, as germinal tissue constitutes only 2% of the male total body weight and has a lower fat content than ovaries. Female germinal tissue can account for as much as 15% of the body weight. No major fat deposits other than those in germinal tissue were found in pike, which also had a low muscle fat content, suggesting that the importance of roe elimination in removing pollutants may be greater in pike than in salmonids. Uptake of persistent pollutants can vary greatly within a species, owing to differences in sex, age, and so forth, as well as between species, owing to differences in fat deposition strategies.

  16. Stocking impact and temporal stability of genetic composition in a brackish northern pike population (Esox lucius L.), assessed using microsatellite DNA analysis of historical and contemporary samples.

    PubMed

    Larsen, P F; Hansen, M M; Nielsen, E E; Jensen, L F; Loeschcke, V

    2005-08-01

    During the last decade, brackish northern pike populations in Denmark have been subject to stocking programmes, using nonindigenous pike from freshwater lakes, in order to compensate for drastic population declines. The present study was designed to investigate the genetic impact of stocking freshwater pike into a brackish pike population in Stege Nor, Denmark. We analysed polymorphism at eight microsatellite loci in samples representing the indigenous Stege Nor population prior to stocking (ie from 1956 to 1957), along with a sample of the contemporary Stege Nor population and samples from the three populations used for stocking. Despite large numbers of stocked fry, the results from both individual and population level admixture analyses demonstrated extremely poor performance and <1% introgression of stocked freshwater pike into the brackish pike population. Furthermore, pairwise F(ST) estimates between samples demonstrated close genetic relationship among temporal samples from Stege Nor, indicating temporal stability over the last 45 years. We also estimated the effective population size (N(e)) of pike in Stege Nor and applied a test for recent population bottlenecks. The harmonic mean of N(e) was relatively high (>250), but there were indications of bottlenecks in all samples and populations. We ascribe this finding to historical rather than recent bottlenecks, possibly dating back to founder events associated with postglacial recolonisation.

  17. The genome and linkage map of the northern pike (Esox lucius): conserved synteny revealed between the salmonid sister group and the Neoteleostei.

    PubMed

    Rondeau, Eric B; Minkley, David R; Leong, Jong S; Messmer, Amber M; Jantzen, Johanna R; von Schalburg, Kristian R; Lemon, Craig; Bird, Nathan H; Koop, Ben F

    2014-01-01

    The northern pike is the most frequently studied member of the Esociformes, the closest order to the diverse and economically important Salmoniformes. The ancestor of all salmonids purportedly experienced a whole-genome duplication (WGD) event, making salmonid species ideal for studying the early impacts of genome duplication while complicating their use in wider analyses of teleost evolution. Studies suggest that the Esociformes diverged from the salmonid lineage prior to the WGD, supporting the use of northern pike as a pre-duplication outgroup. Here we present the first genome assembly, reference transcriptome and linkage map for northern pike, and evaluate the suitability of this species to provide a representative pre-duplication genome for future studies of salmonid and teleost evolution. The northern pike genome sequence is composed of 94,267 contigs (N50 = 16,909 bp) contained in 5,688 scaffolds (N50 = 700,535 bp); the total scaffolded genome size is 878 million bases. Multiple lines of evidence suggest that over 96% of the protein-coding genome is present in the genome assembly. The reference transcriptome was constructed from 13 tissues and contains 38,696 transcripts, which are accompanied by normalized expression data in all tissues. Gene-prediction analysis produced a total of 19,601 northern pike-specific gene models. The first-generation linkage map identifies 25 linkage groups, in agreement with northern pike's diploid karyotype of 2N = 50, and facilitates the placement of 46% of assembled bases onto linkage groups. Analyses reveal a high degree of conserved synteny between northern pike and other model teleost genomes. While conservation of gene order is limited to smaller syntenic blocks, the wider conservation of genome organization implies the northern pike exhibits a suitable approximation of a non-duplicated Protacanthopterygiian genome. This dataset will facilitate future studies of esocid biology and empower ongoing examinations of the Atlantic salmon and rainbow trout genomes by facilitating their comparison with other major teleost groups.

  18. The contribution of ventricular apicobasal and transmural repolarization patterns to the development of the T wave body surface potentials in frogs (Rana temporaria) and pike (Esox lucius).

    PubMed

    Vaykshnorayte, Marina A; Azarov, Jan E; Tsvetkova, Alena S; Vityazev, Vladimir A; Ovechkin, Alexey O; Shmakov, Dmitry N

    2011-05-01

    The study aimed at the simultaneous determination of the transmural and apicobasal differences in the repolarization timing and the comparison of the contributions of these two repolarization gradients to the development of the body surface T wave potentials in animals with the single heart ventricle (fishes and amphibians). Unipolar potentials were measured on the body surface, epicardium and in the intramural (subepicardial, Epi; midmyocardial; and subendocardial, Endo) ventricular layers of 9 pike and 8 frogs. Activation times, repolarization times and activation-recovery intervals were determined. A transmural gradient in repolarization durations in frogs (Endo>Epi, P<0.024) corresponds to the gradient in repolarization times. No significant transmural difference in repolarization duration is observed in pike that produces a repolarization sequence from Endo to Epi (Endo

  19. Spatial relations of mercury contents in Pike (Esox lucius) and sediments concentration of the Anzali wetland, along the southern shores of the Caspian Sea, Iran.

    PubMed

    Zamani-Ahmadmahmoodi, Rasool; Bakhtiari, Alireza Riyahi; Rodríguez Martín, José Antonio

    2014-07-15

    In recent decades, the Anzali wetland has been threatened and destroyed by environmental pollution from several sources. The purpose of this study was to determine the possible relationships between mercury concentrations in Pike and their respective sediments within the assumed multiple activity center scales of Pike (100, 250 and 500 m in radius). To gain a better understanding spatial distribution pattern of Hg in sediments and to pursue the main purpose of this study, kriging (geostatistic spatial interpolation method) was applied. Poor relationships were found between mercury concentrations of Pike and sediments within the assumed multiple activity center scales of Pike. The mercury sediment influence diminished with the increasing radii of assumed activity centers. The results of the present study indicate that fish and sediment mercury concentrations in western parts of the Anzali wetland were low in comparison with the concentrations reported in the literature from other regions.

  20. Mass spectrometric characterization of a biotechnologically produced full-length mechano growth factor (MGF) relevant for doping controls.

    PubMed

    Thevis, Mario; Thomas, Andreas; Geyer, Hans; Schänzer, Wilhelm

    2014-12-01

    Since Goldspink and colleagues identified the expression of the mRNA of an insulin-like growth factor 1 (IGF-1) isoform in response to mechanical stress in 1996, substantial research into the so-called mechano growth factor and its modus operandi followed until today. Promising preclinical results were obtained by using the synthetic, 24-amino acid residues spanning peptide translated from the exons 4-6 of IGF-1Ec (which was later referred to as the mechano growth factor (MGF) peptide), particularly with regard to increased muscle myoblast proliferation. Consequently, the MGF peptide represented a promising drug candidate for the treatment of neuromuscular disorders; however, its misuse potential in sport was also identified shortly thereafter, and the substance (or class of substances) has been considered prohibited according to the regulations of the World Anti-Doping Agency (WADA) since 2005. While various MGF peptide versions have been known to sports drug testing authorities, the occurrence of a 'full-length MGF' as offered via illicit channels to athletes or athletes' managers was reported in 2014, arguably being undetectable in doping controls. An aliquot of the product was obtained and the content characterized by state-of-the-art analytical approaches including gel electrophoretic and mass spectrometric (top-down and bottom-up) sequencing approaches. Upon full characterization, its implementation into modified routine doping controls using ultrafiltration, immunoaffinity-based isolation, and nanoliquid chromatography-high resolution/high accuracy mass spectrometry was established. A protein with a monoisotopic molecular mass of 12264.9 Da and a sequence closely related to IGF-1Ec (lacking the signal- and propeptide moiety) was identified. The C-terminus was found to be modified by the elimination of the terminal lysine and a R109H substitution. With the knowledge of the compound's composition, existing doping control assays targeting peptide hormones such

  1. Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

    SciTech Connect

    Imanishi, Tadashi; Itoh, Takeshi; Suzuki, Yutaka; O'Donovan, Claire; Fukuchi, Satoshi; Koyanagi, Kanako O.; Barrero, Roberto A.; Tamura, Takuro; Yamaguchi-Kabata, Yumi; Tanino, Motohiko; Yura, Kei; Miyazaki, Satoru; Ikeo, Kazuho; Homma, Keiichi; Kasprzyk, Arek; Nishikawa, Tetsuo; Hirakawa, Mika; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Ashurst, Jennifer; Jia, Libin; Nakao, Mitsuteru; Thomas, Michael A.; Mulder, Nicola; Karavidopoulou, Youla; Jin, Lihua; Kim, Sangsoo; Yasuda, Tomohiro; Lenhard, Boris; Eveno, Eric; Suzuki, Yoshiyuki; Yamasaki, Chisato; Takeda, Jun-ichi; Gough, Craig; Hilton, Phillip; Fujii, Yasuyuki; Sakai, Hiroaki; Tanaka, Susumu; Amid, Clara; Bellgard, Matthew; de Fatima Bonaldo, Maria; Bono Hidemasa; Bromberg, Susan K.; Brookes, Anthony J.; Bruford, Elspeth; Carninci Piero; Chelala, Claude; Couillault, Christine; de Souza, Sandro J.; Debily, Marie-Anne; Devignes, Marie-Dominique; Dubchak, Inna; Endo, Toshinori; Estreicher, Anne; Eyras, Eduardo; Fukami-Kobayashi, Kaoru; Gopinath, Gopal R.; Graudens, Esther; Hahn, Yoonsoo; Han, Michael; Han, Ze-Guang; Hanada, Kousuke; Hanaoka, Hideki; Harada, Erimi; Hashimoto, Katsuyuki; Hinz, Ursula; Hirai, Momoki; Hishiki, Teruyoshi; Hopkinson, Ian; Imbeaud, Sandrine; Inoko, Hidetoshi; Kanapin, Alexander; Kaneko, Yayoi; Kasukawa, Takeya; Kelso, Janet; Kersey, Paul; Kikuno Reiko; Kimura, Kouichi; Korn, Bernhard; Kuryshev, Vladimir; Makalowska, Izabela; Makino Takashi; Mano, Shuhei; Mariage-Samson, Regine; Mashima, Jun; Matsuda, Hideo; Mewes, Hans-Werner; Minoshima, Shinsei; Nagai, Keiichi; Nagasaki, Hideki; Nagata, Naoki; Nigam, Rajni; Ogasawara, Osamu; Ohara, Osamu; Ohtsubo, Masafumi; Okada, Norihiro; Okido, Toshihisa; Oota, Satoshi; Ota, Motonori; Ota, Toshio; Otsuki, Tetsuji; Piatier-Tonneau, Dominique; Poustka, Annemarie; Ren, Shuang-Xi; Saitou, Naruya; Sakai, Katsunaga; Sakamoto, Shigetaka; Sakate, Ryuichi; Schupp, Ingo; Servant, Florence; Sherry, Stephen; Shiba Rie; et al.

    2004-01-15

    The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4 percent of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5 percent of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for nonprotein-coding RNA

  2. Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis

    PubMed Central

    Foolen, Jasper; Shiu, Jau-Ye; Mitsi, Maria; Zhang, Yang; Chen, Christopher S.; Vogel, Viola

    2016-01-01

    Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. ‘compact and adsorbed to collagen’ versus ‘extended and fibrillar’ fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin’s contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen

  3. Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

    PubMed Central

    Imanishi, Tadashi; Itoh, Takeshi; Suzuki, Yutaka; O'Donovan, Claire; Fukuchi, Satoshi; Koyanagi, Kanako O; Barrero, Roberto A; Tamura, Takuro; Yamaguchi-Kabata, Yumi; Tanino, Motohiko; Yura, Kei; Miyazaki, Satoru; Ikeo, Kazuho; Homma, Keiichi; Kasprzyk, Arek; Nishikawa, Tetsuo; Hirakawa, Mika; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Ashurst, Jennifer; Jia, Libin; Nakao, Mitsuteru; Thomas, Michael A; Mulder, Nicola; Karavidopoulou, Youla; Jin, Lihua; Kim, Sangsoo; Yasuda, Tomohiro; Lenhard, Boris; Eveno, Eric; Suzuki, Yoshiyuki; Yamasaki, Chisato; Takeda, Jun-ichi; Gough, Craig; Hilton, Phillip; Fujii, Yasuyuki; Sakai, Hiroaki; Tanaka, Susumu; Amid, Clara; Bellgard, Matthew; Bonaldo, Maria de Fatima; Bono, Hidemasa; Bromberg, Susan K; Brookes, Anthony J; Bruford, Elspeth; Carninci, Piero; Chelala, Claude; Couillault, Christine; de Souza, Sandro J.; Debily, Marie-Anne; Devignes, Marie-Dominique; Dubchak, Inna; Endo, Toshinori; Estreicher, Anne; Eyras, Eduardo; Fukami-Kobayashi, Kaoru; R. Gopinath, Gopal; Graudens, Esther; Hahn, Yoonsoo; Han, Michael; Han, Ze-Guang; Hanada, Kousuke; Hanaoka, Hideki; Harada, Erimi; Hashimoto, Katsuyuki; Hinz, Ursula; Hirai, Momoki; Hishiki, Teruyoshi; Hopkinson, Ian; Imbeaud, Sandrine; Inoko, Hidetoshi; Kanapin, Alexander; Kaneko, Yayoi; Kasukawa, Takeya; Kelso, Janet; Kersey, Paul; Kikuno, Reiko; Kimura, Kouichi; Korn, Bernhard; Kuryshev, Vladimir; Makalowska, Izabela; Makino, Takashi; Mano, Shuhei; Mariage-Samson, Regine; Mashima, Jun; Matsuda, Hideo; Mewes, Hans-Werner; Minoshima, Shinsei; Nagai, Keiichi; Nagasaki, Hideki; Nagata, Naoki; Nigam, Rajni; Ogasawara, Osamu; Ohara, Osamu; Ohtsubo, Masafumi; Okada, Norihiro; Okido, Toshihisa; Oota, Satoshi; Ota, Motonori; Ota, Toshio; Otsuki, Tetsuji; Piatier-Tonneau, Dominique; Poustka, Annemarie; Ren, Shuang-Xi; Saitou, Naruya; Sakai, Katsunaga; Sakamoto, Shigetaka; Sakate, Ryuichi; Schupp, Ingo; Servant, Florence; Sherry, Stephen; Shiba, Rie; Shimizu, Nobuyoshi; Shimoyama, Mary; Simpson, Andrew J; Soares, Bento; Steward, Charles; Suwa, Makiko; Suzuki, Mami; Takahashi, Aiko; Tamiya, Gen; Tanaka, Hiroshi; Taylor, Todd; Terwilliger, Joseph D; Unneberg, Per; Veeramachaneni, Vamsi; Watanabe, Shinya; Wilming, Laurens; Yasuda, Norikazu; Yoo, Hyang-Sook; Stodolsky, Marvin; Makalowski, Wojciech; Go, Mitiko; Nakai, Kenta; Takagi, Toshihisa; Kanehisa, Minoru; Sakaki, Yoshiyuki; Quackenbush, John; Okazaki, Yasushi; Hayashizaki, Yoshihide; Hide, Winston; Chakraborty, Ranajit; Nishikawa, Ken; Sugawara, Hideaki; Tateno, Yoshio; Chen, Zhu; Oishi, Michio; Tonellato, Peter; Apweiler, Rolf; Okubo, Kousaku; Wagner, Lukas; Wiemann, Stefan; Strausberg, Robert L; Isogai, Takao; Auffray, Charles; Nomura, Nobuo; Sugano, Sumio

    2004-01-01

    The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In

  4. The Helios Prototype flying wing stretches almost the full length of the 300-foot-long hangar at NAS

    NASA Technical Reports Server (NTRS)

    2002-01-01

    The Helios Prototype flying wing stretches almost the full length of the 300-foot-long hangar at NASA's Dryden flight Research Center, Edwards, California. The 247-foot span solar-powered aircraft, resting on its ground maneuvering dolly, was on display for a visit of NASA Administrator Sean O'Keefe and other NASA officials on January 31, 2002. The unique solar-electric flying wing reached an altitude of 96,863 feet during an almost 17-hour flight near Hawaii on August 13, 2001, a world record for sustained horizontal flight by a non-rocket powered aircraft. Developed by AeroVironment, Inc., under NASA's Environmental Research Aircraft and Sensor Technology (ERAST) project, the Helios Prototype is the forerunner of a planned fleet of slow-flying, long duration, high-altitude uninhabited aerial vehicles (UAV) which can serve as 'atmospheric satellites,' performing Earth science missions or functioning as telecommunications relay platforms in the stratosphere.

  5. Near Full-Length Genomic Sequences of Two Novel HIV-1 Recombinant Forms Identified in Shenzhen, China.

    PubMed

    Jia, Dijing; Zhao, Jin; Li, Tianyi; Sun, Changrong; Li, Hanping; Zheng, Chenli; Chen, Lin; Liu, Yongjian; Liu, Siyang; Zhuang, Daomin; Wang, Xiaolin; Bao, Zuoyi; Li, Jingyun; Li, Lin

    2017-01-01

    Most HIV subtypes prevalent in China can be found in Shenzhen, including CRF07_BC, CRF01_AE, CRF08_BC, CRF55_01B, and subtype B. Multiple subtypes spreading in the same population always lead to the emergence of unique recombinant strains. Here, we report two unique recombinant forms (SZ44LS7251 and SZ95LS8027) of HIV-1 identified in a heterosexual population. Recombinant analyses were fulfilled based on the near full-length genomes. Both strains comprise subtypes B, C, and CRF01_AE. Phylogenetic analysis reveals that SZ44LS7251 is the second generation recombination originated from CRF55_01B andCRF07_BC, whereas SZ95LS8027 comprises CRF01_AE and CRF07_BC.The emergence of second generation recombination of HIV with complicated genomic structures supposed that high ratio of super infections or coinfections might happen in the Shenzhen area.

  6. Full-length protein extraction protocols and gel-based downstream applications in formalin-fixed tissue proteomics.

    PubMed

    Tanca, Alessandro; Uzzau, Sergio; Addis, Maria Filippa

    2015-01-01

    Archival formalin-fixed, paraffin-embedded (FFPE) tissue repositories and their associated clinical information can represent a valuable resource for tissue proteomics. In order to make these tissues available for protein biomarker discovery and validation studies, dedicated sample preparation procedures overcoming the intermolecular cross-links introduced by formalin need to be implemented. This chapter describes a full-length protein extraction protocol optimized for downstream gel-based proteomics applications. Using the procedures detailed here, SDS-PAGE, western immunoblotting, GeLC-MS/MS, 2D-PAGE, and 2D-DIGE can be carried out on FFPE tissues. Technical tips, critical aspects, and drawbacks of the method are presented and discussed.

  7. Ski is involved in transcriptional regulation by the repressor and full-length forms of Gli3

    PubMed Central

    Dai, Ping; Shinagawa, Toshie; Nomura, Teruaki; Harada, Jun; Kaul, Sunil C.; Wadhwa, Renu; Khan, Md Matiullah; Akimaru, Hiroshi; Sasaki, Hiroshi; Colmenares, Clemencia; Ishii, Shunsuke

    2002-01-01

    Transcription factor Glioblastoma-3 (Gli3) is cleaved in the anterior region of the limb bud to generate its repressor form. In contrast, Sonic hedgehog (Shh) signaling from the posterior zone of polarizing activity blocks Gli3 processing and then induces the expression of Gli3 target genes, including Gli1. Here we report that the Ski corepressor binds to Gli3 and recruits the histone deacetylase complex. The Gli3-mediated repression was impaired by anti-Ski antibody and in Ski-deficient fibroblasts, and Shh-induced Gli1 gene transcription mediated by full-length Gli3 was inhibited by Ski. Furthermore, a Ski mutation enhanced the digit abnormalities caused by the Gli3 gene mutation. Thus, Ski plays an important role in pattern formation. PMID:12435627

  8. Quantification of the full length leptin receptor (OB-Rb) in human brown and white adipose tissue.

    PubMed

    Kutoh, E; Boss, O; Levasseur, F; Giacobino, J P

    1998-01-01

    Levels of expression of the leptin receptor (OB-R) splice variants have been studied in human omental white and perirenal brown adipose tissues by reverse transcription-PCR. The level of mRNA expression of the full length form (OB-Rb) was approximately 15% of that of the sum of all splice variants in white or brown adipose tissue. In an attempt to quantify the gene expression of OB-Rb in human white adipose tissue, a quantitative competitive PCR technique was developed, using oligonucleotide primers designed for OB-Rb and an internal standard for a "MIMIC" competition strategy. The levels of expression of OB-Rb mRNA in the omental fat of lean and obese patients were compared and no difference could be observed between the two groups. The quantitative RT-PCR technique allows for a fast and accurate measurement of the expression of the OB-Rb mRNA in small tissue samples.

  9. Fluorescence anisotropy microplate assay to investigate the interaction of full-length steroid receptor coactivator-1a with steroid receptors

    PubMed Central

    Zhang, Chen; Nordeen, Steven K.; Shapiro, David J.

    2013-01-01

    Estrogens, acting via estrogen receptor (ER) play key roles in growth, differentiation and gene regulation in the reproductive, central nervous and skeletal systems. ER-mediated gene transcription contributes to the development and spread of breast, uterine, and liver cancer. Steroid receptor coactivator-1a (SRC1a) belongs to the P160 family of coactivators, which is the best known of the many coactivators implicated in ER-mediated transactivation. Binding of full-length P160 coactivators to steroid receptors has been difficult to investigate in vitro. This chapter details how to investigate the interaction of SRC1a with ER using the fluorescence anisotropy/polarization microplate assay (FAMA). PMID:23436375

  10. Contribution of intertwined loop to membrane association revealed by Zika virus full-length NS1 structure.

    PubMed

    Xu, Xiaoying; Song, Hao; Qi, Jianxun; Liu, Yuqian; Wang, Haiyuan; Su, Chao; Shi, Yi; Gao, George F

    2016-10-17

    The association of Zika virus (ZIKV) infections with microcephaly and neurological diseases has highlighted an emerging public health concern. Here, we report the crystal structure of the full-length ZIKV nonstructural protein 1 (NS1), a major host-interaction molecule that functions in flaviviral replication, pathogenesis, and immune evasion. Of note, a long intertwined loop is observed in the wing domain of ZIKV NS1, and forms a hydrophobic "spike", which can contribute to cellular membrane association. For different flaviviruses, the amino acid sequences of the "spike" are variable but their common characteristic is either hydrophobic or positively charged, which is a beneficial feature for membrane binding. Comparative studies with West Nile and Dengue virus NS1 structures reveal conserved features, but diversified electrostatic characteristics on both inner and outer faces. Our results suggest different mechanisms of flavivirus pathogenesis and should be considered during the development of diagnostic tools.

  11. The Helios Prototype flying wing stretches almost the full length of the 300-foot-long hangar at NAS

    NASA Technical Reports Server (NTRS)

    2002-01-01

    The Helios Prototype flying wing stretches almost the full length of the 300-foot-long hangar at NASA's Dryden flight Research Center, Edwards, California. The 247-foot span solar-powered aircraft, resting on its ground maneuvering dolly, was on display for a visit of NASA Administrator Sean O'Keefe and other NASA officials on January 31, 2002. The unique solar-electric flying wing reached an altitude of 96,863 feet during an almost 17-hour flight near Hawaii on August 13, 2001, a world record for sustained horizontal flight by a non-rocket powered aircraft. Developed by AeroVironment, Inc., under NASA's Environmental Research Aircraft and Sensor Technology (ERAST) project, the Helios Prototype is the forerunner of a planned fleet of slow-flying, long duration, high-altitude uninhabited aerial vehicles (UAV) which can serve as 'atmospheric satellites,' performing Earth science missions or functioning as telecommunications relay platforms in the stratosphere.

  12. Cloning and expression of full-length Trichoderma reesei cellobiohydrolase I cDNAs in Escherichia coli.

    PubMed

    Laymon, R A; Adney, W S; Mohagheghi, A; Himmel, M E; Thomas, S R

    1996-01-01

    The process of converting lignocellulosic biomass to ethanol via fermentation depends on developing economic sources of cellulases. Trichoderma reesei cellobiohydrolase (CBH) I is a key enzyme in the fungal cellulase system; however, specific process application requirements make modification of the enzyme by site-directed mutagenesis (SDM) an attractive goal. To undertake SDM investigations, an efficient, cellulase-free host is required. To test the potential of Escherichia coli as a host, T. reesei CBH I cDNA was expressed in E. coli strain GI 724 as a C-terminal fusion to thermostable thioredoxin protein. Full-length expression of CBH I was subsequently verified by molecular weight, Western blot analysis, and activity on soluble substrates.

  13. The longest mitochondrial RNA editing PPR protein MEF12 in Arabidopsis thaliana requires the full-length E domain

    PubMed Central

    Härtel, Barbara; Zehrmann, Anja; Verbitskiy, Daniil; Takenaka, Mizuki

    2013-01-01

    Mitochondrial RNA editing factor 12 (MEF12) was identified in a screen for editing defects of a chemically mutated plant population in Arabidopsis thaliana. The MEF12 editing protein is required for the C to U change of nucleotide nad5-374. The MEF12 polypeptide is characterized by an exceptionally long stretch of 25 pentatricopeptide repeats (PPR) and a C-terminal extension domain. Editing is lost in mutant plants with a stop codon in the extending element. A T-DNA insertion substituting the 10 C-terminal amino acids of the extension domain reduces RNA editing to about 20% at the target site in a mutant plant. These results support the importance of the full-length extension module for functional RNA editing in plant mitochondria. PMID:23845994

  14. A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

    SciTech Connect

    Pierce, Anson; Wei, Rochelle; Halade, Dipti; Yoo, Si-Eun; Ran, Qitao; Richardson, Arlan

    2010-11-05

    Research highlights: {yields} Development of mouse overexpressing native human HSF1 in all tissues including CNS. {yields} HSF1 overexpression enhances heat shock response at whole-animal and cellular level. {yields} HSF1 overexpression protects from polyglutamine toxicity and favors aggresomes. {yields} HSF1 overexpression enhances proteostasis at the whole-animal and cellular level. -- Abstract: The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenic mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1{sup +/0}) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1{sup +/0} mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1{sup +/0} cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1{sup +/0} cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.

  15. Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits

    PubMed Central

    Trossbach, S V; Bader, V; Hecher, L; Pum, M E; Masoud, S T; Prikulis, I; Schäble, S; de Souza Silva, M A; Su, P; Boulat, B; Chwiesko, C; Poschmann, G; Stühler, K; Lohr, K M; Stout, K A; Oskamp, A; Godsave, S F; Müller-Schiffmann, A; Bilzer, T; Steiner, H; Peters, P J; Bauer, A; Sauvage, M; Ramsey, A J; Miller, G W; Liu, F; Seeman, P; Brandon, N J; Huston, J P; Korth, C

    2016-01-01

    Disrupted-in-schizophrenia 1 (DISC1) is a mental illness gene first identified in a Scottish pedigree. So far, DISC1-dependent phenotypes in animal models have been confined to expressing mutant DISC1. Here we investigated how pathology of full-length DISC1 protein could be a major mechanism in sporadic mental illness. We demonstrate that a novel transgenic rat model, modestly overexpressing the full-length DISC1 transgene, showed phenotypes consistent with a significant role of DISC1 misassembly in mental illness. The tgDISC1 rat displayed mainly perinuclear DISC1 aggregates in neurons. Furthermore, the tgDISC1 rat showed a robust signature of behavioral phenotypes that includes amphetamine supersensitivity, hyperexploratory behavior and rotarod deficits, all pointing to changes in dopamine (DA) neurotransmission. To understand the etiology of the behavioral deficits, we undertook a series of molecular studies in the dorsal striatum of tgDISC1 rats. We observed an 80% increase in high-affinity DA D2 receptors, an increased translocation of the dopamine transporter to the plasma membrane and a corresponding increase in DA inflow as observed by cyclic voltammetry. A reciprocal relationship between DISC1 protein assembly and DA homeostasis was corroborated by in vitro studies. Elevated cytosolic dopamine caused an increase in DISC1 multimerization, insolubility and complexing with the dopamine transporter, suggesting a physiological mechanism linking DISC1 assembly and dopamine homeostasis. DISC1 protein pathology and its interaction with dopamine homeostasis is a novel cellular mechanism that is relevant for behavioral control and may have a role in mental illness. PMID:26754951

  16. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences.

    PubMed

    Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences.

  17. Full-Length, Glycosylated NSP4 Is Localized to Plasma Membrane Caveolae by a Novel Raft Isolation Technique▿

    PubMed Central

    Storey, Stephen M.; Gibbons, Thomas F.; Williams, Cecelia V.; Parr, Rebecca D.; Schroeder, Friedhelm; Ball, Judith M.

    2007-01-01

    Rotavirus NSP4, initially characterized as an endoplasmic reticulum intracellular receptor, is a multifunctional viral enterotoxin that induces diarrhea in murine pups. There have been recent reports of the secretion of a cleaved NSP4 fragment (residues 112 to 175) and of the association of NSP4 with LC3-positive autophagosomes, raft membranes, and microtubules. To determine if NSP4 traffics to a specific subset of rafts at the plasma membrane, we isolated caveolae from plasma membrane-enriched material that yielded caveola membranes free of endoplasmic reticulum and nonraft plasma membrane markers. Analyses of the newly isolated caveolae from rotavirus-infected MDCK cells revealed full-length, high-mannose glycosylated NSP4. The lack of Golgi network-specific processing of the caveolar NSP4 glycans supports studies showing that NSP4 bypasses the Golgi apparatus. Confocal imaging showed the colocalization of NSP4 with caveolin-1 early and late in infection, elucidating the temporal and spatial NSP4-caveolin-1 association during infection. These data were extended with fluorescent resonance energy transfer analyses that confirmed the NSP4 and caveolin-1 interaction in that the specific fluorescently tagged antibodies were within 10 nm of each other during infection. Cells transfected with NSP4 showed patterns of staining and colocalization with caveolin-1 similar to those of infected cells. This study presents an endoplasmic reticulum contaminant-free caveola isolation protocol; describes the presence of full-length, endoglycosidase H-sensitive NSP4 in plasma membrane caveolae; provides confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins; and provides a final plasma membrane destination for Golgi network-bypassing NSP4 transport. PMID:17376898

  18. RNA transcripts of full-length cDNA clones of rabbit hepatitis E virus are infectious in rabbits.

    PubMed

    Cossaboom, Caitlin M; Huang, Yao-Wei; Yugo, Danielle M; Kenney, Scott P; Piñeyro, Pablo; Matzinger, Shannon R; Heffron, C Lynn; Pierson, F William; Meng, Xiang-Jin

    2014-11-07

    Hepatitis E virus (HEV), the causative agent of hepatitis E, is a single-stranded positive-sense RNA virus belonging to the family Hepeviridae. At least four genotypes of the family infect humans: genotypes 1 and 2 are transmitted to humans through contaminated water, while genotypes 3 and 4 are zoonotic and have animal reservoirs. A novel strain of HEV recently identified in rabbits is a distant member of genotype 3, and thus poses a potential risk of zoonotic transmission to humans. The objective of this study was to construct and characterize an infectious cDNA clone of the rabbit HEV. Two full-length cDNA clones of rabbit HEV, pT7g-rabHEV and pT7-rabHEV, were constructed and their infectivity was tested by in vitro transfection of Huh7 human liver cells and by direct intrahepatic inoculation of rabbits with capped RNA transcripts. Results showed that positive signal for rabbit HEV protein was detected by an immunofluorescence assay with a HEV-specific antibody in Huh7 human liver cells transfected with capped RNA transcripts from the two full-length cDNA clones. Rabbits intrahepatically inoculated with capped RNA transcripts from each of the two clones developed active HEV infection as evidenced by seroconversion to anti-HEV antibodies, and detection of rabbit HEV RNA in sera and feces of inoculated animals. The availability of a rabbit HEV infectious cDNA clone now affords us the ability to delineate the mechanism of HEV replication and cross-species infection in a small animal model.

  19. Full-Length cDNA Cloning, Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries

    PubMed Central

    Liu, Nan-Nan; Liu, Zeng-Shan; Hu, Pan; Zhang, Ying; Lu, Shi-Ying; Li, Yan-Song; Yang, Yong-Jie; Zhang, Dong-Song; Zhou, Yu; Ren, Hong-Lin

    2016-01-01

    Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5′-untranslated region (UTR) of 24 bp, a 3′-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future. PMID:27483239

  20. Impairment of Mitochondria in Adult Mouse Brain Overexpressing Predominantly Full-Length, N-Terminally Acetylated Human α-Synuclein

    PubMed Central

    Sarafian, Theodore A.; Ryan, Christopher M.; Souda, Puneet; Masliah, Eliezer; Kar, Upendra K.; Vinters, Harry V.; Mathern, Gary W.; Faull, Kym F.; Whitelegge, Julian P.; Watson, Joseph B.

    2013-01-01

    While most forms of Parkinson’s Disease (PD) are sporadic in nature, a small percentage of PD have genetic causes as first described for dominant, single base pair changes as well as duplication and triplication in the α-synuclein gene. The α-synuclein gene encodes a 140 amino acid residue protein that interacts with a variety of organelles including synaptic vesicles, lysosomes, endoplasmic reticulum/Golgi vesicles and, reported more recently, mitochondria. Here we examined the structural and functional interactions of human α-synuclein with brain mitochondria obtained from an early, pre-manifest mouse model for PD over-expressing human α-synuclein (ASOTg). The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria. No selective interaction of human α-synuclein with mitochondrial electron transport complexes cI-cV was detected. Monomeric human α-synuclein plus carboxyl terminally truncated forms were the predominant isoforms detected in ASOTg brain mitochondria by 2-dimensional PAGE (Native/SDS) and immunoblotting. Oligomers or fibrils were not detected with amyloid conformational antibodies. Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation. Overall the study showed that accumulation of full-length, N-terminally acetylated human α-synuclein was sufficient to disrupt brain mitochondrial function in adult mice. PMID:23667637

  1. Characterization of Full-Length Enterovirus 71 Strains from Severe and Mild Disease Patients in Northeastern China

    PubMed Central

    Bao, Wanguo; Zhao, Ke; Niu, Junqi; Yu, Xiao-Fang; Zhang, Wenyan

    2012-01-01

    Human enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD) has been a leading cause of childhood infection in China since 2008. Epidemic and molecular characteristics of HFMD have been examined in many areas of China, including the central and southern regions. However, clinical and genetic characterization of EV71 in the northeastern region of China is scarce. In this study, a series of analyses were performed on seven full-length EV71 sequences from HFMD patients who had either severe or mild disease. We have determined that these seven circulating EV71 viruses from Changchun, China are actually complex recombinant viruses involving multiple type A human enterovirus (HEV). Classified as EV71 subtype C4 (EV71 C4), these Changchun EV71 viruses contain genetic recombination events between the CA4, CA5, EV71B4 and EV71C1 strains. Most of the structural protein region (P1) of these viruses resembled that of the prototype EV71 C1 strains. The non-structural protein domains (P2 and P3) showed a high degree of similarity with CA4, CA5 and EV71 B4 in different regions. The 5′UTR had unclassified recombination,while partial 3D region of these viruses showed a high degree of similarity to CA16. Phylogenetic analysis of full-length or partial sequences of isolates from severe or mild disease patients in Changchun always formed a single cluster in various phylogenetic analyses of different genomic regions, suggesting that all seven strains originated from one single common ancestor. There was no correlation between viral genomic sequence and virulence. Thus, we found that circulating recombinant forms of EV71 are prevalent among HFMD patients in Northeastern China. The existence of a unique cluster of EV71 related viruses in Northeast China has important implications for vaccine development that would address the increasing prevalence of HFMD. PMID:22479324

  2. Full length nucleotide sequences of 30 common SLC44A2 alleles encoding human neutrophil antigen-3 (HNA-3)

    PubMed Central

    Chen, Qing; Srivastava, Kshitij; Ardinski, Stefanie C.; Lam, Kevin; Huvard, Michael J.; Schmid, Pirmin; Flegel, Willy A.

    2015-01-01

    Background HNA-3a alloantibodies can cause severe transfusion-related acute lung injury (TRALI). The frequency of the single nucleotide polymorphisms (SNPs) indicative of the two clinically relevant HNA-3a/b antigens are known in many populations. In the present study, we determined the full length nucleotide sequence of common SLC44A2 alleles encoding the choline transporter-like protein-2 (CTL2) that harbors HNA-3a/b antigens. Study design and methods A method was devised to determine the full length coding sequence and adjacent intron sequences from genomic DNA by 8 polymerase chain reaction (PCR) amplifications covering all 22 SLC44A2 exons. Samples from 200 African American, 96 Caucasian, 2 Hispanic and 4 Asian blood donors were analyzed. We developed a decision tree to determine alleles (confirmed haplotypes) from the genotype data. Results A total of 10 SNPs were detected in the SLC44A2 coding sequence. The non-coding sequences harbored an additional 28 SNPs (1 in the 5’-untranslated region (UTR); 23 in the introns; and 4 in the 3’-UTR). No SNP indicative of a non-functional allele was detected. The nucleotide sequences for 30 SLC44A2 alleles (haplotypes) were confirmed. There may be 66 haplotypes among the 604 chromosomes screened. Conclusions We found 38 SNPs, including 1 novel SNP, in 8192 nucleotides covering the coding sequence of the SLC44A2 gene among 302 blood donors. Population frequencies of these SNPs were established for African Americans and Caucasians. Because alleles encoding HNA-3b are more common than non-functional SLC44A2 alleles, we confirmed our previous postulate that African American donors are less likely to form HNA-3a antibodies compared to Caucasians. PMID:26437811

  3. 3.5Å cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein

    PubMed Central

    Yu, Xuekui; Jin, Lei; Jih, Jonathan; Shih, Chiaho; Hong Zhou, Z.

    2013-01-01

    The capsid shell of infectious hepatitis B virus (HBV) is composed of 240 copies of a single protein called HBV core antigen (HBc). An atomic model of a core assembled from truncated HBc was determined previously by X-ray crystallography. In an attempt to obtain atomic structural information of HBV core in a near native, non-crystalline environment, we reconstructed a 3.5Å-resolution structure of a recombinant core assembled from full-length HBc by cryo electron microscopy (cryoEM) and derived an atomic model. The structure shows that the 240 molecules of full-length HBc form a core with two layers. The outer layer, composed of the N-terminal assembly domain, is similar to the crystal structure of the truncated HBc, but has three differences. First, unlike the crystal structure, our cryoEM structure shows no disulfide bond between the Cys61 residues of the two subunits within the dimer building block, indicating such bond is not required for core formation. Second, our cryoEM structure reveals up to four more residues in the linker region (amino acids 140-149). Third, the loops in the cryoEM structures containing this linker region in subunits B and C are oriented differently (~30° and ~90°) from their counterparts in the crystal structure. The inner layer, composed of the C-terminal arginine-rich domain (ARD) and the ARD-bound RNAs, is partially-ordered and connected with the outer layer through linkers positioned around the two-fold axes. Weak densities emanate from the rims of positively charged channels through the icosahedral three-fold and local three-fold axes. We attribute these densities to the exposed portions of some ARDs, thus explaining ARD’s accessibility by proteases and antibodies. Our data supports a role of ARD in mediating communication between inside and outside of the core during HBV maturation and envelopment. PMID:24039702

  4. Identification, Molecular Cloning, and Analysis of Full-Length Hepatitis C Virus Transmitted/Founder Genotypes 1, 3, and 4

    PubMed Central

    Stoddard, Mark B.; Li, Hui; Wang, Shuyi; Saeed, Mohsan; Andrus, Linda; Ding, Wenge; Jiang, Xinpei; Learn, Gerald H.; von Schaewen, Markus; Wen, Jessica; Goepfert, Paul A.; Hahn, Beatrice H.; Ploss, Alexander; Rice, Charles M.

    2015-01-01

    ABSTRACT Hepatitis C virus (HCV) infection is characterized by persistent replication of a complex mixture of viruses termed a “quasispecies.” Transmission is generally associated with a stringent population bottleneck characterized by infection by limited numbers of “transmitted/founder” (T/F) viruses. Characterization of T/F genomes of human immunodeficiency virus type 1 (HIV-1) has been integral to studies of transmission, immunopathogenesis, and vaccine development. Here, we describe the identification of complete T/F genomes of HCV by single-genome sequencing of plasma viral RNA from acutely infected subjects. A total of 2,739 single-genome-derived amplicons comprising 10,966,507 bp from 18 acute-phase and 11 chronically infected subjects were analyzed. Acute-phase sequences diversified essentially randomly, except for the poly(U/UC) tract, which was subject to polymerase slippage. Fourteen acute-phase subjects were productively infected by more than one genetically distinct virus, permitting assessment of recombination between replicating genomes. No evidence of recombination was found among 1,589 sequences analyzed. Envelope sequences of T/F genomes lacked transmission signatures that could distinguish them from chronic infection viruses. Among chronically infected subjects, higher nucleotide substitution rates were observed in the poly(U/UC) tract than in envelope hypervariable region 1. Fourteen full-length molecular clones with variable poly(U/UC) sequences corresponding to seven genotype 1a, 1b, 3a, and 4a T/F viruses were generated. Like most unadapted HCV clones, T/F genomes did not replicate efficiently in Huh 7.5 cells, indicating that additional cellular factors or viral adaptations are necessary for in vitro replication. Full-length T/F HCV genomes and their progeny provide unique insights into virus transmission, virus evolution, and virus-host interactions associated with immunopathogenesis. PMID:25714714

  5. Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries.

    PubMed

    Liu, Nan-Nan; Liu, Zeng-Shan; Lu, Shi-Ying; Hu, Pan; Li, Yan-Song; Feng, Xiao-Li; Zhang, Shou-Yin; Wang, Nan; Meng, Qing-Feng; Yang, Yong-Jie; Tang, Feng; Xu, Yun-Ming; Zhang, Wen-Hui; Guo, Xing; Chen, Xiao-Feng; Zhou, Yu; Ren, Hong-Lin

    2015-04-15

    Peroxiredoxin 6 (Prdx6), an important antioxidant enzyme that can eliminate reactive oxygen species (ROS) to maintain homeostasis, is a bifunctional protein that possesses the activities of both glutathione peroxidase and phospholipase A2. In this study, a novel full-length Prdx6 cDNA (OaPrdx6) was cloned from Sheep (Ovis aries) using rapid amplification of cDNA ends (RACE). The full-length cDNA of OaPrdx6 was 1753bp containing a 5'-untranslated region (UTR) of 93bp, a 3'-UTR of 985bp with a poly(A) tail, and an open reading frame (ORF) of 675bp encoding a protein of 224 amino acid residues with a predicted molecular weight of 25.07kDa. The recombinant protein OaPrdx6 was expressed and purified, and its DNA protection activity was identified. In order to analyze the Prdx6 protein expression in tissues from O. aries, monoclonal antibodies against OaPrdx6 were prepared. Western blotting results indicated that OaPrdx6 protein could be detected in heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node and white blood cells, and the highest expression was found in lung while the lowest expression in muscle. Compared to the normal sheep group, the mRNA transcription level of Prdx6 in buffy coat was up-regulated in the group infected with a virulent field strain of Brucella melitensis, and down-regulated in the group inoculated with a vaccine strain S2 of brucellosis. The results indicated that Prdx6 was likely to be involved in the host immune responses against Brucella infection, and probably regarded as a molecular biomarker for distinguishing between animals infected with virulent Brucella infection and those inoculated with vaccine against brucellosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Full-Length cDNA Cloning, Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries.

    PubMed

    Liu, Nan-Nan; Liu, Zeng-Shan; Hu, Pan; Zhang, Ying; Lu, Shi-Ying; Li, Yan-Song; Yang, Yong-Jie; Zhang, Dong-Song; Zhou, Yu; Ren, Hong-Lin

    2016-07-28

    Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5'-untranslated region (UTR) of 24 bp, a 3'-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future.

  7. Full-length enriched cDNA libraries and ORFeome analysis of sugarcane hybrid and ancestor genotypes.

    PubMed

    Nishiyama, Milton Yutaka; Ferreira, Savio Siqueira; Tang, Pei-Zhong; Becker, Scott; Pörtner-Taliana, Antje; Souza, Glaucia Mendes

    2014-01-01

    Sugarcane is a major crop used for food and bioenergy production. Modern cultivars are hybrids derived from crosses between Saccharum officinarum and Saccharum spontaneum. Hybrid cultivars combine favorable characteristics from ancestral species and contain a genome that is highly polyploid and aneuploid, containing 100-130 chromosomes. These complex genomes represent a huge challenge for molecular studies and for the development of biotechnological tools that can facilitate sugarcane improvement. Here, we describe full-length enriched cDNA libraries for Saccharum officinarum, Saccharum spontaneum, and one hybrid genotype (SP803280) and analyze the set of open reading frames (ORFs) in their genomes (i.e., their ORFeomes). We found 38,195 (19%) sugarcane-specific transcripts that did not match transcripts from other databases. Less than 1.6% of all transcripts were ancestor-specific (i.e., not expressed in SP803280). We also found 78,008 putative new sugarcane transcripts that were absent in the largest sugarcane expressed sequence tag database (SUCEST). Functional annotation showed a high frequency of protein kinases and stress-related proteins. We also detected natural antisense transcript expression, which mapped to 94% of all plant KEGG pathways; however, each genotype showed different pathways enriched in antisense transcripts. Our data appeared to cover 53.2% (17,563 genes) and 46.8% (937 transcription factors) of all sugarcane full-length genes and transcription factors, respectively. This work represents a significant advancement in defining the sugarcane ORFeome and will be useful for protein characterization, single nucleotide polymorphism and splicing variant identification, evolutionary and comparative studies, and sugarcane genome assembly and annotation.

  8. Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes

    PubMed Central

    Becker, Scott; Pörtner-Taliana, Antje; Souza, Glaucia Mendes

    2014-01-01

    Sugarcane is a major crop used for food and bioenergy production. Modern cultivars are hybrids derived from crosses between Saccharum officinarum and Saccharum spontaneum. Hybrid cultivars combine favorable characteristics from ancestral species and contain a genome that is highly polyploid and aneuploid, containing 100–130 chromosomes. These complex genomes represent a huge challenge for molecular studies and for the development of biotechnological tools that can facilitate sugarcane improvement. Here, we describe full-length enriched cDNA libraries for Saccharum officinarum, Saccharum spontaneum, and one hybrid genotype (SP803280) and analyze the set of open reading frames (ORFs) in their genomes (i.e., their ORFeomes). We found 38,195 (19%) sugarcane-specific transcripts that did not match transcripts from other databases. Less than 1.6% of all transcripts were ancestor-specific (i.e., not expressed in SP803280). We also found 78,008 putative new sugarcane transcripts that were absent in the largest sugarcane expressed sequence tag database (SUCEST). Functional annotation showed a high frequency of protein kinases and stress-related proteins. We also detected natural antisense transcript expression, which mapped to 94% of all plant KEGG pathways; however, each genotype showed different pathways enriched in antisense transcripts. Our data appeared to cover 53.2% (17,563 genes) and 46.8% (937 transcription factors) of all sugarcane full-length genes and transcription factors, respectively. This work represents a significant advancement in defining the sugarcane ORFeome and will be useful for protein characterization, single nucleotide polymorphism and splicing variant identification, evolutionary and comparative studies, and sugarcane genome assembly and annotation. PMID:25222706

  9. An anatomical study of the full-length phrenic nerve and its blood supply: clinical implications for endoscopic dissection.

    PubMed

    Jiang, Su; Xu, Wen-Dong; Shen, Yun-Dong; Xu, Jian-Guang; Gu, Yu-Dong

    2011-12-01

    For surgeries aimed at the dissection of full-length phrenic nerve, a full appreciation of its trajectory, blood supply and correlation with adjacent anatomical structures is necessary, especially for endoscopic manipulations. A fresh cadaver study was conducted with the purpose of avoiding surgical complications and ensuring further efficacy and efficiency of endoscopic manipulations. Ten fresh adult cadavers were dissected. Special attention was paid to the topography of the origin, the trajectory of the phrenic nerve, and its anatomic communication with the surrounding vessels and organs. In the second side of the cadavers, thoracic endoscopic manipulations and observations were also performed. The full length of the phrenic nerve was 24.6 ± 1.7 and 30.6 ± 1.8 cm on the right and left side, respectively; the blood supply of the phrenic nerve in the thoracic cavity came exclusively from the pericardiacophrenic artery; the distance between the origin of the pericardiacophrenic artery and that of the internal thoracic artery ranged from 0.5 to 5.2 cm on the right side, and from 1.4 to 5.6 cm on the left; most of the pericardiacophrenic veins intermingled with small vessels of pericardium and pleura, forming a venous network and joining the innominate vein. Endoscopic dissection of the thoracic phrenic nerve together with the accompanying pericardiacophrenic artery can be performed. Extreme attention should be paid during surgery to a section of about 6 cm in length of the artery originating from the internal thoracic artery, while the accompanying veins do not require to be spared.

  10. Short-read assembly of full-length 16S amplicons reveals bacterial diversity in subsurface sediments.

    PubMed

    Miller, Christopher S; Handley, Kim M; Wrighton, Kelly C; Frischkorn, Kyle R; Thomas, Brian C; Banfield, Jillian F

    2013-01-01

    In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries), or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing). Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the "long tail" of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change.

  11. Identification, molecular cloning, and analysis of full-length hepatitis C virus transmitted/founder genotypes 1, 3, and 4.

    PubMed

    Stoddard, Mark B; Li, Hui; Wang, Shuyi; Saeed, Mohsan; Andrus, Linda; Ding, Wenge; Jiang, Xinpei; Learn, Gerald H; von Schaewen, Markus; Wen, Jessica; Goepfert, Paul A; Hahn, Beatrice H; Ploss, Alexander; Rice, Charles M; Shaw, George M

    2015-02-24

    Hepatitis C virus (HCV) infection is characterized by persistent replication of a complex mixture of viruses termed a "quasispecies." Transmission is generally associated with a stringent population bottleneck characterized by infection by limited numbers of "transmitted/founder" (T/F) viruses. Characterization of T/F genomes of human immunodeficiency virus type 1 (HIV-1) has been integral to studies of transmission, immunopathogenesis, and vaccine development. Here, we describe the identification of complete T/F genomes of HCV by single-genome sequencing of plasma viral RNA from acutely infected subjects. A total of 2,739 single-genome-derived amplicons comprising 10,966,507 bp from 18 acute-phase and 11 chronically infected subjects were analyzed. Acute-phase sequences diversified essentially randomly, except for the poly(U/UC) tract, which was subject to polymerase slippage. Fourteen acute-phase subjects were productively infected by more than one genetically distinct virus, permitting assessment of recombination between replicating genomes. No evidence of recombination was found among 1,589 sequences analyzed. Envelope sequences of T/F genomes lacked transmission signatures that could distinguish them from chronic infection viruses. Among chronically infected subjects, higher nucleotide substitution rates were observed in the poly(U/UC) tract than in envelope hypervariable region 1. Fourteen full-length molecular clones with variable poly(U/UC) sequences corresponding to seven genotype 1a, 1b, 3a, and 4a T/F viruses were generated. Like most unadapted HCV clones, T/F genomes did not replicate efficiently in Huh 7.5 cells, indicating that additional cellular factors or viral adaptations are necessary for in vitro replication. Full-length T/F HCV genomes and their progeny provide unique insights into virus transmission, virus evolution, and virus-host interactions associated with immunopathogenesis. Hepatitis C virus (HCV) infects 2% to 3% of the world

  12. Construction of phosphorylation interaction networks by text mining of full-length articles using the eFIP system.

    PubMed

    Tudor, Catalina O; Ross, Karen E; Li, Gang; Vijay-Shanker, K; Wu, Cathy H; Arighi, Cecilia N

    2015-01-01

    Protein phosphorylation is a reversible post-translational modification where a protein kinase adds a phosphate group to a protein, potentially regulating its function, localization and/or activity. Phosphorylation can affect protein-protein interactions (PPIs), abolishing interaction with previous binding partners or enabling new interactions. Extracting phosphorylation information coupled with PPI information from the scientific literature will facilitate the creation of phosphorylation interaction networks of kinases, substrates and interacting partners, toward knowledge discovery of functional outcomes of protein phosphorylation. Increasingly, PPI databases are interested in capturing the phosphorylation state of interacting partners. We have previously developed the eFIP (Extracting Functional Impact of Phosphorylation) text mining system, which identifies phosphorylated proteins and phosphorylation-dependent PPIs. In this work, we present several enhancements for the eFIP system: (i) text mining for full-length articles from the PubMed Central open-access collection; (ii) the integration of the RLIMS-P 2.0 system for the extraction of phosphorylation events with kinase, substrate and site information; (iii) the extension of the PPI module with new trigger words/phrases describing interactions and (iv) the addition of the iSimp tool for sentence simplification to aid in the matching of syntactic patterns. We enhance the website functionality to: (i) support searches based on protein roles (kinases, substrates, interacting partners) or using keywords; (ii) link protein entities to their corresponding UniProt identifiers if mapped and (iii) support visual exploration of phosphorylation interaction networks using Cytoscape. The evaluation of eFIP on full-length articles achieved 92.4% precision, 76.5% recall and 83.7% F-measure on 100 article sections. To demonstrate eFIP for knowledge extraction and discovery, we constructed phosphorylation-dependent interaction

  13. Construction of phosphorylation interaction networks by text mining of full-length articles using the eFIP system

    PubMed Central

    Tudor, Catalina O.; Ross, Karen E.; Li, Gang; Vijay-Shanker, K.; Wu, Cathy H.; Arighi, Cecilia N.

    2015-01-01

    Protein phosphorylation is a reversible post-translational modification where a protein kinase adds a phosphate group to a protein, potentially regulating its function, localization and/or activity. Phosphorylation can affect protein–protein interactions (PPIs), abolishing interaction with previous binding partners or enabling new interactions. Extracting phosphorylation information coupled with PPI information from the scientific literature will facilitate the creation of phosphorylation interaction networks of kinases, substrates and interacting partners, toward knowledge discovery of functional outcomes of protein phosphorylation. Increasingly, PPI databases are interested in capturing the phosphorylation state of interacting partners. We have previously developed the eFIP (Extracting Functional Impact of Phosphorylation) text mining system, which identifies phosphorylated proteins and phosphorylation-dependent PPIs. In this work, we present several enhancements for the eFIP system: (i) text mining for full-length articles from the PubMed Central open-access collection; (ii) the integration of the RLIMS-P 2.0 system for the extraction of phosphorylation events with kinase, substrate and site information; (iii) the extension of the PPI module with new trigger words/phrases describing interactions and (iv) the addition of the iSimp tool for sentence simplification to aid in the matching of syntactic patterns. We enhance the website functionality to: (i) support searches based on protein roles (kinases, substrates, interacting partners) or using keywords; (ii) link protein entities to their corresponding UniProt identifiers if mapped and (iii) support visual exploration of phosphorylation interaction networks using Cytoscape. The evaluation of eFIP on full-length articles achieved 92.4% precision, 76.5% recall and 83.7% F-measure on 100 article sections. To demonstrate eFIP for knowledge extraction and discovery, we constructed phosphorylation

  14. Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3).

    PubMed

    Akhmedzhanov, Maksat; Scrochi, Mariela; Barrandeguy, Maria; Vissani, Aldana; Osterrieder, Nikolaus; Damiani, Armando Mario

    2017-01-15

    Equine herpesvirus type 3 (EHV-3) is the causal agent of equine coital exanthema, a disease characterized by pox-like lesions on the penis of stallions and the vulva of mares. Although the complete genomic sequence of EHV-3 has been recently made available, its genomic content remains poorly characterized and the molecular mechanisms of disease development not yet elucidated. In an attempt to facilitate genetic manipulation of EHV-3, we describe here the construction of a full-length infectious bacterial artificial chromosome (BAC) clone of EHV-3. Mini-F vector sequences were inserted into the intergenic region between ORF19 and ORF20 (UL41 and UL40, respectively) of EHV-3 strain C175 by homologous recombination in equine dermal cells (NBL-6). DNA of the resulting recombinant virus was electroporated into E. coli and a full-length EHV-3 BAC clone was recovered. Virus reconstituted after transfection of the EHV-3 BAC into NBL-6 cells showed growth properties in vitro that were indistinguishable from those of the parental virus. To assess the feasibility of mutagenesis of the cloned EHV-3 genome, recombinant viruses targeting the glycoprotein E (gE) gene were generated using Red recombination in E. coli and in vitro growth properties of the recombinant viruses were evaluated. We first repaired the gE (ORF74) coding region, since the parental virus used for BAC cloning specifies a truncated version of the gene, and then created gE-tagged and gE-null versions of the virus. Our results demonstrated that: (i) EHV-3 can be efficiently cloned as a BAC allowing easy manipulation of its genome; (ii) gE is dispensable for EHV-3 growth in vitro and is expressed as a product of approximately 110-kDa in infected cells; (iii) viruses having a deletion compromising gE expression or with a truncation of the cytoplasmic and transmembrane domains are significantly compromised with regard cell-to-cell spread. The cloning of EHV-3 as a BAC simplifies future studies to identify the role

  15. Rapid hepatic clearance of full length CCN-2/CTGF: a putative role for LRP1-mediated endocytosis.

    PubMed

    Gerritsen, K G F; Bovenschen, N; Nguyen, T Q; Sprengers, D; Koeners, M P; van Koppen, A N; Joles, J A; Goldschmeding, R; Kok, R J

    2016-12-01

    CCN-2 (connective tissue growth factor; CTGF) is a key factor in fibrosis. Plasma CCN-2 has biomarker potential in numerous fibrotic disorders, but it is unknown which pathophysiological factors determine plasma CCN-2 levels. The proteolytic amino-terminal fragment of CCN-2 is primarily eliminated by the kidney. Here, we investigated elimination and distribution profiles of full length CCN-2 by intravenous administration of recombinant CCN-2 to rodents. After bolus injection in mice, we observed a large initial distribution volume (454 mL/kg) and a fast initial clearance (120 mL/kg/min). Immunosorbent assay and immunostaining showed that CCN-2 distributed mainly to the liver and was taken up by hepatocytes. Steady state clearance in rats, determined by continuous infusion of CCN-2, was fast (45 mL/kg/min). Renal CCN-2 clearance, determined by arterial and renal vein sampling, accounted for only 12 % of total clearance. Co-infusion of CCN-2 with receptor-associated protein (RAP), an antagonist of LDL-receptor family proteins, showed that RAP prolonged CCN-2 half-life and completely prevented CCN-2 internalization by hepatocytes. This suggests that hepatic uptake of CCN-2 is mediated by a RAP-sensitive mechanism most likely involving LRP1, a member of the LDL-receptor family involved in hepatic clearance of various plasma proteins. Surface plasmon resonance binding studies confirmed that CCN-2 is an LRP1 ligand. Co-infusion of CCN-2 with an excess of the heparan sulphate-binding protamine lowered the large initial distribution volume of CCN-2 by 88 % and reduced interstitial staining of CCN-2, suggesting binding of CCN-2 to heparan sulphate proteoglycans (HSPGs). Protamine did not affect clearance rate, indicating that RAP-sensitive clearance of CCN-2 is HSPG independent. In conclusion, unlike its amino-terminal fragment which is cleared by the kidney, full length CCN-2 is primarily eliminated by the liver via a fast RAP-sensitive, probably LRP1-dependent

  16. A Full-Length Plasmodium falciparum Recombinant Circumsporozoite Protein Expressed by Pseudomonas fluorescens Platform as a Malaria Vaccine Candidate

    PubMed Central

    Li, Xiangming; Coelho-dos-Reis, Jordana G. A.; Funakoshi, Ryota; Giardina, Steve; Jin, Hongfan; Retallack, Diane M.; Haverstock, Ryan; Allen, Jeffrey R.; Vedvick, Thomas S.; Fox, Christopher B.; Reed, Steven G.; Ayala, Ramses; Roberts, Brian; Winram, Scott B.; Sacci, John; Tsuji, Moriya; Zavala, Fidel; Gutierrez, Gabriel M.

    2014-01-01

    The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy

  17. Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas.

    PubMed

    Li, Xing-Xia; Yu, Wen-Chao; Cai, Zhong-Qiang; He, Cheng; Wei, Na; Wang, Xiao-Tong; Yue, Xi-Qing

    2016-01-01

    The shell of the pearl oyster (Pinctada fucata) mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas) is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM) is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM) and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization.

  18. Cloning, high level expression, purification, and crystallization of the full length Clostridium botulinum neurotoxin type E light chain.

    PubMed

    Agarwal, Rakhi; Eswaramoorthy, Subramaniam; Kumaran, Desigan; Dunn, John J; Swaminathan, Subramanyam

    2004-03-01

    The catalytic activity of the highly potent botulinum neurotoxins are confined to their N-terminal light chains ( approximately 50kDa). A full-length light chain for the type E neurotoxin with a C-terminal 6x His-tag, BoNT/E-LC, has been cloned in a pET-9c vector and over-expressed in BL21 (DE3) cells. BoNT/E-LC was purified to homogeneity by affinity chromatography on Ni-NTA agarose followed by exclusion chromatography using a Superdex-75 sizing column. The purified protein has very good solubility and can be stored stably at -20 degrees C; however, it seems to undergo auto-proteolysis when stored at temperature #10878;4-10 degrees C. BoNT/E-LC is active on its natural substrate, the synaptosomal associated 25kDa protein, SNAP-25, indicating that it retains a native-like conformation and therefore can be considered as a useful tool in studying the structure/function of the catalytic light chain. Recombinant BoNT/E-LC has been crystallized under five different conditions and at various pHs. Crystals diffract to better than 2.1A.

  19. Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

    SciTech Connect

    Krieg, A.M.; Gourley, M.F.; Steinberg, A.D. )

    1991-05-01

    Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells.

  20. [Cloning of full-length coding sequence of tree shrew CD4 and prediction of its molecular characteristics].

    PubMed

    Tian, Wei-Wei; Gao, Yue-Dong; Guo, Yan; Huang, Jing-Fei; Xiao, Chang; Li, Zuo-Sheng; Zhang, Hua-Tang

    2012-02-01

    The tree shrews, as an ideal animal model receiving extensive attentions to human disease research, demands essential research tools, in particular cellular markers and monoclonal antibodies for immunological studies. In this paper, a 1 365 bp of the full-length CD4 cDNA encoding sequence was cloned from total RNA in peripheral blood of tree shrews, the sequence completes two unknown fragment gaps of tree shrews predicted CD4 cDNA in the GenBank database, and its molecular characteristics were analyzed compared with other mammals by using biology software such as Clustal W2.0 and so forth. The results showed that the extracellular and intracellular domains of tree shrews CD4 amino acid sequence are conserved. The tree shrews CD4 amino acid sequence showed a close genetic relationship with Homo sapiens and Macaca mulatta. Most regions of the tree shrews CD4 molecule surface showed positive charges as humans. However, compared with CD4 extracellular domain D1 of human, CD4 D1 surface of tree shrews showed more negative charges, and more two N-glycosylation sites, which may affect antibody binding. This study provides a theoretical basis for the preparation and functional studies of CD4 monoclonal antibody.

  1. Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication

    PubMed Central

    Rustiguel, Joane K.; Soares, Ricardo O. S.; Meisburger, Steve P.; Davis, Katherine M.; Malzbender, Kristina L.; Ando, Nozomi; Dias-Baruffi, Marcelo; Nonato, Maria Cristina

    2016-01-01

    Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins. PMID:27642006

  2. DPP4 truncated GM-CSF & IL-3 manifest distinct receptor binding & regulatory functions compared to their full length forms.

    PubMed

    O'Leary, H A; Capitano, M; Cooper, S; Mantel, C; Boswell, H S; Kapur, R; Ramdas, B; Chan, R; Deng, L; Qu, C-K; Broxmeyer, H E

    2017-03-27

    Dipeptidylpeptidase 4 (DPP4/CD26) enzymatically cleaves select penultimate amino acids of proteins, including colony stimulating factors (CSFs), and has been implicated in cellular regulation. To better understand the role of DPP4 regulation of hematopoiesis, we analyzed the activity of DPP4 on the surface of immature blood cells and then comparatively assessed the interactions and functional effects of full-length (FL) and DPP4 truncated factors [(T)-GM-CSF and- IL-3] on both in vitro and in vivo models of normal and leukemic cells. T-GM-CSF and T-IL-3 had enhanced receptor binding, but decreased CSF activity, compared to their FL forms. Importantly, T-GM-CSF and T-IL-3 significantly, and reciprocally, blunted receptor binding and myeloid progenitor cell proliferation activity of both FL-GM-CSF and FL-IL-3 in vitro and in vivo. Similar effects were apparent in vitro using cluster forming cells from patients with Acute Myeloid Leukemia (AML) regardless of cytogenetic or molecular alterations and in vivo utilizing animal models of leukemia. This suggests that DPP4 T-molecules have modified binding and functions compared to their FL counterparts and may serve regulatory roles in normal and malignant hematopoiesis.Leukemia accepted article preview online, 27 March 2017. doi:10.1038/leu.2017.98.

  3. Construction and characterization of a full-length cDNA infectious clone of emerging porcine Senecavirus A.

    PubMed

    Chen, Zhenhai; Yuan, Fangfeng; Li, Yanhua; Shang, Pengcheng; Schroeder, Robin; Lechtenberg, Kelly; Henningson, Jamie; Hause, Benjamin; Bai, Jianfa; Rowland, Raymond R R; Clavijo, Alfonso; Fang, Ying

    2016-10-01

    A full-length cDNA infectious clone, pKS15-01-Clone, was constructed from an emerging Senecavirus A (SVA; strain KS15-01). To explore the potential use as a viral backbone for expressing marker genes, the enhanced green fluorescent protein (EGFP)-tagged reporter virus (vKS15-01-EGFP) was generated using reverse genetics. Compared to the parental virus, the pKS15-01-Clone derived virus (vKS15-01-Clone) replicated efficiently in vitro and in vivo, and induced similar levels of neutralizing antibody and cytokine responses in infected animals. In contrast, the vKS15-01-EGFP virus showed impaired growth ability and induced lower level of immune response in infected animals. Lesions on the dorsal snout and coronary bands were observed in all pigs infected by parental virus KS15-01, but not in pigs infected with vKS15-01-Clone or vKS15-01-EGFP viruses. These results demonstrated that the infectious clone and EGFP reporter virus could be used as important tools in further elucidating the SVA pathogenesis and development of control measures.

  4. Analysis of full-length genomes of porcine teschovirus (PTV) and the effect of purifying selection on phylogenetic trees.

    PubMed

    Villanova, Fabiola; Cui, Shangjin; Ai, Xia; Leal, Élcio

    2016-05-01

    To study the outcome of natural selection using phylogenetic trees, we analyzed full-length genome sequences of porcine teschovirus (PTV). PTV belongs to the family Picornaviridae and has a positive-stranded RNA genome, the replication of which is carried out by the error-prone viral RNA-dependent RNA polymerase. The viral RNA encodes a single polyprotein that is cleaved into structural (i.e., L, VP4, VP2, VP3 and VP1) and nonstructural proteins (i.e., 2A, 2B, 2C, 3A, 3B, and 3C). A high degree of genetic diversity was found based on the pairwise nucleotide distances and on the mean ratio of the number of nonsynonymous (dN) and synonymous (dS) substitutions (dN/dS) in the structural genes. Conversely, the diversity of the nonstructural genes was lower. The differences in genetic diversity between the structural and nonstructural genomic regions were likely due to strong purifying selection; consequently, the estimates of phylogenies were also discordant among these genes. In particular, maximum-likelihood and Bayesian methods generated short-branched trees when loci that are under strong purifying selection were used. These findings indicate that even in an RNA virus with an intrinsically high mutation rate, a strong purifying selection will curb genetic diversity and should be considered an important source of bias in future studies based on phylogenetic methods.

  5. Aminoglycosides restore full-length type VII collagen by overcoming premature termination codons: therapeutic implications for dystrophic epidermolysis bullosa.

    PubMed

    Cogan, Jon; Weinstein, Jacqueline; Wang, Xinyi; Hou, Yingping; Martin, Sabrina; South, Andrew P; Woodley, David T; Chen, Mei

    2014-10-01

    Patients with recessive dystrophic epidermolysis bullosa (RDEB) have severe, incurable skin fragility, blistering, and multiple skin wounds due to mutations in the gene encoding type VII collagen (C7), the major component of anchoring fibrils mediating epidermal-dermal adherence. Nearly 10-25% of RDEB patients carry nonsense mutations leading to premature stop codons (PTCs) that result in truncated C7. In this study, we evaluated the feasibility of using aminoglycosides to suppress PTCs and induce C7 expression in two RDEB keratinocyte cell lines (Q251X/Q251X and R578X/R906) and two primary RDEB fibroblasts (R578X/R578X and R163X/R1683X). Incubation of these cells with aminoglycosides (geneticin, gentamicin, and paromomycin) resulted in the synthesis and secretion of a full-length C7 in a dose-dependent and sustained manner. Importantly, aminoglycoside-induced C7 reversed the abnormal RDEB cell phenotype and incorporated into the dermal-epidermal junction of skin equivalents. We further demonstrated the general utility of aminoglycoside-mediated readthrough in 293 cells transiently transfected with expression vectors encoding 22 different RDEB nonsense mutations. This is the first study demonstrating that aminoglycosides can induce PTC readthrough and restore functional C7 in RDEB caused by nonsense mutations. Therefore, aminoglycosides may have therapeutic potential for RDEB patients and other inherited skin diseases caused by nonsense mutations.

  6. Sequencing and analysis of 10967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis

    SciTech Connect

    Morin, R D; Chang, E; Petrescu, A; Liao, N; Kirkpatrick, R; Griffith, M; Butterfield, Y; Stott, J; Barber, S; Babakaiff, R; Matsuo, C; Wong, D; Yang, G; Smailus, D; Brown-John, M; Mayo, M; Beland, J; Gibson, S; Olson, T; Tsai, M; Featherstone, R; Chand, S; Siddiqui, A; Jang, W; Lee, E; Klein, S; Prange, C; Myers, R M; Green, E D; Wagner, L; Gerhard, D; Marra, M; Jones, S M; Holt, R

    2005-10-31

    Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection initiative. Here we present an analysis of 10967 clones (8049 from X. laevis and 2918 from X. tropicalis). The clone set contains 2013 orthologs between X. laevis and X. tropicalis as well as 1795 paralog pairs within X. laevis. 1199 are in-paralogs, believed to have resulted from an allotetraploidization event approximately 30 million years ago, and the remaining 546 are likely out-paralogs that have resulted from more ancient gene duplications, prior to the divergence between the two species. We do not detect any evidence for positive selection by the Yang and Nielsen maximum likelihood method of approximating d{sub N}/d{sub S}. However, d{sub N}/d{sub S} for X. laevis in-paralogs is elevated relative to X. tropicalis orthologs. This difference is highly significant, and indicates an overall relaxation of selective pressures on duplicated gene pairs. Within both groups of paralogs, we found evidence of subfunctionalization, manifested as differential expression of paralogous genes among tissues, as measured by EST information from public resources. We have observed, as expected, a higher instance of subfunctionalization in out-paralogs relative to in-paralogs.

  7. Endothelial progenitor cell-dependent angiogenesis requires localization of the full-length form of uPAR in caveolae.

    PubMed

    Margheri, Francesca; Chillà, Anastasia; Laurenzana, Anna; Serratì, Simona; Mazzanti, Benedetta; Saccardi, Riccardo; Santosuosso, Michela; Danza, Giovanna; Sturli, Niccolò; Rosati, Fabiana; Magnelli, Lucia; Papucci, Laura; Calorini, Lido; Bianchini, Francesca; Del Rosso, Mario; Fibbi, Gabriella

    2011-09-29

    Endothelial urokinase-type plasminogen activator receptor (uPAR) is thought to provide a regulatory mechanism in angiogenesis. Here we studied the proangiogenic role of uPAR in endothelial colony-forming cells (ECFCs), a cell population identified in human umbilical blood that embodies all of the properties of an endothelial progenitor cell matched with a high proliferative rate. By using caveolae-disrupting agents and by caveolin-1 silencing, we have shown that the angiogenic properties of ECFCs depend on caveolae integrity and on the presence of full-length uPAR in such specialized membrane invaginations. Inhibition of uPAR expression by antisense oligonucleotides promoted caveolae disruption, suggesting that uPAR is an inducer of caveolae organization. Vascular endothelial growth factor (VEGF) promoted accumulation of uPAR in ECFC caveolae in its undegraded form. We also demonstrated that VEGF-dependent ERK phosphorylation required integrity of caveolae as well as caveolar uPAR expression. VEGF activity depends on inhibition of ECFC MMP12 production, which results in impairment of MMP12-dependent uPAR truncation. Further, MMP12 overexpression in ECFC inhibited vascularization in vitro and in vivo. Our data suggest that intratumor homing of ECFCs suitably engineered to overexpress MMP12 could have the chance to control uPAR-dependent activities required for tumor angiogenesis and malignant cells spreading.

  8. Computational insights into the inhibition and destabilization of morin on the oligomer of full-length human islet amyloid polypeptide.

    PubMed

    Wang, Qianqian; Zhou, Shuangyan; Wei, Wei; Yao, Xiaojun; Liu, Huanxiang; Hu, Zhide

    2015-11-21

    The aggregation of human islet amyloid polypeptide (hIAPP) is closely related with the occurrence of type 2 diabetes (T2D). Natural flavonoid morin was confirmed to not only inhibit the amyloid formation of hIAPP, but disaggregate its preformed amyloid fibrils. In this study, with the goal of elucidating the molecular mechanism of inhibition and destabilization of morin on the full-length hIAPP(1-37) oligomer, molecular dynamics simulations were performed for hIAPP(1-37) pentamer in the presence and absence of morin. The obtained results show that during the protein-inhibitor interaction, morin can notably alter the structural properties of hIAPP(1-37) pentamer, such as morphology, solvent accessible surface area and secondary structure. Moreover, we identified three possible binding sites of morin on hIAPP, all of which located near the amyloidogenic region of this protein. From the binding free energy calculations, we found that Site II was the most possible one. Further conformational analysis together with energy decomposition showed that the residues His18, Phe23 and Ile26 play a key role in the binding with morin by hydrogen bond, π-π and hydrophobic interactions. The proposal of the theoretical mechanism of morin against hIAPP aggregation will provide valuable information for the development of new drugs to inhibit hIAPP aggregation.

  9. Collection and comparative analysis of 1888 full-length cDNAs from wild rice Oryza rufipogon Griff. W1943.

    PubMed

    Lu, Tingting; Yu, Shuliang; Fan, Danlin; Mu, Jie; Shangguan, Yingying; Wang, Zixuan; Minobe, Yuzo; Lin, Zhixin; Han, Bin

    2008-10-01

    A huge amount of cDNA and EST resources have been developed for cultivated rice species Oryza sativa; however, only few cDNA resources are available for wild rice species. In this study, we isolated and completely sequenced 1888 putative full-length cDNA (FLcDNA) clones from wild rice Oryza rufipogon Griff. W1943 for comparative analysis between wild and cultivated rice species. Two cDNA libraries were constructed from 3-week-old leaf samples under either normal or cold-treated conditions. Homology searching of these cDNA sequences revealed that >96.8% of the wild rice cDNAs were matched to the cultivated rice O. sativa ssp. japonica cv. Nipponbare genome sequence. However, <22% of them were fully matched to the cv. Nipponbare genome sequence. The comparative analysis showed that O. rufipogon W1943 had greater similarity to O. sativa ssp. japonica than to ssp. indica cultivars. In addition, 17 novel rice cDNAs were identified, and 41 putative tissue-specific expression genes were defined through searching the rice massively parallel signature-sequencing database. In conclusion, these FLcDNA clones are a resource for further function verification and could be broadly utilized in rice biological studies.

  10. Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas

    PubMed Central

    Li, Xing-Xia; Yu, Wen-Chao; Cai, Zhong-Qiang; He, Cheng; Wei, Na

    2016-01-01

    The shell of the pearl oyster (Pinctada fucata) mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas) is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM) is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM) and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization. PMID:27703977

  11. Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication

    NASA Astrophysics Data System (ADS)

    Rustiguel, Joane K.; Soares, Ricardo O. S.; Meisburger, Steve P.; Davis, Katherine M.; Malzbender, Kristina L.; Ando, Nozomi; Dias-Baruffi, Marcelo; Nonato, Maria Cristina

    2016-09-01

    Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

  12. Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication.

    PubMed

    Rustiguel, Joane K; Soares, Ricardo O S; Meisburger, Steve P; Davis, Katherine M; Malzbender, Kristina L; Ando, Nozomi; Dias-Baruffi, Marcelo; Nonato, Maria Cristina

    2016-09-19

    Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

  13. [Analysis of the molecular characteristics and cloning of full-length coding sequence of interleukin-2 in tree shrews].

    PubMed

    Huang, Xiao-Yan; Li, Ming-Li; Xu, Juan; Gao, Yue-Dong; Wang, Wen-Guang; Yin, An-Guo; Li, Xiao-Fei; Sun, Xiao-Mei; Xia, Xue-Shan; Dai, Jie-Jie

    2013-04-01

    While the tree shrew (Tupaia belangeri chinensis) is an excellent animal model for studying the mechanisms of human diseases, but few studies examine interleukin-2 (IL-2), an important immune factor in disease model evaluation. In this study, a 465 bp of the full-length IL-2 cDNA encoding sequence was cloned from the RNA of tree shrew spleen lymphocytes, which were then cultivated and stimulated with ConA (concanavalin). Clustal W 2.0 was used to compare and analyze the sequence and molecular characteristics, and establish the similarity of the overall structure of IL-2 between tree shrews and other mammals. The homology of the IL-2 nucleotide sequence between tree shrews and humans was 93%, and the amino acid homology was 80%. The phylogenetic tree results, derived through the Neighbour-Joining method using MEGA5.0, indicated a close genetic relationship between tree shrews, Homo sapiens, and Macaca mulatta. The three-dimensional structure analysis showed that the surface charges in most regions of tree shrew IL-2 were similar to between tree shrews and humans; however, the N-glycosylation sites and local structures were different, which may affect antibody binding. These results provide a fundamental basis for the future study of IL-2 monoclonal antibody in tree shrews, thereby improving their utility as a model.

  14. An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences.

    PubMed

    Pollier, Jacob; González-Guzmán, Miguel; Ardiles-Diaz, Wilson; Geelen, Danny; Goossens, Alain

    2011-01-01

    cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is a commonly used technique for genome-wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequence information are obtained for a large number of differentially expressed gene tags. However, most of the gene tags do not correspond to full-length (FL) coding sequences, which is a prerequisite for subsequent functional analysis. A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incomplete cDNAs. The method was applied to screen for the FL open reading frames of a selection of 163 cDNA-AFLP tags from three different medicinal plants, leading to the identification of 109 (67%) FL clones. Furthermore, the protocol allows for the use of multiple probes in a single hybridization event, thus significantly increasing the throughput when screening for rare transcripts. The presented strategy offers an efficient method for the conversion of incomplete expressed sequence tags (ESTs), such as cDNA-AFLP tags, to FL-coding sequences.

  15. Mapping of chimpanzee full-length cDNAs onto the human genome unveils large potential divergence of the transcriptome.

    PubMed

    Sakate, Ryuichi; Suto, Yumiko; Imanishi, Tadashi; Tanoue, Tetsuya; Hida, Munetomo; Hayasaka, Ikuo; Kusuda, Jun; Gojobori, Takashi; Hashimoto, Katsuyuki; Hirai, Momoki

    2007-09-01

    The genetic basis of the phenotypic difference between human and chimpanzee is one of the most actively pursued issues in current genomics. Although the genomic divergence between the two species has been described, the transcriptomic divergence has not been well documented. Thus, we newly sequenced and analyzed chimpanzee full-length cDNAs (FLcDNAs) representing 87 protein-coding genes. The number of nucleotide substitutions and sites of insertions/deletions (indels) was counted as a measure of sequence divergence between the chimpanzee FLcDNAs and the human genome onto which the FLcDNAs were mapped. Difference in transcription start/termination sites (TSSs/TTSs) and alternative splicing (AS) exons was also counted as a measure of structural divergence between the chimpanzee FLcDNAs and their orthologous human transcripts (NCBI RefSeq). As a result, we found that transposons (Alu) and repetitive segments caused large indels, which strikingly increased the average amount of sequence divergence up to more than 2% in the 3'-UTRs. Moreover, 20 out of the 87 transcripts contained more than 10% structural divergence in length. In particular, two-thirds of the structural divergence was found in the 3'-UTRs, and variable transcription start sites were conspicuous in the 5'-UTRs. As both transcriptional and translational efficiency were supposed to be related to 5'- and 3'-UTR sequences, these results lead to the idea that the difference in gene regulation can be a major cause of the difference in phenotype between human and chimpanzee.

  16. Genomic integration of the full-length dystrophin coding sequence in Duchenne muscular dystrophy induced pluripotent stem cells.

    PubMed

    Farruggio, Alfonso P; Bhakta, Mital S; du Bois, Haley; Ma, Julia; P Calos, Michele

    2017-04-01

    The plasmid vectors that express the full-length human dystrophin coding sequence in human cells was developed. Dystrophin, the protein mutated in Duchenne muscular dystrophy, is extraordinarily large, providing challenges for cloning and plasmid production in Escherichia coli. The authors expressed dystrophin from the strong, widely expressed CAG promoter, along with co-transcribed luciferase and mCherry marker genes useful for tracking plasmid expression. Introns were added at the 3' and 5' ends of the dystrophin sequence to prevent translation in E. coli, resulting in improved plasmid yield. Stability and yield were further improved by employing a lower-copy number plasmid origin of replication. The dystrophin plasmids also carried an attB site recognized by phage phiC31 integrase, enabling the plasmids to be integrated into the human genome at preferred locations by phiC31 integrase. The authors demonstrated single-copy integration of plasmid DNA into the genome and production of human dystrophin in the human 293 cell line, as well as in induced pluripotent stem cells derived from a patient with Duchenne muscular dystrophy. Plasmid-mediated dystrophin expression was also demonstrated in mouse muscle. The dystrophin expression plasmids described here will be useful in cell and gene therapy studies aimed at ameliorating Duchenne muscular dystrophy.

  17. Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

    PubMed

    Sawada, R; Ohashi, K; Anaguchi, H; Okazaki, H; Hattori, M; Minato, N; Naruto, M

    1990-04-01

    To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.

  18. The full-length cell-cell fusogen EFF-1 is monomeric and upright on the membrane

    NASA Astrophysics Data System (ADS)

    Zeev-Ben-Mordehai, Tzviya; Vasishtan, Daven; Siebert, C. Alistair; Grünewald, Kay

    2014-05-01

    Fusogens are membrane proteins that remodel lipid bilayers to facilitate membrane merging. Although several fusogen ectodomain structures have been solved, structural information on full-length, natively membrane-anchored fusogens is scarce. Here we present the electron cryo microscopy three-dimensional reconstruction of the Caenorhabditis elegans epithelial fusion failure 1 (EFF-1) protein natively anchored in cell-derived membrane vesicles. This reveals a membrane protruding, asymmetric, elongated monomer. Flexible fitting of a protomer of the EFF-1 crystal structure, which is homologous to viral class-II fusion proteins, shows that EFF-1 has a hairpin monomeric conformation before fusion. These structural insights, when combined with our observations of membrane-merging intermediates between vesicles, enable us to propose a model for EFF-1 mediated fusion. This process, involving identical proteins on both membranes to be fused, follows a mechanism that shares features of SNARE-mediated fusion while using the structural building blocks of the unilaterally acting class-II viral fusion proteins.

  19. Two distinct trimeric conformations of natively membrane-anchored full-length herpes simplex virus 1 glycoprotein B

    PubMed Central

    Zeev-Ben-Mordehai, Tzviya; Vasishtan, Daven; Hernández Durán, Anna; Vollmer, Benjamin; White, Paul; Prasad Pandurangan, Arun; Siebert, C. Alistair; Topf, Maya

    2016-01-01

    Many viruses are enveloped by a lipid bilayer acquired during assembly, which is typically studded with one or two types of glycoproteins. These viral surface proteins act as the primary interface between the virus and the host. Entry of enveloped viruses relies on specialized fusogen proteins to help merge the virus membrane with the host membrane. In the multicomponent herpesvirus fusion machinery, glycoprotein B (gB) acts as this fusogen. Although the structure of the gB ectodomain postfusion conformation has been determined, any other conformations (e.g., prefusion, intermediate conformations) have so far remained elusive, thus restricting efforts to develop antiviral treatments and prophylactic vaccines. Here, we have characterized the full-length herpes simplex virus 1 gB in a native membrane by displaying it on cell-derived vesicles and using electron cryotomography. Alongside the known postfusion conformation, a novel one was identified. Its structure, in the context of the membrane, was determined by subvolume averaging and found to be trimeric like the postfusion conformation, but appeared more condensed. Hierarchical constrained density-fitting of domains unexpectedly revealed the fusion loops in this conformation to be apart and pointing away from the anchoring membrane. This vital observation is a substantial step forward in understanding the complex herpesvirus fusion mechanism, and opens up new opportunities for more targeted intervention of herpesvirus entry. PMID:27035968

  20. 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.

  1. Full length genome sequence of Tioman virus, a novel paramyxovirus in the genus Rubulavirus isolated from fruit bats in Malaysia.

    PubMed

    Chua, K B; Wang, L-F; Lam, S K; Eaton, B T

    2002-07-01

    A novel paramyxovirus in the genus Rubulavirus, named Tioman virus (TiV), was isolated in 1999 from a number of pooled urine samples of Island Flying Foxes (Pteropus hypomelanus) during the search for the reservoir host of Nipah virus. TiV is antigenically related to Menangle virus (MenV) that was isolated in Australia in 1997 during disease outbreak in pigs. Sequence analysis of the full length genome indicated that TiV is a novel member of the genus Rubulavirus within the subfamily Paramyxovirinae, family Paramyxoviridae. However, there are several features of TiV which make it unique among known paramyxoviruses and rubulaviruses in particular: (1) TiV, like MenV, uses the nucleotide G as a transcriptional initiation site, rather than the A residue used by all other known paramyxoviruses; (2) TiV uses C as the +1 residue for all intergenic regions, a feature not seen for rubulaviruses but common for all other members within the subfamily Paramyxovirinae; (3) Although the attachment protein of TiV has structural features that are conserved in other rubulaviruses, it manifests no overall sequence homology with members of the genus, lacks the sialic acid-binding motif N-R-K-S-C-S and has only two out of the six highly conserved residues known to be important for the catalytic activity of neuraminidase.

  2. Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch.

    PubMed

    Manz, Christoph; Kobitski, Andrei Yu; Samanta, Ayan; Keller, Bettina G; Jäschke, Andres; Nienhaus, G Ulrich

    2017-09-18

    S-adenosyl-L-methionine (SAM) ligand binding induces major structural changes in SAM-I riboswitches, through which gene expression is regulated via transcription termination. Little is known about the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I riboswitch. Therefore, we have explored its conformational energy landscape as a function of Mg(2+) and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling analysis. We resolved four conformational states both in the presence and the absence of SAM and determined their Mg(2+)-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds coexist, and SAM binding only gradually shifts the populations toward terminator states. We observed a pronounced acceleration of conformational transitions upon SAM binding, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.

  3. VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones✰

    PubMed Central

    Filippone, Claudia; Zhi, Ning; Wong, Susan; Lu, Jun; Kajigaya, Sachiko; Gallinella, Giorgio; Kakkola, Laura; Venermo, Maria S Söderlund; Young, Neal S.; Brown, Kevin E.

    2008-01-01

    Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus. PMID:18252260

  4. Full-length PGC-1α salvages the phenotype of a mouse model of human neuropathy through mitochondrial proliferation.

    PubMed

    Rona-Voros, Krisztina; Eschbach, Judith; Vernay, Aurélia; Wiesner, Diana; Schwalenstocker, Birgit; Geniquet, Pauline; Mousson De Camaret, Bénédicte; Echaniz-Laguna, Andoni; Loeffler, Jean-Philippe; Ludolph, Albert C; Weydt, Patrick; Dupuis, Luc

    2013-12-20

    Increased mitochondrial mass, commonly termed mitochondrial proliferation, is frequently observed in many human diseases directly or indirectly involving mitochondrial dysfunction. Mitochondrial proliferation is thought to counterbalance a compromised energy metabolism, yet it might also be detrimental through alterations of mitochondrial regulatory functions such as apoptosis, calcium metabolism or oxidative stress. Here, we show that prominent mitochondrial proliferation occurs in Cramping mice, a model of hereditary neuropathy caused by a mutation in the dynein heavy chain gene Dync1h1. The mitochondrial proliferation correlates with post-prandial induction of full-length (FL) and N-terminal truncated (NT) isoforms of the transcriptional co-activator PGC-1α. The selective knock-out of FL-PGC-1α isoform, preserving expression and function of NT-PGC-1α, led to a complete reversal of mitochondrial proliferation. Moreover, FL-PGC-1α ablation potently exacerbated the mitochondrial dysfunction and led to severe weight loss. Finally, FL-PGC-1α ablation triggered pronounced locomotor dysfunction, tremors and inability to rear in Cramping mice. In summary, endogenous FL-PGC-1α activates mitochondrial proliferation and salvages neurological and metabolic health upon disease. NT-PGC-1α cannot fulfil this protective action. Activation of this endogenous salvage pathway might thus be a valuable therapeutic target for diseases involving mitochondrial dysfunction.

  5. REAL-Select: full-length antibody display and library screening by surface capture on yeast cells.

    PubMed

    Rhiel, Laura; Krah, Simon; Günther, Ralf; Becker, Stefan; Kolmar, Harald; Hock, Björn

    2014-01-01

    We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1:1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells.

  6. An Adenoviral Vaccine Encoding Full-Length Inactivated Human HER2 Exhibits Potent Immunogenicty and Enhanced Therapeutic Efficacy Without Oncogenicity

    PubMed Central

    Hartman, Zachary; Wei, Junping; Osada, Takuya; Glass, Oliver; Lei, Gangjun; Yang, Xiao-Yi; Peplinski, Sharon; Kim, Dong-Wan; Xia, Wenle; Spector, Neil; Marks, Jeffrey; Barry, William; Hobeika, Amy; Devi, Gayathri; Amalfitano, Andrea; Morse, Michael A.; Lyerly, H. Kim; Clay, Timothy M.

    2010-01-01

    Purpose Overexpression of the breast cancer oncogene HER2 correlates with poor survival. Current HER2-directed therapies confer limited clinical benefits and most patients experience progressive disease. Because refractory tumors remain strongly HER2+, vaccine approaches targeting HER2 have therapeutic potential, but wild type (wt) HER2 cannot safely be delivered in imunogenic viral vectors because it is a potent oncogene. We designed and tested several HER2 vaccines devoid of oncogenic activity to develop a safe vaccine for clinical use. Experimental Design We created recombinant adenoviral vectors expressing the extracellular domain of HER2 (Ad-HER2-ECD), ECD plus the transmembrane domain (Ad-HER2-ECD-TM) and full length HER2 inactivated for kinase function (Ad-HER2-ki) and determined their immunogenicity and anti-tumor effect in wild type (WT) and HER2 tolerant mice. To assess their safety, we compared their effect on the cellular transcriptome, cell proliferation, anchorage-dependent growth, and transformation potential in vivo. Results Ad-HER2-ki was the most immunogenic vector in WT animals, retained immunogenicity in HER2-transgenic tolerant animals, and showed strong therapeutic efficacy in treatment models. Despite being highly expressed, HER2-ki protein was not phosphorylated and did not produce an oncogenic gene signature in primary human cells. And, in contrast to HER2-wt, cells overexpressing HER2-ki were less proliferative, displayed less anchorage independent growth and were not transformed in vivo. Conclusions Vaccination with mutationally inactivated, non-oncogenic Ad-HER2-ki results in robust polyclonal immune responses to HER2 in tolerant models, which translates into strong and effective anti-tumor responses in vivo. Ad-HER2-ki is thus a safe and promising vaccine for evaluation in clinical trials. PMID:20179231

  7. Identification, cross-taxon transferability and application of full-length cDNA SSR markers in Phyllostachys pubescens.

    PubMed

    Lin, Yuan; Lu, Jiang-Jie; Wu, Miao-Dan; Zhou, Ming-Bing; Fang, Wei; Ide, Yuji; Tang, Ding-Qin

    2014-01-01

    Current databases of Phyllostachys pubescens full-length cDNAs (FL-cDNAs) provide a rich source of sequences for the development of potential FL-cDNA simple sequence repeat (SSR) markers. We screened 10,608 P. pubescens cDNAs, discovering 1614 SSRs in 1382 SSR-containing FL-cDNAs. The SSRs were more abundant within transposable elements (TEs) than expressed sequence tags (ESTs) and genome survey sequences (GSSs), and specific dinucleotide repeats tended to associate with particular TE families: (TA)n with En/Spm and (CT)n with Mutator. A selected panel of 100 FL-cDNAs containing type I SSRs yielded 68 functional SSR markers with an average polymorphism information content (PIC) value of 0.12, among which 22 loci contained polymorphisms. These markers became less transferrable (83.1% → 69.9% → 49.3%) but more polymorphic (79.4% → 92.3% → 92.8%) with increasing phylogenetic distance (intra-genus → intra-subtribe → intra-family). Transferability and polymorphism also depended on the location of the marker, with those located in the coding region being more transferrable (69.1%) and less polymorphic (89.4%) than those in the 5'-UTR (63.4% transferable, 90.7% polymorphic) and the 3'-UTR (61.8% transferable, 91.4% polymorphic). As proof of principle, we were able to use our FL-cDNA SSR markers to identify the parental stocks in interspecific hybrids of bamboo within and beyond P. pubescens, and estimate the outcrossing rate for P. pubescens. Our research should facilitate molecular breeding in bamboo species where original genetic markers are scarce.

  8. Full-length genomic analysis of porcine rotavirus strains isolated from pigs with diarrhea in Northern Italy.

    PubMed

    Monini, Marina; Zaccaria, Guendalina; Ianiro, Giovanni; Lavazza, Antonio; Vaccari, Gabriele; Ruggeri, Franco M

    2014-07-01

    Group A rotaviruses (RVA) cause acute dehydrating diarrhea in young of man and many animal species, including pigs. Swine RVA has an important economic impact on the farming industry, and pigs represent a potential reservoir for zoonotic transmission of RVA to humans. To investigate the genetic diversity of porcine RVA strains in Italy and identify their possible zoonotic characteristics, 25 RVA-positive feces were collected from diarrheic pigs in Northern Italy, in 2009-2010; all viral strains were characterized by G and P genotyping RT-PCR. Three samples were selected for full genome sequencing. Sequencing of the NSP3 genes of all samples was also performed. Rotavirus diagnosis was carried out by ELISA and electron microscopy. RT-PCR and Sanger sequencing were performed in a one-tube format, using primer sets specific for each of the 11 genome segments. Analysis of the G (VP7) and P (VP4) genotypes showed that all strains identified were typical porcine RVAs (G4, G5, G9; P[6], P[13], P[23]). Full-length genome sequencing was performed on selected G9 isolates. Most segments belonged to the genotype constellation 1 (Wa-like), which is shared by most human RVA strains, but gene types such as I5 (VP6) and A8 (NSP1), which are typical of porcine and rare among human RVAs, were also detected. We identified RVA strains showing the T7 genotype, an NSP3 gene type that was previously reported in unusual strains of possible porcine or bovine origin from children with diarrhea. Recent reports suggested that G9 RVA may have been introduced from swine to human populations involving gene reassortment events. The observation that some of the RVA genotypes from swine in Italy were similar to viruses characterized in children underlines the importance of animal RVA surveillance, to clarify and monitor the role of animals as genetic reservoirs of emerging RVA strains pathogenic for humans.

  9. Bitis gabonica (Gaboon viper) snake venom gland: toward a catalog for the full- length transcripts (cDNA) and proteins

    PubMed Central

    Francischetti, Ivo M. B.; My-Pham, Van; Harrison, Jim; Garfield, Mark K.; Ribeiro, José M. C.

    2010-01-01

    The venom gland of the snake Bitis gabonica (Gaboon viper) was used for the first time to construct a unidirectional cDNA phage library followed by high-throughput sequencing and bioinformatic analysis. Hundreds of cDNAs were obtained and clustered into contigs. We found mostly novel full-length cDNA coding for metalloproteases (P-II and P-III classes), Lys49-phospholipase A2, serine proteases with essential mutations in the active site, Kunitz protease inhibitors, several C-type lectins, bradykinin-potentiating peptide, vascular endothelial growth factor, nucleotidases and nucleases, nerve growth factor, and L-amino acid oxidases. Two new members of the recently described short coding region family of disintegrin, displaying RGD and MLD motifs are reported. In addition, we have identified for the first time a cytokine-like molecule and a multi-Kunitz protease inhibitor in snake venoms. The CLUSTAL alignment and the unrooted cladograms for selected families of B. gabonica venom proteins are also presented. A significant number of sequences were devoid of database matches, suggesting that their biologic function remains to be identified. This paper also reports the N-terminus of the 15 most abundant venom proteins and the sequences matching their corresponding transcripts. The electronic version of this manuscript, available on request, contains spreadsheets with hyperlinks to FASTA-formatted files for each contig and the best match to the GenBank and Conserved Domain Databases, in addition to CLUSTAL alignments of each contig. We have thus generated a comprehensive catalog of the B. gabonica venom gland, containing for each secreted protein: i) the predicted molecular weight, ii) the predicted isoelectric point, iii) the accession number, and iv) the putative function. The role of these molecules is discussed in the context of the envenomation caused by the Gaboon viper. PMID:15276202

  10. EAVK "segment c" sequence confers Ca(2+)-dependent changes to the kinetics of full length human Ano1.

    PubMed

    Strege, Peter R; Gibbons, Simon J; Mazzone, Amelia; Bernard, Cheryl E; Beyder, Arthur; Farrugia, Gianrico

    2017-03-23

    Anoctamin1 (Ano1, TMEM16A) is a calcium-activated chloride channel specifically expressed in interstitial cells of Cajal (ICC) of the gastrointestinal (GI) tract muscularis propria. Ano1 is necessary for normal electrical slow waves and ICC proliferation. The full length human Ano1 sequence includes an additional exon, exon "0," at the N-terminus. Ano1 with exon "0" (Ano1(0)) had a lower EC50 for intracellular calcium ([Ca(2+)]i) and faster chloride current (ICl) kinetics. The Ano1 alternative splice variant with segment "c" encoding exon 13 expresses on the first intracellular loop four additional amino acid residues, EAVK, which alter ICl at low [Ca(2+)]i Exon 13 is expressed in 75-100% of Ano1 transcripts in most human tissues but only 25% in human stomach. Our aim was to determine the effect of EAVK deletion on Ano1(0) ICl parameters. By voltage-clamp electrophysiology, we examined ICl in HEK293 cells transiently expressing Ano1(0) with or without the EAVK sequence (Ano1(0)ΔEAVK). The EC50 values of activating and deactivating ICl for [Ca(2+)]i was 438±7 and 493±9 nM for Ano1(0) but higher for Ano1(0)ΔEAVK at 746±47 and 761±26 nM, respectively. Meanwhile, the EC50 values for the ratio of instantaneous to steady-state ICl were not different between variants. Congruently, the time constant of activation was slower for Ano1(0)ΔEAVK than Ano1(0) currents at intermediate [Ca(2+)]i These results suggest that EAVK decreases the calcium sensitivity of Ano1(0) current activation and deactivation by slowing activation kinetics. Differential expression of EAVK in human stomach may function as a switch to increase sensitivity to [Ca(2+)]i via faster gating of Ano1.

  11. Cloning and functional characterization of the ovine Hormone Sensitive Lipase (HSL) full-length cDNAs: an integrated approach.

    PubMed

    Lampidonis, Antonis D; Argyrokastritis, Alexandros; Stravopodis, Dimitrios J; Voutsinas, Gerassimos E; Ntouroupi, Triantafyllia G; Margaritis, Lukas H; Bizelis, Iosif; Rogdakis, Emmanuel

    2008-06-15

    Hormone Sensitive Lipase (HSL) is a highly regulated enzyme that mediates lipolysis in adipocytes. HSL enzymatic activity is increased by adrenergic agonists, such as catecholamines and glucagons, which induce cyclic AMP (cAMP) intracellular production, subsequently followed by the activation of Protein Kinase A (PKA) and its downstream signalling cascade reactions. Since HSL constitutes the key enzyme in the regulation of lipid stores and the only enzyme being subjected to hormonal regulation [in terms of the recently identified Adipose Triglyceride Lipase (ATGL)], the ovine Hormone Sensitive Lipase (ovHSL) full-length cDNA clones were isolated, using a Polymerase Chain Reaction-based (PCR) strategy. The two isolated isoforms ovHSL-A and ovHSL-B contain two highly homologous Open Reading Frame (ORF) regions of 2.089 Kb and 2.086 Kb, respectively, the latter having been missed the 688th triplet coding for glutamine (DeltaQ(688)). The putative 695 and 694 amino acid respective sequences bear strong homologies with other HSL protein family members. Southern blotting analysis revealed that HSL is represented as a single copy gene in the ovine genome, while Reverse Transcription-PCR (RT-PCR) approaches unambiguously dictated its variable transcriptional expression profile in the different tissues examined. Interestingly, as undoubtedly corroborated by both RT-PCR and Western blotting analysis, ovHSL gene expression is notably enhanced in the adipose tissue during the fasting period, when lipolysis is highly increased in ruminant species. Based on the crystal structure of an Archaeoglobus fulgidus enzyme, a three-dimensional (3D) molecular model of the ovHSL putative catalytic domain was constructed, thus providing an inchoative insight into understanding the enzymatic activity and functional regulation mechanisms of the ruminant HSL gene product(s).

  12. Innocuous full-length botulinum neurotoxin targets and promotes the expression of lentiviral vectors in central and autonomic neurons.

    PubMed

    O'Leary, V B; Ovsepian, S V; Raghunath, A; Huo, Q; Lawrence, G W; Smith, L; Dolly, J O

    2011-07-01

    Fragments of botulinum neurotoxin (BoNT) have been explored as potential targeting moieties and carriers of biomolecules into neurons, although with lower binding and translocation efficiency compared with intact proteins. This study exploits a detoxified recombinant form of full-length BoNT/B (BoTIM/B) fused with core streptavidin (CS-BoTIM/B) for lentiviral targeting to central and autonomic neurons. CS-BoTIM/B underwent an activity-dependent entry into cultured spinal cord neurons. Coupling CS-BoTIM/B to biotinylated lentivirus-encoding green fluorescent protein (GFP) endowed considerable neuron selectivity to the vector as evident from the preferential expression of the reporter in neurons co-cultured with skeletal muscle cells. CS-BoTIM/B-guided lentiviral transduction with the expression of a SNARE protein, SNAP-25 (S25), rendered non-susceptible to proteolysis by three BoNT serotypes, yielded a sizable decrease in cleaved S25 upon exposure of spinal cord neurons to these toxins. This was accompanied by synaptic transmission being spared from blockade by BoNT/A or BoNT/E, reflecting adequate translation and functional competence of recombinant multi-toxin-resistant S25. The augmented neurotropism conveyed on the lentivirus by CS-BoTIM/B was also demonstrated in vivo through enhanced expression of a reporter in intramural ganglionic neurons in the rat trachea, after injection of the targeted GFP-encoding lentivirus. Thus, a novel and realistic prospect for gene therapy of peripheral neuropathies is offered in this study through lentiviral targeting to neurons by CS-BoTIM/B.

  13. Comparison of Newly Assembled Full Length HIV-1 Integrase With Prototype Foamy Virus Integrase: Structure-Function Prospective

    PubMed Central

    Dayer, Mohammad Reza

    2016-01-01

    Background Drug design against human immunodeficiency virus type 1 (HIV-1) integrase through its mechanistic study is of great interest in the area in biological research. The main obstacle in this area is the absence of the full-length crystal structure for HIV-1 integrase to be used as a model. A complete structure, similar to HIV-1 of a prototype foamy virus integrase in complex with DNA, including all conservative residues, is available and has been extensively used in recent investigations. Objectives The aim of this study was to determine whether the above model is precisely representative of HIV-1 integrase. This would critically determine the success of any designed drug using the model in deactivation of integrase and AIDS treatment. Materials and Methods Primarily, a new structure for HIV-1 was constructed, using a crystal structure of prototype foamy virus as the starting structure. The constructed structure of HIV-1 integrase was simultaneously simulated with a prototype foamy virus integrase on a separate occasion. Results Our results indicate that the HIV-1 system behaves differently from the prototype foamy virus in terms of folding, hydration, hydrophobicity of binding site and stability. Conclusions Based on our findings, we can conclude that HIV-1 integrase is vastly different from the prototype foamy virus integrase and does not resemble it, and the modeling output of the prototype foamy virus simulations could not be simply generalized to HIV-1 integrase. Therefore, our HIV-1 model seems to be more representative and more useful for future research. PMID:27540450

  14. Characterizing solar-type stars from full-length Kepler data sets using the Asteroseismic Modeling Portal

    NASA Astrophysics Data System (ADS)

    Creevey, O. L.; Metcalfe, T. S.; Schultheis, M.; Salabert, D.; Bazot, M.; Thévenin, F.; Mathur, S.; Xu, H.; García, R. A.

    2017-05-01

    The Kepler space telescope yielded unprecedented data for the study of solar-like oscillations in other stars. The large samples of multi-year observations posed an enormous data analysis challenge that has only recently been surmounted. Asteroseismic modeling has become more sophisticated over time, with better methods gradually developing alongside the extended observations and improved data analysis techniques. We apply the latest version of the Asteroseismic Modeling Portal (AMP) to the full-length Kepler data sets for 57 stars, comprising planetary hosts, binaries, solar-analogs, active stars, and for validation purposes, the Sun. From an analysis of the derived stellar properties for the full sample, we identify a variation of the mixing-length parameter with atmospheric properties. We also derive a linear relation between the stellar age and a characteristic frequency separation ratio. In addition, we find that the empirical correction for surface effects suggested by Kjeldsen and coworkers is adequate for solar-type stars that are not much hotter (Teff≲6200 K) or significantly more evolved (log g≳4.2, ⟨ Δν ⟩≳80 μHz) than the Sun. Precise parallaxes from the Gaia mission and future observations from TESS and PLATO promise to improve the reliability of stellar properties derived from asteroseismology. Tables A.1-A.3 are also available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (http://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/601/A67

  15. Comparison of Next-Generation Sequencing Technologies for Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes

    PubMed Central

    Thomson, Emma; Ip, Camilla L. C.; Badhan, Anjna; Christiansen, Mette T.; Adamson, Walt; Ansari, M. Azim; Breuer, Judith; Brown, Anthony; Bowden, Rory; Bonsall, David; Da Silva Filipe, Ana; Hinds, Chris; Hudson, Emma; Klenerman, Paul; Lythgow, Kieren; Mbisa, Jean L.; McLauchlan, John; Myers, Richard; Piazza, Paolo; Roy, Sunando; Trebes, Amy; Sreenu, Vattipally B.; Witteveldt, Jeroen; Simmonds, Peter

    2016-01-01

    Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance. PMID:27385709

  16. Computational Study on Full-length Human Ku70 with Double Stranded DNA: Dynamics, Interactions and Functional Implications

    NASA Technical Reports Server (NTRS)

    Hu, Shaowen; Cucinotta, Francis A.

    2009-01-01

    The Ku70/80 heterodimer is the first repair protein in the initial binding of double-strand break (DSB) ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. In this study we constructed a full-length human Ku70 structure based on its crystal structure, and performed 20 ns conventional molecular dynamic (CMD) simulations on this protein and several other complexes with short DNA duplexes of different sequences. The trajectories of these simulations indicated that, without the topological support of Ku80, the residues in the bridge and C-terminal arm of Ku70 are more flexible than other experimentally identified domains. We studied the two missing loops in the crystal structure and predicted that they are also very flexible. Simulations revealed that they make an important contribution to the Ku70 interaction with DNA. Dislocation of the previously studied SAP domain was observed in several systems, implying its role in DNA binding. Targeted molecular dynamic (TMD) simulation was also performed for one system with a far-away 14bp DNA duplex. The TMD trajectory and energetic analysis disclosed detailed interactions of the DNA-binding residues during the DNA dislocation, and revealed a possible conformational transition for a DSB end when encountering Ku70 in solution. Compared to experimentally based analysis, this study identified more detailed interactions between DNA and Ku70. Free energy analysis indicated Ku70 alone is able to bind DNA with relatively high affinity, with consistent contributions from various domains of Ku70 in different systems. The functional implications of these domains in the processes of Ku heterodimerization and DNA damage recognition and repair can be characterized in detail based upon this analysis.

  17. Quench propagation study for the BNL-built, full-length, 50mm aperture SSC model dipoles

    SciTech Connect

    Muratore, J.; Anerella, M.; Cottingham, G.

    1993-09-01

    As part of the program to build and test SSC 50mm aperture prototype dipole magnets, a series of seven full-length dipoles were built and tested at BNL. Important part of the testing program was the study of quench propagation velocity and hot spot temperature over a range of experimental conditions in order to characterize the safety of the conductor during quenches experienced under different circumstances. Such studies are important tools in design, implementation, and verification of quench protection strategies in superconducting accelerator magnets. This investigation was facilitated by artificially inducing quenches under controlled experimental conditions with spot heaters placed at carefully chosen locations on the magnet coils. Such studies were done as part of the 15m-long magnet test program and were performed on five of the magnets in the series. All were equipped with spot heaters on an inner coil, and two of these also had spot heaters on an outer coil. Therefore, in addition to the studies in the inner coils, it was also possible to study quench propagation in the outer coils, where slower quench velocities and higher conductor temperatures are expected, in comparison to that in the inner coils. In spontaneous quenches, where there may be no voltage taps, it is not possible to measure the conductor hot spot temperature. It is straightforward to measure the number of MIITs generated, since only the magnet current and voltage need be measured. The concept of MIITs then becomes a valuable diagnostic tool which can characterize the temperature behavior of a conductor during quench and can be used to determine limits for safe operation of the coil. With spot heaters placed at known locations and closely bracketed by voltage taps, hot spot temperature can be measured. Research such as is described in this paper is therefore important in order to determine the validity of the MIITs approach and to establish a correlation between temperature and MIITs.

  18. Computational Study on Full-length Human Ku70 with Double Stranded DNA: Dynamics, Interactions and Functional Implications

    NASA Technical Reports Server (NTRS)

    Hu, Shaowen; Cucinotta, Francis A.

    2009-01-01

    The Ku70/80 heterodimer is the first repair protein in the initial binding of double-strand break (DSB) ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. In this study we constructed a full-length human Ku70 structure based on its crystal structure, and performed 20 ns conventional molecular dynamic (CMD) simulations on this protein and several other complexes with short DNA duplexes of different sequences. The trajectories of these simulations indicated that, without the topological support of Ku80, the residues in the bridge and C-terminal arm of Ku70 are more flexible than other experimentally identified domains. We studied the two missing loops in the crystal structure and predicted that they are also very flexible. Simulations revealed that they make an important contribution to the Ku70 interaction with DNA. Dislocation of the previously studied SAP domain was observed in several systems, implying its role in DNA binding. Targeted molecular dynamic (TMD) simulation was also performed for one system with a far-away 14bp DNA duplex. The TMD trajectory and energetic analysis disclosed detailed interactions of the DNA-binding residues during the DNA dislocation, and revealed a possible conformational transition for a DSB end when encountering Ku70 in solution. Compared to experimentally based analysis, this study identified more detailed interactions between DNA and Ku70. Free energy analysis indicated Ku70 alone is able to bind DNA with relatively high affinity, with consistent contributions from various domains of Ku70 in different systems. The functional implications of these domains in the processes of Ku heterodimerization and DNA damage recognition and repair can be characterized in detail based upon this analysis.

  19. A simple strategy for the purification of native recombinant full-length human RPL10 protein from inclusion bodies.

    PubMed

    Pereira, Larissa M; Silva, Luana R; Alves, Joseane F; Marin, Nélida; Silva, Flavio Sousa; Morganti, Ligia; Silva, Ismael D C G; Affonso, Regina

    2014-09-01

    The L10 ribosomal protein (RPL10) plays a role in the binding of the 60 S and 40 S ribosomal subunits and in mRNA translation. The evidence indicates that RPL10 also has multiple extra-ribosomal functions, including tumor suppression. Recently, the presence of RPL10 in prostate and ovarian cancers was evaluated, and it was demonstrated to be associated with autistic disorders and premature ovarian failure. In the present work, we successfully cloned and expressed full-length human RPL10 (hRPL10) protein and isolated inclusion bodies containing this protein that had formed under mild growth conditions. The culture produced 376mg of hRPL10 protein per liter of induced bacterial culture, of which 102.4mg was present in the soluble fraction, and 25.6mg was recovered at approximately 94% purity. These results were obtained using a two-step process of non-denaturing protein extraction from pelleted inclusion bodies. We studied the characteristics of this protein using circular dichroism spectroscopy and by monitoring the changes induced by the presence or absence of zinc ions using fluorescence spectrometry. The results demonstrated that the protein obtained using these non-conventional methods retained its secondary and tertiary structure. The conformational changes induced by the incorporation of zinc suggested that this protein could interact with Jun or the SH3 domain of c-yes. The results suggested that the strategy used to obtain hRPL10 is simple and could be applied to obtaining other proteins that are susceptible to degradation. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Isolation and characterization of a full length cDNA for dentatorubral-pallidoluysian atrophy (DRPLA) gene

    SciTech Connect

    Oyake, M.; Onodera, O.; Ikeuchi, T.

    1994-09-01

    Hereditary dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant spinocerebellar degeneration characterized by anticipation and variable combination of symptoms including myoclonus, epilepsy, cerebellar ataxia, choleoathetosis, and dementia. Recently, we discovered that DRPLA is caused by unstable expansion of a CAG repeat of a B37 gene on chromosome 12. To characterize functions of the DRPLA gene product, we isolated several cDNA clones for the DRPLA gene from human adult and fetus brain cDNA libraries, using an oligonucleotide flanking the CAG repeat. The cDNA spans 4247 bp in length and there is only an open reading frame coding for 986 amino acids. The CAG repeat, which is expanded in DRPLA, is located 291 bp downstream from the initiation methionine and encodes a polyglutamine tract. The deduced amino acid sequence from amino acids residues 582 to 707 has a high homology to published human hippocampus derived expressed sequence (M78755) located at chromosome 1p (63.8% identity), and 3{prime}-untranslated region of the DRPLA cDNA revealed homology to the mouse small nuclear RNA U7 gene (X54165). Northern blot analysis revealed a 4.7 knt transcript which is widely expressed in various tissues including heart, lung, kidney, placenta, skeletal muscle, and brain. In human adult brain, the transcript was broadly expressed including amygdala, caudate nucleus, corpus callosum, hippocampus, hypothalamus, substantia nigra, subthalamic nucleus and thalamus, and was not specific to the dentatorubral-pallidoluysian system. The availability of a full length cDNA will be highly useful for analyzing the pathogenesis of this unique neurodegenerative disease as well as for analyzing other CAG repeat related neurodegenerative diseases.

  1. Structure and Function of the First Full-Length Murein Peptide Ligase (Mpl) Cell Wall Recycling Protein

    PubMed Central

    Das, Debanu; Hervé, Mireille; Feuerhelm, Julie; Farr, Carol L.; Chiu, Hsiu-Ju; Elsliger, Marc-André; Knuth, Mark W.; Klock, Heath E.; Miller, Mitchell D.; Godzik, Adam; Lesley, Scott A.; Deacon, Ashley M.; Mengin-Lecreulx, Dominique; Wilson, Ian A.

    2011-01-01

    Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In Gram-negative bacteria, ∼30–60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships. PMID:21445265

  2. The Photoinitiated Reaction Pathway of Full-length Cyanobacteriochrome Tlr0924 Monitored Over 12 Orders of Magnitude*

    PubMed Central

    Hauck, Anna F. E.; Hardman, Samantha J. O.; Kutta, Roger J.; Greetham, Gregory M.; Heyes, Derren J.; Scrutton, Nigel S.

    2014-01-01

    The coupling of photochemistry to protein chemical and structural change is crucial to biological light-activated signaling mechanisms. This is typified by cyanobacteriochromes (CBCRs), members of the phytochrome superfamily of photoreceptors that exhibit a high degree of spectral diversity, collectively spanning the entire visible spectrum. CBCRs utilize a basic E/Z isomerization of the bilin chromophore as the primary step in their photocycle, which consists of reversible photoconversion between two photostates. Despite intense interest in these photoreceptors as signal transduction modules a complete description of light-activated chemical and structural changes has not been reported. The CBCR Tlr0924 contains both phycocyanobilin and phycoviolobilin chromophores, and these two species photoisomerize in parallel via spectrally and kinetically equivalent intermediates before the second step of the photoreaction where the reaction pathways diverge, the loss of a thioether linkage to a conserved cysteine residue occurs, and the phycocyanobilin reaction terminates in a red-absorbing state, whereas the phycoviolobilin reaction proceeds more rapidly to a final green-absorbing state. Here time-resolved visible transient absorption spectroscopy (femtosecond to second) has been used, in conjunction with time-resolved IR spectroscopy (femtosecond to nanosecond) and cryotrapping techniques, to follow the entire photoconversion of the blue-absorbing states to the green- and red-absorbing states of the full-length form of Tlr0924 CBCR. Our analysis shows that Tlr0924 undergoes an unprecedented long photoreaction that spans from picoseconds to seconds. We show that the thermally driven, long timescale changes are less complex than those reported for the red/far-red photocycles of the related phytochrome photoreceptors. PMID:24817121

  3. Full-length and internally deleted forms of interleukin-7 are present in horse (Equus caballus) lymph node tissue.

    PubMed

    Cook, R Frank; Cook, Sheila J; Even, Deborah L; Schaffer, Catherine; Issel, Charles J

    2008-09-15

    Horse IL-7 (HIL-7) cDNA was isolated from adult lymph node tissue by reverse transcription polymerase chain reaction (RT-PCR) using oligonucleotide primers based on horse genomic sequences (The Broad Institute). In addition, to the full-length (FL) 531bp reading frame encoding 176 amino acids, shorter open-reading frames of 477, 396 and 264bp were also amplified. Nucleotide sequence analysis of these RT-PCR products demonstrated they were homologous except the shorter species were missing internal sequences consistent with multiple RNA splicing events. Consequently, the shorter open-reading frames were re-named splice variant (SV) 1 (477bp), 2 (396bp) and 3 (264bp). Organization of the horse IL-7 is predicted to be similar to that in humans with exon 5 deleted from SV1, exons 3, 5 deleted from SV2 and exons 3, 4, and 5 missing from SV3. Each of these open-reading frames has the potential to be stably expressed as demonstrated using a polyclonal antiserum against human IL-7 to visualize the protein products produced when the FL HIL-7 and each SV were molecularly cloned into pCI and transfected in brefeldin A treated HEK 293 cells. Furthermore, addition of supernatants to horse PBMC from HEK cells transfected (without brefeldin A treatment) with pCI HIL-7 FL, pCI HIL-7SV1, pCI HIL-7SV2 and pCI IL-7SV3 all induced significant incorporation of (3)H-thymidine in the presence of sub-stimulatory amounts of concanavalin A compared to supernatants from mock-transfected cells. Therefore, all isoforms of horse IL-7 described in this report have the ability to stimulate proliferative responses in ex vivo horse PBMC cultures.

  4. Aph-1 associates directly with full-length and C-terminal fragments of gamma-secretase substrates.

    PubMed

    Chen, Allen C; Guo, Lucie Y; Ostaszewski, Beth L; Selkoe, Dennis J; LaVoie, Matthew J

    2010-04-09

    Gamma-secretase is a ubiquitous, multiprotein enzyme composed of presenilin, nicastrin, Aph-1, and Pen-2. It mediates the intramembrane proteolysis of many type 1 proteins, plays an essential role in numerous signaling pathways, and helps drive the pathogenesis of Alzheimer disease by excising the hydrophobic, aggregation-prone amyloid beta-peptide from the beta-amyloid precursor protein. A central unresolved question is how its many substrates bind and enter the gamma-secretase complex. Here, we provide evidence that both the beta-amyloid precursor protein holoprotein and its C-terminal fragments, the immediate substrates of gamma-secretase, can associate with Aph-1 at overexpressed as well as endogenous protein levels. This association was observed using bi-directional co-immunoprecipitation in multiple systems and detergent conditions, and an beta-amyloid precursor protein-Aph-1 complex was specifically isolated following in situ cross-linking in living cells. In addition, another endogenous canonical gamma-substrate, Jagged, showed association of both its full-length and C-terminal fragment forms with Aph-1. We were also able to demonstrate that this interaction with substrates was conserved across the multiple isoforms of Aph-1 (beta, alphaS, and alphaL), as they were all able to bind beta-amyloid precursor protein with similar affinity. Finally, two highly conserved intramembrane histidines (His-171 and His-197) within Aph-1, which were recently shown to be important for gamma-secretase activity, are required for efficient binding of substrates. Taken together, our data suggest a dominant role for Aph-1 in interacting with gamma-secretase substrates prior to their processing by the proteolytic complex.

  5. Analysis of SERPING1 expression on hereditary angioedema patients: quantitative analysis of full-length and exon 3 splicing variants.

    PubMed

    de la Cruz, Rocío Mena; López-Lera, Alberto; López-Trascasa, Margarita

    2012-01-30

    Hereditary angioedema (HAE) due to C1-inhibitor (C1-Inh) deficiency is an autosomal dominant disease caused by mutations in the SERPING1 locus. According to protein levels in plasma, two HAE phenotypes have been described: Type I, with low circulating protein levels in plasma, and Type II, where the protein is present but dysfunctional. Although more than 200 mutations have been described to date, studies on the molecular basis of this autosomic dominant trait are scarce. Previous studies demonstrated that C1-Inh mRNA expression was decreased in HAE patients. Herein, we have confirmed these findings in a large series of Spanish patients. Moreover, when our data were analyzed taking into account the type of mutation carried by the patient (i.e., missense, frameshift,…), significant differences were amongst the control, nonsense and splicing mutations groups (P<0.05). By opposite, no differences in C1-Inh mRNA expression were found between the control and HAE Type II groups, nor between treated and untreated patients groups, although a significant difference was observed between controls and untreated HAE Type I patients. An alternative splicing event has been described in the SERPING1 locus resulting in two different transcripts: the full-length and a shorter variant with skipping of exon 3. In order to investigate a possible role for this splicing in HAE, we quantified both mRNA variants in a series of 28 patients. No statistical differences were found in the expression of both variants between controls and patients when compared. However, a separate analysis considering each type of mutation evidenced a significant decrease (P: 0.0156) in the expression of the exon 3 skipping variant in those HAE Type I patients carrying nonsense mutations. Besides, median of the full variant's copy number was statistically decreased on the splicing group when compared with either stop and/or missense groups. The results of these studies provide new data about C1 inhibitor

  6. BAY 81-8973, a full-length recombinant factor VIII: results from an International comparative laboratory field study.

    PubMed

    Kitchen, S; Beckmann, H; Katterle, Y; Bruns, S; Tseneklidou-Stoeter, D; Maas Enriquez, M

    2016-05-01

    BAY 81-8973 is a full-length, unmodified, recombinant human factor VIII (FVIII) with the same primary amino acid sequence as sucrose-formulated recombinant FVIII but produced with certain more advanced manufacturing technologies. This global laboratory study evaluated variability in measurement of BAY 81-8973 using one-stage and chromogenic assays compared with antihaemophilic factor (recombinant) plasma/albumin-free method (rAHF-PFM; Advate(®) ) under assay conditions routinely used in clinical laboratories. BAY 81-8973 or rAHF-PFM was spiked into FVIII-deficient plasma at 0.043 (low), 0.375 (medium) and 0.865 (normal) IU mL(-1) . Participating laboratories analysed blinded samples and normal plasma in triplicate using their routine assay, reagents and standards. Results were analysed for intra- and interlaboratory variability. Forty-one laboratories in 11 countries participated in the study. One-stage assay and chromogenic assays were used by 40 and 10 laboratories, respectively; 9 laboratories used both assays. Intralaboratory variability was <11% for both assays and both products at all concentrations. Interlaboratory variability was highest at the low concentration in the chromogenic and one-stage assay for BAY 81-8973 (60.0% and 33.7%, respectively) and rAHF-PFM (51.0% and 30.8%) and was lowest at the normal concentration (BAY 81-8973, 5.4% and 14.0%; rAHF-PFM, 5.8% and 12.4%), which was similar to the plasma control (6.6% and 10.3%). The chromogenic:one-stage assay ratio ranged from 0.95 (low concentration) to 1.10 (normal concentration) for BAY 81-8973 and 0.96-1.18 for rAHF-PFM. BAY 81-8973 can be accurately measured in plasma using the one-stage and chromogenic assays routinely used in clinical laboratories without a product-specific standard. © 2016 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

  7. Potency of Full- Length MGF to Induce Maximal Activation of the IGF-I R Is Similar to Recombinant Human IGF-I at High Equimolar Concentrations

    PubMed Central

    Janssen, Joseph A. M. J. L.; Hofland, Leo J.; Strasburger, Christian J.; van den Dungen, Elisabeth S. R.; Thevis, Mario

    2016-01-01

    Aims To compare full-length mechano growth factor (full-length MGF) with human recombinant insulin-like growth factor-I (IGF-I) and human recombinant insulin (HI) in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor (IR-A) and the human insulin receptor-B (IR-B), respectively. In addition, we tested the stimulatory activity of human MGF and its stabilized analog Goldspink-MGF on the IGF-IR. Methods The effects of full-length MGF, IGF-I, human mechano growth factor (MGF), Goldspink-MGF and HI were compared using kinase specific receptor activation (KIRA) bioassays specific for IGF-I, IR-A or IR-B, respectively. These assays quantify activity by measuring auto-phosphorylation of the receptor upon ligand binding. Results IGF-IR: At high equimolar concentrations maximal IGF-IR stimulating effects generated by full-length MGF were similar to that of IGF-I (89-fold vs. 77-fold, respectively). However, EC50 values of IGF-I and full-length MGF for the IGF-I receptor were 0.86 nmol/L (95% CI 0.69–1.07) and 7.83 nmol/L (95% CI: 4.87–12.58), respectively. No IGF-IR activation was observed by human MGF and Goldspink-MGF, respectively. IR-A/IR-B: At high equimolar concentrations similar maximal IR-A stimulating effects were observed for full -length MGF and HI, but maximal IR-B stimulation achieved by full -length MGF was stronger than that by HI (292-fold vs. 98-fold). EC50 values of HI and full-length MGF for the IR-A were 1.13 nmol/L (95% CI 0.69–1.84) and 73.11 nmol/L (42.87–124.69), respectively; for IR-B these values were 1.28 nmol/L (95% CI 0.64–2.57) and 35.10 nmol/L (95% 17.52–70.33), respectively. Conclusions Full-length MGF directly stimulates the IGF-IR. Despite a higher EC50 concentration, at high equimolar concentrations full-length MGF showed a similar maximal potency to activate the IGF-IR as compared to IGF-I. Further research is needed to understand the actions of full-length MGF in vivo and to define the

  8. Identification and isolation of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from guarana (Paullinia cupana).

    PubMed

    Figueirêdo, L C; Faria-Campos, A C; Astolfi-Filho, S; Azevedo, J L

    2011-06-21

    The current intense production of biological data, generated by sequencing techniques, has created an ever-growing volume of unanalyzed data. We reevaluated data produced by the guarana (Paullinia cupana) transcriptome sequencing project to identify cDNA clones with complete coding sequences (full-length clones) and complete sequences of genes of biotechnological interest, contributing to the knowledge of biological characteristics of this organism. We analyzed 15,490 ESTs of guarana in search of clones with complete coding regions. A total of 12,402 sequences were analyzed using BLAST, and 4697 full-length clones were identified, responsible for the production of 2297 different proteins. Eighty-four clones were identified as full-length for N-methyltransferase and 18 were sequenced in both directions to obtain the complete genome sequence, and confirm the search made in silico for full-length clones. Phylogenetic analyses were made with the complete genome sequences of three clones, which showed only 0.017% dissimilarity; these are phylogenetically close to the caffeine synthase of Theobroma cacao. The search for full-length clones allowed the identification of numerous clones that had the complete coding region, demonstrating this to be an efficient and useful tool in the process of biological data mining. The sequencing of the complete coding region of identified full-length clones corroborated the data from the in silico search, strengthening its efficiency and utility.

  9. Large-scale collection and analysis of full-length cDNAs from Brachypodium distachyon and integration with Pooideae sequence resources.

    PubMed

    Mochida, Keiichi; Uehara-Yamaguchi, Yukiko; Takahashi, Fuminori; Yoshida, Takuhiro; Sakurai, Tetsuya; Shinozaki, Kazuo

    2013-01-01

    A comprehensive collection of full-length cDNAs is essential for correct structural gene annotation and functional analyses of genes. We constructed a mixed full-length cDNA library from 21 different tissues of Brachypodium distachyon Bd21, and obtained 78,163 high quality expressed sequence tags (ESTs) from both ends of ca. 40,000 clones (including 16,079 contigs). We updated gene structure annotations of Brachypodium genes based on full-length cDNA sequences in comparison with the latest publicly available annotations. About 10,000 non-redundant gene models were supported by full-length cDNAs; ca. 6,000 showed some transcription unit modifications. We also found ca. 580 novel gene models, including 362 newly identified in Bd21. Using the updated transcription start sites, we searched a total of 580 plant cis-motifs in the -3 kb promoter regions and determined a genome-wide Brachypodium promoter architecture. Furthermore, we integrated the Brachypodium full-length cDNAs and updated gene structures with available sequence resources in wheat and barley in a web-accessible database, the RIKEN Brachypodium FL cDNA database. The database represents a "one-stop" information resource for all genomic information in the Pooideae, facilitating functional analysis of genes in this model grass plant and seamless knowledge transfer to the Triticeae crops.

  10. Generation of a reliable full-length cDNA of infectiousTembusu virus using a PCR-based protocol.

    PubMed

    Liang, Te; Liu, Xiaoxiao; Cui, Shulin; Qu, Shenghua; Wang, Dan; Liu, Ning; Wang, Fumin; Ning, Kang; Zhang, Bing; Zhang, Dabing

    2016-02-02

    Full-length cDNA of Tembusu virus (TMUV) cloned in a plasmid has been found instable in bacterial hosts. Using a PCR-based protocol, we generated a stable full-length cDNA of TMUV. Different cDNA fragments of TMUV were amplified by reverse transcription (RT)-PCR, and cloned into plasmids. Fragmented cDNAs were amplified and assembled by fusion PCR to produce a full-length cDNA using the recombinant plasmids as templates. Subsequently, a full-length RNA was transcribed from the full-length cDNA in vitro and transfected into BHK-21 cells; infectious viral particles were rescued successfully. Following several passages in BKH-21 cells, the rescued virus was compared with the parental virus by genetic marker checks, growth curve determinations and animal experiments. These assays clearly demonstrated the genetic and biological stabilities of the rescued virus. The present work will be useful for future investigations on the molecular mechanisms involved in replication and pathogenesis of TMUV. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Original reverse transcription polymerase chain reaction method to obtain the full-length cDNA of rice tungro spherical virus.

    PubMed

    Perrin, Y; Hull, R

    1999-05-01

    A two-step reverse transcription reaction combined with long PCR was developed in order to obtain the full-length cDNA from the 12.2 kbp genomic RNA of rice tungro spherical virus. A first step reverse transcription, performed at 45 degrees C using a reverse transcriptase deprived of RNase H activity, allowed the synthesis of a nearly full-length cDNA of 11.7 kbp. A second step reaction, carried out at 65 degrees C using a thermostable polymerase, was necessary to destabilise secondary structures present at the 5' extremity of the RNA template which hampered the reverse transcription reaction in this region. The full-length cDNA obtained by the two-step reverse transcription was amplified successfully by long PCR and subsequently cloned into a plasmid vector. The cloned cDNA showed toxicity and proved to be unstable when amplified in E. coli.

  12. Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE).

    PubMed

    Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Watanabe, Kousuke; Emoto, Noriko; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2011-07-01

    Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.

  13. Detection of infectious viral particles in plant protoplasts inoculated with transcripts of full-length shallot virus X cDNA.

    PubMed

    Vishnichenko, V K; Zavriev, S K

    2001-01-01

    Flexible filamentous shallot virus X (ShVX) particles were detected in extracts of Beta vulgaris protoplasts inoculated with transcripts from a full-length ShVX cDNA. Extracts from ShVX-infected protoplast were infectious for ShVX-healthy shallot seedlings. Western blot analysis of inoculated plants revealed the accumulation of the ShVX coat protein, while electron microscopy confirmed the presence of ShVX virions. The results suggest that the in vitro RNA transcripts from full-length ShVX cDNA give rise to infectious viral particles.

  14. Cloning Full-Length cDNAs from Vascular Tissues and Cells by Rapid Amplification of cDNA Ends (RACE) and RT-PCR.

    PubMed

    Shen

    1999-01-01

    The isolation of full-length cDNAs remains a frequent task undertaken in many laboratories. A full-length cDNA is often desirable for one of the following purposes: 1) to complete the sequence of a partial cDNA cloned by library screenings or the yeast one- or two-hybrid system; 2) to derive the cDNA sequence encoding a protein, based on peptide sequences; 3) to obtain the sequence of a reported cDNA for functional analysis or expression studies; and 4) to define exon/intron boundaries of a cloned gene or determine transcription start site(s) of a promoter.

  15. General Lucius D. Clay: A Case Study of Strategic Leadership and Vision

    DTIC Science & Technology

    1991-04-05

    developmental positions that prepared him to create and implement a strategic vision and to lead the effort to establ ish post-war Germany. A review of mi l...military history and biporaph’ is invaluable in this regard. ( emphasis added) [I] It is in that spirit that this study of General Lucius D. Clay has...those general officers in positions as high as corps commanders. ;t this level the leader has less direct control and influence over subordinates. Work

  16. Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

    SciTech Connect

    Deymier, Martin J.; Claiborne, Daniel T.; Ende, Zachary; Ratner, Hannah K.; Kilembe, William; Hunter, Eric

    2014-11-15

    The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor.

  17. Full-length coding sequence for 12 bovine viral diarrhea virus isolates from persistently infected cattle in a feedyard in Kansas

    USDA-ARS?s Scientific Manuscript database

    We report here the full-length coding sequence of 12 bovine viral diarrhea virus (BVDV) isolates from persistently infected cattle from a feedyard in southwest Kansas, USA. These 12 genomes represent the three major genotypes (BVDV 1a, 1b, and 2a) of BVDV currently circulating in the United States....

  18. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    PubMed

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding.

  19. Genome-wide comparisons of phylogenetic similarities between partial genomic regions and the full-length genome in Hepatitis E virus genotyping.

    PubMed

    Wang, Shuai; Wei, Wei; Luo, Xuenong; Cai, Xuepeng

    2014-01-01

    Besides the complete genome, different partial genomic sequences of Hepatitis E virus (HEV) have been used in genotyping studies, making it difficult to compare the results based on them. No commonly agreed partial region for HEV genotyping has been determined. In this study, we used a statistical method to evaluate the phylogenetic performance of each partial genomic sequence from a genome wide, by comparisons of evolutionary distances between genomic regions and the full-length genomes of 101 HEV isolates to identify short genomic regions that can reproduce HEV genotype assignments based on full-length genomes. Several genomic regions, especially one genomic region at the 3'-terminal of the papain-like cysteine protease domain, were detected to have relatively high phylogenetic correlations with the full-length genome. Phylogenetic analyses confirmed the identical performances between these regions and the full-length genome in genotyping, in which the HEV isolates involved could be divided into reasonable genotypes. This analysis may be of value in developing a partial sequence-based consensus classification of HEV species.

  20. Integration of the full-length HPV16 genome in cervical cancer and Caski and Siha cell lines and the possible ways of HPV integration.

    PubMed

    Xu, Feng; Cao, Meng; Shi, Qinfeng; Chen, Hongwei; Wang, Yili; Li, Xu

    2015-04-01

    Integration of high-risk human papillomavirus (HPV) into the host genome is a key event for cervical carcinogenesis. Different methods have been used to explore the physical states of the HPV genome to reveal the mechanisms for malignant transformation of the infected cells. Consensus has been reached that, although variable portions of the HPV genome are deleted in the integrated HPV sequences, common disruption of the viral E2 gene has been demonstrated in different studies. The head-to-tail concatemers of the full-length HPV16 genome is another typical integration pattern of HPV16, typically found in Caski cell lines, but its prevalence in cervical cancer has never been tested. Here, by introducing a modified PCR, we identified this head-to-tail concatemers of full-length HPV genomes in advanced cervical cancer with HPV16 single positive. Our results show that more than half of the cases contain this integrated head-to-tail concatemers of full-length HPV16 genomes. Further studies in two cervical cell lines, Caski cells and Siha cells, revealed a correlation between the prevalence of the spliced variants of integrated HPV16 sequences and the full-length transcription of the integrated head-to-tail concatemers of the full-length HPV16 genome. Based on these results, we propose that HPV16 integrated into host cells by two mechanisms: one mechanism is shared by other DNA virus and cause integration of the head-to-tail concatemers of the viral genome; another is related to the reverse transcription process, which the integrated HPV sequence is generated by the reverse transcription of the viral mRNA.

  1. Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

    PubMed

    Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

    2014-02-01

    Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy.

  2. Identification of stress-tolerance-related transcription-factor genes via mini-scale Full-length cDNA Over-eXpressor (FOX) gene hunting system.

    PubMed

    Fujita, Miki; Mizukado, Saho; Fujita, Yasunari; Ichikawa, Takanari; Nakazawa, Miki; Seki, Motoaki; Matsui, Minami; Yamaguchi-Shinozaki, Kazuko; Shinozaki, Kazuo

    2007-12-14

    Recently, we developed a novel system known as Full-length cDNA Over-eXpressor (FOX) gene hunting [T. Ichikawa, M. Nakazawa, M. Kawashima, H. Iizumi, H. Kuroda, Y. Kondou, Y. Tsuhara, K. Suzuki, A. Ishikawa, M. Seki, M. Fujita, R. Motohashi, N. Nagata, T. Takagi, K. Shinozaki, M. Matsui, The FOX hunting system: an alternative gain-of-function gene hunting technique, Plant J. 48 (2006) 974-985], which involves the random overexpression of a normalized Arabidopsis full-length cDNA library. While our system allows large-scale collection of full-length cDNAs for gene discovery, we sought to downsize it to analyze a small pool of full-length cDNAs. As a model system, we focused on stress-inducible transcription factors. The full-length cDNAs of 43 stress-inducible transcription factors were mixed to create a transgenic plant library. We screened for salt-stress-resistant lines in the T1 generation and identified a number of salt-tolerant lines that harbored the same transgene (F39). F39 encodes a bZIP-type transcription factor that is identical to AtbZIP60, which is believed to be involved in the endoplasmic reticulum stress response. Microarray analysis revealed that a number of stress-inducible genes were up-regulated in the F39-overexpressing lines, suggesting that AtbZIP60 is involved in stress signal transduction. Thus, our mini-scale FOX system may be used to screen for genes with valuable functions, such as transcription factors, from a small pool of genes that show similar expression profiles.

  3. Do additional full-length radiographs of the humerus and forearm improve the decision making in children with supracondylar humerus fractures?

    PubMed

    Bloom, Tamir; Seigerman, Daniel A; Zhao, Caixia; Sabharwal, Sanjeev

    2016-09-01

    We sought to determine the diagnostic utility of additional full-length radiographs of the forearm and humerus for pediatric supracondylar humerus fractures. A pediatric orthopedic surgeon and a senior orthopedic resident individually reviewed the initial humerus, forearm, and elbow radiographs of 55 children with a supracondylar humerus fracture and recommended definitive treatment (operative vs. nonoperative) on the basis of the modified Gartland classification. Interobserver agreements for classification and the recommended treatment were highest for the elbow radiographs (weighted κ=0.92). All disagreements in the recommended treatment were in fractures classified as Gartland type I versus II fractures. Although two children (4%) had an ipsilateral distal forearm fracture, selective versus routine use of additional full-length radiographs in children with a supracondylar humerus fracture needs to be evaluated further.

  4. Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends.

    PubMed

    Schaefer, B C

    1995-05-20

    Rapid amplification of cDNA ends (RACE) is a polymerase chain reaction (PCR)-based technique which was developed to facilitate the cloning of full-length cDNA 5'- and 3'-ends after a partial cDNA sequence has been obtained by other methods. While RACE can yield complete sequences of cDNA ends in only a few days, the RACE procedure frequently results in the exclusive amplification of truncated cDNA ends, undermining efforts to generate full-length clones. Many investigators have suggested modifications to the RACE protocol to improve the effectiveness of the technique. Based on first-hand experience with RACE, a critical review of numerous published variations of the key steps in the RACE method is presented. Also included is a detailed, effective protocol based on RNA ligase-mediated RACE/reverse ligation-mediated PCR, as well as a demonstration of its utility.

  5. Complete sequence and development of a full-length infectious clone of an Ohio isolate of Maize dwarf mosaic virus (MDMV).

    PubMed

    Stewart, L R; Bouchard, R; Redinbaugh, M G; Meulia, T

    2012-05-01

    Maize dwarf mosaic virus (MDMV) is an important and widespread aphid-transmitted virus of maize. It is a member of the genus Potyvirus in the family Potyviridae with a monopartite (+) ssRNA genome. Here we report the complete genome sequence and construction and testing of infectious clones of an Ohio isolate of MDMV. Full-length MDMV cDNA was cloned into the vector pSPORT. Full-length cDNA PCR-amplified from the vector constructs were used as template for in vitro transcription, and transcripts were inoculated to maize seeds by vascular puncture inoculation. Plants inoculated by this procedure showed symptoms typical of MDMV infection, and infection was confirmed by RT-PCR and mechanical transmission to new plants.

  6. TypeLoader: A fast and efficient automated workflow for the annotation and submission of novel full-length HLA alleles.

    PubMed

    Surendranath, V; Albrecht, V; Hayhurst, J D; Schöne, B; Robinson, J; Marsh, S G E; Schmidt, A H; Lange, V

    2017-07-01

    Recent years have seen a rapid increase in the discovery of novel allelic variants of the human leukocyte antigen (HLA) genes. Commonly, only the exons encoding the peptide binding domains of novel HLA alleles are submitted. As a result, the IPD-IMGT/HLA Database lacks sequence information outside those regions for the majority of known alleles. This has implications for the application of the new sequencing technologies, which deliver sequence data often covering the complete gene. As these technologies simplify the characterization of the complete gene regions, it is desirable for novel alleles to be submitted as full-length sequences to the database. However, the manual annotation of full-length alleles and the generation of specific formats required by the sequence repositories is prone to error and time consuming. We have developed TypeLoader to address both these facets. With only the full-length sequence as a starting point, Typeloader performs automatic sequence annotation and subsequently handles all steps involved in preparing the specific formats for submission with very little manual intervention. TypeLoader is routinely used at the DKMS Life Science Lab and has aided in the successful submission of more than 900 novel HLA alleles as full-length sequences to the European Nucleotide Archive repository and the IPD-IMGT/HLA Database with a 95% reduction in the time spent on annotation and submission when compared with handling these processes manually. TypeLoader is implemented as a web application and can be easily installed and used on a standalone Linux desktop system or within a Linux client/server architecture. TypeLoader is downloadable from http://www.github.com/DKMS-LSL/typeloader. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. The Impact of Full-Length, Trimeric and Globular Adiponectin on Lipolysis in Subcutaneous and Visceral Adipocytes of Obese and Non-Obese Women.

    PubMed

    Wedellova, Zuzana; Kovacova, Zuzana; Tencerova, Michaela; Vedral, Tomas; Rossmeislova, Lenka; Siklova-Vitkova, Michaela; Stich, Vladimir; Polak, Jan

    2013-01-01

    Contribution of individual adiponectin isoforms to lipolysis regulation remains unknown. We investigated the impact of full-length, trimeric and globular adiponectin isoforms on spontaneous lipolysis in subcutaneous abdominal (SCAAT) and visceral adipose tissues (VAT) of obese and non-obese subjects. Furthermore, we explored the role of AMPK (5'-AMP-activated protein kinase) in adiponectin-dependent lipolysis regulation and expression of adiponectin receptors type 1 and 2 (AdipoR1 and AdipoR2) in SCAAT and VAT. Primary adipocytes isolated from SCAAT and VAT of obese and non-obese women were incubated with 20 µg/ml of: A) full-length adiponectin (physiological mixture of all adiponectin isoforms), B) trimeric adiponectin isoform or C) globular adiponectin isoform. Glycerol released into media was used as a marker of lipolysis. While full-length adiponectin inhibited lipolysis by 22% in non-obese SCAAT, globular isoform inhibited lipolysis by 27% in obese SCAAT. No effect of either isoform was detected in non-obese VAT, however trimeric isoform inhibited lipolysis by 21% in obese VAT (all p<0.05). Trimeric isoform induced Thr172 p-AMPK in differentiated preadipocytes from a non-obese donor, while globular isoform induced Ser79 p-ACC by 32% (p<0.05) and Ser565 p-HSL by 52% (p = 0.08) in differentiated preadipocytes from an obese donor. AdipoR2 expression was 17% and 37% higher than AdipoR1 in SCAAT of obese and non-obese groups and by 23% higher in VAT of obese subjects (all p<0.05). In conclusion, the anti-lipolytic effect of adiponectin isoforms is modified with obesity: while full-length adiponectin exerts anti-lipolytic action in non-obese SCAAT, globular and trimeric isoforms show anti-lipolytic activity in obese SCAAT and VAT, respectively.

  8. Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

    PubMed

    Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

    2010-05-07

    Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

  9. Cost-Effective Sequencing of Full-Length cDNA Clones Powered by a De Novo-Reference Hybrid Assembly

    PubMed Central

    Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka

    2010-01-01

    Background Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. Methodology We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence ∼800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. Conclusions The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only ∼US$3 per clone, demonstrating a significant advantage over previous approaches. PMID:20479877

  10. Investigating Microbial Eukaryotic Diversity from a Global Census: Insights from a Comparison of Pyrotag and Full-Length Sequences of 18S rRNA Genes

    PubMed Central

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

    2014-01-01

    Next-generation DNA sequencing (NGS) approaches are rapidly surpassing Sanger sequencing for characterizing the diversity of natural microbial communities. Despite this rapid transition, few comparisons exist between Sanger sequences and the generally much shorter reads of NGS. Operational taxonomic units (OTUs) derived from full-length (Sanger sequencing) and pyrotag (454 sequencing of the V9 hypervariable region) sequences of 18S rRNA genes from 10 global samples were analyzed in order to compare the resulting protistan community structures and species richness. Pyrotag OTUs called at 98% sequence similarity yielded numbers of OTUs that were similar overall to those for full-length sequences when the latter were called at 97% similarity. Singleton OTUs strongly influenced estimates of species richness but not the higher-level taxonomic composition of the community. The pyrotag and full-length sequence data sets had slightly different taxonomic compositions of rhizarians, stramenopiles, cryptophytes, and haptophytes, but the two data sets had similarly high compositions of alveolates. Pyrotag-based OTUs were often derived from sequences that mapped to multiple full-length OTUs at 100% similarity. Thus, pyrotags sequenced from a single hypervariable region might not be appropriate for establishing protistan species-level OTUs. However, nonmetric multidimensional scaling plots constructed with the two data sets yielded similar clusters, indicating that beta diversity analysis results were similar for the Sanger and NGS sequences. Short pyrotag sequences can provide holistic assessments of protistan communities, although care must be taken in interpreting the results. The longer reads (>500 bp) that are now becoming available through NGS should provide powerful tools for assessing the diversity of microbial eukaryotic assemblages. PMID:24814788

  11. Structure of the full-length insecticidal protein Cry1Ac reveals intriguing details of toxin packaging into in vivo formed crystals

    PubMed Central

    Evdokimov, Artem G; Moshiri, Farhad; Sturman, Eric J; Rydel, Timothy J; Zheng, Meiying; Seale, Jeffrey W; Franklin, Sonya

    2014-01-01

    For almost half a century, the structure of the full-length Bacillus thuringiensis (Bt) insecticidal protein Cry1Ac has eluded researchers, since Bt-derived crystals were first characterized in 1965. Having finally solved this structure we report intriguing details of the lattice-based interactions between the toxic core of the protein and the protoxin domains. The structure provides concrete evidence for the function of the protoxin as an enhancer of native crystal packing and stability. PMID:25139047

  12. Mass spectrometry defines the C-terminal dimerization domain and enables modeling of the structure of full-length OmpA

    PubMed Central

    Marcoux, Julien; Politis, Argyris; Rinehart, Dennis; Marshall, David P.; Wallace, Mark I.; Tamm, Lukas K.; Robinson, Carol V.

    2014-01-01

    SUMMARY The transmembrane domain of the Outer membrane protein A (OmpA) from Escherichia coli is an excellent model for structural and folding studies of β-barrel membrane proteins. However, full-length OmpA resists crystallographic efforts and the link between its function and tertiary structure remains controversial. Here we use site directed mutagenesis and mass spectrometry of different constructs of OmpA, released in the gas phase from detergent micelles, to define the minimal region encompassing the C-terminal dimer interface. Combining knowledge of the location of the dimeric interface with molecular modeling and ion mobility data allows us to propose a low-resolution model for the full-length OmpA dimer. Our model of the dimer is in remarkable agreement with experimental ion mobility data, with none of the unfolding or collapse observed for full-length monomeric OmpA, implying that dimer formation stabilises the overall structure and prevents collapse of the flexible linker that connects the two domains. PMID:24746938

  13. Bovine adenovirus type 3 virions cannot be rescued in vivo after full-length viral genome transfection in the absence of detectable polypeptide IX.

    PubMed

    Zhang, Peng; Xue, Qinghong; Ma, Jing; Ren, Jingjing; Xia, Shuili; Zhang, Lu; Wang, Wenbin; Tikoo, Suresh K; Du, Enqi

    2017-06-30

    Bovine adenovirus type 3 (BAdV3) is being used in the development of potential vehicles for gene therapy and vectored vaccine. To that end, a more comprehensive description of BAdV3 biology is essential. In this study, we focused on the role of pIX in BAdV3 virion rescue after full-length BAdV3 genome transfection. Initially, pIX deletion or initiation codon mutation abolished the production of progeny virions, which suggested that pIX was essential for the rescue of BAdV3 containing a full-length genome. Moreover, through transfection of a panel of pIX mutant BAdV3 genomes, we observed that the conserved N-terminus and the putative leucine zipper element (PLZP) were essential for virion rescue, whereas the C-terminus following the coiled-coil domain was non-essential. In addition, swap of the PLZP element and its following region of BAdV3 pIX to corresponding domains of human adenovirus type 5 (HAdV5) did not affect virion production, whereas swap of the entire pIX abolished production of progeny virions. We suggest that failure of the full-length BAdV3 pIX swap might be due to species specificity of its N-terminus region before the PLZP element.

  14. Bovine adenovirus type 3 virions cannot be rescued in vivo after full-length viral genome transfection in the absence of detectable polypeptide IX

    PubMed Central

    Zhang, Peng; Xue, Qinghong; Ma, Jing; Ren, Jingjing; Xia, Shuili; Zhang, Lu; Wang, Wenbin

    2017-01-01

    Bovine adenovirus type 3 (BAdV3) is being used in the development of potential vehicles for gene therapy and vectored vaccine. To that end, a more comprehensive description of BAdV3 biology is essential. In this study, we focused on the role of pIX in BAdV3 virion rescue after full-length BAdV3 genome transfection. Initially, pIX deletion or initiation codon mutation abolished the production of progeny virions, which suggested that pIX was essential for the rescue of BAdV3 containing a full-length genome. Moreover, through transfection of a panel of pIX mutant BAdV3 genomes, we observed that the conserved N-terminus and the putative leucine zipper element (PLZP) were essential for virion rescue, whereas the C-terminus following the coiled-coil domain was non-essential. In addition, swap of the PLZP element and its following region of BAdV3 pIX to corresponding domains of human adenovirus type 5 (HAdV5) did not affect virion production, whereas swap of the entire pIX abolished production of progeny virions. We suggest that failure of the full-length BAdV3 pIX swap might be due to species specificity of its N-terminus region before the PLZP element. PMID:27586461

  15. Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56 419 completely sequenced and manually annotated full-length cDNAs

    PubMed Central

    Takeda, Jun-ichi; Suzuki, Yutaka; Nakao, Mitsuteru; Barrero, Roberto A.; Koyanagi, Kanako O.; Jin, Lihua; Motono, Chie; Hata, Hiroko; Isogai, Takao; Nagai, Keiichi; Otsuki, Tetsuji; Kuryshev, Vladimir; Shionyu, Masafumi; Yura, Kei; Go, Mitiko; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Wiemann, Stefan; Nomura, Nobuo; Sugano, Sumio; Gojobori, Takashi; Imanishi, Tadashi

    2006-01-01

    We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56 419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37 670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants. PMID:16914452

  16. Structural snapshots of full-length Jak1, a transmembrane gp130/IL-6/IL-6Rα cytokine receptor complex, and the receptor-Jak1 holocomplex

    PubMed Central

    Lupardus, Patrick J.; Skiniotis, Georgios; Rice, Amanda J.; Thomas, Christoph; Fischer, Suzanne; Walz, Thomas; Garcia, K. Christopher

    2011-01-01

    Summary The shared cytokine receptor gp130 signals as a homodimer or heterodimer through activation of Janus kinases (Jaks) associated with the receptor intracellular domains. Here we reconstitute, in parts and whole, the full-length gp130 homodimer in complex with the cytokine interleukin-6 (IL-6), its alpha receptor (IL-6Rα) and Jak1, for electron microscopy imaging. We find that the full-length gp130 homodimer complex has intimate interactions between the trans- and juxtamembrane segments of the two receptors, appearing to rigidify the connection between the extra- and intracellular regions. 2D averages and 3D reconstructions of full-length Jak1 reveal a three-lobed structure comprised of FERM-SH2, pseudokinase and kinase modules possessing extensive inter-segmental flexibility that likely facilitates allosteric activation. Single-particle imaging of the gp130/IL-6/IL-6Rα/Jak1 holocomplex shows Jak1 associated with the membrane proximal intracellular regions of gp130, abutting the would-be inner leaflet of the cell membrane. Jak1 association with gp130 is enhanced by the presence of a membrane environment. PMID:21220115

  17. Delivery of full-length factor VIII using a piggyBac transposon vector to correct a mouse model of hemophilia A.

    PubMed

    Matsui, Hideto; Fujimoto, Naoko; Sasakawa, Noriko; Ohinata, Yasuhide; Shima, Midori; Yamanaka, Shinya; Sugimoto, Mitsuhiko; Hotta, Akitsu

    2014-01-01

    Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice. piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.

  18. The full-length E1-circumflexE4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression

    SciTech Connect

    Wilson, Regina; Ryan, Gordon B.; Knight, Gillian L.; Laimins, Laimonis A.; Roberts, Sally . E-mail: s.roberts@bham.ac.uk

    2007-06-05

    Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1-circumflexE4 protein. To determine the role of E1-circumflexE4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1-circumflexE4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1-circumflexE4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1-circumflexE4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1-circumflexE4 expression, and to HPV16 where only C-terminal truncations in E1-circumflexE4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1-circumflexE4 proteins.

  19. Reconstitution of an E box-binding Myc:Max complex with recombinant full-length proteins expressed in Escherichia coli.

    PubMed

    Farina, Anthony; Faiola, Francesco; Martinez, Ernest

    2004-04-01

    The c-Myc oncoprotein (Myc) is a DNA sequence-specific transcription factor that regulates transcription of a wide variety of genes involved in the control of cell growth, proliferation, differentiation, and apoptosis and its deregulated expression is implicated in many types of human cancer. Myc has an N-terminal transcription activation domain (TAD) that interacts with various coactivators and a C-terminal basic-helix-loop-helix-leucine zipper (bHLHZip) domain required for E box-specific DNA-binding and heterodimerization with its obligatory bHLHZip protein partner Max. The analysis of the mechanisms by which the Myc:Max complex regulates transcription at the molecular level in vitro has been hampered by the difficulty in obtaining highly pure recombinant Myc:Max heterodimers that contain full-length Myc with its complete TAD domain and that have sequence-specific DNA-binding activity. Here, we describe a simple method to reconstitute recombinant Myc:Max complexes from highly purified full-length proteins expressed in Escherichia coli that are soluble and highly active in E box-specific DNA-binding in vitro. The reconstituted Myc:Max complexes are stable and lack Max:Max homodimers. This procedure should facilitate the characterization of the DNA-binding and transcription activation functions of full-length Myc:Max complexes in vitro and in particular the role of Myc TAD-interacting cofactors and Myc:Max post-translational modifications.

  20. Reconstitution of an E box-binding Myc:Max complex with recombinant full-length proteins expressed in Escherichia coli

    PubMed Central

    Farina, Anthony; Faiola, Francesco; Martinez, Ernest

    2014-01-01

    The c-Myc oncoprotein (Myc) is a DNA sequence-specific transcription factor that regulates transcription of a wide variety of genes involved in the control of cell growth, proliferation, differentiation, and apoptosis and its deregulated expression is implicated in many types of human cancer. Myc has an N-terminal transcription activation domain (TAD) that interacts with various coactivators and a C-terminal basic-helix-loop-helix-leucine zipper (bHLHZip) domain required for E box-specific DNA-binding and heterodimerization with its obligatory bHLHZip protein partner Max. The analysis of the mechanisms by which the Myc:Max complex regulates transcription at the molecular level in vitro has been hampered by the difficulty in obtaining highly pure recombinant Myc:Max heterodimers that contain full-length Myc with its complete TAD domain and that have sequence-specific DNA-binding activity. Here, we describe a simple method to reconstitute recombinant Myc:Max complexes from highly purified full-length proteins expressed in Escherichia coli that are soluble and highly active in E box-specific DNA-binding in vitro. The reconstituted Myc:Max complexes are stable and lack Max:Max homodimers. This procedure should facilitate the characterization of the DNA-binding and transcription activation functions of full-length Myc:Max complexes in vitro and in particular the role of Myc TAD-interacting cofactors and Myc:Max post-translational modifications. PMID:15003254

  1. Full-length RAG1 promotes contact with coding and intersignal sequences in RAG protein complexes bound to recombination signals paired in cis

    PubMed Central

    Kumar, Sushil; Swanson, Patrick C.

    2009-01-01

    The RAG proteins initiate V(D)J recombination by mediating synapsis and cleavage of two different antigen receptor gene segments through interactions with their flanking recombination signal sequences (RSS). The protein–DNA complexes that support this process have mainly been studied using RAG–RSS complexes assembled using oligonucleotide substrates containing a single RSS that are paired in trans to promote synapsis. How closely these complexes model those formed on longer, more physiologically relevant substrates containing RSSs on the same DNA molecule (in cis) remains unclear. To address this issue, we characterized discrete core and full-length RAG protein complexes bound to RSSs paired in cis. We find these complexes support cleavage activity regulated by V(D)J recombination's ‘12/23 rule’ and exhibit plasticity in RSS usage dependent on partner RSS composition. DNA footprinting studies suggest that the RAG proteins in these complexes mediate more extensive contact with sequences flanking the RSS than previously observed, some of which are enhanced by full-length RAG1, and associated with synapsis and efficient RSS cleavage. Finally, we demonstrate that the RAG1 C-terminus facilitates hairpin formation on long DNA substrates, and full-length RAG1 promotes hairpin retention in the postcleavage RAG complex. These results provide new insights into the mechanism of physiological V(D)J recombination. PMID:19233873

  2. Full-length RAG1 promotes contact with coding and intersignal sequences in RAG protein complexes bound to recombination signals paired in cis.

    PubMed

    Kumar, Sushil; Swanson, Patrick C

    2009-04-01

    The RAG proteins initiate V(D)J recombination by mediating synapsis and cleavage of two different antigen receptor gene segments through interactions with their flanking recombination signal sequences (RSS). The protein-DNA complexes that support this process have mainly been studied using RAG-RSS complexes assembled using oligonucleotide substrates containing a single RSS that are paired in trans to promote synapsis. How closely these complexes model those formed on longer, more physiologically relevant substrates containing RSSs on the same DNA molecule (in cis) remains unclear. To address this issue, we characterized discrete core and full-length RAG protein complexes bound to RSSs paired in cis. We find these complexes support cleavage activity regulated by V(D)J recombination's '12/23 rule' and exhibit plasticity in RSS usage dependent on partner RSS composition. DNA footprinting studies suggest that the RAG proteins in these complexes mediate more extensive contact with sequences flanking the RSS than previously observed, some of which are enhanced by full-length RAG1, and associated with synapsis and efficient RSS cleavage. Finally, we demonstrate that the RAG1 C-terminus facilitates hairpin formation on long DNA substrates, and full-length RAG1 promotes hairpin retention in the post-cleavage RAG complex. These results provide new insights into the mechanism of physiological V(D)J recombination.

  3. Delivery of Full-Length Factor VIII Using a piggyBac Transposon Vector to Correct a Mouse Model of Hemophilia A

    PubMed Central

    Matsui, Hideto; Fujimoto, Naoko; Sasakawa, Noriko; Ohinata, Yasuhide; Shima, Midori; Yamanaka, Shinya; Sugimoto, Mitsuhiko; Hotta, Akitsu

    2014-01-01

    Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice.piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII. PMID:25126862

  4. A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq.

    PubMed

    Burke, Catherine M; Darling, Aaron E

    2016-01-01

    The bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision. We describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection. This method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution.

  5. A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq

    PubMed Central

    Darling, Aaron E.

    2016-01-01

    Background The bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision. Results We describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection. Conclusions This method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution. PMID:27688981

  6. Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning.

    PubMed

    Deymier, Martin J; Claiborne, Daniel T; Ende, Zachary; Ratner, Hannah K; Kilembe, William; Allen, Susan; Hunter, Eric

    2014-11-01

    The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual׳s diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens.

  7. A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

    PubMed Central

    Marques, M Carmen; Alonso-Cantabrana, Hugo; Forment, Javier; Arribas, Raquel; Alamar, Santiago; Conejero, Vicente; Perez-Amador, Miguel A

    2009-01-01

    Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new EST collection denotes an

  8. Correlation of the level of full-length CFTR transcript with pulmonary phenotype in patients carrying R117H and 1342-1,-2delAG mutations

    SciTech Connect

    Hamosh, A.; Cutting, G.R.; Oates, R.; Amos, J.

    1994-09-01

    The R117H mutation occurs on two chromosome backgrounds, one associated with a 7 thymidine tract (7T-R11H) in the splice-acceptor site of intron 8, the other with a 5 thymidine tract (5T-R117H). We examined exon 9 splicing efficiency in 5 patients of genotype R117H/{delta}F508 and one carrying 1342-1,-2delAG{delta}F508, an obligate exon 9 slice site mutation. Four patients carried R117H on a 7T background -- three adult men with congenital bilateral absence of the vas deferens and one adolescent female with pancreatitis and borderline sweat chloride concentration. The patient with R117H on a 5T background had pancreatic sufficient CF (PS-CF). The 1342-1,-2delAG patient has classic pancreatic insufficient CF (PI-CF). cDNA was synthesized from total RNA extracted from nasal epithlial cells and analyzed for CFTR splicing by 35 cycle PCR using primers in exon 7 and 11. The quantity of full length transcript derived from the R117H or {delta}F508 alleles was assessed by allele-specific oligonucleotide hybridization. While 91.4% of transcript from the 5T-R117H allele was full-length, only 42.2% of CFTR transcript from the 5T-R117H allele was full length. Since CBAVD patients have no lung disease and PS-CF patients do, this indicates that the threshold of developing CF lung disease is crossed when the amount of CFTR transcript bearing R117H is reduced by half. Interestingly, 17.1% of transcript derived from the 1342-1,-2delAG allele (or 8.6% of total CFTR transcript) was normal and full length. This suggests that up to 9% of full length wild-type CFTR transcript may be inadequate to escape the lung disease of CF and that a 9 thymidine tract followed by AAC (the result of the AG deletion) can be used as a splice donor with 2-9% efficiency.

  9. Assessment of adaptive evolution between wheat and rice as deduced from full-length common wheat cDNA sequence data and expression patterns.

    PubMed

    Kawaura, Kanako; Mochida, Keiichi; Enju, Akiko; Totoki, Yasushi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Kai, Chikatoshi; Kawai, Jun; Hayashizaki, Yoshihide; Seki, Motoaki; Shinozaki, Kazuo; Ogihara, Yasunari

    2009-06-18

    Wheat is an allopolyploid plant that harbors a huge, complex genome. Therefore, accumulation of expressed sequence tags (ESTs) for wheat is becoming particularly important for functional genomics and molecular breeding. We prepared a comprehensive collection of ESTs from the various tissues that develop during the wheat life cycle and from tissues subjected to stress. We also examined their expression profiles in silico. As full-length cDNAs are indispensable to certify the collected ESTs and annotate the genes in the wheat genome, we performed a systematic survey and sequencing of the full-length cDNA clones. This sequence information is a valuable genetic resource for functional genomics and will enable carrying out comparative genomics in cereals. As part of the functional genomics and development of genomic wheat resources, we have generated a collection of full-length cDNAs from common wheat. By grouping the ESTs of recombinant clones randomly selected from the full-length cDNA library, we were able to sequence 6,162 independent clones with high accuracy. About 10% of the clones were wheat-unique genes, without any counterparts within the DNA database. Wheat clones that showed high homology to those of rice were selected in order to investigate their expression patterns in various tissues throughout the wheat life cycle and in response to abiotic-stress treatments. To assess the variability of genes that have evolved differently in wheat and rice, we calculated the substitution rate (Ka/Ks) of the counterparts in wheat and rice. Genes that were preferentially expressed in certain tissues or treatments had higher Ka/Ks values than those in other tissues and treatments, which suggests that the genes with the higher variability expressed in these tissues is under adaptive selection. We have generated a high-quality full-length cDNA resource for common wheat, which is essential for continuation of the ongoing curation and annotation of the wheat genome. The data

  10. Genetic characterization of human herpesvirus type 1: Full-length genome sequence of strain obtained from an encephalitis case from India.

    PubMed

    Bondre, Vijay P; Sankararaman, Vasudha; Andhare, Vijaysinh; Tupekar, Manisha; Sapkal, Gajanan N

    2016-11-01

    Human herpes simplex virus 1 (HSV-1) is the most common cause of sporadic encephalitis in humans that contributes to >10 per cent of the encephalitis cases occurring worldwide. Availability of limited full genome sequences from a small number of isolates resulted in poor understanding of host and viral factors responsible for variable clinical outcome. In this study genetic relationship, extent and source of recombination using full-length genome sequence derived from a newly isolated HSV-1 isolate was studied in comparison with those sampled from patients with varied clinical outcome. Full genome sequence of HSV-1 isolated from cerebrospinal fluid (CSF) of a patient with acute encephalitis syndrome (AES) by inoculation in baby hamster kidney-21 (BHK-21) cells was determined using next-generation sequencing (NGS) technology. Phylogenetic analysis of the newly generated sequence in comparison with 33 additional full-length genomes defined genetic relationship with worldwide distributed strains. The bootscan and similarity plot analysis defined recombination crossovers and similarities between newly isolated Indian HSV-1 with six Asian and a total of 34 worldwide isolated strains. Mapping of 376,332 reads amplified from HSV-1 DNA by NGS generated full-length genome of 151,024 bp from newly isolated Indian HSV-1. Phylogenetic analysis classified worldwide distributed strains into three major evolutionary lineages correlating to their geographic distribution. Lineage 1 containing strains were isolated from America and Europe; lineage 2 contained all the strains from Asian countries along with the North American KOS and RE strains whereas the South African isolates were distributed into two groups under lineage 3. Recombination analysis confirmed events of recombination in Indian HSV-1 genome resulting from mixing of different strains evolved in Asian countries. Our results showed that the full-length genome sequence generated from an Indian HSV-1 isolate shared close

  11. Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.

    PubMed

    Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2015-12-01

    Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

  12. Gene VI of figwort mosaic virus (caulimovirus group) functions in posttranscriptional expression of genes on the full-length RNA transcript.

    PubMed

    Gowda, S; Wu, F C; Scholthof, H B; Shepherd, R J

    1989-12-01

    Experimental evidence for a molecular function for gene VI of the caulimoviruses is presented. Based on experiments with the figwort mosaic virus (FMV), it appears that gene VI has a role in the posttranscriptional expression of the closely packed genes (VII and I-V), which appear on the larger, full-length RNA transcript of this virus. Gene VI with its flanking 5'/3' expression signals included as a separate plasmid during electroporation of DNA into protoplasts of Nicotiana edwardsonii shows an unusual type of transactivation of a chloramphenicol acetyltransferase (CAT) gene fused at its 5' end to a small open reading frame (gene VII) of the long 5' leader of the full-length RNA transcript of the FMV genome. The level of activity of the CAT gene is increased up to 20-fold over the activity of control plasmids when gene VI is included in the electroporation mixture. Mutagenesis of the coding portions of gene VI of pGS1 RVI, a transactivating plasmid used in the electroporation experiments, demonstrated that it was probably the polypeptide product of gene VI that was responsible for the transactivating effect. Experiments with various portions of the 5' leader of the large, full-length RNA of FMV showed that the coding region of gene VII is necessary for the transactivation event. Clones of cauliflower mosaic virus (CaMV) or FMV with intact gene VI were found to reciprocally transactivate gene VII-CAT fusions (FMV) or gene I-CAT fusions (CaMV) located downstream of the 5' leader sequences of either viral genome.

  13. Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

    PubMed Central

    Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2015-01-01

    Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure. PMID:26633465

  14. New Primers Targeting Full-Length Ciliate 18S rRNA Genes and Evaluation of Dietary Effect on Rumen Ciliate Diversity in Dairy Cows.

    PubMed

    Zhang, Jun; Zhao, Shengguo; Zhang, Yangdong; Sun, Peng; Bu, Dengpan; Wang, Jiaqi

    2015-12-01

    Analysis of the full-length 18S rRNA gene sequences of rumen ciliates is more reliable for taxonomical classification and diversity assessment than the analysis of partial hypervariable regions only. The objective of this study was to develop new oligonucleotide primers targeting the full-length 18S rRNA genes of rumen ciliates, and to evaluate the effect of different sources of dietary fiber (corn stover or a mixture of alfalfa hay and corn silage) and protein (mixed rapeseed, cottonseed, and/or soybean meals) on rumen ciliate diversity in dairy cows. Primers were designed based on a total of 137 previously reported ciliate 18S rRNA gene sequences. The 3'-terminal sequences of the newly designed primers, P.1747r_2, P.324f, and P.1651r, demonstrated >99% base coverage. Primer pair D (P.324f and P.1747r_2) was selected for the cloning and sequencing of ciliate 18S rRNA genes because it produced a 1423-bp amplicon, and did not amply the sequences of other eukaryotic species, such as yeast. The optimal species-level cutoff value for distinguishing between the operational taxonomic units of different ciliate species was 0.015. The phylogenetic analysis of full-length ciliate 18S rRNA gene sequences showed that distinct ciliate profiles were induced by the different sources of dietary fiber and protein. Dasytricha and Entodinium were the predominant genera in the ruminal fluid of dairy cattle, and Dasytricha was significantly more abundant in cows fed with corn stover than in cows fed with alfalfa hay and corn silage.

  15. One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system

    PubMed Central

    Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

    2008-01-01

    The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Jun × 4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems. PMID:18329890

  16. Sensitive Multiplexed Quantitative Analysis of Autoantibodies to Cancer Antigens with Chemically S-Cationized Full-Length and Water-Soluble Denatured Proteins.

    PubMed

    Futami, Junichiro; Nonomura, Hidenori; Kido, Momoko; Niidoi, Naomi; Fujieda, Nao; Hosoi, Akihiro; Fujita, Kana; Mandai, Komako; Atago, Yuki; Kinoshita, Rie; Honjo, Tomoko; Matsushita, Hirokazu; Uenaka, Akiko; Nakayama, Eiichi; Kakimi, Kazuhiro

    2015-10-21

    Humoral immune responses against tumor-associated antigens (TAAs) or cancer/testis antigens (CTAs) aberrantly expressed in tumor cells are frequently observed in cancer patients. Recent clinical studies have elucidated that anticancer immune responses with increased levels of anti-TAA/CTA antibodies improve cancer survival rates. Thus, these antibody levels are promising biomarkers for diagnosing the efficiency of cancer immunotherapy. Full-length antigens are favored for detecting anti-TAA/CTA antibodies because candidate antigen proteins contain multiple epitopes throughout their structures. In this study, we developed a methodology to prepare purified water-soluble and full-length antigens by using cysteine sulfhydryl group cationization (S-cationization) chemistry. S-Cationized antigens can be prepared from bacterial inclusion bodies, and they exhibit improved protein solubility but preserved antigenicity. Anti-TAA/CTA antibodies detected in cancer patients appeared to recognize linear epitopes, as well as conformational epitopes, and because the frequency of cysteine side-residues on the epitope-paratope interface was low, any adverse effects of S-cationization were virtually negligible for antibody binding. Furthermore, S-cationized antigen-immobilized Luminex beads could be successfully used in highly sensitive quantitative-multiplexed assays. Indeed, patients with a more broadly induced serum anti-TAA/CTA antibody level showed improved progression-free survival after immunotherapy. The comprehensive anti-TAA/CTA assay system, which uses S-cationized full-length and water-soluble recombinant antigens, may be a useful diagnostic tool for assessing the efficiency of cancer immunotherapy.

  17. One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system.

    PubMed

    Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

    2008-05-01

    The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Junx4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems.

  18. Large Scale Full-Length cDNA Sequencing Reveals a Unique Genomic Landscape in a Lepidopteran Model Insect, Bombyx mori

    PubMed Central

    Suetsugu, Yoshitaka; Futahashi, Ryo; Kanamori, Hiroyuki; Kadono-Okuda, Keiko; Sasanuma, Shun-ichi; Narukawa, Junko; Ajimura, Masahiro; Jouraku, Akiya; Namiki, Nobukazu; Shimomura, Michihiko; Sezutsu, Hideki; Osanai-Futahashi, Mizuko; Suzuki, Masataka G; Daimon, Takaaki; Shinoda, Tetsuro; Taniai, Kiyoko; Asaoka, Kiyoshi; Niwa, Ryusuke; Kawaoka, Shinpei; Katsuma, Susumu; Tamura, Toshiki; Noda, Hiroaki; Kasahara, Masahiro; Sugano, Sumio; Suzuki, Yutaka; Fujiwara, Haruhiko; Kataoka, Hiroshi; Arunkumar, Kallare P.; Tomar, Archana; Nagaraju, Javaregowda; Goldsmith, Marian R.; Feng, Qili; Xia, Qingyou; Yamamoto, Kimiko; Shimada, Toru; Mita, Kazuei

    2013-01-01

    The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes. PMID:23821615

  19. Bacterially Expressed Full-Length Recombinant Plasmodium falciparum RH5 Protein Binds Erythrocytes and Elicits Potent Strain-Transcending Parasite-Neutralizing Antibodies

    PubMed Central

    Reddy, K. Sony; Pandey, Alok K.; Singh, Hina; Sahar, Tajali; Emmanuel, Amlabu; Chitnis, Chetan E.; Chauhan, Virander S.

    2014-01-01

    Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an essential merozoite ligand that binds with its erythrocyte receptor, basigin. PfRH5 is an attractive malaria vaccine candidate, as it is expressed by a wide number of P. falciparum strains, cannot be genetically disrupted, and exhibits limited sequence polymorphisms. Viral vector-induced PfRH5 antibodies potently inhibited erythrocyte invasion. However, it has been a challenge to generate full-length recombinant PfRH5 in a bacterial-cell-based expression system. In this study, we have produced full-length recombinant PfRH5 in Escherichia coli that exhibits specific erythrocyte binding similar to that of the native PfRH5 parasite protein and also, importantly, elicits potent invasion-inhibitory antibodies against a number of P. falciparum strains. Antibasigin antibodies blocked the erythrocyte binding of both native and recombinant PfRH5, further confirming that they bind with basigin. We have thus successfully produced full-length PfRH5 as a functionally active erythrocyte binding recombinant protein with a conformational integrity that mimics that of the native parasite protein and elicits potent strain-transcending parasite-neutralizing antibodies. P. falciparum has the capability to develop immune escape mechanisms, and thus, blood-stage malaria vaccines that t