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Sample records for balanus amphitrite cdna

  1. Studies on some ecological aspects of Balanus amphitrite (Cirripedia: Thoracica)

    Digital Repository Service at National Institute of Oceanography (India)

    Desai, D.V.

    The year round breeding capability of Balanus amphitrite indicates a potential for continuous recruitment. The recruitment pattern however indicated a lull during monsoon. The study site experiences increased land run off lowering the salinity...

  2. Influence of bacterial exopolymers and the adult extract of Balanus amphitrite and Cthamalus sp. on cyprid metamorphosis of Balanus amphitrite

    Digital Repository Service at National Institute of Oceanography (India)

    Anil, A.C.; Khandeparker, L.; Mitbavkar, S.; Wagh, A.B.

    play a major role in the recruitment of larvae of fouling organisms, probably by providing inducing/inhibitory chemical cues. In this experiment, the influence of the extract of adult Balanus amphitrite and the expolymers of the bacteria colonising...

  3. Identification of Balanus amphitrite larvae from field zooplankton using species-specific primers

    Digital Repository Service at National Institute of Oceanography (India)

    Gaonkar, C.C.; Khandeparker, L.; Desai, D.V.; Anil, A.C.

    Identification of marine invertebrate larvae using morphological characters is laborious and complicated by phenotypic plasticity. Balanus amphitrite is a dominant barnacle, important in the context of intertidal ecology and biofouling of manmade...

  4. Influence of food concentration, temperature and salinity on the larval development of Balanus amphitrite

    Digital Repository Service at National Institute of Oceanography (India)

    Anil, A.C.; Kurian, J.

    Influence of food concentration (0.5, 1 and 2 x 10 sup(5) cell ml sup(-1) of Skeletonema costatum), temperature (20 and 30 degrees C) and salinity (15, 25 and 35 ppt) on the larval development of Balanus amphitrite (Cirripedia: Thoracica...

  5. Epibiotic community on the acorn barnacle (Balanus amphitrite) from a monsoon-influenced tropical estuary

    Digital Repository Service at National Institute of Oceanography (India)

    Sahoo, G.; Khandeparker, L.

    The epibiotic communities (diatoms and metazoans) on the outer surfaces of the shell of the barnacle Balanus amphitrite (BSh) and its opercular valves (the scutum and tergum; BST) were investigated on a monthly basis for 1 year in a tropical monsoon-influenced...

  6. Influence of bacterial exopolymers, conspecific adult extract and salinity on the cyprid metamorphosis of Balanus amphitrite (Cirripedia: Thoracica)

    Digital Repository Service at National Institute of Oceanography (India)

    Anil, A.C.; Khandeparker, R.

    The influence of bacterial exopolymers and conspecific adult extract of Balanus amphitrite on metamorphosis of cyprid larvae at different salinities has been evaluated through laboratory assay. The bacterial exopolymers (epm) extracted from...

  7. Exploration and metamorphosis in Balanus amphitrite Darwin (Cirripedia ; Thoracica) cyprids: significance of sugars and adult extract

    Digital Repository Service at National Institute of Oceanography (India)

    Khandeparker, L.; Anil, A.C.; Raghukumar, S.

    present in the adults is thought to be responsible for this behavior (Knight-Jones, 1953; Knight-Jones and Crisp, 3 1953; Crisp and Meadows, 1963). Clare et al. (1995) reported the involvement of cyclic AMP in the pheromonal modulation of barnacle... of the fourth antennular segment of Balanus amphitrite amphitrite (Crustacea: Cirripedia). J Mar. Biol. Ass. U. K. 74, 967-970. Clare, A.S., Thomas, R.F., Rittschof, D., 1995. Evidence for the involvement of cyclic AMP in the pheromonal modulation...

  8. Influence of temperature on the starvation threshold of nauplii of barnacle Balanus amphitrite (Cirripedia: Thoracica)

    Digital Repository Service at National Institute of Oceanography (India)

    Desai, D.V.; Anil, A.C.

    of starvation factor in recruitment ecology8. Among barnacles, Balanus amphitrite has gained prominence in studies relevant to larval metamorphosis, influence of different chemical cues and mechanism of their perception. Selection of this species... for his support and encouragement, Dr. A. B. Wagh for his interest in initiating this investigation. We are thankful to Dr. N. B. Bhosle, Head, MCMRD as well as other colleagues. We acknowledge the assistance extended by Mr. S Naik, Mr. N Prabhu and Mr. P...

  9. Effect of Sublethal Concentrations of Fuel Oil on the Behavior and Survival of Larvae and Adults of the Barnacle Balanus Amphitrite Amphitrite (Darwin)

    OpenAIRE

    Hashim, Amna A.

    2010-01-01

    The sublethal effect of petroleum hydrocarbons (fuel oil) was investigated using different developmental stages of Balanus amphitrite amphitrite. 5, 10, 15 and 20 ppm concentrations of the water soluble fraction of the fuel oil were applied and found to have significant effect on the behavior and survival of the organism. Larval stage II was more sensitive than the other stages and was affected even with the lowest concentration of hydrocarbons (5 ppm). Nutritional activity of the adults ...

  10. Comparative Proteome and Phosphoproteome Analyses during Cyprid Development of the Barnacle Balanus ( =Amphibalanus ) amphitrite

    KAUST Repository

    Zhang, Yu

    2010-06-04

    The barnacle Balanus amphitrite (=Amphibalanus amphitrite) is a major marine biofouling invertebrate worldwide. It has a complex life cycle during which the larva (called a nauplius) molts six times before transforming into the cyprid stage. The cyprid stage in B. amphitrite is the critical stage for the larval decision to attach and metamorphose. In this study, proteome and phosphoproteome alterations during cyprid development/aging and upon treatment with the antifouling agent butenolide were examined with a two-dimensional electrophoresis (2-DE) multiplexed fluorescent staining approach. Optimized protein separation strategies, including solution-phase isoelectric fractionation and narrow-pH-range 2-DE, were used in a proteomic analysis. Our results show that the differential regulation of the target proteins is highly dynamic on the levels of both protein expression and posttranslational modification. Two groups of proteins, stress-associated and energy metabolism-related proteins, are differentially expressed during cyprid development. Comparison of the control and treatment groups suggests that butenolide exerts its effects by sustaining the expression levels of these proteins. Altogether, our data suggest that proteins involved in stress regulation and energy metabolism play crucial roles in regulating larval attachment and metamorphosis of B. amphitrite. © 2010 American Chemical Society.

  11. Transcriptomic analysis of neuropeptides and peptide hormones in the barnacle Balanus amphitrite: evidence of roles in larval settlement.

    KAUST Repository

    Yan, Xing-Cheng

    2012-10-02

    The barnacle Balanus amphitrite is a globally distributed marine crustacean and has been used as a model species for intertidal ecology and biofouling studies. Its life cycle consists of seven planktonic larval stages followed by a sessile juvenile/adult stage. The transitional processes between larval stages and juveniles are crucial for barnacle development and recruitment. Although some studies have been conducted on the neuroanatomy and neuroactive substances of the barnacle, a comprehensive understanding of neuropeptides and peptide hormones remains lacking. To better characterize barnacle neuropeptidome and its potential roles in larval settlement, an in silico identification of putative transcripts encoding neuropeptides/peptide hormones was performed, based on transcriptome of the barnacle B. amphitrite that has been recently sequenced. Potential cleavage sites andstructure of mature peptides were predicted through homology search of known arthropod peptides. In total, 16 neuropeptide families/subfamilies were predicted from the barnacle transcriptome, and 14 of them were confirmed as genuine neuropeptides by Rapid Amplification of cDNA Ends. Analysis of peptide precursor structures and mature sequences showed that some neuropeptides of B. amphitrite are novel isoforms and shared similar characteristics with their homologs from insects. The expression profiling of predicted neuropeptide genes revealed that pigment dispersing hormone, SIFamide, calcitonin, and B-type allatostatin had the highest expression level in cypris stage, while tachykinin-related peptide was down regulated in both cyprids and juveniles. Furthermore, an inhibitor of proprotein convertase related to peptide maturation effectively delayed larval metamorphosis. Combination of real-time PCR results and bioassay indicated that certain neuropeptides may play an important role in cypris settlement. Overall, new insight into neuropeptides/peptide hormones characterized in this study shall

  12. Quantitative proteomics study of larval settlement in the barnacle Balanus amphitrite

    KAUST Repository

    Chen, Zhang-Fan

    2014-02-13

    Barnacles are major sessile components of the intertidal areas worldwide, and also one of the most dominant fouling organisms in fouling communities. Larval settlement has a crucial ecological effect not only on the distribution of the barnacle population but also intertidal community structures. However, the molecular mechanisms involved in the transition process from the larval to the juvenile stage remain largely unclear. In this study, we carried out comparative proteomic profiles of stage II nauplii, stage VI nauplii, cyprids, and juveniles of the barnacle Balanus amphitrite using label-free quantitative proteomics, followed by the measurement of the gene expression levels of candidate proteins. More than 700 proteins were identified at each stage; 80 were significantly up-regulated in cyprids and 95 in juveniles vs other stages. Specifically, proteins involved in energy and metabolism, the nervous system and signal transduction were significantly up-regulated in cyprids, whereas proteins involved in cytoskeletal remodeling, transcription and translation, cell proliferation and differentiation, and biomineralization were up-regulated in juveniles, consistent with changes associated with larval metamorphosis and tissue remodeling in juveniles. These findings provided molecular evidence for the morphological, physiological and biological changes that occur during the transition process from the larval to the juvenile stages in B. amphitrite. © 2014 Chen et al.

  13. Expression of Calmodulin and Myosin Light Chain Kinase during Larval Settlement of the Barnacle Balanus amphitrite

    KAUST Repository

    Chen, Zhang-Fan

    2012-02-13

    Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus (= Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca 2+/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite. © 2012 Chen et al.

  14. Comparison of nutritional status of field and laboratory reared Balanus amphitrite Darwin (Cirripedia: Thoracica) larvae and implication of starvation

    Digital Repository Service at National Institute of Oceanography (India)

    Desai, D.V.; Anil, A.C.

    Comparison of nutritional status of field and laboratory reared Balanus amphitrite Darwin (Cirripedia: Thoracica) larvae and implication of starvation Dattesh V. Desai, A.C. Anil * Marine Corrosion and Materials Research Division, National Institute... (Thorson, 1950; Crisp, 1976; Olson and Olson, 1989; Anil et al., 1995). Starvation will lead to nutritional stress, which can kill the larvae directly or may increase or decrease the duration of larval developmental stages (Knowlton, 1974; Sandifer...

  15. Effects of poly-ether B on proteome and phosphoproteome expression in biofouling Balanus amphitrite cyprids

    KAUST Repository

    Dash, Swagatika

    2012-04-01

    Biofouling is ubiquitous in marine environments, and the barnacle Balanus amphitrite is one of the most recalcitrant and aggressive biofoulers in tropical waters. Several natural antifoulants that were claimed to be non-toxic have been isolated in recent years, although the mechanism by which they inhibit fouling is yet to be investigated. Poly-ether B has shown promise in the non-toxic inhibition of larval barnacle attachment. Hence, in this study, multiplex two-dimensional electrophoresis (2-DE) was applied in conjunction with mass spectrometry to investigate the effects of poly-ether B on barnacle larvae at the molecular level. The cyprid proteome response to poly-ether B treatment was analyzed at the total proteome and phosphoproteome levels, with 65 protein and 19 phosphoprotein spots found to be up- or down-regulated. The proteins were found to be related to energy-metabolism, oxidative stress, and molecular chaperones, thus indicating that poly-ether B may interfere with the redox-regulatory mechanisms governing the settlement of barnacle larvae. The results of this study demonstrate the usefulness of the proteomic technique in revealing the working mechanisms of antifouling compounds. © 2012 Copyright Taylor and Francis Group, LLC.

  16. Transcriptome and proteome dynamics in larvae of the barnacle Balanus Amphitrite from the Red Sea

    KAUST Repository

    Chandramouli, Kondethimmanahalli

    2015-12-15

    Background The barnacle Balanus amphitrite is widely distributed in marine shallow and tidal waters, and has significant economic and ecological importance. Nauplii, the first larval stage of most crustaceans, are extremely abundant in the marine zooplankton. However, a lack of genome information has hindered elucidation of the molecular mechanisms of development, settlement and survival strategies in extreme marine environments. We sequenced and constructed the genome dataset for nauplii to obtain comprehensive larval genetic information. We also investigated iTRAQ-based protein expression patterns to reveal the molecular basis of nauplii development, and to gain information on larval survival strategies in the Red Sea marine environment. Results A nauplii larval transcript dataset, containing 92,117 predicted open reading frames (ORFs), was constructed and used as a reference for the proteome analysis. Genes related to translation, oxidative phosphorylation and cytoskeletal development were highly abundant. We observed remarkable plasticity in the proteome of Red Sea larvae. The proteins associated with development, stress responses and osmoregulation showed the most significant differences between the two larval populations studied. The synergistic overexpression of heat shock and osmoregulatory proteins may facilitate larval survival in intertidal habitats or in extreme environments. Conclusions We presented, for the first time, comprehensive transcriptome and proteome datasets for Red Sea nauplii. The datasets provide a foundation for future investigations focused on the survival mechanisms of other crustaceans in extreme marine environments.

  17. Response of cyprid specific genes to natural settlement cues in the barnacle Balanus (=Amphibalanus) amphitrite

    KAUST Repository

    Li, Honglei

    2010-06-01

    Quantitative real-time PCR was used to further our understanding of the molecular processes involved in the attachment and metamorphosis of larval barnacles. We report the effects of natural settlement cues (microbial biofilms and conspecific settlement-inducing factor) on the expression profiles of six barnacle cyprid specific (bcs) genes in cyprids of the barnacle Balanus (=Amphibalanus) amphitrite Darwin. Genes bcs-1 to bcs-5 all showed marked decreases in their expression between initial cyprid attachment and the completion of metamorphosis, whereas bcs-6 showed significant up-regulation. Generally, settlement cues exerted no significant effect on the decreasing trend of bcs-1 to bcs-5 expression during attachment and metamorphosis. However, the expression of bcs-6 increased prior to cyprid attachment in response to both settlement cues. This elevated expression of bcs-6 gene indicates the importance and key regulatory role of this specific gene to larval attachment and metamorphosis in this barnacle species. © 2010 Elsevier B.V. All rights reserved.

  18. Algal epibiosis on Megabalanus tintinnabulum and its role in segregation of the Balanus amphitrite population

    Digital Repository Service at National Institute of Oceanography (India)

    Eswaran, R.; Khandeparker, L.

    in intergeneric interactions with the algae was examined in laboratory experiments. For this, the influence that extracts of algae, extracts of algae-associated bacteria and natural leachants from M. tintinnabulum exerted on cyprid metamorphosis of B. amphitrite...

  19. The Mode of Action of Isocyanide in Three Aquatic Organisms, Balanus amphitrite, Bugula neritina and Danio rerio

    KAUST Repository

    Zhang, Yi-Fan

    2012-09-18

    Isocyanide is a potential antifouling compound in marine environments. In this study, we investigated its mode of action in three aquatic organisms. Two of them, the bryozoan Bugula neritina and the barnacle Balanus amphitrite, are major marine fouling invertebrates, and the other organism is the non-target species zebrafish Danio rerio. In the swimming larvae of B. neritina, isocyanide did not affect the total attachment rate (≤50 µg ml^(−1)), but it did change the attachment site by increasing the percentage of attachment on the bottom of the container rather than on the wall or air-water inter-surface. Isocyanide binds several proteins in B. neritina as identified via SDS-PAGE-LC-MS/MS: 1) a 30 kD protein band containing two proteins similar to voltage dependent anion channels (VDAC), which control the direct coupling of the mitochondrial matrix to the energy maintenance of the cytosol and the release of apoptogenic factors from mitochondria of mammalian cells; and 2) an unknown 39 kD protein. In B. amphitrite cyprids, the isocyanide binding protein were 1) a protein similar to NADH-ubiquinone oxidoreductase, which is the “entry enzyme” of oxidative phosphorylation in mitochondria; and 2) cytochrome P450. In Danio rerio embryos, isocyanide caused “wavy” notochords, hydrocephalus, pericardial edema, poor blood circulation, and defects in pigmentation and hematopoiesis, which phenocopied copper deficiency. This is the first report on isocyanide binding proteins in fouling organisms, as well as the first description of its phenotype and potential toxicology in zebrafish.

  20. The effect of butenolide on behavioral and morphological changes in two marine fouling species, the barnacle Balanus amphitrite and the bryozoan Bugula neritina

    KAUST Repository

    Zhang, Yi Fan

    2011-05-23

    Butenolide [5-octylfuran-2(5H)-one] is a very promising antifouling compound. Here, the effects of butenolide on larval behavior and histology are compared in two major fouling organisms, viz. cypris larvae of Balanus amphitrite and swimming larvae of Bugula neritina. Butenolide diminished the positive phototactic behavior of B. amphitrite (EC50=0.82 μg ml(-1)) and B. neritina (EC50=3 μg ml(-1)). Its effect on the attachment of cyprids of B. amphitrite was influenced by temperature, and butenolide increased attachment of larvae of B. neritina to the bottom of the experimental wells. At concentrations of 4 μg ml(-1) and 10 μg ml(-1), butenolide decreased attachment of B. amphitrite and B. neritina, respectively, but the effects were reversible within a certain treatment time. Morphologically, butenolide inhibited the swelling of secretory granules and altered the rough endoplasmic reticulum (RER) in the cement gland of B. amphitrite cyprids. In B. neritina swimming larvae, butenolide reduced the number of secretory granules in the pyriform-glandular complex.

  1. Larval development, sensory mechanisms and physiological adaptions in acorn barnacles with special reference to Balanus amphitrite

    Digital Repository Service at National Institute of Oceanography (India)

    Anil, A.C.; Khandeparker, L.; Desai, D.V.; Baragi, L.V.; Gaonkar, C.

    is greatest on the instar II nauplii (Anil et al., 1995). It was further observed that food availability and temperature jointly determine energy allocation for development and the influence varied with naupliar instars. The influence of salinity (15, 25... compared to other species of cirripede with simplified settlement behavior. The maneuverable cyprid antennule in B. amphitrite consists of four jointed segments, is furnished with numerous chemo- and mechanoreceptor organs, and is supported by the most...

  2. Physiological responses to hypoxia and anoxia in Balanus amphitrite (Cirripedia: Thoracica)

    Digital Repository Service at National Institute of Oceanography (India)

    Desai, D.V.; Prakash, S.

    -mediated toxicity (Halliwell 1978, Halliwell & Gutteridge 1986, Pryor & Godber 1991, Winston & Di Giulio 1991, Ahmad 1995). B. amphitrite is regularly exposed in the intertidal area of the study region to tidal fluctuations from 0.25 to 2.5m. Being an intertidal... #................... 17 REFERENCES Ahmad S (1995) Antioxidant mechanism of enzymes and proteins. In: S. Ahmad (ed) Oxidative stress and Antioxidant defenses in Biology, Chapman and Hall, New York, p 238-272 Anger K, Dawirs RR (1981) Influence of starvation...

  3. Reversible anti-settlement activity against Amphibalanus (= Balanus ) amphitrite, Bugula neritina , and Hydroides elegans by a nontoxic pharmaceutical compound, mizolastine

    KAUST Repository

    Zhou, Xiaojian

    2009-11-01

    Mizolastine, an antihistamine pharmaceutical, was found to significantly inhibit larval settlement of the barnacle Amphibalanus (=Balanus) amphitrite, the bryozoan Bugula neritina, and the polychaete Hydroides elegans with EC50 values of 4.2, 11.2, and 4.1 mg ml-1, respectively. No toxicity against the larvae of these three species was observed at the concentration range tested during incubations with mizolastine. To determine whether the anti-settlement activity of mizolastine is reversible, recovery bioassays using these three species were conducted. More than 70% of the larvae that had been exposed for 4 h to mizolastine at concentrations four-fold greater than their respective EC50 values completed normal metamorphosis. The results of the recovery bioassay provide evidence that the antisettlement effect of mizolastine is reversible in addition to being nontoxic. The anti-settlement activities of several intermediates of the synthesis process of mizolastine were also examined. One of the intermediates, 2-chloro-1-(4- fluorobenzyl)-1H-benzo[d]imidazole, inhibited larval settlement and metamorphosis with low toxicity. These results may improve the understanding of the key functional group responsible for the anti-settlement activity of mizolastine. © 2009 Taylor & Francis.

  4. Metamorphosis in the cirripede crustacean Balanus amphitrite

    DEFF Research Database (Denmark)

    Maruzzo, Diego; Aldred, Nick; Clare, Anthony S.;

    2012-01-01

    ecology of this species and a platform for studying the factors that control its metamorphosis. Metamorphosis in B. amphitrite involves a complex sequence of events: cementation, epidermis separation from the cypris cuticle, degeneration of cypris musculature, rotation of the thorax inside the mantle...... settlement biology has been intensively studied. By contrast, surprisingly few papers have dealt with the critical series of metamorphic events from cementation of the cyprid to the substratum until the appearance of a suspension feeding juvenile. This metamorphosis is both ontogenetically complex and...

  5. iTRAQ-Based Proteomic Profiling of the Barnacle Balanus amphitrite in Response to the Antifouling Compound Meleagrin

    KAUST Repository

    Han, Zhuang

    2013-05-03

    Marine biofouling refers to the unwanted accumulation of fouling organisms, such as barnacles, on artificial surfaces, resulting in severe consequences for marine industries. Meleagrin is a potential nontoxic antifoulant that is isolated from the fungus Penicillium sp.; however, its mechanistic effect mode of action on larval settlement remains unknown. Here, we applied iTRAQ coupled with 2D LC-MS/MS proteomic analysis to investigate the effect of meleagrin on the proteomic expression profile of cyprid development and aging in the barnacle Balanus amphitrite. Fifty proteins were differentially expressed in response to treatment with meleagrin, among which 26 proteins were associated with cyprid development/aging and 24 were specifically associated with the meleagrin treatment. The 66 proteins that were associated with aging only remained unaltered during exposure to meleagrin. Using KEGG analysis, those proteins were assigned to several groups, including metabolic pathways, ECM-receptor interactions, and the regulation of the actin cytoskeleton. Among the 24 proteins that were not related to the development/aging process, expression of the cyprid major protein (CMP), a vitellogenin-like protein, increased after the meleagrin treatment, which suggested that meleagrin might affect the endocrine system and prevent the larval molting cycle. With the exception of the chitin binding protein that mediates the molting process and ATPase-mediated energy processes, the majority of proteins with significant effects in previous studies in response to cyprid treatment with butenolide and polyether B remained unchanged in the present study, suggesting that meleagrin may exhibit a different mechanism. © 2013 American Chemical Society.

  6. The regulatory role of the NO/cGMP signal transduction cascade during larval attachment and metamorphosis of the barnacle Balanus (=Amphibalanus) amphitrite

    KAUST Repository

    Zhang, Y.

    2012-08-01

    The barnacle Balanus amphitrite is among the most dominant fouling species on intertidal rocky shores in tropical and subtropical areas and is thus a target organism in antifouling research. After being released from adults, the swimming nauplius undertakes six molting cycles and then transforms into a cyprid. Using paired antennules, a competent cyprid actively explores and selects a suitable substratum for attachment and metamorphosis (collectively known as settlement). This selection process involves the reception of exogenous signals and subsequent endogenous signal transduction. To investigate the involvement of nitric oxide (NO) and cyclic GMP (cGMP) during larval settlement of B. amphitrite, we examined the effects of an NO donor and an NO scavenger, two nitric oxide synthase (NOS) inhibitors and a soluble guanylyl cyclase (sGC) inhibitor on settling cyprids. We found that the NO donor sodium nitroprusside (SNP) inhibited larval settlement in a dose-dependent manner. In contrast, both the NO scavenger carboxy-PTIO and the NOS inhibitors aminoguanidine hemisulfate (AGH) and S-methylisothiourea sulfate (SMIS) significantly accelerated larval settlement. Suppression of the downstream guanylyl cyclase (GC) activity using a GC-selective inhibitor ODQ could also significantly accelerate larval settlement. Interestingly, the settlement inhibition effects of SNP could be attenuated by ODQ at all concentrations tested. In the developmental expression profiling of NOS and sGC, the lowest expression of both genes was detected in the cyprid stage, a crucial stage for the larval decision to attach and metamorphose. In summary, we concluded that NO regulates larval settlement via mediating downstream cGMP signaling.

  7. Three-dimensional computer-aided reconstruction of FMRFamide immunopositive neuron distribution in the ventral ganglion of the barnacle Balanus amphitrite (Cirripedia, Crustacea

    Directory of Open Access Journals (Sweden)

    L Gallus

    2009-12-01

    Full Text Available We have implemented a simple program to solve three of the problems related to 3D reconstruction (3D-Rec of soft tissues: alignment of sections, distortions, and estimation of the spatial position of elements of interest inside the tissues. As a model, we chose the distribution of FMRFamide-like immunopositive neurons in the ventral ganglion of the barnacle Balanus amphitrite collected during different seasonal periods. Images of immunostained sections were acquired by means of a CCDcamera- equipped microscope and a PC and the reference points were taken inside the sections. The FMRFamide-like immunopositive neurons detected in the barnacle ventral ganglion were grouped into four different classes according to size, shape and staining intensity. More numerous FMRFamide- like immunopositive neurons were detected in the autumn-collected barnacle than in the summer counterpart. The two 3D reconstructions obtained from transverse and longitudinal ventral ganglion sections were efficaciously compared after 90° rotation of one of them. Comparison of these two 3D-Rec suggests the presence of at least two groups of FMRFamide-like immunopositive neurons that are seasonally-related and probably involved in reproduction.

  8. Cryoperservation of Balanus amphitrite nauplii

    Digital Repository Service at National Institute of Oceanography (India)

    Anil, A.C.; Tulaskar, A.S.; Khandeparker, D.C.; Wagh, A.B.

    ) (Fig. 3b) revealed Practical aspects of Ice-free cryopreservation. In a decrease in the rate from 020 to 0507C. ‘‘Future Developments in Blood Banking’’ (C. Th. However, the transfer temperature experiment Smit-sibinga, P. C. Das, and T. J. Greenwalt...

  9. Starvation threshold of Balanus amphitrite larvae in relation to temperature

    Digital Repository Service at National Institute of Oceanography (India)

    Anil, A.C.; Desai, D.V.

    naupliar instars and the influence of the different environmental stresses that regulate it. From the antifouling assay perspective, it is also important to produce cyprids at energy level that mimic the stock in nature. In pursuit of this gobal...

  10. Relevance of biofilm bacteria in modulating the larval metamorphosis of Balanus amphitrite

    Digital Repository Service at National Institute of Oceanography (India)

    Khandeparker, L.; Anil, A; Raghukumar, S.

    that the water borne and the surface associated cues from the bacteria function differentially in mediating larval metamorphosis. Understanding the complexities involved in such interactions and identification of the factors governing them would be a step ahead....

  11. Reproduction in Balanus amphitrite Darwin (Cirripedia: Thoracica); influence of temperature and food concentration

    Digital Repository Service at National Institute of Oceanography (India)

    Desai, D.V.; Anil, A.C.; Venkat, K.

    Biol Ecol 19:227-232 Barnes H, Crisp DJ (1956) Evidence of self-fertilization in certain species of barnacles. J Mar Biol Ass U K 35:631-639 Charnov EL (1987) Sexuality and hermaphroditism in barnacles: a natural selection approach. In: Southward...

  12. Influence of temperature and salinity on larval development of Balanus amphitrite: Implications in fouling ecology

    Digital Repository Service at National Institute of Oceanography (India)

    Anil, A.C.; Chiba, K.; Okamoto, K.; Kurokura, H.

    stream_size 8 stream_content_type text/plain stream_name Mar_Ecol_Prog_Ser_118_159.pdf.txt stream_source_info Mar_Ecol_Prog_Ser_118_159.pdf.txt Content-Encoding ISO-8859-1 Content-Type text/plain; charset=ISO-8859-1 ...

  13. Influence of diatom exopolymers and biofilms on metamorphosis in the barnacle Balanus amphitrite

    Digital Repository Service at National Institute of Oceanography (India)

    Patil, J.S.; Anil, A.C.

    relatively weaker effects than Navicula spp. Axenic diatom films also facilitated larval metamorphosis (depending on species and cell density) in some cases, suggesting that the cues provided by the diatoms themselves can also mediate invertebrate larval...

  14. Quantification of the energy required for the destruction of Balanus Amphitrite larva by ultrasonic treatment

    Digital Repository Service at National Institute of Oceanography (India)

    Seth, N.; Chakravarty, P.; Khandeparker, L.; Anil, A.C.; Pandit, A.B.

    organism. Since the power used and treatment time for disinfection are economically, and practically, the most important parameters, the energy required to pulverize the larvae into pieces less than equal to 30 mm was determined as a function...

  15. Studies on some cues regulating metamorphosis of the larvae of Balanus amphitrite (Cirripedia: Thoracica)

    Digital Repository Service at National Institute of Oceanography (India)

    Khandeparker, L.

    -filtered (0.22 ?m), UV- irradiated seawater. 2.2.4 Visualization of footprints The sugar-treated, as well as non-treated cyprids, were siphoned out and introduced individually into six-well plates (Corning- 430343) coated with 50 ?g ml-1 AE and to non... introduced into wells of 24-well plates coated with AE and to non-coated multiwells (Corning-430262) along with 2 ml of autoclaved, filtered seawater at 35? (Maki et al. 1990). The above treatments were repeated employing a single cyprid (single cyprid...

  16. Role of conspecific cues and sugars in the settlement of cyprids of the barnacle, Balanus amphitrite

    Digital Repository Service at National Institute of Oceanography (India)

    Khandeparker, L.; Anil, A.C.

    the settlement significantly at a lower concentration, that is 10 C08 M without any extract, whereas, D-glucose showed a reduced effect. In the present investigation, we examined whether the influence of different sugars is modulated by different proteinextracts...,A.,Dennis,B.,Julian,L.,Martin,R.,Keith,R.&James, D.W. (1994). Molecular biology of the cell, 3rd edn. New York: GarlandPublishing, Taylorand FrancisGroup. Clare, A.S. & Matsumura, K. (2000). Nature and perception of barnacle settlement pheromones. Biofouling 15, 57–71. Dobretsov...

  17. Significance of biofilm proteins in modulating cyprid metamorphosis of Balanus amphitrite (Cirripedia: Thoracica)

    Digital Repository Service at National Institute of Oceanography (India)

    Khandeparker, L.; KrishnaKumar, S.

    The role of biofilm proteins in modulating cyprid metamorphosis is not well understood. In the present investigation this possibility was explored by disrupting biofilm proteins using commercially available protease. The influence of natural biofilm...

  18. Settlement and recruitment of the barnacle Balanus amphitrite from a tropical environment influenced by monsoons

    Digital Repository Service at National Institute of Oceanography (India)

    Gaonkar, C.A.; Anil, A.C.

    , with two-phase life cycle, and understanding the determinants of recruitment is a complex problem. Many processes which operate during the pelagic pre-settlement phase and benthic post-settlement phase at different spatial and temporal scales can determine...

  19. Worldwide genetic differentiation in the common fouling barnacle, Amphibalanus amphitrite

    KAUST Repository

    Chen, Hsi-Nien

    2014-10-21

    © 2014, © 2014 Taylor & Francis. Amphibalanus amphitrite is a common fouling barnacle distributed globally in tropical and subtropical waters. In the present study, the genetic (mitochondrial cytochrome oxidase subunit I) and morphological differentiation in A. amphitrite from 25 localities around the world were investigated. The results revealed three clades within A. amphitrite with a genetic divergence of ~ 4% among clades, whereas there were no diagnostic morphological differences among clades. Clade 1 is widely distributed in both temperate and tropical waters, whereas Clade 3 is currently restricted to the tropical region. The deep divergence among clades suggests historical isolation within A. amphitrite; thus, the present geographical overlaps are possibly a result of the combined effects of rising sea level and human-mediated dispersals. This study highlights the genetic differentiation that exists in a common, widely distributed fouling organism with great dispersal potential; future antifouling research should take into account the choice of lineages.

  20. Characterisation of the bacteria associated with barnacle, Balanus amphitrite, shell and their role in gregarious settlement of cypris larvae

    Digital Repository Service at National Institute of Oceanography (India)

    BacchettiDeGregoris, T.; Khandeparker, L.; Anil, A.C.; Mesbahi, E.; Burgess, J.G.; Clare, A.S.

    induce permanent attachment in a variety of taxa, including the alga Ulva sp. (Joint et al., 2000), the coral Acropora microphthalma (Webster et al., 2004) and the polychaete Hydroides elegans (Lau et al., 2003). However, the literature regarding... of primers). A taxonomical analysis of the BAB and RB sequences was obtained using BLAST. Results and Discussion Stable associations between bacteria and animals are widely distributed in both marine and terrestrial ecosystems. Many...

  1. Inhibitory effect of Indian sponge extracts on bacterial strains and larval settlement of the barnacle, Balanus Amphitrite

    Digital Repository Service at National Institute of Oceanography (India)

    LimnaMol, V.P.; Raveendran, T.V.; Abhilash, K.R.; Parameswaran, P.S.

    The total ban imposed on TBT based antifouling paints, since 2008, has triggered the search for Natural Product Antifoulants (NPAs) as a promising alternative for the development of environmentally benign coatings. In view of this, thirty six...

  2. Notes on the ultrastructure of the setae on the fourth antennulary segment of the Balanus amphitrite cyprid (Crustacea : Cirripedia : Thoracica)

    DEFF Research Database (Denmark)

    Lagersson, Niklas; Garm, Anders Lydik; Høeg, Jens Thorvald

    2003-01-01

    This study presents electron microscopy (SEM, TEM) observations on the putative settlement receptors of the fourth antennulary segments. The TEM data from all nine setae are from the outer dendritic segment. Eight of them have morphological characteristics, indicating bimodal sensory properties...

  3. Recruitment of the barnacle Balanus amphitrite Darwin in a tropical estuary: implications of environmental perturbation, reproduction and larval ecology

    Digital Repository Service at National Institute of Oceanography (India)

    Desai, D.V.; Anil, A.C.

    variations in recruitment, larval abundance, development and reproduction. Adult conditioning and inter-brood variations were important factors in the larval ecology of this organism. The results indicate that the impulsive release of larvae during breaks...

  4. Larval development and metamorphosis in Balanus amphitrite Darwin (Cirripedia; Thoracica): Significance of food concentration, temperature and nucleic acids

    Digital Repository Service at National Institute of Oceanography (India)

    Anil, A.C.; Desai, D.V.; Khandeparker, L.

    incorporated during the earlier planktotrophic naupliar development. Nucleic acids play a major role in growth and development. It has been shown that Ž. the RNArDNA ratio is an indicator of nutritional condition Clemmesen, 1996 , and has been routinely used... for estimating growth rates or nutritional condition of larval fish in the field. Only a few studies have applied this technique to crustaceans and their larvae Ž Dagg and Littlepage, 1972; Sulkin et al., 1975; Anger and Hirche, 1990; Wagner et al., . 1998...

  5. The impact of food type, temperature and starvation on larval development of Balanus amphitrite Darwin (Cirripedia: Thoracica)

    Digital Repository Service at National Institute of Oceanography (India)

    Desai, D.V.; Anil, A.C.

    that with development the utility of food types changes. The differences in the carbon gain can be attributed to differences in grazing rate due to variations in the size of the diatom cells, larval intersetular distance, diatom sinking rate and the photo-taxic behavior...

  6. The effects of a serine protease, Alcalase®, on the adhesives of barnacle cyprids (Balanus amphitrite)

    NARCIS (Netherlands)

    Aldred, Nick; Phang, In Yee; Conlan, Sheelagh L.; Clare, Anthony S.; Vancso, G. Julius

    2008-01-01

    Barnacles are a persistent fouling problem in the marine environment, although their effects (eg reduced fuel efficiency, increased corrosion) can be reduced through the application of antifouling or fouling-release coatings to marine structures. However, the developments of fouling-resistant coatin

  7. Factors regulating the production of different inducers in Pseudomonas aeruginosa with reference to larval metamorphosis in Balanus amphitrite

    Digital Repository Service at National Institute of Oceanography (India)

    Khandeparker, L.; Anil, A.C.; Raghukumar, S.

    + Denitrification + Gelatin liquefaction + Starch hydrolysis – O-F glucose Oxidative Arginine dihydrolase + Alkaline phosphatase heat resistance + Litmus milk (peptonization) + Utilization of: Glucose + D-xylose – D-ribose + Mannitol + Cellobiose – D-mannose – L.... The settlement assays were carried out using Corning-430343 6-well multiwells. The mul- tiwells were inoculated with BF, CS, fractions, bacter- ial EPS and bacterial extract. They were also assessed along with AE (3 replicates for each of the combination...

  8. Effects of Toxic Leachate from Commercial Plastics on Larval Survival and Settlement of the Barnacle Amphibalanus amphitrite.

    Science.gov (United States)

    Li, Heng-Xiang; Getzinger, Gordon J; Ferguson, P Lee; Orihuela, Beatriz; Zhu, Mei; Rittschof, Daniel

    2016-01-19

    Plastic pollution represents a major and growing global problem. It is well-known that plastics are a source of chemical contaminants to the aquatic environment and provide novel habitats for marine organisms. The present study quantified the impacts of plastic leachates from the seven categories of recyclable plastics on larval survival and settlement of barnacle Amphibalanus (=Balanus) amphitrite. Leachates from plastics significantly increased barnacle nauplii mortality at the highest tested concentrations (0.10 and 0.50 m(2)/L). Hydrophobicity (measured as surface energy) was positively correlated with mortality indicating that plastic surface chemistry may be an important factor in the effects of plastics on sessile organisms. Plastic leachates significantly inhibited barnacle cyprids settlement on glass at all tested concentrations. Settlement on plastic surfaces was significantly inhibited after 24 and 48 h, but settlement was not significantly inhibited compared to the controls for some plastics after 72-96 h. In 24 h exposure to seawater, we found larval toxicity and inhibition of settlement with all seven categories of recyclable commercial plastics. Chemical analysis revealed a complex mixture of substances released in plastic leachates. Leaching of toxic compounds from all plastics should be considered when assessing the risks of plastic pollution. PMID:26667586

  9. Predicting the Invasion Pathway of Balanus perforates in Korean Seawaters

    Directory of Open Access Journals (Sweden)

    Keun-Hyung Choi

    2013-03-01

    Full Text Available The European Common Barnacle Balanus perforatus Brugiére (Crustacea, Cirripedia has been introduced into the east coast of Korea, presumably via the ballast water of ships. The species has since been spreading along both the northern and southern coast to the east, most likely due to alongshore currents. We predicted the potential range expansion of Balanus perforatus in Korean waters using Genetic Algorithm for Rule-set Prediction (GARP, an environmental niche modeling technique. The results show that much of the southern coastal waters of Korea could be colonized by the spread of the nonindigenous species, but that the west coast is unlikely to be invaded. More sampling on the west coast would enhance the predictability of the model. To our knowledge, this is the first report of its kind for predicting marine nonindigenous species in Korean waters using GARP modeling.

  10. Interactive effect of elevated pCO2 and temperature on the larval development of an inter-tidal organism, Balanus amphitrite Darwin (Cirripedia: Thoracica)

    Digital Repository Service at National Institute of Oceanography (India)

    Baragi, L.V.; Anil, A.C.

    of the suitable food type (Barnes and Barnes, 1958). The growth rate of an organism is also influenced by the food quality and quantity (Moyse, 1963). Several studies have reported that the rising pCO2/reduced pH change the nutritional quality (Burkhardt et al... on the nutritional quality of the diatom (Chaetoceros calcitrans) which is used as feed for the rearing of barnacle larvae. In this experiment the growth of this diatom was measured in terms of cell numbers; and the nutritional quality was measured in terms...

  11. An original procedure for balanus repair with transposition of the testis

    Directory of Open Access Journals (Sweden)

    D. G. Kurbatov

    2016-01-01

    Full Text Available The paper presents the unique clinical experience of successful sexual rehabilitation of a patient who has undergone penile amputation for cancer. Complex reconstruction of all parts of the lost organ, by using known methods and those proposed for the first time in global practice (balanus repair with transposition of the testis, was performed in the patient.

  12. Distal histidine conformational flexibility in dehaloperoxidase from Amphitrite ornata

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zuxu; de Serrano, Vesna; Betts, Laurie; Franzen, Stefan; (NCSU); (UNC)

    2009-01-28

    The enzyme dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a heme protein which has a globin fold but can function as both a hemoglobin and a peroxidase. As a peroxidase, DHP is capable of converting 2,4,6-trihalophenols to the corresponding 2,6-dihaloquinones in the presence of hydrogen peroxide. As a hemoglobin, DHP cycles between the oxy and deoxy states as it reversibly binds oxygen for storage. Here, it is reported that the distal histidine, His55, exhibits conformational flexibility in the deoxy form and is consequently observed in two solvent-exposed conformations more than 9.5 {angstrom} away from the heme. These conformations are analogous to the open conformation of sperm whale myoglobin. The heme iron in deoxy ferrous DHP is five-coordinate and has an out-of-plane displacement of 0.25 {angstrom} from the heme plane. The observation of five-coordinate heme iron with His55 in a remote solvent-exposed conformation is consistent with the hypothesis that His55 interacts with heme iron ligands through hydrogen bonding in the closed conformation. Since His55 is also displaced by the binding of 4-iodophenol in an internal pocket, these results provide new insight into the correlation between heme iron ligation, molecular binding in the distal pocket and the conformation of the distal histidine in DHP.

  13. Nitric oxide inhibits larval settlement in Amphibalanus amphitrite cyprids by repressing muscle locomotion and molting

    KAUST Repository

    Zhang, Gen

    2015-08-28

    Nitric oxide (NO) is a universal signaling molecule and plays a negative role in the metamorphosis of many biphasic organisms. Recently, the NO/NO (cyclic guanosine monophosphate) signaling pathway was reported to repress larval settlement in the barnacle Amphibalanus amphitrite. To understand the underlying molecular mechanism, we analyzed changes in the proteome of A. amphitrite cyprids in response to different concentrations of the NO donor sodium nitroprusside (SNP; 62.5, 250 and 1000 μM) using a label-free proteomics method. Compared with the control, the expression of 106 proteins differed in all three treatments. These differentially expressed proteins were assigned to 13 pathways based on KEGG pathway enrichment analysis. SNP treatment stimulated the expression of heat shock proteins and arginine kinase, which are functionally related to NO synthases, increased the expression levels of glutathione transferases for detoxification, and activated the iron-mediated fatty acid degradation pathway and the citrate cycle through ferritin. Moreover, NO repressed the level of myosins and cuticular proteins, which indicated that NO might inhibit larval settlement in A. amphitrite by modulating the process of muscle locomotion and molting.

  14. Community composition, structure, and interrelationships in the marine intertidal Endocladia muricata – Balanus glandula association in Monterey Bay, California

    NARCIS (Netherlands)

    Glynn, Peter W.

    1965-01-01

    Studies of the community composition, structure and species interrelationships of the Endocladia-Balanus association were carried out on the rocky shores at the Hopkins Marine Station, Pacific Grove, California, over the period 1959—1961. The organisms making up this biotic association form a horizo

  15. cDNA: 34099 [

    Lifescience Database Archive (English)

    Full Text Available H. sapiens + Hs.15282 Homo sapiens cDNA FLJ44214 fis, clone THYMU3003309, moderately similar to ... Homo sapiens sarcoma antigen (SAGE ) gnl|UG|Hs#S16886502 AK126202 23/5622_34099.png ...

  16. cDNA: 43397 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.30035 Mus musculus adult male corpora quadrigemina cDNA, RIKEN full-length enri ... FOLATE DEHYDROGENASE (EC 1.5.1.6) (10-FTHFDH) (FBP-CI ) homolog [Rattus norvegicus], full insert sequence ...

  17. cDNA: 33377 [

    Lifescience Database Archive (English)

    Full Text Available H. sapiens + Hs.181243 Homo sapiens full open reading frame cDNA clone RZPDo834H102D for gene AT ... F4, activating transcription factor 4 (tax -responsive enhancer element B67); complete cds; wi ...

  18. cDNA: 17527 [

    Lifescience Database Archive (English)

    Full Text Available H. sapiens + Hs.134229 Homo sapiens cDNA FLJ44146 fis, clone THYMU2027734, weakly similar to Hom ... o sapiens SA hypertension -associated homolog (rat) (SAH) gnl|UG|Hs#S16886570 ...

  19. cDNA: 47992 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.228067 Mus musculus 15 days embryo male testis cDNA, RIKEN full-length enriched ... lone:8030476B22 product:hypothetical Mitochondrial energy ... transfer proteins (carrier protein) containing pro ...

  20. cDNA: 40711 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.41523 Mus musculus adult male thymus cDNA, RIKEN full-length enriched library, ... lone:5830492N08 product:hypothetical Mitochondrial energy ... transfer proteins (carrier protein) containing pro ...

  1. cDNA: 47994 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.228067 Mus musculus 0 day neonate eyeball cDNA, RIKEN full-length enriched libr ... lone:E130118D21 product:hypothetical Mitochondrial energy ... transfer proteins (carrier protein) containing pro ...

  2. cDNA: 47991 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.228067 Mus musculus adult male diencephalon cDNA, RIKEN full-length enriched li ... lone:9330189G22 product:hypothetical Mitochondrial energy ... transfer proteins (carrier protein) containing pro ...

  3. cDNA: 36928 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.240850 Mus musculus adult male medulla oblongata cDNA, RIKEN full-length enrich ... MA AMPLIFIED SEQUENCE 1 (NOVEL AMPLIFIED IN BREAST CANCER ... 1) (AMPLIFIED AND OVEREXPRESSED IN BREAST CANCER ) ...

  4. cDNA: 36927 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.240850 Mus musculus adult male stomach cDNA, RIKEN full-length enriched library ... MA AMPLIFIED SEQUENCE 1 (NOVEL AMPLIFIED IN BREAST CANCER ... 1) (AMPLIFIED AND OVEREXPRESSED IN BREAST CANCER ) ...

  5. cDNA: 40220 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.207654 Mus musculus adult male olfactory brain ... cDNA, RIKEN full-length enriched ... library, clone:6430704M03 product:similar to BRAIN ... PROTEIN (FRAGMENT) [Homo sapiens], full insert seq ...

  6. cDNA: 52275 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.275648 Mus musculus 11 days embryo head cDNA, RIKEN full-length enriched librar ... y, clone:6230400I01 product:SIMILAR TO ENIGMA ... (LIM DOMAIN PROTEIN) homolog [Homo sapiens], full ...

  7. cDNA: 52278 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.275648 Mus musculus adult male tongue cDNA, RIKEN full-length enriched library, ... clone:2310073F10 product:SIMILAR TO ENIGMA ... (LIM DOMAIN PROTEIN) homolog [Homo sapiens], full ...

  8. cDNA: 52277 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.275648 Mus musculus 18-day embryo whole body cDNA, RIKEN full-length enriched l ... ibrary, clone:1110003B01 product:SIMILAR TO ENIGMA ... (LIM DOMAIN PROTEIN) homolog [Homo sapiens], full ...

  9. cDNA: 52276 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.275648 Mus musculus adult male brain cDNA, RIKEN full-length enriched library, ... clone:0710007K04 product:SIMILAR TO ENIGMA ... (LIM DOMAIN PROTEIN) homolog [Homo sapiens], full ...

  10. Antifouling Activity of Simple Synthetic Diterpenoids against Larvae of the Barnacle Balanus albicostatus Pilsbry

    Directory of Open Access Journals (Sweden)

    Dan-Qing Feng

    2010-11-01

    Full Text Available Five new pimarane diterpenoids 1-5 were synthesized using ent-8(14-pimarene-15R,16-diol as starting material. The structures were elucidated by means of extensive NMR and MS analysis. The antifouling activity against larval settlement of the barnacle Balanus albicostatus were evaluated using capsaicin as a positive control. Compounds 1-3 and 5 showed more potent antifouling activity than capsaicin. Compound 5, which exhibited almost the same antifouling activity as starting material, showed better stability than starting material. These compounds all showed antifouling activity in a non-toxic way against larval settlement of the barnacle B. albicostatus. Analysis of structure-activity relationships (SAR demonstrated that the substituents on the C-15 and C-16 position of pimarane diterpenoid were responsible for the antifouling activity.

  11. Nest structure and notes on the social behavior of Augochlora amphitrite (Schrottky (Hymenoptera, Halictidae

    Directory of Open Access Journals (Sweden)

    Milagros Dalmazzo

    2012-05-01

    Full Text Available The nesting biology of Augochlora (Augochlora amphitrite (Schrottky in a natural reserve in the Province of Buenos Aires, Argentina, is described. The species nests in decaying wood. Two types of nest architecture were found, which differed according to the substrate where they were built, either soft or hard wood. Nests in soft wood had the cells grouped in clusters surrounded by a cavity, and the clusters were supported by a varying number of pillars. Nests constructed in decomposing portions of cracks in otherwise hard wood had the cells constructed against the walls, without any pillars or surrounding cavity. Cells of both types of nests were oriented in all directions, without any detectable pattern. Measurements and characteristics of the nests are tabulated and compared to those known for other species of Augochlora s. str. Behavioral observations of active nests are indicative of a social division of tasks in A. amphitrite. Such observations include nests with several females, some of which were never observed outside the nests, females with different degrees of wear and of ovary development, and at least one female that actively collected pollen which had much worn mandibles and wings, and undeveloped ovaries, all characteristics of the worker caste in social halictids.

  12. cDNA: 53887 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.196480 Mus musculus adult male testis cDNA, RIKEN full-length enriched library, ... uct:DNA Segment, Chr 15 Massachusetts Institute of Technology ... 260, full insert sequence gnl|UG|Mm#S10837764 AK07 ...

  13. cDNA: 53885 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.196480 Mus musculus adult male testis cDNA, RIKEN full-length enriched library, ... uct:DNA Segment, Chr 15 Massachusetts Institute of Technology ... 260, full insert sequence gnl|UG|Mm#S10837547 AK07 ...

  14. cDNA: 12295 [

    Lifescience Database Archive (English)

    Full Text Available H. sapiens + Hs.267288 Homo sapiens full open reading ... frame cDNA clone RZPDo834G0212D for gene C ... 6orf55, chromosome 6 open reading ... frame 55; complete cds, incl. stopcodon gnl|UG|Hs# ...

  15. cDNA: 16610 [

    Lifescience Database Archive (English)

    Full Text Available H. sapiens + Hs.62595 Homo sapiens full open reading ... frame cDNA clone RZPDo834H088D for gene C9o ... rf9, chromosome 9 open reading ... frame 9; complete cds, incl. stopcodon gnl|UG|Hs#S ...

  16. cDNA: 3940 [

    Lifescience Database Archive (English)

    Full Text Available H. sapiens - Hs.7188 Homo sapiens cDNA PSEC0078 fis, clone NT2RP2004036, moderately similar to M ... -Sema F=a factor in neural network ... development. gnl|UG|Hs#S4806431 AK075388 2/887_394 ...

  17. cDNA: 41587 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.30133 Mus musculus 18-day embryo whole body cDNA, RIKEN full-length enriched li ... brary, clone:1110004A14 product:ethanol ... induced 6, full insert sequence gnl|UG|Mm#S9085518 ...

  18. cDNA: 39377 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.7091 Mus musculus adult pancreas islet cells cDNA, RIKEN fu ll-length enriched l ... R BETA SUBUNIT) (SSR-BETA) homolog [Homo sapiens], fu ... ... gnl|UG|Mm#S10839939 AK050505 3/6436_39377.png ...

  19. cDNA: 46817 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.30092 Mus musculus adult male cerebellum cDNA, RIKEN fu ll-length enriched libra ... 1 PRECURSOR (EC 3.4.21.-) homolog [Homo sapiens], fu ... ... gnl|UG|Mm#S10838326 AK075719 8/7837_46817.png ...

  20. cDNA: 56670 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083407 AK006184 17/9809_56670.png ...

  1. cDNA: 56898 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083407 AK006184 17/9810_56898.png ...

  2. cDNA: 56671 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083172 AK006562 17/9809_56671.png ...

  3. cDNA: 56677 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083407 AK006184 17/9809_56677.png ...

  4. cDNA: 56899 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083172 AK006562 17/9810_56899.png ...

  5. cDNA: 46327 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.292517 Mus musculus 12 days embryo spinal ganglion cDNA, RIKEN fu ll-length enri ... PHA CHAIN) (PHERS) (CML33) homolog [Homo sapiens], fu ... ... gnl|UG|Mm#S10835133 AK084031 8/7824_46327.png ...

  6. cDNA: 56891 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083407 AK006184 17/9810_56891.png ...

  7. cDNA: 56678 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083172 AK006562 17/9809_56678.png ...

  8. cDNA: 41699 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.246636 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... e/G-protein beta WD-40 repeats containing protein, fu ... ... gnl|UG|Mm#S9075317 AK016965 5/6970_41699.png ...

  9. cDNA: 56892 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083172 AK006562 17/9810_56892.png ...

  10. cDNA: 36690 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.345070 Mus musculus 16 days embryo head cDNA, RIKEN fu ll-length enriched librar ... SPLICEOSOME ASSOCIATED PROTEIN 49) [Homo sapiens], fu ... ... gnl|UG|Mm#S10835972 AK081584 2/6005_36690.png ...

  11. cDNA: 49729 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.286963 Mus musculus 10 days lactation, adult female mammary gland cDNA, RIKEN f ... d library, clone:D730027I09 product:similar to LAK-4P ... [Homo sapiens], full insert sequence gnl|UG|Mm#S10 ...

  12. cDNA: 40377 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.154312 Mus musculus 0 day neonate kidney cDNA, RIKEN full-length enriched libra ... ry, clone:D630023P19 product:HYPERTENSION ... RELATED PROTEIN 1, full insert sequence gnl|UG|Mm# ...

  13. cDNA: 45098 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.334199 Mus musculus adult male aorta and vein cDNA, RIKEN full-length enriched ... library, clone:A530074J19 product:SA rat hypertension -associated homolog, full insert sequence gnl|UG|Mm ...

  14. cDNA: 40378 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.154312 Mus musculus 7 days embryo whole body cDNA, RIKEN full-length enriched l ... ibrary, clone:C430046A10 product:HYPERTENSION ... RELATED PROTEIN 1, full insert sequence gnl|UG|Mm# ...

  15. Characterization of two 20kDa-cement protein (cp20k homologues in Amphibalanus amphitrite.

    Directory of Open Access Journals (Sweden)

    Li-Sheng He

    Full Text Available The barnacle, Amphibalanus amphitrite, is a common marine fouling organism. Understanding the mechanism of barnacle adhesion will be helpful in resolving the fouling problem. Barnacle cement is thought to play a key role in barnacle attachment. Although several adult barnacle cement proteins have been identified in Megabalanus rosa, little is known about their function in barnacle settlement. In this study, two homologous 20k-cement proteins (cp20k in Amphibalanus amphitrite, named Bamcp20k-1 and Bamcp20k-2, were characterized. The two homologues share primary sequence structure with proteins from other species including Megabalanus rosa and Fistulobalanus albicostatus. The conserved structure included repeated Cys domains and abundant charged amino acids, such as histidine. In this study we demonstrated that Bamcp20k-1 localized at the α secretory cells in the cyprid cement gland, while Bamcp20k-2 localized to the β secretory cells. The differential localizations suggest differential regulation for secretion from the secretory cells. Both Bamcp20k-1 and Bamcp20k-2 from cyprids dissolved in PBS. However, adult Bamcp20k-2, which was dominant in the basal shell of adult barnacles, was largely insoluble in PBS. Solubility increased in the presence of the reducing reagent Dithiothreitol (DTT, suggesting that the formation of disulfide bonds plays a role in Bamcp20k-2 function. In comparison, Bamcp20k-1, which was enriched in soft tissue, could not be easily detected in the shell and base by Western blot and easily dissolved in PBS. These differential solubilities and localizations indicate that Bamcp20k-1 and Bamcp20k-2 have distinct functions in barnacle cementing.

  16. Characterization of Two 20kDa-Cement Protein (cp20k) Homologues in Amphibalanus amphitrite

    KAUST Repository

    He, Li-Sheng

    2013-05-22

    The barnacle, Amphibalanus amphitrite, is a common marine fouling organism. Understanding the mechanism of barnacle adhesion will be helpful in resolving the fouling problem. Barnacle cement is thought to play a key role in barnacle attachment. Although several adult barnacle cement proteins have been identified in Megabalanus rosa, little is known about their function in barnacle settlement. In this study, two homologous 20k-cement proteins (cp20k) in Amphibalanus amphitrite, named Bamcp20k-1 and Bamcp20k-2, were characterized. The two homologues share primary sequence structure with proteins from other species including Megabalanus rosa and Fistulobalanus albicostatus. The conserved structure included repeated Cys domains and abundant charged amino acids, such as histidine. In this study we demonstrated that Bamcp20k-1 localized at the α secretory cells in the cyprid cement gland, while Bamcp20k-2 localized to the β secretory cells. The differential localizations suggest differential regulation for secretion from the secretory cells. Both Bamcp20k-1 and Bamcp20k-2 from cyprids dissolved in PBS. However, adult Bamcp20k-2, which was dominant in the basal shell of adult barnacles, was largely insoluble in PBS. Solubility increased in the presence of the reducing reagent Dithiothreitol (DTT), suggesting that the formation of disulfide bonds plays a role in Bamcp20k-2 function. In comparison, Bamcp20k-1, which was enriched in soft tissue, could not be easily detected in the shell and base by Western blot and easily dissolved in PBS. These differential solubilities and localizations indicate that Bamcp20k-1 and Bamcp20k-2 have distinct functions in barnacle cementing. © 2013 He et al.

  17. Hydrocarbon pollutants alter short-term recruitment in the barnacle Balanus eburneus

    International Nuclear Information System (INIS)

    We examined barnacle recruitment to oil-treated clay tiles at two coastal and two protected sites along the Louisiana coast. Tiles exposed to crude oil and its water-soluble fraction (WSF) had diminished barnacle recruitment after 3 weeks, in comparison with non-treated tiles. Effects were most obvious at the exposed coastal sites. After a 6-week immersion, however, treatment effects were no longer consistent. In later experiments, Balanus eburneus recruitment was higher on weathered WSF tiles (exposed, and leached in seawater before immersion in the field) than on WSF tiles, but not different from control tiles. Hydrocarbons depress recruitment initially, but may be removed later by leaching or by hydrocarbon-degrading microbes, and the remaining biofilm may facilitate recruitment. While barnacle recruitment was generally higher at exposed sites, as expected from the literature, local factors caused significant variation in recruitment within exposure categories as well. Barnacles also grow to larger sizes at open-coast sites, probably because of increased water flow and planktonic food delivery. Naupliar abundances varied tremendously among sites and dates and were poor predictors of eventual recruitment. (author)

  18. siRNA transfection in larvae of the barnacle Amphibalanus amphitrite

    KAUST Repository

    Zhang, G.

    2015-06-25

    RNA interference (RNAi) provides an efficient and specific technique for functional genomic studies. Yet, no successful application of RNAi has been reported in barnacles. In this study, siRNA against p38 MAPK was synthesized and then transfected into A. amphitrite larvae at either the nauplius or cyprid stage, or at both stages. Effects of siRNA transfection on the p38 MAPK level were hardly detectable in the cyprids when they were transfected at the nauplius stage. In contrast, larvae that were transfected at the cyprid stage showed lower levels of p38 MAPK than the blank and reagent controls. However, significantly decreased levels of phosphorylated p38 MAPK (pp38 MAPK) and reduced settlement rates were observed only in ‘double transfections’, in which larvae were exposed to siRNA solution at both the nauplius and cyprid stages. A relatively longer transfection time and more larval cells directly exposed to siRNA might explain the higher efficiency of double transfection experiments.

  19. cDNA: 27908 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available H. sapiens - Hs.406397 Homo sapiens cDNA FLJ45472 fis, clone BRSTN2016918, highly similar to Gli ... al fibrillary acidic protein, astrocyte ... gnl|UG|Hs#S16883914 AK128790 17/4578_27908.png ...

  20. cDNA: 57843 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.1441 Mus musculus adult male testis cDNA, RIKEN full-length enriched library, c ... lone:4930519F16 product:hypothetical Serine proteases , trypsin family/Chymotrypsin serine protease famil ...

  1. cDNA: 55929 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.244143 Mus musculus adult male small intestine cDNA, RIKEN full-length enriched ... rary, clone:2010001P08 product:hypothetical Serine proteases , trypsin family containing protein, full insert se ...

  2. cDNA: 44740 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.29756 Mus musculus adult male testis cDNA, RIKEN full-length enriched library, ... 2 product:hypothetical Eukaryotic thiol (cysteine) proteases ... active site containing protein, full insert sequen ...

  3. cDNA: 43675 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.333219 Mus musculus 8 days embryo whole body cDNA, RIKEN full-length enriched l ... 0 product:hypothetical Eukaryotic thiol (cysteine) proteases ... active site containing protein, full insert sequen ...

  4. cDNA: 53523 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.277582 Mus musculus 16 days neonate cerebellum cDNA, RIKEN full-length enriched ... rary, clone:9630053E09 product:hypothetical Serine proteases , subtilase family/Leucine-rich repeat containing p ...

  5. cDNA: 42035 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.274255 Mus musculus adult male urinary bladder cDNA, RIKEN full-length enriched ... rary, clone:9530081K03 product:hypothetical Serine proteases , trypsin family containing protein, full insert se ...

  6. cDNA: 43679 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.333219 Mus musculus 10 days neonate cerebellum cDNA, RIKEN full-length enriched ... 0 product:hypothetical Eukaryotic thiol (cysteine) proteases ... active site containing protein, full insert sequen ...

  7. cDNA: 54640 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.212157 Mus musculus adult male kidney cDNA, RIKEN full-length enriched library, ... 1 product:hypothetical Eukaryotic thiol (cysteine) proteases ... active site containing protein, full insert sequen ...

  8. cDNA: 44744 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.29756 Mus musculus 0 day neonate thymus cDNA, RIKEN full-length enriched librar ... 1 product:hypothetical Eukaryotic thiol (cysteine) proteases ... active site containing protein, full insert sequen ...

  9. cDNA: 35986 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.122430 Mus musculus adult male corpora quadrigemina cDNA, RIKEN full-length enr ... 0341B09 product:hypothetical Ubiquitin-conjugating enzymes ... containing protein, full insert sequence gnl|UG|Mm ...

  10. cDNA: 35981 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.122430 Mus musculus adult retina cDNA, RIKEN full-length enriched library, clon ... 0014I21 product:hypothetical Ubiquitin-conjugating enzymes ... containing protein, full insert sequence gnl|UG|Mm ...

  11. cDNA: 35985 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.122430 Mus musculus adult male testis cDNA, RIKEN full-length enriched library, ... 0440H19 product:hypothetical Ubiquitin-conjugating enzymes ... containing protein, full insert sequence gnl|UG|Mm ...

  12. MKK3 Was Involved in Larval Settlement of the Barnacle Amphibalanus amphitrite through Activating the Kinase Activity of p38MAPK

    KAUST Repository

    Zhang, Gen

    2013-07-29

    The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway. © 2013 Zhang et al.

  13. cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data Description of data contents Phred's quality score. PHD format, one file to a single cDNA data, and co...ription Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive ...

  14. Levels of chromium contamination in the estuary of the Iraja river (Guanabara Bay) and experimental incorporation of 51Cr in barnacles (Balanus sp)

    International Nuclear Information System (INIS)

    Levels were determined of chromium contamination in the estuary of Iraja River, produced by an electroplating industry located 3 km upstream the study area. Uptake-and release kinetics of Cr(VI) and Cr(III) in barnacles (Balanus sp.) were studied. Samples of barnacles and suspended particles from Guanabara Bay were analysed. Chromium concentrations (dry weight) ranged from not detectable (ND) to 154,66 μg/g for soft tissues and from ND to 423,76 μg/g for suspended particles. Mean of maximum concentrations of chromium in samples from Guanabara Bay are 3 and 4 times above those of identical samples from control area (Coroa Grande). Soft tissues presented a concentration factor (CF) of 103 related to chromium available in suspended particles. 51Cr(VI) is preferentiably incorparated by soft tissues (biological half life being 100 days). Chromium uptake by Balanus sp from solution is as significant as it is from particulate matter available in sea water from experimental sets. CF for Cr(VI) in soft tissues in laboratory conditions was 102 related to 51Cr present in sea water. Environmental chromium contamination was found to be of the same order of magnitude or above levels reported for other areas subjected to industrial impacts. Barnacles appear to be able to accumulate chromium in soft tissues from the available metal in the environment. Cr(VI) is the critical form, being greatly accumulated in soft tissues of barnacles, that act as a long-term integrator of this metal. For Cr(III), this organism can only be regarded as an instantaneous indicator of environmental contamination of chromium attached to suspended particles. (M.A.)

  15. cDNA: 13750 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available H. sapiens + Hs.183373 Homo sapiens full open reading frame cDNA clone RZPDo834C1012D for gene HIP ... IP-55, src homology 3 domain-containing protein HIP -55; complete cds, incl. stopcodon gnl|UG|Hs#S20347 ...

  16. cDNA: 46042 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.290868 Mus musculus 12 days embryo spinal ganglion cDNA, RIKEN full-length enri ... , clone:D130020P04 product:target of myb1 homolog (chicken ), full insert sequence gnl|UG|Mm#S10839663 AK05124 ...

  17. cDNA: 37672 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.315626 Mus musculus adult male thymus cDNA, RIKEN full-length enriched library, ... 10 product:MODULATOR OF ANTIGEN RECEPTOR SIGNALING MARS , full insert sequence gnl|UG|Mm#S10847064 AK030877 ...

  18. cDNA: 37677 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.315626 Mus musculus adult retina cDNA, RIKEN full-length enriched library, clon ... 21 product:MODULATOR OF ANTIGEN RECEPTOR SIGNALING MARS , full insert sequence gnl|UG|Mm#S9072626 AK020837 ...

  19. cDNA: 37673 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.315626 Mus musculus 2 days neonate thymus thymic cells cDNA, RIKEN full-length ... 24 product:MODULATOR OF ANTIGEN RECEPTOR SIGNALING MARS , full insert sequence gnl|UG|Mm#S10833741 AK088672 ...

  20. cDNA: 53525 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.277582 Mus musculus 10 days neonate cerebellum cDNA, RIKEN full-length enriched ... rary, clone:B930059G06 product:hypothetical Serine proteases , subtilase family/Leucine-rich repeat containing p ...

  1. cDNA: 53519 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.277582 Mus musculus 0 day neonate eyeball cDNA, RIKEN full-length enriched libr ... ary, clone:E130306I01 product:hypothetical Serine proteases , subtilase family/Leucine-rich repeat containing p ...

  2. cDNA: 57842 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.1441 Mus musculus adult male spinal cord cDNA, RIKEN full-length enriched libra ... ry, clone:A330093C04 product:hypothetical Serine proteases , trypsin family/Chymotrypsin serine protease famil ...

  3. cDNA: 35980 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.122430 Mus musculus 0 day neonate cerebellum cDNA, RIKEN full-length enriched l ... 0027A22 product:hypothetical Ubiquitin-conjugating enzymes ... containing protein, full insert sequence gnl|UG|Mm ...

  4. cDNA: 35899 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.337238 Mus musculus 13 days embryo liver cDNA, RIKEN full-length enriched libra ... 0010F15 product:hypothetical Ubiquitin-conjugating enzymes ... containing protein, full insert sequence gnl|UG|Mm ...

  5. cDNA: 35982 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.122430 Mus musculus 14 days embryo thymus cDNA, RIKEN full-length enriched libr ... 0401J04 product:hypothetical Ubiquitin-conjugating enzymes ... containing protein, full insert sequence gnl|UG|Mm ...

  6. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    2000-10-03

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties.

  7. Construction of cDNA Library from Populus euphratica

    Institute of Scientific and Technical Information of China (English)

    Yu Guangjun; Wang Yiqin; Shen Xin

    2003-01-01

    In order to isolate and clone salt-tolerance involved genes of Populus euphratica, we constructed a cDNA library from salt-treated leaves of P. euphratica. In the experiment, double strand cDNA were synthesized by a beads-based method. The syntheses of the first strand and the second strand cDNA, adapter ligation and restriction reaction for releasing cDNA were all conducted on the beads. The double strand cDNA were released from magnetic beads by digestion with NotI, and cDNA fragments smaller than 500 bp and residual adapters were removed through cDNA size fractionation columns. Finally, double strand cDNA were directionally cloned intoλExcell vector. The results show that the primary titer of the cDNA library is 7.46×106 pfu per mL and the packaging efficiency reaches 1.47×107 recombinants per μg DNA. λDNA extracted from two clones of plaque were digested by EcoR I and NotI, both of the clones contained inserts larger than 900 bp. These results show that the cDNA library of salt-treated P. euphratica leaves has been successfully constructed.

  8. Influence of heme environment structure on dioxygen affinity for the dual function Amphitrite ornata hemoglobin/dehaloperoxidase. Insights into the evolutional structure-function adaptations

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Shengfang; Sono, Masanori; Wang, Chunxue; Du, Jing; Lebioda, Lukasz; Dawson, John H. [SC

    2014-05-15

    Sea worm, Amphitrite ornata, has evolved its globin (an O2 carrier) also to serves as a dehaloperoxidase (DHP) to detoxify haloaromatic pollutants generated by competing species. A previous mutagenesis study by our groups on both DHP and sperm whale myoglobin (SW Mb) revealed some structural factors that influence the dehaloperoxidase activities (significantly lower for Mb) of both proteins. Using an isocyanide/O2 partition constant measurement method in this study, we have examined the effects of these structural factors on the O2 equilibrium constants (KO2) of DHP, SW Mb, and their mutants. A clear trend of decreasing O2 affinity and increasing catalytic activity along with the increase in the distal His Nε–heme iron distance is observed. An H93K/T95H Mb double mutant mimicking the DHP proximal His positioning exhibited markedly enhanced O2 affinity, confirming the essential effect of proximal His rotation on the globin function of DHP. For DHP, the L100F, T56G and M86E variants showed the effects of distal volume, distal His flexibility and proximal electronic push, respectively, on the O2 affinity. This study provides insights into how DHP has evolved its heme environment to gain significantly enhanced peroxidase capability without compromising its primary function as an O2 carrier.

  9. Influence of heme environment structure on dioxygen affinity for the dual function Amphitrite ornata hemoglobin/dehaloperoxidase. Insights into the evolutional structure-function adaptations.

    Science.gov (United States)

    Sun, Shengfang; Sono, Masanori; Wang, Chunxue; Du, Jing; Lebioda, Lukasz; Dawson, John H

    2014-03-01

    Sea worm, Amphitrite ornata, has evolved its globin (an O(2) carrier) also to serves as a dehaloperoxidase (DHP) to detoxify haloaromatic pollutants generated by competing species. A previous mutagenesis study by our groups on both DHP and sperm whale myoglobin (SW Mb) revealed some structural factors that influence the dehaloperoxidase activities (significantly lower for Mb) of both proteins. Using an isocyanide/O(2) partition constant measurement method in this study, we have examined the effects of these structural factors on the O(2) equilibrium constants (KO2) of DHP, SW Mb, and their mutants. A clear trend of decreasing O(2) affinity and increasing catalytic activity along with the increase in the distal His N(ε)-heme iron distance is observed. An H93K/T95H Mb double mutant mimicking the DHP proximal His positioning exhibited markedly enhanced O(2) affinity, confirming the essential effect of proximal His rotation on the globin function of DHP. For DHP, the L100F, T56G and M86E variants showed the effects of distal volume, distal His flexibility and proximal electronic push, respectively, on the O(2) affinity. This study provides insights into how DHP has evolved its heme environment to gain significantly enhanced peroxidase capability without compromising its primary function as an O(2) carrier. PMID:24440609

  10. Cloning of Drosophila choline acetyltransferase cDNA.

    OpenAIRE

    Itoh, N; Slemmon, J.R.; Hawke, D.H.; Williamson, R.; Morita, E.; Itakura, K; Roberts, E; Shively, J. E.; Crawford, G D; Salvaterra, P M

    1986-01-01

    Choline acetyltransferase (EC 2.3.1.6) is the biosynthetic enzyme for the neurotransmitter acetylcholine. To isolate choline acetyltransferase cDNA clones, a cDNA library was constructed from poly(A)+ RNA of Drosophila melanogaster heads, these being one of the richest known sources of the enzyme. By screening the cDNA library with a mixture of three different monoclonal antibodies to Drosophila choline acetyltransferase, we isolated 14 positive clones. Only 1 of these clones was identified t...

  11. 5'-end sequences of budding yeast full-length cDNA clones - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones Data detail Data name 5'-end sequence...s of budding yeast full-length cDNA clones Description of data contents cDNA sequence...e Update History of This Database Site Policy | Contact Us 5'-end sequences of budding yeast full-length cDNA clones - Budding yeast cDNA sequencing project | LSDB Archive ...

  12. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

    Konieczny, A; Jensen, E O; Marcker, K A;

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... specific for nodules were selected by differential colony hybridization using 32P-labeled cDNA synthesized either from nodule poly(A)+ RNA or from poly(A)+ RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  13. cDNA cloning and sequencing of ostrich Growth hormone

    Directory of Open Access Journals (Sweden)

    Doosti Abbas

    2012-01-01

    Full Text Available In recent years, industrial breeding of ostrich (Struthio camelus has been widely developed in Iran. Growth hormone (GH is a peptide hormone that stimulates growth and cell reproduction in different animals. The aim of this study was to clone and sequence the ostrich growth hormone gene in E. coli, done for the first time in Iran. The cDNA that encodes ostrich growth hormone was isolated from total mRNA of the pituitary gland and amplified by RT-PCR using GH specific PCR primers. Then GH cDNA was cloned by T/A cloning technique and the construct was transformed into E. coli. Finally, GH cDNA sequence was submitted to the GenBank (Accession number: JN559394. The results of present study showed that GH cDNA was successfully cloned in E. coli. Sequencing confirmed that GH cDNA was cloned and that the length of ostrich GH cDNA was 672 bp; BLAST search showed that the sequence of growth hormone cDNA of the ostrich from Iran has 100% homology with other records existing in GenBank.

  14. High-Throughput Plasmid cDNA Library Screening

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, SusanE.

    2006-05-24

    Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.

  15. RECOGNITION OF CDNA MICROARRAY IMAGE USING FEEDFORWARD ARTIFICIAL NEURAL NETWORK

    Directory of Open Access Journals (Sweden)

    R. M. Farouk

    2014-07-01

    Full Text Available The complementary DNA (cDNA sequence considered th e magic biometric technique for personal identification. Microarray image processing used fo r the concurrent genes identification. In this pape r, we present a new method for cDNA recognition based on the artificial neural network (ANN. We have segmented the location of the spots in a cDNA micro array. Thus, a precise localization and segmenting of a spot are essential to obtain a more exact intensity measurement, leading to a more accurate gene expression measurement. The segmented cDNA microarr ay image resized and used as an input for the proposed artificial neural network. For matching an d recognition, we have trained the artificial neura l network. Recognition results are given for the gall eries of cDNA sequences . The numerical results sho w that, the proposed matching technique is an effecti ve in the cDNA sequences process. The experimental results of our matching approach using different da tabases shows that, the proposed technique is an effective matching performance.

  16. RECOGNITION OF CDNA MICROARRAY IMAGE USING FEEDFORWARD ARTIFICIAL NEURAL NETWORK

    Directory of Open Access Journals (Sweden)

    R. M. Farouk

    2014-09-01

    Full Text Available The complementary DNA (cDNA sequence considered the magic biometric technique for personal identification. Microarray image processing used for the concurrent genes identification. In this paper, we present a new method for cDNA recognition based on the artificial neural network (ANN. We have segmented the location of the spots in a cDNA microarray. Thus, a precise localization and segmenting of a spot are essential to obtain a more exact intensity measurement, leading to a more accurate gene expression measurement. The segmented cDNA microarray image resized and used as an input for the proposed artificial neural network. For matching and recognition, we have trained the artificial neural network. Recognition results are given for the galleries of cDNA sequences . The numerical results show that, the proposed matching technique is an effective in the cDNA sequences process. The experimental results of our matching approach using different databases shows that, the proposed technique is an effective matching performance.

  17. Amphitrite ornata Dehaloperoxidase (DHP): Investigations of Structural Factors That Influence the Mechanism of Halophenol Dehalogenation Using ;Peroxidase-like; Myoglobin Mutants and ;Myoglobin-like; DHP Mutants

    Energy Technology Data Exchange (ETDEWEB)

    Du, Jing; Huang, Xiao; Sun, Shengfang; Wang, Chunxue; Lebioda, Lukasz; Dawson, John H. (SC)

    2012-05-14

    Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O{sub 2} transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity {approx}10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of 'peroxidase-like' Mb mutants and 'Mb-like' DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned {approx}0.3 and {approx}0.8 {angstrom}, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the 'DHP-like' position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the

  18. Amphitrite ornata dehaloperoxidase (DHP): investigations of structural factors that influence the mechanism of halophenol dehalogenation using "peroxidase-like" myoglobin mutants and "myoglobin-like" DHP mutants.

    Science.gov (United States)

    Du, Jing; Huang, Xiao; Sun, Shengfang; Wang, Chunxue; Lebioda, Lukasz; Dawson, John H

    2011-09-27

    Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O(2) transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity ∼10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of "peroxidase-like" Mb mutants and "Mb-like" DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned ∼0.3 and ∼0.8 Å, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the "DHP-like" position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the molecular evolution of the dual-function enzyme DHP. PMID

  19. Cloning and expression analysis of MBLL cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The mbl (muscleblind) gene of Drosophila encodes a nuclear protein which contains two Cys3His motifs. The mutation of mbl gene will disturb the differentiation of all the Drosophila's photoreceptors. Primers have been designed according to human EST086139, which is highly homologous to mbl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designated MBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys3His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology to Drosophila's mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show that MBLL is a widely expressed gene, but the expression amounts differ in these tissues.

  20. cDNA microarray screening in food safety

    International Nuclear Information System (INIS)

    The cDNA microarray technology and related bioinformatics tools presents a wide range of novel application opportunities. The technology may be productively applied to address food safety. In this mini-review article, we present an update highlighting the late breaking discoveries that demonstrate the vitality of cDNA microarray technology as a tool to analyze food safety with reference to microbial pathogens and genetically modified foods. In order to bring the microarray technology to mainstream food safety, it is important to develop robust user-friendly tools that may be applied in a field setting. In addition, there needs to be a standardized process for regulatory agencies to interpret and act upon microarray-based data. The cDNA microarray approach is an emergent technology in diagnostics. Its values lie in being able to provide complimentary molecular insight when employed in addition to traditional tests for food safety, as part of a more comprehensive battery of tests

  1. Restriction landmark cDNA scanning (RLCS): a novel cDNA display system using two-dimensional gel electrophoresis.

    Science.gov (United States)

    Suzuki, H; Yaoi, T; Kawai, J; Hara, A; Kuwajima, G; Wantanabe, S

    1996-01-01

    We have developed a new method, designated restriction landmark cDNA scanning (RLCS), which displays many cDNA species quantitatively and simultaneously as two-dimensional gel spots. In this method cDNA species of uniform length were prepared for each mRNA species using restriction enzymes. After the restriction enzyme sites were radiolabeled as landmarks, the labeled fragments were subjected to high resolution two-dimensional gel electrophoresis. In analyses of cDNA samples from adult mouse liver and brain (cerebral cortex, cerebellum and brain stem) we detected approximately 500 and >1000 discrete gel spots respectively of various intensities at a time. The spot patterns of the three brain regions were very similar, although not identical, but were quite different from the pattern for the liver. RNA blot hybridization analysis using several cloned spot DNAs as probes showed that differences in intensity of the spots among RLCS profiles correlated well with expression levels of the corresponding mRNA species in the brain regions. Because the spots and their intensities reflect distinct mRNA species and their expression level respectively, the RLCS is a novel cDNA display system which provides a great deal of information and should be useful for systematic documentation of differentially expressed genes. PMID:8628652

  2. Rice8987 g_array: cDNA information: 4866 [

    Lifescience Database Archive (English)

    Full Text Available g_4866 SS3984 >CELZC13_4(U67953|pid:g1519683) Caenorhabditis elegans cosmid ZC13; weak similarit ... y to EGF-like domain cysteine pattern signature (PS :PS 00022); coded for by C. elegans cDNA yk70d5.3; c ...

  3. cDNA sequence for human erythrocyte ankyrin

    International Nuclear Information System (INIS)

    The cDNA for human erythrocyte ankyrin has been isolated from a series of overlapping clones obtained from a reticulocyte cDNA library. The composite cDNA sequence has a large open reading frame of 5636 base pairs (bp) with the complete coding sequence for a polypeptide of 1879 amino acids with a predicted molecular mass of 206 kDa. The derived amino acid sequence contained 194 residues that were identical to those obtained by direct amino acid sequencing of 11 ankyrin proteolytic peptides. The primary sequence contained 23 highly homologous repeat units of 33 amino acids within the 90-kDa band 3 binding domain. Two cDNA clones showed evidence of apparent mRNA processing, resulting in the deletions of 486 bp and 135 bp, respectively. The 486-bp deletion resulted in the removal of a 16-kDa highly acidic peptide, and the smaller deletion had the effect of altering the COOH terminus of the molecule. Radiolabeled ankyrin cDNAs recognized two erythroid message sizes by RNA blot analysis, one of which was predominantly associated with early erythroid cell types. An ankyrin message was also observed in RNA from the human cerebellum by the same method. The ankyrin gene is assigned to chromosome 8 using genomic DNA from a panel of sorted human chromosomes

  4. cDNA expression cloning in mammalian cells.

    Science.gov (United States)

    Hoffman, B J

    2001-05-01

    This unit contains protocols for expression cloning in mammalian cells. Either calcium phosphate- or liposome-mediated transfection of mammalian cells, or virus infection and liposome-mediated transfection are used to screen pools derived from a cDNA library. cDNA pools are prepared for cloning from library-transformed E. coli grown in liquid culture medium or on antibiotic-containing selection plates. Results of screening assays for expression can be detected using autoradiography of dishes of cultured cells to identify clones, direct visualization of radiolabeled cells on emulsion-coated and developed chamber slides, detection and quantification of gene activity by a functional (transport) assay with scintillation counting, or detection using a filter-based assay for binding of radioligand to membranes or whole cells. The most critical step of any cDNA cloning project is the establishment of the screening protocol. Therefore, the bioassay for the gene product must be established prior to executing any of these protocols, including construction of the cDNA library. PMID:18428491

  5. The function analysis of full-length cDNA sequence from IRM-2 mouse cDNA library

    International Nuclear Information System (INIS)

    Objective: To identify the function of full-length cDNA sequence from IRM-2 mouse cDNA library. Methods: Full-length cDNA products were amplified by PCR from IRM-2 mouse cDNA library according to twenty-one pieces of expressed sequence tag. The expression of full-length cDNAs were detected after mouse embryonic fibroblasts were exposed to 6.5 Gy γ-ray radiation. And the effect on the growth of radiosensitivity cells AT5B1VA transfected with full-length cDNAs was investigated. Results: The expression of No.4, 5 and 2 full-length cDNAs from IRM-2 mouse were higher than that of parental ICR and 615 mouse after mouse embryonic fibroblasts irradiated with γ-ray radiation. And the survival rate of AT5B1VA cells transfected with No.4, 5 and 2 full-length cDNAs was high. Conclusion: No.4, 5 and 2 full-length cDNAs of IRM-2 mouse are of high radioresistance. (authors)

  6. Construction of cDNA representational difference analysis based on two cDNA libraries and identification of garlic inducible expression genes in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong Li; Lin Yang; Jian-Tao Cui; Wen-Mei Li; Rui-Fang Guo; You-Yong Lu

    2002-01-01

    AIM: To elucidate molecular mechanism of chemopreventiveefficacies of garlic against human gastric cancer (HGC):METHODS: HGC cell line BGC823 was treated with Allitridi (akind of garlic extract) and Allitridi-treated and parentalBGC823 cDNA librarles were constructed respectively byusing λZAP Ⅱ vector. cDNA Representatinal DifferenceAnalysis (cDNA RDA) was perfonmed using BamH Ⅰ cutting-site and abundant ~DNA messages provided by the Iibrarles.Northern blot analysls was applied to identifythe obtaineddifference prnducts.RESULTS: Two specific cDNA fragments were obtained andcharacterized to be derived from homo sapiens folatereceptorα (FRα) gene and calcyclin gene respectively.Northern blot results showed a 4-fold increase in FRα geneexpression level and 9-fold increase in calcyclin mRNA levelin BGC823 cells after Allilridi treatment for 72 h.CONCLUSION: The method of cDNA RDA based on cDNAlibraries combines the high specificity of cDNA RDA withabundant cDNA messages in cDNA library; this expands theapplication of cDNA library and increases the specificity ofcDNA RDA. Up-regulstion of FRα gene and calcyclin geneexpressions induced by Allitridi provide valuable molecularevidence for theefficacy of garlic in treating HGC as well asother diseases.

  7. Analysis of proteins encoded by full-length cDNA sequence from IRM-2 mouse

    International Nuclear Information System (INIS)

    Objective: To screen and isolate radioresistance-related genes from IRM-2 mouse. Methods: Full-length cDNA products were amplified by PCR from IRM-2 mouse cDNA library according to twenty-one pieces of expressed sequence tags. The property of proteins encoded by full-length cDNA were analyzed by comparing with GenBank database. Results: Five pieces of full-length cDNA which were not the same source as the known mice genes were found out from IRM-2 mouse cDNA library.Amino acid sequence and property of proteins encoded by these five pieces of full-length cDNA were obtained. Conclusion: Proteins encoded by full-length cDNA imply that unknown radioresistance-related genes may exist in IR M-2 mouse. (authors)

  8. Cloning and prokaryotic expression of HGLP cDNA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A novel cDNA, HGLP, which encodes a G- protein coupled receptor (GPCR) like protein, has been isolated and cloned. The coding region of the human HGLP predicts a 7-transmembrane region protein with motifs of rhodopsin-like GPCR superfamily. Northern blot analysis reveals a 3-kb transcript in various human tissues examined. The N- and C-terminal coding regions of HGLP, which are deduced as non-transmembrane regions, have been amplified by PCR and cloned into pET30a+ vector. Then the recombinant proteins are highly expressed in E. coli.

  9. Molecular cloning of human interferon cDNA.

    OpenAIRE

    Taniguchi, T.; Fujii-Kuriyama, Y; Muramatsu, M

    1980-01-01

    A hybrid plasmid, TpIF319, has been shown to contain the sequence for human fibroblast interferon mRNA [Taniguchi, T., Sakai, M., Fujii-Kuriyama, Y., Muramatsu, M., Kobayashi, S. & Sudo, T. (1979) Proc. Jpn. Acad. 55, Ser. B, 464-469]. This conclusion was confirmed by a hybridization-translation assay, using rabbit globin mRNA and its cDNA-containing plasmid as a control. Plasmid TpIF319 was used as probe and another recombinant plasmid, TpIF319-13, whose cDNA insert consists of about 800 bas...

  10. Molecular cloning of cDNA for human prothymosin alpha.

    OpenAIRE

    Goodall, G J; Dominguez, F.; Horecker, B L

    1986-01-01

    A cDNA library was constructed from human spleen mRNA and screened for clones containing cDNAs coding for prothymosin alpha. A clone containing a 503-base-pair insert including the entire coding sequence for the translated portion of the mRNA was isolated. The deduced amino acid sequence confirms and completes the partial sequence of human prothymosin alpha determined by protein sequencing methods. The presence of an initiator codon immediately preceding the codon for the NH2-terminal serine ...

  11. Automation of cDNA Synthesis and Labelling Improves Reproducibility

    Directory of Open Access Journals (Sweden)

    Daniel Klevebring

    2009-01-01

    Full Text Available Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.

  12. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen;

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 k...

  13. Molecular cloning of growth hormone encoding cDNA of Indian major carps by a modified rapid amplification of cDNA ends strategy

    Indian Academy of Sciences (India)

    T Venugopal; S Mathavan; T J Pandian

    2002-06-01

    A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in Escherichia coli. These sequences show 96–98% homology to each other and are about 85% homologous to that of common carp. Besides, an attempt has been made for the first time to describe a 3-D model of the fish GH protein.

  14. Cloning and expression of cDNA for salmon growth hormone in Escherichia coli

    OpenAIRE

    Sekine, Susumu; Mizukami, Tamio; Nishi, Tatsunari; Kuwana, Yoshihisa; Saito, Akiko; Sato, Moriyuki; Itoh, Seiga; Kawauchi, Hiroshi

    1985-01-01

    cDNA clones encoding chum salmon (Oncorhynchus keta) growth hormone (sGH) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A)+ RNA. Synthetic oligodeoxynucleotide mixtures based on amino acid residues 23-28 of sGH were used as hybridization probes to select recombinant plasmids carrying the sGH coding sequence. The complete nucleotide sequence of sGH cDNA has been determined. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putativ...

  15. Sequence analysis of a cDNA coding for a pancreatic precursor to somatostatin.

    OpenAIRE

    Taylor, W.L.; Collier, K J; Deschenes, R J; Weith, H L; Dixon, J. E.

    1981-01-01

    A synthetic oligonucleotide having the sequence d(T-T-C-C-A-G-A-A-G-A-A) deduced from the amino acid sequence Phe-Phe-Trp-Lys of somatostatin-14 was used to prime the synthesis of a cDNA from channel catfish (Ictalurus punctatus) pancreatic poly(A)-RNA. The major product of this reaction was a cDNA fragment of 565 nucleotides. Chemical sequence analysis of the cDNA fragment revealed that it was complementary to a mRNA coding for somatostatin. The 565-nucleotide cDNA hybridizes strongly with a...

  16. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

    International Nuclear Information System (INIS)

    The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [32P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

  17. THE CLONING OF HRNT-1 USING A COMBINATION OF cDNA LIBRARY SCREENING WITH BIOTIN-LABELED PROBE AND RAPID AMPLIFICATION OF cDNA ENDS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To clone the human counterpart of rat ZA73, EST cloned from rat tracheal epithelial (RTE) neoplastic transformed cell model induced by (a-particles radiation by using mRNA differential display. Methods: According to the sequence of rat ZA73, a probe was biotin-labeled to screen human cDNA library, and then the gene sequence was extended by RACE (rapid amplification of cDNA ends). Result: Human gene HRNT-1 (GenBank Accession Number: AF223393) is 4.256 kb in length, with an ORF located in the region between 254 and 3013 bp. 5' UTS (untranslated sequences) is 253 bp, 3' UTS is 1243 bp. Conclusion: The combination of cDNA library screening with biotin-labeled probes and RACE is an effective method to clone full-length cDNA, especially for sequences longer than 2 kb.

  18. Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

    Indian Academy of Sciences (India)

    Vikas Anathy; Thayanithy Venugopal; Ramanathan Koteeswaran; Thavamani J Pandian; Sinnakaruppan Mathavan

    2001-09-01

    A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence of H. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.

  19. Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

    Indian Academy of Sciences (India)

    Vikas Anathy; Thayanithy Venugopal; Ramanathan Koteeswaran; Thavamani J Pandian; Sinnakaruppan Mathavan

    2013-03-01

    A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence of H. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.

  20. Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA

    International Nuclear Information System (INIS)

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-Km, low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. The authors isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus

  1. Vector sequences - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project Vector sequences Data detail Data name Vector sequences Description of data contents Vector seq...wnload License Update History of This Database Site Policy | Contact Us Vector sequences - Budding yeast cDNA sequencing project | LSDB Archive ... ...uences used for sequencing. Multi FASTA format. 7 entries. Data file File name: vec

  2. Construction and characterization of a normalized whole-life-cycle cDNA library of rice

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A cDNA library with genomic complete coverage is a powerful tool for functional genomic studies. For studying the functions of rice genes on a large scale, a normalized whole-life-cycle cDNA library is constructed based on the strategy of saturation hybridization with genomic DNA using rice cultivar Minghui 63, an elite restorer line for a number of rice hybrids that are widely cultivated in China. This library consists of cDNA from 15 directionally cloned cDNA libraries constructed with different tissues from 9 developmental stages. For normalization, the denatured plasmids purified from the 15 directionally cloned libraries are mixed and hybridized with saturated genomic DNA labeled with magnetic beads in two complementary systems. Well-matched plasmids are captured from the hybridized genomic DNA and electroporated into competent DH10B E. coli for construction of the normalized whole-life-cycle cDNA library. This library consists of 62000 clones with an average insert length about 1.4 kb. Inverse Northern blotting shows that this cDNA library included many rarely expressed genes and tissue-specific genes. Sequencing of 10750 cDNA clones of this library reveals 6399 unique ESTs (expressed sequence tags), indicating that the non-redundancy of the library is about 59.5%. This library has been used to make cDNA microarrays for functional genomic studies.

  3. Molecular cloning and characterization of duck hepatitis virus cDNA

    International Nuclear Information System (INIS)

    The aim was to determine the complete nucleotide sequence of duck hepatitis virus (DHV) and compare this sequence with that of the other picornaviruses. The DHV cDNA has been cloned, which will enable us to demonstrate the genome structure of the DHV RNA and the translation mechanisms of the viral protein. Also, we can use the cDNA probes to detect DHV in clinical samples. DHV cDNA has been cloned with a size of the insert fragments of 0.5-0.7 kb. The cDNA clones were authenticated by northern hybridization. The availability of cloned DHV cDNA should make it possible to deduce RNA and amino acid sequences, both for use as a probe of clinical specimens and as to the presence of DHV genome

  4. Cloning and bioinformatics analysis of cDNA encoding cattle Smad4 gene

    Institute of Scientific and Technical Information of China (English)

    Xiaohui ZHANG; Shangzhong XU; Xue GAO; Hongyan REN; Jinbao CHEN

    2008-01-01

    The cDNA of cattle Smad4 gene was cloned by RT-PCR, 3' RACE and 5' RACE and got a 3503-bp full-long cDNA sequence. The cloned cattle Smad4 cDNA sequence had been send to GenBank and got an accession number: DQ494856. Cattle Smad4 gene consists of 12 exons and codes 553 amino acids. Cattle Smad4 cDNA shares 99%, 96%, 95%, 91% and 91% similarity in nucleic acid sequences, and 99%, 98%, 98%, 99% and 98% sim-ilarity in amino acid sequences with sheep, pig, human, rat and mouse, respectively. Smad4 cDNA was found in the testes, pancreas, liver, small intestine, ovary, lymph, car-diac muscle, skeleton muscle and thymus gland, which indicated that Smad4 was broadly expressed in cattle.

  5. CDNA library from the Latex of Hevea brasiliensis

    Directory of Open Access Journals (Sweden)

    Wilaiwan Chotigeat

    2010-12-01

    Full Text Available Latex from Hevea brasiliensis contains 30-50% (w/w of natural rubber (cis-1,4-polyisoprene, the important rawmaterial for many rubber industries. We have constructed a cDNA library from the latex of H. brasiliensis to investigate theexpressed genes and molecular events in the latex. We analyzed 412 expressed sequence tags (ESTs. More than 90% of theEST clones showed homology to previously described sequences in public databases. Functional classification of the ESTsshowed that the largest category were proteins of unknown function (30.1%, 11.4% of ESTs encoded for rubber synthesisrelatedproteins (RS and 8.5% for defense or stress related proteins (DS. Those with no significant homology to knownsequences (NSH accounted for 8.7%, primary metabolism (PM and gene expression and RNA metabolism were 7.8% and6.6%, respectively. Other categories included, protein synthesis-related proteins (6.6%, chromatin and DNA metabolism(CDM 3.9%, energy metabolism (EM 3.4%, cellular transport (CT 3.2%, cell structure (CS 3.2%, signal transduction (ST2.2%, secondary metabolism (SM 1.7%, protein fate (PF 2.2%, and reproductive proteins (RP 0.7%.

  6. Random rapid amplification of cDNA ends (RRACE) allows for cloning of multiple novel human cDNA fragments containing (CAG)n repeats.

    Science.gov (United States)

    Carney, J P; McKnight, C; VanEpps, S; Kelley, M R

    1995-04-01

    We describe a new technique for isolating cDNA fragments in which (i) either a partial sequence of the cDNA is known or (ii) a repeat sequence is utilized. We have used this technique, termed random rapid amplification of cDNA ends (random RACE), to isolate a number of trinucleotide repeat (CAG)n-containing genes. Using the random RACE (RRACE) technique, we have isolated over a hundred (CAG)n-containing genes. The results of our initial analysis of ten clones indicate that three are identical to previously cloned (CAG)n-containing genes. Three of our clones matched with expressed sequence tags, one of which contained a CA repeat. The remaining four clones did not match with any sequence in GenBank. These results indicate that this approach provides a rapid and efficient method for isolating trinucleotide repeat-containing cDNA fragments. Finally, this technique may be used for purposes other than cloning repeat-containing cDNA fragments. If only a partial sequence of a gene is known, our system, described here, provides a rapid and efficient method for isolating a fragment of the gene of interest. PMID:7536696

  7. XPB mediated retroviral cDNA degradation coincides with entry to the nucleus

    International Nuclear Information System (INIS)

    Retroviruses must integrate their cDNA to a host chromosome, but a significant fraction of retroviral cDNA is degraded before integration. XPB and XPD are part of the TFIIH complex which mediates basal transcription and DNA nucleotide excision repair. Retroviral infection increases when XPB or XPD are mutant. Here we show that inhibition of mRNA or protein synthesis does not affect HIV cDNA accumulation suggesting that TFIIH transcription activity is not required for degradation. Other host factors implicated in the stability of cDNA are not components of the XPB and XPD degradation pathway. Although an increase of retroviral cDNA in XPB or XPD mutant cells correlates with an increase of integrated provirus, the integration efficiency of pre-integration complexes is unaffected. Finally, HIV and MMLV cDNA degradation appears to coincide with nuclear import. These results suggest that TFIIH mediated cDNA degradation is a nuclear host defense against retroviral infection.

  8. RICD: A rice indica cDNA database resource for rice functional genomics

    Directory of Open Access Journals (Sweden)

    Zhang Qifa

    2008-11-01

    Full Text Available Abstract Background The Oryza sativa L. indica subspecies is the most widely cultivated rice. During the last few years, we have collected over 20,000 putative full-length cDNAs and over 40,000 ESTs isolated from various cDNA libraries of two indica varieties Guangluai 4 and Minghui 63. A database of the rice indica cDNAs was therefore built to provide a comprehensive web data source for searching and retrieving the indica cDNA clones. Results Rice Indica cDNA Database (RICD is an online MySQL-PHP driven database with a user-friendly web interface. It allows investigators to query the cDNA clones by keyword, genome position, nucleotide or protein sequence, and putative function. It also provides a series of information, including sequences, protein domain annotations, similarity search results, SNPs and InDels information, and hyperlinks to gene annotation in both The Rice Annotation Project Database (RAP-DB and The TIGR Rice Genome Annotation Resource, expression atlas in RiceGE and variation report in Gramene of each cDNA. Conclusion The online rice indica cDNA database provides cDNA resource with comprehensive information to researchers for functional analysis of indica subspecies and for comparative genomics. The RICD database is available through our website http://www.ncgr.ac.cn/ricd.

  9. Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA

    International Nuclear Information System (INIS)

    Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. λ Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC)

  10. Genetic instability of Japanese encephalitis virus cDNA clones propagated in Escherichia coli.

    Science.gov (United States)

    Zheng, Xuchen; Tong, Wu; Liu, Fei; Liang, Chao; Gao, Fei; Li, Guoxin; Tong, Guangzhi; Zheng, Hao

    2016-04-01

    The genetic instability of Flavivirus cDNA clones in transformed bacteria is a common phenomenon. Herein, a cDNA fragment of the nucleotide (nt) 1-2913 of the genome of a flavivirus, Japanese encephalitis virus (JEV), was used to investigate factors that caused the instability of cDNA clones. Several cDNA fragments with different 5'- or 3'-termini of the 2913-nt cDNA were obtained by PCR amplification or restriction enzyme digestion and cloned into a pCR-Blunt II-TOPO vector. All the cDNA fragments were stably propagated at 25 °C. However, the 5'-untranslated region and half of the 3'-E gene could cause the instability of the 2913-nt cDNA at 37 °C. The 5'-terminus sequences of the 2913-nt fragment were subjected to testing of the prokaryotic promoter activity by luciferase assay and Western blot. The sequences of 54-120 nt of the JEV genome exhibited high prokaryotic promoter activity at 37 °C, and the activity declined markedly at 25 °C. These findings revealed that the high prokaryotic promoter activity of the 54-120 nt sequences of the JEV genome together with expression of JEV structural genes determined the instability of a JEV cDNA clone. Growth at room temperature may reduce the prokaryotic promoter activity of 5'-sequences of the JEV genome and could represent an effective way to improve the stability of flavivirus cDNA clones in host bacteria. PMID:26888374

  11. Expression cloning of a cDNA encoding the bovine histamine H1 receptor.

    OpenAIRE

    Yamashita, M; Fukui, H; Sugama, K; Horio, Y; Ito, S.; Mizuguchi, H.; Wada, H

    1991-01-01

    A functional cDNA clone for the histamine H1 receptor was isolated from a cDNA library of bovine adrenal medulla by a combination of molecular cloning in an expression vector and electrophysiological assay in Xenopus oocytes. The H1 receptor cDNA encodes a protein of 491 amino acids (Mr 55,954) with seven putative transmembrane domains, illustrating the similarity to other receptors that couple with guanine nucleotide-binding regulatory proteins (G protein-coupled receptors). The sequence hom...

  12. Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs.

    OpenAIRE

    Meyers, G; Tautz, N.; Becher, P; Thiel, H J; Kümmerer, B M

    1997-01-01

    After cDNA cloning of the genome of bovine viral diarrhea virus (BVDV) isolate CP7, a full-length cDNA clone was constructed. RNA transcribed in vitro from this construct was shown to direct the generation of infectious BVDV upon transfection into bovine cells. To confirm the de novo generation of infectious BVDV from cloned cDNA a genetically tagged virus was constructed. In comparison with parental BVDV, the recombinant virus was slightly retarded in growth. The NS2 coding region of the CP7...

  13. Molecular cloning of human interleukin 2 cDNA and its expression in E. coli.

    OpenAIRE

    Devos, René; Plaetinck, Geert; Cheroutre, Hilde; Simons, Guus; Degrave, Wim,; Tavernier, Jan; Remaut, Erik; Fiers, Walter

    1983-01-01

    A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal seq...

  14. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon [College of Medicine, Univ. of Korea, Seoul (Korea, Republic of)

    2003-07-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  15. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    International Nuclear Information System (INIS)

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology

  16. cDNA cloning and gene expression of ascorbate oxidase in tobacco.

    Science.gov (United States)

    Kato, N; Esaka, M

    1996-02-01

    A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Nothern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant. PMID:8624413

  17. Quantitative Transcript Analysis in Plants: Improved First-strand cDNA Synthesis

    Institute of Scientific and Technical Information of China (English)

    Nai-Zhong XIAO; Lei BA; Preben Bach HOLM; Xing-Zhi WANG; Steve BOWRA

    2005-01-01

    The quantity and quality of first-strand cDNA directly influence the accuracy of transcriptional analysis and quantification. Using a plant-derived α-tubulin as a model system, the effect of oligo sequence and DTT on the quality and quantity of first-strand cDNA synthesis was assessed via a combination of semi-quantitative PCR and real-time PCR. The results indicated that anchored oligo dT significantly improved the quantity and quality of α-tubulin cDNA compared to the conventional oligo dT. Similarly, omitting DTT from the first-strand cDNA synthesis also enhanced the levels of transcript. This is the first time that a comparative analysis has been undertaken for a plant system and it shows conclusively that small changes to current protocols can have very significant impact on transcript analysis.

  18. Construction of infectious cDNA clones for RNA viruses: Turnip crinkle virus.

    Science.gov (United States)

    Ryabov, Eugene V

    2008-01-01

    Reverse genetic approach is widely used in virology as it makes possible direct identification of viral gene function and uses RNA genomes as vectors. Production of infectious cDNA clones is an essential step in developing a reverse genetic system for an RNA virus. Here, we present rapid method for generation of infectious cDNA clone for Turnip crinkle virus (TCV). The infectious cDNA clone could be used for production of in vitro transcripts with the T7 RNA polymerase which could be used for infection of plants or plant cell protoplasts. The procedure described here includes purification of TCV, viral RNA extraction, reverse transcription, PCR amplification of the full-length cDNA copy of TCV linked to a T7 RNA polymerase promoter, cloning into a plasmid vector, in vitro transcription, and selection of infectious clones. PMID:18370276

  19. Construction and analysis of full-lengh and normalized cDNA libraries from citrus

    OpenAIRE

    Marqués, M.Carmen; Pérez-Amador, Miguel A.

    2012-01-01

    We have developed an integrated method to generate a normalized cDNA collection enriched in full-length and rare transcripts from citrus, using different species and multiple tissues and developmental stages. Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. In this regard, the availability of full-length cDNA clones facilitates functional an...

  20. Cloning and expression of human neuron-specific enolase cDNA in Escherichia coli.

    Science.gov (United States)

    Pavlov, K A; Gurina, O I; Antonova, O M; Semenova, A V; Chekhonin, V P

    2011-12-01

    cDNA fragment encoding neuron-specific enolase was amplified from the cDNA library of human brain. Then the fragment was cloned for expression in E. coli using the vector pET28-a. High level of neuron-specific enolase expression was confirmed by SDS-PAAG electrophoresis and immunochemical identity by immunoblot analysis. The constructed producer strain is the cheapest source of neuron-specific enolase suitable for the use in diagnostic applications. PMID:22808461

  1. Expression of hepatitis A virus cDNA in Escherichia coli: antigenic VP1 recombinant protein.

    OpenAIRE

    Ostermayr, R; von der Helm, K; Gauss-Müller, V; Winnacker, E L; Deinhardt, F.

    1987-01-01

    The genome of hepatitis A virus (HAV) was reverse transcribed into cDNA and molecularly cloned. cDNA clones coding for the capsid protein VP1 that carries the major HAV antigen were cloned into the expression vector pUR290 and expressed in Escherichia coli. The recombinant fusion protein reacted in an immunoblot with rabbit anti-HAV serum, suggesting that it possesses HAV antigenicity.

  2. Construction and partial characterization of two recombinant cDNA clones for procollagen from chicken cartilage.

    OpenAIRE

    Vuorio, E; Sandell, L; Kravis, D; Sheffield, V C; Vuorio, T; Dorfman, A.; Upholt, W B

    1982-01-01

    Type II procollagen mRNA has been partially purified from embryonic chick sternal cartilage by guanidine hydrochloride extraction, sucrose gradient sedimentation and Sepharose 4B chromatography. Double stranded cDNA was synthesized using AMV reverse transcriptase and E. coli DNA polymerase I, tailed using terminal transferase and inserted into the Pst I site of pBR322. Two putative type II procollagen cDNA clones have been characterized. Both plasmids hybridize to 2 sternal RNA species, a maj...

  3. First characterization of infectious cDNA clones of Olive mild mosaic virus

    OpenAIRE

    Cardoso, Joana M.S.; Félix, M. Rosário; Clara, M.Ivone; Oliveira, Solange

    2012-01-01

    Full-length cDNA clones of an Olive mild mosaic virus (OMMV) isolate were constructed in order to find infectious cDNA clones. The sequencing of three individual full-length clones revealed some differences between them. In vitro transcription of these clones was performed and the effect of spontaneous mutations in the biological behaviour of the in vitro transcripts was evaluated by symptomatology, RNA accumulation and virus replication in inoculated plants. In vitro synthesized RNA from one...

  4. Cloning vectors for expression of cDNA libraries in mammalian cells.

    OpenAIRE

    Murphy, A.J.; Efstratiadis, A

    1987-01-01

    We have constructed a series of compound cloning vectors (lambda ZD vectors), each consisting of phage lambda arms carrying a modified version of the retroviral expression vector pZIP-neoSV (x)1. cDNA, inserted into a cloning site present in the retroviral vector component, is cloned with high efficiency using the lambda system. A cDNA library in plasmids is then released by homologous recombination between the retroviral long terminal repeats. Retroviral transduction is achieved by transient...

  5. Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones

    OpenAIRE

    Halgren, Robert G.; Fielden, Mark R.; Fong, Cora J.; Zacharewski, Timothy R

    2001-01-01

    This report documents the error rate in a commercially distributed subset of the IMAGE Consortium mouse cDNA clone collection. After isolation of plasmid DNA from 1189 bacterial stock cultures, only 62.2% were uncontaminated and contained cDNA inserts that had significant sequence identity to published data for the ordered clones. An agarose gel electrophoresis pre-screening strategy identified 361 stock cultures that appeared to contain two or more plasmid species. Is...

  6. Mouse Peroxisomal Protein cDNA Cloning and Characterization of its Intraclleular Localization

    Directory of Open Access Journals (Sweden)

    Somayeh Tanhaie

    2009-01-01

    Full Text Available Objective: The aim of this study was to clone peroxisomal protein (PEP cDNA in a mammalianexpression vector in a chimeric cDNA type, with enhanced green fluorescent protein(EGFP cDNA. To investigate the intracellular localization of PEP protein linked to EGFPmarker, the constructed plasmid was used for transfection into the chinese hamster ovary(CHO cells.Materials and Methods: Total RNA was extracted from the heart tissue of an adult mouse.PEP cDNA was constructed using reverse transcriptase and was amplified with specific primerscovering the entire length of ORF. RT-PCR products containing PEP cDNA were treatedby enzymatic digestion and inserted into the pEGFP-C1 downstream of EGFP cDNA and wereused for transformation into bacterial competent cells. The positive colonies which showedinserted PEP cDNA were selected for plasmid preparations and additional analysis was performedto ensure that PEP cDNA was inserted properly. Finally, to confirm the intracellularlocalization of EGFP-PEP, CHO cells were transfected with the constructed plasmid.Results: Our results confirmed amplification and cloning of the expected product. PEP cDNAencompasses 630 bp which encodes 209 amino acid residues. Bioinformatics analyses haveshown the presence of a fibronectin type III domain (31-114 a.a. and two hydrophobic domains(12-32 a.a. and 152-169 a.a., respectively. Because of the presence of serine, Lysine,leucine (SKI in the C-terminal of the related protein, transfection data showed peroxisomallocalization of PEP as was similar to the catalase.Conclusion: Taken together these data showed that PEP is a peroxisomal protein. Howeverthe importance of its fibronectin type III and two hydrophobic domains should be assessedby further experiments.

  7. Characterization of Expressed Sequence Tags From a Gallus gallus Pineal Gland cDNA Library

    OpenAIRE

    Stefanie Hartman; Greg Touchton; Jessica Wynn; Tuoyu Geng; Chong, Nelson W.; Ed Smith

    2005-01-01

    The pineal gland is the circadian oscillator in the chicken, regulating diverse functions ranging from egg laying to feeding. Here, we describe the isolation and characterization of expressed sequence tags (ESTs) isolated from a chicken pineal gland cDNA library. A total of 192 unique sequences were analysed and submitted to GenBank; 6% of the ESTs matched neither GenBank cDNA sequences nor the newly assembled chicken genomic DNA sequence, three ESTs aligned with sequences d...

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

    Science.gov (United States)

    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. PMID:15640053

  9. The effect of column purification on cDNA indirect labelling for microarrays

    Directory of Open Access Journals (Sweden)

    Kiss John Z

    2007-06-01

    Full Text Available Abstract Background The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. Results We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Conclusion Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive

  10. cDNA2Genome: A tool for mapping and annotating cDNAs

    Directory of Open Access Journals (Sweden)

    Suhai Sandor

    2003-09-01

    Full Text Available Abstract Background In the last years several high-throughput cDNA sequencing projects have been funded worldwide with the aim of identifying and characterizing the structure of complete novel human transcripts. However some of these cDNAs are error prone due to frameshifts and stop codon errors caused by low sequence quality, or to cloning of truncated inserts, among other reasons. Therefore, accurate CDS prediction from these sequences first require the identification of potentially problematic cDNAs in order to speed up the posterior annotation process. Results cDNA2Genome is an application for the automatic high-throughput mapping and characterization of cDNAs. It utilizes current annotation data and the most up to date databases, especially in the case of ESTs and mRNAs in conjunction with a vast number of approaches to gene prediction in order to perform a comprehensive assessment of the cDNA exon-intron structure. The final result of cDNA2Genome is an XML file containing all relevant information obtained in the process. This XML output can easily be used for further analysis such us program pipelines, or the integration of results into databases. The web interface to cDNA2Genome also presents this data in HTML, where the annotation is additionally shown in a graphical form. cDNA2Genome has been implemented under the W3H task framework which allows the combination of bioinformatics tools in tailor-made analysis task flows as well as the sequential or parallel computation of many sequences for large-scale analysis. Conclusions cDNA2Genome represents a new versatile and easily extensible approach to the automated mapping and annotation of human cDNAs. The underlying approach allows sequential or parallel computation of sequences for high-throughput analysis of cDNAs.

  11. cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

    International Nuclear Information System (INIS)

    Recently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis

  12. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones and quality ...scores Data detail Data name 5'-end sequences of budding yeast full-length cDNA clones and quality scores De...from the budding yeast full-length cDNA library by the vector-capping method, the sequence quality score gen...s accession only. Sequence 5'-end sequence data of budding yeast full-length cDNA clones. FASTA format. Quality Phred's quality... Update History of This Database Site Policy | Contact Us 5'-end sequences of budding yeast full-length cDNA clones and quality

  13. Digital analysis of cDNA abundance; expression profiling by means of restriction fragment fingerprinting

    Directory of Open Access Journals (Sweden)

    Regenbogen Johannes

    2002-03-01

    Full Text Available Abstract Background Gene expression profiling among different tissues is of paramount interest in various areas of biomedical research. We have developed a novel method (DADA, Digital Analysis of cDNA Abundance, that calculates the relative abundance of genes in cDNA libraries. Results DADA is based upon multiple restriction fragment length analysis of pools of clones from cDNA libraries and the identification of gene-specific restriction fingerprints in the resulting complex fragment mixtures. A specific cDNA cloning vector had to be constructed that governed missing or incomplete cDNA inserts which would generate misleading fingerprints in standard cloning vectors. Double stranded cDNA was synthesized using an anchored oligo dT primer, uni-directionally inserted into the DADA vector and cDNA libraries were constructed in E. coli. The cDNA fingerprints were generated in a PCR-free procedure that allows for parallel plasmid preparation, labeling, restriction digest and fragment separation of pools of 96 colonies each. This multiplexing significantly enhanced the throughput in comparison to sequence-based methods (e.g. EST approach. The data of the fragment mixtures were integrated into a relational database system and queried with fingerprints experimentally produced by analyzing single colonies. Due to limited predictability of the position of DNA fragments on the polyacrylamid gels of a given size, fingerprints derived solely from cDNA sequences were not accurate enough to be used for the analysis. We applied DADA to the analysis of gene expression profiles in a model for impaired wound healing (treatment of mice with dexamethasone. Conclusions The method proved to be capable of identifying pharmacologically relevant target genes that had not been identified by other standard methods routinely used to find differentially expressed genes. Due to the above mentioned limited predictability of the fingerprints, the method was yet tested only with

  14. Cloning and characterization of a cDNA encoding human differentiation antigen 5D4

    Institute of Scientific and Technical Information of China (English)

    马凤蓉; 朱立平; 汪燚; 赵方萄; 史耕先; 李波; 李国燕; 张淑珍; 王讯

    2000-01-01

    A 1 846 bp cDNA is isolated from a human tonsil cell λgt 11 cDNA library (ATCC No. 37546) with mAb 5D4 reactive strongly with human B cell line 3D5, but weakly with human B cell line Daudi and human T cell line Jurkat as a probe. RT-PCR also shows a strong reaction in 3D5 cell and a weak reaction in Daudi and Jurkat cell for 5D4 mRNA. There is an open reading frame from 88 to 1 209 bp in 5D4 cDNA encoding a 374 AA protein. Both the Northern blot analysis and the two consecutive stop codens before start coden demonstrate that the cDNA is a full-length cDNA. Secondary structure prediction suggests that there are a region from 295 to 334 AA in the protein with strong hydrophobicity and a transmembrane helix region with high score from 313 to 334 AA with an orientation from the inside to the outside of the cell.

  15. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S. (Istituto Nazionale Neurologico C. Besta, Milan (Italy)); Rocchi, M. (Istituto G. Gaslini, Genoa (Italy))

    1991-01-15

    The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH{sub 2}-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH{sub 2}-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids.

  16. CLONING AND EXPRESSION OF A cDNA SEQUENCE FOR HUMAN THIOREDOXIN

    Institute of Scientific and Technical Information of China (English)

    Liu Qingyong(刘庆勇); Ruan Xiyun(阮喜云); Liu Xiaogong(刘效恭); Ji Zongzheng(纪宗正); Dang Jiangong; Nan Xunyi(南勋义); Wang Quanying(王全颖); Yang Guangxiao(杨广笑)

    2003-01-01

    Objective To clone and determine the sequence and expression of a cDNA segment for human thioredoxin. Methods The cDNA segment of thioredoxin was obtained through amplification by RT-PCR cloning from 143 (TK-) human osteosarcoma cell. The amplified products were cloned into pGEM-T Easy vector and sequenced. Then the expressed vector pBV220-hTRX was constructed and transformed into E.coli strain DH5α for hTRX expression. The hTRX was purified by DEAE-Sephadex A-50 column and the activity of recombinant hTRX was determined by the insulin disulfide reduction assay. Results Comparison of cDNA sequence of the cloned fragments with that of the reported hTRX (GenBank J04026) demonstrated that there were two differences compared to the reported cDNA sequence for hTRX at bp180 and bp284, and the amino acids enceoded altered respectively, but motif of the sequence was identical to that of the reported hTRX. The recombinant hTRX can catalyze insulin reduction by DTT. Conclusion The successful cloning and expression of hTRX cDNA formed a basis for further study on biological functions and utilization of hTRX.

  17. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

    International Nuclear Information System (INIS)

    The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH2-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH2-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids

  18. Cloning and sequencing of complete -crystallin cDNA from embryonic lens of Crocodylus palustris

    Indian Academy of Sciences (India)

    Raman Agrawal; Reena Chandrashekhar; Anurag Kumar Mishra; Jetty Ramadevi; Yogendra Sharma; Ramesh K Aggarwal

    2002-06-01

    -Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete -crystallin cDNA from the embryonic lens of Crocodylus palustris and establish it to be identical to the -enolase gene from non-lenticular tissues. Quantitatively, the -crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile -crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5′- and 3′-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete -crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5′-and 3′-ends respectively. Further, it was found to be identical to its putative counterpart enzyme -enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of -crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.

  19. Study on Wusan Granule Anti-tumor Related Target Gene Screened by Cdna Microarray

    Institute of Scientific and Technical Information of China (English)

    YOU Zi-li; SHI Jin-ping; CHEN Hai-hong

    2006-01-01

    To screen Wusan Granule anti-tumor related target gene using cDNA microarray technique, both mRNA from Lewis lung carcinoma tissues treated by Wusan Granule and untreated control are reversibly transcribed to prepare cDNA probes which are labeled by Cy5 and Cy3. Then, the probes are hybridized to the mice cDNA microarray type MGEC-20S. After hybridization, the cDNA microarray is scanned by ScanArray 3 000 scanner and the data is analyzed by ImaGene 3 software to screen the differentially expressed genes. There are 45 differentially expressed genes including 18 known genes and 27 unknown genes between the two groups, and among them, 20 elevated genes and 25 reduced genes are identified. Additionally, the genes related to invasion and metastasis of malignant carcinomas are down-regulated and the genes related to apoptosis are up-regulated. The cDNA microarray technique is a high-throughput approach to screen the Wusan Granule anti-tumor related target genes, which allow us to explore the molecular biological mechanism on a genomic scale.

  20. Molecular cloning and sequencing of a cDNA encoding partial putative molt-inhibiting hormone from Penaeus chinensis

    Science.gov (United States)

    Wang, Zai-Zhao; Xiang, Jian-Hai

    2002-09-01

    Total RNA was extracted from eyestalks of shrimp Penaeus chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymerase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A specific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 base pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  1. Construction of a Plant Transformation-ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

    Institute of Scientific and Technical Information of China (English)

    Wan-Song Ni; Zhi-Yong Lei; Xi Chen; David J. Oliver; Cheng-Bin Xiang

    2007-01-01

    Salt cress (Thellungiella halophila), a close relative of the model plant Arabidopsis thaliana L., is an extremophile that is adapted to harsh saline environments. To mine salt-tolerance genes from this species, we constructed an entry cDNA library from the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombinationassisted cDNA library construction method that is completely free of manipulations involving restriction enzymes and DNA ligase. This cDNA library construction procedure is significantly simplified and the quality of the cDNA library is improved. This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for plant transformation and expression via recombination-assisted cloning. The library is plant transformation ready and is used to transform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.

  2. MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING PARTIAL PUTATIVE MOLT-INHIBITING HORMONE FROM PENAEUS CHINENSIS

    Institute of Scientific and Technical Information of China (English)

    王在照; 相建海

    2002-01-01

    Total RNA was extracted from eyestalks of shrimp Penaeue chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymerase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A specific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 base pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  3. MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING PARTIAL PUTATIVE MOLT-INHIBITING HORMONE FROM PENAEUS CHINENSIS

    Institute of Scientific and Technical Information of China (English)

    王在照; 相建海

    2002-01-01

    Total RNA was extracted from eyestalks of shrimp Penaeus chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymer ase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A s pecific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 ba se pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  4. cDNA macroarray for analysis of gene expression profiles in prostate cancer

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Early diagnosis and timely treatment are important for improving therapeutic efficiency of prostate cancer. DNA array is a new bio-technology for disease diagnosis. This study was conducted to diagnose prostate cancer with cDNA macroarray and analysis gene expression profiles of some selective genes in prostate cancer.Methods Total RNA was isolated from patients with prostate cancer and from normal people, and poly(A) RNA was further purified. Then it was analyzed for differentially expressed genes in prostate cancer and normal prostate by cDNA macroarray system.Results There were different expressions in the nine prostate-associated specific genes in prostate cancer as compared with normal prostate, in which, 7 were significantly upregulated and 2 were down-regulated.Conclusion As a diagnostic approach at molecular level, the cDNA macroarray is an effectively diagnostic method for prostate cancer.

  5. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    Science.gov (United States)

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury. PMID:26345909

  6. Isolation and Characterization of Phytoene Desaturase cDNA from Stigma of Crocus sativus

    Institute of Scientific and Technical Information of China (English)

    Bai Jie(白洁); Xu Ying; Tang Lin; Zeng Yu; Feng Yun; Wang Shenghua; Chen Fang

    2004-01-01

    Phytoene desaturase (PDS) has recently been identified as an important enzyme in carotenoid biosynthesis pathway. A cDNA clone encoding phytoene desaturase gene is isolated from stigma of saffron (Crocus sativus L.) using RT-PCR technique. Sequence analysis shows 83% similarity to Narcissus pseudonarcissus, 79% to Zea mays, 78% to Arabidopsis thaliana, 77% to Lycopersicon esculentum. A new full-length cDNA is obtained by 5'-RACE and 3' -RACE techniques. The cDNA is 2149bp long with an open reading frame of 1697bp, which encodes a polypeptide of 565 amino acids. Southern analysis shows that the PDS gene is a single copy in saffron. Northern blot analysis shows higher expression level of PDS gene in stigma and anther than in leaves and stem.

  7. Cloning and sequencing of human intestinal alkaline phosphatase cDNA

    International Nuclear Information System (INIS)

    Partial protein sequence data obtained on intestinal alkaline phosphatase indicated a high degree of homology with the reported sequence of the placental isoenzyme. Accordingly, placental alkaline phosphatase cDNA was cloned and used as a probe to clone intestinal alkaline phosphatase cDNA. The latter is somewhat larger (3.1 kilobases) than the cDNA for the placental isozyme (2.8 kilobases). Although the 3' untranslated regions are quite different, there is almost 90% homology in the translated regions of the two isozymes. There are, however, significant differences at their amino and carboxyl termini and a substitution of an alanine in intestinal alkaline phosphatase for a glycine in the active site of the placental isozyme

  8. Molecular Cloning and Bacterial Expression of Germacrene A Synthase cDNA from Crepidiastrum sonchifolium

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Germacrene A synthase(GAS) catalyzes the biosynthesis of germacrene A, which is a key precursor for sesquiterpene lactones. Cloning of a novel full-length cDNA encoding GAS from the medicinal plant Crepidiastrum sonchifolium(designated CsGAS) is reported in this study. The cDNA is 1837 bp long and contains a 1680-bp open reading frame encoding a 559 amino-acid protein. The functional expression of the cDNA in Escherichia coli, as an N-terminal thioredoxin fusion protein, with the pET32a vector yielding a recombinant enzyme. Sequence analysis was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco, and the effect of possible involvement of a number of amino acids in sesquiterpene synthase on product specificity was also discussed.

  9. Design and Screening of M13 Phage Display cDNA Libraries

    Directory of Open Access Journals (Sweden)

    Yuliya Georgieva

    2011-02-01

    Full Text Available The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS will be presented.

  10. Cloning, sequencing, and expression of cDNA for human β-glucuronidase

    International Nuclear Information System (INIS)

    The authors report here the cDNA sequence for human placental β-glucuronidase (β-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH2-terminal amino acid sequence determined for human spleen β-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human β-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human β-glucuronidase, demonstrate the existence of two populations of mRNA for β-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length

  11. Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family.

    OpenAIRE

    Grove, G; Zarlengo, R P; Timmerman, K P; Li, N Q; Tam, M F; Tu, C P

    1988-01-01

    We have isolated from a constructed lambda gt11 expression library two classes of cDNA clones encoding the entire sequence of the maize GSH S-transferases GST I and GST III. Expression of a full-length GST I cDNA in E. coli resulted in the synthesis of enzymatically active maize GST I that is immunologically indistinguishable from the native GST I. Another GST I cDNA with a truncated N-terminal sequence is also active in heterospecific expression. Our GST III cDNA sequence differs from the ve...

  12. Cloning and expression of cDNA for anti-müllerian hormone.

    OpenAIRE

    Picard, J Y; Benarous, R; Guerrier, D.; Josso, N; Kahn, A.

    1986-01-01

    Messenger RNA, prepared from fetal bovine testicular tissue, was used to construct a cDNA library in lambda gt11 phage. The library was screened with an antibody probe directed against bovine anti-Müllerian hormone and three positive clones were isolated. Cross-hybridizing cDNA inserts carried by clones 4 and 5 (1.2 and 0.08 kilobases long, respectively) code for a fragment of authentic anti-Müllerian hormone, as shown by the ability of the anti-epitope antibodies eluted from fusion protein 4...

  13. Cloning and expression of a novel human HCUTA cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Copper is one of the most important trace elements to life. Human HCUTA is a novel cDNA encoding a 156aa protein, which may participate in human copper tolerance system. The HCUTA protein is highly similar to protein CUT A1 of E. coli. The whole opening reading frame of HCUTA cDNA was amplified by PCR and cloned into pET28a + express vector, and the HCUTA protein was effectively expressed in E. coli BL21 (DE3).

  14. Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis)

    OpenAIRE

    Soumen Naskar; Deb, Sitangsu M.; Saket K. Niranjan; Subodh Kumar; Deepak Sharma; Durgam Sakaram; Arjava Sharma

    2012-01-01

    In the present study, water buffalo MHC (Bubu)-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with import...

  15. Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis)

    OpenAIRE

    Naskar, Soumen; Deb, Sitangsu M.; Saket K. Niranjan; Kumar, Subodh; Sharma, Deepak; Sakaram, Durgam; Sharma, Arjava

    2012-01-01

    In the present study, water buffalo MHC (Bubu)-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with important f...

  16. cDNA for R-cognin: homology with a multifunctional protein.

    OpenAIRE

    Krishna Rao, A S; Hausman, R E

    1993-01-01

    Retina cognin (R-cognin) is a developmentally regulated 50-kDa protein that was isolated from chicken embryo retina cell membranes. It mediates the adhesion and reaggregation in vitro of retina cells from chicken and mouse embryos, but not of cells from other tissues, and may be involved in neuronal differentiation. We report here the cloning of a cDNA for R-cognin. A chicken embryo retina cDNA library was constructed in lambda gt11 vector and was screened with polyclonal R-cognin antiserum, ...

  17. Molecular cloning of a cDNA for the chicken progesterone receptor B antigen.

    OpenAIRE

    Zarucki-Schulz, T; Kulomaa, M S; Headon, D R; N.L. Weigel; Baez, M; Edwards, D.P.; McGuire, W L; Schrader, W T; O'Malley, B W

    1984-01-01

    A cDNA for the chicken progesterone receptor B subunit antigen (Mr, 108,000) has been isolated from a cDNA library prepared from size-selected chicken oviduct poly(A)+RNA. A specific monoclonal antibody raised against hen progesterone receptor B subunit (alpha PR-B) was used to screen the library. Recombinant clones reacting with the antibody by virtue of antigen expression were used in hybrid-selected translation. A single clone, pPRB-1, hybridized specifically to a mRNA that yielded a Mr 10...

  18. Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver catalase.

    OpenAIRE

    Furuta, S.; Hayashi, H; Hijikata, M; Miyazawa, S.; Osumi, T; Hashimoto, T.

    1986-01-01

    We have isolated five cDNA clones for rat liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6). These clones overlapped with each other and covered the entire length of the mRNA, which had been estimated to be 2.4 kilobases long by blot hybridization analysis of electrophoretically fractionated RNA. Nucleotide sequencing was carried out on these five clones and the composite nucleotide sequence of catalase cDNA was determined. The 5' noncoding region contained 83 b...

  19. Construction of cDNA libraries: focus on protists and fungi.

    Science.gov (United States)

    Rodríguez-Ezpeleta, Naiara; Teijeiro, Shona; Forget, Lise; Burger, Gertraud; Lang, B Franz

    2009-01-01

    Sequencing of cDNA libraries is an efficient and inexpensive approach to analyze the protein-coding portion of a genome. It is frequently used for surveying the genomes of poorly studied eukaryotes, and is particularly useful for species that are not easily amenable to genome sequencing, because they are nonaxenic and/or difficult to cultivate. In this chapter, we describe protocols that have been applied successfully to construct and normalize a variety of cDNA libraries from many different species of free-living protists and fungi, and that require only small quantities of cell material. PMID:19277563

  20. cDNA sequence of a new chicken embryonic rho-globin.

    OpenAIRE

    Roninson, I B; Ingram, V M

    1981-01-01

    In order to use specific DNA probes for the study of developmentally regulated gene expression, we have prepared cDNA clones corresponding to chicken embryonic globins by inserting cDNA.mRNA hybrids into the Pst I site of the plasmid pBR322 by using poly(dG) and poly(dC) linkers. The nucleotide sequence of the insert of one clone, representing a nearly full-length copy of an embryonic beta-like globin cDNA, has been determined. The amino acid sequence of the globin encoded by this insert is i...

  1. Systematic sequencing of cDNA clones using the transposon Tn5

    OpenAIRE

    Shevchenko, Yuriy; Bouffard, Gerard G.; Butterfield, Yaron S.N.; Blakesley, Robert W.; Hartley, James L.; Young, Alice C.; Marco A. Marra; Jones, Steven J M; Touchman, Jeffrey W.; Green, Eric D.

    2002-01-01

    In parallel with the production of genomic sequence data, attention is being focused on the generation of comprehensive cDNA-sequence resources. Such efforts are increasingly emphasizing the production of high-accuracy sequence corresponding to the entire insert of cDNA clones, especially those presumed to reflect the full-length mRNA. The complete sequencing of cDNA clones on a large scale presents unique challenges because of the generally small, yet heterogeneous, sizes of the cloned inser...

  2. Molecular cloning of cDNA encoding the small subunit of Drosophila transcription initiation factor TFIIF.

    OpenAIRE

    Gong, D W; Mortin, M A; Horikoshi, M; Nakatani, Y

    1995-01-01

    Transcription initiation factor TFIIF is a tetramer consisting of two large subunits (TFIIF alpha or RAP74) and two small subunits (TFIIF beta or RAP30). We report here the molecular cloning of a Drosophila cDNA encoding TFIIF beta. The cDNA clone contains an open-reading frame encoding a 277 amino acid polypeptide having a calculated molecular mass of 32,107 Da. Comparison of the deduced amino acid sequence with the corresponding sequences from vertebrates showed only 50% identity, with four...

  3. Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization.

    OpenAIRE

    Foster, D C; Sprecher, C A; Grant, F J; Kramer, J M; Kuijper, J L; Holly, R D; Whitmore, T E; Heipel, M D; Bell, L A; Ching, A F

    1994-01-01

    Thrombopoietin (TPO), a lineage-specific cytokine affecting the proliferation and maturation of megakaryocytes from committed progenitor cells, is believed to be the major physiological regulator of circulating platelet levels. Recently we have isolated a cDNA encoding a ligand for the murine c-mpl protooncogene and shown it to be TPO. By employing a murine cDNA probe, we have isolated a gene encoding human TPO from a human genomic library. The TPO locus spans over 6 kb and has a structure si...

  4. Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

    Institute of Scientific and Technical Information of China (English)

    HUANG Jun-hua; LI Yong-feng; HE Fan; LI Dan; SUN Yuan; HAN Wen; QIU Hua-ji

    2013-01-01

    The reverse genetics for classical swine fever virus (CSFV) is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the low-copy vector pOK12, producing pOKShimen-RzT . Direct transfection of pOKShimen-RzT into PK/T7 cells, a PK-15-derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.

  5. Brain tubulin and actin cDNA sequences: isolation of recombinant plasmids.

    OpenAIRE

    Ginzburg, I.(Sobolev Institute of Mathematics and Novosibirsk State University, 630090, Novosibirsk, Russia); de Baetselier, A; Walker, M D; Behar, L; Lehrach, H; Frischauf, A M; Littauer, U Z

    1980-01-01

    Rat brain mRNA enriched for tubulin and actin sequences was used to prepare double stranded cDNA. A library of recombinant clones was constructed by inserting the dsDNA into the Pst1 site of pBR322 plasmid and transformation of E. coli chi 1776 host. Clones bearing sequences coding for tubulin and actin were identified and characterized.

  6. Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes

    International Nuclear Information System (INIS)

    Several DNA topoisomerase II partial cDNA clones obtained from a human Raji-HN2 cDNA library were sequenced and two classes of nucleotide sequences were found. One member of the first class, SP1, was identical to an internal fragment of human HeLa cell Topo II cDNA described earlier. A member of the second class, SP11, shared extensive nucleotide (75%) and predicted peptide (92%) sequence similarities with the first two-thirds of HeLa Topo II. Each class of cDNAs hybridized to unique, nonoverlapping restriction enzyme fragments of genomic DNA from several human cell lines. Synthetic 24-mer oligonucleotide probes specific for each cDNA class hybridized to 6.5-kilobase mRNAs; furthermore, hybridization of probe specific for one class was not blocked by probe specific for the other. Antibodies raised against a synthetic SP1-encoded dodecapeptide specifically recognized the 170-kDa form of Topo II, while antibodies raised against the corresponding SP11-encoded dodecapeptide, or a second unique SP11-encoded tridecapeptide, selectively recognized the 180-kDa form of Topo II. These data provide genetic and immunochemical evidence for two Topo II isozymes

  7. Cloning and Sequence Analysis of cDNA Encoding MRJP3 of Apis cerana cerana

    Institute of Scientific and Technical Information of China (English)

    SU Song-kun; ZHNEG Huo-qing; CHEN Sheng-lu; ZHONG Bo-xiong; Stefan Albert

    2005-01-01

    By screening the worker (Apis cerana cerana) heads cDNA library using a fragment of the mrjp3 gene ofApis cerana as probe, 120 positive clones were obtained. The clone containing A. cerana cerana MRJP3 (AccMRJP3) cDNA was selected. Based on the sequencing of the inserts of the positive clone, a sequence of AccMRJP3 cDNA which is 1 887 bp long including a poly (A) tail was obtained. The AccMRJP3 cDNA encompassed an open-reading frame (ORF) with 1 779 bp encoding 593 amino acids. The un-translated regions (UTR) of the 5' end and 3' end are 46 bp and 160 bp in length,respectively. Similar to AmMRJP3 and AdMRJP3, the putative AccMRJP3 also has a repetitive region. The comparison of the repetitive region of AccMRJP3, AmMRJP3 and AdMRJP3 shows some differences between them.

  8. Isolation of 24 novel cDNA fragments from microdis—sected human chromosome band

    Institute of Scientific and Technical Information of China (English)

    ZHANGMIN; LONGYU; 等

    1998-01-01

    The strategy of isolating the band0specific expression fragments from a probe pool generated by human chromosome microdissection was reported.A chromosome 14q 24.3 band-specific single copy DNA pool was constructed based on this probe pool.Using total DNA of the pool as probe to hybridize the human marrow cDNA library,68 primary positive clones were selected from 5×105 cDNA clones.Among these primary clones,32 secondary clones were obtained after second-round screening and designed as cFD14-1-32.Finally,24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization.Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but cann't hybridize to 17q11-12 DNA,Partial sequences of 13 fragments of them were sequenced and idenfified as novel cDNA sequences,and these sequences were proved to have some homology with known genes in NCBI database.Analysis of expression spectrum of cFD 14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.

  9. GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN CDNA MICROARRAY ANALYSES

    Science.gov (United States)

    GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN cDNA MICROARRAY ANALYSESB.S. Pukazhenthi1, J. C. Rockett2, M. Ouyang3, D.J. Dix2, J.G. Howard1, P. Georgopoulos4, W.J. J. Welsh3 and D. E. Wildt11Department of Reproductiv...

  10. Molecular cloning and sequence analysis of cDNA encoding human prostatic acid phosphatase.

    Science.gov (United States)

    Vihko, P; Virkkunen, P; Henttu, P; Roiko, K; Solin, T; Huhtala, M L

    1988-08-29

    lambda gt11 clones encoding human prostatic acid phosphatase (PAP) (EC 3.1.3.2) were isolated from human prostatic cDNA libraries by immunoscreening with polyclonal antisera. Sequence data obtained from several overlapping clones indicated that the composite cDNAs contained the complete coding region for PAP, which encodes a 354-residue protein with a calculated molecular mass of 41,126 Da. In the 5'-end, the cDNA codes for a signal peptide of 32 amino acids. Direct protein sequencing of the amino-terminus of the mature protein and its proteolytic fragments confirmed the identity of the predicted protein sequence. PAP has no apparent sequence homology to other known proteins. However, both the cDNA clones coding for human placental alkaline phosphatase and PAP have an alu-type repetitive sequence about 900 nucleotides downstream from the coding region in the 3'-untranslated region. Two of our cDNA clones differed from others at the 5'-ends. RNA blot analysis indicated mRNA of 3.3 kb. We are continuing to study whether acid phosphatases form a gene family as do alkaline phosphatases. PMID:2842184

  11. CLONING AND CHARACTERIZATION OF CDNA ENCODING GIARDIA LAMBLIA d-GIARDIN

    Science.gov (United States)

    A cDNA coding for d-giardin was cloned from Giardia lamblia trophozoites in order to localize the protein and study its function in mediating surface attachment. Recombinant d-giardin antigen was produced in Escherichia coli as a poly-histidine fusion protein and was purified by affinity chromatogr...

  12. Purification, characterization, and cDNA cloning of opine dehydrogenases from the polychaete rockworm Marphysa sanguinea.

    Science.gov (United States)

    Endo, Noriyuki; Kan-no, Nobuhiro; Nagahisa, Eizoh

    2007-06-01

    Alanopine dehydrogenase (AlDH) and three isoforms of strombine/alanopine dehydrogenase (St/AlDH) were purified from muscle tissue of the polychaete rockworm Marphysa sanguinea. The four enzymes, which can be distinguished by the isoelectric point, are monomeric 42 kDa proteins, possess similar pH-activity profiles, and display specificity for pyruvate and NAD(H). The three isoforms of St/AlDH show equivalent Km and Vmax for glycine and L-alanine and for D-strombine and meso-alanopine. Free amino acid levels in the muscle and D-strombine accumulation in vivo during muscle activity suggest that St/AlDHs function physiologically as StDH. AlDH shows specificity for L-alanine and meso-alanopine, but not for glycine or D-strombine. The amino acid sequences of AlDH and one of the St/AlDH isoforms were determined by a combination of amino acid sequence analysis and cDNA cloning. St/AlDH cDNA consisted of 1586 bp nucleotides that encode a 399-residue protein (43,346.70 Da), and AlDH cDNA consisted of 1587 bp nucleotides that encode a 399-residue protein (43,886.68 Da). The two amino acid sequences deduced from the cDNA displayed 67% amino acid identity, with greatest similarity to that of tauropine dehydrogenase from the polychaete Arabella iricolor. PMID:17350870

  13. Molecular Cloning and Sequencing of Channel Catfish, Ictalurus punctatus, Cathepsin H and L cDNA

    Science.gov (United States)

    Cathepsin H and L, a lysosomal cysteine endopeptidase of the papain family, are ubiquitously expressed and involve in antigen processing. In this communication, the channel catfish cathepsin H and L transcripts were sequenced and analyzed. Total RNA from tissues was extracted and cDNA libraries we...

  14. cDNA array screening differentially expressed gene in liver regeneration

    Institute of Scientific and Technical Information of China (English)

    强晖; 谢渭芬; 张忠兵; 张新; 张兴荣; 陈岳祥; 杨秀疆

    2003-01-01

    Objective: To screen the differentially expressed gene in liver regeneration with cDNA array and gain insight of the pathogenesis of the acute hepatic failure. Methods: Acute liver failure model in Sprague-Dawley (SD) rat was induced by 95% hepatectomy. The differential expression profiles in regenerating rat liver were studied by a cDNA array representing 1176 cDNA clusters. Some genes were randomly selected from a pool of differentially expressed genes and subjected to RT-PCR to further confirm the result from microarray hybridization. Results: The alterations of transcription levels were noted in the expressions of 188 genes. There were 138 genes with their expression up-regulated such as growth factors, PCNA, ribosomal proteins, IL6 and CDKs which were associated with cell cycle regulation, stress, metabolism and proliferation. Conclusion: cDNA array is a powerful tool to explore the gene expression of acute liver failure and is potential for the diagnosis and treatment of liver regeneration.

  15. Detection of reverse transcriptase termination sites using cDNA ligation and massive parallel sequencing

    DEFF Research Database (Denmark)

    Kielpinski, Lukasz J; Boyd, Mette; Sandelin, Albin;

    2013-01-01

    Detection of reverse transcriptase termination sites is important in many different applications, such as structural probing of RNAs, rapid amplification of cDNA 5' ends (5' RACE), cap analysis of gene expression, and detection of RNA modifications and protein-RNA cross-links. The throughput of...

  16. Next-generation cDNA screening for oncogene and resistance phenotypes.

    Directory of Open Access Journals (Sweden)

    Nobuaki Shindoh

    Full Text Available There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo.

  17. Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

    Science.gov (United States)

    van Tegelen, Léon J.P.; Moreno, Paolo R.H.; Croes, Anton F.; Verpoorte, Robert; Wullems, George J.

    1999-01-01

    Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The Km values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus. PMID:9952467

  18. Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

    DEFF Research Database (Denmark)

    Cowell, G M; Kønigshøfer, E; Danielsen, E M;

    1988-01-01

    The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250...

  19. [Construction and sequencing of full-length cDNA of peste des petits ruminants virus].

    Science.gov (United States)

    Zhai, Jun-Jun; Dou, Yong-Xi; Zhang, Hai-Rui; Mao, Li; Meng, Xue-Lian; Luo, Xuo-Nong; Cai, Xue-Peng

    2010-07-01

    To develop a reverse genetics system of Peste des petits ruminants virus(PPRV), five pairs of oligonucleotide primers were designed on the basis of the full-length genomic sequence of PPRV Nigeria 75/ 1 strain. Using RT-PCR technique, five over-lapping cDNA fragments, designated as JF1, JF2, JF3, JF4 and JF5, respectively, were amplified, followed by cloning into pcDNA3.1(+)vector. An AscI restriction enzyme site and a T7 promoter sequence were introduced immediately upstream of 5'-end, while a PacI restriction enzyme site was engineered downstream of 3'-end. Using pok12 as a plasmid vector, the full-length cDNA clone pok12-PPRV of Nigeria 75/1 was assembled by connecting the five cDNA fragments via the unique restriction endonuclease site of PPRV genome. The resultant nucleotide sequence of the PPRV Nigeria 75/1 strain in the study was compared with other members of genus morbillivirus, and phylogenetic analysis was used to examine the evolutionary relationships. The results showed that PPRV Nigeria 75/ 1 was antigenically closely related to Rinderpest virus and Measles virus. Successful construction of full-length cDNA clone of PPRV Nigeria 75/1 strain lays the basis rescuing PPRV effectively and enables further research of PPRV at molecular level. PMID:20836386

  20. Rescue of Newcastle disease virus from cloned cDNA using an RNA polymerase II promoter.

    Science.gov (United States)

    Li, Bao-Yu; Li, Xue-Rui; Lan, Xi; Yin, Xiang-Pin; Li, Zhi-Yong; Yang, Bin; Liu, Ji-Xing

    2011-06-01

    A new system was developed to improve the efficiency and simplify the procedure of recovery of Newcastle disease virus (NDV) from cloned cDNA. A full-length cDNA clone of mesogenic NDV vaccine strain Mukteswar was assembled from five subgenomic cDNA fragments and cloned into a plasmid allowing transcription driven by cellular RNA polymerase II. The full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences, resulted in the synthesis of antigenomic RNA with exact termini. Without supplying T7 RNA polymerase, infectious NDV could be generated efficiently in some eukaryotic cell lines by simultaneous transcription of antigenomic RNA from the full-length plasmid and expression of NP, P and L proteins from helper plasmids introduced by cotransfection. The efficiency of recovery with the conventional T7 promoter system based on BRS-T7 cells and the cytomegalovirus (CMV) promoter system was compared, and the results demonstrate that the new system facilitates the generation of recombinant NDV and more efficient than the T7 rescue system using BRS-T7. PMID:21327786

  1. Characterization of antigen-expressing Plasmodium falciparum cDNA clones that are reactive with parasite inhibitory antibodies.

    Science.gov (United States)

    Horii, T; Bzik, D J; Inselburg, J

    1988-07-01

    A Plasmodium falciparum (FCR3 strain) lambda gt11 cDNA expression library was constructed from trophozoite and schizont poly(A) RNA and was screened immunologically with a pooled human immune serum from Nigeria to form a gene bank of 288 positive clones. The gene bank was subsequently screened with parasite inhibitory mouse monoclonal antibodies (mMAb) and with individual human Liberian sera. Two mMAb, 43E5 and 5H10, strongly reacted with 8 and 3 cDNA clones, respectively. Several of those clones also weakly cross-reacted with the other mMAb. Two of those weakly cross-reactive clones, cDNA#366 and cDNA#22, were shown to be located in different chromosomal regions of the parasite by Southern hybridization and so appeared to represent two different parasite genes. The genomic organization of both cDNA#366 and cDNA#22 sequences were identical in the FCR3 and the Honduras-1 strain. The nucleotide sequence of cDNA#366 and the amino acid sequence it coded for were homologous to a partial DNA and amino acid sequence previously reported for a P. falciparum (Camp strain) exoantigen designated p126. The mRNA for cDNA#366 appeared to represent an abundant message in blood stage trophozoites and schizonts. PMID:2456465

  2. A NEW METHOD TO CONSTRUCT A FULL-LENGTH cDNA LIBRARY OF HUMAN NORMAL BLADDER TISSUE

    Institute of Scientific and Technical Information of China (English)

    成瑜; 李旭; 陈葳; 杨玉琮; 赵乐

    2003-01-01

    Objective Using template-switch mechanism at the 5'-end of mRNA technique (SMART) to construct a full-length cDNA library of human normal bladder tissue. Methods The novel procedures used the template-switching activity of powerscript reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, 5 cycles of PCR were performed using a modified oligo(dT) primer and an anchor primer to enrich the full-length cDNA population with 1.0 g human normal bladder poly(A)+RNA, then double-strand cDNA was synthesized. After digestion with sfiI and size-fractionation by CHROMA SPIN-400 columns, double-strand cDNA was ligated into λTripIEx2 vector and was packaged. We determined the titer of the primary library and the percentage of recombinant clones and finally amplified the library. Results The titer of the cDNA library constructed was 2.1×106 pfu*mL-1, and the amplified cDNA library was 6×1011 pfu*mL-1, the percentage of recombination clones was 99%. Conclusion Using SMART technique helps us to construct full-length cDNA library with high efficiency and high capacity which lays solid foundation for screening target genes of bladder diseases with probes and antibodies.

  3. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase.

    Science.gov (United States)

    Finocchiaro, G; Taroni, F; Rocchi, M; Martin, A L; Colombo, I; Tarelli, G T; DiDonato, S

    1991-01-01

    We have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase (CPTase; palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21), an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH2-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH2-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hamster somatic cell hybrids. Images PMID:1988962

  4. Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene

    International Nuclear Information System (INIS)

    A human umbilical vein endothelial cell cDNA library in λgt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, λHTm10 and λHTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A) + RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of λHTm10. One isolate, λHTm15, contained a 3693 base pair cDNA insert with an apparent 5'-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3'-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The organization of thrombomodulin is similar to that of the low-density lipoprotein receptor, and the protein is homologous to a large number of other proteins that also contain EGF-like domains, including factor VII, factor IX, factor X, factor XII, protein C, tissue plasminogen activator, and urokinase. The gene for thrombomodulin has been localized to chromosome 20 by hybridization of cDNA probes to purified human chromosomes

  5. Cloning and expression of a novel chicken sulfotransferase cDNA regulated by GH.

    Science.gov (United States)

    Cao, H; Agarwal, S K; Burnside, J

    1999-03-01

    We have used mRNA differential display to compare gene expression in normal and GH receptor-deficient dwarf chickens, and report here the characterization of one differentially expressed gene, which shows significant sequence identity to the sulfotransferase gene family. Partial cDNA clones were isolated from a chicken liver cDNA library and an additional sequence was obtained using 5' rapid amplification of cDNA ends. A complete cDNA probe hybridizes to three transcripts (2.4, 2.0 and 1.45 kb) on Northern blots of chicken liver RNA, which differ in the length of the 3' untranslated region. All three transcripts are expressed at higher levels in normal vs dwarf chickens, as expected for a GH-regulated gene. The expression of this sulfotransferase mRNA was also detected in skeletal muscle, but not other tissues. The administration of GH to chickens increased the hepatic expression within 1 h, suggesting this sulfotransferase could be directly regulated by GH. Sulfotransferase activity, using estradiol or corticosterone as substrate, is detected in cells transfected with an expression vector containing the full-length cDNA. The sequence of this sulfotransferase does not show significant similarity with any subfamily of the sulfotransferases and its endogenous substrate is presently unknown. However, we speculate that GH activation of sulfotransferase activity could play a role in reducing concentrations of growth-antagonistic steroid hormones in GH target tissues. These results demonstrate the usefulness of differential display in this model system to identify genes that play a role in mediating GH action. PMID:10076195

  6. Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library

    OpenAIRE

    Bhore, Subhash J.; Kassim, Amelia; Loh, Chye Ying; Shah, Farida H

    2010-01-01

    It is well known that the nutritional quality of the American oil-palm (Elaeis oleifera) mesocarp oil is superior to that of African oil-palm (Elaeis guineensis Jacq. Tenera) mesocarp oil. Therefore, it is of important to identify the genetic features for its superior value. This could be achieved through the genome sequencing of the oil-palm. However, the genome sequence is not available in the public domain due to commercial secrecy. Hence, we constructed a cDNA library and generated expres...

  7. Generation of EST and cDNA Microarray Resources for the Study of Bovine Immunobiology*

    Directory of Open Access Journals (Sweden)

    Coussens PM

    2003-03-01

    Full Text Available Recent developments in expressed sequence tag (EST and cDNA microarray technology have had a dramatic impact on the ability of scientists to study the responses of thousands of genes to external stimuli, such as infection, nutrient flux, and stress. To date however, these studies have largely been limited to human and rodent systems. Despite the tremendous potential benefit of EST and cDNA microarray technology to studies of complex problems in domestic animal species, a lack of integrated resources has precluded application of these technologies to domestic species. To address this problem, the Center for Animal Functional Genomics (CAFG at Michigan State University has developed a normalized bovine total leukocyte (BOTL cDNA library, generated EST clones from this library, and printed cDNA microarrays suitable for studying bovine immunobiology. Our data revealed that the normalization procedure successfully reduced highly abundant cDNA species while enhancing the relative percentage of clones representing rare transcripts. To date, a total of 932 EST sequences have been generated from this library (BOTL and the sequence information plus BLAST results made available through a web-accessible database http://gowhite.ans.msu.edu. Cluster analysis of the data indicates that a total of 842 unique cDNAs are present in this collection, reflecting a low redundancy rate of 9.7%. For creation of first generation cDNA microarrays, inserts from 720 unique clones in this library were amplified and microarrays were produced by spotting each insert or amplicon 3 times on glass slides in a 48-patch arrangement with 64 total spots (including blanks and positive controls per patch. To test our BOTL microarray, we compared gene expression patterns of concanavalin A stimulated and unstimulated peripheral blood mononuclear cells (PBMCs. In total, hybridization signals on over 90 amplicons showed upregulation (>3× in response to Con A stimulation, relative to

  8. Human tissue factor: cDNA sequence and chromosome localization of the gene

    International Nuclear Information System (INIS)

    A human placenta cDNA library in λgt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, λHTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of λHTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of ∼ 3.2 kilobases in poly(A) + RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased several fold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of λHTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(a) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser. The 3'-noncoding region contains an inverted Alu family repetitive sequence. The tissue factor gene was localized to chromosome 1 by hybridization of the cDNA insert of λHTF8 to flow-sorted human chromosomes

  9. Cloning of cDNA Encoding GRA1 Protein of Tachyzoite Toxoplasma Gondii Local Isolate

    Directory of Open Access Journals (Sweden)

    Erma Sulistyaningsih

    2015-10-01

    Full Text Available Gene encoding GRA1 protein is potent DNA-vaccine candidate against toxoplasmosis. The aim of the researchwas to clone the gene encoding GRA1 protein of tachyzoite Toxoplasma gondii local isolate by DNA recombinanttechnology. Tachyzoite was grown in Balb/c mice in vivo. Messenger RNA was isolated from total RNA and itwas used to synthesis cDNA. Complementary DNA encoding GRA1 protein of tachyzoite Toxoplasma gondii localisolate was amplified and cloned in a prokaryote cloning vector. The recombinant GRA1-encoding gene was thendigesting using EcoRI restriction endonuclease and sequencing. The result showed that the recombinant GRA1-encoding gene consisted of DNA sequences encoding all signal peptide and mature peptide of GRA1 protein.Alignment of recombinant GRA1 sequence to gene encoding GRA1 protein of Toxoplasma gondii RH isolate showed100% homologous.Keywords: GRA1 protein, Toxoplasma gondii, tachyzoite, cloning, cDNA

  10. Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    Soumen Naskar

    2012-01-01

    Full Text Available In the present study, water buffalo MHC (Bubu-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

  11. Identification of brassinosteroid responsive genes in Arabidopsis by cDNA array

    Institute of Scientific and Technical Information of China (English)

    胡玉欣; 汪政科; 王永红; 包方; 李凝; 彭镇华; 李家洋

    2001-01-01

    We have systematically monitored brassinosteroid (BR) responsive genes in a BR-deficient mutant det2 suspension culture of Arabidopsis by using a cDNA array approach. Among 13000 cDNA clones arrayed on filters, 53 BR responsive clones were identified and designated BRR1-BRR53. Sequence analysis of 43 clones showed that 19 clones are novel genes, 3 clones are genes involved in the control of cell division, 4 clones are genes related to plant stress responses, 4 clones are transcriptional factor or signal transduction component genes, and 3 clones are genes involved in RNA splicing or structure forming. In addition, we also found that BR regulated the transcription of genes related to many physiological processes, such as photoreaction, ion transportation and some metabolic processes. These findings present molecular evidence that BR plays an essential role in plant growth and development.

  12. Peptidomics combined with cDNA library unravel the diversity of centipede venom

    DEFF Research Database (Denmark)

    Rong, Mingqiang; Yang, Shilong; Wen, Bo;

    2015-01-01

    extensive diversity of centipede toxins and provide powerful tools to understand the capture and defense weapon of centipede. BIOLOGICAL SIGNIFICANCE: Peptide toxins from venomous animal have attracted increasing attentions due to their extraordinary chemical and pharmacological diversity. Centipedes are......UNLABELLED: Centipedes are one of the oldest venomous arthropods using toxin as their weapon to capture prey. But little attention was focused on them and only few centipede toxins were demonstrated with activity on ion channels. Therefore, more deep works are needed to understand the diversity of...... centipede venom. In the present study, we use peptidomics combined with cDNA library to uncover the diversity of centipede Scolopendra subspinipes mutilans L. Koch. 192 peptides were identified by LC-MS/MS and 79 precursors were deduced by cDNA library. Surprisingly, the signal peptides of centipede toxins...

  13. Human kidney amiloride-binding protein: cDNA structure and functional expression

    International Nuclear Information System (INIS)

    Phenamil, an analog of amiloride, is a potent blocker of the epithelial Naplus channel. It has been used to purify the porcine kidney amiloride-binding protein. Synthetic oligonucleotides derived from partial sequences have been used to screen a human kidney cDNA library and to isolate the cDNA encoding the human amiloride-binding protein. The primary structure was deduced from the DNA sequence analysis. The protein is 713 residues long, with a 19-amino acid signal peptide. The mRNA was expressed in 293-S and NIH 3T3 cells, yielding a glycoprotein (i) that binds amiloride and amiloride analogs with affinities similar to the amiloride receptor associated with the apical Naplus channel in pig kidney membranes and (ii) that is immunoprecipitated with monoclonal antibodies raised against pig kidney amiloride-binding protein

  14. Consistent errors in first strand cDNA due to random hexamer mispriming.

    Directory of Open Access Journals (Sweden)

    Thomas P van Gurp

    Full Text Available Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.

  15. Using a cDNA microarray to study cellular gene expression altered by Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    徐永忠; 谢建平; 李瑶; 乐军; 陈建平; 淳于利娟; 王洪海

    2003-01-01

    Objective To examine the global effects of Mycobacterium tuberculosis (M.tuberculosis) infection on macrophages. Methods The gene expression profiling of macrophage U937, in response to infection with M.tuberculosis H37Ra, was monitored using a high-density cDNA microarray. Results M.tuberculosis infection caused 463 differentially expressed genes, of which 366 genes are known genes registered in the Gene Bank. These genes function in various cellular processes including intracellular signalling, cytoskeletal rearrangement, apoptosis, transcriptional regulation, cell surface receptors, cell-mediated immunity as well as a variety of cellular metabolic pathways, and may play key roles in M.tuberculosis infection and intracellular survival. Conclusions M.tuberculosis infection alters the expression of host-cell genes, and these genes will provide a foundation for understanding the infection process of M.tuberculosis. The cDNA microarray is a powerful tool for studying pathogen-host cell interaction.

  16. CDNA cloning, characterization and expression of an endosperm-specific barley peroxidase

    DEFF Research Database (Denmark)

    Rasmussen, Søren Kjærsgård; Welinder, K.G.; Hejgaard, J.

    1991-01-01

    amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed......A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C-terminal...

  17. Identification of auxin responsive genes in Arabidopsis by cDNA array

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The plant hormone auxin influences a variety of developmental and physiological processes. But the mechanism of its action is quite unclear. In order to identify and analyze the expression of auxin responsive genes, a cDNA array approach was used to screen for genes with altered expression from Arabidopsis suspension culture after IAA treatment and was identified 50 differentially expressed genes from 13824 cDNA clones. These genes were related to signal transduction, stress responses, senescence, photosynthesis, protein biosynthesis and transportation. The results provide the molecular evidence that auxin influences a variety of physiological processes and pave a way for further investigation of the mechanism of auxin action. Furthermore,we found that the expression of a ClpC (regulation subunit of Clp protease) was repressed by exogenous auxin, but increased in dark-induced senescing leaves. This suggests that ClpC may be a senescence-associated gene and can be regulated by auxin.

  18. Construction of cDNA Library of Pyrocystis lunula(Pyrophyta)

    Institute of Scientific and Technical Information of China (English)

    SUI Zhenghong; Klaus V.Kowallik

    2004-01-01

    Complementary DNA library of a dinoflagellate Pyrocystis lunula was constructed for the purpose of expression sequence tags analysis. The RNA isolated from this alga was about 20 μg g-1 net cells, and the band intensity ratio of 28 S/18 S in electrophoresis pattern was nearly 1 to 1. Different cDNA/vector molar ratios were exploited in the ligating reaction to be optimized. The clones produced by cDNA/vector molar ratio of 3.75 to 1 were desirable, most of whose inserts were longer than 300 bp. The recombinants insert length of the unfractionation cDNA library was largely shorter than 500 bp. However, in the fractionation library made from high molecule weight cDNA parts, over seventy percent of the recombinants contained inserts longer than 1 kb, some of which were even longer than 3 kb. Operating concerns were discussed at the end.

  19. Cdna cloning and expression analyses of the isoflavone reductase-like gene of dendrobium officinale

    International Nuclear Information System (INIS)

    The full length of the isoflavone reductase-like gene (IRL) cDNA of Dendrobium officinale was cloned by using reverse transcription (RT) PCR combined with cDNA library, the IRL function was identified by Bioinformatics and prokaryotic expression analyses, and the IRL expression levels in the organs and tissues of D. officinale plants with different ages were determined by using real-time quantitative PCR (RT-qPCR). The results indicated that the full length of the cDNA of D. officinale IRL, DoIRL, was 1238 bp (accession no. KJ661023). Its open reading frame (ORF) was 930 bp which encoded 309 amino acids with a predicted molecular mass of 34 kDa, the 5 untranslated region (UTR) was 61 bp and the 3 UTR containing a poly (A) tail was 247 bp. The deduced amino acid sequence of DoIRL, DoIRL, was forecast to contain a NAD(P)H-binding motif (GGTGYIG) in the N-terminal region, two conserved N-glycosylation sites, a conserved nitrogen metabolite repression regulator (NmrA) domain and a phenylcoumaran benzylic ether reductase (PCBER) domain, to hold the nearest phylogenetic relationship with the PCBER of Striga asiatica, and to share both 73% identity with the isoflavone reductases-like (IRLs) of Cucumis sativus and Striga asiatica. In Escherichia coli 'BL21' cells, the DoIRL cDNA expression produced a protein band holding the predicted molecular mass of 34 kDa. DoIRL expressed in all organs and tissues of D. officinale plants with different ages at comparatively low levels, and the expression level in the leaves of the two-year-old plants was the highest. (author)

  20. Cloning by insertional mutagenesis of a cDNA encoding Caenorhabditis elegans kinesin heavy chain.

    OpenAIRE

    Patel, N; Thierry-Mieg, D.; Mancillas, J R

    1993-01-01

    An additional genetic locus in Caenorhabditis elegans, unc-116, was identified in a screen for mutations resulting in defective locomotion. unc-116 was cloned by use of a transposon insertion mutant and the physical and genetic map of the genome. The cDNA sequence predicts an 815-amino acid protein. Based upon sequence comparison and secondary structure predictions, unc-116 encodes all three domains of the kinesin heavy chain: the motor, stalk, and tail. While the motor and tail domains have ...

  1. Identification and molecular characterization of numerous Histomonas meleagridis proteins using a cDNA library

    OpenAIRE

    Bilic, I.; LEBERL, M.; Hess, M.

    2009-01-01

    Histomonas meleagridis is a protozoan parasite of various galliform birds causing a type of enterohepatitis termed histomonosis or ‘blackhead disease’. Due to the ban of chemotherapeutic substances and an increase in free-range poultry production, histomonosis is currently a re-emerging disease. So far limited molecular knowledge is available. In the present work, mRNAs coding for antigenic proteins of H. meleagridis were identified. For this purpose, a cDNA expression library was constructed...

  2. Characterization of mouse Clpp protease cDNA, gene, and protein

    DEFF Research Database (Denmark)

    Andresen, B S; Corydon, T J; Wilsbech, M;

    2000-01-01

    characterized the mouse Clpp cDNA sequence, the organization of the mouse gene, the chromosomal localization, and the tissue-specific expression pattern. Moreover. the cellular localization and processing of mouse Clpp was studied by overexpression in transfected eukaryotic cells. Our results indicate that...... mouse and human Clpp have similar roles, and they provide the molecular basis for establishing a Clpp knockout mouse and to study its phenotype, thereby shedding light on a possible role of Clpp in human disease....

  3. Detection of HIV cDNA Point Mutations with Rolling-Circle Amplification Arrays

    OpenAIRE

    Zhongwei Wu; Zuhong Lu; Quanjun Liu; Lingwei Wu

    2010-01-01

    In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplifica...

  4. Analysis of gene expression profile of pancreatic carcinoma using CDNA microarray

    Institute of Scientific and Technical Information of China (English)

    ZhiJun Tan; Xian-Gui Hu; Gui-Song Cao; Yan Tang

    2003-01-01

    AIM: To identify new diagnostic markers and drug targets,the gene expression profiles of pancreatic cancer were compared with that of adjacent normal tissues utilizing cDNA microarray analysis.METHODS: cDNA probes were prepared by labeling mRNA from samples of six pancreatic carcinoma tissues with Cy5dUTP and mRNA from adjacent normal tissues with Cy3dUTP respectively through reverse transcription. The mixed probes of each sample were then hybridized with 12 800cDNA arrays (12 648 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3 000scanner (General Scanning, Inc.). The values of CyS-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Differentially expressed genes were screened according to the criterion that the absolute value of natural logarithm of the ratio of Cy5-dUTP to Cy3-dUTP was greater-than 0.69.RESETS: Among 6 samples investigated, 301 genes, which accounted for 2.38% of genes on the microarry slides,exhibited differentially expression at least in 5. There were 166 over-expressed genes including 136 having been registered in Genebank, and 135 under-expressed genes including 79 in Genebank in cancerous tissues.CONCLUSION: Microarray analysis may provide invaluable information on disease pathology, progression, resistance to treatment, and response to cellular microenvironments of pancreatic carcinoma and ultimately may lead to improving early diagnosis and discovering innovative therapeutic approaches for cancer.

  5. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase.

    OpenAIRE

    Finocchiaro, G; Taroni, F; Rocchi, M; Martin, A.L.; Colombo, I; Tarelli, G T; DiDonato, S

    1991-01-01

    We have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase (CPTase; palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21), an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA enco...

  6. Cloning of cDNA Encoding GRA1 Protein of Tachyzoite Toxoplasma Gondii Local Isolate

    OpenAIRE

    Erma Sulistyaningsih; Sukarti Moeljopawiro; Jarot Subandono; Wayan T. Artama

    2015-01-01

    Gene encoding GRA1 protein is potent DNA-vaccine candidate against toxoplasmosis. The aim of the researchwas to clone the gene encoding GRA1 protein of tachyzoite Toxoplasma gondii local isolate by DNA recombinanttechnology. Tachyzoite was grown in Balb/c mice in vivo. Messenger RNA was isolated from total RNA and itwas used to synthesis cDNA. Complementary DNA encoding GRA1 protein of tachyzoite Toxoplasma gondii localisolate was amplified and cloned in a prokaryote cloning vector. The recom...

  7. Rice8987 g_array: cDNA information: 6928 [RMOS[Archive

    Lifescience Database Archive (English)

    Full Text Available g_6928 EG0853 >T1J1_6(AF128393|pid:g4325345) Arabidopsis thaliana BAC T1J1; similar to thioredox ... in-like proteins (Pfam: PF00085, Score=42 .9, E=1.4e-11, N=1); contains similarity to dihydro ... orotases (Pfam: PF00744, Score=154.9, E=1.4e-42 , N=1); coded for by A. thaliana cDNA W43503; coded ...

  8. Recombinant Expression of Pleurocidin cDNA Using the Pichia pastoris Expression System

    OpenAIRE

    Olive-Jean Burrowes; Gill Diamond; Tung-Ching Lee

    2005-01-01

    This research utilized the Pichia pastoris expression system for recombinant expression of cDNA of pleurocidin, a small (2.7 kd) antimicrobial peptide isolated from winter flounder (Pleuronectes americanus). The Pichia vector contains the alcohol oxidase gene promoter (AOX 1), which under the induction of methanol allows for expression of heterologous protein gene inserted downstream in the vector. Two strains of P pastoris were used as host cells, the wild type (P pastoris X-33(mut+)) and th...

  9. Induction of pigmentation in mouse fibroblasts by expression of human tyrosinase cDNA

    OpenAIRE

    1989-01-01

    A distinguishing characteristic of cells of the melanocyte lineage is the expression of the melanosomal enzyme tyrosinase that catalyzes the synthesis of the pigment melanin. A tyrosinase cDNA clone, designated BBTY-1, was isolated from a library constructed from the pigmented TA99+/CF21+ melanoma cell line SK-MEL-19. Expression of BBTY-1 in mouse L929 fibroblasts led to synthesis and expression of active tyrosinase, and, unexpectedly, to stable production of melanin. Melanin was synthesized ...

  10. Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells

    DEFF Research Database (Denmark)

    Jensen, T G; Andresen, B S; Bross, P; Jensen, U B; Holme, E; Kølvraa, S; Gregersen, N; Bolund, L

    1992-01-01

    polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild...

  11. Expression cloning of cDNA encoding a seven-helix receptor from human placenta with affinity for opioid ligands

    OpenAIRE

    1992-01-01

    Here we report the expression cloning of cDNA encoding a putative opioid receptor from a human placenta cDNA library. Placental opioid receptors are of the kappa type. As the dynorphin opioid peptides are kappa-selective, a dynorphin ligand was used in an affinity-enrichment (panning) procedure to select transiently transfected COS-7 cells expressing kappa receptor binding sites. The cloned cDNA encodes a 440-residue protein of the seven-helix guanine nucleotide-binding protein (G-protein)-co...

  12. Cloning and expression of a cDNA encoding ribosomal protein S4 from Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A cDNA clone, pS4, has been isolated from a cDNA library prepared from rice anthers of about 1.0 mm in length. DNA sequence analysis and database search show that the cDNA encodes a protein which is highly homologous to eukaryotic 80S ribosomal protein subunit 4 (S4). Northern hybridization indicates that this gene expresses in all tissues analyzed although the expression level varies and it cannot be induced by mechanical wounding in leaves. Southern blot analysis demonstrates that this rice S4 gene is from a multigene family.

  13. MOLECULAR-CLONING AND CHARACTERIZATION OF A CDNA FOR THE BETA-SUBUNIT OF HUMAN ALCOHOL-DEHYDROGENASE

    OpenAIRE

    Duester, G; Hatfield, G.; Buhler, R; Hempel, J; Jornvall, H; Smith, M.

    1984-01-01

    Human alcohol dehydrogenase (ADH) is encoded by at least five genes that fall into three classes. The class I ADH genes encode the three closely related alpha, beta, and gamma polypeptides. Molecular genetic analysis of class I ADH genes has been initiated by isolating a cDNA clone from a human adult liver cDNA library. A synthetic oligonucleotide mixture encoding a portion of the beta subunit of ADH was used as an in situ hybridization probe for the cDNA library. One positively hybridizing c...

  14. Cloning and chromosomal localization of a human kidney cDNA involved in cystine, dibasic, and neutral amino acid transport.

    OpenAIRE

    Lee, W S; Wells, R G; Sabbag, R V; Mohandas, T K; Hediger, M A

    1993-01-01

    We have recently cloned, sequenced, and characterized a rat kidney cDNA (D2) that stimulates cystine as well as dibasic and neutral amino acid transport. In order to evaluate the role of this protein in human inherited diseases such as cystinuria, we have isolated a human D2 clone (D2H) by low stringency screening of a human kidney cDNA library using the radiolabeled D2 insert as a probe. The D2H cDNA is 2284 nucleotides long and encodes a 663 amino acid protein that is 80% identical to the r...

  15. Recombinant selection by microinjection: a simple cDNA cloning procedure for production of exclusively sense RNA transcripts

    OpenAIRE

    Digweed, M; Günthert, U

    1989-01-01

    A new strategy for cDNA cloning is presented, designed particularly for identification of recombinants by functional analysis, after microinjection into somatic cells. First-strand synthesis is primed by the oligodeoxyribonucleotide: (formula; see text) After second-strand synthesis and blunting, double-stranded cDNA is formed, which carries restriction sites for NotI and ApaI downstream from the coding sequence. The cDNA is ligated into a plasmid, between two promoters for phage T7 and T3 RN...

  16. cDNA cloning and expression of an apoptosis-related gene, human TFAR15 gene

    Institute of Scientific and Technical Information of China (English)

    王玉刚; 刘洪涛; 张颖妹; 马大龙

    1999-01-01

    By means of cDNA-RDA method. some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these tragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1218 nueleotides and encodes 212 amino acids. The putative protein protein product of TFAR15 is partially homologous to C. elegans protein C14A4. 11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung. and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-(?) cells after GM-CSF withdrawal. In vitro analysis showed that the recombinant TFAR15 protein co(?)ld inhibit the natural cell death of 293 cells, an embryonic kidney cell

  17. Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil.

    Science.gov (United States)

    Fang, Changxun; Xu, Tiecheng; Ye, Changliang; Huang, Likun; Wang, Qingshui; Lin, Wenxiong

    2014-02-01

    Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al₂(SO₄)₃ were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al₂(SO₄)₃ for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses. PMID:24078111

  18. Detection of HIV cDNA Point Mutations with Rolling-Circle Amplification Arrays

    Directory of Open Access Journals (Sweden)

    Zhongwei Wu

    2010-01-01

    Full Text Available In this paper we describe an isothermal rolling-circle amplification (RCA protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP.The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

  19. cDNA cloning and expression of a collectin from red-spotted grouper (Epinephelus akaara)

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhiwen; DING Shaoxiong; WANG Ying; MAO Yong; SU Yongquan; WANG Jun

    2009-01-01

    Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens. We obtained the complete cDNA of a C-type lectin (EALec1) from Epinephelus akaara using RACE. The complete EALec1 cDNA sequence was 827 bp. The 5-UTR and 3-UTR were 28 bp and 151 bp, respectively, in length. The sequence also contained a polyadenylation signal AATAAA and a poly(A) tail. The EALec1 cDNA encodes polypeptides with 215 amino acids, including a signal peptide of 31 amino acids. The protein has a cysteine-rich region at the N terminal, a collagenous region characterized by G-X-Y repeats, a neck region, and a typical carbohydrate-recognition domain (CRD), indicating that EALec1 is a collectin. The key recognition positions of this CRD are EPD, isolated for the first time in fish. These are likely the interim types, between mannan-binding lectin and galactose-binding lectin. We evaluated the expression pattern of EALec1 in 12 different tissues using RT-PCR. EALec1 was expressed in all tissues, though at different levels. In addition, we inserted EALec1 into an expression vector (pET-28a) for transformation into the BL21 engineering bacteria. Based on enzyme digestion and sequencing of the positive clone, we successfully constructed the EALec1 recombinant expression vector.

  20. Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana.

    Science.gov (United States)

    Villand, P; Olsen, O A; Kleczkowski, L A

    1993-12-01

    PCR amplification of cDNA prepared from poly(A)+ RNA from aerial parts of Arabidopsis thaliana, using degenerate nucleotide primers based on conserved regions between the large and small subunits of ADP-glucose pyrophosphorylase (AGP), yielded four different cDNAs of ca. 550 nucleotides each. Based on derived amino acid sequences, the identities between the clones varied from 49 to 69%. Sequence comparison to previously published cDNAs for AGP from various species and tissues has revealed that three of the amplified cDNAs (ApL1, ApL2 and ApL3) correspond to the large subunit of AGP, and one cDNA (ApS) encodes the small subunit of AGP. Both ApL1 and ApS were subsequently found to be present in a cDNA library made from Arabidopsis leaves. All four PCR products are encoded by single genes, as found by genomic Southern analysis. PMID:8292792

  1. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

  2. cDNA cloning and expression of a collectin from red-spotted grouper ( Epinephelus akaara)

    Science.gov (United States)

    Zhang, Zhiwen; Ding, Shaoxiong; Wang, Ying; Mao, Yong; Su, Yongquan; Wang, Jun

    2009-09-01

    Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens. We obtained the complete cDNA of a C-type lectin (EALec1) from Epinephelus akaara using RACE. The complete EALec1 cDNA sequence was 827 bp. The 5-UTR and 3-UTR were 28 bp and 151 bp, respectively, in length. The sequence also contained a polyadenylation signal AATAAA and a poly(A) tail. The EALec1 cDNA encodes polypeptides with 215 amino acids, including a signal peptide of 31 amino acids. The protein has a cysteine-rich region at the N terminal, a collagenous region characterized by G-X-Y repeats, a neck region, and a typical carbohydrate-recognition domain (CRD), indicating that EALec1 is a collectin. The key recognition positions of this CRD are EPD, isolated for the first time in fish. These are likely the interim types, between mannan-binding lectin and galactose-binding lectin. We evaluated the expression pattern of EALec1 in 12 different tissues using RT-PCR. EALec1 was expressed in all tissues, though at different levels. In addition, we inserted EALec1 into an expression vector (pET-28a) for transformation into the BL21 engineering bacteria. Based on enzyme digestion and sequencing of the positive clone, we successfully constructed the EALec1 recombinant expression vector.

  3. Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger

    Directory of Open Access Journals (Sweden)

    Chang-Qing Liu, Tao-Feng Lu, Bao-Gang Feng, Dan Liu, Wei-Jun Guan, Yue-Hui Ma

    2010-01-01

    Full Text Available In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×106 pfu/ml and 1.62×109 pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers.

  4. Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

    DEFF Research Database (Denmark)

    Lundwall, Åke B; Wetsel, Rick A; Kristensen, Torsten;

    1985-01-01

    clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence......We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA...... obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication...

  5. Secreted form of the human interleukin 2 receptor encoded by an anchor minus cDNA

    International Nuclear Information System (INIS)

    The DNA sequence encoding all of the putative intracytoplasmic domain and most of the trans-membrane domain of the huma IL 2 receptor was detected from a full length receptor cDNA. After expression in mouse L cells, the resultant anchor minus cDNA was found to direct the synthesis of a secreted rather than membrane-associated form of the IL 2 receptor. The secreted receptor protein (44,000 to 46,000 M/sub r/) retained the capacity to bind both IL 2 and the monoclonal anti-Tac antibody, as evidenced by (1) retention on IL 2 and anti-Tac affinity columns, (2) inhibition of [3H]-antiTac binding to HUT 102B2 cells, and (3) partial inhibition of IL 2-induced CTLL proliferation. Removal of these domains from the IL 2 receptor did not alter the post-translational processing or rate of export of the truncated receptor protein. These data confirm the proposed membrane orientation of the IL 2 receptor (NH2 terminus out, COOH terminus in) and underscore the anchoring function of this carboxy terminal receptor segment. The availability of such anchor minus receptor cDNA constructs may facilitate purification of large quantities of receptor protein for further analysis of receptor structure, valency, and localization of the IL 2 binding site(s)

  6. Cloning and roles of goldfish maternal factor β-Catenin cDNA in embryonic development

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jingpu; WANG Weixian; ZHU Shaoxia

    2004-01-01

    Interaction between nucleus and cytoplasm has been focused in the field of animal embryonic development, in which study of maternal factors is required positively. Β-Catenin, an important maternal factor in early embryogenesis, has been analyzed in its expression pattern and functions in this paper. We have cloned goldfish β-Catenin cDNA gene and compared it with zebrafish β-Catenin cDNA. High homology was found in cDNA and in amino acid sequences between them, 93% (2227/2384 bp) and 98.5% (768/780 aa) respectively. The expression pattern of β-Catenin by in situ hybridization and the roles of β-Catenin on embryonic development by co-injection of anti-sense RNA and reporter gene, EGFP have been investigated in the whole process of goldfish embryonic development. The results suggest that β-Catenin presents dynamic distribution, mainly locates at body axis, dorsal tissues, head and tail structures after being fertilized. The loss of β-Catenin activity would cause serious destruction of embryo in dorsal tissues and in anteroposterior axes, and leads embryos to die before larva get hatched.

  7. Cloning and characterization of a cDNA coding for mouse placental alkaline phosphatase

    International Nuclear Information System (INIS)

    Mouse alkaline phosphatase was partially purified from placenta. Data obtained by immunoblotting analysis suggested that the primary structure of this enzyme has a much greater homology to that of human and bovine liver ALPs than to the human placental isozyme. Therefore, a full-length cDNA encoding human liver-type ALP was used as a probe to isolate the mouse placental ALP cDNA. The cloned mouse cDNA is 2459 base pairs long and is composed of an open reading frame encoding a 524-amino acid polypeptide that contains a putative signal peptide of 17 amino acids. Homology at the amino acid level of the mouse placental ALP is 90% to the human liver isozyme but only 55% to the human placental counterpart. RNA blot hybridization results indicate that the mouse placental ALP is encoded by a gene identical to the gene expressed in mouse liver, kidney, and teratocarcinoma stem cells. This gene is therefore evolutionarily highly conserved in mouse and human

  8. Molecular cloning of a full-length cDNA for ECBP21 from Angelica dahurica

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1-25 amino acid sequence is a predicted signal peptide and the other 26-216 amino acid sequence is a mature peptide. The 26-45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca2+- dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.

  9. Gene expression profile analysis in astaxanthin-induced Haematococcus pluvialis using a cDNA microarray.

    Science.gov (United States)

    Eom, Hyunsuk; Lee, Choul-Gyun; Jin, EonSeon

    2006-05-01

    The unicellular green alga Haematococcus pluvialis (Volvocales) is known for the ketocarotenoid astaxanthin (3, 3'-dihydroxy-beta, beta-carotene-4, 4'-dione) accumulation, which is induced under unfavorable culture conditions. In this work, we used cDNA microarray analysis to screen differentially expressed genes in H. pluvialis under astaxanthin-inductive culture conditions, such as combination of cell exposure to high irradiance and nutrient deprivation. Among the 965 genes in the cDNA array, there are 144 genes exhibiting differential expression (twofold changes) under these conditions. A significant decrease in the expression of photosynthesis-related genes was shown in astaxanthin-accumulating cells (red cells). Defense- or stress-related genes and signal transduction genes were also induced in the red cells. A comparison of microarray and real-time PCR analysis showed good correlation between the differentially expressed genes by the two methods. Our results indicate that the cDNA microarray approach, as employed in this work, can be relied upon and used to monitor gene expression profiles in H. pluvialis. In addition, the genes that were differentially expressed during astaxanthin induction are suitable candidates for further study and can be used as tools for dissecting the molecular mechanism of this unique pigment accumulation process in the green alga H. pluvialis. PMID:16320067

  10. Paramyosin from the parasitic mite Sarcoptes scabiei: cDNA cloning and heterologous expression.

    Science.gov (United States)

    Mattsson, J G; Ljunggren, E L; Bergström, K

    2001-05-01

    The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies. PMID:11393829

  11. Identification and cloning of the cDNA of a Rb-associated protein RAP140a

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Rb exerts important physiological functions in cell-cycle control, gene expression, cell differentiation, apoptosis, development and tumorigenesis by interacting with many cellular proteins. Using human partial Rb as bait, we screened a human fetal brain cDNA library through yeast two-hybrid system and obtained six novel cDNA fragments. Among them, one cDNA fragment corre-sponds to two different transcripts, 7 kb and 9 kb in Northern blot analysis. These two transcripts showed uniform distribution in various human tissues. We cloned the full-length cDNA of a 7.2 kb transcript through three times PCR amplifications. It was named RAP140a and predicted to encode a 1 233 amino acids hydrophilic protein. RAP140a was mapped to chromosome 3p13-p14.1. RAP140a may be functionally related to the intracellular translocation of Rb or other proteins.

  12. Identification and cloning of the cDNA of a Rb-associated protein RAP140a

    Institute of Scientific and Technical Information of China (English)

    李权; 闻宏; 敖世洲

    2000-01-01

    Rb exerts important physiological functions in cell-cycle control, gene expression, cell differentiation, apoptosis, development and tumorigenesis by interacting with many cellular proteins. Using human partial Rb as bait, we screened a human fetal brain cDNA library through yeast two-hybrid system and obtained six novel cDNA fragments. Among them, one cDNA fragment corresponds to two different transcripts, 7 kb and 9 kb in Northern blot analysis. These two transcripts showed uniform distribution in various human tissues. We cloned the full-length cDNA of a 7.2 kb transcript through three times PCR amplifications. It was named RAP140a and predicted to encode a 1 233 amino acids hydrophilic protein. RAP140a was mapped to chromosome 3p13-p14.1. RAP140a may be functionally related to the intracellular translocation of Rb or other proteins.

  13. Two human cDNA molecules coding for the Duchenne muscular dystrophy (DMD) locus are highly homologous

    Energy Technology Data Exchange (ETDEWEB)

    Rosenthal, A.; Speer, A.; Billwitz, H. (Zentralinstitut fuer Molekularbiologie, Berlin-Buch (Germany Democratic Republic)); Cross, G.S.; Forrest, S.M.; Davies, K.E. (Univ. of Oxford (England))

    1989-07-11

    Recently the complete sequence of the human fetal cDNA coding for the Duchenne muscular dystrophy (DMD) locus was reported and a 3,685 amino acid long, rod-shaped cytoskeletal protein (dystrophin) was predicted as the protein product. Independently, the authors have isolated and sequenced different DMD cDNA molecules from human adult and fetal muscle. The complete 12.5 kb long sequence of all their cDNA clones has now been determined and they report here the nucleotide (nt) and amino acid (aa) differences between the sequences of both groups. The cDNA sequence comprises the whole coding region but lacks the first 110 nt from the 5{prime}-untranslated region and the last 1,417 nt of the 3{prime}-untranslated region. They have found 11 nt differences (approximately 99.9% homology) from which 7 occurred at the aa level.

  14. cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity

    International Nuclear Information System (INIS)

    The hst gene was originally identified as a transforming gene in DNAs from human stomach cancers and from a noncancerous portion of stomach mucosa by DNA-mediated transfection assay using NIH3T3 cells. cDNA clones of hst were isolated from the cDNA library constructed from poly(A)+ RNA of a secondary transformant induced by the DNA from a stomach cancer. The sequence analysis of the hst cDNA revealed the presence of two open reading frames. When this cDNA was inserted into an expression vector containing the simian virus 40 promoter, it efficiently induced the transformation of NIH3T3 cells upon transfection. It was found that one of the reading frames, which coded for 206 amino acids, was responsible for the transforming activity

  15. Construction and identification of subtracted cDNA library in bone marrow cells of radon-exposed mice

    International Nuclear Information System (INIS)

    Objective: To construct and identify subtracted cDNA library in bone marrow cells of mice exposed to radon inhalation. Methods: Adult male BALB/c mice, weighing 18-22 g, were placed in a multi- functional radon chamber. One group of mice was exposed to radon up to the accumulative dose of 105 work level month (WLM). The control group of mice was housed in a room with an accumulative dose of 1 WLM. To construct a subtracted cDNA library enriched with differentially expressed genes, the SMART technique and the suppression subtractive hybridization were performed. The obtained forward and reverse cDNA fragments were directly inserted into pMD18-T vector and transformed into E. coli JM109. The inserting cDNA fragments were screened by the blue-and-white blot screening and nested PCR of bacterium liquid. Results: The 244 of 285 white bacteria clones obtained randomly were positive clones contained 100-1100 bp inserted cDNA fragments. Conclusions: The forward and reverse subtracted cDNA library in bone marrow cells of mice exposed to radon inhalation is successfully constructed. (authors)

  16. Molecular cloning of a human glycophorin B cDNA: nucleotide sequence and genomic relationship to glycophorin A

    International Nuclear Information System (INIS)

    The authors describe the isolation and nucleotide sequence of a human glycophorin B cDNA. The cDNA was identified by differential hybridization of synthetic oligonucleotide probes to a human erythroleukemic cell line (K562) cDNA library constructed in phage vector λgt10. The nucleotide sequence of the glycophorin B cDNA was compared with that of a previously cloned glycophorin A cDNA. The nucleotide sequences encoding the NH2-terminal leader peptide and first 26 amino acids of the two proteins are nearly identical. This homologous region is followed by areas specific to either glycophorin A or B and a number of small regions of homology, which in turn are followed by a very homologous region encoding the presumed membrane-spanning portion of the proteins. They used RNA blot hybridization with both cDNA and synthetic oligonucleotide probes to prove our previous hypothesis that glycophorin B is encoded by a single 0.5- to 0.6-kb mRNA and to show that glycophorins A and B are negatively and coordinately regulated by a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. They established the intron/exon structure of the glycophorin A and B genes by oligonucleotide mapping; the results suggest a complex evolution of the glycophorin genes

  17. Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a P-Glycoprotein cDNA.

    Directory of Open Access Journals (Sweden)

    Kristen M Pluchino

    Full Text Available The efflux transporter P-glycoprotein (P-gp is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.

  18. cDNA cloning, prokaryotic expression and purification of rat α-synuclein

    Institute of Scientific and Technical Information of China (English)

    Xin LI; Yao-Hua LI; Jun-Yan HAN; Shun YU; Biao CHEN

    2006-01-01

    Objective To clone the cDNA of rat α-Syn gene, investigate its prokaryotic expression and produce purified recombinant rat α-Syn protein. Methods Rat α-Syn cDNA was amplified from the rat brain total RNA by RT-PCR and was cloned into pGEX-4T-1, a prokaryotie expressing vector. The recombinant plasmid containing rat α-Syn gene was transformed into E. Coli BL21 to express a fusion protein with rat α-Syn protein tagged by glutathione-S-transferase (GST). The fusion protein was then cleaved by thrombin during passing through the GST-agarose 4B column to release the recombinant rat α-Syn protein. The recombinant rat α-Syn protein was further purified using Superdex S200 gel filtration.Results DNA sequencing confirmed that the cloned cDNA contained 420 base pairs encoding 140 amino acids, which was identical to the reported amino acid sequence of rat α-Syn. After transformation, the recombinant plasmid pGEX-raSyn expressed a soluble protein that was inducible by IPTG. The purified recombinant protein was shown to be single band on SDS-PAGE, with a molecular size of around 18000, which was identical to the reported molecular size of rat α-Syn.Western blot analysis demonstrated that the recombinant protein was recognized by specific antibody against α-Syn. Conclusion The rat α-Syn gene was successfully expressed in prokaryotic expression system and highly purified rat α-Syn recombinant protein was produced.

  19. ANALYSIS OF GENES ASSOCIATED WITH LYMPHATIC METASTASIS IN PANCREATIC CARCINOMA USING cDNA MICROARRAY

    Institute of Scientific and Technical Information of China (English)

    谭志军; 胡先贵; 曹贵松; 唐岩

    2003-01-01

    Objective: To identify new markers for prediction of lymph node metastasis. Methods: cDNA probes were prepared by labeling mRNA from samples of four pancreatic carcinoma tissues with Cy5-dUTP and mRNA from adjacent normal tissues with Cy3-dUTP respectively through reverse transcription. The mixed probes of each sample were then hybridized with 4,096 cDNA arrays (4,000 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Genes that differentially expresses in each cancerous tissue were sought out according to the standard that the absolute value of natural logarithm of the ratio of Cy5 to Cy3 is greater than 0.69, i. e., more than 2 times change of gene expression, and the signal value of either Cy3 and Cy5 need to be greater than 600. Then, the genes differently expressed in cancer with and without lymphatic metastasis were screened out for further analysis. Results: Among 2 samples with lymphatic metastasis and 2 samples without metastasis, 56 genes, which accounted for 1.40% of genes on the microarray slides, exhibited differentially expression in cancerous tissues with lymphatic metastasis. There were 32 over-expressed genes including 11 having been registered in Genebank, and 24 under-expressed genes including 3 in Genebank. Conclusion: Microarray analysis may provide invaluable information to identify specific gene expression profile of lymphatic metastasis in pancreatic cancer.

  20. Screening for candidate genes related to breast cancer with cDNA microarray analysis

    Institute of Scientific and Technical Information of China (English)

    Yu-Juan Xiang; Zhi-Gang Yu; Ming-Ming Guo; Qin-Ye Fu; Zhong-Bing Ma; De-Zong Gao; Qiang Zhang; Yu-Yang Li; Liang Li; Lu Liu; Chun-Miao Ye

    2015-01-01

    Objective: The aim of this study was to reveal the exact changes during the occurrence of breast cancer to explore significant new and promising genes or factors related to this disease. Methods: We compared the gene expression profiles of breast cancer tissues with its uninvolved normal breast tissues as controls using the cDNA microarray analysis in seven breast cancer patients. Further, one representative gene, named IFI30, was quanti-tatively analyzed by real-time PCR to confirm the result of the cDNA microarray analysis. Results: A total of 427 genes were identified with significantly differential expression, 221 genes were up-regulated and 206 genes were down-regulated. And the result of cDNA microarray analysis was validated by detection of IFI30 mRNA level changes by real-time PCR. Genes for cell proliferation, cell cycle, cell division, mitosis, apoptosis, and immune response were enriched in the up-regulated genes, while genes for cell adhesion, proteolysis, and transport were significantly enriched in the down-regulated genes in breast cancer tissues compared with normal breast tissues by a gene ontology analysis. Conclusion: Our present study revealed a range of differentially expressed genes between breast cancer tissues and normal breast tissues, and provide candidate genes for further study focusing on the pathogenesis and new biomarkers for breast cancer. Copyright © 2015, Chinese Medical Association Production. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

  1. Cloning and expression of cDNA for human poly(ADP-ribose)polymerase

    International Nuclear Information System (INIS)

    cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma λgt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly(ADP-ribose) polymerase. To confirm that the 1.3-kb insert from λgt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents

  2. A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs

    Directory of Open Access Journals (Sweden)

    I. da Silva

    2002-08-01

    Full Text Available In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8 showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries. Plants submitted to stress (wounding, virus infection and ethylene treatment presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

  3. Full-length cDNA cloning and structural characterization of preproinsulin in Alligator sinensis.

    Science.gov (United States)

    Zhang, R; Zhang, S Z; Li, E; Wang, C; Wang, C L; Wu, X B

    2014-01-01

    Insulin is an important endocrine hormone that plays a critical physiological role in regulating metabolism and glucostasis in vertebrates. In this study, the complete cDNA of Alligator sinensis preproinsulin gene was cloned for the first time by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods; the amino acid sequence encoded and protein structure were analyzed. The full-length of preproinsulin cDNA sequence consists of 528 base pairs (bp), comprising a 34-bp 5'-untranslated region, a 170-bp 3'-untranslated region and an open reading frame that is 324 bp in length. The open reading frame encodes a 107-amino acid preproinsulin with a molecular weight of approximately 12,153.8 Da, theoretical isoelectric point of 5.68, aliphatic index of 92.06, and grand average of hydropathicity of -0.157, from which a signal peptide, a B-chain, a C-peptide, and an A-chain are derived. Online analysis suggested that the deduced preproinsulin amino acid sequence contains a transmembrane region, and that it has a signal peptide whose cleavage site occurs between alanine 24 and alanine 25. Comparative analysis of preproinsulin amino acid sequences indicated that the A-chain and B-chain sequences of preproinsulins are highly conserved between reptiles and birds, and that the preproinsulin amino acid sequence of Alligator sinensis shares 89% similarity to that of Chelonia mydas, but low similarity of 48-63% to those of mammals and fishes. The phylogenetic tree constructed using the neighbor-joining method revealed that preproinsulin of Alligator sinensis had high homology with reptiles and birds, such as Chelonia mydas, Gallus gallus, and Columba livia. PMID:25366775

  4. Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration

    Directory of Open Access Journals (Sweden)

    Bryant Susan V

    2009-01-01

    Full Text Available Abstract Background Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR and denervated (DL forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. Results Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa. Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST contigs from the Ambystoma EST database more than doubled (3935 to 9411 the number of non-redundant human-A. mexicanum orthologous sequences. Conclusion Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

  5. Generation and Analysis of Full-length cDNA Sequences from Elephant Shark (Callorhinchus milii)

    KAUST Repository

    Kodzius, Rimantas

    2009-03-17

    Cartilaginous fishes are the oldest living group of jawed vertebrates and therefore is an important group for understanding the evolution of vertebrate genomes including the human genome. Our laboratory has proposed elephant shark (C. milii) as a model cartilaginous fish genome because of its relatively small genome size (910 Mb). The whole genome of C. milii is being sequenced (first cartilaginous fish genome to be sequenced completely). To characterize the transcriptome of C. milii and to assist in annotating exon-intron boundaries, transcriptional start sites and alternatively spliced transcripts, we are generating full-length cDNA sequences from C. milii.

  6. Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

    Directory of Open Access Journals (Sweden)

    Murwantoko M

    2015-11-01

    Full Text Available Rhoptry protein belongs to an excretory and secretory antigens (ESAs that play an important role during active penetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targeted cell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasite successfully enter the cell target then Granule (GRA proteins are responsible for the formation of parasitophorus vacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently, this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone and sequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique. Total ribonucleic acid (RNA was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA was used as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor from Riboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinant plasmid was transformed into E. coli (XL1-Blue. The transformed E. coli XL-1 Blue were plated on LB agar containing X-Gal, IPTG and ampicillin. Recombinant clones (white colony were picked up and grown up in the LB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order to identify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolated using alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid was cut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward and M13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretory

  7. Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

    Directory of Open Access Journals (Sweden)

    Wayan T. Artama

    2015-10-01

    Full Text Available Rhoptry protein belongs to an excretory and secretory antigens (ESAs that play an important role during activepenetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targetedcell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasitesuccessfully enter the cell target then Granule (GRA proteins are responsible for the formation of parasitophorusvacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently,this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone andsequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique.Total ribonucleic acid (RNA was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA wasused as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor fromRiboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinantplasmid was transformed into E. coli (XL1-Blue. The transformed E. coli XL-1 Blue were plated on LB agarcontaining X-Gal, IPTG and ampicillin. Recombinant clones (white colony were picked up and grown up in theLB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order toidentify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolatedusing alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid wascut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward andM13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretoryand secretory

  8. Construction of Rat Calcineurin A α cDNA Recombinant Adenovirus Vector and Its Identification

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Rat calcineurin (CaN) A α isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemiareperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG-FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared.The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results veri fied that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A α (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.

  9. Significant overexpression of oligophrenin-1 in colorectal tumors detected by cDNA microarray analysis.

    Science.gov (United States)

    Pinheiro, N A; Caballero, O L; Soares, F; Reis, L F; Simpson, A J

    2001-10-22

    The human oligophrenin-1 gene is ubiquitously expressed at low levels and expressed at high levels in the developing neuroepithelium of the neural tube. Mutations in this gene have been related to the X-linked mental retardation. Using cDNA microarrays, we found evidence that oligophrenin-1 is strongly up-regulated in colorectal tumors. Semiquantitative reverse transcriptase polymerase chain reaction confirmed this finding. Thus, a well-known nervous system-associated human gene transcript may also be an important colorectal tumor marker and potential therapeutic target. PMID:11595131

  10. A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication.

    OpenAIRE

    Grafi, G; R.J. Burnett; Helentjaris, T; Larkins, B A; DeCaprio, J A; Sellers, W R; Kaelin, W G

    1996-01-01

    Retinoblastoma (RB-1) is a tumor suppressor gene that encodes a 105-kDa nuclear phosphoprotein. To date, RB genes have been isolated only from metazoans. We have isolated a cDNA from maize endosperm whose predicted protein product (ZmRb) shows homology to the "pocket" A and B domains of the Rb protein family. We found ZmRb behaves as a pocket protein based on its ability to specifically interact with oncoproteins encoded by DNA tumor viruses (E7, T-Ag, E1A). ZmRb can interact in vitro and in ...

  11. Cloning and sequencing of Indian Water buffalo (Bubalus bubalis) interleukin-3 cDNA

    KAUST Repository

    Sugumar, Thennarasu

    2011-12-12

    Full-length cDNA (435 bp) of the interleukin-3(IL-3) gene of the Indian water buffalo was amplified by reverse transcriptase-polymerase chain reaction and sequenced. This sequence had 96% nucleotide identity and 92% amino acid identity with bovine IL-3. There are 10 amino acid substitutions in buffalo compared with that of bovine. The amino acid sequence of buffalo IL-3 also showed very high identity with that of other ruminants, indicating functional cross-reactivity. Structural homology modelling of buffalo IL-3 protein with human IL-3 showed the presence of five helical structures.

  12. Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing

    OpenAIRE

    Hafner, Markus; Renwick, Neil; Farazi, Thalia A.; Mihailovi, Aleksandra; Pena, John T.G.; Tuschl, Thomas

    2012-01-01

    The characterization of post-transcriptional gene regulation by small regulatory (20–30 nt) RNAs, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for characterizing small RNAs is their identification and quantification across different developmental stages, and in normal and disease tissues, as well as model cell lines. Here we present a step-by-step protocol for generating barcoded small RNA cDNA libraries compatible with Illumina HiSeq se...

  13. Cloned hepatitis delta virus cDNA is infectious in the chimpanzee.

    OpenAIRE

    Sureau, C; Taylor, J; Chao, M; Eichberg, J W; Lanford, R E

    1989-01-01

    A head-to-tail trimer of a full-length cDNA clone of the hepatitis delta virus (HDV) genome was examined for infectivity by direct inoculation into the liver of a chimpanzee that was already infected with hepatitis B virus. Five weeks after inoculation, a marked elevation of serum alanine aminotransferase activity was observed, followed by the appearance of high levels of HDV RNA and antigen in both liver and serum and a high level of viral particles in the serum. A transient suppression of h...

  14. Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor.

    OpenAIRE

    Dobner, P R; Barber, D L; Villa-Komaroff, L; McKiernan, C

    1987-01-01

    Cloned cDNAs encoding neurotensin were isolated from a cDNA library derived from primary cultures of canine enteric mucosa cells. Nucleotide sequence analysis has revealed the primary structure of a 170-amino acid precursor protein that encodes both neurotensin and the neurotensin-like peptide neuromedin N. The peptide-coding domains are located in tandem near the carboxyl terminus of the precursor and are bounded and separated by the paired, basic amino acid residues Lys-Arg. An additional c...

  15. A cDNA clone from Zea mays endosperm sucrose synthetase mRNA.

    OpenAIRE

    Geiser, M.; Döring, H P; Wöstemeyer, J; Behrens, U.; Tillmann, E; Starlinger, P.

    1980-01-01

    A cDNA clone for maize endosperm sucrose synthetase of 62o nucleotide pairs length was obtained by cloning double stranded DNA obtained from the total maize endosperm poly(A) RNA in pBR322, and identifying the appropriate clone by hybrid-promoter translation. In Southern blotting to genomic BamHI-digested DNA, a single band only of approximately 20 Kb lights up, indicating that the sucrose synthetase gene is unique, or that closely linked copies are located on this DNA fragment.

  16. The characterization of cDNA clones coding for wheat storage proteins.

    OpenAIRE

    Bartels, D.; Thompson, R D

    1983-01-01

    Poly(A)+ RNA isolated from the developing wheat endosperm var. Chinese Spring, has been used as template for the construction of a cDNA library. Within the library, clones have been identified by in vitro translation of hybrid-selected mRNA which encode alpha/beta gliadin related sequences and gamma-gliadin related sequences. The DNA sequence of one such clone has been determined and it shows homology with that of a clone encoding a barley storage protein, B-hordein. The sequence includes a t...

  17. Rat beta casein cDNA: sequence analysis and evolutionary comparisons.

    OpenAIRE

    Blackburn, D E; Hobbs, A A; Rosen, J. M.

    1982-01-01

    The complete sequence of a 1072 nucleotide rat beta-casein cDNA insertion in the hybrid plasmid pC beta 23 has been determined. Primer extension was employed to determine the sequence of an additional 82 5'-terminal nucleotides in beta-casein mRNA. Rat beta-casein mRNA consists of a 696 nucleotide coding region, flanked by 52 nucleotide 5' and 406 nucleotide 3' noncoding regions, including a 40 nucleotide poly(A) tail. The derived 216 amino acid sequence of rat beta-casein was compared to the...

  18. Rapid and Efficient cDNA Library Screening by Self-Ligation ofInverse PCR Products (SLIP)

    Energy Technology Data Exchange (ETDEWEB)

    Hoskins, Roger A.; Stapleton, Mark; George, Reed A.; Yu, Charles; Wan, Kenneth H.; Carlson, Joseph W.; Celniker, Susan E.

    2005-04-22

    The production of comprehensive cDNA clone collections is an important goal of the human and model organism genome projects. cDNA sequences are used to determine the structures of transcripts, including splice junctions, polyadenylation sites, and 5' and 3' untranslated regions (UTRs). cDNA collections are also valuable resources for functional studies of genes and proteins. Expressed Sequence Tag (EST)sequencing is the method of choice for recovering cDNAs representing a majority of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a library at random, so it realizes diminishing returns as the project progresses. To drive cDNA collections toward completion new methods are needed to recover cDNAs representing specific genes and alternative transcripts, including transcripts with low expression levels. We describe a simple and effective inverse-PCR-based method for screening plasmid libraries to recover intact cDNAs for specific transcripts. We tested the method by screening libraries used in our Drosophila EST projects for 153 transcription factor genes that were not yet represented by full-length cDNAs. We recovered target-specific clones for 104 of the genes: 46 exactly match, 30 improve and 28partially match current gene annotations. Successful application of the screening method depends on cDNA library complexity and quality of the gene models. The approach should be effective for improving cDNA collections for other model organisms and the human. It also provides a simple and rapid method for isolating cDNAs of interest in any system for which plasmid cDNA libraries and complete or partial gene sequences are available.

  19. Screening of a peanut (Arachis hypogaea L.) cDNA library to isolate a Bowman-Birk trypsin inhibitor clone.

    Science.gov (United States)

    Boateng, Judith A; Viquez, Olga M; Konan, Koffi N; Dodo, Hortense W

    2005-03-23

    Peanut crop losses due to insect and pest infestation cost peanut farmers nearly 20% of their annual yields. The conventional use of chemicals to combat this problem is costly and toxic to humans and livestock and leads to the development of resistance by target insects. Transgenic plants expressing a trypsin inhibitor gene in tobacco and cowpea have proven to be efficient for resistance against insects. Therefore, a transgenic peanut overexpressing a trypsin inhibitor gene could be an alternative solution to the use of toxic chemicals. Five Bowman-Birk trypsin inhibitor (BBTI) proteins were previously isolated from peanut. However, to date, neither cDNA nor genomic DNA sequences are available. The objective of this research was to screen a peanut cDNA library to isolate and sequence at least one full-length peanut BBTI cDNA clone. Two heterologous oligonucleotides were constructed on the basis of a garden pea (Pisum sativa) trypsin inhibitor nucleotide sequence and used as probes to screen a peanut lambda gt-11 cDNA library. Two positive and identical cDNA clones were isolated, subcloned into a pBluescript vector, and sequenced. Sequence analysis revealed a full-length BBTI cDNA of about 243 bp, with a start codon ATG at position +1 and a stop codon TGA at position +243. In the 3' end, two poly adenylation signals (AATAAA) were identified at positions +261 and +269. The isolated cDNA clone encodes a protein of 80 amino acid residues including a leader sequence of 11 amino acids. The deduced amino acid sequence is 100% identical to published sequences of peanut BBTI AI, AII, BI, and BIII and 81% identical to BII. PMID:15769131

  20. Development of a cDNA array for chicken gene expression analysis

    Directory of Open Access Journals (Sweden)

    Burt David

    2005-02-01

    Full Text Available Abstract Background The application of microarray technology to functional genomic analysis in the chicken has been limited by the lack of arrays containing large numbers of genes. Results We have produced cDNA arrays using chicken EST collections generated by BBSRC, University of Delaware and the Fred Hutchinson Cancer Research Center. From a total of 363,838 chicken ESTs representing 24 different adult or embryonic tissues, a set of 11,447 non-redundant ESTs were selected and added to an existing collection of clones (4,162 from immune tissues and a chicken bursal cell line (DT40. Quality control analysis indicates there are 13,007 useable features on the array, including 160 control spots. The array provides broad coverage of mRNAs expressed in many tissues; in addition, clones with expression unique to various tissues can be detected. Conclusions A chicken multi-tissue cDNA microarray with 13,007 features is now available to academic researchers from genomics@fhcrc.org. Sequence information for all features on the array is in GenBank, and clones can be readily obtained. Targeted users include researchers in comparative and developmental biology, immunology, vaccine and agricultural technology. These arrays will be an important resource for the entire research community using the chicken as a model.

  1. Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus

    International Nuclear Information System (INIS)

    Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35 S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.

  2. cDNA cloning and expression of earthworm fibrinolytic enzyme component A

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Earthworm fibrinolytic enzyme component A (EFEa), a protein with dual fibrinolytic activity, is one of the major therapeutically important earthworm fibrinolytic enzyme components. The cDNA fragment encoded the mature protein was cloned from earthworm (Eisenia fetida) by the RT-PCR technique. The deduced amino acid sequence of the EFE component A shows high homology with some members of serine proteases trypsin family, and the amino acid residues constituting the active sites are conserved in the EFEa as compared with the other proteins of the trypsin family. The cDNA fragment was subcloned into the expression vector pQE31 and pMAL-c2X of E. coli. The resulting expression plasmids, pQE-efea and pMAL-efea, were used to transform the E. coli strain M15. Recombinant protein bands corresponding with calculated molecular weights were induced. The induced His6-EFEa fusion protein with pQE- efea was accumulated into inclusion body, while the induced MBP-EFEa fusion protein with pMAL-efea was soluble and showed fibrinolytic activities.

  3. Identification and cDNA cloning of a protein abundantly expressed during apple fruit development.

    Science.gov (United States)

    Yamada, K; Mori, H; Yamaki, S

    1999-02-01

    A 60 kDa protein (MF-60) abundantly appearing in matured apple fruit was detected by SDS-PAGE of the soluble protein. It was partially purified through Butyl-Toyopearl and DEAE-cellulose. Its partial amino acid sequences were determined to isolate a full-length cDNA. MF-60 cDNA (mf-60) consisting of 1,825 bp containing an open reading frame of 1,524 bp and encoding a 54.2 kDa polypeptide. The deduced polypeptide of mf-60 has 81.1% identity to turgor-responsive protein 26 g from wilted garden pea shoot. Northern blot and Western blot analyses showed that the levels of the protein and the transcript of MF-60 changed in parallel through the developmental season; they were very low in young fruit at 36 DAF and 60 DAF, started to increase at 85 DAF, and then remained at a higher level from 114 DAF to 176 DAF. These results suggested that MF-60 functions are connected with fruit development but not with the fruit ripening induced by ethylene. PMID:10202815

  4. Radioactive cDNA microarrys for gene expression profiles in antidepressant therapy

    International Nuclear Information System (INIS)

    Using radioactive cDNA microarray, we investigated a pattern of gene regulation under treatment of antidepressant on patients of depressive disoder. Basic microarray technology was performed as previously described in our research. The bioinformatic selection of human cDNAs, which is specifically designed for psychiatry, neurology, and signal transduction, were arrayed on nylon membranes. Using with 33P-labeled probes, this method provided highly sensitive gene expression profiles of our interest including brain receptors, drug metabolism, and cellular signalings. Gene expression profiles were also classified into several categories in accordance with the gene-regulation of antidepressant. The gene profiles of our interest were significantly up- (16 genes, >2.0 of Z-ratio) or down- (24 genes, <-2.0 of Z ratio) regulated when compared the good responsed group with the bad-responsed one. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology

  5. Gene-expression profiling of human mononuclear cells from welders using cDNA microarray.

    Science.gov (United States)

    Rim, Kyung Taek; Park, Kun Koo; Kim, Yang Ho; Lee, Yong Hwan; Han, Jeong Hee; Chung, Yong Hyun; Yu, Il Je

    2007-08-01

    A toxicogenomic chip developed to detect welding-related diseases was tested and validated for field trials. To verify the suitability of the microarray, white blood cells (WBC) or whole blood was purified and characterized from 20 subjects in the control group (average work experience of 7 yr) and 20 welders in the welding-fume exposed group (welders with an average work experience of 23 yr). Two hundred and fifty-three rat genes homologous to human genes were obtained and spotted on the chip slide. Meanwhile, a human cDNA chip spotted with 8600 human genes was also used to detect any increased or decreased levels of gene expression among the welders. After comparing the levels of gene expression between the control and welder groups using the toxicogenomic chips, 103 genes were identified as likely to be specifically changed by welding-fume exposure. Eighteen of the 253 rat genes were specifically changed in the welders, while 103 genes from the human cDNA chip were specifically changed. The genes specifically expressed by the welders were associated with inflammatory responses, toxic chemical metabolism, stress proteins, transcription factors, and signal transduction. In contrast, there was no significant change in the genes related to short-term welding-fume exposure, such as tumor necrosis factor (TNF)-alpha and interleukin. In conclusion, if further validation studies are conducted, the present toxicogenomic gene chips could be used for the effective monitoring of welding-fume-exposure-related diseases among welders. PMID:17654244

  6. Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method

    Directory of Open Access Journals (Sweden)

    Wikström Lilian

    2005-04-01

    Full Text Available Abstract Background Neural stem cells (NSCs can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression. Results To address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability. Conclusions We demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis.

  7. Human type VII collagen: cDNA cloning and chromosomal mapping of the gene

    Energy Technology Data Exchange (ETDEWEB)

    Parente, M.G.; Chung, L.C.; Ryynaenen, J.; Monli Chu; Uitto, J. (Thomas Jefferson Univ., Philadelphia, PA (United States)); Woodley, D.T.; Wynn, K.C.; Bauer, E.A. (Stanford Univ., CA (United States)); Mattei, M.G. (Institute National de la Sante et de la Recherche Medicale, Marseille (France))

    1991-08-15

    A human keratinocyte cDNA expression library in bacteriophage {lambda}gt11 was screened with the purified IgG fraction of serum from a patient with epidermolysis bullosa acquisita, which had a high titer of anti-type VII collagen antibodies. Screening of {approx}3 {times} 10{sup 5} plaques identified 8 positive clones, the largest one (K-131) being {approx}1.9 kilobases in size. Dideoxynucleotide sequencing of K-131 indicated that it consisted of 1875 base pairs and contained an open reading frame coding for a putative N-terminal noncollagenous domain of 439 amino acids and a collagenous domain was characterized by repeating Gly-Xaa-Yaa sequences that were interrupted in several positions by insertions or deletions of 1-3 amino acids. The deduced amino acid sequence also revealed a peptide segment that had a high degree of identity with a published type VII collagen protein sequence. The results mapped the COL7A1 to the locus 3p21. The cDNA clones characterized in this study will be valuable for understanding the protein structure and gene expression of type VII collagen present in anchoring fibrils and its aberrations in the dystrophic forms of heritable epidermolysis bullosa.

  8. Rice bicoid-related cDNA sequence and its expression during early embryogenesis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation.To clone and characterize the rice bicoid-related genes,one cDNA clone,Rb24 (EMBL accession number: AJ2771380),was isolated by screening of rice unmature seed cDNA library.Sequence analysis indicates that Rb24 contains a putative amino acid sequence,which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity,75% similarity) and involves a lys-9 in putative helix 3.Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner.The transcript was detected strongly in young panicles,but less in young leaves and roots.This results are further confirmed with paraffin section in situ hybridization.The signal is intensive in rice globular embryo and located at the apical tip of the embryo,then,along with the development of embryo,the signal is getting reduced and transfers into both sides of embryo.The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

  9. Molecular cloning of a cDNA encoding the human Sm-D autoantigen

    International Nuclear Information System (INIS)

    Antibodies to the Sm-D polypeptide antigen are closely associated with the rheumatic disease systemic lupus erythematosus. Sm-D exists in the cell as one of the core proteins of the small nuclear ribonucleoprotein complexes implicated in RNA processing. The authors have isolated a cDNA clone, D45-2, coding for the Sm-D human nuclear antigen by screening a human B-lymphocyte cDNA library with synthetic oligonucleotide probes. The 1633-base-pair clone contains an open reading frame (ORF) 357 nucleotides long, capable of encoding a 13,282-dalton polypeptide. The Sm-D coding region is initiated at an AUG codon downstream from a sequence with excellent match to the consensus for the eukaryotic ribosome-binding site. The Sm-D ORF is preceded by a 150-nucleotide-long untranslated leader and followed by a 1126-nucleotide-long untranslated region containing four putative poly(A) signals. The predicted amino acid sequence reveals a (Gly-Arg)9 repeated motif at the C terminus, which may constitute one of the Sm-D immunoreactive determinants. Moreover, this C terminus shows interesting features: (i) a good homology to protamines as expected for a nucleic acid binding protein and (ii) a striking similarity to a region in the Epstein-Barr nuclear antigen

  10. Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor

    Energy Technology Data Exchange (ETDEWEB)

    Dobner, P.R.; Barber, D.L.; Villa-Komaroff, L.; McKiernan, C.

    1987-05-01

    Cloned cDNAs encoding neurotensin were isolated from a cDNA library derived from primary cultures of canine enteric mucosa cells. Nucleotide sequence analysis using /sup 32/P-labeled nucleotides, has revealed the primary structure of a 170-amino acid precursor protein that encodes both neurotensin and the neurotensin-like peptide neuromedin N. The peptide-coding domains are located in tandem near the carboxyl terminus of the precursor and are bounded and separated by the paired, basic amino acid residues Lys-Arg. An additional coding domain, resembling neuromedin N, occurs immediately after an Arg-Arg basic amino acid pair located in the central region of the precursor. Additional amino acid homologies suggest that tandem duplications have contributed to the structure of the gene. RNA blot analysis, using the cloned cDNA probe, has revealed several mRNA species ranging in size from 500 to 980 nucleotides in the canine enteric mucosa. In contrast a single RNA species of 1500 nucleotides was detected in bovine hypothalamus poly-(A)/sup +/ RNA. The ability of the canine probe to cross-hybridize with bovine mRNA suggest that this probe can be used to isolate neurotensin/neuromedin N genes from other mammalian species.

  11. Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor

    International Nuclear Information System (INIS)

    Cloned cDNAs encoding neurotensin were isolated from a cDNA library derived from primary cultures of canine enteric mucosa cells. Nucleotide sequence analysis using 32P-labeled nucleotides, has revealed the primary structure of a 170-amino acid precursor protein that encodes both neurotensin and the neurotensin-like peptide neuromedin N. The peptide-coding domains are located in tandem near the carboxyl terminus of the precursor and are bounded and separated by the paired, basic amino acid residues Lys-Arg. An additional coding domain, resembling neuromedin N, occurs immediately after an Arg-Arg basic amino acid pair located in the central region of the precursor. Additional amino acid homologies suggest that tandem duplications have contributed to the structure of the gene. RNA blot analysis, using the cloned cDNA probe, has revealed several mRNA species ranging in size from 500 to 980 nucleotides in the canine enteric mucosa. In contrast a single RNA species of 1500 nucleotides was detected in bovine hypothalamus poly-(A)+ RNA. The ability of the canine probe to cross-hybridize with bovine mRNA suggest that this probe can be used to isolate neurotensin/neuromedin N genes from other mammalian species

  12. A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

    Science.gov (United States)

    Wang, W.; Takezawa, D.; Poovaiah, B. W.

    1996-01-01

    A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

  13. Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning, expression and substrate specificity analysis.

    Science.gov (United States)

    Jiang, Jingwei; Zhou, Zunchun; Dong, Ying; Sun, Hongjuan; Chen, Zhong; Yang, Aifu; Gao, Shan; Wang, Bai; Jiang, Bei; Guan, Xiaoyan

    2014-02-01

    Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses. PMID:24355405

  14. Radioactive cDNA microarrys for gene expression profiles in antidepressant therapy

    Energy Technology Data Exchange (ETDEWEB)

    Lee, M. S.; Han, B. J.; Cha, J. H.; Ryu, Y. M.; Shin, E. K.; Park, J. H.; Park, Y. H.; Kim, M. K. [Korea University Medical College, Seoul (Korea, Republic of)

    2002-07-01

    Using radioactive cDNA microarray, we investigated a pattern of gene regulation under treatment of antidepressant on patients of depressive disoder. Basic microarray technology was performed as previously described in our research. The bioinformatic selection of human cDNAs, which is specifically designed for psychiatry, neurology, and signal transduction, were arrayed on nylon membranes. Using with 33P-labeled probes, this method provided highly sensitive gene expression profiles of our interest including brain receptors, drug metabolism, and cellular signalings. Gene expression profiles were also classified into several categories in accordance with the gene-regulation of antidepressant. The gene profiles of our interest were significantly up- (16 genes, >2.0 of Z-ratio) or down- (24 genes, <-2.0 of Z ratio) regulated when compared the good responsed group with the bad-responsed one. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  15. Establishment and characterization of a chimeric infectious cDNA clone of classical swine fever virus.

    Science.gov (United States)

    Zhao, T S; Xia, Y H

    2016-01-01

    Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. There are two important CSFV strains in China, Shimen and hog cholera lapinized virus (HCLV). Shimen strain is highly virulent while HCLV, also referred to as C-strain, is a live attenuated vaccine strain considered to be one of the most effective and safest live vaccines. In this study, a chimeric infectious cDNA clone of CSFV named pT7SM-c was engineered by replacing the Erns genomic region of an infectious clone of CSFV Shimen strain, pT7SM, with the same region obtained from HCLV. RNA transcripts of pT7SM-c containing an engineered EcoRI site that served as a genetic marker were directly infectious in PK15 cells. The rescued virus vT7SM-c showed similar growth kinetics and cytopathic effect with the parental virus vT7SM in the cells. The chimeric infectious cDNA clone can be used as a practical tool for further studying of the virulence, protein function and pathogenesis of CSFV through genetic manipulation. PMID:27265471

  16. Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Baozhong, E-mail: bmeng@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Venkataraman, Srividhya; Li, Caihong; Wang, Weizhou [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Dayan-Glick, Cathy; Mawassi, Munir [The Plant Pathology Department-The Virology Unit, Plant Protection Institute, Agricultural Research Organization, The Volcani Center, Bet-Dagan 50250 (Israel)

    2013-01-20

    Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35 S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.

  17. Cloning a cDNA encoding an alternatively spliced protein of BRCA2-associated factor 35.

    Science.gov (United States)

    Wang, Chiang; McCarty, Ida M; Balazs, Louisa; Li, Yi; Steiner, Mitchell S

    2002-07-01

    Inheritance of mutations in the breast cancer susceptibility gene, BRCA2, predisposes humans to breast and ovarian cancers. Inherited mutations in the BRCA2 gene are also known to cause susceptibility to prostate cancer. BRCA2 protein exists in a large multi-protein complex from which a novel structural DNA binding protein BRCA2-associated factor 35 (BRAF35) has been isolated. We have cloned a novel cDNA encoding an alternatively spliced protein of BRAF35, designated as BRAF25. BRAF25 transcript is present in various human cells. We have precisely mapped the BRAF25 cDNA sequence to the genomic chromosome 19 sequence. Analysis of the predicted sequence of BRAF25 identified a protein of 215 amino acids. BRAF25 contains a truncated high mobility group domain, a kinesin-like coiled-coil domain and multiple Src homology 2 (SH2) motifs. Western blot analysis using antibodies specific for BRAF25 revealed the presence of BRAF25 in human prostate cancer cells. PMID:12083779

  18. Modification of pGH cDNA using the first intron and adenovirus-mediated expression in CHO cells

    Institute of Scientific and Technical Information of China (English)

    李秀锦; 仲飞; 齐顺章

    2003-01-01

    Objective This study was conducted to investigate the function of the first intron of porcine growth hormone (pGH) gene in the gene expression.Methods PCR method was used to amplify the first intron from pig genomic DNA. The intron was then inserted into pGH cDNA to construct pGH cDNA-intron (pGH cDNA-in). The recombinant adenoviruses containing pGH cDNA and pGH cDNA-in genes under control of CMV promoter were generated by homologous recombination method in HEK 293 cells respectively. The effect of the first intron on gene expression was evaluated by comparing the expression levels of pGH cDNA-in and pGH cDNA mediated by adenovirus vectors in CHO cells.Results The expression level of pGH cDNA containing the first intron increased by 117%, which was significantly higher than that of pGH cDNA without the intron (P<0.001). Conclusion The first intron of pGH gene has the function to improve pGH gene expression.

  19. Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations.

    Science.gov (United States)

    Oikonomopoulos, Spyros; Wang, Yu Chang; Djambazian, Haig; Badescu, Dunarel; Ragoussis, Jiannis

    2016-01-01

    To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules. PMID:27554526

  20. Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations

    Science.gov (United States)

    Oikonomopoulos, Spyros; Wang, Yu Chang; Djambazian, Haig; Badescu, Dunarel; Ragoussis, Jiannis

    2016-01-01

    To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules. PMID:27554526

  1. Cloning, expression, and mapping of GDP-D-mannose pyrophosphorylase cDNA from tomato (Lycopersicon esculentum).

    Science.gov (United States)

    Zou, Li-Ping; Li, Han-Xia; Ouyang, Bo; Zhang, Jun-Hong; Ye, Zhi-Biao

    2006-08-01

    GDP-D-mannose pyrophosphorylase (GMP, EC 2.7.7.22) catalyzes the synthesis of GDP-D-mannose and represents the first committed step in plant ascorbic acid biosynthesis. Using potato GMP cDNA sequence as a querying probe, 65 highly homologous tomato ESTs were obtained from dbEST of GenBank and the putative cDNA sequence of tomato GMP was assembled. The full-length GMP cDNA of tomato was cloned by RACE-PCR with primers designed according to the assembled cDNA sequence. The full-length cDNA sequence contained a complete open reading frame (ORF) of 1,086 bp, which encoded 361 amino acid residues. This gene was designated as LeGMP (GenBank accession No. AY605668). Homology analysis of LeGMP showed a 96% identity with potato GMP and the deduced amino acid showed 99%, 97%, 91% and 89% homology with GMP from potato, tobacco, alfalfa and Arabidopsis thaliana, respectively. Northern blot analysis showed that LeGMP was constitutively expressed in roots, stems, leaves, flowers and fruits of tomato; but the expression levels varied. LeGMP was mapped to 3-D using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii. PMID:16939010

  2. The construction of a recombinant cDNA library representative of the poly(A)+ mRNA population from normal human lymphocytes.

    OpenAIRE

    Woods, D.; Crampton, J.; Clarke, B.; Williamson, R

    1980-01-01

    A recombinant library has been constructed using the plasmid pAT153 and double stranded cDNA prepared from normal human lymphocyte poly(A)+ RNA. Transformation conditions were optimized to yield approximately 200,000 recombinants per microgram of double stranded cDNA. Statistical analysis as well as sequence complexity analysis of the inserted sequences indicates that the cDNA library is representative of > 99% of the poly(A)+ RNA present in the normal human lymphocyte.

  3. Cloning and expression of a cDNA covering the complete coding region of the P32 subunit of human pre-mRNA splicing factor SF2

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Rasmussen, H H;

    1993-01-01

    We have cloned and expressed a cDNA encoding the 32-kDa subunit (P32) of the human pre-mRNA splicing factor, SF2. This cDNA extends beyond the 5'-end of a previously reported cDNA [Krainer et al., Cell 66 (1991) 383-394]. Importantly, our fragment includes an ATG start codon which was absent from...

  4. An Infectious cDNA Clone of Zika Virus to Study Viral Virulence, Mosquito Transmission, and Antiviral Inhibitors.

    Science.gov (United States)

    Shan, Chao; Xie, Xuping; Muruato, Antonio E; Rossi, Shannan L; Roundy, Christopher M; Azar, Sasha R; Yang, Yujiao; Tesh, Robert B; Bourne, Nigel; Barrett, Alan D; Vasilakis, Nikos; Weaver, Scott C; Shi, Pei-Yong

    2016-06-01

    The Asian lineage of Zika virus (ZIKV) has recently caused epidemics and severe disease. Unraveling the mechanisms causing increased viral transmissibility and disease severity requires experimental systems. We report an infectious cDNA clone of ZIKV that was generated using a clinical isolate of the Asian lineage. The cDNA clone-derived RNA is infectious in cells, generating recombinant ZIKV. The recombinant virus is virulent in established ZIKV mouse models, leading to neurological signs relevant to human disease. Additionally, recombinant ZIKV is infectious for Aedes aegypti and thus provides a means to examine virus transmission. The infectious cDNA clone was further used to generate a luciferase ZIKV that exhibited sensitivity to a panflavivirus inhibitor, highlighting its potential utility for antiviral screening. This ZIKV reverse genetic system, together with mouse and mosquito infection models, may help identify viral determinants of human virulence and mosquito transmission as well as inform vaccine and therapeutic strategies. PMID:27198478

  5. Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system

    International Nuclear Information System (INIS)

    CD28 (Tp44) is a human T-cell-specific homodimer surface protein that may participate in T-cell activation. The authors have isolated a cDNA clone encoding CD28 by a simple and highly efficient cloning strategy based on transient expression in COS cells. Central to this strategy is the use of an efficient method to prepare large plasmid cDNA libraries. The libraries are introduced into COS cells, where transient expression of surface antigen allows the isolation of cDNAs by way of monoclonal antibody binding. The CD28 cDNA encodes a highly glycosylated membrane protein with homology to the immunoglobulin superfamily and directs the production of a homodimer in transfected COS cells

  6. Human CSF-1: molecular cloning and expression of 4-kb cDNA encoding the human urinary protein

    Energy Technology Data Exchange (ETDEWEB)

    Wong, G.G.; Temple, P.A.; Leary, A.C.; Witek-Giannotti, J.S.; Yang, Y.; Ciarletta, A.B.; Chung, M.; Murtha, P.; Kriz, R.; Kaufman, R.J.; Ferenz, C.R.

    1987-03-20

    A 4-kilobase complementary DNA (cDNA) encoding human macrophage-specific colony-stimulating factor (CSF-1) was isolated. When introduced into mammalian cells, this cDNA directs the expression of CSF-1 that is structurally and functionally indistinguishable from the natural human urinary CSF-1. Direct structural analysis of both the recombinant CSF-1 and the purified human urinary protein revealed that these species contain a sequence of at least 40 amino acids at their carboxyl termini which are not found in the coding region of a 1.6-kilobase CSF-1 cDNA that was previously described. These results demonstrate that the human CSF-1 gene can be expressed to yield at least two different messenger RNA species that encode distinct but related forms of CSF-1.

  7. Characterization of cDNA encoding human placental anticoagulant protein (PP4): Homology with the lipocortin family

    International Nuclear Information System (INIS)

    A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 106 independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA the authors identified 9 other recombinants encoding a protein with considerable similarity (74%) to PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I

  8. Cloning of low dose radiation induced gene RIG1 by RACE based on non-cloned cDNA library

    International Nuclear Information System (INIS)

    Objective: To obtain full-length cDNA of radiation induced new gene RIG1 based on its EST fragment. Methods: Based on non-cloned cDNA library, enhanced nested RACE PCR and biotin-avidin labelled probe for magnetic bead purification was used to obtain full-length cDNA of RIG1. Results: About 1 kb of 3' end of RIG1 gene was successfully cloned by this set of methods and cloning of RIG1 5' end is proceeding well. Conclusion: The result is consistent with the design of experiment. This set of protocol is useful for cloning of full-length gene based on EST fragment

  9. Preparation of cDNA libraries for high-throughput RNA sequencing analysis of RNA 5′ ends

    Science.gov (United States)

    Vvedenskaya, Irina O.; Goldman, Seth R.; Nickels, Bryce E.

    2015-01-01

    Summary We provide a detailed protocol for preparing cDNA libraries suitable for high throughput sequencing that are derived specifically from the 5′ ends of RNA (5′ specific RNA-seq). The protocol describes how cDNA libraries for 5′ specific RNA-seq can be tailored to analyze specific classes of RNAs based upon the phosphorylation status of the 5′ end. Thus, the analysis of cDNA libraries generated by these methods provides information regarding both the sequence and phosphorylation status of the 5′ ends of RNAs. 5′ specific RNA-seq can be used to analyze transcription initiation and post-transcriptional processing of RNAs with single base pair resolution on a genome-wide level. PMID:25665566

  10. Construction and Characterization of cDNA Library from Water-Stressed Plantlets Regenerated in vitro of Populus hopeiensis

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    In order to isolate and clone water-stress-responsive genes, total RNA was extracted from water-stressed plantlets regenerated in vitro of Populus hopeiensis using a QIAGEN RNeasy Plant Mini Kit. CDNA, synthesized by LD-PCR with the SMART cDNA Library Construction Kit, was in vitro packaged into a phage λTriplEx2 vector. The resulting primary library and amplified library have a titer of 1.68×106 and 1.69×109 pfu·mL-1 respectively. The combination ratio reached 98.8% and the average size of inserts was about 800 bp. In addition, the percentage of inserted fragments (> 400 bp) was approximately 90%. The results indicate that a cDNA library has been successfully constructed.

  11. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

    International Nuclear Information System (INIS)

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

  12. Synthesis of human placental CDNA and demonstration of the expression of M-CSF in that tissue

    Directory of Open Access Journals (Sweden)

    Elahy E

    1998-08-01

    Full Text Available Macrophage colony stimulating factor (M-CSF has previously been shown to affect the differentiation of cells of the mono-nuclear phagocytic line. More recent studies indicate that M-CSF may have a role in pregnancy. In the present study, the expression of M-CSF in the human placenta was demonstrated. Placental mRNA was isolated and used as template for synthesis of complementary DNA (cDNA. The presence of M-CSF related sequences in the cDNA was shown by PCR and RT-PCR reactions in which M-CSF specific primers were used. In addition, it was shown that a 2.4 kb cDNA after electrophoresis and transfer to a nylon filter, hybridized with a digoxygenin labeled M-CSF specific probe.

  13. Molecular cloning and sequence analysis of growth hormone cDNA of Neotropical freshwater fish Pacu (Piaractus mesopotamicus

    Directory of Open Access Journals (Sweden)

    Janeth Silva Pinheiro

    2008-01-01

    Full Text Available RT-PCR was used for amplifying Piaractus mesopotamicus growth hormone (GH cDNA obtained from mRNA extracted from pituitary cells. The amplified fragment was cloned and the complete cDNA sequence was determined. The cloned cDNA encompassed a sequence of 543 nucleotides that encoded a polypeptide of 178 amino acids corresponding to mature P. mesopotamicus GH. Comparison with other GH sequences showed a gap of 10 amino acids localized in the N terminus of the putative polypeptide of P. mesopotamicus. This same gap was also observed in other members of the family. Neighbor-joining tree analysis with GH sequences from fishes belonging to different taxonomic groups placed the P. mesopotamicus GH within the Otophysi group. To our knowledge, this is the first GH sequence of a Neotropical characiform fish deposited in GenBank.

  14. Evaluation of normalization methods for cDNA microarray data by k-NN classification

    Directory of Open Access Journals (Sweden)

    Myers Connie

    2005-07-01

    Full Text Available Abstract Background Non-biological factors give rise to unwanted variations in cDNA microarray data. There are many normalization methods designed to remove such variations. However, to date there have been few published systematic evaluations of these techniques for removing variations arising from dye biases in the context of downstream, higher-order analytical tasks such as classification. Results Ten location normalization methods that adjust spatial- and/or intensity-dependent dye biases, and three scale methods that adjust scale differences were applied, individually and in combination, to five distinct, published, cancer biology-related cDNA microarray data sets. Leave-one-out cross-validation (LOOCV classification error was employed as the quantitative end-point for assessing the effectiveness of a normalization method. In particular, a known classifier, k-nearest neighbor (k-NN, was estimated from data normalized using a given technique, and the LOOCV error rate of the ensuing model was computed. We found that k-NN classifiers are sensitive to dye biases in the data. Using NONRM and GMEDIAN as baseline methods, our results show that single-bias-removal techniques which remove either spatial-dependent dye bias (referred later as spatial effect or intensity-dependent dye bias (referred later as intensity effect moderately reduce LOOCV classification errors; whereas double-bias-removal techniques which remove both spatial- and intensity effect reduce LOOCV classification errors even further. Of the 41 different strategies examined, three two-step processes, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, all of which removed intensity effect globally and spatial effect locally, appear to reduce LOOCV classification errors most consistently and effectively across all data sets. We also found that the investigated scale normalization methods do not reduce LOOCV classification error. Conclusion Using LOOCV error of k-NNs as the

  15. Evaluation of normalization methods for cDNA microarray data by k-NN classification

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wei; Xing, Eric P; Myers, Connie; Mian, Saira; Bissell, Mina J

    2004-12-17

    Non-biological factors give rise to unwanted variations in cDNA microarray data. There are many normalization methods designed to remove such variations. However, to date there have been few published systematic evaluations of these techniques for removing variations arising from dye biases in the context of downstream, higher-order analytical tasks such as classification. Ten location normalization methods that adjust spatial- and/or intensity-dependent dye biases, and three scale methods that adjust scale differences were applied, individually and in combination, to five distinct, published, cancer biology-related cDNA microarray data sets. Leave-one-out cross-validation (LOOCV) classification error was employed as the quantitative end-point for assessing the effectiveness of a normalization method. In particular, a known classifier, k-nearest neighbor (k-NN), was estimated from data normalized using a given technique, and the LOOCV error rate of the ensuing model was computed. We found that k-NN classifiers are sensitive to dye biases in the data. Using NONRM and GMEDIAN as baseline methods, our results show that single-bias-removal techniques which remove either spatial-dependent dye bias (referred later as spatial effect) or intensity-dependent dye bias (referred later as intensity effect) moderately reduce LOOCV classification errors; whereas double-bias-removal techniques which remove both spatial- and intensity effect reduce LOOCV classification errors even further. Of the 41 different strategies examined, three two-step processes, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, all of which removed intensity effect globally and spatial effect locally, appear to reduce LOOCV classification errors most consistently and effectively across all data sets. We also found that the investigated scale normalization methods do not reduce LOOCV classification error. Using LOOCV error of k-NNs as the evaluation criterion, three double

  16. THE CLONING OF HRNT-1 USING A COMBINATION OF cDNA LIBRARY SCREENING WITH BIOTIN-LABELED PROBE AND RAPID AMPLIFICATION OF cDNA ENDS

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Kai-tai

    2001-01-01

    [1]Tom S, Andrew PR. Human Molecular Genetics [M]. John Wiley & Sons, Inc. United States of America 1996; 335.[2]Zhao Yong-liang, Jin Cui-zhen, Wu De-chang et al. Neoplastic transformation and cytogenetic changes of rat tracheal epithelial cells induced by a-particles irradiation [J]. Chin Med Sci J 1997; 12:202.[3]Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE [J]. Methods Enzymol 1993; 218:340.[4]Frederick A, Roger B. Current Protocols in Molecular Biology [M]. John Wiley & Sons, Inc. United States of America 1998; 2.1.1.[5]Roux KH. Optimization and troubleshooting in PCR [J]. PCR Methods Appl 1995; 4:5158.[6]Sambrook, J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual [M]. 2nd Ed. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989; 54.[7]Zhang Y, Frohman MA. Using rapid amplification of cDNA ends (RACE) to obtain full-length cDNAs [J]. Methods Mol Biol 1997; 69:61.[8]Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE [J]. Methods Enzymol 1993; 218:340.[9]Iqbal S, Robinson J, Deere D, et al. Efficiency of the polymerase chain reaction amplification of the uid gene for detection of Escherichia coli in contaminated water [J]. Lett Appl Microbiol 1997; 24:498.[10]Schunck B, Kraft W, Truyen U. A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces [J]. J Virol Methods 1995; 55:427.

  17. Construction and selection of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential

    Institute of Scientific and Technical Information of China (English)

    Li Hou; Jan-Wu Tang; Xiao-Nan Cui; Bo Wang; Bo Song; Lei Sun

    2004-01-01

    AIM: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential.METHODS: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver. cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5 α competent cells. Individual clones were randomly selected and used for PCR amplification.Vector DNA from positive clones was isolated for sequencing.RESULTS: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13 ,ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes.CONCLUSION: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential.

  18. Isolation and characterization of human cDNA clones encoding the α and the α' subunits of casein kinase II

    International Nuclear Information System (INIS)

    Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two α or α' subunits (or one of each) and two β subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell λgt 10 library using cDNA clones isolated from Drosophila melanogasten. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 close was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 by (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of α and α' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the α and α' subunits of casein kinase II. These studies show that there are two distinct catalytic subunits for casein II (α and α') and that the sequence of these subunits is largely conserved between the bovine and the human

  19. Molecular cloning of the genome of a cardiotropic Coxsackie B3 virus: full-length reverse-transcribed recombinant cDNA generates infectious virus in mammalian cells.

    OpenAIRE

    Kandolf, R; Hofschneider, P H

    1985-01-01

    The molecular cloning of double-stranded cDNA synthesized from the single-stranded RNA genome of the cardiotropic Coxsackie B3 virus (Nancy strain) is reported. Full-length reverse-transcribed cloned viral cDNA of approximately equal to 7500 nucleotides generated infectious antigenically identical Coxsackie B3 virus upon transfection of recombinant plasmid DNA into mammalian cells, demonstrating the molecular cloning of a biologically active viral cDNA copy. Furthermore, the cloned cDNA is ch...

  20. Isolation and Cloning of cDNA of Gene Encoding for Metallothionein Type 2 from Soybean [Glycine max (L.) (Merrill)] cv. Slamet

    OpenAIRE

    UTUT WIDYASTUTI; YASSIER ANWAR; SUHARSONO

    2009-01-01

    Metallothionein has an important role in the detoxification of metal ions. It has a low molecular weight and contains cysteine-rich residue. The objective of this research is to isolate and clone the cDNA of gene encoding for metallothionein from soybean [Glycine max (L.) (Merrill)] cv Slamet (GmMt2). We had successfully isolated total RNA by reverse transcription and synthesized total cDNA from total RNA as template. cDNA of GmMt2 had been isolated from total cDNA by PCR. It was successfully...

  1. Characterization of the mRNA and cloned cDNA specifying the third component of mouse complement.

    OpenAIRE

    Domdey, H; Wiebauer, K; Kazmaier, M; Müller, V.; Odink, K.; Fey, G

    1982-01-01

    Eighteen cDNA clones containing inserts specific for the third component of complement (C3) have been derived from high molecular weight mouse liver mRNA. The inserts span 4,600 nucleotides of the C3 coding sequence, including the 3' end of C3 mRNA. The length of C3 mRNA was determined to be 5,100 +/- 200 nucleotides, including a poly(A)-containing tail of mean length 170 nucleotides. From cDNA sequence analysis of the 5'-proximal region of C3 mRNA, the NH2-terminal amino acid sequence of the...

  2. Identification of glucocorticoid-induced genes in rat hepatoma cells by isolation of cloned cDNA sequences.

    OpenAIRE

    Feinberg, R. F.; Sun, L. H.; Ordahl, C P; Frankel, F R

    1983-01-01

    The expression of specific cellular genes in M1.19 rat hepatoma cells involves glucocorticoid regulation by mechanisms that are not well understood. To approach this problem we cloned cDNA prepared from dexamethasone-induced poly(A)-RNA and used a comparative colony hybridization method to identify recombinant clones containing hormone-regulated sequences. Two such cDNA clones, p1394 and p255, hybridize to a homogeneous RNA species of 900 nucleotides that is present in high abundance in 24-hr...

  3. Characterization of cDNA encoding a human sperm membrane protein related to A4 amyloid protein.

    OpenAIRE

    Yan, Y C; Bai, Y.(Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China); Wang, L.F.; Miao, S Y; Koide, S S

    1990-01-01

    A rat testis lambda gt11 cDNA library was screened with a monoclonal antibody raised against a human sperm membrane protein designated YWK-II. A clone was found with a cDNA insert composed of 1837 base pairs that contained an open reading frame coding for 191 amino acid residues. The deduced polypeptide contained a segment with high homology to the transmembrane-cytoplasmic domains of the A4 amyloid protein found in brain plaques of Alzheimer disease patients. A sequence of basic amino acid r...

  4. Nucleotide sequence of a cDNA clone carrying the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus.

    OpenAIRE

    Koener, J F; Passavant, C W; Kurath, G; Leong, J

    1987-01-01

    The nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined from a cDNA clone containing the entire coding region. The G-protein cDNA is 1,609 nucleotides long (excluding the polyadenylic acid) and encodes a protein of 508 amino acids. The predicted amino acid sequence was compared with that of the glycoprotein of the Indiana and New Jersey serotypes of vesicular stomatitis virus and with the glycoprotein of rabies virus, using a com...

  5. Development of cDNA probes for typing group A bovine rotaviruses on the basis of VP4 specificity.

    OpenAIRE

    Parwani, A V; Rosen, B I; McCrae, M A; Saif, L. J.

    1992-01-01

    Dot and Northern (RNA) blot hybridization assays were developed for the P typing of group A bovine rotaviruses (BRV) by using cDNA probes prepared from gene segment 4. The probes were prepared by polymerase chain reaction amplification of hyperdivergent regions (nucleotides 211 to 686) of BRV strain UK, IND, NCDV, and Cr VP4 cDNA by using specific oligonucleotide primers. The probes were P type specific (VP4) and exhibited little or no cross-reactivity with double-stranded RNA from heterologo...

  6. Recovery of Virulent and RNase-Negative Attenuated Type 2 Bovine Viral Diarrhea Viruses from Infectious cDNA Clones

    OpenAIRE

    Meyer, Christiane; von Freyburg, Martina; Elbers, Knut; Meyers, Gregor

    2002-01-01

    Cloned cDNA derived from the genome of the virulent type 2 bovine viral diarrhea virus (BVDV) strain NY'93/C was sequenced and served for establishment of the infectious cDNA clone pKANE40A. Virus recovered from pKANE40A exhibited growth characteristics similar to those of wild-type BVDV NY'93/C and proved to be clinically indistinguishable from the wild-type virus in animal experiments. A virus mutant in which the RNase residing in the viral glycoprotein Erns was inactivated, revealed an att...

  7. Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli

    OpenAIRE

    Mohammad Hadi Sekhavati; Mojtaba Tahmoorespur; Kamran Ghaedi; Kianoush Dormiani, Pharm; Mohammad Reza Nassiri; Yahya Khazaie; Mahboubeh Foruzanfar; Morteza Hosseini; Mohammad Hossein Nasr Esfahani

    2013-01-01

    Objective: The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein. Materials and Methods: In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 (DE3). Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro fu...

  8. Analysis of common bean (Phaseolus vulgaris L., genotype BAT93 calmodulin cDNA using computational tools

    Directory of Open Access Journals (Sweden)

    Kassim Amelia

    2015-01-01

    Full Text Available Background: Common bean (Phaseolus vulgaris L. is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day and 20-day old bean-pod-tissues. Altogether, 5972 cDNA clones were isolated to have ESTs. While processing ESTs, we found a transcript for calmodulin (CaM gene. It is an important gene that encodes for a calcium-binding protein and known to express in all eukaryotic cells. Hence, this study was undertaken to analyse and annotate it. Objective: The objective of this study was to analyze and annotate P. vulgaris CaM (PvCaM gene cDNA and its deduced protein (amino acids sequence. Materials and Methods: Both strands of PvCaM cDNA clone were sequenced using M13 forward and reverse primer to elucidate the nucleotide sequence. The cDNA sequence and deduced protein sequence were analyzed and annotated using bioinformatics tools available online. The secondary structures and three-dimensional (3D structure of PvCaM protein were predicted using the Phyre automatic fold recognition server. Results: Results showed that PvCaM cDNA is 818 bp in length. The cDNA analysis results showed that it contains an open reading frame that encodes for 149 amino acid residues. The deduced protein sequence analysis results showed the presence of conserved domains required for CaM function. The predicted secondary structures and 3D structure are analogous to the Solanum tuberosum CaM. Conclusions: This study analyzed and annotated PvCaM cDNA and protein. However, in order to obtain a complete understanding of PvCaM protein, further study on its expression, structure and regulation is

  9. Synthesis of human placental CDNA and demonstration of the expression of M-CSF in that tissue

    OpenAIRE

    Elahy E; Arvan R

    1998-01-01

    Macrophage colony stimulating factor (M-CSF) has previously been shown to affect the differentiation of cells of the mono-nuclear phagocytic line. More recent studies indicate that M-CSF may have a role in pregnancy. In the present study, the expression of M-CSF in the human placenta was demonstrated. Placental mRNA was isolated and used as template for synthesis of complementary DNA (cDNA). The presence of M-CSF related sequences in the cDNA was shown by PCR and RT-PCR reactions in which M-C...

  10. Analysis of common bean (Phaseolus vulgaris L., genotype BAT93) calmodulin cDNA using computational tools

    Science.gov (United States)

    Amelia, Kassim; Singh, Jasvin; Shah, Farida Habib; Bhore, Subhash J.

    2015-01-01

    Background: Common bean (Phaseolus vulgaris L.) is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs) is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day and 20-day old bean-pod-tissues. Altogether, 5972 cDNA clones were isolated to have ESTs. While processing ESTs, we found a transcript for calmodulin (CaM) gene. It is an important gene that encodes for a calcium-binding protein and known to express in all eukaryotic cells. Hence, this study was undertaken to analyse and annotate it. Objective: The objective of this study was to analyze and annotate P. vulgaris CaM (PvCaM) gene cDNA and its deduced protein (amino acids) sequence. Materials and Methods: Both strands of PvCaM cDNA clone were sequenced using M13 forward and reverse primer to elucidate the nucleotide sequence. The cDNA sequence and deduced protein sequence were analyzed and annotated using bioinformatics tools available online. The secondary structures and three-dimensional (3D) structure of PvCaM protein were predicted using the Phyre automatic fold recognition server. Results: Results showed that PvCaM cDNA is 818 bp in length. The cDNA analysis results showed that it contains an open reading frame that encodes for 149 amino acid residues. The deduced protein sequence analysis results showed the presence of conserved domains required for CaM function. The predicted secondary structures and 3D structure are analogous to the Solanum tuberosum CaM. Conclusions: This study analyzed and annotated PvCaM cDNA and protein. However, in order to obtain a complete understanding of PvCaM protein, further study on its expression, structure and regulation is essential. PMID

  11. The identification of specific cDNA clones from tall and dwarf rice plants

    International Nuclear Information System (INIS)

    Full text: The use of dwarfing genes in rice breeding has proceeded for several years without a clear understanding of the genetic, hormonal and physiological mechanisms involved. This issue was addressed by focussing on the isolation of specific clones from tall- and dwarf-derived cDNA libraries. The materials used include near-isogenic lines of the tall rice cultivar 'Shiokari', differing at the DGWG or 'Tanginbozu' dwarfing gene loci. Also used were tall and dwarf 'Ginbozu' rice, the latter having been induced by treatment with 5-azacytidine, a potent demethylating agent. Subtractive and differential hybridisation have, to date, identified several candidate tall- and dwarf-specific clones. Their further characterisation is currently underway. (author)

  12. Molecular cloning and expression of a cDNA encoding Lon protease from rice (Oryza sativa).

    Science.gov (United States)

    Su, Wei; Lin, Changfa; Wu, Jiaxue; Li, Kegui; He, Guangming; Qian, Xiaoyin; Wei, Chun; Yang, Jinshui

    2006-06-01

    The ATP-dependent Lon protease is a highly conserved enzyme that is present in archeae, eubacteria, and eukaryotes, and plays an important role in intracellular protein degradation. We have isolated a Lon protease gene, OsLon1, from Oryza sativa. The cDNA contained a 2,655 bp ORF. Comparative analysis showed that OsLon1 shared significant similarity with the previously reported Lon proteases from maize, Arabidopsis, human, and bacteria. Tissue expression pattern analysis revealed that OsLon1 was highly expressed in young leaves, mature leaves, and leaf sheaths but only weakly in young roots, mature roots, and young panicles. The OsLon1 gene was successfully expressed in E. coli and the detected protein size, about 120 kDa, matched the expected molecular mass of the His-tagged OsLon1 protein. PMID:16786279

  13. Analysis of Gene Expression Profile in Lung Adenosquamous Carcinoma Using cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    YANG Fei; YANG Jiong; JIANG Man; YE Bo; ZHANG Yu-xia; CHEN Hong-lei; XIA Dong; LIU Ming-qiu

    2004-01-01

    Gene expression profile of the lung adenosquamous carcinoma was characterized by using cDNA microarray chip containing 4 096 human genes. Among target genes, 508 differentially expressed genes were identified in adenosquamous carcinoma of the lung, 232 genes were overexpressed and 276 genes were underexpressed. Among them, 92 genes are cell signals transduction genes, 34 genes are proto-oncogenes and tumor suppressor genes or cell cycle related genes or cell apoptosis related genes, 29 genes are cell skeleton genes, 28 genes are DNA synthesis, repair and recombination genes, 12 genes are DNA binding and transcription genes. These genes may be associated with the occurence and development of adenosquamous carinome of the lung.

  14. cDNA cloning and characterization of two trehalases from Spodoptera litura (Lepidoptera; Noctuidade).

    Science.gov (United States)

    Zou, Q; Wei, P; Xu, Q; Zheng, H Z; Tang, B; Wang, S G

    2013-01-01

    The oriental leafworm moth, Spodoptera litura, is a major agricultural pest in southeast Asia and nearby Pacific regions. Two distinct trehalases have been identified in insects: soluble trehalase (Treh1) and membrane-bound trehalase (Treh2), although there is currently no information on these genes in S. litura. To characterize the distribution and function of treh, cDNAs of Treh proteins were cloned from S. litura. SpoliTreh1 cDNA has an open reading frame of 1758 nucleotides, which encodes a protein of 585 amino acids, with a predicted mass of approximately 67.07 kDa and an isoelectric point of 4.86. SpoliTreh2 cDNA has an open reading frame of 2325 nucleotides, encoding a protein of 645 amino acids, a mass of approximately 73.62 kDa, and an isoelectric point of 5.90. Northern blotting analysis revealed that SpoliTreh1 transcripts are in the midgut, fat body, tracheae, and epidermis, but not in the brain and Malpighian tubules of S. litura larvae, whereas SpoliTreh2 transcripts were found in all 6 tissues. SpoliTreh1 transcripts were highly expressed in the fat body of the pre-pupal stage, and SpoliTreh2 transcripts were highly expressed in the fat body of 3-day-old larvae of the 6th instar and during the 1st 6 days of the pupal stage, except the 2nd day. Both SpoliTreh1 and SpoliTreh2 were highly expressed in third-instar larvae. PMID:23613237

  15. Nucleotide sequence of cloned cDNA for human sphingolipid activator protein 1 precursor

    International Nuclear Information System (INIS)

    Two cDNA clones encoding prepro-sphingolipid activator protein 1 (SAP-1) were isolated from a λ gt11 human hepatoma expression library using polyclonal antibodies. These had inserts of ≅ 2 kilobases (λ-S-1.2 and λ-S-1.3) and both were both homologous with a previously isolated clone (λ-S-1.1) for mature SAP-1. The authors report here the nucleotide sequence of the longer two EcoRI fragments of S-1.2 and S-1.3 that were not the same and the derived amino acid sequences of mature SAP-1 and its prepro form. The open reading frame encodes 19 amino acids, which are colinear with the amino-terminal sequence of mature SAP-1, and extends far beyond the predicted carboxyl terminus of mature SAP-1, indicating extensive carboxyl-terminal processing. The nucleotide sequence of cDNA encoding prepro-SAP-1 includes 1449 bases from the assigned initiation codon ATG at base-pair 472 to the stop codon TGA at base-pair 1921. The first 23 amino acids coded after the initiation ATG are characteristic of a signal peptide. The calculated molecular mass for a polypeptide encoded by 1449 bases is ≅ 53 kDa, in keeping with the reported value for pro-SAP-1. The data indicate that after removal of the signal peptide mature SAP-1 is generated by removing an additional 7 amino acids from the amino terminus and ≅ 373 amino acids from the carboxyl terminus. One potential glycosylation site was previously found in mature SAP-1. Three additional potential glycosylation sites are present in the processed carboxyl-terminal polypeptide, which they designate as P-2

  16. Analysis of cDNA libraries from developing seeds of guar (Cyamopsis tetragonoloba (L. Taub

    Directory of Open Access Journals (Sweden)

    Dixon Richard A

    2007-11-01

    Full Text Available Abstract Background Guar, Cyamopsis tetragonoloba (L. Taub, is a member of the Leguminosae (Fabaceae family and is economically the most important of the four species in the genus. The endosperm of guar seed is a rich source of mucilage or gum, which forms a viscous gel in cold water, and is used as an emulsifier, thickener and stabilizer in a wide range of foods and industrial applications. Guar gum is a galactomannan, consisting of a linear (1→4-β-linked D-mannan backbone with single-unit, (1→6-linked, α-D-galactopyranosyl side chains. To better understand regulation of guar seed development and galactomannan metabolism we created cDNA libraries and a resulting EST dataset from different developmental stages of guar seeds. Results A database of 16,476 guar seed ESTs was constructed, with 8,163 and 8,313 ESTs derived from cDNA libraries I and II, respectively. Library I was constructed from seeds at an early developmental stage (15–25 days after flowering, DAF, and library II from seeds at 30–40 DAF. Quite different sets of genes were represented in these two libraries. Approximately 27% of the clones were not similar to known sequences, suggesting that these ESTs represent novel genes or may represent non-coding RNA. The high flux of energy into carbohydrate and storage protein synthesis in guar seeds was reflected by a high representation of genes annotated as involved in signal transduction, carbohydrate metabolism, chaperone and proteolytic processes, and translation and ribosome structure. Guar unigenes involved in galactomannan metabolism were identified. Among the seed storage proteins, the most abundant contig represented a conglutin accounting for 3.7% of the total ESTs from both libraries. Conclusion The present EST collection and its annotation provide a resource for understanding guar seed biology and galactomannan metabolism.

  17. Update History of This Database - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us ...Budding yeast cDNA sequencing project Update History of This Database Date Update contents 2010/03/29 Buddin...tio About This Database Database Description Download License Update History of This Database Site Policy | Contact Us Update History

  18. Isolation of a cDNA for HSF 2: Evidence for two heat shock factor genes in humans

    Energy Technology Data Exchange (ETDEWEB)

    Schuetz, T.J.; Gallo, G.J.; Sheldon, L.; Kingston, R.E. (Massachusetts General Hospital, Boston (United States) Harvard Medical School, Boston, MA (United States)); Tempst, P. (Harvard Medical School, Boston, MA (United States))

    1991-08-15

    The heat shock response is transcriptionally regulated by an evolutionarily conserved protein termed heat shock factor (HSF). The authors report the purification to homogeneity and the partial peptide sequence of HSF from HeLa cells. The peptide sequence was used to isolate a human cDNA with a predicted open reading frame that has homology to the DNA binding domains of both Saccharomyces cerevisiae and Drosophila HSFs. The cDNA directs the synthesis of a protein that binds to the heat shock element with specificity identical to HeLa HSF and stimulates transcription from a heat shock promoter. The expressed protein cross-reacts with anti-HSF antibodies. Surprisingly, however, this cDNA does not encode all of the peptides obtained from purified HeLa HSF. These peptides are encoded by a distinct human cDNA. HSF1. It therefore appears that there is a human heat shock factor gene family and that at least two separate but related HSF proteins regulate the stress response in humans.

  19. cDNA cloning and characterization of a mannose-binding lectin from Zingiber officinale Roscoe (ginger) rhizomes

    Indian Academy of Sciences (India)

    Zhonghai Chen; Guoyin Kai; Xiaojun Liu; Juan Lin; Xiaofen Sun; Kexuan Tang

    2005-03-01

    Using RNA extracted from Zingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA of Z. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zoa was 746 bp and contained a 510 bp open reading frame (ORF) encoding a lectin precursor of 169 amino acids with a signal peptide. ZOA was a mannose-binding lectin with three typical mannose-binding sites (QDNY). Semi-quantitative RT-PCR analysis revealed that zoa expressed in all the tested tissues of Z. officinale including leaf, root and rhizome, suggesting it to be a constitutively expressing form. ZOA protein was successfully expressed in Escherichia coli with the molecular weight expected. To our knowledge, this is the first mannose-binding lectin cDNA cloned from the family Zingiberaceae. Our results demonstrate that monocot mannose-binding lectins also occur within the family Zingiberaceae.

  20. Expression cloning of a cDNA encoding the murine interleukin 4 receptor based on ligand binding

    Energy Technology Data Exchange (ETDEWEB)

    Harada, N.; Castle, B.E.; Gorman, D.M.; Itoh, A.; Schreurs, J.; Barrett, R.L.; Howard, M.; Miyajima, A. (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA (USA))

    1990-02-01

    Interleukin 4 (IL-4) is a potent mediator of growth and differentiation for various lymphoid and myeloid cells. To isolate a cDNA encoding the murine IL-4 receptor, the authors have developed an expression cloning method that uses biotinylated ligand as a probe and that may be generally applicable to cloning of receptor genes. COS-7 cells transiently transfected with the cloned full-length cDNA bind murine IL-4 specifically with a K{sub d} = 165 pM. Crosslinking of {sup 125}I-labeled IL-4 to COS-7 cells transfected with the cDNA reveals binding to proteins of 120-140 kDa. IL-4-responsive cells also express IL-4-binding proteins of 120-140 kDa but show additional bands at 60-70 kDa; the relationship of the smaller proteins to the larger ones is unclear. The nucleotide sequence indicates that the full-length cDNA encodes 810 amino acids including the signal sequence. While no consensus sequence for protein kinases is present in the cytoplasmic domain, a sequence comparison with the erythropoietin receptor, the IL-6 receptor, and the {beta} chain of the IL-2 receptor reveals a significant homology in the extracellular domain, indicating that the IL-4 receptor is a member of a cytokine receptor family.

  1. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F

    DEFF Research Database (Denmark)

    Helin, K; Lees, J A; Vidal, M;

    1992-01-01

    The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3...

  2. Efficient expression of human factor Ⅸ cDNA in livermediated by hydrodynamics-based plasmid administration

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor Ⅸ (hFⅨ) cDNA in 2.2 mL Ringer's solution into mice within 7 s. The peak level of expression of hFⅨ was 2921 ng/mL in mouse plasma. The hFⅨ cDNA expression increased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFⅨ cDNA was detected in various organs, but the highest level of gene expression appeared in liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage, which was rapidly repaired within 3-10 d. These results suggested that high-level expression of hFⅨ cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is now further used for gene therapy and gene function study in our lab.

  3. Integrated statistical analysis of cDNA microarray and NIR spectroscopic data applied to a hemp dataset

    NARCIS (Netherlands)

    Reijmers, T.H.; Maliepaard, C.A.; Broeck, van den H.C.; Kessler, R.W.; Toonen, M.A.J.; Voet, van der H.

    2005-01-01

    Both cDNA microarray and spectroscopic data provide indirect information about the chemical compounds present in the biological tissue under consideration. In this paper simple univariate and bivariate measures are used to investigate correlations between both types of high dimensional analyses. A l

  4. Development of a porcine skeletal muscle cDNA microarray: analysis of differential transcript expression in phenotypically distinct muscles

    Directory of Open Access Journals (Sweden)

    Stear Michael

    2003-03-01

    Full Text Available Abstract Background Microarray profiling has the potential to illuminate the molecular processes that govern the phenotypic characteristics of porcine skeletal muscles, such as hypertrophy or atrophy, and the expression of specific fibre types. This information is not only important for understanding basic muscle biology but also provides underpinning knowledge for enhancing the efficiency of livestock production. Results We report on the de novo development of a composite skeletal muscle cDNA microarray, comprising 5500 clones from two developmentally distinct cDNA libraries (longissimus dorsi of a 50-day porcine foetus and the gastrocnemius of a 3-day-old pig. Clones selected for the microarray assembly were of low to moderate abundance, as indicated by colony hybridisation. We profiled the differential expression of genes between the psoas (red muscle and the longissimus dorsi (white muscle, by co-hybridisation of Cy3 and Cy5 labelled cDNA derived from these two muscles. Results from seven microarray slides (replicates correctly identified genes that were expected to be differentially expressed, as well as a number of novel candidate regulatory genes. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray. Conclusion We have developed a porcine skeletal muscle cDNA microarray and have identified a number of candidate genes that could be involved in muscle phenotype determination, including several members of the casein kinase 2 signalling pathway.

  5. Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-)

    International Nuclear Information System (INIS)

    Glucose-6-phosphate dehydrogenase A(-) is a common variant in Blacks that causes sensitivity to drug- and infection-induced hemolytic anemia. A cDNA library was constructed from Epstein-Barr virus-transformed lymphoblastoid cells from a male who was G6PD A(-). One of four cDNA clones isolated contained a sequence not found in the other clones nor in the published cDNA sequence. Consisting of 138 bases and coding 46 amino acids, this segment of cDNA apparently is derived from the alternative splicing involving the 3' end of intron 7. Comparison of the remaining sequences of these clones with the published sequence revealed three nucleotide substitutions: C33 → G, G202 → A, and A376 → G. Each change produces a new restriction site. Genomic DNA from five G6PD A(-) individuals was amplified by the polymerase chain reaction. The findings of the same mutation in G6PD A(-) as is found in G6PD A(+) strongly suggests that the G6PD A(-) mutation arose in an individual with G6PD A(+), adding another mutation that causes the in vivo instability of this enzyme protein

  6. Measuring number of fluorophores labelling cDNA strands, in solution, with Fluorescence Correlation Spectroscopy and photobleaching

    CERN Document Server

    Delon, Antoine; Lambert, Emeline; Lerbs-Mache, Silva; Mache, Régis; Derouard, Jacques; Motto-Ros, Vincent; Galland, Rémi

    2009-01-01

    We present different approaches that aim at determining, in solution, the brightness and the number of Alexa Fluor 647 molecules labelling the C bases of two sequences of cDNA, corresponding to two transcripts of different sizes, a short and a long transcript (123 and 306 base long, with 45 and 74 dCTP residues, respectively). In each case, the Alexa labeled bases have been incorporated during reverse-transcription. Two kinds of experiments have been performed and combined: photobleaching and Fluorescence Correlation Spectroscopy (together with the factorial cumulant analysis method). As a result, we show that the photobleaching cross-section of labelled cDNA strands is about half that of free Alexa in aqueous solution, while their brightness is about twice. The factorial cumulant analysis put into evidence the fact that the brightness of cDNA strands varies from molecule to molecule, due to the statistical distribution of the number of Alexa fluorophores labelling cDNA. Our measurements are consistent with a...

  7. A mesocarp-and species-specific cDNA clone from oil palm encodes for sesquiterpene synthase.

    Science.gov (United States)

    Shah; Cha

    2000-05-29

    The differential display method was used to isolate cDNAs corresponding to transcripts that accumulate during the period of lipid synthesis, 12-20 weeks after anthesis (WAA) in the mesocarp of two oil palms, Elaeis oleifera and Elaeis guineensis, Tenera. DNA-free total RNA from mesocarp and kernel of E. guineensis, Tenera and E. oleifera (15 WAA) were used to obtain differential gene expression patterns between these tissues from the two species. In this report, we describe the isolation and characterization of a specific cDNA clone, MO1 (434 bp) which was shown to be mesocarp-specific as well as species-specific for E. oleifera Sequencing of this fragment showed homology to the enzyme sesquiterpene synthase. Its longer cDNA clone, pMO1 (1072 bp), isolated from a 15-week E. oleifera mesocarp cDNA library confirmed that it encodes for sesquiterpene synthase. The complete sequence of 1976 bp was obtained using 5'RACE method. Northern hybridization showed that MO1 and pMO1 mRNA transcripts are highly expressed only in the mesocarp of E. oleifera from 5 to 20 WAA. No expression was detected in the kernel (12-17 WAA) and vegetative tissues of both species nor in the mesocarp of E. guineensis. This is the first communication to document on the isolation and characterisation of a mesocarp-and species-specific cDNA clone from oil palm. PMID:10729614

  8. Pattern analysis approach reveals restriction enzyme cutting abnormalities and other cDNA library construction artifacts using raw EST data

    Directory of Open Access Journals (Sweden)

    Zhou Sun

    2012-05-01

    Full Text Available Abstract Background Expressed Sequence Tag (EST sequences are widely used in applications such as genome annotation, gene discovery and gene expression studies. However, some of GenBank dbEST sequences have proven to be “unclean”. Identification of cDNA termini/ends and their structures in raw ESTs not only facilitates data quality control and accurate delineation of transcription ends, but also furthers our understanding of the potential sources of data abnormalities/errors present in the wet-lab procedures for cDNA library construction. Results After analyzing a total of 309,976 raw Pinus taeda ESTs, we uncovered many distinct variations of cDNA termini, some of which prove to be good indicators of wet-lab artifacts, and characterized each raw EST by its cDNA terminus structure patterns. In contrast to the expected patterns, many ESTs displayed complex and/or abnormal patterns that represent potential wet-lab errors such as: a failure of one or both of the restriction enzymes to cut the plasmid vector; a failure of the restriction enzymes to cut the vector at the correct positions; the insertion of two cDNA inserts into a single vector; the insertion of multiple and/or concatenated adapters/linkers; the presence of 3′-end terminal structures in designated 5′-end sequences or vice versa; and so on. With a close examination of these artifacts, many problematic ESTs that have been deposited into public databases by conventional bioinformatics pipelines or tools could be cleaned or filtered by our methodology. We developed a software tool for Abnormality Filtering and Sequence Trimming for ESTs (AFST, http://code.google.com/p/afst/ using a pattern analysis approach. To compare AFST with other pipelines that submitted ESTs into dbEST, we reprocessed 230,783 Pinus taeda and 38,709 Arachis hypogaea GenBank ESTs. We found 7.4% of Pinus taeda and 29.2% of Arachis hypogaea GenBank ESTs are “unclean” or abnormal, all of which could be cleaned

  9. Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit

    DEFF Research Database (Denmark)

    Christensen, C B; Jørgensen, L; Jensen, A T;

    2000-01-01

    Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L...

  10. Molecular cloning and sequence analysis of the cDNA for thyroid-stimulating hormone beta subunit of Muscovy duck.

    Science.gov (United States)

    Hsieh, Y L; Chatterjee, A; Lee, G; Yuh-Lin Yu, J

    2000-12-01

    The cDNA-encoding thyroid-stimulating hormone beta subunit (TSHbeta) of the Muscovy duck (Cairina moschata) was cloned and sequenced to investigate the phylogenic diversity and evolution of TSH molecules. Oligonucleotide primers were designed and used for reverse transcription and polymerase chain reaction amplification of a TSHbeta cDNA fragment from the total cellular RNA of pituitary glands. The remaining sequence was completed by rapid amplification of cDNA ends. The nucleotide sequence of Muscovy duck TSHbeta cDNA includes 66 bp of 5'-untranslated region (UTR), 402 bp of coding region, and 82 bp of 3'-UTR, followed by an 18-bp poly(A)(+) tract. The total number of amino acids deduced from the cDNA of duck TSHbeta is 134, with a signal peptide of 19 amino acids and a mature protein of 115 amino acids. The homologies of the amino acid sequence of Muscovy duck TSHbeta compared with those of chicken, quail, mammals, amphibian (frog), and teleosts are 97, 98, 68-69, 56, and 42-47%, respectively. To test for tissue specificity of the duck TSHbeta cDNA, total cellular RNA samples from brain, liver, pituitary gland, testis, and thyroid gland were analyzed by Northern blotting. A band, approximately 700 bases, was found in the pituitary gland alone. The pituitary tissue fragments were treated for 24 h in serum-free medium containing thyrotropin-releasing hormone (TRH), triiodothyronine (T(3)), or thyroxine (T(4)). TRH increased and T(3) and T(4) decreased TSHbeta mRNA levels. This study demonstrated that the amino acid sequence of TSHbeta subunits is highly conserved among birds, exhibiting a high degree of inter-order homology, and that hypothalamic TRH upregulates the TSHbeta mRNA expression in duck. PMID:11121298

  11. Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine.

    Directory of Open Access Journals (Sweden)

    Utut Widyastuti Suharsono

    2008-11-01

    Full Text Available Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE.

  12. Isolation and characterization of a full length cDNA for dentatorubral-pallidoluysian atrophy (DRPLA) gene

    Energy Technology Data Exchange (ETDEWEB)

    Oyake, M.; Onodera, O.; Ikeuchi, T. [Niigata Univ. (Japan)] [and others

    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.

  13. Efficacy and toxicity of self-polishing biocide-free antifouling paints.

    Science.gov (United States)

    Löschau, Margit; Krätke, Renate

    2005-11-01

    The ban on harmful substances in antifouling paints requires the development of new antifouling strategies. Alternatives should be as effective as conventional paints but of lower toxicity. In the present study two commercially available, self-polishing antifouling paints were examined in order to get information on their antifouling properties and toxicological potential. Efficacy was shown in settlement assays with the marine barnacle species Balanus amphitrite, however, efficacy was related to toxic effects observed on target and non-target organisms. Toxicity of the paint extracts was concentration-dependent and differed according to the paint and the species investigated. Toxicity could at least partially be attributed to zinc leached from the paints. Effects of a water-soluble paint were more pronounced in larvae of B. amphitrite, Artemia salina and in the green algae Dunaliella tertiolecta. Embryos of the freshwater species Danio rerio and Vibrio fisheri were more affected by a paint based on organic solvents. PMID:15955603

  14. Gene expression in retinoic acid-induced neural tube defects A cDNA mieroarray analysis

    Institute of Scientific and Technical Information of China (English)

    Xiaodong Long; Zhong Yang; Yi Zeng; Hongli Li; Yangyun Han; Chao You

    2009-01-01

    BACKGROUND: Neural tube defects can be induced by abnormal factors in vivo or in vitro during development. However, the molecular mechanisms of neural tube defect induction, and the related gene expression and regulation are still unknown.OBJECTIVE: To compare the differences in gene expression between normal embryos and those with neural tube defects.DESIGN, TIME AND SETTING: A neural development study was performed at the Department of Neurobiology, Third Military Medical University of Chinese PLA between January 2006 and October 2007.MATERIALS: Among 120 adult Kunming mice, 60 pregnant mice were randomly and evenly divided into a retinoic acid group (n = 30) and a normal control group (n =30). The retinoic acid was produced by Sigma, USA, the gene microarray by the Amersham Pharmacia Company, Hong Kong, and the gene sequence was provided by the Incyte database, USA.METHODS: Retinoic acid was administered to prepare models of neural tube defects, and corn oil was similady administered to the normal control group. Total RNA was extracted from embryonic tissue of the two groups using a Trizol kit, and a cDNA microarray containing 1 100 known genes was used to compare differences in gene expression between the normal control group and the retinoic acid group on embryonic (E) clay 10.5 and 11.5. Several differentially expressed genes were randomly selected from the two groups for Northern blotting, to verify the results of the cDNA microarray.MAIN OUTCOME MEASURES: Morphological changes and differential gene expression between the normal control group and the retinoic acid group.RESULTS: Anatomical microscopy demonstrated that an intact closure of the brain was formed in the normal mouse embryos by days E10.5 and E11.5. The cerebral appearance was full and smooth, and the surface of the spine was intact. However, in the retinoic acid group on days E10.5 and E11.5, there were more dead embryos. Morphological malformations typically included non-closure at the top of

  15. A System-Wide Approach to Identify the Mechanisms of Barnacle Attachment: Toward the Discovery of New Antifouling Compounds

    KAUST Repository

    Al-Aqeel, Sarah

    2015-11-01

    Biofouling is a significant economic problem, particularly for marine and offshore oil industries. The acorn barnacle (Amphibalanus (Balanus) amphitrite) is the main biofouling organism in marine environments. Environmental conditions, the physiology of the biofouling organism, the surrounding microbial community, and the properties of the substratum can all influence the attachment of biofouling organisms to substrates. My dissertation investigated the biological processes involved in B. amphitrite development and attachment in the unique environment of the Red Sea, where the average water surface temperature is 34°C and the salinity reaches 41‰. I profiled the transcriptome and proteome of B. amphitrite at different life stages (nauplius II, nauplius VI, and cyprid) and identified 65,784 expressed contigs and 1387 expressed proteins by quantitative proteomics. During the planktonic stage, genes related to osmotic stress, salt stress, the hyperosmotic response, and the Wnt signaling pathway were strongly up-regulated, hereas genes related to the MAPK pathway, lipid metabolism, and cuticle development were down-regulated. In the transition from the nauplius VI to cyprid stages, there was up-regulation of genes involved in blood coagulation, cuticle development, and eggshell formation, and down-regulation of genes in the nitric oxide pathway, which stimulates the swimming and feeding responses of marine invertebrates. This system-wide integrated approach elucidated the development and attachment pathways important in B. amphitrite. Enzymes and metabolites in these pathways are potential molecular targets for the development of new antifouling compounds.

  16. Optimization of cDNA microarrays procedures using criteria that do not rely on external standards

    Directory of Open Access Journals (Sweden)

    Beisvag Vidar

    2007-10-01

    Full Text Available Abstract Background The measurement of gene expression using microarray technology is a complicated process in which a large number of factors can be varied. Due to the lack of standard calibration samples such as are used in traditional chemical analysis it may be a problem to evaluate whether changes done to the microarray procedure actually improve the identification of truly differentially expressed genes. The purpose of the present work is to report the optimization of several steps in the microarray process both in laboratory practices and in data processing using criteria that do not rely on external standards. Results We performed a cDNA microarry experiment including RNA from samples with high expected differential gene expression termed "high contrasts" (rat cell lines AR42J and NRK52E compared to self-self hybridization, and optimized a pipeline to maximize the number of genes found to be differentially expressed in the "high contrasts" RNA samples by estimating the false discovery rate (FDR using a null distribution obtained from the self-self experiment. The proposed high-contrast versus self-self method (HCSSM requires only four microarrays per evaluation. The effects of blocking reagent dose, filtering, and background corrections methodologies were investigated. In our experiments a dose of 250 ng LNA (locked nucleic acid dT blocker, no background correction and weight based filtering gave the largest number of differentially expressed genes. The choice of background correction method had a stronger impact on the estimated number of differentially expressed genes than the choice of filtering method. Cross platform microarray (Illumina analysis was used to validate that the increase in the number of differentially expressed genes found by HCSSM was real. Conclusion The results show that HCSSM can be a useful and simple approach to optimize microarray procedures without including external standards. Our optimizing method is highly

  17. Isolation of cDNA from Jacaratia mexicana encoding a mexicain-like cysteine protease gene.

    Science.gov (United States)

    Ramos-Martínez, Erick M; Herrera-Ramírez, Alejandra C; Badillo-Corona, Jesús Agustín; Garibay-Orijel, Claudio; González-Rábade, Nuria; Oliver-Salvador, María Del Carmen

    2012-07-01

    Cysteine proteases (CPs) from the C1 family, which are similar to papain, can be found in animals and plants, as well as some viruses and prokaryotes. These enzymes have diverse physiological functions and are thus very attractive for science and industry. Jacaratia mexicana, a member of the Caricaceae plant family, contains several CPs, the principal being mexicain, found to favorably compete against papain for many industrial applications due to its high stability and specific activity. In this study, leaves of J. mexicana were used to isolate a CP-coding gene, similar to those that code for mexicain and chymomexicain. By using rapid amplification of cDNA ends (RACE) as well as oligonucleotide design from papain-like conserved amino acids (aa), a sequence of 1404 bp consisting of a 5' terminal untranslated region (UTR) of 153 bp, a 3' terminal UTR of 131 bp, with a polyadenylation (poly(A)) signal sequence and a poly(A) tail, and an open reading frame (ORF) of 1046 bp, was obtained by overlapping three partial sequences. Two full-length cDNA sequences that encode for mexicain-like proteases were cloned from mRNA (JmCP4 and JmCP5). JmCP4 is predicted to have an ORF of 1044 bp, which codifies for polypeptides that have a 26 aa signal peptide region, a 108 aa propeptide region and a mature enzyme of 214 aa. A 969 bp fragment (JmCP5) encodes for a partial sequence of a CP gene, without the signal peptide region but with a full-length propeptide region. The sequence analysis showed that this protease presented a high similarity to other plant CPs from J. mexicana, Vasconcellea cundinamarcensis, Vasconcellea stipulata, and Carica papaya, among others, mainly at the conserved catalytic site. Obtaining the sequence of this CP gene from J. mexicana provides an alternative for production in a standard system and could be an initial step towards the commercialization of this enzyme. PMID:22543019

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

    Science.gov (United States)

    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. PMID:24381103

  19. Calamenenes - aromatic bicyclic sesquiterpenes - from the Indian gorgonian Subergorgia reticulata (Ellis and Solander, 1786)

    Digital Repository Service at National Institute of Oceanography (India)

    LimnaMol, V.P.; Raveendran, T.V.; Naik, B.G.; Kunnath, R.J.; Parameswaran, P.S.

    ; Nakashima et al., 2002; Nishizawa, Inoue, Sastrapradja, & Hayashi, 1983; Salmoun et al., 2007). Thus, (+)-2-hydroxy calamenene, a potent fish toxin was obtained from the seeds of Dysoxylum acutangulum and D. alliaceum (Nishizawa et al., 1983). The more..., Zygadlo, & Demo, 2007). Compounds 1-3 also inhibited settlement of cyprids of the fouling barnacle, Balanus amphitrite (EC 50 of 4.4, 7.8 and 0.03µg/mL, respectively) with therapeutic ratios (T.R.) of 7.3, 4.6 and 799, respectively. Of these, compound 3...

  20. Chemical constituents of soft coral Sarcophyton infundibuliforme from the South China Sea

    KAUST Repository

    Wang, Chang Yun

    2011-08-01

    Chemical investigation on soft coral Sarcophyton infundibuliforme collected from the South China Sea led to the isolation and identification of 14 secondary metabolites, including ten cembrene diterpenoids (1-10), one α-tocopheryl quinone derivative (11), one prostaglandin (12), one lipid (13) and one carotinoid (14). Their structures were determined by extensive analysis of their spectroscopic data. All of these metabolites were isolated from this species for the first time. Diterpenoids 1, 2, 7 and 10 showed potent antifouling activity against the larval settlement of barnacle Balanus amphitrite. © 2011 Elsevier Ltd.

  1. Alkaloids and Sesquiterpenes from the South China Sea Gorgonian Echinogorgia pseudossapo

    Directory of Open Access Journals (Sweden)

    Shu-Hua Qi

    2011-11-01

    Full Text Available Five zoanthoxanthin alkaloids (1–5 and four sesquiterpenes (6–9 were isolated from the South China Sea gorgonian Echinogorgia pseudossapo. Their structures were determined on the bases of extensive spectroscopic analyses, including 1D and 2D NMR data. Among them, pseudozoanthoxanthins III and IV (1–2, 8-hydroxy-6β-methoxy-14-oxooplop-6,12-olide (6 and 3β-methoxyguaian-10(14-en-2β-ol (7 were new, 1 and 3 showed mild anti-HSV-1 activity, and 7 showed significant antilarval activity towards Balanus amphitrite larvae.

  2. ESTS from skin and PBMC cDNA subtractive library of alpaca

    International Nuclear Information System (INIS)

    Full text: As an effort to map and identify genes and genetic markers that influence the fibre quality in alpacas, cDNA subtractive libraries of Alpaca (Vicugna pacos) were constructed in order to find differentially expressed genes in skin. Skin and blood samples were removed from six adult Alpaca (1.5 year old). Total RNA was extracted using Trizol (Invitrogen) and mRNA was purified using the Gene Elute mRNA purification kit (Sigma). Suppression PCR was used to construct the library using mRNA from skin as a tester and the mRNA from PBMC as a driver. The subtracted PCR products were inserted into the TA cloning vector and the ligation reaction was transformed into TOP10 E. coli cells. Randomly selected clones were sequenced and a total of 2280 high quality 5' end sequences were generated. Clustering analysis using StackPACK version 2.2.0 resulted in 1075 unique transcripts, consisting of 347 consensi and 728 singletons. BLAST analysis of the generated sequences revealed skin associated transcripts such as hair keratin 6A, keratin 10, keratin KA27, keratin 34, wool keratin microfibril type I, and collagen. A total of 27 microsatellite loci were also uncovered. Further work is in progress to generate more sequences in order to build an EST database of differentially expressed genes from Alpaca skin and PBMC, and for the generation of genetic molecular markers such as microsatellites and SNP for Alpaca. (author)

  3. A human cDNA library for high-throughput protein expression screening.

    Science.gov (United States)

    Büssow, K; Nordhoff, E; Lübbert, C; Lehrach, H; Walter, G

    2000-04-01

    We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses. PMID:10777659

  4. Identification and molecular characterization of numerous Histomonas meleagridis proteins using a cDNA library.

    Science.gov (United States)

    Bilic, I; Leberl, M; Hess, M

    2009-04-01

    SUMMARYHistomonas meleagridis is a protozoan parasite of various galliform birds causing a type of enterohepatitis termed histomonosis or 'blackhead disease'. Due to the ban of chemotherapeutic substances and an increase in free-range poultry production, histomonosis is currently a re-emerging disease. So far limited molecular knowledge is available. In the present work, mRNAs coding for antigenic proteins of H. meleagridis were identified. For this purpose, a cDNA expression library was constructed from a mono-eukaryotic culture of H. meleagridis. The library was screened with polyclonal rabbit serum raised against purified H. meleagridis trophozoites. Polyclonal rabbit serum specifically recognized the same major H. meleagridis antigens as chicken and turkey sera originating from animal trials, but displayed a significantly lower bacteria-dependent background signal. After 2 rounds of screening, a total of 95 positive clones were sequenced. Bioinformatics analyses were performed on nucleotide and deduced amino acid sequences, identifying 37 unique clones. Based on the homology to other protozoan parasites, mostly Trichomonas vaginalis, the clones were grouped according to functional aspects: structural proteins, possible surface proteins, oxygen reducing proteins, ribosomal proteins, protein kinases and various other intracellular proteins. PMID:19154645

  5. Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas

    Institute of Scientific and Technical Information of China (English)

    Haibo Wang; Duoduo Yuan; Rong Xu; Cheng-Wu Chi

    2011-01-01

    Chymotrypsin C is a bifunctional secretory-type serine protease in pancreas; besides proteolytical activity, it also exhibits a calcium-decreasing activity in serum, In this study, we purified activated chymotrypsin C from porcine pancreas, and identified its three active forms. Active chymotrypsin C was found to be different in the length of its 13-residue activation peptide due to carboxydipeptidase (present in the pancreas) degradation or autolysis of the activated chymotrypsin C itself, resulting in the removal of several C-terminus residues from the activation peptide. After limited chymotrypsin C cleavage with endopeptidase Lys C, several purified peptides were partially sequenced, and the entire cDNA sequence for porcine chymotrypsin C was cloned. Recombinant chymotrypsinogen C was successfully expressed in Escherichia coli cells as inclusion bodies. After refolding and activation with trypsin, the comparison of the recombinant chymotrypsin C with the natural form showed that their proteolytic and calcium-decreasing activities were at the same level. The successful expression of chymotrypsin C gene paves the way to further mutagenic structurefunction studies.

  6. Construction and characterization of an infectious cDNA clone of Echovirus 25.

    Science.gov (United States)

    Hou, Wangheng; Yang, Lisheng; Li, Shuxuan; Yu, Hai; Xu, Longfa; He, Delei; Chen, Mengyuan; He, Shuizhen; Ye, Xiangzhong; Que, Yuqiong; Shih, James Wai Kuo; Cheng, Tong; Xia, Ningshao

    2015-07-01

    Echovirus 25 (E-25) is a member of the enterovirus family and a common pathogen that induces hand, foot, and mouth disease (HFMD), meningitis, skin rash, and respiratory illnesses. In this study, we constructed and characterized an infectious full-length E-25 cDNA clone derived from the XM0297 strain, which was the first subgenotype D6 strain isolated in Xiamen, China. The 5'-Untranslated Regions (5'-UTR), P3 (3A-3B, 3D) and P3 (3C) regions of this E-25 (XM0297) strain were highly similar to EV-B77, E-16 and E-13, respectively. Our data demonstrate that the rescued E-25 viruses exhibited similar growth kinetics to the prototype virus strain XM0297. We observed the rescued viral particles using transmission electron microscope (TEM) and found them to possess an icosahedral structure, with a diameter of approximately 30 nm. The cross neutralization test demonstrated that the E-25 (XM0297) strain immune serum could not neutralize EV-A71, CV-A16 or CV-B3; likewise, the EV-A71 and CV-A16 immune serum could not neutralize E-25 (XM0297). The availability of this infectious clone will greatly enhance future virological investigations and possible vaccine development against E-25. PMID:26004198

  7. Cloning and Expression of the cDNA of a Murine Soluble Fas

    Institute of Scientific and Technical Information of China (English)

    胡中波; 邹萍; 李爱香; 肖娟; 仲照东; 刘凌波

    2002-01-01

    Summary: In order to regulate the apoptosis induced by Fas-FasL system, a soluble isoform of mouse Fas was cloned from thymocytes of immature mice with the primers designed according to the full-length Fas cDNA sequence in the GeneBank. It was directionally inserted into the intermedium vector pUC19. DNA sequencing proved that it was consistent with the expected sequence.Then it was subcloned into the eukaryotic expression vector pCA13, which was used to construct the recombinant vector pCA13-FasC. By lipofectamine (LF2000)-mediated transfection, pCA13FasC was transfected into the 293 cells. RT-PCR and Western blot indicated that the murine soluble Fas C protein was expressed in the 293 cells. Apoptosis inducing test showed that the expression of this murine Fas C could block the Fas-induced apoptosis, which confirmed the biological activity of the recombinant Fas C.

  8. cDNA cloning and expression analysis of a mannose-binding lectin from Pinellia pedatisecta

    Indian Academy of Sciences (India)

    Juan Lin; Xuanwei Zhou; Shi Gao; Xiaojun Liu; Weisheng Wu; Xiaofen Sun; Kexuan Tang

    2007-03-01

    Pinellia pedatisecta agglutinin (PPA) is a very basic protein that accumulates in the tuber of P. pedatisecta. PPA is a hetero-tetramer protein of 40 kDa, composed of two polypeptide chains A (about 12 kDa) and two polypeptides chains B (about 12 kDa). The full-length cDNA of PPA was cloned from P. pedatisecta using SMART RACE-PCR technology; it was 1146 bp and contained a 771 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acid residues with a 24 amino acid signal peptide. The PPA precursor contained 3 mannose-binding sites (QXDXNXVXY) and two conserved domains of 43% identity, PPA-DOM1 (polypeptides A) and PPA-DOM2 (polypeptides B). PPA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families such as Araceae, Alliaceae, Iridaceae, Liliaceae, Amaryllidaceae and Bromeliaceae. Southern blot analysis indicated that ppa belonged to a multi-copy gene family. Expression pattern analysis revealed that ppa expressed in most tested tissues, with high expression being found in spadix, spathe and tuber. Cloning of the ppa gene not only provides a basis for further investigation of its structure, expression and regulatory mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.

  9. Analysis of expressed sequence tags from a fetal human heart cDNA library

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, D.M.; Fung, Y.W.; Wang, R.X.; Laurenssen, C.M. [Univ. of Toronto, Ontario (Canada)] [and others

    1995-11-20

    Single-pass sequencing of randomly selected cDNA clones to generate expressed sequence tags (ESTs) has been widely used to identify novel genes and to study gene expression in a variety of tissues. We have generated 2244 ESTs from a human fetal heart library (Gen-Bank Accession Nos. R30692-30774 and R56965-58824), which we present in this report. Of these, 51.7% showed no homology to known genes or were similar only to other ESTs, while 48.4% demonstrated homology to known transcripts. A total of 764 ESTs corresponding to known genes were used to study gene expression patterns in the fetal heart and to analyze differences in these patterns from those observed in the adult heart. These analyses demonstrate the utility of ESTs and sequence-tagged clones in comparative studies of gene expression in the cardiovascular system, and they reveal that differential gene expression underlies the structural and functional characteristics of the developing heart. 48 refs., 1 fig., 3 tabs.

  10. Identifying and mapping cDNA fragments related to rice photoperiod sensitive genic male sterility

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The differentially expressed cDNA fragments have been obtained by differential screening with cDNA-RAPD technique in photoperiod sensitive genic male sterile (PGMS) rice.Some of them have been reassessed with Northern blot hybridization,from which a PGMS-related positive fragment,RPG43,has been identified.Further analysis on RPG43 with Southern blot and RAPD indicates that the fragment is a single-copy sequence and its mRNA has been processed after transcription.Sequence analysis reveals that RPG43 is 744 bp in length and contains a 60 bp region (from 126th to 185th bp) showing 72% homology to a human DNA sequence,pac pDJ- 356d6,on chromosome 11.So it is a new sequence found in plant and its GenBank access number is AF126027.In addition,RPG43 has been mapped to a position 3.8 cM away from RFLP marker R1553 on chromosome 5 of rice.

  11. Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence

    International Nuclear Information System (INIS)

    The sequence and structure of human testis-specific L-lactate dehydrogenase [LDHC4, LDHX; (L)-lactate:NAD+ oxidoreductase, EC 1.1.1.27] has been derived from analysis of a complementary DNA (cDNA) clone comprising the complete protein coding region of the enzyme. From the deduced amino acid sequence, human LDHC4 is as different from rodent LDHC4 (73% homology) as it is from human LDHA4 (76% homology) and porcine LDHB4 (68% homology). Subunit homologies are consistent with the conclusion that the LDHC gene arose by at least two independent duplication events. Furthermore, the lower degree of homology between mouse and human LDHC4 and the appearance of this isozyme late in evolution suggests a higher rate of mutation in the mammalian LDHC genes than in the LDHA and -B genes. Comparison of exposed amino acid residues of discrete anti-genic determinants of mouse and human LDHC4 reveals significant differences. Knowledge of the human LDHC4 sequence will help design human-specific peptides useful in the development of a contraceptive vaccine

  12. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. No obvious bands were detected in other examined tissues. The GGPPS gene was located on human chromosome 1q43 by Radiation Hybrid mapping method. It was proved that there was a putative predisposing gene for prostate cancer in this region, and that analogs of GGPP can inhibit the geranylgeranylation of p21rap protein in PC-3 prostate cancer cell lines. These facts suggest that GGPPS may be one of the candidate genes for prostate cancer.

  13. Molecular characteristics and expression of calmodulin cDNA from the freshwater pearl mussel, Hyriopsis schlegelii.

    Science.gov (United States)

    Zeng, L-G; Wang, J-H; Li, Y-J; Sheng, J-Q; Gu, Q; Hong, Y-J

    2012-01-01

    Calmodulin (CaM) is a multifunctional intracellular calcium ion receptor protein that participates in a range of cellular processes, including calcium metabolism in mussels. To investigate the role of CaM in freshwater mollusk shell calcium metabolism, the full-length CaM cDNA was isolated from the freshwater pearl mussel, Hyriopsis schlegelii (referred to as hsCaM) using SMART RACE technology. The full-length hsCaM was 855 bp in size, containing a 70-bp 5'-untranslated sequence, a 447-bp open reading frame, a 309-bp 3'-untranslated sequence, and a 26-nucleotide long poly(A) tail. The hsCaM mRNA expression in different mussel tissues was examined using real-time PCR. The hsCaM mRNA was found to be ubiquitously expressed, but far more abundant in the gill, foot, and mantle than in the posterior adductor muscle. Real-time PCR was also used to determine hsCaM mRNA expression levels in mantle tissues of H. schlegelii at different ages. No significant differences between one-, two-, and three-year-old mussels were detected, but expression increased in four-year-old mussels and then decreased in five-year-old mussels. CaM appears to be involved in calcium regulation of the mantle in four-year-old mussels, which may secrete more mother of pearl during pearl culture. PMID:22290464

  14. Existence of homologous sequences corresponding to cDNA of the ver

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The presence of DNA homologues corresponding to verc203 (vernalization-related cDNA clone) was investigated by molecular hybridization techniques. The genes were detected in 16 plant species that cover 12 subclasses of the Takhtajan system of angiosperms classification including diverse model species. The results of Southern blot analysis showed a low copy number of this gene existed in rice, wheat, barley and Arabidopsis. The hybridization result of PCR products demonstrated the conservation of the gene corresponding to ver203 in diverse plants. The phylogenetic tree of the ver203 gene in tested plants was supported by evolution relationship of species. The ver203 gene expressed in a vernalized plumule winter wheat, instead of the root. And the endosperm before the treatment was essential for the ver203 expression during vernalization in wheat. In Arabidopsis thaliana, the pattern of expression showed that the gene corresponding to ver203 was expressed at low temperature for 14 days. Gibberellin (GA3) may accelerate the expression of ver203 gene in Arabidopsis exposed to low temperature. However, it could not replace vernalization treatment to initiate the gene expression.

  15. Identification of cDNA clones expressing immunodiagnostic antigens from Trichinella spiralis

    Energy Technology Data Exchange (ETDEWEB)

    Zarlenga, D.; Gamble, H.R.

    1987-05-01

    A cDNA expression library was built in lambda gt11 phage using poly A mRNA isolated from Trichinella spiralis muscle stage larvae. This library was screened with rabbit antibodies to parasite excretory-secretory (ES) products and greater than 180 clones were isolated. Thirteen clones producing highly immunogenic protein antigens were plaque purified and rescreened with pig antisera to T.spiralis, Trichuris suis or Ascaris suum to identify clones producing epitopes specific to T.spiralis ES products, only. Two clones, TsAc-2 and TsAc-8, which displayed strong interactions with pig antisera to T. spiralis were lysogenized in E. coli Y1089 and the protein extracted. Western blots of the crude fusion proteins revealed molecular weights of 133 kD and 129 kD, respectively. Northern blot analysis of total RNA with TSP labelled cDNA:lambda gt11 probes indicated single RNA transcripts for each clone with molecular sizes corresponding to 800-850 nucleotides. dscDNA inserts were estimated by southern blot analysis to be 500 bp and 340 bp, respectively, with no cross-hybridization observed between the cloned sequences. Dot blots using pig sera to screen crude fusion protein preparations, total bacterial protein (negative controls) and crude worm extract or ES products from T.spiralis, T.suis and A.suum (positive controls) corroborated the specificity and sensitivity of these clones as potential diagnostic antigens for swine trichinellosis.

  16. Identification of cDNA clones expressing immunodiagnostic antigens from Trichinella spiralis

    International Nuclear Information System (INIS)

    A cDNA expression library was built in lambda gt11 phage using poly A mRNA isolated from Trichinella spiralis muscle stage larvae. This library was screened with rabbit antibodies to parasite excretory-secretory (ES) products and greater than 180 clones were isolated. Thirteen clones producing highly immunogenic protein antigens were plaque purified and rescreened with pig antisera to T.spiralis, Trichuris suis or Ascaris suum to identify clones producing epitopes specific to T.spiralis ES products, only. Two clones, TsAc-2 and TsAc-8, which displayed strong interactions with pig antisera to T. spiralis were lysogenized in E. coli Y1089 and the protein extracted. Western blots of the crude fusion proteins revealed molecular weights of 133 kD and 129 kD, respectively. Northern blot analysis of total RNA with 32P labelled cDNA:lambda gt11 probes indicated single RNA transcripts for each clone with molecular sizes corresponding to 800-850 nucleotides. dscDNA inserts were estimated by southern blot analysis to be 500 bp and 340 bp, respectively, with no cross-hybridization observed between the cloned sequences. Dot blots using pig sera to screen crude fusion protein preparations, total bacterial protein (negative controls) and crude worm extract or ES products from T.spiralis, T.suis and A.suum (positive controls) corroborated the specificity and sensitivity of these clones as potential diagnostic antigens for swine trichinellosis

  17. Effects of aspirin on metastasis-associated gene expression detected by cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    Xue-qin GAO; Jin-xiang HAN; Hai-yan HUANG; Shi YAN; Chang-zheng SONG; Hai-nan HUANG

    2004-01-01

    AIM: To investigate the effect of aspirin on the metastasis-associated gene expression in 3AO ovarian cancer cells.METHODS: 3AO cells were treated with aspirin at the concentration of 1.2 mmol/L for 16 and 48 h, respectively.The total RNA was extracted with Trizol reagents and reverse transcribed with Superscript II and hybridized with cDNA microarray (containing oncogenes, tumor suppressor genes, signal transduction pathway molecules, adhesive molecules, growth factors and ESTs) fabricated in our lab. After normalization, the ratio of gene expression of aspirin treated to untreated 3AO cells being either 2 fold up higher or 0.5 fold down (lower) were defined as differential expression. Semi-quantitative RT-PCR was used to validate the microarray results. RESULTS: Among the 447 metastasis-associated genes, 4 genes were up-regulated and 14 genes were down-regulated in 3AO cells treated with aspirin for 16 h compared with untreated cells. While 24 genes were up-regulated and 10 genes were down-regulated in cells treated with aspirin for 48 h. Several up or down-regulated gene expression changes continued from 16 h to 48 h. CONCLUSION: Aspirin might exert its anti-metastasis effects on ovarian cancer by affecting metastasis-associated gene expression.

  18. cDNA identification, comparison and phylogenetic aspects of lombricine kinase from two oligochaete species.

    Science.gov (United States)

    Doumen, Chris

    2010-06-01

    Creatine kinase and arginine kinase are the typical representatives of an eight-member phosphagen kinase family, which play important roles in the cellular energy metabolism of animals. The phylum Annelida underwent a series of evolutionary processes that resulted in rapid divergence and radiation of these enzymes, producing the greatest diversity of the phosphagen kinases within this phylum. Lombricine kinase (EC 2.7.3.5) is one of such enzymes and sequence information is rather limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two oligochaete species, the California blackworm (Lumbriculus variegatus) and the sludge worm (Tubifex tubifex). The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases, including two additional lombricine kinase sequences extracted from DNA databases and provide further insights in the evolution and position of these enzymes within the phosphagen kinase family. The data confirms the presence of a deleted region within the flexible loop (the GS region) of all six examined lombricine kinases. A phylogenetic analysis of these six lombricine kinases clearly positions the enzymes together in a small subcluster within the larger creatine kinase (EC 2.7.3.2) clade. PMID:20230902

  19. Serine Protease Variants Encoded by Echis ocellatus Venom Gland cDNA: Cloning and Sequencing Analysis

    Directory of Open Access Journals (Sweden)

    S. S. Hasson

    2010-01-01

    Full Text Available Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent.

  20. Serine protease variants encoded by Echis ocellatus venom gland cDNA: cloning and sequencing analysis.

    Science.gov (United States)

    Hasson, S S; Mothana, R A; Sallam, T A; Al-balushi, M S; Rahman, M T; Al-Jabri, A A

    2010-01-01

    Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs) from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization) the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent. PMID:20936075

  1. Characterization of porcine ENO3: genomic and cDNA structure, polymorphism and expression

    Directory of Open Access Journals (Sweden)

    Xiong Yuanzhu

    2008-09-01

    Full Text Available Abstract In this study, a full-length cDNA of the porcine ENO3 gene encoding a 434 amino acid protein was isolated. It contains 12 exons over approximately 5.4 kb. Differential splicing in the 5'-untranslated sequence generates two forms of mRNA that differ from each other in the presence or absence of a 142-nucleotide fragment. Expression analysis showed that transcript 1 of ENO3 is highly expressed in liver and lung, while transcript 2 is highly expressed in skeletal muscle and heart. We provide the first evidence that in skeletal muscle expression of ENO3 is different between Yorkshire and Meishan pig breeds. Furthermore, real-time polymerase chain reaction revealed that, in Yorkshire pigs, skeletal muscle expression of transcript 1 is identical at postnatal day-1 and at other stages while that of transcript 2 is higher. Moreover, expression of transcript 1 is lower in skeletal muscle and all other tissue samples than that of transcript 2, with the exception of liver and kidney. Statistical analysis showed the existence of a polymorphism in the ENO3 gene between Chinese indigenous and introduced commercial western pig breeds and that it is associated with fat percentage, average backfat thickness, meat marbling and intramuscular fat in two different populations.

  2. [Characterization of cDNA of T-cell receptor beta chain in rainbow trout].

    Science.gov (United States)

    Partula, S; Fellah, J S; de Guerra, A; Charlemagne, J

    1994-08-01

    Using a two-step PCR strategy, we have cloned several cDNA segments encoding the T-cell receptor beta chain in a Teleost fish, the rainbow trout (Oncorhynchus mykiss). The nine clones analyzed encode identical N-terminal-truncated V beta regions which present limited sequence similarities with several mammalian TcR V beta chains, from residue Tyr-35 to residue Ser-95. These V beta regions are followed by V beta-D beta-J beta-like regions which are different in all the sequenced clones, and by identical C beta regions. The trout C beta domain (156 amino acids) is most related to the chicken and to amphibian (axolotl) C beta domains but no cysteine residue appears in the hinge region. Like in other vertebrate C beta s, the TM region carries a positively charged lysine residue (Lys-271). The intracytoplasmic domain is virtually absent. The possibility to analyze the structure, expression and diversity of a T-cell receptor chain in a Teleost fish model will be important for our future understanding of the evolution of specific immune recognition in vertebrates. PMID:7882160

  3. Biochemical analysis of myeloid antigens and cDNA expression of gp55 (CD14)

    International Nuclear Information System (INIS)

    The authors have analyzed the reactivity patterns of the myeloid panel of mAbs by immunoprecipitation and SDS-PAGE analysis of 125I-labelled membrane extracts and surface binding to transfected COS7 and L-cells expressing specific gene products. Forty-seven of the 150 myeloid antibodies precipitated recognizable protein products. Fifteen mAbs reacted with molecules of 160-170kDa, four reacted with molecules of 220kDa, two reacted with molecules of 43kDa, and five reacted with molecules of 130- 140kDa. Several others reacted with protein products that did not fall into any of these categories. By surface binding to COS7 cells transfected with a specific gp55 (CD14) cDNA clone, 20 mAbs were shown to have reactivity with the gp55 molecule. Similarly, surface binding to transfected L-cells expressing a 130-140kDa (CD31) product demonstrated that two mAbs had a unique specificity

  4. Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

    International Nuclear Information System (INIS)

    Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors)

  5. Potential markers of tongue tumor progression selected by cDNA microarray.

    Science.gov (United States)

    Carinci, F; Lo Muzio, L; Piattelli, A; Rubini, C; Chiesa, F; Ionna, F; Palmieri, A; Maiorano, E; Pastore, A; Laino, G; Dolci, M; Pezzetti, F

    2005-01-01

    Squamous cell carcinoma (SCC), the most frequent malignant tumor of the oral cavity, generally exhibits a poor prognosis and metastases are the main cause of death. This tumor often arises from pre-malignant lesions. To date, it is difficult to predict if and which pre-malignant lesions may progress into oral SCC using traditional methods. For these reasons, several studies are trying to identify markers useful in the progression of pre-malignant lesions and tumors. To define the genetic expression profile of tongue tumor progression we compared 9 dysplasias (DS), 8 tumors without metastasis (TWM), 11 metastasizing SCCs (MT) of the tongue, and a baseline of 11 normal tissues by using cDNA microarray containing 19.2 K clones. We initially applied hierarchical agglomerative clustering based on information from all 6026 clones. Results were obtained by performing a two steps analysis: a Significance Analysis of Microarray (SAM) and a Gene Ontology search. One hundred and five clones have statistically significant different expression levels (FDR ADAMTS2 and cathepsin O). Additionally, under-expressed genes encoded apoptosis-related proteins (PDCD4 and CASP4). In conclusion, we identified several genes differentially expressed in tumor progression which can potentially help in better classifying pre-malignant lesions and tongue SCCs. PMID:16164832

  6. Monitoring the Expression Profiles of Cereal Crops Seedlings by Using Rice cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    SUN Liang-xian; DONG Hai-tao; LI De-bao

    2004-01-01

    Through exploiting the high homology of cereal crop genes, membranous cDNA microarrays containing 3 311 unique rice transcripts (including 1 639 endosperm-derived transcripts and 1 672 mature stem-derived transcripts) were used for monitoring the expression profiles of 1-leaf stage seedlings of 4 cereal crop species: rice, maize, sorghum and barley. After hybridizing with [α-33p] labeled probes, 73.6 % of the arrayed genes generated reliable signals in all of the four cereal crops. Further analysis revealed that among the arrayed genes, a higher percentage of the endosperm-derived transcripts (86.6 %) expressed than that of the mature stem-derived genes (60.9 %), indicating that most of the endosperm expressed genes functioned in young seedlings while considerable amount of mature stem tissue expressed genes did not express. These results also inferred that some genes might function only at certain developmental stages. By comparing the obtained profiles, 84 genes were identified constantly expressed in all the four cereal crops. Many housekeeping genes, such as polyubiquitin, ubiquitin conjugating enzyme and ribosomal proteins were included in this catalogue. The experiment also identified 14 rice seedling specifically expressed genes, including 3 biotic and abiotic stress induced genes and 1 apoptosis suppressor encoding gene Bax inhibitor-1. This investigation provided invaluable information for comparative genomics of gramineae members.

  7. Monitoring the Expression Profiles of Cereal Crops Seedlings by Using Rice cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    SUNLiang-xian; DONGHai-tao; LIDe-bao

    2004-01-01

    Through exploiting the high homology of cereal crop genes, membranous cDNA microarrays containing 3311 unique rice transcripts (including 1 639 cndosperm-derived transcripts and 1 672 mature stem-derived transcripts) were used for monitoring the expression profiles of l-leaf stage seedlings of 4 cereal crop species: rice, maize, sorghum and barley. After hybridizing with [P] labeled probes, 73.6 % of the arrayed genes generated reliable signals in all of the four cereal crops. Further analysis revealed that among the arrayed genes, a higher percentage of the endosperm-derived transcripts (86.6%) expressed than that of the mature stem-derived genes (60.9 %), indicating that most of the endosperm expressed genes functioned in young seedlings whilc considerable amount of mature stem tissue expressed genes did not express. These results also inferred that some genes might function only at certain developmental stages. By comparing the obtained profdes, 84 genes were identified constantly expressed in all the four cereal crops. Many housekeeping genes, such as polyubiquitin, ubiquitin conjugating enzyme and ribosomal proteins were included in this catalogue. The experiment also identified 14 rice seedling specifically expressed genes, including 3 biotic and abiotic stress induced genes and 1 apoptosis suppressor encoding gene Bax inhibitor-1. This investigation provided invaluable information for comparative genomics of gramineae members.

  8. Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project

    Science.gov (United States)

    Fernández, Paula; Paniego, Norma; Lew, Sergio; Hopp, H Esteban; Heinz, Ruth A

    2003-01-01

    Background Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. Results Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4). The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60%) did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences putatively related to responses

  9. Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project

    Directory of Open Access Journals (Sweden)

    Heinz Ruth A

    2003-09-01

    Full Text Available Abstract Background Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. Results Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4. The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60% did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences

  10. ISOLATION AND IDENTIFICATION OF cDNA FRAGMENTS AND FULL-LENGTH cDNA DIFFERENTIALLY EXPRESSEDIN HUMAN GLIOBLASTOMA CELL LINE BT-325 VERSUS ALL-TRANS RETINOIC ACID INDUCTION

    Institute of Scientific and Technical Information of China (English)

    金虎林; 胡松年; 李光涛; 涂纯; 袁建刚; 强伯勤

    2000-01-01

    Objective. To investigate the differentiation process of the human glioblastoma cells. Methods. Differential display reverse transcribed-PCR(DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325. Routine method of cDNA library screening was performed to clone full-length cDNA. Results. Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered, amplified and cloned. Of them,46 ESTs were sequenced and delivered into the GenBank. The homology comparison us-ing BLAST algorithm revealed that 22ESFs are highly homologous with the known genes and many of them play impor-tant roles in the cell differentiation progress. A dot-blot hybridization was conducted to certify the differemiation expres-sion. The result showed that 27 EST clones are expressed at different level in control and all-traus retinoic acid treated BT-325 cells. A full-length cDNA was cloned using the EST-HGBB098.Conclusion. DDRT-PCR was a simple and effective method to segally analyze the differentially expressed genes.

  11. ISOLATION AND IDENTIFICATION OF cDNA FRAGMENTS AND FULL-LENGTH cDNA DIFFERENTIALLY EXPRESSED IN HUMAN GLIOBLASTOMA CELL LINE BT-325 VERSUS ALL-TRANS RETINOIC ACID INDUCTION

    Institute of Scientific and Technical Information of China (English)

    金虎林; 胡松年; 李光涛; 涂纯; 袁建刚; 强伯勤

    2000-01-01

    Objective. To investigate the differentiation process of the human glioblastoma cells. Methods. Differential display reverse transcribed-PCR(DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325. Routine method of cDNA library screening was performed to clone full-length cDNA. Results. Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered, amplified and cloned. Of them,46 ESTs were sequenced and delivered into the GenBank. The homology comparison us ing BLAST algorithm revealed that 22ESTs are highly homologous with the known genes and many of them play impor tant roles in the cell differentiation progress. A dot-blot hybridization was conducted to certify the differentiation expres sion. The result showed that 27 EST clones are expressed at different level in control and all-trans retinoi c acid treated BT-325 cells. A full-length cDNA was cloned using the ES T-HGBB098. Conclusion. DDRT-PCR was a simple and effective method to serially analyze the differentially expressed genes.

  12. Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

    International Nuclear Information System (INIS)

    Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A)+ RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A)+ RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A)+ RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding

  13. Molecular Cloning of Myostatin Partial cDNA of Beijing Duck and Its Expression in Breast Muscle

    Institute of Scientific and Technical Information of China (English)

    WANG Yong-sheng; HOU Shui-sheng; HUANG Wei; KANG Jun-mei

    2006-01-01

    In this experiment, 500 bp cDNA of myostatin gene was cloned from a Beijing duck's breast. The duck myostatin gene was found to have 98, 96, 95, 88, and 87% sequence similarity at the cDNA level with domestic goose, chicken, domestic pigeon, human, and pig, respectively. The predicted amino acid sequence has an overall similarity with a comparable region of turkey 99%, domestic goose 98%, and chicken 99%. Conserved domains of deduced amino acids showed that it belonged to the TGF-beta family. Myostatin expression in breast muscle was higher at 28, 35, and 42 days than at 7, 14, and 21 days. The pattern of myostatin expression was closely parallel to the trend of breast muscle growth, suggesting that myostatin might play an important role in breast muscle development. It was possible to postulate that myostatin may be a major determinant of muscle mass in breast muscle, as shown in other species.

  14. Sequence of MET protooncogene cDNA has features characteristic of the tyrosine kinase family of growth-factor receptors

    International Nuclear Information System (INIS)

    The authors isolated overlapping cDNA clones corresponding to the major MET protooncogene transcript. The cDNA nucleotide sequence contained an open reading frame of 1408 amino acids with features characteristic of the tyrosine kinase family of growth factor receptors. These features include a putative 24-amino acid signal peptide and a candidate, hybrophobic, membrane-spanning segment of 23 amino acids, which defines an extracellular domain of 926 amino acids that could serve as a ligand-binding domain. A putative intracellular domain 435 amino acids long shows high homology with the SRC family of tyrosine kinases and within the kinase domain is most homologous with the human insulin receptor (44%) and v-abl (41%). Despite these similarities, however, they found no apparent sequence homology to other growth factor receptors in the putative ligand-binding domain. They conclude from the results that the MET protooncogene is a cell-surface receptor for an as-yet-unknown ligand

  15. Screening a Novel Human Breast Cancer-Associated Antigen from a cDNA Expression Library of Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    Shuhua Yang; Lin Zhang; Ruifang Niu; Defa Wang; Yurong Shi; Xiyin Wei; Yi Yang

    2005-01-01

    OBJECTIVE The aim of this research was to clone and express the antigen of the previously prepared monoclonal antibody named M4G3.METHODS Western blots were used to screen a breast cancer cell line that overexpresses the M4G3-associated antigen. A λ zap cDNA expression library of breast cancer cells was constructed and screened using M4G3 as a probe to clone the antigen. The positive clones were subcloned and identified by homologous comparison using BLAST.RESULTS The λ zap cDNA expression library had 1.0x106 independent clones. Fifteen positive clones were isolated following 3 rounds of immunoscreening and identified as being from Mycoplasma pulmonis.CONCLUSION The specific antigen that matched the monoclonal M4G3 antibody is an unknown protein of M. pulmonis. This work is helpful for the further study of the association of M. pulmonis infection with breast cancer.

  16. cDNA Cloning and Molecular Modeling of Procerain B, a Novel Cysteine Endopeptidase Isolated from Calotropis procera

    OpenAIRE

    Singh, Abhay Narayan; Yadav, Prity; Dubey, Vikash Kumar

    2013-01-01

    Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimen...

  17. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    International Nuclear Information System (INIS)

    A λgt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-125I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degree C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested

  18. cDNA probes for detection of specific dsRNAs from the fungal pathogen, Monosporascus cannonballus.

    Science.gov (United States)

    Batten, J S; Scholthof, K B; Miller, M E; Martyn, R D

    2000-02-01

    Monosporascus cannonballus is an ascomycete fungus that is the causative agent of Monosporascus root rot/vine decline, a serious disease of muskmelon and watermelon. Double-stranded RNA (dsRNA) was identified in approximately 60% of M. cannonballus isolates recovered from infected muskmelon plants in 1993. After repeated laboratory transfer on culture media, the majority of the isolates harboring dsRNAs developed degenerate culture phenotypes and showed reduced virulence (hypovirulence) to muskmelon. Initially, dsRNA purification and cDNA synthesis were attempted in three M. cannonballus isolates harboring dsRNAs. However, numerous difficulties were encountered due to the stable, double-stranded nature of the dsRNAs and contamination of the preparations by fungal rRNA. Several purification and cDNA protocols were evaluated and eventually modified into methods that were ultimately highly effective for cloning dsRNAs from M. cannonballus. The cDNAs derived from purified dsRNA preparations were cloned into a pUC119 plasmid vector and amplified in Escherichia coli. Nine cDNA clones were identified that are specific for medium-sized (ca. 3 kbp) dsRNAs associated with M. cannonballus isolate Ca91-17(96+). The methods used to make the cDNA clones of the dsRNAs in M. cannonballus may be useful for those working on fungal dsRNAs. In addition, these cDNAs may be useful for identifying dsRNAs associated with the hypovirulence phenotype. PMID:10680971

  19. Mouse protein arrays from a TH1 cell cDNA library for antibody screening and serum profiling

    OpenAIRE

    Gutjahr, C.; Murphy, D.; Lueking, A.; Koenig, A.; Janitz, M; O'Brien, J.; Korn, B. (Bernhard); S. Horn; Lehrach, H; Cahill, D.

    2005-01-01

    The mouse is the premier genetic model organism for the study of disease and development. We describe the establishment of a mouse T helper cell type 1 (TH1) protein expression library that provides direct access to thousands of recombinant mouse proteins, in particular those associated with immune responses. The advantage of a system based on the combination of large cDNA expression libraries with microarray technology is the direct connection of the DNA sequence information from a particula...

  20. Random sequencing of an induced Taxus cell cDNA library for identification of clones involved in Taxol biosynthesis

    OpenAIRE

    Jennewein, Stefan; Wildung, Mark R.; Chau, MyDoanh; Walker, Kevin; Croteau, Rodney

    2004-01-01

    Biosynthesis of the anticancer drug Taxol involves 19 enzymatic steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methylerythritol phosphate pathway for isoprenoid precursor supply. To gain further insight about Taxol biosynthesis relevant to the improved production of this drug and to draw inferences about the organization, regulation, and origins of this complex natural product pathway, random sequencing of a cDNA library derived from Taxus...

  1. Stabilization of a Full-Length Infectious cDNA Clone of Transmissible Gastroenteritis Coronavirus by Insertion of an Intron

    OpenAIRE

    González, José M; Pénzes, Zoltan; Almazán, Fernando; Calvo, Enrique; Enjuanes, Luis

    2002-01-01

    The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in Escherichia coli cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different po...

  2. Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project

    OpenAIRE

    Heinz Ruth A; Lew Sergio; Hopp H; Paniego Norma; Fernández Paula

    2003-01-01

    Abstract Background Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative str...

  3. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  4. Analysis of differences of gene expressions in keloid and normal skin with the aid of cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    Chen Wei; Fu Xiaobing; Sun Xiaoqing; Sun Tongzhu; Zhao Zhili; Yang Yinhui; Sheng Zhiyong

    2003-01-01

    Background: Microarray analysis is a popular tool to investigate the function of genes that are responsible for the phenotype of the disease. Keloid is a intricate lesion which is probably modulated by interplay of many genes. We ventured to study the differences of gene expressions between keloids and normal skins with the aid of cDNA microarray in order to explore the molecular mechanism underlying keloid formation. Methods: The PCR products of 8400 human genes were spotted on a chip in array. The DNAs were then fixed on the glass plate by a series of treatments. Total RNAs was isolated from freshly excised human keloids and normal skin, and then was purified to mRNA by Oligotex. Both the mRNA from keloids and normal skin was reversely transcribed to cDNAs with the incorporations of fluorescent dUTP, for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing, the cDNA microarray was scanned for the fluorescent signals to display the differences between two kinds of tissues. Results: Among 8400 human genes, there were 402 genes (4.79%) with different expression levels between the keloids and normal skins in all cases, 250were up-regulated (2.98%) and 152 down-regulated (1.81%). Analyses of collagen, fibronectin, proteoglycan,growth factors and apoptosis related molecule gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with published biochemical and clinical observations of keloids. Conclusions: DNA microarray technology is an effective technique in screening for differences in gene expression between keloid and normal skin. Many genes are involved in the formation of keloids. Further analysis of the obtained genes will help understand the molecular mechanism of keloid formation.

  5. Generation and Analysis of Expressed Sequence Tags (ESTs) from Muscle Full-Length cDNA Library of Wujin Pig

    Institute of Scientific and Technical Information of China (English)

    ZHAO Su-mei; LIU Yong-gang; PAN Hong-bing; ZHANG Xi; GE Chang-rong; JIA Jun-jing; GAO Shi-zheng

    2014-01-01

    Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identiifed in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1076 bp, and the cDNA fullness ratio was 86.2%. A total of 1058 unique sequences with 342 contigs (32.3%) and 716 singleton (67.7%) expressed sequence tags (EST) were obtained by clustering and assembling. Meanwhile, 826 (78.1%) ESTs were categorized as known genes, and 232 (21.9%) ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index ofSus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank (accession numbers: EU650784-EU650788, GE843306, GH228978-GH229100). The abundantly expressed genes in porcine muscle tissue were related to muscle ifber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.

  6. Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

    OpenAIRE

    Saxena, A.; Padmanabha, R; Glover, C V

    1987-01-01

    Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic...

  7. Cloning, tissue expression pattern characterization and chromosome localization of human peptide methionine sulfoxide reductase cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Oxidation and reduction of some amino acids are one of the molecular mechanisms for regulating the function of proteins. The oxidation of methionine (Met) to methionine sulfoxide (Met(O)) results in decreasing or loss of the biological activity of related proteins. It was found that peptide methionine sulfoxide reductase (msrA) can reduce Met(O) to Met and therefore restored the biological function of the oxidized proteins. To reveal the methionine oxidation-reduction mechanism in human body, in this study, the cDNA sequence of bovine msrA was used as an information-probe to screen the human EST database. Based on a contig assembled from homologous ESTs, a 1 256-bp human MSRA cDNA was cloned from several human cDNA libraries. The cDNA contains an open reading frame (ORF) of 705 bp in length, which encodes 235 amino acid residues. Homology comparison revealed that human MSRA shares 88% and 61% identities with bovine and Escherichia coli msrA protein respectively. Expression pattern analysis revealed a single 1.6-kb transcript of human MSRA in most human tissues and with highest expression in kidney. By radiation hybrid panel mapping, the gene was localized to human chromosome 8p22-23 between markers D8S518 and D8S550. There are 2 human inherited diseases Keratolytic Winter Erythema and Microcephaly related genes in this region, it is inferred that human MSRA might be the candidate of the two diseases.

  8. Analysis of common bean (Phaseolus vulgaris L., genotype BAT93) calmodulin cDNA using computational tools

    OpenAIRE

    Kassim Amelia; Jasvin Singh; Farida Habib Shah; Subhash J Bhore

    2015-01-01

    Background: Common bean (Phaseolus vulgaris L.) is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs) is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day an...

  9. Human liver mitochondrial carnitine palmitoyltransferase I: characterization of its cDNA and chromosomal localization and partial analysis of the gene.

    OpenAIRE

    Britton, C H; Schultz, R.A.; Zhang, B; Esser, V; Foster, D W; McGarry, J D

    1995-01-01

    Using the cDNA for rat liver mitochondrial carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) as a probe, we isolated its counterpart as three overlapping clones from a human liver cDNA library. Both the nucleotide sequence of the human cDNA and the predicted primary structure of the protein (773 aa) proved to be very similar to those of the rat enzyme (82% and 88% identity, respectively). The CPT I mRNA size was also found to be the same (approximately 4.7 kb) in both species. Screening o...

  10. Introduction by molecular cloning of artifactual inverted sequences at the 5' terminus of the sense strand of bovine parathyroid hormone cDNA.

    OpenAIRE

    Weaver, C A; Gordon, D. F.; Kemper, B

    1981-01-01

    To study the structure and function of the gene for parathyroid hormone, we obtained recombinant plasmids containing bovine parathyroid hormone cDNA. The nucleotide sequence at the 5' terminus (relative to the sense strand) of the cDNA insert in a recombinant plasmid, pPTHi4, was different from that previously reported for the bovine parathyroid hormone cDNA insert of another recombinant plasmid, pPTHm1 [Kronenberg, H. M., McDevitt, B. F., Majzoub, J. A., Nathans, J., Sharp, P. A., Potts, J. ...

  11. Primary structure of cucumber (Cucumis sativus) ascorbate oxidase deduced from cDNA sequence: homology with blue copper proteins and tissue-specific expression.

    OpenAIRE

    Ohkawa, J; Okada, N; Shinmyo, A; Takano, M.

    1989-01-01

    cDNA clones for ascorbate oxidase were isolated from a cDNA library made from cucumber (Cucumis sativus) fruit mRNA. The library was screened with synthetic oligonucleotides that encode the NH2-terminal sequence of this enzyme. Nucleotide sequence analysis of the cloned cDNA inserts revealed a 1761-base-pair open reading frame that encoded an NH2-terminal signal peptide of 33 amino acids and a mature enzyme of 554 amino acids (Mr, 62,258). The amino acid sequence deduced from nucleotide seque...

  12. Molecular cloning of cDNA and analysis of protein secondary structure of Candida albicans enolase, an abundant, immunodominant glycolytic enzyme.

    OpenAIRE

    Sundstrom, P; Aliaga, G R

    1992-01-01

    We isolated and sequenced a clone for Candida albicans enolase from a C. albicans cDNA library by using molecular genetic techniques. The 1.4-kbp cDNA encoded one long open reading frame of 440 amino acids which was 87 and 75% similar to predicted enolases of Saccharomyces cerevisiae and enolases from other organisms, respectively. The cDNA included the entire coding region and predicted a protein of molecular weight 47,178. The codon usage was highly biased and similar to that found for the ...

  13. Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA.

    OpenAIRE

    Ruggli, N; Tratschin, J D; Mittelholzer, C.; Hofmann, M A

    1996-01-01

    The complete nucleotide sequence of the genome of classical swine fever virus (CSFV) strain Alfort/187 was determined from three cDNA libraries constructed by cloning of DNA fragments obtained from independent sets of reverse transcription and PCR. The cDNA fragments were then assembled and inserted downstream of a T7 promoter in a P15A-derived plasmid vector to obtain the full-length cDNA clone pA187-1. The first nucleotide of the CSFV genome was positioned at the transcription start site of...

  14. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee

    OpenAIRE

    Yanagi, Masayuki; Purcell, Robert H.; Emerson, Suzanne U.; Bukh, Jens

    1997-01-01

    We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5′ and 3′ termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase–PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA tra...

  15. Fluorescent cDNA microarray hybridization reveals complexity and heterogeneity of cellular genotoxic stress responses.

    Science.gov (United States)

    Amundson, S A; Bittner, M; Chen, Y; Trent, J; Meltzer, P; Fornace, A J

    1999-06-17

    The fate of cells exposed to ionizing radiation (IR) may depend greatly on changes in gene expression, so that an improved view of gene induction profiles is important for understanding mechanisms of checkpoint control, repair and cell death following such exposures. We have used a quantitative fluorescent cDNA microarray hybridization approach to identify genes regulated in response to 7-irradiation in the p53 wild-type ML-1 human myeloid cell line. Hybridization of the array to fluorescently-labeled RNA from treated and untreated cells was followed by computer analysis to derive relative changes in expression levels of the genes present in the array, which agreed well with actual quantitative changes in expression. Forty-eight sequences, 30 not previously identified as IR-responsive, were significantly regulated by IR. Induction by IR and other stresses of a subset of these genes, including the previously characterized CIP1/ WAF1, MDM2 and BAX genes, as well as nine genes not previously reported to be IR-responsive, was examined in a panel of 12 human cell lines. Responses varied widely in cell lines with different tissues of origin and different genetic backgrounds, highlighting the importance of cellular context to genotoxic stress responses. Two of the newly identified IR-responsive genes, FRA-1 and ATF3, showed a p53-associated component to their IR-induction, and this was confirmed both in isogenic human cell lines and in mouse thymus. The majority of the IR-responsive genes, however, showed no indication of p53-dependent regulation, representing a potentially important class of stress-responsive genes in leukemic cells. PMID:10380890

  16. A peroxiredoxin cDNA from Taiwanofungus camphorata: role of Cys31 in dimerization.

    Science.gov (United States)

    Huang, Chih-Yu; Chen, Yu-Ting; Wen, Lisa; Sheu, Dey-Chyi; Lin, Chi-Tsai

    2014-01-01

    Peroxiredoxins (Prxs) play important roles in antioxidant defense and redox signaling pathways. A Prx isozyme cDNA (TcPrx2, 745 bp, EF552425) was cloned from Taiwanofungus camphorata and its recombinant protein was overexpressed. The purified protein was shown to exist predominantly as a dimer by sodium dodecyl sulfate-polyacrylamide gel electrolysis in the absence of a reducing agent. The protein in its dimeric form showed no detectable Prx activity. However, the protein showed increased Prx activity with increasing dithiothreitol concentration which correlates with dissociation of the dimer into monomer. The TcPrx2 contains two Cys residues. The Cys(60) located in the conserved active site is the putative active peroxidatic Cys. The role of Cys(31) was investigated by site-directed mutagenesis. The C31S mutant (C(31) → S(31)) exists predominantly as a monomer with noticeable Prx activity. The Prx activity of the mutant was higher than that of the corresponding wild-type protein by nearly twofold at 12 μg/mL. The substrate preference of the mutant was H2O2 > cumene peroxide > t-butyl peroxide. The Michaelis constant (K M) value for H2O2 of the mutant was 0.11 mM. The mutant enzyme was active under a broad pH range from 6 to 10. The results suggest a role of Cys(31) in dimerization of the TcPrx2, a role which, at least in part, may be involved in determining the activity of Prx. The C(31) residue does not function as a resolving Cys and therefore the TcPrx2 must follow the reaction mechanism of 1-Cys Prx. This TcPrx2 represents a new isoform of Prx family. PMID:24194195

  17. The murine biglycan: Complete cDNA cloning, genomic organization, promoter function, and expression

    Energy Technology Data Exchange (ETDEWEB)

    Wegrowski, Y.; Pillarisetti, J.; Danielson, K.G.; Iozzo, R.V. [Thomas Jefferson Univ., Philadelphia, PA (United States); Suzuki, S. [Univ. of Southern California, Los Angeles, CA (United States)

    1995-11-01

    Biglycan is a ubiquitous chondroitin/dermatan sulfate proteoglycan that belongs to a growing family of proteins harboring leucine-rich repeats. We have cloned and sequenced the cDNA containing the complete murine biglycan, elucidated its genomic organization, and demonstrated functional promoter activity of its 5{prime} flanking region. The deduced biglycan protein core was highly conserved across species. However, the mouse biglycan (Bgn) gene was significantly larger than the human counterpart, primarily because of a large > 4.5-kb intron between exons 1 and 2. The mouse Bgn gene spanned over 9.5 kb of continuous DNA and comprised 8 exons, with a perfectly conserved intron/exon organization vis-a-vis the human counterpart. The promoter region was enriched in GC dinucleotide and contained numerous cis-acting elements including binding sites for SP-1, AP-1, and AP-2 factors. It lacked TATA and CAAT boxes typical of housekeeping genes. In support of this, primer extension analysis showed the existence of multiple transcription start sites. Transient cell transfection assays with a construct comprising the 548 hp upstream of the major transcription start site fused to the chloramphenicol acetyl transferase reporter gene showed functional promoter activity. Internal and 5{prime} deletion constructs showed that the distal promoter of the Bgn gene was required for full transcriptional activity. In contrast to the homologous proteoglycan decorin, the highest expression of biglycan mRNA was observed in lung, liver, and spleen of adult mouse and the lowest in skin, heart, and kidney. These results will be useful for the study of biglycan gene regulation and for the generation of mice with targeted null mutation of the Bgn gene. 56 refs., 7 figs., 1 tab.

  18. The full-ORF clone resource of the German cDNA Consortium

    Directory of Open Access Journals (Sweden)

    Michel Guenter

    2007-10-01

    Full Text Available Abstract Background With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. Results Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen. A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. Conclusion The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available

  19. Recovery of infectious pariacoto virus from cDNA clones and identification of susceptible cell lines.

    Science.gov (United States)

    Johnson, K N; Ball, L A

    2001-12-01

    Pariacoto virus (PaV) is a nodavirus that was recently isolated in Peru from the Southern armyworm, Spodoptera eridania. Virus particles are non enveloped and about 30 nm in diameter and have T=3 icosahedral symmetry. The 3.0-A crystal structure shows that about 35% of the genomic RNA is icosahedrally ordered, with the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that underlie the inner surface of the capsid. The PaV genome comprises two single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor alpha. In order to apply molecular genetics to the structure and assembly of PaV, we identified susceptible cell lines and developed a reverse genetic system for this virus. Cell lines that were susceptible to infection by PaV included those from Spodoptera exigua, Helicoverpa zea and Aedes albopictus, whereas cells from Drosophila melanogaster and Spodoptera frugiperda were refractory to infection. To recover virus from molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were cotranscribed by T7 RNA polymerase in baby hamster kidney cells that expressed T7 RNA polymerase. Lysates of these cells were infectious both for cultured cells from Helicoverpa zea (corn earworm) and for larvae of Galleria mellonella (greater wax moth). The combination of infectious cDNA clones, cell culture infectivity, and the ability to produce milligram amounts of virus allows the application of DNA-based genetic methods to the study of PaV structure and assembly. PMID:11711613

  20. Mole ghrelin: cDNA cloning, gene expression, and diverse molecular forms in Mogera imaizumii.

    Science.gov (United States)

    Satou, Motoyasu; Kaiya, Hiroyuki; Nishi, Yoshihiro; Shinohara, Akio; Kawada, Shin-Ichiro; Miyazato, Mikiya; Kangawa, Kenji; Sugimoto, Hiroyuki

    2016-06-01

    Here, we describe cDNA cloning and purification of the ghrelin gene sequences and ghrelin peptides from the Japanese true mole, Mogera imaizumii. The gene spans >2.9kbp, has four exons and three introns, and shares structural similarity with those of terrestrial animals. Mature mole ghrelin peptide was predicted to be 28 amino acids long (GSSFLSPEHQKVQQRKESKKPPSKPQPR) and processed from a prepropeptide of 116 amino acids. To further elucidate molecular characteristics, we purified ghrelin peptides from mole stomach. By mass spectrometry, we found that the mole ghrelin peptides had higher ratios of the odd-number fatty acids (C9 and C11 as much as C8) attached to the third serine residue than other vertebrate ghrelin. Truncated forms of ghrelins such as [1-27], [1-19], [1-16] and [1-15], and that lacked the 14th glutamine residue (des-Gln14 ghrelin) were produced in the stomach. Marked expression of ghrelin mRNA in lung was observed as in stomach and brain. Phylogenetic analysis indicated that the branch of M. imaizumii has slightly higher dN/dS ratios (the nucleotide substitution rates at non-synonymous and synonymous sites) than did other eulipotyphlans. Peptide length was positively correlated with human ghrelin receptor activation, whereas the length of fatty-acyl chains showed no obvious functional correlation. The basal higher luciferase activities of the 5'-proximal promoter region of mole ghrelin were detected in ghrelin-negative C2C12 cells and hypoxic culture conditions impaired transcriptional activity. These results indicated that moles have acquired diverse species of ghrelin probably through distinctive fatty acid metabolism because of their food preferences. The results provide a gateway to understanding ghrelin metabolism in fossorial animals. PMID:27102942

  1. Normal uniform mixture differential gene expression detection for cDNA microarrays

    Directory of Open Access Journals (Sweden)

    Raftery Adrian E

    2005-07-01

    Full Text Available Abstract Background One of the primary tasks in analysing gene expression data is finding genes that are differentially expressed in different samples. Multiple testing issues due to the thousands of tests run make some of the more popular methods for doing this problematic. Results We propose a simple method, Normal Uniform Differential Gene Expression (NUDGE detection for finding differentially expressed genes in cDNA microarrays. The method uses a simple univariate normal-uniform mixture model, in combination with new normalization methods for spread as well as mean that extend the lowess normalization of Dudoit, Yang, Callow and Speed (2002 1. It takes account of multiple testing, and gives probabilities of differential expression as part of its output. It can be applied to either single-slide or replicated experiments, and it is very fast. Three datasets are analyzed using NUDGE, and the results are compared to those given by other popular methods: unadjusted and Bonferroni-adjusted t tests, Significance Analysis of Microarrays (SAM, and Empirical Bayes for microarrays (EBarrays with both Gamma-Gamma and Lognormal-Normal models. Conclusion The method gives a high probability of differential expression to genes known/suspected a priori to be differentially expressed and a low probability to the others. In terms of known false positives and false negatives, the method outperforms all multiple-replicate methods except for the Gamma-Gamma EBarrays method to which it offers comparable results with the added advantages of greater simplicity, speed, fewer assumptions and applicability to the single replicate case. An R package called nudge to implement the methods in this paper will be made available soon at http://www.bioconductor.org.

  2. Gene expression profile of esophageal cancer in North East India by cDNA microarray analysis

    Institute of Scientific and Technical Information of China (English)

    Indranil Chattopadhyay; Sujala Kapur; Joydeep Purkayastha; Rupkumar Phukan; Amal Kataki; Jagadish Mahanta; Sunita Saxena

    2007-01-01

    AIM: To identify alterations in genes and molecular functional pathways in esophageal cancer in a high incidence region of India where there is a widespread use of tobacco and betel quid with fermented areca nuts.METHODS: Total RNA was isolated from tumor and matched normal tissue of 16 patients with esophageal squamous cell carcinoma. Pooled tumor tissue RNA was labeled with Cy3-dUTP and pooled normal tissue RNA was labeled with Cy5-dUTP by direct labeling method.The labeled probes were hybridized with human 10K cDNA chip and expression profiles were analyzed by Genespring GX V 7.3 (Silicon Genetics).RESULTS: Nine hundred twenty three genes were differentially expressed. Of these, 611 genes were upregulated and 312 genes were downregulated. Using stringent criteria (P ≤ 0.05 and ≥ 1.5 fold change),127 differentially expressed genes (87 upregulated and 40 downregulated) were identified in tumor tissue. On the basis of Gene Ontology, four different molecular functional pathways (MAPK pathway,G-protein coupled receptor family, ion transport activity,and serine or threonine kinase activity) were most significantly upregulated and six different molecular functional pathways (structural constituent of ribosome,endopeptidase inhibitor activity, structural constituent of cytoskeleton, antioxidant activity, acyl group transferase activity, eukaryotic translation elongation factor activity)were most significantly downregulated.CONCLUSION: Several genes that showed alterations in our study have also been reported from a high incidence area of esophageal cancer in China. This indicates that molecular profiles of esophageal cancer in these two different geographic locations are highly consistent.

  3. Amino acid sequence of the serine-repeat antigen (SERA) of Plasmodium falciparum determined from cloned cDNA.

    Science.gov (United States)

    Bzik, D J; Li, W B; Horii, T; Inselburg, J

    1988-09-01

    We report the isolation of cDNA clones for a Plasmodium falciparum gene that encodes the complete amino acid sequence of a previously identified exported blood stage antigen. The Mr of this antigen protein had been determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis analysis, by different workers, to be 113,000, 126,000, and 140,000. We show, by cDNA nucleotide sequence analysis, that this antigen gene encodes a 989 amino acid protein (111 kDa) that contains a potential signal peptide, but not a membrane anchor domain. In the FCR3 strain the serine content of the protein was 11%, of which 57% of the serine residues were localized within a 201 amino acid sequence that included 35 consecutive serine residues. The protein also contained three possible N-linked glycosylation sites and numerous possible O-linked glycosylation sites. The mRNA was abundant during late trophozoite-schizont parasite stages. We propose to identity this antigen, which had been called p126, by the acronym SERA, serine-repeat antigen, based on its complete structure. The usefulness of the cloned cDNA as a source of a possible malaria vaccine is considered in view of the previously demonstrated ability of the antigen to induce parasite-inhibitory antibodies and a protective immune response in Saimiri monkeys. PMID:2847041

  4. GTP cyclohydrolase I feedback regulatory protein is a pentamer of identical subunits. Purification, cDNA cloning, and bacterial expression.

    Science.gov (United States)

    Yoneyama, T; Brewer, J M; Hatakeyama, K

    1997-04-11

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by tetrahydrobiopterin and also mediates the stimulatory effect of phenylalanine on the enzyme activity. To characterize the molecular structure of GFRP, we have purified it from rat liver using an efficient step of affinity chromatography and isolated cDNA clones, based on partial amino acid sequences of peptides derived from purified GFRP. Comparison between the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of purified GFRP showed that the mature form of GFRP consists of 83 amino acid residues with a calculated Mr of 9,542. The isolated GFRP cDNA was expressed in Escherichia coli as a fusion protein with six consecutive histidine residues at its N terminus. The fusion protein was affinity-purified and digested with thrombin to remove the histidine tag. The resulting recombinant GFRP showed kinetic properties similar to those of GFRP purified from rat liver. Cross-linking experiments using dimethyl suberimidate indicated that GFRP was a pentamer of 52 kDa. Sedimentation equilibrium measurements confirmed the pentameric structure of GFRP by giving an average Mr of 49,734, which is 5 times the calculated molecular weight of the recombinant GFRP polypeptide. Based on the pentameric structure of GFRP, we have proposed a model for the quaternary structure of GFRP and GTP cyclohydrolase I complexes. PMID:9092499

  5. Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

    Science.gov (United States)

    Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

    1992-01-01

    We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

  6. Cloning and cDNA sequence of the regulator subunit of cAMP-dependent protein kinase from Dictyostelium discoideum

    International Nuclear Information System (INIS)

    cDNA clones encoding the regulatory subunit of the cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from Dictyostelium discoideum were isolated by immunoscreening of a cDNA library constructed in the expression vector λgt11, using autoradiography. High-affinity cAMP binding activity was detected in extracts from bacteria lysogenized with these clones. Nucleotide sequence analysis of three overlapping clones allowed the determination of a 1195-base-pair cDNA sequence coding for the entire regulatory subunit and containing nontranslated 5' and 3' sequences. The open reading frame codes for a protein of 327 amino acids, with molecular weight 36,794. The regulatory subunit from Dictyostelium shares a high degree of homology with its mammalian counterparts, but is lacking the NH2-terminal domain required for the association of regulatory subunits into dimers in other eukaryotes. On the basis of the comparison of the regulatory subunits from Dictyostelium, yeast, and bovine tissues, a model for the evolution of these proteins is proposed

  7. Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine

    International Nuclear Information System (INIS)

    O6-Methylguanine-DNA methyltransferase a unique DNA repair protein present in most organisms, removes the carcinogenic and mutagenic adduct O6-alkylguanine from DNA by stoichiometrically accepting the alkyl group on a cysteine residue in a suicide reaction. The mammalian protein is highly regulated in both somatic and germ-like cells. In addition, the toxicity of certain alkylating drugs in tumor and normal cells is inversely related to the levels of this protein. The cDNA of the human gene, henceforth named MGMT, has been cloned in an expression vector on the basis of its rescue of a methyltransferase-deficient (ada-) Escherichia coli host. A 22-kDa active methyltransferase encoded entirely by the cDNA contains an amino acid sequence of 61 residues that bears 60-65% similarity with segments of E. coli methyltransferase which encompass the alkyl-acceptor residues. The human cDNA has no sequence similarity with the ada and ogt genes, due in part to differences in codon usage, and shows no detectable homology with E. coli genomic DNA. However, it hybridizes with distinct restriction fragments of human, mouse, and rat DNAs. The lack of methyltransferase observed in many human cell lines is due to the absence of the MGNT gene or to lack of synthesis and/or stability of its 0.95-kilobase poly(A)+ RNA transcript

  8. Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence

    International Nuclear Information System (INIS)

    Secretory protein I (SP-I), also referred to as chromogranin A, is an acidic glycoprotein that has been found in every tissue of endocrine and neuroendocrine origin examined but never in exocrine or epithelial cells. Its co-storage and co-secretion with peptide hormones and neurotransmitters suggest that it has an important endocrine or secretory function. The authors have isolated cDNA clones from a bovine pituitary λgt11 expression library using an antiserum to parathyroid SP-I. The largest clone (SP4B) hybridized to a transcript of 2.1 kilobases in RNA from parathyroid, pituitary, and adrenal medulla. Immunoblots of bacterial lysates derived from SP4B lysognes demonstrated specific antibody binding to an SP4B/β-galactosidase fusion protein (160 kDa) with a cDNA-derived component of 46 kDa. Radioimmunoassay of the bacterial lystates with SP-I antiserum yielded parallel displacement curves of 125I-labeled SP-I by the SP4B lysate and authentic SP-I. SP4B contains a cDNA of 1614 nucleotides that encodes a 449-amino acid protein (calculated mass, 50 kDa). The nucleotide sequences of the pituitary SP-I cDNA and adrenal medullary SP-I cDNAs are nearly identical. Analysis of genomic DNA suggests that pituitary, adrenal, and parathyroid SP-I are products of the same gene

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

    International Nuclear Information System (INIS)

    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 x 106 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J. (Mount Sinai School of Medicine, New York, NY (USA))

    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.

  11. Molecular cloning, sequence analysis and expression in Escherichia coli of Camelus dromedarius glucose-6-phosphate dehydrogenase cDNA.

    Science.gov (United States)

    Saeed, Hesham Mahmoud; Alanazi, Mohammad Saud; Abduljaleel, Zainularifeen; Al-Amri, Abdullah; Khan, Zahid

    2012-06-01

    This study determined the full length sequence of glucose-6-phosphate dehydrogenase cDNA (G6PD) from the Arabian camel Camelus dromedarius using reverse transcription polymerase chain reaction. The C. dromedarius G6PD has an open reading frame of 1545 bp, and the cDNA encodes a protein of 515 amino acid residues with a molecular weight of 59.0 KDa. The amino acid sequence showed the highest identity with Equus caballus (92%) and Homo sapiens (92%). The G6PD cDNA was cloned and expressed into Escherichia coli as a fusion protein and was purified in a single chromatographic step using nickel affinity gel column. The purity and the molecular weight of the enzyme were checked on SDS-PAGE and the purified enzyme showed a single band on the gel with a molecular weight of 63.0 KDa. The specific activity of G6PD was determined to be 289.6 EU/mg protein with a fold purification of 95.45 and yield of 56.8%. PMID:22538316

  12. Accuracy of cDNA microarray methods to detect small gene expression changes induced by neuregulin on breast epithelial cells

    Directory of Open Access Journals (Sweden)

    Ahmed Sharlin

    2004-07-01

    Full Text Available Abstract Background cDNA microarrays are a powerful means to screen for biologically relevant gene expression changes, but are often limited by their ability to detect small changes accurately due to "noise" from random and systematic errors. While experimental designs and statistical analysis methods have been proposed to reduce these errors, few studies have tested their accuracy and ability to identify small, but biologically important, changes. Here, we have compared two cDNA microarray experimental design methods with northern blot confirmation to reveal changes in gene expression that could contribute to the early antiproliferative effects of neuregulin on MCF10AT human breast epithelial cells. Results We performed parallel experiments on identical samples using a dye-swap design with ANOVA and an experimental design that excludes systematic biases by "correcting" experimental/control hybridization ratios with control/control hybridizations on a spot-by-spot basis. We refer to this approach as the "control correction method" (CCM. Using replicate arrays, we identified a decrease in proliferation genes and an increase in differentiation genes. Using an arbitrary cut-off of 1.7-fold and p values Conclusions We validated two experimental design paradigms for cDNA microarray experiments capable of detecting small (

  13. Identification of differentially expressed genes of Xanthomonas axonopodis pv. citri by representational difference analysis of cDNA

    Directory of Open Access Journals (Sweden)

    Angela Mehta

    2005-03-01

    Full Text Available Xanthomonas axonopodis pv. citri is a phytopathogenic bacterium responsible for citrus canker, a serious disease which causes severe losses in citriculture around the world. In this study we report the differential expression of X. axonopodis pv. citri in response to specific treatments by using Representational Difference Analysis of cDNA (cDNA RDA. cDNAs from X. axonopodis pv. citri cultured in the presence of leaf extract of the host plant (Citrus sinensis, in vivo, as well as in the complex medium were hybridized against cDNA of the bacterium grown in the minimal medium. Sequencing of the difference products obtained after the second and third hybridizations revealed a total of 37 distinct genes identified by homology searches in the genome of X. axonopodis pv. citri. These genes were distributed in different functional categories, including genes that encode hypothetical proteins, genes involved in metabolism, cellular processes and pathogenicity, and mobile genetic elements. Most of these genes are likely related to growth and/or acquisition of nutrients in specific treatments whereas others might be important for the bacterium pathogenicity.

  14. Hypoxia-induced regulation of MAPK phosphatase-1 as identified by subtractive suppression hybridization and cDNA microarray analysis.

    Science.gov (United States)

    Seta, K A; Kim, R; Kim, H W; Millhorn, D E; Beitner-Johnson, D

    2001-11-30

    Subtractive suppression hybridization was used to generate a cDNA library enriched in cDNA sequences corresponding to mRNA species that are specifically up-regulated by hypoxia (6 h, 1% O(2)) in the oxygen-responsive pheochromocytoma cell line. The dual specificity protein-tyrosine phosphatase MAPK phosphatase-1 (MKP-1) was highly represented in this library. Clones were arrayed on glass slides to create a hypoxia-specific cDNA microarray chip. Microarray, northern blot, and western blot analyses confirmed that MKP-1 mRNA and protein levels were up-regulated by hypoxia by approximately 8-fold. The magnitude of the effect of hypoxia on MKP-1 was approximately equal to that induced by KCl depolarization and much larger than the effects of either epidermal growth factor or nerve growth factor on MKP-1 mRNA levels. In contrast to the calcium-dependent induction of MKP-1 by KCl depolarization, the effect of hypoxia on MKP-1 persisted under calcium-free conditions. Cobalt and deferoxamine also increased MKP-1 mRNA levels, suggesting that hypoxia-inducible factor proteins may play a role in the regulation of MKP-1 by hypoxia. Pretreatment of cells with SB203580, which inhibits p38 kinase activity, significantly reduced the hypoxia-induced increase in MKP-1 RNA levels. Thus, hypoxia robustly increases MKP-1 levels, at least in part through a p38 kinase-mediated mechanism. PMID:11577072

  15. Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Leustek, T; Murillo, M; Cervantes, M

    1994-01-01

    ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana. APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast). Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein. The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S. cerevisiae, with which it is 25% identical. Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase. Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts. Several genomic DNA fragments that hybridize with the APS1 probe were identified. The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb). PMID:8058839

  16. Construction of infectious cDNA clones of PRRSV:Separation of coding regions for nonstructural and structural proteins

    Institute of Scientific and Technical Information of China (English)

    YUAN ShiShan; WEI ZuZhang

    2008-01-01

    Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the causative agent of the ongoing"porcine high fever syndrome" in China, is capable of genetic and antigenic mutations at high frequency. How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great Interest. We developed an infectious cDNA clone of an attenuated strain of Type Ⅱ PRRSV, and further manipulated the infectious cDNA clone by inserting polylinker between ORF1 and ORF2, encoding for nonstructural- or structural-protein, respectively. The cDNA was generated from the cell-attenuated virus strain, APRRS, via RT-PCR, and followed by nucleotide sequencing and molecular cloning. The full-length of the APRRS genomic RNA was determined as 15521 nucleotides in length excluding poly(A) tail, which has a 99.7% nucleotide identity with that of PRRSV Nsp strain, also a vaccine strain. Based on the nucleotide sequencing results, the full-length cDNA clone was assembled in pBlueScript vector, under the control of T7 promoter at the immediate 5' terminus of genome. To discern the rescued viruses from that of parental virus, a Mlu I restriction site was engineered into ORF5 coding region. Upon trensfection of the in vitro transcripts of both the original and MIu I-tagged cDNAs into MA-104 cells, typical PRRSV cytopathic effects were observed. The rescued viruses from the full-length cDNA clones displayed the same virological and molecular properties. Subsequently,PCR-based mutagenesis was conducted to separate the coding regions between PRRSV nonstructural genes, ORF1, and structural proteins, ORF2-ORF7. The synthetic RNA of such mutant clone, pCSA,was infectious and the rescued virus shared similar properties with that of the parental virus. This study provided a valuable tool for development of chimeric PRRSV as vaccine candidate offering crose-protection to various genetically diversified PRRSV strains, and a platform for further development of PRRSV as a gene

  17. Cloning of UGT1A9 cDNA from liver tissues and its expression in CHL cells

    Institute of Scientific and Technical Information of China (English)

    Xin Li; Ying-Nian Yu; Ge-Jian Zhu; Yu-Li Qian

    2001-01-01

    AIM: To clone the cDNA of UGT1 A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGTI A9. METHODS: cDNA of UGT1A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E. CoIl DH5α. The inserted fragment, verified by DNA sequencing, wes subcloned into the Hind III/Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9. CHL cells were transfected with the resultant recombinants, pREP9- UGT1A9, and selected by G418 (400 mg. L-1) for one month. The surviving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in medium containing G418 to obtain samples for UGT1 A9 assays. The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPLC. RESULTS: The sequence of the cDNA segment cloned,which was 1666 bp in length, was identical to that released by Gene Bank (GenBank accession number: AF056188) in coding region. The recombinant constructed, pREP9- UGT1 A9, contains the entire coding region, along with 18bp of the 5' and 55 bp of the 3′ untranslated region of the UGT1A9 cDNA, respectively. The cell lines established expressed the protein of UGT1 A9, and the enzyme activity towards propranolol in S9 protein was found to be 101 ± 24pmol.min-1 .mg-1 protein (n = 3), but was not detectable in parental CHL cells. CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chinese human liver and transfected into CHL ceils. The CHL-UGT1A9 cell lines established efficiently expressed the protein of UGT1A9 for the further enzyme study of drug glucuronidation.

  18. Construction of infectious cDNA clones of PRRSV: Separation of coding regions for nonstructural and structural proteins

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the causative agent of the ongoing "porcine high fever syndrome" in China, is capable of genetic and antigenic mutations at high fre- quency. How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great interest. We developed an infectious cDNA clone of an attenuated strain of Type II PRRSV, and further manipulated the infectious cDNA clone by inserting polylinker between ORF1 and ORF2, en- coding for nonstructural- or structural-protein, respectively. The cDNA was generated from the cell-attenuated virus strain, APRRS, via RT-PCR, and followed by nucleotide sequencing and molecular cloning. The full-length of the APRRS genomic RNA was determined as 15521 nucleotides in length excluding poly(A) tail, which has a 99.7% nucleotide identity with that of PRRSV Nsp strain, also a vac- cine strain. Based on the nucleotide sequencing results, the full-length cDNA clone was assembled in pBlueScript vector, under the control of T7 promoter at the immediate 5′ terminus of genome. To dis- cern the rescued viruses from that of parental virus, a Mlu I restriction site was engineered into ORF5 coding region. Upon transfection of the in vitro transcripts of both the original and Mlu I-tagged cDNAs into MA-104 cells, typical PRRSV cytopathic effects were observed. The rescued viruses from the full-length cDNA clones displayed the same virological and molecular properties. Subsequently, PCR-based mutagenesis was conducted to separate the coding regions between PRRSV nonstructural genes, ORF1, and structural proteins, ORF2-ORF7. The synthetic RNA of such mutant clone, pCSA, was infectious and the rescued virus shared similar properties with that of the parental virus. This study provided a valuable tool for development of chimeric PRRSV as vaccine candidate offering cross-protection to various genetically diversified PRRSV strains, and a platform for further develop- ment of

  19. Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Dräger, Carolin;

    2015-01-01

    infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique......-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from...... clone must necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones...

  20. Construction of cDNA libraries by blunt-end ligation: high-frequency cloning of long cDNAs from filamentous fungi.

    Science.gov (United States)

    Teeri, T T; Kumar, V; Lehtovaara, P; Knowles, J

    1987-07-01

    A simplified cDNA synthesis and cloning method, suitable for efficient generation of cDNA libraries at frequencies up to 10(6) clones/micrograms mRNA, is described. Routine synthesis of transcripts of well over 4 kb is facilitated by the use of high-quality RNA template isolated from materials rich in RNases. Laborious cloning steps, like tailing or addition of linkers, can be omitted by the use of efficient blunt-end ligation to plasmid vectors, and rapid verification as well as characterization of the clones is possible by double-stranded plasmid sequencing. Using this method we have constructed several cDNA libraries of different filamentous fungi and show here the synthesis and cloning of cDNA copies larger than 1.8 kb corresponding to three Trichoderma reesei cellulases. PMID:2823635

  1. Cloning a Full-length cDNA Encoding UDP-glucose Pyrophosphorylase from Amorpha fruticosa by PCR-based Methods

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A method based on degenerate Oligo-primed polymerase chain reaction (PCR) and random amplification of cDNA end (RACE) PCR for cloning a full-length cDNA is described. An Amorpha fruticosa cDNA clone encoding UDP-glucose pyrophosphorylase (UGP), a key enzyme producing UDP-glucose in the synthesis of sucrose and cell ulose, is cloned by using this method. We design 5' RACE primers based on UGP A1 fragment, which obtains from degenerate PCR. Inverse PCR and nested PCR enable cloning of the remainder 5' and 3' end fragments of the gene. The deduced amino acid sequence exhibits significant homology with the other UGP genes cloned. This method is more simple and inexpensive than screening cDNA library, and can be easily adapted to clone other genes.

  2. Partial Purification and Characterization of Bacillus thuringiensis Cry1A Toxin Receptor A from Heliothis virescens and Cloning of the Corresponding cDNA

    OpenAIRE

    Oltean, Daniela I.; Pullikuth, Ashok K; Lee, Hyun-Ku; Gill, Sarjeet S.

    1999-01-01

    Although extensively studied, the mechanism of action of insecticidal Bacillus thuringiensis Cry toxins remains elusive and requires further elucidation. Toxin receptors in the brush border membrane demand particular attention as they presumably initiate the cascade of events leading to insect mortality after toxin activation. The 170-kDa Cry1Ac toxin-binding aminopeptidase from the tobacco budworm (Heliothis virescens) was partially purified, and its corresponding cDNA was cloned. The cDNA e...

  3. Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof

    OpenAIRE

    Nolden, T.; Pfaff, F; Nemitz, S.; Freuling, C. M.; Höper, D.; Müller, T; Stefan Finke

    2016-01-01

    Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T re...

  4. In vitro recombination cloning of entire cDNA libraries in Arabidopsis thaliana and its application to the yeast two-hybrid system

    OpenAIRE

    Bürkle, L.; Meyer, S.; Dortay, H.; Lehrach, H; Heyl, A.

    2005-01-01

    In the postgenomic era many experiments rely on the availability of transcript sequence for cloning. As these clones usually originate from cDNA libraries, the quality of these libraries is crucial. If a good library is generated it is desirable to use a versatile cloning system suitable for many different kinds of applications. The cloning systems based on in vitro recombination proves fitting for this task. However, the use of this method for shuttling entire cDNA libraries between differen...

  5. Retroviral transfer of a murine cDNA for multidrug resistance confers pleiotropic drug resistance to cells without prior drug selection.

    OpenAIRE

    Guild, B C; Mulligan, R C; Gros, P.; Housman, D.E.

    1988-01-01

    We have constructed a retrovirus expression vector that carries the murine mdr cDNA transcribed under the control of the human H4 histone promoter to examine the feasibility of efficiently transferring a multidrug resistance phenotype to cells without requiring drug selection. This approach will facilitate the transfer of mdr cDNA to hematopoietic progenitor cells for the study of multidrug resistance in vivo. The retrovirus vector pHmdr has been used for transmission and expression of the md...

  6. Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase: a new approach in PCR-mediated analysis of mRNA sequences.

    OpenAIRE

    Schmidt, W. M.; Mueller, M W

    1996-01-01

    Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase is a polymerase chain reaction (PCR)-mediated technique that was developed to facilitate cloning and direct sequence analysis of complete 5'-terminal unknown coding regions of rare RNA molecules. In contrast with standard tailing protocols using dNTPs as the substrate, ribo-tailing of cDNA ends is easily controllable, self-limited (from two to four rNMP incorporations) and highly efficient (>98%). B...

  7. cDNA clone for the alpha-chain of human beta-hexosaminidase: deficiency of alpha-chain mRNA in Ashkenazi Tay-Sachs fibroblasts.

    OpenAIRE

    Myerowitz, R; Proia, R L

    1984-01-01

    We have isolated a cDNA clone containing sequences complementary to mRNA encoding the alpha-chain of the lysosomal enzyme beta-hexosaminidase. RNA from a human lung fibroblast strain, IMR90, was enriched for beta-hexosaminidase messenger by polysome immunoselection with antiserum against beta-hexosaminidase A. This preparation was used to construct cDNA recombinant plasmids by the Okayama-Berg vector primer procedure. After transformation of Escherichia coli, 385 ampicillin-resistant colonies...

  8. An annotated genetic map of loblolly pine based on microsatellite and cDNA markers

    Directory of Open Access Journals (Sweden)

    Wimalanathan Kokulapalan

    2011-01-01

    Full Text Available Abstract Background Previous loblolly pine (Pinus taeda L. genetic linkage maps have been based on a variety of DNA polymorphisms, such as AFLPs, RAPDs, RFLPs, and ESTPs, but only a few SSRs (simple sequence repeats, also known as simple tandem repeats or microsatellites, have been mapped in P. taeda. The objective of this study was to integrate a large set of SSR markers from a variety of sources and published cDNA markers into a composite P. taeda genetic map constructed from two reference mapping pedigrees. A dense genetic map that incorporates SSR loci will benefit complete pine genome sequencing, pine population genetics studies, and pine breeding programs. Careful marker annotation using a variety of references further enhances the utility of the integrated SSR map. Results The updated P. taeda genetic map, with an estimated genome coverage of 1,515 cM(Kosambi across 12 linkage groups, incorporated 170 new SSR markers and 290 previously reported SSR, RFLP, and ESTP markers. The average marker interval was 3.1 cM. Of 233 mapped SSR loci, 84 were from cDNA-derived sequences (EST-SSRs and 149 were from non-transcribed genomic sequences (genomic-SSRs. Of all 311 mapped cDNA-derived markers, 77% were associated with NCBI Pta UniGene clusters, 67% with RefSeq proteins, and 62% with functional Gene Ontology (GO terms. Duplicate (i.e., redundant accessory and paralogous markers were tentatively identified by evaluating marker sequences by their UniGene cluster IDs, clone IDs, and relative map positions. The average gene diversity, He, among polymorphic SSR loci, including those that were not mapped, was 0.43 for 94 EST-SSRs and 0.72 for 83 genomic-SSRs. The genetic map can be viewed and queried at http://www.conifergdb.org/pinemap. Conclusions Many polymorphic and genetically mapped SSR markers are now available for use in P. taeda population genetics, studies of adaptive traits, and various germplasm management applications. Annotating mapped

  9. Cloning and Bioimformatic Analysis of Full Length cDNA of LHY Gene from Cicer arietinum L.%鹰嘴豆 LHY 基因 cDNA 克隆及生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    韩慧; 王希东; 姚正培; 夏木斯亚·卡坎

    2014-01-01

    从鹰嘴豆中克隆生物节律钟 LHY 基因的 cDNA 全长序列,进行序列信息学分析.通过同源克隆策略,利用 RT-PCR 技术获得核心片段,结合5′-RACE 和3′-RACE 技术,克隆得到鹰嘴豆生物节律钟基因 LHY 的 cD-NA 全长序列,其核苷酸序列长度为3061 bp,包括2220 bp 的完整开放阅读框(ORF),编码739个氨基酸.验证后命名为 CarLHY 基因,获得基因登录号为 KJ558378.生物信息学研究表明 CarLHY 基因 cDNA 序列与其他植物 LHY 基因具有较高的相似性;预测 CarLHY 蛋白不具有跨膜结构;为转录因子,定位于细胞核中;不具备信号肽.对 CarLHY 蛋白功能结构域预测表明,蛋白质核心结构存在符合转录因子与 DNA 结合的常见功能域 HTH.蛋白系统进化树显示,与大豆分子进化距离最近,其次是黑杨、拟南芥.%The sequence analysis was carried out to investigate the full-length cDNA sequence of biothythm clock gene LHY cloned from Cicer arietinum Linn with 5′-RACE and 3′-RACE.RT-PCR tech-nology was used to obtain core fragment with the homologous cloning strategy.The cDNA sequence was 3061 bp,and the open reading frame (ORF)was 2 220 bp,encoded 739 amino acids.It was named CarLHY after checked.The registration number was KJ558378 in Genebank datebase.The research of biological in-formation science indicated that cDNA sequence of CarLHY gene and other plant LHY gene have higher similarity.It was predicted that CarLHY protein have no transmembrane structure.It was speculated that CarLHY protein have no transmembrane spiral;It was a transcriptional factors and located in the nucleus. It has no signal peptide.CarLHY protein functional domains forecast indicated that there were transcription factor and HTH domain binded to DNA in protein core structure.The phylogenetic tree showed that CarL-HY and Glycine LHY are closest in molecular evolution distance,followed by Populus LHY and Arabii-dopsis thalana LHY gene.

  10. Construction of a full-length cDNA library of Solen grandis dunker and identification of defense- and immune-related genes

    Science.gov (United States)

    Sun, Guohua; Liu, Xiangquan; Ren, Lihua; Yang, Jianmin; Wei, Xiumei; Yang, Jialong

    2013-11-01

    The basic genetic characteristics, important functional genes, and entire transcriptome of Solen grandis Dunker were investigated by constructing a full-length cDNA library with the `switching mechanism at the 5'-end of the RNA transcript' (SMART) technique. Total RNA was isolated from the immune-relevant tissues, gills and hemocytes, using the Trizol reagent, and cDNA fragments were digested with Sfi I before being ligated to the pBluescript II SK* vector. The cDNA library had a titer of 1048 cfu μL-1 and a storage capacity of 1.05×106 cfu. Approximately 98% of the clones in the library were recombinants, and the fragment lengths of insert cDNA ranged from 0.8 kb to 3.0 kb. A total of 2038 expressed sequence tags were successfully sequenced and clustered into 965 unigenes. BLASTN analysis showed that 240 sequences were highly similar to the known genes (E-value 80%), accounting for 25% of the total unigenes. According to the Gene Ontology, these unigenes were related to several biological processes, including cell structure, signal transport, protein synthesis, transcription, energy metabolism, and immunity. Fifteen of the identified sequences were related to defense and immunity. The full-length cDNA sequence of HSC70 was obtained. The cDNA library of S. grandis provided a useful resource for future researches of functional genomics related to stress tolerance, immunity, and other physiological activities.

  11. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs

    International Nuclear Information System (INIS)

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA

  12. Isolation of an insulin-like growth factor II cDNA with a unique 5' untranslated region from human placenta

    International Nuclear Information System (INIS)

    Human insulin-like growth factor II (IGF-II) cDNA from a placental library was isolated and sequenced. The 5' untranslated region (5'-UTR) sequence of this cDNA differs completely from that of adult human liver and has considerable base sequence identity to the same region of an IGF-II cDNA of a rat liver cell line, BRL-3A. Human placental poly(A)+ RNA was probed with either the 5'-UTR of the isolated human placental IGF-II cDNA or the 5'-UTR of the IGF-II cDNA obtained from adult human liver. No transcripts were detected by using the 5'-UTR of the adult liver IGF-II as the probe. In contrast, three transcripts of 6.0, 3.2, and 2.2 kilobases were detected by using the 5'-UTR of the placental IGF-II cDNA as the probe or the probe from the coding sequence. A fourth IGF-II transcript of 4.9 kilobases presumably containing a 5'-UTR consisting of a base sequence dissimilar to that of either IGF-II 5'-UTR was apparent. Therefore, IGF-II transcripts detected may be products of alternative splicing as their 5'-UTR sequence is contained within the human IGF-II gene or they may be a consequence of alternative promoter utilization in placenta

  13. Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici

    Directory of Open Access Journals (Sweden)

    Chen Xianming

    2007-06-01

    Full Text Available Abstract Background Puccinia striiformis is a plant pathogenic fungus causing stripe rust, one of the most important diseases on cereal crops and grasses worldwide. However, little is know about its genome and genes involved in the biology and pathogenicity of the pathogen. We initiated the functional genomic research of the fungus by constructing a full-length cDNA and determined functions of the first group of genes by sequence comparison of cDNA clones to genes reported in other fungi. Results A full-length cDNA library, consisting of 42,240 clones with an average cDNA insert of 1.9 kb, was constructed using urediniospores of race PST-78 of P. striiformis f. sp. tritici. From 196 sequenced cDNA clones, we determined functions of 73 clones (37.2%. In addition, 36 clones (18.4% had significant homology to hypothetical proteins, 37 clones (18.9% had some homology to genes in other fungi, and the remaining 50 clones (25.5% did not produce any hits. From the 73 clones with functions, we identified 51 different genes encoding protein products that are involved in amino acid metabolism, cell defense, cell cycle, cell signaling, cell structure and growth, energy cycle, lipid and nucleotide metabolism, protein modification, ribosomal protein complex, sugar metabolism, transcription factor, transport metabolism, and virulence/infection. Conclusion The full-length cDNA library is useful in identifying functional genes of P. striiformis.

  14. Molecular Cloning of a Novel cDNA From Mus Muscular BALB/c Mice Encoding Glycosyl Hydrolase Family 1: A Homolog of HumanLactase-Phlorizin Hydrolase

    Institute of Scientific and Technical Information of China (English)

    WEI HE; ZHEN-YU JI; CHENG-YU HUANG

    2006-01-01

    Objective To study the mechanism of lactose intolerance (LI) by cloning the mouse lactase cDNA and recombining a vector. Methods Total murine RNA was isolated from the small intestine of a 4-week-old BALB/c mouse (♂).Gene-specific primers were designed and synthesized according to the cDNA sequences of lactase-phlorizin hydrolase (LPH) in human, rat, and rabbit. A coding sequence (CDS) fragment was obtained using RT-PCR, and inserted into a clone vector pNEB-193, then the cDNA was sequenced and analyzed using bioinformatics. Results The cDNA from the BALB/c mouse with 912 bp encoding 303 amino acid residues. Analysis of the deduced amino acid sequence using bioinformatics revealed that this cDNA shared extensive sequence homology with human LPH containing a conserved glycosy1 hydrolase family 1 motif important for regulating lactase intolerance. Conclusion BALB/c mouse LPH cDNA (GenBank accession No: AY751548) provides a necessary foundation for study of the biological function and regulatory mechanism of the lactose intolerance in mice.

  15. Isolation and Cloning of cDNA of Gene Encoding for Metallothionein Type 2 from Soybean [Glycine max (L. (Merrill] cv. Slamet

    Directory of Open Access Journals (Sweden)

    UTUT WIDYASTUTI

    2009-07-01

    Full Text Available Metallothionein has an important role in the detoxification of metal ions. It has a low molecular weight and contains cysteine-rich residue. The objective of this research is to isolate and clone the cDNA of gene encoding for metallothionein from soybean [Glycine max (L. (Merrill] cv Slamet (GmMt2. We had successfully isolated total RNA by reverse transcription and synthesized total cDNA from total RNA as template. cDNA of GmMt2 had been isolated from total cDNA by PCR. It was successfully inserted into pGEM-T Easy plasmid, and the recombinant plasmids were introduced into Escherichia coli strain DH5α. Sequence analysis by using T7 and SP6 primers showed that the length of PCR-isolated fragment is 257 bp containing 246 bp completed sequence of Mt2 cDNA encoding for 81 amino acids. Enzyme restriction analysis showed that GmMt2 does not contain any restriction sites found in the multi cloning sites of pGEM-T easy. Nucleotide and amino acid alignment analysis using BLAST program showed that GmMt2 is similar with completed cDNA of AtMt2A from Arabidopsis thaliana (L. Heynh. Amino acid sequence analysis showed that the motifs of Cys sequence of GmMT2 are Cys-Cys, Cys-X-Cys, and Cys-X-X-Cys.

  16. Identification of brassinosteroid responsive genes in Arabidopsis by cDNA array

    Institute of Scientific and Technical Information of China (English)

    HU; Yuxin; (

    2001-01-01

    [1]Grove, M. D., Spencer, G. F., Rohwedder, W. K. et al., Brassinolide, a plant growth-promoting steroid isolated from Brassica napus pollen, Nature, 1979, 281: 216-217.[2]Mandava, N. B., Plant growth-promoting brassinosteroids, Annu. Rev. Plant Physiol. Plant Mol. Biol., 1988, 39: 23-52.[3]Clouse, S. D., Sasse, J. M., Brassinosteroids: essential regulators of plant growth and development, Annu. Rev. Plant Physiol. Plant Mol. Biol., 1998, 49: 427-451.[4]Altmann, T., Recent advances in brassinosteroid molecular genetics, Curr. Opin. Plant Biol., 1998, 1: 378-383.[5]Aharoni, A., Keizer, L. C. P., Bouwmeester, H. J. et al., Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarray, Plant Cell, 2000, 12: 647-661.[6]Reymond, P., Weber, H., Damond, M. et al., Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis, Plant Cell, 2000, 12: 707-719.[7]Hu, Y., Han, C., Mou, Z. et al., Monitoring gene expression by cDNA array, Chin. Sci. Bull., 1999, 44: 441-444.[8]Fujioka, S., Li, J., Choi, Y. H. et al., The Arabidopsis deetiolated2 mutant is blocked early in brassinosteroid biosynthesis, Plant Cell, 1997, 9: 1951-1962.[9]Wadsworth, G. J., Redinbaugh, M. G., Scandalios, J. G., A procedure for small-scale isolation of plant RNA suitable for RNA blot analysis, Anal. Biochem., 1988, 172: 279-283.[10]Church, G. M., Gilbert, W., Genomic sequencing, Proc. Natl. Acad. Sci. USA, 1984, 81: 1991-1995.[11]Huntley, R. P., Murray, J. A. H., The plant cell cycle, Curr. Opin. Plant Biol., 1999, 2: 440-446.[12]Riou-Khamlichi, C., Huntley, R., Jacqmard, A. et al., Cytokinin activation of Arabidopsis cell division through a D-type cyclin, Science, 1999, 283: 1541-1544.[13]Hu, Y., Bao, F., Li, J., Promotive effect of brassinosteroids on cell division involves a distinct CycD3-induction pathway, Plant J., 2000, 24: 693-701.[14]Hirayama, T., Shinozaki, K., A

  17. Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments.

    Science.gov (United States)

    Suhardiman, Maman; Kramyu, Jarin; Narkpuk, Jaraspim; Jongkaewwattana, Anan; Wanasen, Nanchaya

    2015-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent for a swine disease affecting the pig industry worldwide. Infection with PRRSV leads to reproductive complications, respiratory illness, and weak immunity to secondary infections. To better control PRRSV infection, novel approaches for generating control measures are critically needed. Here, in vitro Gibson assembly (GA) of viral genomic cDNA fragments was tested for its use as a quick and simple method to recover infectious PRRSV in cell culture. GA involves the activities of T5-exonuclease, Phusion polymerase, and Taq ligase to join overlapping cDNA fragments in an isothermal condition. Four overlapping cDNA fragments covering the entire PRRSV genome and one vector fragment were used to create a plasmid capable of expressing the PRRSV genome. The assembled product was used to transfect a co-culture of 293T and MARC-145 cells. Supernatants from the transfected cells were then passaged onto MARC-145 cells to rescue infectious virus particles. Verification and characterization of the recovered virus confirmed that the GA protocol generated infectious PRRSV that had similar characteristics to the parental virus. This approach was then tested for the generation of a chimeric virus. By replacing one of the four genomic fragments with that of another virus strain, a chimeric virus was successfully recovered via GA. In conclusion, this study describes for the first time the use of GA as a simple, yet powerful tool for generating infectious PRRSV needed for studying PRRSV biology and developing novel vaccines. PMID:25300804

  18. cDNA Cloning, Expression and Characterization of an Allergenic 60s Ribosomal Protein of Almond (Prunus dulcis

    Directory of Open Access Journals (Sweden)

    Abolhassani Mohsen

    2009-06-01

    Full Text Available Tree nuts, including almond (prunus dulcis are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.

  19. Difference in gene expression of macrophage between normal spleen and portal hypertensive spleen idendified by cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    Feng Yan; Xiao-Min Wang

    2007-01-01

    AIM: To identify the difference in gene expression of microphage (Mφ) between normal spleen and portal hypertensive spleen using cDNA microarrays and find new gene functions associated with hypersplenism in portal hypertension.METHODS: The Biostar-H140s chip containing 14112 spots of cDNAs were used to investigate the difference of the expression. The total RNA extracted from macrophages isolated from both normal spleen and portal hypertensive spleen was reversely transcribed to cDNA with the incorporation of fluorescent (cy3 and cy5) labeled dCTP to prepare the hybridization probes.After hybridization, the gene chip was scanned for the fluorescent intensity. The differentially expressed genes were screened. That was repeated three times,and only the genes which had differential expression in all three chips were considered to be associated with hypersplenism in portal hypertension.RESULTS: Eight hundred and ninety-six, 1330 and 898 genes were identified to be differentially expressed in three chips, respectively. One hundred and twenty-one genes (0.86%) were identified to be differentially expressed in all three chips, including 21 up-regulated genes and 73 down-regulated genes. The differentially expressed genes were related to ionic channel and transport protein, cyclin, cytoskeleton, cell receptor, cell signal conduct, metabolism, immune, and so on. These genes might be related to the hypersplenism in portal hypertension.CONCLUSION: The investigations based on cDNA microarray can screen differentially expressed genes of macrophages between normal spleen and portal hypertensive spleen, thus may provide a new idea in studying the pathogenesis of hypersplenism in portal hypertension.

  20. Characterization of a Pinus pinaster cDNA encoding an auxin up-regulated putative peroxidase in roots.

    Science.gov (United States)

    Charvet-Candela, V; Hitchin, S; Reddy, M S; Cournoyer, B; Marmeisse, R; Gay, G

    2002-03-01

    As part of a study to identify host plant genes regulated by fungal auxin during ectomycorrhiza formation, we differentially screened a cDNA library constructed from roots of auxin-treated Pinus pinaster (Ait.) Sol. seedlings. We identified three cDNAs up-regulated by auxin. Sequence analysis of one of these cDNAs, PpPrx75, revealed the presence of an open reading frame of 216 amino acids with the characteristic consensus sequences of plant peroxidases. The deduced amino acid sequence showed homology with Arabidopsis thaliana (L.) Heynh., Arachis hypogaea L. and Stylosanthes humilis HBK cationic peroxidases. Amino acid sequence identities in the conserved domains of plant peroxidases ranged from 60 to 100%. In PpPrx75, there are five cysteine residues and one histidine residue that are found at conserved positions among other peroxidases. A potential glycosylation site (NTS) is present in the deduced sequence. Phylogenetic analysis showed that PpPrx75 is closely related to two A. thaliana peroxidases. The PpPrx75 cDNA was induced by active auxins, ethylene, abscisic acid and quercetin, a flavonoid possibly involved in plant-microorganism interactions. Transcript accumulation was detected within 3 h following root induction by auxin, and the amount of mRNA increased over the following 24 h. The protein synthesis inhibitor cycloheximide did not inhibit indole-3-acetic acid-induced transcript accumulation, suggesting that PpPrx75 induction is a primary (direct) response to auxin. This cDNA can be used to study expression of an auxin-regulated peroxidase during ectomycorrhiza formation. PMID:11874719