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Sample records for balanus amphitrite cdna

  1. Balanus amphitrite or Amphibalanus amphitrite?

    DEFF Research Database (Denmark)

    Clare, Anthony S; Høeg, Jens Thorvald

    2008-01-01

    In a recent revision of the Balanidae based on morphological systematics, the much studied fouling species Balanus amphitrite was renamed Amphibalanus amphitrite. Here, the case is made for retaining the former nomenclature. Taxonomists are urged to exercise caution before introducing new formal ...

  2. 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.

    –140 (1997) ARTICLE NO. CY961991 Cryopreservation of Balanus amphitrite nauplii A. C. ANIL, A. S. TULASKAR, D. C. KHANDEPARKAR, AND A. B. WAGH Marine Corrosion and Materials Research Division, National Institute of Oceanography, Dona Paula, Goa 403 004, India... it possible to establish a larval bank of fouling organisms and thereby make the foul-commercial or research objectives, is often de- pendent on a natural source of brood stock. ing larvae available for antifouling studies at any time of the year...

  3. Construction of an adult barnacle (Balanus amphitrite cDNA library and selection of reference genes for quantitative RT-PCR studies

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    Burgess J Grant

    2009-06-01

    Full Text Available Abstract Background Balanus amphitrite is a barnacle commonly used in biofouling research. Although many aspects of its biology have been elucidated, the lack of genetic information is impeding a molecular understanding of its life cycle. As part of a wider multidisciplinary approach to reveal the biogenic cues influencing barnacle settlement and metamorphosis, we have sequenced and annotated the first cDNA library for B. amphitrite. We also present a systematic validation of potential reference genes for normalization of quantitative real-time PCR (qRT-PCR data obtained from different developmental stages of this animal. Results We generated a cDNA library containing expressed sequence tags (ESTs from adult B. amphitrite. A total of 609 unique sequences (comprising 79 assembled clusters and 530 singlets were derived from 905 reliable unidirectionally sequenced ESTs. Bioinformatics tools such as BLAST, HMMer and InterPro were employed to allow functional annotation of the ESTs. Based on these analyses, we selected 11 genes to study their ability to normalize qRT-PCR data. Total RNA extracted from 7 developmental stages was reverse transcribed and the expression stability of the selected genes was compared using geNorm, BestKeeper and NormFinder. These software programs produced highly comparable results, with the most stable gene being mt-cyb, while tuba, tubb and cp1 were clearly unsuitable for data normalization. Conclusion The collection of B. amphitrite ESTs and their annotation has been made publically available representing an important resource for both basic and applied research on this species. We developed a qRT-PCR assay to determine the most reliable reference genes. Transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, although other genes also performed well and could prove useful to normalize gene expression studies.

  4. 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.

    Balanus amphitrite in nature on the cyprid metamorphosis of B. amphitrite has been examined. The experiments were carried out in polystyrene multiwells. A similar set of experiments was also repeated with the extract of Cthamalus sp. which co...

  5. On the roles of serotonin and dopamine in the settlement of the cyprids of the barnacle Balanus amphitrite (= Amphibalanus amphitrite

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    L. Gallus

    2011-01-01

    Full Text Available In the cyprid of Balanus amphitrite (=Amphibalanus amphitrite was investigated by settlement tests the role of serotonin, related substances and dopamine. The results indicate an activity of serotonin in B. amphitrite cyprid as settlement inhibitors.

  6. 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.

    promotion of Balanus amphitrite. The barnacle cyprid, while exploring some surfaces, leaves behind 'footprints' of temporary adhesive. In the absence of AE, sugar-treated cyprids did not deposit footprints. Concurrently, the sugar-treated cyprids deposited...

  7. 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.

    An evaluation of starvation threshold of the newly hatched Balanus amphitrite nauplii (instar, II) has been made at three different temperatures (5, 15 and 25°C). Earlier studies on starvation in crustacean larvae demonstrated Point of No Return...

  8. 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.C.; Raghukumar, S.

    Balanus amphitrite, on its larval metamorphosis. The effect of multispecies bacterial film was also assessed. The production of different molecules by the bacteria was influenced by the nutrient media under which they were grown. It was observed...

  9. 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.

    Ultrasonic treatment, a relatively less explored technology in water disinfection, was used to quantify the energy required for the destruction of larvae of barnacle Balanus amphitrite, which is a major marine fouling and a potential invasive...

  10. 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.

    ) films and EPS(greater than 1000 molecular weight) on metamorphosis in the acorn barnacle Balanus amphitrite Darwin, a dominant fouling organism, using axenic and non-axenic films, and free and biofilm EPS, of 5 species of pennate diatoms: Amphora...

  11. Desenvolvimento larvário de Balanídeos em laboratório - Balanus amphitrite (Var. amphitrite Larval development of Balanids reared in laboratory: Balanus amphitrite var. amphitrite

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    Dyrce Lacombe

    1972-01-01

    Full Text Available É descrita a técnicade criação de balanus amphitrite var. amphitrite em laboratório, sob condições controladas, que permitem observações das mudas, do número de estágios e, ainda são apresentadas as características de cada estágio. Balanus amphitrite amphitrite é criado pela primeira vez em laboratório com sucesso. Dos 200 Nauplii obtidos da eclosão dos ovos das lamelas dos adultos, conseguiu-se a fixação de 120 cypris e o crescimento de 78 Balanus pelo período de três meses. A alimentação usada foi Cyclotella nana, proveniente do New York Aquarium. A fase larvária de Balanus amphitrite amphitrite consiste de seis estágios de Nauplii e um estágio de Cypris. A disposição, forma, tamanho, número de setas, sétulas e espinhos diferem entre os 6 estágios. São apresentados todos os detalhes de microanatomia de cada estágio. A setação é critério valioso para a identificação dos Nauplii e pode ser usada para separar certos estágios especiais. A duração dos seis estágios é a seguinte: o primeiro estágio leva, aproximadamente, de 15 a 20 minutos; 2º estágio de 2 até 4 dias a partir do nascimento dos Nauplii; 3º estágio vai até ao 6º dia; 4º estágio é encontrado a partir do 7º dia de eclosão; 5º estágio varia entre o 9º ao 11º dia e, finalmente, o 6º estágio até o aparecimento do Cypris que se faz no 12º dia de criação. O tempo necessário para o completo desenvolvimento larvários em laboratório estende-se de 12 até 14 dias, aproximadamente. O estudo da metamorfose do Cypris, e o crescimento dos Balanídeos foi realizado por um período de tres meses no frasco de criação.We described the technique for the creation of the Nauplii of Balanus amphitrite amphitrite under controlled laboratory condictions, which permits observations of the moltings, the number of stages, and the characteristics of each stages. It was for the firt time that Balanus amphitrite amphitrite was reared in

  12. 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.

    The impact of diatom food species (Chaetoceros calcitrans and Skeletonema costatum), temperature and starvation on the larval development of Balanus amphitrite was evaluated. Starvation threshold levels for different ages of larvae (0- to 5-day...

  13. 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.

    Energy content in the non-feeding (lecithotrophic) cyprid larvae of B. amphitrite plays an important role in determining its metamorphic characteristics. In this context it is important to understand the energy transformed to this stage from...

  14. 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.

    . Ecol. (Evol. Persp.), vol.35(4); 2014; 492-505 Algal epibiosis on Megabalanus tintinnabulum and its role in segregation of Balanus amphitrite population Ranjith E and Lidita Khandeparker* Council of Scientific and Industrial Research... & BPA were maintained on ZMA and BPA slants as described by Konya et al. (1995). The isolated bacteria (Table I) were identified following ‘Bergey’s manual of systematic bacteriology (Holt et al. 1994) and manual for the identification of medical...

  15. Chemical Component and Proteomic Study of the Amphibalanus (= Balanus amphitrite Shell.

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    Gen Zhang

    Full Text Available As typical biofoulers, barnacles possess hard shells and cause serious biofouling problems. In this study, we analyzed the protein component of the barnacle Amphibalanus (= Balanus amphitrite shell using gel-based proteomics. The results revealed 52 proteins in the A. Amphitrite shell. Among them, 40 proteins were categorized into 11 functional groups based on KOG database, and the remaining 12 proteins were unknown. Besides the known proteins in barnacle shell (SIPC, carbonic anhydrase and acidic acid matrix protein, we also identified chorion peroxidase, C-type lectin-like domains, serine proteases and proteinase inhibitor proteins in the A. Amphitrite shell. The sequences of these proteins were characterized and their potential functions were discussed. Histology and DAPI staining revealed living cells in the shell, which might secrete the shell proteins identified in this study.

  16. 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.

  17. 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

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

    Directory of Open Access Journals (Sweden)

    Xing-Cheng Yan

    Full Text Available 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

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

    Science.gov (United States)

    Yan, Xing-Cheng; Chen, Zhang-Fan; Sun, Jin; Matsumura, Kiyotaka; Wu, Rudolf S S; Qian, Pei-Yuan

    2012-01-01

    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

  20. Repetitive testing of TBTO, Sea-Nine 211 and farnesol using Balanus Amphitrite (Darwin) cypris larvae: variability in larval sensitivity

    NARCIS (Netherlands)

    Willemsen, P.R.; Overbeke, J.C.; Suurmond, A.

    1998-01-01

    Settlement inhibition assays with mass cultured cypris larvae of the barnacle Balanus amphitrite are widely used in (anti)fouling research. In this study, TBTO, Sea-Nine 211 and farnesol were tested repeatedly using multiple batches of larvae to study variability in larval sensitivity. There were si

  1. Can body burden in the barnacle Balanus amphitrite indicate seasonal variation in cadmium concentrations?

    Science.gov (United States)

    da Silva, Eduardo Teixeira; Ridd, Michael; Klumpp, David

    2005-10-01

    Two three-month sampling programs measuring Cd in the environment and a biomonitor ( Balanus amphitrite) were undertaken in the austral winter of 2002 and summer of 2004 in Ross Creek, North Queensland, Australia. The objective was to test whether the burden of Cd in the biomonitor responded to any variation in the dissolved and particulate phase Cd concentrations in Ross Creek, caused by rainfall variation. The barnacles from the most Cd contaminated site were exposed to a total Cd concentration twice that in winter (93.6 ng L -1) than in summer (45.6 ng L -1). However, no significant variation was identified for the Cd concentration in the biomonitor between winter (8.4 mg kg -1) and summer (7.4 mg kg -1). A budget analysis based on a bioenergetic kinetic model indicated that Cd flux from food contributes >80% to the Cd concentration in B. amphitrite. A sensitivity analysis showed that physiological characteristics of the biomonitor are the key parameters controlling Cd accumulation in B. amphitrite, rather than the metal concentration in the dissolved or particulate phases. These two model's outcomes suggest that a tight coupling between Cd in the biomonitor and its availability in the environment does not occur.

  2. Presence and distribution of serotonin immunoreactivity in the cyprids of the barnacle Balanus amphitrite

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    L Gallus

    2009-06-01

    Full Text Available In this work, the presence and distribution of serotonin in the cyprid of the barnacle Balanus amphitrite were investigated by immunohistochemical methods. Serotonin-like immunoreactive neuronal cell bodies were detected in the central nervous system only. Various clusters of immunoreactive neuronal cell bodies are distributed in the brain (protocerebrum, deutocerebrum, optical lobes, and at least, four pairs of neuronal cell bodies were detected in the centrally positioned neuropil of the posterior ganglion. Rich plexuses of immunoreactive nerve fibers in the neuropil area were also observed. Furthermore, bundles of strongly immunoreactive nerve fibers surrounding the gut wall were localized, and immunoreactive nerve terminals in the antennules and compound eyes were observed. These data demonstrate the presence of a serotonin-like immunoreactive substance in the barnacle cyprids; furthermore, its immunolocalization in the cephalic nerve terminals allows us to postulate the involvement of this bioactive molecule in substrate recognition during the settlement process.

  3. Video observation of surface exploration in cyprids of Balanus amphitrite

    DEFF Research Database (Denmark)

    Maruzzo, Diego; Conlan, Sheelagh; Aldred, Nick;

    2011-01-01

    Video microscopy of cyprids of Balanus amphitrite was used to monitor the action of antennular setae during the exploratory behaviour prior to attachment. In addition, SEM was used to provide a revised description of all antennular setae for that species. The videos describe if a particular seta...... touches the substratum and the area it can cover during surface exploration. On the fourth segment, the plumose terminal setae A and B are never in contact with the substratum, lack a terminal pore and it is argued that they sense hydrodynamic forces. The aesthetasc-like terminal seta D is likewise held...... free in the water at all times and it is speculated that it senses dissolved substances, but, since it contains a scolopale rod, it must also have a mechano-receptive function. All remaining antennular setae on the second, third and fourth segments have a terminal pore and it is argued...

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

    -based Polymerase Chain Reaction (PCR) method for the specific identification of the barnacle, B. amphitrite, from the heterogeneous zooplankton sample. This method is reliable and accurate thereby overcoming taxonomic ambiguity. Sequence alignment of the 18S rRNA...

  10. 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.

    amphitrite, whereas monospecies biofilms did not. The treatment of biofilms with protease resulted in a decline in metamorphosis, except in B. brevis, where although metamorphosis was negated initially, it increased with the protease concentration...

  11. 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.

  12. The mode of action of isocyanide in three aquatic organisms, Balanus amphitrite, Bugula neritina and Danio rerio.

    Directory of Open Access Journals (Sweden)

    Yi-Fan Zhang

    Full Text Available 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.

  13. 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.

  14. 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.

  15. Antifouling effect of bioactive compounds from marine sponge Acanthella elongata and different species of bacterial film on larval attachment of Balanus amphitrite (cirripedia, crustacea

    Directory of Open Access Journals (Sweden)

    Viswambaran Ganapiriya

    2012-06-01

    Full Text Available The antifouling activity of bioactive compounds from marine sponge Acanthella elongata (Dendy and five species of bacterial biofilm were studied. Larvae of Balanus amphitrite (Cyprids and nauplii were used to monitor the settlement inhibition and the extent to which inhibition was due to toxicity. The crude extract and partially purified fractions of A.elongata showed significant inhibition over the settlement individually, and with the interaction of bacterial species. No bacterial film stimulated the barnacle settlement. The high but variable levels of antifouling activity in combination with less amount of toxicity showed the potential of these metabolites in environmentally-friendly antifouling preparations.

  16. 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.

  17. 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.

    amphitrite. The larvae were also field-reared using micro-enclosures. Laboratory-reared larvae were larger in size than the field-reared larvae. An increase in size, DNA content and instar index of the starved II instar larvae was observed indicating...

  18. 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.

  19. 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.

  20. Influence of elevated temperature, pCO2, and nutrients on larva-biofilm interaction: Elucidation with acorn barnacle, Balanus amphitrite Darwin (Cirripedia: Thoracica)

    Science.gov (United States)

    Baragi, Lalita V.; Anil, Arga Chandrashekar

    2017-02-01

    Selection of optimal habitat by larvae of sessile organism is influenced by cues offered by the biofilm. Ocean warming and acidification are likely to enforce changes in the biofilm community and inturn influence the settlement process. Hence, we evaluated the influence of biofilm (multispecies and unialgal) and diet-mediated changes on the settlement of Balanus amphitrite cyprids (presettlement non-feeding larval stage) under different combinations of temperature (28, 30, 32 and 34 °C), pCO2 (400, 750 and 1500 μatm) and nutrient (unenriched and f/2 enriched). Nutrient enrichment enhanced the diatom and bacterial abundance at ambient temperature (30 °C) and pCO2 (400 μatm), which inturn increased larval settlement. Elevated pCO2 (750 and 1500 μatm) had no direct effect but a variable cascading effect on the settlement via biofilm-mediated changes was observed, depending on the type of biofilm. In contrast, elevated temperature (32 and 34 °C), either individually or in combination with elevated pCO2 had direct negative effect on settlement. However, biofilm-mediated changes compensated this negative effect. The larval settlement was also influenced by changes in the larval diet. Under elevated temperature and pCO2, cyprids raised with a feed (Chaetoceros calcitrans) from ambient temperature and pCO2 were of poor quality (lower RNA:DNA ratio, lower protein synthetic capacity) and yielded lower settlement. However, cyprids raised with a feed from elevated temperature and pCO2 were of better quality (higher RNA:DNA ratio, higher protein synthetic capacity) and yielded higher settlement. Overall, the observations from the present study provide insights into the significance of biotic interactions on the coastal biofouling communities under future climatic scenario and emphasise the need for future experiments on these aspects.

  1. Quantifying the exploratory behaviour of Amphibalanus amphitrite cyprids.

    Science.gov (United States)

    Chaw, Kuan Chun; Birch, William R

    2009-10-01

    The behavioural response of cypris larvae from A. amphitrite (=Balanus amphitrite) exploring three model glass surfaces is quantified by close-range microscopy. Step length and step duration measurements reveal a response to both surface properties and flow. Without flow, 2-day-old cyprids took larger steps with shorter step duration on hydrophilic glass surfaces (bare and NH2-treated) vs hydrophobic glass (CH3-treated). These parameters suggest a more detailed, local inspection of hydrophobic surfaces and a more extensive exploration for hydrophilic surfaces. Cyprids under flow took longer steps and exhibited shorter probing times on hydrophobic glass. On hydrophilic glass, cyprids increased their step duration under flow. This active response is attributed to drag and lift forces challenging the cyprids' temporary anchoring to the substratum. Seven-day-old cyprids showed almost no discrimination between the model surfaces. Microscopic-scale observation of cyprid exploration is expected to provide new insights into interactions between cyprids and surfaces.

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

    Digital Repository Service at National Institute of Oceanography (India)

    Desai, D.V.

    been made by several researchers in view of addressing antifouling issues. The larval rearing experiments indicated that larvae in the field experience a nutritional stress. However this stress did not translate proportionately to the well-fed larvae...

  3. Transcriptome and Proteome Studies Reveal Candidate Attachment Genes during the Development of the Barnacle Amphibalanus Amphitrite

    KAUST Repository

    Al-Aqeel, Sarah

    2016-09-21

    The acorn barnacle, Balanus amphitrite, is the main biofouling organism in marine environments. In the present study we profiled the transcriptome and proteome of B. amphitrite at different life stages (nauplius II, nauplius VI, and cyprid) from the Red Sea, where the average water surface temperature is 34°C and the salinity reaches 41%. We identified 65,784 expressed contigs, and a total of 1387 expressed proteins measured by quantitative proteomics. We found that osmotic stress, salt stress, hyperosmotic response and the Wnt signaling pathway were strongly up-regulated during the planktonic stage, while the MAPK pathway, lipid metabolism, and cuticle development genes were down-regulated. In the transition stage between the nauplius VI and the cyprid, genes that are involved in blood coagulation, cuticle development and eggshell formation were highly up-regulated, while the nitric oxide pathway, which stimulates the swimming and feeding response in marine invertebrates, was down-regulated. We are able to report for the first time that sound sensory system proteins are highly abundant in the nauplius VI stage, implying that these proteins are good targets for the development of new antifouling compounds. The results presented here together with the new genome-wide datasets for a non-model specie represent an important resource for the study of biofouling and development. Proteomics data are available via ProteomeXchange with identifier PXD004679.

  4. Transcriptome and proteome studies reveal candidate attachment genes during the development of the barnacle Amphibalanus Amphitrite

    Directory of Open Access Journals (Sweden)

    Sarah Al-Aqeel

    2016-09-01

    Full Text Available The acorn barnacle, Balanus amphitrite, is the main biofouling organism in marine environments. In the present study we profiled the transcriptome and proteome of B. amphitrite at different life stages (nauplius II, nauplius VI and cyprid from the Red Sea, where the average water surface temperature is 34°C and the salinity reaches 41‰. We identified 65,784 expressed contigs, and a total of 1,387 expressed proteins measured by quantitative proteomics. We found that osmotic stress, salt stress, hyperosmotic response and the Wnt signaling pathway were strongly up-regulated during the planktonic stage, while the MAPK pathway, lipid metabolism, and cuticle development genes were down-regulated. In the transition stage between the nauplius VI and the cyprid, genes that are involved in blood coagulation, cuticle development and eggshell formation were highly up-regulated, while the nitric oxide pathway, which stimulates the swimming and feeding response in marine invertebrates, was down-regulated. We are able to report for the first time that sound sensory system proteins are highly abundant in the nauplius VI stage, implying that these proteins are good targets for the development of new antifouling compounds. The results presented here together with the new genome-wide datasets for a non-model specie represent an important resource for the study of biofouling and development.

  5. 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.

    collected at different tidal levels was observed. Enzyme activity increased with increase in the duration of tidal exposure, suggesting a capability to adapt to stress in different niches. Nauplii showed antioxidant defense (increased enzyme activity) under...

  6. 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 ...

  7. 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.

    of Phycology, 23(3), 434-441. Ianora, A., A. Miralto (2010) Toxigenic effects of diatoms on grazers, phytoplankton and other microbes: a review. Ecotoxicology, 19(3), 493-511. Jackson, D., S.P. Leys, V.F. Hinman, R. Woods, M.F. Lavin, B.M. Degnan (2002...

  8. 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.

    obligatory cross-fertilizing hermaphrodite, self-fertilization was observed. The rise in temperature from 20 to 30°C resulted in a longer interbreeding interval (6 to 7 days, 200 Artemia ind-1 day-1; 11 to 13 days, 50 Artemia ind-1 day-1). Computed carbon...

  9. 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.

    dark photoperiod. They were monitored for a period of 24h, cyprids that had metamorphosed into juvenile barnacles were counted. Treatment of cyprids with sugars (monosaccharides and disaccharides) The cyprids were immersed for 5min (Neal & Yule, 1996... maintained and monitored as described above. Treatment of cyprids with Concanavalin A In order to show that sugar is bound to cyprid antennules, the cyprids were stained using an FITC-conjugated lectin, Concanavalin A, which binds specifically to D...

  10. 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.

    onto the antennular attachment disc (Nott and Foster, 1969). The exploratory behavior and subsequent settlement of cyprid when subjected simultaneously to sugars and adult extract (AE) showed that sugar-treated cyprids did not deposit foot...-prints in the absence of AE. However, sugar-treated cyprids deposited foot-prints when exposed to adult extract (Khandeparker et al., 2002a). As earlier hypothesized (Yule and Walker, 1987), that sugars in solution adsorb electrostatically through –OH groups to polar...

  11. 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.

    to carry out the assays. Rittschof et al. (1984) have described these methods in detail. 14 2.2.3 Treatment with sugars The cyprids were immersed in seawater containing different concentrations (10-8, 10-7, 10-5 or 10-3M) of D-glucose, D-galactose or D... 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...

  12. 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.

    ) was examined. The mortality rate at 20 degrees C was lower than at 30 degrees C in general. INcrease in food concentration from 0.5 to 1 x 10 sup(5) cells ml sup(-1) . The results indicate that food availability and temperature jointly determine the energy...

  13. 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

    Studies on the settlement and the subsequent recruitment of intertidal organisms are crucial steps in understanding their population structure in a particular bioregion. However, studying the recruitment of intertidal organisms such as barnacles...

  14. Recyclable plastics as substrata for settlement and growth of bryozoans Bugula neritina and barnacles Amphibalanus amphitrite.

    Science.gov (United States)

    Li, Heng-Xiang; Orihuela, Beatriz; Zhu, Mei; Rittschof, Daniel

    2016-11-01

    Plastics are common and pervasive anthropogenic debris in marine environments. Floating plastics provide opportunities to alter the abundance, distribution and invasion potential of sessile organisms that colonize them. We selected plastics from seven recycle categories and quantified settlement of (i) bryozoans Bugula neritina (Linnaeus, 1758) in the lab and in the field, and of (ii) barnacles Amphibalanus (= Balanus) amphitrite (Darwin, 1854) in the field. In the laboratory we cultured barnacles on the plastics for 8 weeks and quantified growth, mortality, and breaking strength of the side plates. In the field all recyclable plastics were settlement substrata for bryozoans and barnacles. Settlement depended on the type of plastic. Fewer barnacles settled on plastic surfaces compared to glass. In the lab and in the field, bryozoan settlement was higher on plastics than on glass. In static laboratory rearing, barnacles growing on plastics were initially significantly smaller than on glass. This suggested juvenile barnacles were adversely impacted by materials leaching from the plastics. Barnacle mortality was not significantly different between plastic and glass surfaces, but breaking strength of side plates of barnacles on polyvinyl chloride (PVC) and polycarbonate (PC) were significantly lower than breakage strength on glass. Plastics impact marine ecosystems directly by providing new surfaces for colonization with fouling organisms and by contaminants shown previously to leach out of plastics and impact biological processes.

  15. 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.

  16. 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; Anil, A.C.

    in monsoon and late pre-monsoon months (June 1998 to August 1998 and April 1999 to September 1999). The results of the dendrogram to identify the pattern in the monthly abundance of cirripede larvae showed three clusters (Figure 1D&I). The first peak... during September 1998 and November 1998 formed one cluster, followed by second peak during October 1998 and March 1999. Rest of the months, which had less number of larvae, grouped separately (Figure 1D&I). Reproduction The percentages of individuals...

  17. 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.

    , the lower molecular-weight fraction of the culture supernatant showed maxi- mum inhibition when the adsorbed (surface-bound) compounds were assessed along with the leachants. Bacterial extract showed the presence of ketonic compounds, and its influence... inhibitory. Fourier transformed infrared spectroscopy analysis revealed that bacteria grown in different nutrient media yielded culture supernatants with different chemical composition, thus altering their ability to induce metamorphosis of cyprids. Maxi- mum...

  18. 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 metamorphosis rate. The interaction between these two variables, however, did not change in any combination, irrespective of the bacterial strain used. The Pseudomonas epm promoted metamorphosis at 15 ppt, but was inhibitory at 45 ppt. The Acinetobacter epm...

  19. 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.

    , antifungal, antimalarial, antituberculosis and anti rat brain nitric oxide synthase activities (Venkateswarlu et al., 1994; Rao et al., 1998; Orabi et al., 2002; Liu et al., 2004); Oceanapia sp. and Iotrochota sp. are known for cytotoxicity (Venkateswarlu...., Fusetani, N. 2010. Natural products as antifouling compounds: recent progress and future perspectives. Biofouling. 26, 223–234. 22. Rao, V.J., Desaiah, D., Vig, P., Venkateswarlu, Y., 1998. Marine biomolecules inhibit rat brain nitric oxide synthase...

  20. 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 w...

  1. Marine biofouling field tests, settlement assay and footprint micromorphology of cyprid larvae of Balanus amphitrite on model surfaces

    NARCIS (Netherlands)

    Phang, In Yee; Chaw, Kuan Chun; Choo, Sue Sok Hui; Kang, Ryan Kok Chuan; Lee, Serina Siew Chen; Birch, William R.; Teo, Serena Lay Ming; Vancso, G. Julius

    2009-01-01

    Atomic force microscopy (AFM), laboratory settlement assays and field tests were used to correlate cyprid footprint (FP) morphology with the behaviour of cyprids on different substrata. AFM imaging under laboratory conditions revealed more porous and larger FPs on glass exposing a CH3-surface than o

  2. 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.

    . Increase in rearing temperature and food concentration positively influenced the size of the larvae. Food concentration influenced the DNA of 3 and 4 naupliar instars and temperature influenced the DNA of 4 and 5 instar nauplii. The RNA content of 4...

  3. 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.

    that differences were present at lower taxonomic levels. The capacity of these communities to influence larval settlement was assessed by growing multispecies biofilms on artificial medium, obtained by extracting nutrients from adult barnacles. Biofilms composed...

  4. Marine biofouling field tests, settlement assay and footprint micromorphology of cyprid larvae of Balanus amphitrite on model surfaces.

    Science.gov (United States)

    Phang, In Yee; Chaw, Kuan Chun; Choo, Sue Sok Hui; Kang, Ryan Kok Chuan; Lee, Serina Siew Chen; Birch, William R; Teo, Serena Lay Ming; Vancso, G Julius

    2009-01-01

    Atomic force microscopy (AFM), laboratory settlement assays and field tests were used to correlate cyprid footprint (FP) morphology with the behaviour of cyprids on different substrata. AFM imaging under laboratory conditions revealed more porous and larger FPs on glass exposing a CH3-surface than on aminosilane functionalised (NH2-) surfaces. The secreted FP volume was found to be similar on both substrata (2.1-2.6 microm(3)). Laboratory settlement assays and marine field tests were performed on three substrata, viz. untreated clean glass, NH2-glass, and CH3-glass. The results distinguished settlement preferences for NH2-glass and untreated glass over CH3-terminated surfaces, suggesting that cyprids favour settling on hydrophilic over hydrophobic surfaces. On combining observations from different length scales, it is speculated that the confined FP size on NH2-glass may induce a higher concentration of the settlement inducing protein complex. Settlement may be further facilitated by a stronger adherence of FP adhesives to the NH2-surface via Coulombic interactions.

  5. 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.

    selected based on present day pCO2 (~400 µatm) and predicted concentration for the year 2100 (~750 µatm) (Stocker et al., 2013). Four treatments were used in this study: (1) Control treatment - ambient temperature (~30 °C) and ambient pCO2 (~400 µatm); (2...) Elevated pCO2 treatment - ambient temperature (~30 °C) and elevated pCO2 (~750 µatm); (3) Elevated temperature treatment - elevated temperature (~34 °C) and ambient pCO2 (~400 µatm); and (4) Synergistic treatment - elevated temperature (~34 °C...

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Preliminary results on the anatomy of the larval musculature of Balanus improvisus (Darwin, 1854) (Crustacea: Cirripedia: Thecostraca) using phalloidin staining in combination with confocal laserscanning microscopy

    DEFF Research Database (Denmark)

    Semmler, Henrike; Høeg, Jens Thorvald; Scholtz, Gerhard;

    2006-01-01

    The anatomy of the larval muscular systems in Balanus improvisus (Darwin, 1854) was investigated by using phalloidin staining to visualize filamentous F-actin in combination with confocal laser scanning microscopy (CLSM). The larval musculature contains an anterior muscle complex associated with ...

  13. Nonmicrobial Nitrophenol Degradation via Peroxygenase Activity of Dehaloperoxidase-Hemoglobin from Amphitrite ornata.

    Science.gov (United States)

    McCombs, Nikolette L; D'Antonio, Jennifer; Barrios, David A; Carey, Leiah M; Ghiladi, Reza A

    2016-05-01

    The marine hemoglobin dehaloperoxidase (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of nitrophenols, an unprecedented nonmicrobial degradation pathway for nitrophenols by a hemoglobin. Using 4-nitrophenol (4-NP) as a representative substrate, the major monooxygenated product was 4-nitrocatechol (4-NC). Isotope labeling studies confirmed that the O atom incorporated was derived exclusively from H2O2, indicative of a peroxygenase mechanism for 4-NP oxidation. Accordingly, X-ray crystal structures of 4-NP (1.87 Å) and 4-NC (1.98 Å) bound to DHP revealed a binding site in close proximity to the heme cofactor. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. The 4-NC product was itself a peroxidase substrate for DHP, leading to the secondary products 5-nitrobenzene-triol and hydroxy-5-nitro-1,2-benzoquinone. DHP was able to react with 2,4-dinitrophenol (2,4-DNP) but was unreactive against 2,4,6-trinitrophenol (2,4,6-TNP). pH dependence studies demonstrated increased reactivity at lower pH for both 4-NP and 2,4-DNP, suggestive of a pH effect that precludes the reaction with 2,4,6-TNP at or near physiological conditions. Stopped-flow UV-visible spectroscopic studies strongly implicate a role for Compound I in the mechanism of 4-NP oxidation. The results demonstrate that there may be a much larger number of nonmicrobial enzymes that are underrepresented when it comes to understanding the degradation of persistent organic pollutants such as nitrophenols in the environment.

  14. Nocturnal Ozone Depletion Events at the Amphitrite Point Observatory on West Vancouver Island

    Science.gov (United States)

    Garner, N.; Brownsey, D. K.; Tokarek, T. W.; Ye, C. Z.; Yordanov, N. R.; Osthoff, H. D.; Schiller, C. L.; Vingarzan, R.

    2015-12-01

    Routine monitoring stations on the West coast of North America serve to monitor baseline levels of criteria pollutants such as ozone (O3) arriving from the Pacific Ocean. In Canada, the Amphitrite Point Observatory (APO) in Ucluelet on the West coast of Vancouver Island has been added to this network to provide regional baseline measurements. Recently, McKendry and coworkers have reported frequent episodes of nocturnal O3 depletion events (ODEs) at APO (range: 5-20 ppbv) that generally correlate with alongshore winds, elevated levels of carbon dioxide (CO2), and low vertical entrainment but whose cause(s) has (have) remained unclear. In this work, results from the Ozone-depleting reactions in a coastal atmosphere (ORCA) campaign, which took place at APO from July 6 - 31, 2015, are presented. In addition to the long-term measurements that include aerosol size distribution and composition measurements, mixing ratios of speciated monoterpenes (e.g., α- and β-pinene, limonene), molecular halogens (i.e., Cl2, I2), halogen oxides (i.e., OIO), plus a full suite of nitrogen oxides (including N2O5, PAN, PPN, ΣPN, ΣAN, HNO3, HONO, and ClNO2) were quantified. Synoptic conditions at the site varied greatly between nights. During westerly flow of relatively clean marine air, O3 was generally conserved at night, indicating that deposition of O3 to the ocean surface is a minor loss pathway. When the air mass originated from other sectors, episodes of nocturnal ODEs were observed on several occasions, in which mixing ratios of biogenic VOCs were enhanced. These included air masses that originated from densely forested areas to the East, air masses polluted by marine traffic emissions from the southeast, and air masses from the NW that have traveled parallel to the coastline. In this sector, the air was likely in contact with terrestrial vegetation via land-sea breeze circulations. The results suggest that nocturnal ODEs at APO are mainly driven by local or regional processes

  15. Coumarins from the Herb Cnidium monnieri and Chemically Modified Derivatives as Antifoulants against Balanus albicostatus and Bugula neritina Larvae

    Directory of Open Access Journals (Sweden)

    Zhan-Chang Wang

    2013-01-01

    Full Text Available In the search for new environmental friendly antifouling (AF agents, four coumarins were isolated from the herbal plant Cnidium monnieri, known as osthole (1, imperatorin (2, isopimpinellin (3 and auraptenol (4. Furthermore, five coumarin derivatives, namely 8-epoxypentylcoumarin (5, meranzin hydrate (6, 2'-deoxymetranzin hydrate (7, 8-methylbutenalcoumarin (8, and micromarin-F (9 were synthesized from osthole. Compounds 1, 2, 4, 7 showed high inhibitory activities against larval settlement of Balanus albicostatus with EC50 values of 4.64, 3.39, 3.38, 4.67 μg mL−1. Compound 8 could significantly inhibit larval settlement of Bugula neritina with an EC50 value of 3.87 μg mL−1. The impact of functional groups on anti-larval settlement activities suggested that the groups on C-5' and C-2'/C-3' of isoamylene chian could affect the AF activities.

  16. Gut fluorescence analysis of barnacle larvae: An approach to quantify the ingested food

    Digital Repository Service at National Institute of Oceanography (India)

    Gaonkar, C.A; Anil, A

    Gut fluorescence analysis can provide a snapshot of ingested food and has been employed in feeding studies of various organisms. In this study we standardized the gut fluorescence method using laboratory-reared barnacle larvae (Balanus amphitrite...

  17. Three-dimensional reconstruction of the naupliar musculature and a scanning electron microscopy atlas of nauplius development of Balanus improvisus (Crustacea

    DEFF Research Database (Denmark)

    Semmler, Henrike; Høeg, Jens Thorvald; Scholtz, Gerhard

    2009-01-01

    An atlas of the naupliar development of the cirripede Balanus improvisus Darwin, 1854 using scanning electron microscopy (SEM) is provided. Existing spikes on the hindbody increase in number with each moult and are an applicable character for identification of the different nauplius stages......, as is the setation pattern of the first antennae. The naupliar musculature of B. improvisus was stained with phalloidin to visualise F-actin, followed by analysis using confocal laser scanning microscopy (CLSM) with subsequent application of 3D imaging software. The larval musculature is already fully established...

  18. Investigating a possible role for the bacterial signal molecules N-acylhomoserine lactones in Balanus improvisus cyprid settlement.

    Science.gov (United States)

    Tait, Karen; Havenhand, Jon

    2013-05-01

    Increased settlement on bacterial biofilms has been demonstrated for a number of marine invertebrate larvae, but the nature of the cue(s) responsible is not well understood. We tested the hypothesis that the bay barnacle Balanus improvisus utilizes the bacterial signal molecules N-acylhomoserine lactones (AHLs) as a cue for the selection of sites for permanent attachment. Single species biofilms of the AHL-producing bacteria Vibrio anguillarum, Aeromonas hydrophila and Sulfitobacter sp. BR1 were attractive to settling cypris larvae of B. improvisus. However, when AHL production was inactivated, either by mutation of the AHL synthetic genes or by expression of an AHL-degrading gene (aiiA), the ability of the bacteria to attract cyprids was abolished. In addition, cyprids actively explored biofilms of E. coli expressing the recombinant AHL synthase genes luxI from Vibrio fischeri (3-oxo-C6-HSL), rhlI from Pseudomonas aeruginosa (C4-HSL/C6-HSL), vanI from V. anguillarum (3-oxo-C10-HSL) and sulI from Sulfitobacter sp. BR1 (C4-HSL, 3-hydroxy-C6-HSL, C8-HSL and 3-hydroxy-C10-HSL), but not E. coli that did not produce AHLs. Finally, synthetic AHLs (C8-HSL, 3-oxo-C10-HSL and C12-HSL) at concentrations similar to those found within natural biofilms (5 μm) resulted in increased cyprid settlement. Thus, B. improvisus cypris exploration of and settlement on biofilms appears to be mediated by AHL-signalling bacteria in the laboratory. This adds to our understanding of how quorum sensing inhibition may be used as for biofouling control. Nonetheless, the significance of our results for larvae settling naturally in the field, and the mechanisms that underlay the observed responses to AHLs, is as yet unknown.

  19. The Story of a Hitchhiker: Population Genetic Patterns in the Invasive Barnacle Balanus(Amphibalanus improvisus Darwin 1854.

    Directory of Open Access Journals (Sweden)

    Anna-Lisa Wrange

    Full Text Available Understanding the ecological and evolutionary forces that determine the genetic structure and spread of invasive species is a key component of invasion biology. The bay barnacle, Balanus improvisus (= Amphibalanus improvisus, is one of the most successful aquatic invaders worldwide, and is characterised by broad environmental tolerance. Although the species can spread through natural larval dispersal, human-mediated transport through (primarily shipping has almost certainly contributed to the current global distribution of this species. Despite its worldwide distribution, little is known about the phylogeography of this species. Here, we characterize the population genetic structure and model dispersal dynamics of the barnacle B. improvisus, and describe how human-mediated spreading via shipping as well as natural larval dispersal may have contributed to observed genetic variation. We used both mitochondrial DNA (cytochrome c oxidase subunit I: COI and nuclear microsatellites to characterize the genetic structure in 14 populations of B. improvisus on a global and regional scale (Baltic Sea. Genetic diversity was high in most populations, and many haplotypes were shared among populations on a global scale, indicating that long-distance dispersal (presumably through shipping and other anthropogenic activities has played an important role in shaping the population genetic structure of this cosmopolitan species. We could not clearly confirm prior claims that B. improvisus originates from the western margins of the Atlantic coasts; although there were indications that Argentina could be part of a native region. In addition to dispersal via shipping, we show that natural larval dispersal may play an important role for further colonisation following initial introduction.

  20. 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.

  1. 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 ...

  2. Coliform accumulation in Amphibalanus amphitrite (Darwin, 1854 (Cirripedia and its use as an organic pollution bioindicator in the estuarine area of Recife, Pernambuco, Brazil

    Directory of Open Access Journals (Sweden)

    CMR Farrapeira

    Full Text Available Samples of water and barnacles Amphibalanus amphitrite were collected from Recife, Brazil, to assess if it accumulates total (TC and thermotolerant coliforms (TTC related with sewage pollution. The Most Probable Number (MPN values and the standard procedures for examination of shellfish were used. Comparatively with the water samples, the highest coliform values came from the barnacles, with TC values ranging from 2.4 × 10(6 MPN.g-1, and TTC ranging from > 2.4 × 10³ to 2.9 × 10(5 MPN.g-1. Barnacles accumulate the TC Ewingella americana, and the TTC Escherichia coli, Enterobacter gergoviae, Enterobacter aerogenes, and Enterobacter sakazakii. The results provided an indication of the level of organic contamination at the sampling locations and that this species could be a good organic pollution bioindicator.

  3. 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.

  4. 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.

  5. Multi-seasonal barnacle (Balanus improvisus) protection achieved by trace amounts of a macrocyclic lactone (ivermectin) included in rosin-based coatings.

    Science.gov (United States)

    Pinori, Emiliano; Berglin, Mattias; Brive, Lena M; Hulander, Mats; Dahlström, Mia; Elwing, Hans

    2011-10-01

    Rosin-based coatings loaded with 0.1% (w/v) ivermectin were found to be effective in preventing colonization by barnacles (Balanus improvisus) both on test panels as well as on yachts for at least two fouling seasons. The leaching rate of ivermectin was determined by mass-spectroscopy (LC/MS-MS) to be 0.7 ng cm(-2) day(-1). This low leaching rate, as deduced from the Higuchi model, is a result of the low loading, low water solubility, high affinity to the matrix and high molar volume of the model biocide. Comparison of ivermectin and control areas of panels immersed in the field showed undisturbed colonisation of barnacles after immersion for 35 days. After 73 days the mean barnacle base plate area on the controls was 13 mm(2), while on the ivermectin coating it was 3 mm(2). After 388 days, no barnacles were observed on the ivermectin coating while the barnacles on the control coating had reached a mean of 60 mm(2). In another series of coated panels, ivermectin was dissolved in a cosolvent mixture of propylene glycol and glycerol formal prior to the addition to the paint base. This method further improved the anti-barnacle performance of the coatings. An increased release rate (3 ng cm(-2) day(-1)) and dispersion of ivermectin, determined by fluorescence microscopy, and decreased hardness of the coatings were the consequences of the cosolvent mixture in the paint. The antifouling mechanism of macrocyclic lactones, such as avermectins, needs to be clarified in further studies. Beside chronic intoxication as ivermectin is slowly released from the paint film even contact intoxication occurring inside the coatings, triggered by penetration of the coating by barnacles, is a possible explanation for the mode of action and this is under investigation.

  6. 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.

  7. 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...... 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...

  8. 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

  9. Differential cDNA cloning by enzymatic degrading subtraction (EDS).

    OpenAIRE

    1994-01-01

    We describe a new method, called enzymatic degrading subtraction (EDS), for the construction of subtractive libraries from PCR amplified cDNA. The novel features of this method are that i) the tester DNA is blocked by thionucleotide incorporation; ii) the rate of hybridization is accelerated by phenol-emulsion reassociation; and iii) the driver cDNA and hybrid molecules are enzymatically removed by digestion with exonucleases III and VII rather than by physical partitioning. We demonstrate th...

  10. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    1994-01-04

    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. GOVERNMENT RIGHTS This application was funded under Department of Energy Contract DE-AC02-76ER01338. The U.S. Government has certain rights under this application and any patent issuing thereon.

  11. 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.

  12. 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.

  13. 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.

  14. Preparation of cDNA libraries from vascular cells.

    Science.gov (United States)

    Lieb, M E; Taubman, M B

    1999-01-01

    The vast majority of past and present efforts in the molecular cloning of expressed sequences involve isolation of clones from cDNA libraries constructed in bacteriophage lambda (1,2). As discussed in Chapter 6 , screening these cDNA libraries using labeled probes remains the most straightforward method to isolate full length cDNAs for which some partial sequence information is known. Although the availability of high quality reagents and kits over the past decade has made the process of library construction increasingly straightforward, generation of high-quality libraries is a task that still requires a fair amount of dedicated effort. Because alternative PCR-based cloning strategies have become increasingly popular alternatives to cDNA library screening, it is useful to consider the advantages and disadvantages of each strategy before embarking on a project to construct a cDNA library (Table 1). In our opinion, it is worthwhile to construct a cDNA library when the transcript of interest is not exceedingly rare (i.e., can readily be detected by Northern blot analysis of total RNA), when multiple cDNAs will need to be cloned over a period of time, and in situations where occasional mutations can not be tolerated (for example, if the cDNA is to be expressed in mammalian cells to examine function). In situations where the transcript of interest is expressed at exceedingly low levels, or when only a single cDNA needs to be cloned, a PCR-based strategy should be considered. When the tissue source is precious (such as a unique clinical specimen), successful construction of a phage library provides a resource that can be amplified and used for multiple cloning projects over many years, but runs the risk of consuming the available RNA if the library construction fails. Table 1 Comparison of Relative Advantages of cDNA Cloning from Lambda Phage Libraries by Plaque Hybridization Compared to Newer PCR- Based Strategies Lambda phage cDNA library PCR-based strategy Freedom

  15. Procedure for normalization of cDNA libraries

    Science.gov (United States)

    Bonaldo, Maria DeFatima; Soares, Marcelo Bento

    1997-01-01

    This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

  16. Rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Yeku, Oladapo; Frohman, Michael A

    2011-01-01

    Rapid Amplification of cDNA ends (RACE) provides an inexpensive and powerful tool to quickly obtain full-length cDNA when the sequence is only partially known. Starting with an mRNA mixture, gene-specific primers generated from the known regions of the gene and non-specific anchors, full-length sequences can be identified in as little as 3 days. RACE can also be used to identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for rapid amplification of 5(') and 3(') cDNA ends using the "new RACE" technique.

  17. 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.

  18. The Cirripedia of Trinidad

    NARCIS (Netherlands)

    Bacon, Peter R.

    1976-01-01

    The cirripede fauna of the island of Trinidad has been little investigated. SOUTHWARD (1962) recorded five species collected in north-west Trinidad for experimental studies; he listed Chthamalus fragilis Darwin on mangroves, Balanus amphitrite var. on mangroves and harbour piles, B. tintinnabulum Li

  19. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    Science.gov (United States)

    A Bioinformatic Strategy to Rapidly Characterize cDNA LibrariesG. Charles Ostermeier1, David J. Dix2 and Stephen A. Krawetz1.1Departments of Obstetrics and Gynecology, Center for Molecular Medicine and Genetics, & Institute for Scientific Computing, Wayne State Univer...

  20. Rescue of mumps virus from cDNA.

    Science.gov (United States)

    Clarke, D K; Sidhu, M S; Johnson, J E; Udem, S A

    2000-05-01

    A complete DNA copy of the genome of a Jeryl Lynn strain of mumps virus (15,384 nucleotides) was assembled from cDNA fragments such that an exact antigenome RNA could be generated following transcription by T7 RNA polymerase and cleavage by hepatitis delta virus ribozyme. The plasmid containing the genome sequence, together with support plasmids which express mumps virus NP, P, and L proteins under control of the T7 RNA polymerase promoter, were transfected into A549 cells previously infected with recombinant vaccinia virus (MVA-T7) that expressed T7 RNA polymerase. Rescue of infectious virus from the genome cDNA was demonstrated by amplification of mumps virus from transfected-cell cultures and by subsequent consensus sequencing of reverse transcription-PCR products generated from infected-cell RNA to verify the presence of specific nucleotide tags introduced into the genome cDNA clone. The only coding change (position 8502, A to G) in the cDNA clone relative to the consensus sequence of the Jeryl Lynn plaque isolate from which it was derived, resulting in a lysine-to-arginine substitution at amino acid 22 of the L protein, did not prevent rescue of mumps virus, even though an amino acid alignment for the L proteins of paramyxoviruses indicates that lysine is highly conserved at that position. This system may provide the basis of a safe and effective virus vector for the in vivo expression of immunologically and biologically active proteins, peptides, and RNAs.

  1. Characterization of the porcine carboxypeptidase E cDNA

    DEFF Research Database (Denmark)

    Hreidarsdôttir, G.E.; Cirera, Susanna; Fredholm, Merete

    2007-01-01

    Carboxypeptidase E (CPE) is an important enzyme responsible for the proteolytic processing of prohormone intermediates. A naturally occurring point mutation, leading to an accumulation of many neuroendocrine peptides has been characterized within exon 5 of the CPE gene in mice. In the present study...... the sequence of the cDNA for the porcine CPE gene including all the coding region and the 3'-UTR region was generated. Comparisons with bovine, human, mouse, and rat CPE cDNA sequences showed that the coding regions of the gene are highly conserved both at the nucleotide and at the amino acid level. A very low...... nonsynonymousl/synonymous substitution ratios between the proteins was found indicating that purifying selection os acting on the CPE gene. A nonsynonymous SNP identified at position 1272 in the transcript resulting in a codon change from TCA (Serine) to TTA (Leucine) was genotyped in the Danish pig populations...

  2. 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.

  3. Construction of a rice immature seeds cDNA library and molecular cloning of oryzacystatin cDNA

    Institute of Scientific and Technical Information of China (English)

    周兆斓; 朱祯; 刘春明; 张海涛; 肖桂芳; 李向辉

    1996-01-01

    Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5’-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5’-end non-coding region and a poly(A) signal AATAAA at the 3’-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3’ region after poly(A) signal.

  4. cDNA library information - Dicty_cDB | 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 Dicty_cDB cDNA library... information Data detail Data name cDNA library information Description of data conten...d - Data analysis method - Number of data entries 14 entries Data item Description cDNA library name Names o...(Slug phase)(S) 4) culminating stages (Morphogenetic phase)(C) 5) sexually fusion-competent KAX3 cells (Gamete phase) (F) cDNA librar...y construction method How to construct cDNA library 1) C

  5. [cDNA library construction from panicle meristem of finger millet].

    Science.gov (United States)

    Radchuk, V; Pirko, Ia V; Isaenkov, S V; Emets, A I; Blium, Ia B

    2014-01-01

    The protocol for production of full-size cDNA using SuperScript Full-Length cDNA Library Construction Kit II (Invitrogen) was tested and high quality cDNA library from meristematic tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was created. The titer of obtained cDNA library comprised 3.01 x 10(5) CFU/ml in avarage. In average the length of cDNA insertion consisted about 1070 base pairs, the effectivity of cDNA fragment insertions--99.5%. The selective sequencing of cDNA clones from created library was performed. The sequences of cDNA clones were identified with usage of BLAST-search. The results of cDNA library analysis and selective sequencing represents prove good functionality and full length character of inserted cDNA clones. Obtained cDNA library from meristematic tissue of finger millet panicle represents good and valuable source for isolation and identification of key genes regulating metabolism and meristematic development and for mining of new molecular markers to conduct out high quality genetic investigations and molecular breeding as well.

  6. Toward a cDNA map of the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Korenberg, J.R.; Chen, X.N. [Cedars-Sinai Research Institute, Los Angeles, CA (United States); Adams, M.D.; Venter, J.C. [Institute for Genomic Research, Gaithersburg, MD (United States)

    1995-09-20

    Advances in the Human Genome Project are shaping the strategies for identifying the 50,000-100,000 human genes. High-resolution genetic maps of the human genome combined with sequencing herald an era of rapid regional definition of disease genes. However, only once their chromosomes band location is known will the systematic partial sequencing of thousands of random cDNA clones provide the reagents for the rapid assessment of the genes responsible for the inherited disorders. We now present an approach to the rapid determination of map position and therefore to the creation of a transcribed map of the human genome. Sensitive fluorescence in situ hybridization has been combined with high-resolution chromosome banding and random cDNA sequencing to 41 cDNAs with an average insert size of < 2 kb to single human chromosome bands. The results provide 15 new genes, with database and functional information, as candidates for human disease. These include the large extracellular single-related kinase (HUMERK), the ERK activator kinase (PRKMK1), a new member of the RAS oncogene family, protein phosphotase 2 regulatory subunit B alpha isoform (PPP2R2A), and a novel human gene with very high homology to a plant membrane transport family. Further, an analysis of expressed genes associated with pseudogenes showed that by using these techniques, it is possible to detect accurately the transcribed locus within a multigene or processed pseudogene family in most cases. These findings suggest that direct cDNA mapping using fluorescence in situ hybridization provides an accurate and rapid approach to the definition of a transcribed map of the human genome. This low-cost, high-resolution (205 Mb) mapping greatly enhances the speed with which these genes can be subsequently assigned to contigs. This assignment provides a necessary first step in understanding the relationship of the genes to both acquired and inherited human diseases. 16 refs., 1 fig., 3 tabs.

  7. 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.

  8. Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends.

    Science.gov (United States)

    Schaefer, B C

    1995-05-20

    Rapid amplification of cDNA ends (RACE) is a polymerase chain reaction (PCR)-based technique which was developed to facilitate the cloning of full-length cDNA 5'- and 3'-ends after a partial cDNA sequence has been obtained by other methods. While RACE can yield complete sequences of cDNA ends in only a few days, the RACE procedure frequently results in the exclusive amplification of truncated cDNA ends, undermining efforts to generate full-length clones. Many investigators have suggested modifications to the RACE protocol to improve the effectiveness of the technique. Based on first-hand experience with RACE, a critical review of numerous published variations of the key steps in the RACE method is presented. Also included is a detailed, effective protocol based on RNA ligase-mediated RACE/reverse ligation-mediated PCR, as well as a demonstration of its utility.

  9. Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Watanabe, Kousuke; Emoto, Noriko; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2011-07-01

    Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.

  10. A drosophila full-length cDNA resource

    Energy Technology Data Exchange (ETDEWEB)

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

    2003-05-09

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

  11. The mechanism of oxidative halophenol dehalogenation by Amphitrite ornata dehaloperoxidase is initiated by H2O2 binding and involves two consecutive one-electron steps: role of ferryl intermediates.

    Science.gov (United States)

    Osborne, Robert L; Coggins, Michael K; Raner, Gregory M; Walla, Mike; Dawson, John H

    2009-05-26

    The enzymatic globin, dehaloperoxidase (DHP), from the terebellid polychaete Amphitrite ornata is designed to catalyze the oxidative dehalogenation of halophenol substrates. In this study, the ability of DHP to catalyze this reaction by a mechanism involving two consecutive one-electron steps via the normal order of addition of the oxidant cosubstrate (H(2)O(2)) before organic substrate [2,4,6-trichlorophenol (TCP)] is demonstrated. Specifically, 1 equiv of H(2)O(2) will fully convert 1 equiv of TCP to 2,6-dichloro-1,4-benzoquinone, implicating the role of multiple ferryl [Fe(IV)O] species. A significant amount of heterolytic cleavage of the O-O bond of cumene hydroperoxide, consistent with transient formation of a Compound I [Fe(IV)O/porphyrin pi-cation radical] species, is observed upon its reaction with ferric DHP. In addition, a more stable high-valent Fe(IV)O-containing DHP intermediate [Compound II (Cpd II) or Compound ES] is characterized by UV-visible absorption and magnetic circular dichroism spectroscopy. Spectral similarities are seen between this intermediate and horse heart myoglobin Cpd II. It is also shown in single-turnover experiments that the DHP Fe(IV)O intermediate is an active oxidant in halophenol oxidative dehalogenation. Furthermore, reaction of DHP with 4-chlorophenol leads to a dimeric product. The results presented herein are consistent with a normal peroxidase order of addition of the oxidant cosubstrate (H(2)O(2)) followed by organic substrate (TCP) and indicate that the enzymatic mechanism of DHP-catalyzed oxidative halophenol dehalogenation involves two consecutive one-electron steps with a dissociable radical intermediate.

  12. Evidence of the direct involvement of the substrate TCP radical in functional switching from oxyferrous O2 carrier to ferric peroxidase in the dual-function hemoglobin/dehaloperoxidase from Amphitrite ornata.

    Science.gov (United States)

    Sun, Shengfang; Sono, Masanori; Du, Jing; Dawson, John H

    2014-08-05

    The coelomic O2-binding hemoglobin dehaloperoxidase (DHP) from the sea worm Amphitrite ornata is a dual-function heme protein that also possesses a peroxidase activity. Two different starting oxidation states are required for reversible O2 binding (ferrous) and peroxidase (ferric) activity, bringing into question how DHP manages the two functions. In our previous study, the copresence of substrate 2,4,6-trichlorophenol (TCP) and H2O2 was found to be essential for the conversion of oxy-DHP to enzymatically active ferric DHP. On the basis of that study, a functional switching mechanism involving substrate radicals (TCP(•)) was proposed. To further support this mechanism, herein we report details of our investigations into the H2O2-mediated conversion of oxy-DHP to the ferric or ferryl ([TCP] TCP and 4-bromophenol (4-BP)] and nonrelevant (ferrocyanide) compounds. At TCP/4-BP (but not ferrocyanide)-triggered conversion of oxy-DHP to ferric DHP. These results and O2 concentration-dependent conversion rates observed in this study demonstrate that substrate TCP triggers the conversion of oxy-DHP to a peroxidase by TCP(•) oxidation of the deoxyferrous state. TCP(•) is progressively generated, by increasingly produced amounts of ferric DHP, upon H2O2 oxidation of TCP catalyzed initially by trace amounts of ferric enzyme present in the oxy-DHP sample. The data presented herein further address the mechanism of how the halophenolic substrate triggers the conversion of hemoglobin DHP into a peroxidase.

  13. Rabbit muscle creatine phosphokinase. CDNA cloning, primary structure and detection of human homologues.

    Science.gov (United States)

    Putney, S; Herlihy, W; Royal, N; Pang, H; Aposhian, H V; Pickering, L; Belagaje, R; Biemann, K; Page, D; Kuby, S

    1984-12-10

    A cDNA library was constructed from rabbit muscle poly(A) RNA. Limited amino acid sequence information was obtained on rabbit muscle creatine phosphokinase and this was the basis for design and synthesis of two oligonucleotide probes complementary to a creatine kinase cDNA sequence which encodes a pentapeptide. Colony hybridizations with the probes and subsequent steps led to isolation of two clones, whose cDNA segments partially overlap and which together encode the entire protein. The primary structure was established from the sequence of two cDNA clones and from independently determined sequences of scattered portions of the polypeptide. The reactive cysteine has been located to position 282 within the 380 amino acid polypeptide. The rabbit cDNA hybridizes to digests of human chromosomal DNA. This reveals a restriction fragment length polymorphism associated with the human homologue(s) which hybridizes to the rabbit cDNA.

  14. Recognition of cDNA microarray image Using Feedforward artificial neural network

    OpenAIRE

    R. M. Farouk; E. M. Badr; M. A. SayedElahl

    2014-01-01

    The complementary DNA (cDNA) sequence is considered to be the magic biometric technique for personal identification. In this paper, we present a new method for cDNA recognition based on the artificial neural network (ANN). Microarray imaging is used for the concurrent identification of thousands of genes. 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 accurate intensity measurement, leading...

  15. 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...

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

    Science.gov (United States)

    Venugopal, T; Mathavan, S; Pandian, T J

    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.

  17. Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells.

    OpenAIRE

    1991-01-01

    The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10(-2)M N,N'-hexamethylene-bis-acetamide (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)30-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-t...

  18. 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.

  19. Preparation of microbial community cDNA for metatranscriptomic analysis in marine plankton.

    Science.gov (United States)

    Stewart, Frank J

    2013-01-01

    High-throughput sequencing and analysis of microbial community cDNA (metatranscriptomics) are providing valuable insight into in situ microbial activity and metabolism in the oceans. A critical first step in metatranscriptomic studies is the preparation of high-quality cDNA. At the minimum, preparing cDNA for sequencing involves steps of biomass collection, RNA preservation, total RNA extraction, and cDNA synthesis. Each of these steps may present unique challenges for marine microbial samples, particularly for deep-sea samples whose transcriptional profiles may change between water collection and RNA preservation. Because bacterioplankton community RNA yields may be relatively low (microbiology research.

  20. A method for generating subtractive cDNA libraries retaining clones containing repetitive elements.

    OpenAIRE

    1997-01-01

    Here we describe a two-stepped photobiotin-based procedure to enrich a target (canine retinal) cDNA library for tissue specific clones without removing those containing repetitive ( SINE ) elements, despite the presence of these elements in the driver population. In a first hybridization excess SINE elements were hybridized to a driver (canine cerebellar) cDNA. In a second hybridization target cDNA was added to this reaction. The resulting cDNA library was enriched for retinal specific clones...

  1. Lysosomal {beta}-mannosidase: cDNA cloning and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Leipprandt, J.R.; Traviss, C.E. [Michigan State Univ., East Lansing, MI (United States)] [and others

    1994-09-01

    Lysosomal {beta}-mannosidase is an exoglycosidase that cleaves the single {beta}-linked mannose residue from the non-reducing end of all N-linked glycoprotein oligosaccharides. Deficiency of this enzyme results in {beta}-mannosidosis, a severe neurodegenerative disease in goats and cattle. The human cases described have a milder, highly variable presentation. Study of the molecular pathology of this disease in ruminants and humans and development of the animal model for gene therapy studies required cloning of the gene for {beta}-mannosidase has been cloned. {beta}-Mannosidase cDNA were obtained from a bovine thyroid cDNA library by screening with mixed oligonucleotides derived from peptide sequences resulting from microsequencing of bovine {beta}-mannosidase peptides. A total of six independent positive clones were identified from 5 x 10{sup 5} plaques, covering about 80% of the C-terminal region. The missing 5{prime} region was obtained using 5{prime} RACE. The full-length construct contains 3852-bp nucleotides, encoding 879 amino acids. The initiation codon is followed by 17 amino acids containing the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrated a 4.2 kb single transcript in various tissues from both normal and affected goats and calves. The mRNA level was decreased in affected {beta}-mannosidosis animals. The gene encoding {beta}-mannosidase was localized on human chromosome 4 by Southern analysis of rodent/human somatic cell hybrids. The mutation in bovine {beta}-mannosidosis has been identified. This is the first report of cloning of the {beta}-mannosidase gene.

  2. 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.

  3. Application of an improved cDNA competition technique to identify prostate cancer-associated gene.

    Science.gov (United States)

    Rinaldy, A R; Steiner, M S

    1999-11-01

    A technique to improve cDNA library screening was developed by using mixed probes derived from two closely related cDNA populations of high-metastatic MAT-LyLu and low-metastatic AT-1 Dunning R3227 rat prostate cancer sublines. The technique required the generation of a cDNA library from each subline followed by polymerase chain reaction (PCR) amplification of the cDNA insert population. The PCR products derived from the first library were radiolabeled and mixed with an excess amount of PCR products from the second library. The mixture and an excess amount of both the lambda and pBluescript DNA were used as a probe to screen the first cDNA library. This mixed probe (designated the competition probe) differentially cross-hybridized with the plaque lift of the screened first cDNA library. Weak radioactive signals indicated the cross-hybridization of cDNA sequences common to the competition probe mixture and the first cDNA library, whereas strong signals implied unhybridized unique or abundant cDNA sequences in the first cDNA library. The reproducibility of this technique was confirmed by showing that the full-length cDNA clones were associated with the phenotype of the screened first cell line. The isolated clones were characterized as rat nucleolar protein, rat mitochondrial genes coding for 16S and 12S rRNAs, and rat tRNAs specific for valine and phenyl-alanine. This result is consistent with the fact that the first cell line, MAT-LyLu, is metabolically more active than are AT-1 cells because of higher gene dosage or amplification of nucleolar and mitochondrial RNA and its associated genes. Another clone which had a strong signal represented a novel gene associated with the MAT-LyLu cancer phenotype.

  4. Protein Aggregation Formed by Recombinant cp19k Homologue of Balanus albicostatus Combined with an 18 kDa N-Terminus Encoded by pET-32a(+ Plasmid Having Adhesion Strength Comparable to Several Commercial Glues.

    Directory of Open Access Journals (Sweden)

    Chao Liang

    Full Text Available The barnacle is well known for its tenacious and permanent attachment to a wide variety of underwater substrates, which is accomplished by synthesizing, secreting and curing a mixture of adhesive proteins termed "barnacle cement". In order to evaluate interfacial adhesion abilities of barnacle cement proteins, the cp19k homologous gene in Balanus albicostatus (Balcp19k was cloned and expressed in Escherichia coli. Here, we report an intriguing discovery of a gel-like super adhesive aggregation produced by Trx-Balcp19k, a recombinant Balcp19k fusion protein. The Trx-Balcp19k consists of an 18 kDa fragment at the N-terminus, which is encoded by pET-32a(+ plasmid and mainly comprised of a thioredoxin (Trx tag, and Balcp19k at the C-terminus. The sticky aggregation was designated as "Trx-Balcp19k gel", and the bulk adhesion strength, biochemical composition, as well as formation conditions were all carefully investigated. The Trx-Balcp19k gel exhibited strong adhesion strength of 2.10 ± 0.67 MPa, which was approximately fifty folds higher than that of the disaggregated Trx-Balcp19k (40 ± 8 kPa and rivaled those of commercial polyvinyl acetate (PVA craft glue (Mont Marte, Australia and UHU glue (UHU GmbH & Co. KG, Germany. Lipids were absent from the Trx-Balcp19k gel and only a trace amount of carbohydrates was detected. We postulate that the electrostatic interactions play a key role in the formation of Trx-Balcp19k gel, by mediating self-aggregation of Trx-Balcp19k based on its asymmetric distribution pattern of charged amino acids. Taken together, we believe that our discovery not only presents a promising biological adhesive with potential applications in both biomedical and technical fields, but also provides valuable paradigms for molecular design of bio-inspired peptide- or protein-based materials.

  5. 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.

  6. 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.

  7. Cloning of a cDNA encoding the smallest neurofilament protein from the rat

    NARCIS (Netherlands)

    J-P. Julien (Jean-Pierre); K. Ramachadran; F.G. Grosveld (Frank)

    1985-01-01

    textabstractWe have cloned a cDNA coding for the smallest rat neurofilament protein. The cDNA is 861 nucleotides long coding for 287 amino acids from the internal alpha-helical region and the carboxy-terminal tail domain of the neurofilament protein. Comparison of the porcine, mouse and rat neurofil

  8. IDENTIFICATION OF DIFFERENTIAL GENES IN OVARIAN CANCER USING REPRESENTATIONAL DIFFERENCE ANALYSIS OF cDNA

    Institute of Scientific and Technical Information of China (English)

    Hong Chen; Min Wang; Xin-yan Wang; Shan Gao; Jun Wang; Xiao-ming Guan

    2005-01-01

    Objective To identify differential genes between normal ovarian epithelium tissue and ovarian epithelial cancer using representational difference analysis of cDNA (cDNA-RDA). Methods cDNA-RDA was performed to identify the differentially expressed sequences between cDNAs from cancer tissue and cDNAs from normal ovarian tissue in the same patient who was in the early stage of ovarian serous cystadeno carcinoma. These differentially expressed fragments were cloned and analyzed, then sequenced and compared with known genes.Results Three differentially expressed cDNA fragments were isolated using cDNA from normal ovarian tissue as tester and cDNA from cancer tissue as driver amplicon by cDNA-RDA. DP Ⅲ-1 and DP Ⅲ-2 cDNA clone showed significant ho mology to the cDNA of alpha actin gene; DPⅢ-3 cDNA clone showed significant homology to the cDNA of transgelin gene. Conclusion cDNA-RDA can be used to sensitively identify the differentially expressed genes in ovarian serous cystadenocarcinoma. Ovarian serous cystadenocarcinoma involves alteration of multiple genes.

  9. 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.

  10. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein.

    OpenAIRE

    Jiang, H P; Serrero, G

    1992-01-01

    We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA ...

  11. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein

    OpenAIRE

    Jiang, Hui-Ping; Serrero, Ginette

    1992-01-01

    We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA ...

  12. 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.

  13. Cloning and analysis of a cDNA encoding acetohydroxy acid isomeroreductase from G2 pea

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Using cDNA representational difference analysis (cDNA RDA) method, we have successfully isolated a gene fragment whose expression was specifically induced by external GA3 application. Screening a G2 pea cDNA library using this fragment as a probe, we obtained a 2036 bp full-length cDNA. It contains a 1746 bp open reading frame and encodes a protein of 581 amino acids with a theoretical molecular weight of 64 ku. It shares high-level sequence identity with AAIR genes from other plant species. This cDNA was cloned into expression vector and recombinant E. coli DH5α cells with remarkable AAIR enzyme activity were obtained.

  14. 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%.

  15. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

    OpenAIRE

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.

  16. 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

    1995-04-03

    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.

  17. Construction of full length cDNA expression library of hepatopancreas of Penaeus monodon

    Institute of Scientific and Technical Information of China (English)

    罗田; 徐洵

    2002-01-01

    --mRNA was isolated from the hepatopancrease of shrimp Penaeus monodon with a PolyATtract System 1000 Kit. By using mRNA as template, double- strand cDNA with EcoR I/Xho I ends was synthesized by using a ZAP Express cDNA Synthesis Kit. The cDNA was inserted into the lambda ZAP Express vector predigested with EcoR I/Xho I, and the recombinant DNA was in vitro packaged into larnbda phage with GigapackⅢ Gold packaging extracts. These recombinant phages were then used to transfect E. coli XLl - Blue MRF', and finally a cDNA expression library was constructed. The library is 7.2 × 105pfu in capacity and its recombination ratio is higher than 99%. The size of the inserted cDNAs was determined by EcoR I/Xho I digestion of 9 phagemids prepared by in vivo excision of plaques selected randomly from amplified cDNA library . The longest inserted cDNA is about 1.6 kb in length. The complete sequence (about 1.2 kb) of actin cDNA was amplified from the library by PCR reveals that this library contains full-length cDNAs of Penaeus mod on hepatopancreas and is available for screening and expression of shrimp genes.

  18. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

  19. [Construction and analysis of subtractive cDNA library associated with multidrug resistance of acute leukemia].

    Science.gov (United States)

    Ji, Lei; Zhang, Wang-Gang; Liu, Jie; Liu, Xin-Ping; Yao, Li-Bo

    2004-08-01

    The study was aimed to construct subtractive cDNA library associated with multidrug resistance (MDR) of acute leukemia for screening genes related to MDR in leukemia. The improved PCR-based subtractive hybridization was performed to clone differential genes between HL-60/VCR and HL-60 cell line. The mRNA of HL-60/VCR and HL-60 cell line were isolated. Then the mRNA of HL-60/VCR group was reversely transcribed into cDNA by Cap-Finder method, and the mRNA of HL-60 was reversely transcribed into cDNA by ordinary method to be marked by biotin for the hybridization next with HL-60/VCR cDNA. After hybridizing, filtrating through the sephacryl S-400 column, absorbing by the magnetic beads, and amplifying by PCR method, the fragments were cloned by T-A method and the cDNA library was constructed. Then the quality of cDNA library was identified by dot-blotting hybridization method. The results showed that after constriction, the library demonstrated its good quality. There was a high proportion of large fragments in this library. From small amount of samples a large amount of candidate fragments could be screened rapidly at once by dot-blotting hybridization. It is concluded that a differentially-expressed subtractive cDNA library in MDR of leukemia with high quality and larger fragments can be efficiently constructed by improving subtractive hybridization and selective PCR method.

  20. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, E.K.; Horton, R.; Bloem, L.; Bach, R.; Williams, K.R.; Guha, A.; Kraus, J.; Lin, T.C.; Nemerson, Y.; Konigsberg, W.H.

    1987-08-01

    Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. lambda 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).

  2. Circular rapid amplification of cDNA ends for high-throughput extension cloning of partial genes.

    Science.gov (United States)

    Fu, Glenn K; Wang, Jonathan T; Yang, Junming; Au-Young, Janice; Stuve, Laura L

    2004-07-01

    The rapid amplification of cDNA ends (RACE) procedure is a widely used PCR-based method to clone the cDNA ends of mRNA transcripts. Current RACE methods often produce a high background of nonspecific PCR products, which can exclude the identification of the target cDNA of interest. We describe here an improved RACE procedure using circular cDNA templates and demonstrate the successful extension cloning of 4406 cDNAs.

  3. A CLINICAL STUDY ON PROSTATE CANCER DIAGNOSIS WITH cDNA MACROARRAY

    Institute of Scientific and Technical Information of China (English)

    ZHONG Wei-de; XIE Ke-ji; WEI Hong-ai; LIU Liang-shi; LIU Wen-hua; JIANG Fu-neng; ZENG Guang-qiao; DAI Qi-shan; HE Hui-chan; BI Xue-cheng; PENG Zhi-qiang

    2005-01-01

    Objective: To diagnose prostatic cancer (CaP) with cDNA macroarray. Methods: Total RNA was isolated from patients with prostate cancer and from normal people, and poly(A) RNA was further purified. Then differentially expressed genes were analysed in CaP and normal prostate by cDNA macroarray system. Results: There were differential expressions of nine prostate-associated specific genes in CaP as compared with normal prostate, among which, 7 were significantly up-regulated and 2 were down-regulated. Conclusion: As a diagnostic approach at molecular level, the cDNA macroarray is supposed to elevate the detection rate of CaP.

  4. Characterization and simulation of cDNA microarray spots using a novel mathematical model

    OpenAIRE

    2007-01-01

    Abstract Background The quality of cDNA microarray data is crucial for expanding its application to other research areas, such as the study of gene regulatory networks. Despite the fact that a number of algorithms have been suggested to increase the accuracy of microarray gene expression data, it is necessary to obtain reliable microarray images by improving wet-lab experiments. As the first step of a cDNA microarray experiment, spotting cDNA probes is critical to determining the quality of s...

  5. Cloning and sequencing of dolphinfish (Coryphaena hippurus, Coryphaenidae) growth hormone-encoding cDNA.

    Science.gov (United States)

    Peduel, A D; Elizur, A; Knibb, W

    1994-01-01

    The cDNA encoding the preprotein growth hormone from the dolphinfish (Coryphaena hippurus) has been cloned and sequenced. The cDNA was derived by reverse transcription of RNA from the pituitary of a young fish using the method known as Rapid Amplification of cDNA Ends (RACE). An oligonucleotide primer corresponding to the 5' region of Pagrus major and the universal RACE primer enabled amplification using the Polymerase Chain Reaction (PCR). The dolphinfish and yellow-tail, Seriola quineqeradiata, are both members of the sub-order Percoidei (Perciforme) and their GH sequences show a high level of homology.

  6. Characterization of the human HOX 7 cDNA and identification of polymorphic markers.

    Science.gov (United States)

    Padanilam, B J; Stadler, H S; Mills, K A; McLeod, L B; Solursh, M; Lee, B; Ramirez, F; Buetow, K H; Murray, J C

    1992-09-01

    cDNA clones for a human HOX 7 gene obtained with homologous clones of Drosophila were used in human gene mapping studies. The human cDNA clone was isolated from a library constructed from human embryonic craniofacial material. The sequence of the cDNA demonstrates significant homology with mouse HOX 7. A search for RFLPs identified MboII and BstEII variants. A CA dinucleotide repeat with 5 alleles was also identified and allowed placement of HOX 7 into a defined linkage map. Evidence for linkage disequilibrium was found with markers tested. These results place the human HOX 7 gene in a defined position on 4p.

  7. 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.

  8. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M;

    1995-01-01

    regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76......(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed......, was determined to be on distal mouse Chromosome (Chr) 9 by analysis of two sets of multilocus crosses....

  9. 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.

  10. Construction and analysis of a subtracted cDNA library of Betula platyphylla female inflorescence

    Institute of Scientific and Technical Information of China (English)

    WEIJi-cheng; YANGChuan-ping; WANGChao; JIANGJing

    2005-01-01

    Female inflorescence of Betula platyphylla was sampled at an interval of each two days to analyze the background of gene expression in floral phase. On the basis of SMART strategy, the driver cDNA was obtained from total RNA of the last sample and the tester cDNA was from that of the others by RT-PCR which were subsequently used to construct a subtracted cDNA library. The result of the ESTs (expression sequence tags) blastX showed that the genes in the subtracted cDNA library could be mainly clustered into 5 groups related to metabolism, transportation and signal transduction, cell cycle, stress response, and regulation. The relationship between gene expression and development was also discussed.

  11. Conversion of cDNA differential display results (DDRT-PCR into quantitative transcription profiles

    Directory of Open Access Journals (Sweden)

    Koopmann Birger

    2005-04-01

    Full Text Available Abstract Background Gene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Commonly used protocols for gel-based transcript profiling are cDNA differential display (DDRT-PCR and cDNA-AFLP. Both methods have been used merely as qualitative gene discovery tools so far. Results We developed procedures for the conversion of cDNA Differential Display data into quantitative transcription profiles. Amplified cDNA fragments are separated on a DNA sequencer and detector signals are converted into virtual gel images suitable for semi-automatic analysis. Data processing consists of four steps: (i cDNA bands in lanes corresponding to samples treated with the same primer combination are matched in order to identify fragments originating from the same transcript, (ii intensity of bands is determined by densitometry, (iii densitometric values are normalized, and (iv intensity ratio is calculated for each pair of corresponding bands. Transcription profiles are represented by sets of intensity ratios (control vs. treatment for cDNA fragments defined by primer combination and DNA mobility. We demonstrated the procedure by analyzing DDRT-PCR data on the effect of secondary metabolites of oilseed rape Brassica napus on the transcriptome of the pathogenic fungus Leptosphaeria maculans. Conclusion We developed a data processing procedure for the quantitative analysis of amplified cDNA fragments separated by electrophoresis. The system utilizes common software and provides an open-end alternative to DNA microarray analysis of the transcriptome. It is expected to work equally well with DDRT-PCR and cDNA-AFLP data and be useful particularly in reseach on organisms for which microarray analysis is not available or economical.

  12. Comprehensive Assessment of Marine Coatings in the Laboratory and Field

    Science.gov (United States)

    2015-03-27

    NAME OF RESPONSIBLE PERSON Dean E. Wendt 19b. TELEPHONE NUMBER (Include area code) 805-756-2988 Standard Form 298 (Rev. 8/98) Prescribed by ANSI...approximately 50% and the percentage of settled larvae was calculated and compared to that of the controls. Laboratory Rearing of Juvenile Barnacles...Newly metamorphosed juvenile barnacles {Balanus amphitrite) were transferred on their respective coatings to growth chambers where they were fed the

  13. Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

    Science.gov (United States)

    Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

    2016-08-01

    Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.

  14. ASSOCIATION OF DIFFERENTIALLY EXPRESSED cDNA FRAGMENT OF FGG WITH HEPATOCELLULAR CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    范秉琳; 朱武凌; 邹国林; 段芳龄

    2002-01-01

    Objective: To identify a cDNA clone from the subtracted library of human hepatocellular carcinoma (HCC). Methods: Suppression subtractive hybridization was used to isolated a panel of genes that are differentially expressed in hepatocellular carcinoma as compared with cirrhotic liver. T/A cloning method was used to construct a subtracted cDNA library. DNA sequencing analysis and Northern blot analysis were also utilized. Results: The cloned cDNA is 787 nucleotides in length and contains an open reading frame of 230 amino acids, which is a cDNA fragment of reported human fibrinogen, gamma polypeptide (FGG). Northern analysis revealed that this gene was overexpressed in two hepatocellular carcinoma cell lines, SMMC-7721 and HepG2. Conclusion: Sequence identity proved the cDNA clone fragment of as FGG gene. Differential expression of the cDNA fragment in HCC suggested that FGG is related to HCC, indicating a new clue for developing a novel diagnostic and prognostic marker.

  15. 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

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

    Directory of Open Access Journals (Sweden)

    Jesionek-Kupnicka Dorota

    2009-08-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion 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.

  17. Cloning and screening of cDNA of Psilgramma menephorn allergen

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To construct a cDNA expression library of Psilgramma menephorn to screen its major allergen so as to provide the basis for producing recombinant allergen vaccine of Psilgramma menephorn. Methods Total RNA was extracted from the whole body of Psilgramma menephorn with Trizol and mRNA was purified with Oligo (dT) Spin-Column. And dscDNA was synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoRⅠ adapters. Then the cDNAs were digested by XhoⅠ, and the fragments less than 400 bp were removed by using GHROMA SPIN-400 column. The remaining fragments longer than 400 bp were ligated with Uni-ZAP XR vector. The recombinants were packaged in vitro and a small portion of the packaged phage was used to infect E.coli XL1-Blue MRF′ for titration. The recombinants were examined by color selection. The size of cDNA inserts and the diversity of library were analyzed by PCR. The library was screened using SPT positive sera from patients with Psilgramma menephorn allergy repeatedly. Results The cDNA expression library consisting of a 5×105 recombinant bacteriophages was constructed with the recombinant ratio of 67%. The average length of recombinant exogenous inserts was about 1.49 kb. Five positive cDNA clones were obtained. Conclusion The constructed cDNA expression library shows appropriate contents and size of cDNA fragments and the related genes of Psilgramma menephorn major allergens were harbored successfully, which lays the foundation for the positive clone identification and further analysis.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

    Science.gov (United States)

    Honda, A; Sugimoto, Y; Namba, T; Watabe, A; Irie, A; Negishi, M; Narumiya, S; Ichikawa, A

    1993-04-15

    A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG in the order of PGE2 = PGE1 > iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

  4. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes.

    Science.gov (United States)

    Yang, G P; Ross, D T; Kuang, W W; Brown, P O; Weigel, R J

    1999-03-15

    Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.

  5. 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.

  6. Molecular cloning and nucleotide sequence of cDNA for human liver arginase

    Energy Technology Data Exchange (ETDEWEB)

    Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

    1987-01-01

    Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated lambda hARG6 and lambda hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying lambda hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes.

  7. Construction of cDNA Library from NPC Tissue and Screening of Antigenic Genes

    Institute of Scientific and Technical Information of China (English)

    Jun Shu; Xiaojuan He; Guancheng Li

    2006-01-01

    To construct cDNA library of nasopharyngeal carcinoma (NPC) and obtain the NPC associated or specific antigens from it, we used a powerful new method to identify the antigens eliciting humoral immune response, which is SEREX (serological identification of antigen by recombinant cDNA expression library). Autologous serum of NPC patient was used to screen the reactive clones in the human NPC tissue cDNA library consisted of 3.64×106 recombinants. The 23 exact positive clones were subcloned to monoclonality and the size of cDNA inserts was identified by PCR. Then the nucleotide sequence of cDNA inserts was determined, and the sequence alignments were performed with BLAST software on GenBank database. They represented 16 different antigens. A detailed sequence analysis showed that 10 of 16 genes were high homologous to genes known in GenBank, such as RPL31,S100 A2, MT2A, etc. However, there were also 6 genes with low homology to genes in GenBank. Furthermore, 3 of 6 genes may be novel genes. The associations of these genes to NPC and the roles that they played in the occurrence and development of NPC should be further revealed.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Profiling gene expression patterns of nasopharyngeal carcinoma and normal nasopharynx tissues with cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    5 μg of total RNAs from normal nasopharynx and nasopharyngeal carcinoma tissue have been labeled with α-32P-dCTP during reverse transcription. The synthesized cDNA probes have been hybridized to high-density cDNA microarray containing 5184 genes or expression sequence tags (ESTs). Then image analysis software has been applied to comparing their expression profiles. Results show that 187 ESTs were of density value above 200 in nasopharyngeal carcinoma tissue while there were 307 such ESTs in normal nasopharynx tissue; 38 ESTs were strongly expressed in nasopharynx, but weakly expressed in nasopharyngeal carcinoma; 48 ESTs were strongly expressed in nasopharyngeal carcinoma, but weakly expressed in normal nasopharynx. These results suggest that there may exist some new differentially expressed genes involved in nasopharyngeal carcinoma development. Furthermore, the results strongly indicate that high-density cDNA microarray is a powerful and efficient tool for large-scale screening differentially expressed genes.

  13. Regulation of human clotting factor IX cDNA expression in transgenic mice

    Institute of Scientific and Technical Information of China (English)

    胡以平; 邱信芳; 薛京伦; 刘祖洞

    1995-01-01

    To study the expression of human dotting factor IX cDNA in transgenic mice,Which is an es-sential work on gene therapy for hemophilia B,3 recombinant constructions containing different lengths ofhuman dotting factor IX cDNA have been introduced into the cultured cells.All of the recombinant constructionswere found to he expressed well in vitro.They were then microinjected into the male pronudei of the fertilizedmouse eggs respectively for generating trahsgenic mice.Unfortunately,none of them was expressed in any transgenicmice.These results show that the expression of the human clotting factor IX cDNA in the transgenic mice canbe determined by cis regulatory element(s).As compared With the results from other related works,it is sug-gested that the cis regulatory element(s)is resided in the 5’-end non-coding region.

  14. Optimization of yeast surface-displayed cDNA library screening for low abundance targets.

    Science.gov (United States)

    Kim, Juh-Yung; Kim, Hyung Kyu; Jang, Hye Jeong; Kim, Eun-Kyung; Kim, Moon Kyu

    2015-04-01

    The yeast surface-displayed cDNA library has been used to identify unknown antigens. However, when unknown target antigens show moderate-to-low abundance, some modifications are needed in the screening process. In this study, a directional random-primed cDNA library was used to increase the number of candidates for the unknown antigen. To avoid the loss of target yeast clones that express proteins at a low frequency in the cDNA library, a comprehensive monitoring system based on magnetic-activated cell sorting, fluorescence-activated cell sorting, and immunofluorescence was established, and a small number of target yeast cells was successfully enriched. These results showed that our optimized method has potential application for identifying rare unknown antigens of the human monoclonal antibody.

  15. 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.

  16. Identification of a Herbicide Safener AD-67 Inducible cDNA in Rice

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A herbicide safener AD-67 inducible cDNA was identified in an indica rice variety 9311 by mRNA differential display. The transcript was increased 6 h after sprayed with the safener solution, and 4 days later, the expression still could be detected. The fragment was recycled from the poly-gel and sequenced, and homologous analysis revealed the cDNA was 100% identical to some ESTs and cDNAs in rice database, and the amino acid sequence was 60-84% homologous to those of the Yippee genes in several eukaryotes. The fragment was extended to the whole long cDNA, and thus a primer pair was designed. RT-PCR analysis for the designed primer supported the induction result.

  17. 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.

  18. 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.

  19. 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.

  20. LD-RTPCR:\tA NEW METHOD FOR LABELLING TRACE cDNA MICROARRAY PROBE

    Institute of Scientific and Technical Information of China (English)

    范保星; 孙敬芬; 梁好; 王升启; 周平坤; 吴德昌

    2002-01-01

    Objective: To explore the usefulness of long distance reverse transcript combining linear amplification (LD-RTPCR) in labeling slight trace probe used for cDNA microarray. Methods: Total RNA from BEP2D cells was extracted and labeled by two different methods, LD-RTPCR with Cy3-dCTP as fluorescent dye and traditionally used RNA reverse transcript (RT) with Cy5-dCTP as fluorescent dye. Then, the probes labeled by two methods were mixed equally and hybridized with the cDNA microarray. Results: Scan and analysis of the microarray showed that the two methods labeled probes had consistent results. Conclusion: LD-RTPCR was proved useful for labeling cDNA microarray probe, especially for limited RNA material.

  1. Analysis of gene expression profile of aspermia using cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    杨波; 高晓康; 王禾; 刘贺亮; 陈宝琦; 秦荣良; 康福霞; 邵国兴; 邵晨

    2003-01-01

    Objective: To identify the differential gene expression profiles between the normal and aspermia human testes utilizing cDNA microarray. Methods: cDNA probes were prepared by labeling mRNA of aspermia testes tissues with Cy5-dUTP and mRNA of normal testes tissues with Cy3-dUTP respectively through reverse transcription. The mixed cDNA probes were then hybridized with 4096 cDNA arrays (4096 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.). 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 2.0 or less-than 0.5. A randomly chosen gene RAP1A was studied by in situ hybridization to evaluate the accuracy of the results. Results: 623 differential expressed genes related to aspermia were found. There were 303 up-expressed genes and 320 down-expressed genes. A distinct up-expressed gene RAP1A was confirmed by in situ hybridization. Conclusions: Screening the differential gene expression profiles between the normal and aspermia human testis by cDNA microarray can be used in the study of aspermia-related genes and the further research due to its properties, RAP1A may play some roles in the development and progression of aspermia.

  2. 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).

  3. cDNA cloning of the beta subunit of teleost thyrotropin.

    OpenAIRE

    Ito, M.; Koide, Y.; Takamatsu, N; Kawauchi, H.; Shiba, T.

    1993-01-01

    cDNA clones encoding the beta subunit of thyrotropin (thyroid-stimulating hormone; TSH) were isolated from a cDNA library made from the pituitaries of immature rainbow trout and sequenced. The precursor of rainbow trout TSH beta consists of 147 aa, which can be cleaved into a signal peptide (20 aa) and a mature protein (127 aa) containing one potential N-glycosylation site and 12 cysteine residues. The protein showed highest homology with human TSH beta (51%) and lesser homology with human fo...

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

    OpenAIRE

    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 designated to be on...

  5. Cloning and screening of cDNA of Psilgramma menephorn allergen

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To construct a cDNA expression library of Psilgramma menephorn to screen its major allergen so as to provide the basis for producing recombinant allergen vaccine of Psilgramma menephorn. Methods Total RNA was extracted from the whole body of Psilgramma menephorn with Trizol and mRNA was purified with Oligo (dT) Spin-Column. And dscDNA was synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoRⅠ adapters. Then the cDNAs were digested by XhoⅠ, and the fra...

  6. Download - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project Download First of all, please read the license of this database. Data ...names and data descriptions are about the downloadable data in this page. They might not correspond to the c...f the data. # Data name File Simple search and download 1 README README_e.html - 2 5'-end sequences of buddi...ng yeast full-length cDNA clones and quality scores yeast_seq_qual.zip (59.9MB) Simple search and download 3...Downlaod via FTP Joomla SEF URLs by Artio About This Database Database Description Download License Update H

  7. 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...

  8. Isolation and cDNA cloning of somatolactin in rabbitfish (Siganus guttatus).

    Science.gov (United States)

    Ayson, F G; de Jesus, E G; Amemiya, Y; Moriyama, S; Hirano, T; Kawauchi, H

    1999-08-01

    We report the isolation and cDNA cloning of somatolactin (SL) from rabbitfish, Siganus guttatus. Rabbitfish SL was isolated from an alkaline extract of the pituitary glands by gel filtration chromatography on Sephadex G-100 and reversed-phase high-performance liquid chromatography. SL was monitored by immunoblotting with flounder SL antiserum. The preparation (yield: 0.86 mg/g wet tissues) contained two immunoreactive bands of 24 and 28 kDa on SDS-PAGE. Overlapping partial cDNA clones corresponding to teleost SLs were amplified by PCR from single-strand cDNA from pituitary glands. Excluding the poly(A) tail, rabbitfish SL cDNA is 1605 bp long. It contains a 693-bp open reading frame encoding a signal peptide of 24 amino acids (aa) and a mature protein of 207 aa. Rabbitfish SL has two possible N-glycosylation sites at positions 11 and 121 and seven half Cys residues. The deduced amino acid sequence shows over 80% identity with those of advanced teleosts like sea bream, red drum, and flounder, 76% with the salmonids, 57% with the eel, and 46% with the goldfish SL.

  9. Isolation of RNA and DNA from leukocytes and cDNA synthesis.

    NARCIS (Netherlands)

    Jansen, J.H.; Reijden, B.A. van der

    2006-01-01

    In this chapter, methods to isolate RNA and DNA from human leukocytes for the subsequent use in molecular diagnostic tests are described. In addition, protocols for cDNA synthesis are given, both for the use in conventional reverse transcription (RT)-polymerase chain reaction (PCR), and for the use

  10. Rapid Amplification of cDNA Ends for RNA Transcript Sequencing in Staphylococcus.

    Science.gov (United States)

    Miller, Eric

    2016-01-01

    Rapid amplification of cDNA ends (RACE) is a technique that was developed to swiftly and efficiently amplify full-length RNA molecules in which the terminal ends have not been characterized. Current usage of this procedure has been more focused on sequencing and characterizing RNA 5' and 3' untranslated regions. Herein is described an adapted RACE protocol to amplify bacterial RNA transcripts.

  11. Data of evolutionary structure change: 1CDNA-1CLMA [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1CDNA-1CLMA 1CDN 1CLM A A KSPEELKGIFEKYAAKE--GDPNQLSKEELKLLLQTEFP...--- LTE-EQIAEFKEAFALFDKDGDGTITTKELGTVMRSL-----GQNPTEAELQDMINEVDADGNGTIDFPEFLSLMARKMKEQDSEEELIEA...line> 1CLM A 1CLMA 1CLM A 1CLMA...fEVID> 2 1CLM A 1CLMA

  12. Observation of intermittency in gene expression on cDNA microarrays

    CERN Document Server

    Peterson, L E

    2002-01-01

    We used scaled factorial moments to search for intermittency in the log expression ratios (LERs) for thousands of genes spotted on cDNA microarrays (gene chips). Results indicate varying levels of intermittency in gene expression. The observation of intermittency in the data analyzed provides a complimentary handle on moderately expressed genes, generally not tackled by conventional techniques.

  13. 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.

  14. 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...

  15. Strategies to enhance immunogenicity of cDNA vaccine encoded antigens by modulation of antigen processing

    NARCIS (Netherlands)

    Platteel, Anouk C M; Marit de Groot, A; Andersen, Peter; Ovaa, Huib; Kloetzel, Peter M; Mishto, Michele; Sijts, Alice J A M

    2016-01-01

    Most vaccines are based on protective humoral responses while for intracellular pathogens CD8(+) T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8(+) T cell priming, hampering vaccine efficacy. The multistage cDNA va

  16. Revised sequence and expression of cyclin B cDNA from the starfish Asterina pectinifera.

    Science.gov (United States)

    Miyake, Y; Deshimaru, S; Toraya, T

    2001-05-01

    Cyclin B cDNA was cloned from the ovary of the starfish Asterina pectinifera and analyzed by RT-PCR and 3'- and 5'-RACE techniques. The cDNA consists of a 0.13-kb upstream untranslated region, a 1.22-kb coding region, and a 0.86-kb downstream untranslated region. The open reading frame encoded a polypeptide of 404 amino acid residues with a calculated molecular weight of 45,692. All the characteristic sequences, such as destruction and cyclin boxes, cyclin B motif, and cytoplasmic retention and nuclear export signals, were found in the newly cloned cyclin B cDNA. The deduced amino acid sequence of the cyclin B cDNA was highly homologous in the middle and carboxy terminal regions to that from mature eggs of the same organism, but quite different in the amino terminal region. Evidence was obtained which suggested that this cyclin B is expressed in immature and maturing oocytes and is the same as that cloned from mature eggs.

  17. Discovery and analysis of pancreatic adenocarcinoma genes using cDNA microarrays

    Institute of Scientific and Technical Information of China (English)

    Gang Jin; Xian-Gui Hu; Kang Ying; Yan Tang; Rui Liu; Yi-Jie Zhang; Zai-Ping Jing; Yi Xie; Yu-Min Mao

    2005-01-01

    AIM: To study the pathogenetic processes and the role of gene expression by microarray analyses in expediting our understanding of the molecular pathophysiology of pancreatic adenocarcinoma, and to identify the novel cancer-associated genes.METHODS: Nine histologically defined pancreatic head adenocarcinoma specimens associated with clinical data were studied. Total RNA and mRNA were isolated and labeled by reverse transcription reaction with Cy5 and Cy3 for cDNA probe. The cDNA microarrays that represent a set of 4 096 human genes were hybridized with labeled cDNA probe and screened for molecular profiling analyses.RESULTS: Using this methodology, 184 genes were screened out for differences in gene expression level after nine couples of hybridizations. Of the 184 genes,87 were upregulated and 97 downregulated, including 11 novel human genes. In pancreatic adenocarcinoma tissue, several invasion and metastasis related genes showed their high expression levels, suggesting that poor prognosis of pancreatic adenocarcinoma might have a solid molecular biological basis.CONCLUSION: The application of cDNA microarray technique for analysis of gene expression patterns is a powerful strategy to identify novel cancer-associated genes, and to rapidly explore their role in clinical pancreatic adenocarcinoma. Microarray profiles provide us new insights into the carcinogenesis and invasive process of pancreatic adenocarcinoma. Our results suggest that a highly organized and structured process of tumor invasion exists in the pancreas.

  18. Avoiding cross hybridization by choosing nonredundant targets on cDNA arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Knudsen, Steen

    2002-01-01

    PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end....

  19. 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 250-555 p...

  20. 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.

  1. Isolation of carrot Argonaute1 from subtractive somatic embryogenesis cDNA library.

    Science.gov (United States)

    Takahata, Kiminori

    2008-03-01

    Carrot Argonaute1 (C-Ago1) was isolated from a subtractive cDNA library to obtain somatic embryogenesis related genes. C-Ago1 has three conserved domains, which are found in all other Argonautes. C-Ago1 has specific expression during somatic embryogenesis, which indicates that microRNA gene expression controlling system is required for somatic embryogenesis.

  2. 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...

  3. Molecular cloning and expression of a novel human cDNA containing CAG repeats.

    Science.gov (United States)

    Takeuchi, T; Chen, B K; Qiu, Y; Sonobe, H; Ohtsuki, Y

    1997-12-19

    A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.

  4. Vector sequences - 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 ...od - Number of data entries 7 entries - Joomla SEF URLs by Artio About This Database Database Description Download License Update His...tory of This Database Site Policy | Contact Us Vector sequences - Budding yeast cDNA sequencing project | LSDB Archive ...

  5. 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.

  6. Cloning, expression and mapping of the full-length cDNA of human CCTβ subunit

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Chaperonins assist the proper folding of target proteins without being a part of the substrates. The eukaryotic cytosolic chaperonin, CCT-Chaperonin Containing TCP-1 (tailless complex polypeptide-1), is mainly involved in the formation of cytoskeletal proteins and is essential for cell viability. Mammalian CCT is commonly a protein complex composed of 7-9 subunit species. We have isolated a novel full-length cDNA from human testis cDNA library. This cDNA of 1935 bp contains a 1605 bp open reading frame (ORF) encoding 535 amino acids (aa). The deduced protein of the cDNA is highly homologous to the CCTβ subunit of saccharomyces cerevisiae, schizosaccharomyces pombe, caenorhabditis elegans and mouse, etc. Especially high homology (97%) is found between the deduced protein and mouse CCTb. On the basis of such high homology, the protein encoded by the new gene was proposed to be a human CCTβ subunit. Northern hybridization showed that human CCTβ gene is expressed as a transcript of about 2.0 kb in various tissues. Overexpression was seen in testis with the expression level 3-24 times of those in other tissues. The CCTβ gene was mapped to human chromosome 12q14 by Radiation Hybrid Mapping. Through homologous search, the 5′-end of the cDNA sequence was found to share intermittent regional homology with the 3′-end of human genomic sequence (U91327). The genomic structure of the 5′-end of CCTβ was also described in detail through comparative analysis.

  7. KLONING cDNA HORMON PERTUMBUHAN DARI IKAN GURAME (Osphronemus gouramy

    Directory of Open Access Journals (Sweden)

    Estu Nugroho

    2016-11-01

    Full Text Available Penelitian mengenai kloning cDNA pengkode hormon pertumbuhan ikan gurame telah dilakukan. Tujuan dari penelitian ini adalah untuk memperoleh sekuens DNA komplemen hormon pertumbuhan sebagai langkah awal dalam rangka pengembangan teknologi rekayasa genetik ikan gurame. Empat buah kelenjar hifopisa ikan gurame digunakan sebagai bahan bakunya dan dilakukan proses ekstraksi RNA total dari kelenjar hipofisa, dilanjutkan dengan sintesis cDNA, amplifikasi PCR, purifikasi fragmen DNA dari gel, ligasi produk PCR dengan vektor kloning, transformasi dan inkubasi bakteri, seleksi koloni bakteri putih, isolasi plasmid, dan sekuensing. Hasil sekuensing menunjukkan bahwa panjang produk amplifikasi PCR adalah 843 bp yang menyandikan 204 asam amino residu dan mengandung sekuens-sekuens yang konserf untuk gen hormon pertumbuhan (GH. Analisis homologi menunjukkan kesamaan sekuens hasil isolasi antara 52,4%--97,6% dengan gen GH ikan lainnya, dengan persentase homologi tertinggi adalah dengan ikan sepat. Dengan demikian dapat disimpulkan bahwa sekuens hasil isolasi merupakan sekuens gen GH. Dari hasil analisis sekuens terlihat bahwa gen GH ikan gurame secara evolusi adalah konserf. Research on cDNA cloning encoded the gouramy growth hormone was conducted. The aim of the research was to get complementary DNA, cDNA, sequences of growth hormone as an initial step to develop genetic engineering of gouramy fish. Four pituitary glands of the gouramy were taken and then processed with total RNA extraction, and continued with cDNA synthesis, PCR amplification, DNA fragment purification from the gel, PCR product legation with cloning vector, transformation and incubation of bacteria, white colony bacteria selection, plasmid isolation and sequencing analysis. Sequencing result showed that the amplified PCR product length had 834 bp, encoding 204 amino acid residue and contained conserve sequence for GH (growth hormone gen. Homolog analysis showed sequence similarity of

  8. Cloning and expression of a cDNA encoding human sterol carrier protein 2

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III (Univ. of Pennsylvania School of Medicine, Philadelphia (United States)); Billheimer, J.T. (E.I. DuPont de Nemours, Inc., Wilmington, DE (United States))

    1991-01-15

    The authors report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP{sub 2}). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP{sub 2} amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP{sub 2}. The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A){sup +} RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP{sub 2} gene in the human genome or that the SCP{sub 2} gene is very large. Coexpression of the SCP{sub 2} cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP{sub 2} plays a role in regulating steroidogenesis, among other possible functions.

  9. Construction and Screening of an Expression cDNA Library from the Triactinomyxon Spores of Myxobolus cerebralis, the causative agent of Salmonid Whirling Diseases

    OpenAIRE

    Soliman, Hatem Mohamed Touhan

    2005-01-01

    The ZAP Express cDNA library was constructed using mRNA extracted from the triactinomyxon spores. First-strand cDNA was synthesized using Moloney Murine leukaemia virus reverse transcriptase. Following second-strand cDNA synthesis, the double-stranded cDNA was digested with Xho I restriction enzyme, cDNA fragments less than 400bp were removed and the remaining cDNA was ligated with the lambda ZAP Express vector. The recombinants were packaged in vitro using Gigapack III gold packaging extract...

  10. 3' rapid amplification of cDNA ends (RACE) walking for rapid structural analysis of large transcripts.

    Science.gov (United States)

    Ozawa, Tatsuhiko; Kondo, Masato; Isobe, Masaharu

    2004-01-01

    The 3' rapid amplification of cDNA ends (3' RACE) is widely used to isolate the cDNA of unknown 3' flanking sequences. However, the conventional 3' RACE often fails to amplify cDNA from a large transcript if there is a long distance between the 5' gene-specific primer and poly(A) stretch, since the conventional 3' RACE utilizes 3' oligo-dT-containing primer complementary to the poly(A) tail of mRNA at the first strand cDNA synthesis. To overcome this problem, we have developed an improved 3' RACE method suitable for the isolation of cDNA derived from very large transcripts. By using the oligonucleotide-containing random 9mer together with the GC-rich sequence for the suppression PCR technology at the first strand of cDNA synthesis, we have been able to amplify the cDNA from a very large transcript, such as the microtubule-actin crosslinking factor 1 (MACF1) gene, which codes a transcript of 20 kb in size. When there is no splicing variant, our highly specific amplification allows us to perform the direct sequencing of 3' RACE products without requiring cloning in bacterial hosts. Thus, this stepwise 3' RACE walking will help rapid characterization of the 3' structure of a gene, even when it encodes a very large transcript.

  11. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

    Institute of Scientific and Technical Information of China (English)

    Yong-Bo Liu; Zhao-Xia Wei; Li Li; Hang-Sheng Li; Hui Chen; Xiao-Wen Li

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.METHODS: Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMARTTM PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder,cDNA xProfiler, Digital GENE Expression Displayer (DGED),and Digital Differential Display (DDD) were used.RESULTS: Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed.CONCLUSION: Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocardnoma.

  12. [Construction of subtractive cDNA libraries of the sporogony stage of Eimeria tenella by suppression subtractive hybridization].

    Science.gov (United States)

    Han, Hong-Yu; Lin, Jiao-Jiao; Zhao, Qi-Ping; Dong, Hui; Jiang, Lian-Lian; Wang, Xin; Han, Jing-Fang; Huang, Bing

    2007-11-01

    In order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver, respectively, the cDNAs from sporozoites of E. tenella was used tester, Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver, the cDNAs from sporulated ooceysts was used tester, one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%, 96% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively, thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with previously described. A total of forty unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with previously described, the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis.

  13. PvuII RFLP detected by a human. beta. ADH cDNA probe

    Energy Technology Data Exchange (ETDEWEB)

    Parsian, A.; Burgess, A.K.; Khan, M.A.; Devor, E.J. (Washington Univ. School of Medicine, St. Louis, MO (USA))

    1989-12-11

    A 0.97 kb cDNA (ADH12) fragment encoding human exons 7, 8, 9 of the ADH{sub 2} gene was isolated from an adult human liver cDNA library. The insert can be excised by Pst I digestion. Pvu II identifies a two-allele polymorphism with bands at 4.4 kb (A{sub 1}) and 3.0 kb (A{sub 2}) and invariant bands at 5.1, 4.0, 2.8, and 2.3 kb. It was localized on Chromosome 4q21-q25 by in situ hybridization. Co-dominant segregation was observed in 18 informative families.

  14. 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.

  15. Cloning and expression of full-length cDNA encoding human vitamin D receptor

    Energy Technology Data Exchange (ETDEWEB)

    Baker, A.R.; McDonnell, D.P.; Hughes, M.; Crisp, T.M.; Mangelsdorf, D.J.; Haussler, M.R.; Pike, J.W.; Shine, J.; O' Malley, B.W. (California Biotechnology Inc., Mountain View (USA))

    1988-05-01

    Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3{prime} noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of {approx} 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.

  16. 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.

  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. 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.

  19. Identification of alternative transcripts using rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Yeku, Oladapo; Scotto-Lavino, Elizabeth; Frohman, Michael A

    2009-01-01

    Many organisms, including humans, have many more proteins than are actually coded for by their genes. This discrepancy is partially explained by the existence of alternative transcripts produced by the same gene. Multiple isoforms of the same gene sometimes perform completely different functions, and as such, knowing the sequence of one of the transcripts is not enough. Rapid Amplification of cDNA Ends (RACE) provides an inexpensive and powerful tool to quickly identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for rapid amplification of 5' and 3' cDNA ends using the "New Race" technique.

  20. 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

    of these methods can be increased by applying massive parallel sequencing technologies.Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3' end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase™). In the following PCR......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...... that do not require formal bioinformatics training. As an example, we apply the method to detection of transcription start sites in mouse liver cells....

  1. 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

  2. Molecular cloning of a cDNA related to vernalization(verc203) in winter wheat

    Institute of Scientific and Technical Information of China (English)

    种康; 谭克辉; 黄华梁; 梁厚果

    1995-01-01

    A cDNA clone related to the vernalization in winter wheat(verc203)was harvested from the en-riched cold-induced cDNA library of 10~4 pfu with differential screening.The insert of verc203 in λ gt10 vector wassubcloned into the sites between BamH Ⅰ and Hind Ⅲ in pUC19 plasmid after being amplified with PCR.the analysis of the Northern blotting with a probe of verc203 indicated that the verc203 has a negative signalfor the control and the devernalized mRNA and a positive signal for the vernalized winter wheat and non-vernalized spring wheat at about 2.6 kb.

  3. 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

    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 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...

  4. cDNA Cloning, Prokaryotic and Eukaryotic Expression and Characterization of Porcine Leukemia Inhibitory Factor

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Molecular cloning of the porcine leukemia inhibitor factor(pLIF) has not been reported. A full-length cDNA encoding pLIF was cloned, expressed and characterized. The full-length porcine LIF cDNA encodes a 202 amino acid protein that has an 84% sequence identity to mouse LIF and 86% sequence identity to human LIF. The deduced amino acid sequence of a pLIF protein contains six conserved consensus N-linked glycosylation sites and six cysteine groups to form potential disulfide bonds. The pLIF was expressed in E coli, as a mature form, and in CHO cells as a secreted form. Both the forms of the recombinant pLIFs can maintain murine embryonic stem cells in an undifferentiated state in a culture. The recombinant pLIFs will be useful in establishing a long-term culture of stable pluripotent porcine embryonic stem cells for further manipulation.

  5. Molecular cloning of GA-suppressed G2 pea genes by cDNA RDA

    Institute of Scientific and Technical Information of China (English)

    朱玉贤; 张翼凤; 李慧英

    1997-01-01

    GA-treated and non-treated G2 pea cDNAs were compared using a newly developed method called cDNA representational difference analysis (cDNA-RDA), and several GA-suppressed mRNAs were found. After cloning of the larger fragments PGAS1-3 ( pea GA-suppressed cDNA 1-3), they were demonstrated to be expressed only in pea tissue not treated with GA3 through Northern analysis. Compared with subtractive hybridization and differ-ential display techniques, this method not only can be easily manipulated but also has a relatively low rate of false posi-tive and is highly repetitive. It is the major progress in molecular cloning techniques.

  6. 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.

  7. Analysis of cDNA sequence, protein structure and expression of parotid secretory protein in pig

    Institute of Scientific and Technical Information of China (English)

    YIN Haifang; FAN Baoliang; ZHAO Zhihui; LIU Zhaoliang; FEI Jing; LI Ning

    2003-01-01

    Parotid secretory protein (PSP) secreted abundantly in saliva, whose function is related with the anti-bacterial effect. The PSP cDNA has been isolated from pig parotid glands by 3′ and 5′ rapid amplification of cDNA end (RACE),based on the conserved signal peptide region among the known mammalian PSP. Theresult of homologous comparison shows that pig PSP and human PSP shares the high identity at the level of the primary, secondary and tertiary protein structure. A search for functionally significant protein motifs revealed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu- X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is important to its function. RT-PCR, Dot blot and Northern blot analysis demonstrated that PSP was strongly expressed in parotid glands, but not in other tissues.

  8. 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.

  9. cDNA cloning and immunological characterization of the rye grass allergen Lol p I.

    Science.gov (United States)

    Perez, M; Ishioka, G Y; Walker, L E; Chesnut, R W

    1990-09-25

    The complete amino acid sequence of two "isoallergenic" forms of Lol p I, the major rye grass (Lolium perenne) pollen allergen, was deduced from cDNA sequence analysis. cDNA clones isolated from a Lolium perenne pollen library contained an open reading frame coding for a 240-amino acid protein. Comparison of the nucleotide and deduced amino acid sequence of two of these clones revealed four changes at the amino acid level and numerous nucleotide differences. Both clones contained one possible asparagine-linked glycosylation site. Northern blot analysis shows one RNA species of 1.2 kilobases. Based on the complete amino acid sequence of Lol p I, overlapping peptides covering the entire molecule were synthesized. Utilizing these peptides we have identified a determinant within the Lol p I molecule that is recognized by human leukocyte antigen class II-restricted T cells obtained from persons allergic to rye grass pollen.

  10. 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.

  11. RAPID SCREENING OF AN ARRAYED cDNA LIBRARY BY IMPROVED PCR-BASED METHOD

    Institute of Scientific and Technical Information of China (English)

    杜光伟; 潘美辉; 袁建刚; 周彦; 强伯勤; 梁植权

    1998-01-01

    Tbe present study reports an improved PCR-based technique that allows quick and effecfive screening of eDNA libraries. First, the eDNA library was arrayed as follows: about 3×106 cDNA clones were multiplied as individual plsques on solid medium in 24-well culture dishes at 1 200 plaque forming units per well.The phage suspension of each well was transferred to an individual micrccentrifuge tube in 72-tube box. Then,box pool, row pools and column pools were set up that respectively represent a 72-tube box,rows and columns within the box. To screen a specific target cDNA,primers specific for novel ESTs obtained in our laboratory were eznployed to conduct PCR in a fiierarchy mode. PCR began with the box pools, resulting in the identification of some positive box pools. Then PCR went down to the row and column pools of the positive box. Tbe intersection of the positive row(s) and column(s) revealed the candidate positive tubes. The specificity of PCR products were meanwhile checked hy restriction enzyme digestion. Finally, hybridization was carried out to get single specific eDNA clones-from the positive tlabes. This PCR-hased technique features high specificity, high efficiency and Les-cost in large-scale cDNA library screening. Our initial implementation of the technique resulted in the isolation of three longer different cDNA clones from a hnman fetal brain eDNA library. Thus this improved technique can serve as an alternative to the time-consuming and laborious conventional hybridization-hased metfiod for screening cDNA library.

  12. Human Vascular Endothelial Growth Factor cDNA Cloning and Expression in Osteoblasts

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Human vascular endothelial growth factor (VEGF) cDNA was amplified by nested polymerase chain reaction method from the HL60 cells. Then a pCD-hVEGF165 recombinant plasmid was constructed. Rabbit osteoblasts were transfected with pCD-hVEGF165 plasmid by lipofectin mediated gene transfer. The transient expressive results were detected by immunohistochemical method. It was observed that the expression of human VEGF gene was detected 72 h after transfecting distinctly.

  13. cDNA cloning and function analysis of two novel erythroid differentiation related genes

    Institute of Scientific and Technical Information of China (English)

    WANG; Xin; (王鑫); WANG; Duncheng; (王敦成); CHEN; Xing; (陈兴),; HU; Meiru; (胡美茹); WANG; Jian'an; (王建安); LI; Yan; (黎燕); GUO; Ning; (郭宁); SHEN; Beifen; (沈倍奋)

    2001-01-01

    Our previous studies showed that some nuclear proteins that were expressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen l-gtll human cDNA expression library of fetal liver in order to obtain the rele-vant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' func-tions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leucine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigat-ed the expression pattern of EDRF1 and EDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid develop-ment, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.

  14. cDNA cloning and function analysis of two novel erythroid differentiation related genes

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Our previous studies showed that some nuclear proteins that wereexpressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen l-gtll human cDNA expression library of fetal liver in order to obtain the rele-vant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' func-tions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leucine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigat-ed the expression pattern of EDRF1 and EDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid develop-ment, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.

  15. Single primer amplification (SPA) of cDNA for microarray expression analysis

    OpenAIRE

    2003-01-01

    The potential of expression analysis using cDNA microarrays to address complex problems in a wide variety of biological contexts is now being realised. A limiting factor in such analyses is often the amount of RNA required, usually tens of micrograms. To address this problem researchers have turned to methods of improving detection sensitivity, either through increasing fluorescent signal output per mRNA molecule or increasing the amount of target available for labelling by use of an amplific...

  16. Obtaining reliable information from minute amounts of RNA using cDNA microarrays

    OpenAIRE

    2002-01-01

    Abstract Background High density cDNA microarray technology provides a powerful tool to survey the activity of thousands of genes in normal and diseased cells, which helps us both to understand the molecular basis of the disease and to identify potential targets for therapeutic intervention. The promise of this technology has been hampered by the large amount of biological material required for the experiments (more than 50 μg of total RNA per array). We have modified an amplification procedu...

  17. cDNA2Genome: A tool for mapping and annotating cDNAs

    OpenAIRE

    Suhai Sandor; Glatting Karl-Heinz; del Val Coral

    2003-01-01

    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 problem...

  18. [cDNA cloning and sequence analysis of pluripotency genes in tree shrews (Tupaia belangeri)].

    Science.gov (United States)

    Wang, Cai-Yun; Ma, Yun-Han; He, Da-Jian; Yang, Shi-Hua

    2013-04-01

    In this paper, partial sequences of the tree shrew (Tupaia belangeri) Klf4, Sox2, and c-Myc genes were cloned and sequenced, which were 382, 612, and 485 bp in length and encoded 127, 204, and 161 amino acids, respectively. Whereas, their cDNA sequence identities with those of human were 89%, 98%, and 89%, respectively. Their phylogenetic tree results indicated different topologies and suggested individual evolutional pathways. These results can facilitate further functional studies.

  19. 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.

  20. Detection of HIV cDNA point mutations with rolling-circle amplification arrays.

    Science.gov (United States)

    Wu, Lingwei; Liu, Quanjun; Wu, Zhongwei; Lu, Zuhong

    2010-01-27

    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.

  1. Isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus) growth hormone.

    Science.gov (United States)

    Ayson, F G; de Jesus, E G; Amemiya, Y; Moriyama, S; Hirano, T; Kawauchi, H

    2000-02-01

    We report the isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus; Teleostei; Perciformes; Siganidae) growth hormone (GH). Rabbitfish GH was extracted from pituitary glands under alkaline conditions, fractionated by gel filtration chromatography on Sephadex G-100, and purified by high-performance liquid chromatography. The fractions containing GH were identified by immunoblotting with bonito GH antiserum. Under nonreducing conditions, the molecular weight of rabbitfish GH is about 19 kDa as estimated by SDS-PAGE. The purified hormone was potent in promoting growth in rabbitfish fry. Weekly intraperitoneal injections of the hormone significantly accelerated growth. This was evident 3 weeks after the start of the treatment, and its effect was still significant 2 weeks after the treatment was terminated. Rabbitfish GH cDNA was cloned to determine its nucleotide sequence. Excluding the poly (A) tail, rabbitfish GH cDNA is 860 base pairs (bp) long. It contained untranslated regions of 94 and 175 bp in the 5' and 3' ends, respectively. It has an open reading frame of 588 bp coding for a signal peptide of 18 amino acids and a mature protein of 178 amino acid residues. Rabbitfish GH has 4 cysteine residues. On the amino acid level, rabbitfish GH shows high identity (71-74%) with GHs of other perciforms, such as tuna, sea bass, yellow tail, bonito, and tilapia, and less (47-49%) identity with salmonid and carp GHs.

  2. cDNA Cloning and Sequence Analysis of Rice Sbel and Sbe3 Genes

    Institute of Scientific and Technical Information of China (English)

    CHENXiu-hua; LIUQiao-quan; WuHsin-kan; WANGZong-yang; GuMing-hong

    2004-01-01

    Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes SheI and Shed encoding SBE Ⅰ and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA libray, derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned SheI and Shed cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of She3 was the same as that of shed (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference,which resulted in changes of two deduced amino acids between the cloned She1 cDNA and the reported she1 (Genbank Accession No. D11082). The cloned SheI and Shed cDNAs make it possible to improve rice starch quality through genetic engineering.

  3. cDNA Cloning and Sequence Analysis of Rice Sbe1 and Sbe3 Genes

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiu-hua; LIU Qiao-quan; WU Hsin-kan; WANG Zong-yang; GU Ming-hong

    2004-01-01

    Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes Sbe1 and Sbe3 encoding SBE I and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA library derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned Sbe1 and Sbe3 cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of Sbe3 was the same as that of sbe3 (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference,which resulted in changes of two deduced amino acids between the cloned Sbe1 cDNA and the reported sbe1 (Genbank Accession No. D11082). The cloned Sbe1 and Sbe3 cDNAs make it possible to improve rice starch quality through genetic engineering

  4. 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.

  5. Development and validation of a bovine macrophage specific cDNA microarray

    Directory of Open Access Journals (Sweden)

    Waddington David

    2006-09-01

    Full Text Available Abstract Background The response of macrophages to danger signals is an important early stage in the immune response. Our understanding of this complex event has been furthered by microarray analysis, which allows the simultaneous investigation of the expression of large numbers of genes. However, the microarray resources available to study these events in livestock animals are limited. Results Here we report the development of a bovine macrophage specific (BoMP cDNA microarray. The BoMP microarray contains 5026 sequence elements (printed in duplicate and numerous controls. The majority of the clones incorporated on the microarray were derived from the BoMP cDNA library generated from bovine myeloid cells subjected to various stimuli, including over 900 sequences unique to the library. Additional clones representing immunologically important genes have been included on the BoMP microarray. The microarray was validated by investigating the response of bovine monocytes to stimulation with interferon-γ and lipopolysaccharide using amplified RNA. At 2 and 16 hours post stimulation 695 genes exhibited statistically significant differential expression, including; 26 sequences unique to the BoMP library, interleukin 6, prion protein and toll-like receptor 4. Conclusion A 5 K cDNA microarray has been successfully developed to investigate gene expression in bovine myeloid cells. The BoMP microarray is available from the ARK-Genomics Centre for Functional Genomics in Farm Animals, UK.

  6. Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA.

    Science.gov (United States)

    Michael, A J; Furze, J M; Rhodes, M J; Burtin, D

    1996-02-15

    A cDNA for a plant ornithine decarboxylase (ODC), a key enzyme in putrescine and polyamine biosynthesis, has been isolated from root cultures of the solanaceous plant Datura stramonium. Reverse transcription-PCR employing degenerate oligonucleotide primers representing conserved motifs from other eukaryotic ODCs was used to isolate the cDNA. The longest open reading frame potentially encodes a peptide of 431 amino acids and exhibits similarity to other eukaryotic ODCs, prokaryotic and eukaryotic arginine decarboxylases (ADCs), prokaryotic meso-diaminopimelate decarboxylases and the product of the tabA gene of Pseudomonas syringae cv. tabaci. Residues involved at the active site of the mouse ODC are conserved in the plant enzyme. The plant ODC does not possess the C-terminal extension found in the mammalian enzyme, implicated in rapid turnover of the protein, suggesting that the plant ODC may have a longer half-life. Expression of the plant ODC in Escherichia coli and demonstration of ODC activity confirmed that the cDNA encodes an active ODC enzyme. This is the first description of the primary structure of a eukaryotic ODC isolated from an organism where the alternative ADC routine to putrescine is present.

  7. Requirements in screening cDNA libraries for new genes and solutions offered by SBH technology

    Energy Technology Data Exchange (ETDEWEB)

    Drmanac, R.; Drmanac, S.; Labat, I.; Stavropoulos, N.

    1993-12-31

    Under different assumptions about the total number of genes, the number of housekeeping and tissue-specific genes, and the difference in the number of mRNAs per cell for functional and nonfunctional genes, significantly different results can be expected from screening random cDNA clones. We have developed gene expression models as a guide for interpretation of experimental results. For statistical, biological, and technical reasons, the search for 100,000 plus genes and discrimination between nonfunctional, housekeeping, and tissue-specific genes requires the analysis of up to 10 million clones from 20 to 50 tissues. Oligonucleotide hybridization of dense clone blots is an inexpensive and fast way to screen such large clone sets. Our preliminary results on control clones and thousands of cDNA clones from an infant brain library demonstrate the feasibility of the method. We present several models of gene expression and analyze the main factors which can influence the hunt for new genes via the screening of random cDNA libraries. The basic steps in the preparation and use of dense DNA dot arrays are described, and some results that demonstrate the feasibility and efficiency of gene inventorying by oligonucleotide hybridization are presented. Furthermore, partial SBH and single-pass gel sequencing are compared and a gene analysis scheme that combines the two approaches is discussed.

  8. 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.

  9. 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.

  10. Coordinate regulation of stromelysin and collagenase genes determined with cDNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Frisch, S.M.; Clark, E.J.; Werb, Z.

    1987-05-01

    Secreted proteinases are required for tumor metastasis, angiogenesis, and tissue remodeling during wound healing and embryonic growth. Thus, the regulation of the genes of secreted proteinases may serve as an interesting model for growth-controlled genes in general. The authors studied the genes of the secreted proteinases stromelysin and collagenase by using molecularly cloned cDNAs from each proteinase. Stromelysin cDNA was cloned by differential screening of a total cDNA library from rabbit synovial cells treated with phorbol 12-myristate 13-acetate, which yielded a clone of 1.2 kilobase pairs; collagenase cDNA was obtained by cloning reverse transcripts of anti-collagenase-immunoadsorbed polysomal mRNA, which yielded a clone of 0.8 kilobase pairs. Stromelysin and collagenase mRNA species of 2.2 and 2.4 kilobases, respectively, were detected on hybridization blots of RNA from phorbol 12-myristate 13-acetate-treated but not untreated rabbit synovial cells. Expression of stromelysin mRNA was also induced in rabbit alveolar macrophages and rabbit brain capillary endothelial cells treated with phorbol 12-myristate 13-acetate. Stromelysin and collagenase mRNA were both induced by phorbol 12-myristate 13-acetate and cytochalasin B at a constant ratio of the two gene products; this suggest coordinate regulation. The fact that induction was blocked after inhibition of protein synthesis by cycloheximide implicates an indirect signal transduction pathway that requires new protein synthesis.

  11. 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

  12. Isolation of a cDNA Encoding a Protease from Perinereis aibuhitensis Grube

    Institute of Scientific and Technical Information of China (English)

    Rong-Gui LI; Dong-Meng QIAN; Dao-Sen GUO; Gui-Cai DU; Zhi-Yong YAN; Bin WANG

    2006-01-01

    The cDNA encoding a protease of Perinereis aibuhitensis Grube (PPA) was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMALPPA. pMAL-PPA was introduced into Escherichia coli BL21(DE3) and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-β-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.

  13. CTL responses to Leishmania mexicana gp63-cDNA vaccine in a murine model.

    Science.gov (United States)

    Ali, S A; Rezvan, H; McArdle, S E; Khodadadi, A; Asteal, F A; Rees, R C

    2009-07-01

    Immunity to Leishmania is believed to be strongly dependent upon the activation of Th1 immune responses, although the exact role of cytotoxic T lymphocytes (CTLs) has not yet been determined. The aims of this study were to establish a suitable cytotoxicity assay to measure CTL activity and to compare immunity induced by Leishmania mexicana gp63 cDNA via i.m. injection and gene gun immunization in the BALB/c mouse model. The CTL activity was evaluated by short-term (51)Cr-release cytotoxicity assays against CT26 tumour cells transfected with L. mexicana gp63 cDNA and dendritic cells (DCs) loaded with soluble Leishmania antigen (SLA) as targets. The results clearly demonstrated that higher protection to L. mexicana infection was induced by gene gun DNA-immunization vs. i.m. injection. Cytotoxic T lymphocyte activity of splenocytes was observed in mice immunized either with L. mexicana gp63 cDNA or SLA and long-lived CTL activity was observed in immunized and/or re-challenged mice but not naïve mice infected with the parasite.

  14. Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu.

    Science.gov (United States)

    Wee, Ching Ching; Roslan, Hairul Azman

    2012-01-01

    Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zinc-binding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group.

  15. [Construction and analysis of subtractive cDNA library of Phellodendron amurense under drought stress].

    Science.gov (United States)

    Wang, Huimei; Wang, Yanbing; Zu, Yuangang; Sun, Lianhui

    2008-02-01

    With cDNA from Phellodendron amurense seedlings treated with drought stress as tester and cDNA from this plant in normal growth as driver, we construct cDNA subtracted library using suppression subtractive hybridization (SSH). In the library, the rate of recombination was 95%, the size of inserts was 300-800 bp. Two hundred and sixty-five new genes were obtained by DNA sequencing 816 positive clones picked randomly, and partitioned to 16 classes after nucleotide Blast and BlastX homological analysis against NT, NR, SWISSPROT, KEGG database. Forty-four drought stress associated genes, such as heat shock protein cognate 70, dehydration responsive protein 22, universal stress protein, metallothionein II, late embryogenesis abundant protein, were obtained, which made 16.6% of the overall genes. These genes included osmotic regulator, signal component regulatory protein and antioxidant enzyme. The research had established a basis for cloning stress resistance genes and further studying genes expression in P. amurense seedlings under drought stress.

  16. 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.

  17. 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.

  18. THE TREE SHREW APOLIPOPROTEIN C-I cDNA: SEQUENCE AND ITS EXPRESSION

    Institute of Scientific and Technical Information of China (English)

    王克勤; 吕新跃; 吴钢; 薛红; 陈保生

    2001-01-01

    A rabbit anti-serum to tree shrew apolipoprotein C-I (apo C-l) was used to screen an expression cDNA li-braDy constructed by us from tree shrew (TS) liver tissue. Two apo C-I cDNA clones were obtained. The longerone consists of 380 nucleotides, including 21 bp and 95 bp at the 5' and 3' end of the non-translated region srespectively, and a 2 64-bp fragment in an open reading frame encoding 88 amino acids prepropeptide which con-ta-ins 26 amino acids of signal peptide and a mature protein (62 amino acids). Comparing the amino-acid se-quence deduced from this cDNA with those of the published mammalian apo C-Is reveals that it shared some struc-tural similarity with zat, mouse and dog apo C-l, but it had 5 more amino acids than that of human and baboon.The expression of apo C-I mRNA in 8 different tissues were also assayed with Northern blot. The results demonstrat-ed that liver had the highest expression, intestine had much less expression and no expression in other tissues,which is much different from human and other species. This study has laid down a good foundation for further study-ing on the function and the stucture of tree shrew apo C-I gene.``

  19. 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.

  20. Isolation and characterization of sequences homologous to the tobacco clone axi 1 (auxin independent) from a Vicia sativa nodule cDNA library

    NARCIS (Netherlands)

    Yalçin-Mendi, Y.; Çetiner, S.; Bisseling, T.

    2001-01-01

    In this research, partial nucleotide sequences of the axi 1 gene, which is related to auxin perception and transduction, isolated from Vicia sativa using cDNA library screening were investigated. Four V. sativa cDNA clones representing homologous of the tobacco axi 1 (auxin independent) cDNA clone w

  1. PG25, a pineal-specific cDNA, cloned by differential display PCR (DDPCR) and rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Wang, X; Brownstein, M J; Young, W S

    1997-05-16

    Synthesis of melatonin in the mammalian pineal gland is regulated by the rhythmic expression of acetyl-CoA: serotonin N-acetyltransferase (SNAT) and other unknown factors. To screen for pineal-specific mRNAs potentially involved in melatonin synthesis and/or regulation, differential display PCR (DDPCR) was employed. We used 80 primer pairs and examined 40 bands of interest. One of the pineal specific clones (relative to brain and eye), PG25, was studied further. Hybridization histochemical and Northern analyses confirmed its tissue specificity. The size of the corresponding mRNA is 2.4 kb. A cDNA (2 kb) containing the coding region was obtained using a long-template PCR-based RACE technique. A data base search indicates that PG25 is highly homologous to a recently identified human lung endothelial cell-specific gene, ESM-1. Interestingly, not only the amino acid sequences but also the cDNA sequences, including the long 3' untranslated regions, are highly similar. This suggests that the conserved 3' untranslated region may carry information to regulate its own expression. Northern analysis revealed that PG25 is also expressed in the rat lung, but at a much lower (10%) level compared to the pineal. Finally, our work shows the feasibility of a fast, integrated PCR-based cloning method for obtaining long, potentially full-length cDNAs with restricted expression in anatomically complex regions of the brain. This protocol combining several existing methodologies is suitable for use with limited tissue sources and uses minimal amounts of isotopes.

  2. Construction of human and mouse brain cDNA libraries and isolation of full-length cDNAs

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    cDNA libraries from aborted human 3-month fetal brain,adult rat and mouse brain were constructed by using a yZAP express cDNA library construction kin.Low molecular weight fragments of the second strand cDNASA were removed by flowing through the Sepharose CL-4B column and the frractionated long,Middle,Short fragments and the combined fragments weire respectively inserted into clone vectors to construct the cDNA libraries of the brain of human 3-month fetus.The 5'ends of 1200 clones from each of human fetal brain cDNA libraries were sequenced.A total of 894 ESTs were obtained and some full-length clones were squenced.By andalyaing the se-quences,12 novel full-length cDNAs were obtained.

  3. Difference in gene expression of macrophage between normal spleen and portal hypertensive spleen idendified by cDNA microarray

    OpenAIRE

    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.

  4. 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.

  5. 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.

  6. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs.

    OpenAIRE

    1988-01-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we 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...

  7. Rapid Amplification of 5′ cDNA End of S. Liaotungensis Choline Monooxygenase Using Inverse PCR RACE

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Based on part of a known cDNA sequence of Suaeda Liaotungensis choline monooxygenase, the authors successfully cloned the 5′ cDNA end of Suaeda Lianotungensis choline monooxygenase using Inverse PCR RACE with a specially designed 5′-phosphated RT primer and two pairs of specific inverse PCR primers. Compared with the anchored PCR RACE, inverse PCR RACE has better specificity and higher amplification.

  8. Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Marcelo Bento (New York, NY); Bonaldo, Maria de Fatima (New York, NY)

    1998-01-01

    This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.

  9. Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

    Energy Technology Data Exchange (ETDEWEB)

    Soares, M.B.; Fatima Bonaldo, M. de

    1998-12-08

    This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods. 25 figs.

  10. ISOLATION AND CLONING OF cDNA OF GENE ENCODING FOR METALLOTHIONEIN TYPE 2 FROM MELASTOMA AFFINE

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    UTUT WIDYASTUT

    2009-01-01

    Full Text Available Metallothionein is an important protein for detoxifying heavy metal ions. h is research was conducted to isolate and clone cDNA of gene encoding for metallothionein type 2 from Melastoma affi ne . Total RNA was isolated from young leaves. Total cDNA was synthesized from the total RNA by reverse transcription. h e MaMt2 cDNA was successfully isolated by PCR technique. h e MaMt2 cDNA was inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into Escherichia coli DH5 α . DNA sequencing analysis showed that this cDNA is full length consisting of 246 pb encoding 81 amino acid residues. h is cDNA is identical to mRNA of AtMt2 from Arabidopsis thaliana. It does not contain any restriction sites found in the cloning sites of pGEM-T Easy. h e deduced protein of MaMT2 contains 14 cysteine residues distributed in the Cys-Cys, Cys-X-Cys, and Cys-X-X-Cys motifs

  11. Preparation of a subtractive cDNA library enriched in cDNAs which expressed at a high level in cultured senescent human fibroblasts.

    Science.gov (United States)

    Tahara, H; Hara, E; Tsuyama, N; Oda, K; Ide, T

    1994-03-30

    Subtracted cDNA library was prepared by subtracting [cDNA from young growing SV40-transformed human fibroblasts] from [cDNA from growing SV40-transformed fibroblasts in extended lifespan]. Isolated cDNA clones which expressed at high level in life-extended transformed cells also expressed at high level in normal senescent fibroblasts but did at low level in growing and growth-arrested young cells. Neither fibronectin nor procollagen cDNA was isolated. This cDNA library is useful for isolation of senescent-specific cDNA species which express at high level in normal senescent cells but at low level in growing and growth-arrested young cells, avoiding growth-arrest-specific cDNAs.

  12. 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/).

  13. Application of restriction display PCR technique in the preparation of cDNA microarray probes

    Institute of Scientific and Technical Information of China (English)

    Zhao-Hui Sun; Wen-Li Ma; Bao Zhang; Yi-Fei Peng; Wen-Ling Zheng

    2005-01-01

    AIM: To develop a simplified and efficient method for the preparation of hepatitis C virus (HCV) cDNA microarray probes.METHODS: With the technique of restriction display PCR (RD-PCR), restriction enzyme Sau3A I was chosen to digest the full-length HCV cDNAs. The products were classified and re-amplified by RD-PCR. We separated the differential genes by polyacrylamide gel electrophoresis and silver staining. Single bands cut out from the polyacrylamide gel were isolated. The third-round PCR was performed using the single bands as PCR template.The RD-PCR fragments were purified and cloned into the pMD18-T vector. The recombinant plasmids were extracted from positive clones, and the target gene fragments were sequenced. The cDNA microarray was prepared by spotting RD-PCR products to the surface of amino-modified glass slides using a robot. We validated the detection of microarray by hybridization and sequence analysis.RESULTS: A total of 24 different cDNA fragments ranging from 200 to 800 bp were isolated and sequenced,which were the specific gene fragments of HCV. These fragments could be further used as probes in microarray preparation. The diagnostic capability of the microarray was evaluated after the washing and scanning steps. The results of hybridization and sequence analysis showed that the specificity, sensitivity, accuracy, reproducibility,and linearity in detecting HCV RNA were satisfactory.CONCLUSION: The RD-PCR technique is of great value in obtaining a large number of size-comparable gene probes, which provides a speedy protocol in generating probes for the preparation of microarrays. Microarray prepared as such could be further optimized and applied in the clinical diagnosis of HCV.

  14. 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.

  15. Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays.

    Directory of Open Access Journals (Sweden)

    Christine M Costello

    2005-08-01

    Full Text Available BACKGROUND: The differential pathophysiologic mechanisms that trigger and maintain the two forms of inflammatory bowel disease (IBD, Crohn disease (CD, and ulcerative colitis (UC are only partially understood. cDNA microarrays can be used to decipher gene regulation events at a genome-wide level and to identify novel unknown genes that might be involved in perpetuating inflammatory disease progression. METHODS AND FINDINGS: High-density cDNA microarrays representing 33,792 UniGene clusters were prepared. Biopsies were taken from the sigmoid colon of normal controls (n = 11, CD patients (n = 10 and UC patients (n = 10. 33P-radiolabeled cDNA from purified poly(A+ RNA extracted from biopsies (unpooled was hybridized to the arrays. We identified 500 and 272 transcripts differentially regulated in CD and UC, respectively. Interesting hits were independently verified by real-time PCR in a second sample of 100 individuals, and immunohistochemistry was used for exemplary localization. The main findings point to novel molecules important in abnormal immune regulation and the highly disturbed cell biology of colonic epithelial cells in IBD pathogenesis, e.g., CYLD (cylindromatosis, turban tumor syndrome and CDH11 (cadherin 11, type 2. By the nature of the array setup, many of the genes identified were to our knowledge previously uncharacterized, and prediction of the putative function of a subsection of these genes indicate that some could be involved in early events in disease pathophysiology. CONCLUSION: A comprehensive set of candidate genes not previously associated with IBD was revealed, which underlines the polygenic and complex nature of the disease. It points out substantial differences in pathophysiology between CD and UC. The multiple unknown genes identified may stimulate new research in the fields of barrier mechanisms and cell signalling in the context of IBD, and ultimately new therapeutic approaches.

  16. Construction of the subtractive cDNA library of injured adult and fetal rabbit skins

    Institute of Scientific and Technical Information of China (English)

    张波; 刘大维; 王正国; 朱佩芳; 周继红; 蒋建新

    2004-01-01

    Objective: Early gestational mammalian fetuses possess the amazing ability to heal cutaneous wounds in a scarless fashion. Over the past years, scientists have been working to decipher the mechanisms underlying this regenerative repair. The remarkable phenotypic differences between fetal and adult healings behoves us to learn their characteristics in genetics, which represents potentially important mechanisms involved in wound repair observed in fetal versus adult tissues. In this sense, it is reasonable to construct subtractive cDNA library for future research.Methods: Middle laparotomy and hysterotomy were performed on pregnant rabbits at 20-day gestation to expose the fetal back, and a longitudinal incision through the skin was made on the back of the fetus. The traumatized fetal skin was harvested 12 hours post-operation, the fetus control and traumatized adult skin specimens were taken at the same time. dscDNA was synthesized from total RNA of skin samples with SMART technology. Taking one of the three samples as Tester respectively and the other two as Drivers, we obtained 1 forward and 2 reverse hybridization products. After being amplified with selective polymerase chain reaction, the products were inserted into a vector, and then transferred into E.coli HB101. The colonies were screened afterwards. Results: The wounded fetuses were alive for a long time even after birth. Every determinant step, such as RNA isolation, cDNA synthesis, Rsa I digestion, adaptor ligation and hybridization, was well-operated. Subtractive efficiency identification demonstrated that the suppression subtractive hybridization (SSH) was successful. Insertion into vector and transferring to E.coli were satisfactory. Conclusions: Instead of classic SSH, an improved SSH with 2 Drivers was applied for the experiment. Results confirmed that the improved program was reasonable and correct in both theory and practice. The subtractive cDNA library we have obtained is going to be used for

  17. 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.

  18. 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

    2014-10-27

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Alkhatib, H.M.; Chen, D.; Cherney, B.; Bhatia, K.; Notario, V.; Giri, C.; Stein, G.; Slattery, E.; Roeder, R.G.; Smulson, M.E.

    1987-03-01

    cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambdagt11 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 lambdagt11 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.

  20. 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.

  1. Mink serum amyloid A protein. Expression and primary structure based on cDNA sequences.

    Science.gov (United States)

    Marhaug, G; Husby, G; Dowton, S B

    1990-06-15

    The nucleotide sequences of two mink serum amyloid A (SAA) cDNA clones have been analyzed, one (SAA1) 776 base pairs long and the other (SAA2) 552 base pairs long. Significant differences were discovered when derived amino acid sequences were compared with data for apoSAA isolated from high density lipoprotein. Previous studies of mink protein SAA and amyloid protein A (AA) suggest that only one SAA isotype is amyloidogenic. The cDNA clone for SAA2 defines the "amyloid prone" isotype while SAA1 is found only in serum. Mink SAA1 has alanine in position 10, isoleucine in positions 24, 67, and 71, lysine in position 27, and proline in position 105. Residue 10 in mink SAA2 is valine while arginine and asparagine are at positions 24 and 27, respectively, all characteristics of protein AA isolated from mink amyloid fibrils. Mink SAA2 also has valine in position 67, phenylalanine in position 71, and amino acid 105 is serine. It remains unknown why these six amino acid substitutions render SAA2 more amyloidogenic than SAA1. Eighteen hours after lipopolysaccharide stimulation, mink SAA mRNA is abundant in liver with relatively minor accumulations in brain and lung. Genes encoding both SAA isotypes are expressed in all three organs while no SAA mRNA was detectable in amyloid prone organs, including spleen and intestine, indicating that deposition of AA from locally synthesized SAA is unlikely. A third mRNA species (2.2 kilobases) was identified and hybridizes with cDNA probes for mink SAA1 and SAA2. In addition to a major primary translation product (molecular mass 14,400 Da) an additional product with molecular mass 28,000 Da was immunoprecipitable.

  2. Cloning and expression analysis of human reticulon 4c cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    RTNs (reticulons) is a gene family related to the growth and differentiation of neuroendocrine cell. This family is composed of several members such as RTN1, RTN2 and RTN3. RTN1 and RTN2 have been proved to have 3 transcripts with different length. Because the RTN1c cDNA was involved in the sologenesis of small cell lung carcinoma (SCLC), it was selected as a bioinformatic probe to clone novel members of RTN family with the electric hybridization assistant new-gene cloning method (EHAC). A 1677-bp cDNA was identified from human brain cDNA library. The cDNA contains an intact open reading frame (ORF) which encodes a protein of 199 amino acids. This deduced protein is highly homologous to RTN1c, RTN2c and RTN3 with identities of 64.4%, 45.8% and 50.0% respectively. This new gene was named RTN4c (GenBank accession number: AF087901). Northern hybridization showed that the full length of RTN4c transcript is about 1.8 kb. It is hardly expressed in heart, placenta, lung, spleen, thymus, testis, ovary, small intestine and peripheral blood white cells; but it is highly expressed in the tissues of skeletal muscle, brain, liver and kidney, and less expressed in the pancreas, prostate and colon. Furthermore, Northern results also showed that there is a 2.3 kb transcript expressed in 14 tissues except liver and skeletal muscle; while another 5.0 kb transcript in brain, skeletal muscle and testis. By the electric hybridization walking, we obtained two full-length contigs with a length of 4632 and 2235 bp respectively. The former encodes a protein with 1192 amino acids and was defined as RTN4a; the latter encodes another protein with 373 amino acids, and was named RTN4b. The RTN4 gene was mapped to human chromosome 2p14-p13 region by the radiation hybridization (RH).

  3. License - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Standard License, as long as you comply with the following conditions: You must attribute this database in t...Budding yeast cDNA sequencing project License to Use This Database Last updated : 2010/02/15 You may use thi... of this database and the requirements you must follow in using this database. The Additional License specif...ecified in the Creative Commons Attribution-Share Alike 2.1 Japan . If you use data from this database, plea...n . The summary of the Creative Commons Attribution-Share Alike 2.1 Japan is found here . With regard to this database, you

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Lunner Sigbjørn

    2009-10-01

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

  6. 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.

  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. 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

  9. 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.

  10. Rat serum amyloid P component. Analysis of cDNA sequence and gene expression.

    Science.gov (United States)

    Dowton, S B; McGrew, S D

    1990-09-01

    cDNA clones for rat serum amyloid P component (SAP) were isolated, and the derived amino acid sequence for pre-SAP was determined from the complete nucleotide sequence. Rat SAP is encoded by approximately 1 kb of mRNA, and the mature SAP protein is predicted to be 208 amino acids long. An increase in hepatic mRNA levels for rat SAP was found after administration of lipopolysaccharide, and SAP mRNA levels in livers of unstimulated male rats were lower than in hepatic RNA from female rats.

  11. Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States); Kaur, G.P. [Temple Univ. Medical School, Philadelphia, PA (United States)] [and others

    1997-05-01

    We have isolated and sequenced a cDNA that encodes an apparent human orthologue of a rat sulfotransferase (ST) cDNA that has been referred to as {open_quotes}ST1C1{close_quotes} - although it was recently recommended that sulfotransferase proteins and cDNAs be abbreviated {open_quotes}SULT.{close_quotes} The new human cDNA was cloned from a fetal liver-spleen cDNA library and had an 888-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 62% identical with that encoded by the rat ST1C1 cDNA and included signature sequences that are conserved in all cytosolic SULT enzymes. Dot blot analysis of mRNA from 50 human tissues indicated that the cDNA was expressed in adult human stomach, kidney, and thyroid, as well as fetal kidney and liver. Northern blot analyses demonstrated that the major SULT1C1 mRNA in those same tissues was 1.4 kb in length. We next determined the partial human SULT1C1 gene sequence for a portion of the 5{prime}-terminus of one intron. That sequence was used to design SULT1C1 gene-specific primers that were used to perform the PCR with DNA from human/rodent somatic cell hybrids to demonstrate that the gene was located on chromosome 2. PCR amplifications performed with human chromosome 2/rodent hybrid cell DNA as template sublocalized SULT1C1 to a region between bands 2q11.1 and 2q11.2. 14 refs., 2 figs.

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

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    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.

  13. 人胎儿骨骼和关节RACE cDNA文库的构建%The construction of rapid amplification of cDNA ends cDNA libraries from human fetal bone and joint

    Institute of Scientific and Technical Information of China (English)

    梁晓媛; 龚瑶琴; 刘奇迹; 李江夏; 陈丙玺; 郭辰虹

    2001-01-01

    目的 建立人胎儿骨骼和关节快速扩增cDNA末端(rapid amplification of cDNA ends,RACE cDNA)文库,为分离骨骼和关节发育相关基因奠定基础。方法 采用改进的异硫氰酸胍-酚-氯仿-异戊醇一步法提取骨骼和关节总RNA,用TaKaRa公司生产的cDNA合成试剂盒合成平末端的双链cDNA,然后与衔接子连接。再用位于双链cDNA末端的通用引物扩增全部cDNA。结果 建立了从骨骼和关节构建RACE cDNA文库的方法,并用该方法成功地构建了人胎儿骨骼和关节RACE cDNA文库。结论 所构建的的利用少量总RNA构建RACE cDNA文库方法切实可行,所构建的文库适用于用RACE方法从中分离骨骼和关节发育相关基因。%Objective To construct rapid amplification cDNA ends(RACE) cDNA libraries from human fetal bone and joint and provide resources for isolation of bone- and joint- specific development-related genes.Methods Total RNA of bone and joint were extracted with the modified single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. The double-stranded end-blunted cDNA were synthesized using TaKaRa's cDNA synthesis kit and ligated to cassette adaptors. All of the cDNA molecules were amplified by a pair of common primers.Results A protocol for RACE cDNA library construction from bone and joint was established and two RACE cDNA libraries from human fetal bone and joint were successfully constructed.Conclusion The protocol of RACE cDNA library construction from limited materials proved to be simple and efficient and the library was suitable for RACE to isolate tissue-specific genes.

  14. Glutamate-gated chloride channel subunit cDNA sequencing of Cochliomyia hominivorax (Diptera: Calliphoridae): cDNA variants and polymorphisms.

    Science.gov (United States)

    Lopes, Alberto Moura Mendes; de Carvalho, Renato Assis; de Azeredo-Espin, Ana Maria Lima

    2014-09-01

    The New World screwworm (NWS) Cochliomyia hominivorax (Coquerel) is one of the major myiasis-causing flies that injures livestock and leads to losses of ~US$ 2.7 billions/year in the Neotropics. Ivermectin (IVM), a macrocyclic lactone (ML), is the most used preventive insecticide for this parasite and targets the glutamate-gated chloride (GLUCLα) channels. Several authors have associated altered GluClα homologues to MLs resistance in invertebrates, although studies about resistance in NWS are limited to other genes. Here, we aimed to characterise the NWS GluClα (ChGluClα) cDNA and to search for alterations associated with IVM resistance in NWS larvae from a bioassay. The open reading frame of the ChGluClα comprised 1,359 bp and encoded a sequence of 452 amino acids. The ChGluClα cDNAs of the bioassay larvae showed different sequences that could be splice variants, which agree with the occurrence of alternative splicing in GluClα homologues. In addition, we found cDNAs with premature stop codons and the K242R SNP, which occurred more frequently in the surviving larvae and was located close to mutation (L256F) involved in ML resistance. Although these alterations were in low frequency, the ChGluClα sequencing will allow further studies to find alterations in the gene of resistant natural populations.

  15. Molecular cloning of a cysteine proteinase cDNA from adult Ancylostoma ceylanicum by the method of rapid amplification of cDNA ends using polymerase chain reaction.

    Science.gov (United States)

    Mieszczanek, J; Kofta, W; Wedrychowicz, H

    2000-12-01

    The hookworm Ancylostoma ceylanicum is a parasite of great importance in human and veterinary medicine. The most promising vaccination trials against hookworm infections are based on antigens belonging to the proteinase family. The aim of the present research was to isolate a cysteine proteinase gene from A. ceylanicum. This was achieved by rapid amplification of cDNA ends using polymerase chain reaction (RACE-PCR). A set of consensus oligonucleotide primers was designed to anneal to the conserved coding regions of cysteine proteinase. The PCR products were cloned and sequenced. The novel sequence displayed a high degree of homology with genes of cysteine proteinases known from other hookworm species. In the coding region the nucleotide identity with accp-1, the cysteine proteinase gene of A. caninum, reaches 84.3%. Analysis of the expression of acey-1. the cysteine proteinase gene of A. ceylanicum, suggests that it is produced exclusively in the gland cells of either adult worms or blood-feeding stages of A. ceylanicum.

  16. Discovery of Phytophthora infestans genes expressed in planta through mining of cDNA libraries.

    Directory of Open Access Journals (Sweden)

    Roberto Sierra

    Full Text Available BACKGROUND: Phytophthora infestans (Mont. de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora--Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta. METHODOLOGY/PRINCIPAL FINDINGS: We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family. CONCLUSIONS/SIGNIFICANCE: We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant--oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta.

  17. Molecular cloning and characterization of a cDNA encoding the Paracoccidioides brasiliensis 135 ribosomal protein.

    Science.gov (United States)

    Jesuino, Rosália S A; Pereira, Maristela; Felipe, M Sueli S; Azevedo, Maristella O; Soares, Célia M A

    2004-06-01

    A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library. Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0. The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes. Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly. The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P. brasiliensis. The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P. brasiliensis. The cDNA was deposited in GenBank under accession number AF416509.

  18. Expression of HNP1cDNA in CHO-dhfr- cells

    Institute of Scientific and Technical Information of China (English)

    LIU Juan; SUN Yong-tao; DU De-wei; WANG Lin-xu; ZHAI Song; WANG Shao-yang; WANG Ding-cheng

    2004-01-01

    To prepare secretary recombinant human neutrophil peptide1 (HNP1)and test its antimicrobialactivity. Methods: The eukaryotic expression vector pcDNA3. 1/V5-His-TOPO-HNP1 was cotransfected with plasmidpDCH1P11 carrying dhfr gene into dhfr- negative CHO (CHO-dhfr-) cells and recombinant protein was verified by ELISA;G418 selective medium was used to screen the stably expressing cell clones followed by serial passages in 5 × 10-8 mol/L and5 × 10-7 mol/L methotrexate (MTX) for gene amplification. Finally 4 cell clones with high expression level were obtainedand confirmed by ELISA, RT-PCR and IFA. The bacteriastatic activity of concentrated supernatants was tested in vitro asthat was almost 200-fold increase than that in G418 selective medium. 303 bp segments were amplified from 4 tably tranfec-tant cloneswhich matched the length of HNP1 cDNA by RT-PCR. Strong fluorescence was visible in cell plasma in the sta-blly transfectant cells by IFA. K-B disc agar diffusion test showed obvious bacteriastatic diffusion on MH plate of E. Coli.Conclusion: HNP1cDNA can be strongly expressed in CHO-dhfr- cells, which supernatants exhibited high inhibitive effectagainst bacteria.

  19. Identification of ABA-responsive genes in rice shoots via Cdna macroarray

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Phytohormone abscisic acid (ABA) was critical for many plant growth and developmental processesincluding seed maturation, germination and response to environmental factors. With the purpose to detectthe possible ABA related signal transduction pathways, we tried to isolate ABA-regulated genes throughcDNA macroarray technology using ABA-treated rice seedling as materials (under treatment for 2, 4, 8 and12 h). Of 6144 cDNA clones tested, 37 differential clones showing induction or suppression for at least onetime, were isolated. Of them 30 and 7 were up- or down-regulated respectively. Sequence analyses revealedthat the putative encoded proteins were involved in different possible processes, including transcription,metabolism and resistance, photosynthesis, signal transduction, and seed maturation. 6 cDNA clones werefound to encode proteins with unknown functions. Regulation by ABA of 7 selected clones relating to signaltransduction or metabolism was confirmed by reverse transcription PCR. In addition, some clones werefurther shown to be regulated by other plant growth regulators including auxin and brassinosteroid, which,however, indicated the complicated interactions of plant hormones. Possible signal transduction pathwaysinvolved in ABA were discussed.

  20. cDNA Cloning of Two Novel T-superfamily Conotoxins from Conus leopardus

    Institute of Scientific and Technical Information of China (English)

    Wei-Hua CHEN; Yu-Hong HAN; Qi WANG; Xiao-Wei MIAO; Ling OU; Xiao-Xia SHAO

    2006-01-01

    The full-length cDNAs of two novel T-superfamily conotoxins, Lp5.1 and Lp5.2, were cloned from a vermivorous cone snail Conus leopardus using 3′/5′-rapid amplification of cDNA ends. The cDNA of Lp5.1 encodes a precursor of 65 residues, including a 22-residue signal peptide, a 28-residue propeptide and a 15-residue mature peptide. Lp5.1 is processed at the common signal site -X-Arg- immediately before the mature peptide sequences. In the case of Lp5.2, the precursor includes a 25-residue signal peptide and a 43-residue sequence comprising the propeptide and mature peptide, which is probably cleaved to yield a 29-residue propeptide and a 14-residue mature toxin. Although these two conotoxins share a similar signal sequence and a conserved disulfide pattern with the known T-superfamily, the pro-region and mature peptides are of low identity, especially Lp5.2 with an identity as low as 10.7% compared with the reference Mr5. 1a.The elucidated cDNAs of these two toxins will facilitate a better understanding of the species distribution,the sequence diversity of T-superfamily conotoxins, the special gene structure and the evolution of these peptides.

  1. Construction of Hypericin Gland-Specific cDNA Library via Suppression Subtractive Hybridization.

    Science.gov (United States)

    Singh, Rupesh Kumar; Hou, Weina; Franklin, Gregory

    2016-01-01

    Hypericin, an important determinant of the pharmacological properties of the genus Hypericum, is considered as a major molecule for drug development. However, biosynthesis and accumulation of hypericin is not well understood. Identification of genes differentially expressed in tissues with and without hypericin accumulation is a useful strategy to elucidate the mechanisms underlying the development of the dark glands and hypericin biosynthesis. Suppression Subtractive Hybridization (SSH) is a unique method for PCR-based amplification of specific cDNA fragments that differ between a control (driver) and experimental (tester) transcriptome. This technique relies on the removal of dsDNA formed by hybridization between a control and test sample, thus eliminating cDNAs of similar abundance, and retaining differentially expressed or variable in sequence cDNAs. In our laboratory we applied this method to identify the genes involved in the development of dark glands and accumulation of hypericin in Hypericum perforatum. Here we describe the complete procedure for the construction of hypericin gland-specific subtracted cDNA library.

  2. Primary analysis of the expressed sequence tags in a pentastomid nymph cDNA library.

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    Full Text Available BACKGROUND: Pentastomiasis is a rare zoonotic disease caused by pentastomids. Despite their worm-like appearance, they are commonly placed into a separate sub-class of the subphylum Crustacea, phylum Arthropoda. However, until now, the systematic classification of the pentastomids and the diagnosis of pentastomiasis are immature, and genetic information about pentastomid nylum is almost nonexistent. The objective of this study was to obtain information on pentastomid nymph genes and identify the gene homologues related to host-parasite interactions or stage-specific antigens. METHODOLOGY/PRINCIPAL FINDINGS: Total pentastomid nymph RNA was used to construct a cDNA library and 500 colonies were sequenced. Analysis shows one hundred and ninety-seven unigenes were identified. In which, 147 genes were annotated, and 75 unigenes (53.19% were mapped to 82 KEGG pathways, including 29 metabolism pathways, 29 genetic information processing pathways, 4 environmental information processing pathways, 7 cell motility pathways and 5 organismal systems pathways. Additionally, two host-parasite interaction-related gene homologues, a putative Kunitz inhibitor and a putative cysteine protease. CONCLUSION/SIGNIFICANCE: We first successfully constructed a cDNA library and gained a number of expressed sequence tags (EST from pentastomid nymphs, which will lay the foundation for the further study on pentastomids and pentastomiasis.

  3. 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.

  4. Infectivity and complete nucleotide sequence of cucumber fruit mottle mosaic virus isolate Cm cDNA.

    Science.gov (United States)

    Rhee, Sun-Ju; Hong, Jin-Sung; Lee, Gung Pyo

    2014-07-01

    Three isolates of cucumber fruit mottle mosaic virus (CFMMV) were collected from melon, cucumber, and pumpkin plants in Korea. A full-length cDNA clone of CFMMV-Cm (melon isolate) was produced and evaluated for infectivity after T7 transcription in vitro (pT7CF-Cmflc). The complete CFMMV genome sequence of the infectious clone pT7CF-Cmflc was determined. The genome of CFMMV-Cm consisted of 6,571 nucleotides and shared high nucleotide sequence identity (98.8 %) with the Israel isolate of CFMMV. Based on the infectious clone pT7CF-Cmflc, a CaMV 35S-promoter driven cDNA clone (p35SCF-Cmflc) was subsequently constructed and sequenced. Mechanical inoculation with RNA transcripts of pT7CF-Cmflc and agro-inoculation with p35SCF-Cmflc resulted in systemic infection of cucumber and melon, producing symptoms similar to those produced by CFMMV-Cm. Progeny virus in infected plants was detected by RT-PCR, western blot assay, and transmission electron microscopy.

  5. cDNA cloning, characterization, and expression analysis of the Rac1 gene from Scophthalmus maximus.

    Science.gov (United States)

    Jia, Airong; Zhang, Xiao-Hua

    2009-09-01

    Rac1 is a small GTP-binding protein that belongs to the Rho small GTPases, which are important signaling molecules that regulate the dynamics of the actin cytoskeleton and mediate changes in cell morphology and motility. The EST sequence of Rac1 from turbot (Scophthalmus maximus L.) was obtained from a subtractive cDNA library previously. In this study, the full-length cDNA sequence of turbot Rac1 was obtained, which was 2420 nucleotides (nt) encoding a protein of 192 amino acids, with a putative molecular weight of 21.3 kDa. At the amino-acid level, turbot Rac1 was highly conserved to previously characterized GTPases of Rac sub-family, and was nearly identical to human Rac1 (95.3% identity). Quantitative real-time PCR demonstrated that the Rac1 was constitutively expressed in all tissues examined, but at different levels. Upon challenge with Vibrio harveyi, the expression level of Rac1 fluctuated in the liver at different time points. In the head kidney, its expression level decreased to the lowest at 4 h, and then increased to the background level at 24 h. The remarkable degree of evolutionary conservation observed in turbot Rac1 primary structure together with its changing in expression level upon challenge suggested a functionally important role for this Rho family member in the immune response.

  6. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rokeach, L.A.; Haselby, J.A.; Hoch, S.O. (Agouron Institute, La Jolla, CA (USA))

    1988-07-01

    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){sub 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.

  8. Nucleotide sequence of cloned cDNA for human pancreatic kallikrein.

    Science.gov (United States)

    Fukushima, D; Kitamura, N; Nakanishi, S

    1985-12-31

    Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed.

  9. Rabbit serum amyloid protein A: expression and primary structure deduced from cDNA sequences.

    Science.gov (United States)

    Rygg, M; Marhaug, G; Husby, G; Dowton, S B

    1991-12-01

    Serum amyloid A protein (SAA), the precursor of amyloid protein A (AA) in deposits of secondary amyloidosis, is an acute phase plasma apolipoprotein produced by hepatocytes. The primary structure of SAA demonstrates high interspecies homology. Several isoforms exist in individual species, probably with different amyloidogenic potential. The nucleotide sequences of two different rabbit serum amyloid A cDNA clones have been analysed, one (corresponding to SAA1) 569 base pairs (bp) long and the other (corresponding to SAA2) 513 bp long. Their deduced amino acid sequences differ at five amino acid positions, four of which are located in the NH2-terminal region of the protein. The deduced amino acid sequence of SAA2 corresponds to rabbit protein AA previously described except for one amino acid in position 22. Eighteen hours after turpentine stimulation, rabbit SAA mRNA is abundant in liver, while lower levels are present in spleen. None of the other extrahepatic organs studied showed any SAA mRNA expression. A third mRNA species (1.9 kb) hybridizing with a single-stranded RNA probe transcribed from the rabbit SAA cDNA, was identified. SAA1 and SAA2 mRNA were found in approximately equal amounts in turpentine-stimulated rabbit liver, but seem to be coordinately decreased after repeated inflammatory stimulation.

  10. cDNA heterogeneity suggests structural variants related to the high-affinity IgE receptor.

    Science.gov (United States)

    Liu, F T; Albrandt, K; Robertson, M W

    1988-08-01

    The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, we identified a number of cDNA clones from a rat basophilic leukemia cell cDNA library. Nucleotide sequencing established four different forms of cDNA: one is nearly identical to the published cDNA; the second differs from the first in the 5' untranslated sequence; the other two forms use either one or the other of the 5'-end sequences as above and lack 163 base pairs in the region coding for the second extracellular domain. RNase protection analysis with radioactive RNA probes established the heterogeneity of rat basophilic leukemia cell mRNA with regard to both the 5' and the internal sequences. Our results suggest the existence of at least four different protein forms related to the alpha subunit of the high-affinity IgE receptor.

  11. 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.

  12. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.

    1987-04-01

    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  13. 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

    2016-08-24

    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.

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

    Directory of Open Access Journals (Sweden)

    Amiruddin Nadzirah

    2012-01-01

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

  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. 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.

  17. Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

    Science.gov (United States)

    Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

    1997-02-01

    Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

  18. Cloning of human brevican cDNA and expression of its mRNA in human glioma

    Institute of Scientific and Technical Information of China (English)

    韩唏; 董艳; 由振东; 何成; 卢亦成

    2003-01-01

    Objective:To clone the cDNA of human brevican secreting isoform and to investigate its mRNA expression in human glioma.Methods:The full-length cDNA of human brevican secreted isoform was cloned from a human ahaplastic astrocytoma by RT-PCR,and the expression of human brevican mRNA in 22 cases of human glioma and 13 cases of non-glial brain tumors were investigated by in situ hybridization.Results:The cDNA which including the whole open reading frame of human brevican secreted isoform was obtained.In situ hybridization showed that brevican positive cells were present in all of the 22 cases of gliomas(100%),whereas none were found in the 13 cases of non-glial and metastasis brain tumors examined.Conclusion:The results suggest that brevican mRNA is highly and specifically expressed in human glioma.

  19. Screening cDNA Libraries Using Partial Probes to Isolate Full-Length cDNAs from Vascular Cells.

    Science.gov (United States)

    Csortos, C; Lazar, V; Garcia, J G

    1999-01-01

    The purpose of screening cDNA libraries is to isolate a particular cDNA clone encoding a mRNA and by implication, a protein, of interest. The screening is based on identification of the desired clone among a large number of recombinant clones within the library selected (1,2). As an example of both the utility and power of library screening, we will relate our own library screening efforts utilized to isolate the nonmuscle high molecular weight myosin light chain kinase isoform from a human umbilical vein endothelial cell cDNA library (3). This unique nonmuscle myosin light chain kinase isoform phosphorylates myosin light chains, thereby playing an essential role in agonist-mediated endothelial cell contraction, paracellular gap formation and increased vascular permeability. We are hopeful that this step-by-step approach will help the reader to understand the discussed methods.

  20. The cDNA and Genomic DNA Organization of a Novel Toxin SHT-I from Spider Ornithoctonus huwena

    Institute of Scientific and Technical Information of China (English)

    Peng QIAO; Xiao-Pan ZUO; Zhi-Fang CHAI; Yong-Hua JI

    2004-01-01

    In present study the full-length cDNA of a novel toxin from the venom gland of spider Ornithoctonus huwena, named as SHT-I, has been cloned using the strategy of rapid amplification of cDNA ends, and then the whole genomic sequence of SHT-I (Selenocosmia huwena toxin-I) was determined using sequence-specific primers synthesized based on the acquired 3' and 5' ends of SHT-I cDNA sequence. It is unexpectedly found that intron was lacking in the genomic sequence of SHT-I. The result might evoke an interesting question whether the gene code of inhibitor cystine-knot peptides from spider venom is distinct from that of those known toxic peptides of scorpion and cone snail.

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

    Energy Technology Data Exchange (ETDEWEB)

    Grundmann, U.; Abel, K.J.; Bohn, H.; Loebermann, H.; Lottspeich, F.; Kuepper, H. (Research Institutes, Postfach (West Germany))

    1988-06-01

    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 10{sup 6} 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.

  2. Nucleic acid (cDNA) and amino acid sequences of alpha-type gliadins from wheat (Triticum aestivum).

    Science.gov (United States)

    Kasarda, D D; Okita, T W; Bernardin, J E; Baecker, P A; Nimmo, C C; Lew, E J; Dietler, M D; Greene, F C

    1984-01-01

    The complete amino acid sequence for an alpha-type gliadin protein of wheat (Triticum aestivum Linnaeus) endosperm has been derived from a cloned cDNA sequence. An additional cDNA clone that corresponds to about 75% of a similar alpha-type gliadin has been sequenced and shows some important differences. About 97% of the composite sequence of A-gliadin (an alpha-type gliadin fraction) has also been obtained by direct amino acid sequencing. This sequence shows a high degree of similarity with amino acid sequences derived from both cDNA clones and is virtually identical to one of them. On the basis of sequence information, after loss of the signal sequence, the mature alpha-type gliadins may be divided into five different domains, two of which may have evolved from an ancestral gliadin gene, whereas the remaining three contain repeating sequences that may have developed independently. Images PMID:6589619

  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. Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica*

    OpenAIRE

    2005-01-01

    To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosoma japonicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes. Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Ampli...

  5. 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.

  6. FY 1999 report on the survey of Research Association for biotechnology development. Trend survey on the structural analysis of full length cDNA; 1999 nendo biotechnology kaihatsu gijutsu kenkyu kumiai chosa hokokusho. Kanzen cho cDNA no kozo kaiseki ni kansuru doko chosa

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    Recognizing that the acquisition and structural analysis of full length cDNA clones are important subjects for connecting genome study and proteomics, survey was made of projects and technical trends in each country. The results of the survey were divided into the following four items: 1) trend of full length cDNA projects overseas; 2) study of full length cDNA in Japan; 3) full length cDNA project in Japan; 4) survey on technical trends of the structural analysis of full length cDNA. In 4), studies were made on the following: trend of technical development on the structural analysis of full length cDNA, trend of patents on the making of full length cDNA library, outline of the technology for the making of full length cDNA library. Countries for survey were the U.S., Japan, Germany, France and the U.K., and patents for survey were Japan open patents, U.S. open patents and WPI patents. For reference, included were seven data on full length cDNA related general remarks in Japanese, full length cDNA library related papers in English, full length cDNA related trend in Japan, etc. (NEDO)

  7. 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.

  8. Cloning Full-Length cDNAs from Vascular Tissues and Cells by Rapid Amplification of cDNA Ends (RACE) and RT-PCR.

    Science.gov (United States)

    Shen

    1999-01-01

    The isolation of full-length cDNAs remains a frequent task undertaken in many laboratories. A full-length cDNA is often desirable for one of the following purposes: 1) to complete the sequence of a partial cDNA cloned by library screenings or the yeast one- or two-hybrid system; 2) to derive the cDNA sequence encoding a protein, based on peptide sequences; 3) to obtain the sequence of a reported cDNA for functional analysis or expression studies; and 4) to define exon/intron boundaries of a cloned gene or determine transcription start site(s) of a promoter.

  9. Targeted rapid amplification of cDNA ends (T-RACE)—an improved RACE reaction through degradation of non-target sequences

    OpenAIRE

    Neil I Bower; Johnston, Ian A

    2010-01-01

    Amplification of the 5' ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3' end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-contain...

  10. Nucleotide sequence of cDNA coding for dianthin 30, a ribosome inactivating protein from Dianthus caryophyllus.

    Science.gov (United States)

    Legname, G; Bellosta, P; Gromo, G; Modena, D; Keen, J N; Roberts, L M; Lord, J M

    1991-08-27

    Rabbit antibodies raised against dianthin 30, a ribosome inactivating protein from carnation (Dianthus caryophyllus) leaves, were used to identify a full length dianthin precursor cDNA clone from a lambda gt11 expression library. N-terminal amino acid sequencing of purified dianthin 30 and dianthin 32 confirmed that the clone encoded dianthin 30. The cDNA was 1153 basepairs in length and encoded a precursor protein of 293 amino acid residues. The first 23 N-terminal amino acids of the precursor represented the signal sequence. The protein contained a carboxy-terminal region which, by analogy with barley lectin, may contain a vacuolar targeting signal.

  11. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    OpenAIRE

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-bindin...

  12. Simple and inexpensive three-step rapid amplification of cDNA 5′ ends using 5′ phosphorylated primers

    OpenAIRE

    Dallmeier, Kai; Neyts, Johan

    2013-01-01

    Rapid amplification of cDNA 5′ ends (5′-RACE) is routinely used for the sequence analysis of the upstream noncoding regions of cellular mRNAs; however, it represents a tedious and cost-intensive procedure. By employing 5′ phosphorylated gene-specific primers for first-strand cDNA synthesis, we cut short the previously established reverse ligation and amplification protocol of Mandl and coworkers (BioTechniques, 1991, vol. 10, pp. 484–486) to a streamlined three-step procedure that no longer d...

  13. Isolation of novel human cDNA (hGMF-gamma) homologous to Glia Maturation Factor-beta gene.

    Science.gov (United States)

    Asai, K; Fujita, K; Yamamoto, M; Hotta, T; Morikawa, M; Kokubo, M; Moriyama, A; Kato, T

    1998-03-13

    A novel full-length human cDNA homologous to Glia Maturation Factor-beta (GMF-beta) gene was isolated. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein sequence of 142 amino acid residues. The deduced amino acid sequences of its putative product is highly homologous to human GMF-beta (82% identity) and named for GMF-gamma. Northern blot analysis indicated that a message of 0.9 kb long, but not 4.1 kb of GMF-beta, is predominantly expressed in human lung, heart, and placenta.

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

    OpenAIRE

    Gray, P W; Barrett, K; Chantry, D; Turner, M.; Feldmann, M

    1990-01-01

    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 TNF alpha with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surf...

  15. Nucleic acid (cDNA) and amino acid sequences of alpha-type gliadins from wheat (Triticum aestivum).

    OpenAIRE

    Kasarda, D.D.; Okita, T W; Bernardin, J. E.; Baecker, P A; Nimmo, C C; Lew, E J; Dietler, M D; Greene, F C

    1984-01-01

    The complete amino acid sequence for an alpha-type gliadin protein of wheat (Triticum aestivum Linnaeus) endosperm has been derived from a cloned cDNA sequence. An additional cDNA clone that corresponds to about 75% of a similar alpha-type gliadin has been sequenced and shows some important differences. About 97% of the composite sequence of A-gliadin (an alpha-type gliadin fraction) has also been obtained by direct amino acid sequencing. This sequence shows a high degree of similarity with a...

  16. 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

  17. Ultrasensitive cDNA detection of dengue virus RNA using electrochemical nanoporous membrane-based biosensor.

    Directory of Open Access Journals (Sweden)

    Varun Rai

    Full Text Available A nanoporous alumina membrane-based ultrasensitive DNA biosensor is constructed using 5'-aminated DNA probes immobilized onto the alumina channel walls. Alumina nanoporous membrane-like structure is carved over platinum wire electrode of 76 µm diameter dimension by electrochemical anodization. The hybridization of complementary target DNA with probe DNA molecules attached inside the pores influences the pore size and ionic conductivity. The biosensor demonstrates linear range over 6 order of magnitude with ultrasensitive detection limit of 9.55×10(-12 M for the quantification of ss-31 mer DNA sequence. Its applicability is challenged against real time cDNA PCR sample of dengue virus serotype1 derived from asymmetric PCR. Excellent specificity down to one nucleotide mismatch in target DNA sample of DENV3 is also demonstrated.

  18. Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase.

    Science.gov (United States)

    Faletto, M B; Koser, P L; Battula, N; Townsend, G K; Maccubbin, A E; Gelboin, H V; Gurtoo, H L

    1988-09-01

    Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.

  19. Expression and Characterization of HGV E2 cDNA in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A 559 base pair fragment of cDNA locating at the putative E2 region of GBV-C/HGV was in-serted into Pichia pastoris expression vector pPICgK in the reading frame of α-factor secreting signal pep-tide. The recombinant expression plasmid pPIC9K-E2 was introduced into P. pastoris GSll5 with electro-poration and recombined with the host genome by homological recombination. The His+Mut+ recombinantyeasts were selected and cultivated in the BMMY medium. After 3 days induction with 0. 5% methanol,the target protein(E2) accumulated up to 30% of total proteins in the supernatant. The expressed E2 pro-tein was proved possessing antigenicity and high specificity with Western blot and ELISA probed with serafrom the immunized rabbits and the patients infected by GBV-C/HGV.

  20. Isolation and characterization of a cDNA encoding a CBF transcription factor from E. globulus.

    Science.gov (United States)

    Gamboa, Maria C; Rasmussen-Poblete, Susana; Valenzuela, Pablo D T; Krauskopf, Erwin

    2007-01-01

    The transcription factors CBF/DREB play an important role during low temperature, drought and high-salt stress in higher plants. In this work, we isolated one full-length CBF cDNA clone from the angiosperm Eucalyptus globulus. The derived peptide sequence reveals that it encodes a transcriptional activator that has all the characteristic motifs present in CBF proteins previously described in Arabidopsis and tomato. RT-PCR analysis shows that EgCBF1 is transiently induced in E. globulus seedlings that had been exposed to low temperature within the first 15 min. These results suggest that the isolated CBF gene participates in the cold responsive pathway of E. globulus.

  1. Generation of Infectious Poliovirus with Altered Genetic Information from Cloned cDNA.

    Science.gov (United States)

    Bujaki, Erika

    2016-01-01

    The effect of specific genetic alterations on virus biology and phenotype can be studied by a great number of available assays. The following method describes the basic protocol to generate infectious poliovirus with altered genetic information from cloned cDNA in cultured cells.The example explained here involves generation of a recombinant poliovirus genome by simply replacing a portion of the 5' noncoding region with a synthetic gene by restriction cloning. The vector containing the full length poliovirus genome and the insert DNA with the known mutation(s) are cleaved for directional cloning, then ligated and transformed into competent bacteria. The recombinant plasmid DNA is then propagated in bacteria and transcribed to RNA in vitro before RNA transfection of cultured cells is performed. Finally, viral particles are recovered from the cell culture.

  2. 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.

  3. Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts.

    Science.gov (United States)

    Tillett, D; Burns, B P; Neilan, B A

    2000-03-01

    A simple, efficient and sensitive RACE-based procedure was developed for the determination of unknown 5' regions from bacterial cDNA. A number of critical modifications were made to the standard RACE method, including the optimization of the RNA extraction, reverse transcription and PCR conditions. This procedure was used to accurately determine the site of transcript initiation and structure of the promoter region of the Helicobacter pylori aspartate carbamoyltransferase gene (pyrB). The technique avoids many of the difficulties associated with established bacterial transcript mapping protocols and can be performed in two days starting with less than 1 microgram of total RNA. The modifications described here have significant potential for the identification of transcript start sites of bacterial genes and non-polyadenylated eukaryotic RNA.

  4. Production of Arrayed and Rearrayed cDNA Libraries for Public Use

    Energy Technology Data Exchange (ETDEWEB)

    Rasmussen, K

    2005-08-29

    Researchers studying genes and their protein products need an easily available source for that gene. The I.M.A.G.E. Consortium at Lawrence Livermore National Laboratory is an important source of such genes in the form of arrayed cDNA libraries. The arrayed clones and associated data are available to the public, free of restriction. Libraries are transformed and titered into 384-well master plates, from which 2-8 copies are made. One copy plate is stored by LLNL while others are sent to sequencing groups, plate distributors, and to the group which contributed the library. Clones found to be unique and/or full-length are rearrayed and also made publicly available. Bioinformatics tools supporting the use of I.M.A.G.E. clones are accessible via the World Wide Web.

  5. [Development of new SSR markers from EST of SSH cDNA libraries on rose fragrance].

    Science.gov (United States)

    Yan, Hui-Jun; Zhang, Hao; Xie, Ji-Rong; Li, Shu-Fa; Jian, Hong-Ying; Qiu, Xian-Qin; Wang, Qi-Gang; Wang, Ji-Hua; Tang, Kai-Xue

    2009-09-01

    The new SSR markers of rose related fragrance were developed based on the SSH cDNA libraries of rose floral scent mutant. In this study, 10 EST-SSRs (2.6%) from 391 ESTs in the libraries were identified. Six EST-SSRs primers were designed to sequence flanking SSRs. The primer pairs designed were screened on the wild-type Jinyindao, which has flowers full of pleasant scent, and the mutant-type Wangriqinghuai without perceivable floral scent. Five primer pairs were amplified effectively in Jinyindao and Wangriqinghuai, and 3 were polymorphic between Jinyindao and Wangriqinghuai. Eighteen rose cultivars including fragrant roses and nonfragrant roses were identified by the five prime pairs. These results proved that EST-SSR markers are effective markers to identify the polymorphism of the rose.

  6. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    赵勇[1; 余龙[2; 高洁[3; 付强[4; 华益民[5; 张宏来[6; 赵寿元[7

    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

  7. Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Dufva, I.H.; Dufva, Hans Martin

    2006-01-01

    Reverse transcription of RNA is an invaluable method for gene expression analysis by real-time PCR or microarray methods. Random primers of varying lengths were compared with respect to their efficiency of priming reverse transcription reactions. The results showed that l5-nucleotide-long random...... with cDNA generated with random hexamers. The increased efficiency of priming using random pentadecamers resulted in reverse transcription of > 80% of the template aRNA, while random hexamers induced reverse transcription of only 40% of the template aRNA. This suggests a better coverage...... of the transcriptome when using random pentadecamers over random hexamers. Using the same amount of aRNA as starting material, random pentadecamer-printed reactions resulted in 11-fold more genes being detected in whole transcriptome DNA microarray experiments than random hexamer-primed reactions. The results indicate...

  8. FPGA based system for automatic cDNA microarray image processing.

    Science.gov (United States)

    Belean, Bogdan; Borda, Monica; Le Gal, Bertrand; Terebes, Romulus

    2012-07-01

    Automation is an open subject in DNA microarray image processing, aiming reliable gene expression estimation. The paper presents a novel shock filter based approach for automatic microarray grid alignment. The proposed method brings up significantly reduced computational complexity compared to state of the art approaches, while similar results in terms of accuracy are achieved. Based on this approach, we also propose an FPGA based system for microarray image analysis that eliminates the shortcomings of existing software platforms: user intervention, increased computational time and cost. Our system includes application-specific architectures which involve algorithm parallelization, aiming fast and automated cDNA microarray image processing. The proposed automated image processing chain is implemented both on a general purpose processor and using the developed hardware architectures as co-processors in a FPGA based system. The comparative results included in the last section show that an important gain in terms of computational time is obtained using hardware based implementations.

  9. Molecular Cloning and Characterization of cDNA Encoding Fibrinolytic Enzyme-3 from Earthworm Eisenia foetida

    Institute of Scientific and Technical Information of China (English)

    Guo-Qing DONG; Xiao-Ling YUAN; Ya-Jun SHAN; Zhen-Hu ZHAO; Jia-Pei CHEN; Yu-Wen CONG

    2004-01-01

    The earthworm fibrinolytic enzyme-3 (EFE-3, GenBank accession No: AY438622), from the earthworm Eiseniafoetida, is a component of earthworm fibrinolytic enzymes. In this study, cDNA encoding the EFE-3 was cloned by RT-PCR. The eDNA contained an open reading frame of 741 nucleotides, which encoded a deduced protein of 247 amino acid residues, including signal sequences. EFE-3 showed a high degree of homology to earthworm (Lumbricus rebullus) proteases F-III-1, F-III-2, and bovine trypsin. The recombinant EFE-3 was expressed in E. coli as inclusion bodies, and the gene encoding the native form of EFE-3 was expressed in COS-7 cells in the medium. Both the refolding product of inclusion bodies and the secreted protease could dissolve the artificial fibrin plate.

  10. GENE EXPRESSION PROFILING OF GANGLIOGLIOMA MALIGNANT PROGRESSION BY cDNA ARRAY

    Institute of Scientific and Technical Information of China (English)

    ZHANG Quan-bin; HUANG Qiang; DONG Jun; WANG Ai-dong; SUN Ji-yong; LAN Qing; HU Geng-xi

    2005-01-01

    Objective: To establish gene expression profiles associated with malignant progression of ganglioglioma. Methods: The primary and two recurrent glioma specimens were collected intraoperatively from the same patient who experienced tumor transformation into anaplastic astrocytoma and glioblastoma multiform for the first and second recurrence respectively. Gene expression was assayed through cDNA array and bioinformatics analysis. Results: A total of 197 differentially expressed genes with differential ratio value more than 3 compared with normal brain tissue were obtained. Among 109 functionally denned genes, those associated with development ranked the first by frequency, followed by genes associated with metabolism, differentiation, signal transduction and so on. As a result of cluster analysis among 368 genes, eleven genes were up regulated with malignant progression, while six genes were down regulated. Conclusion: Gene expression profiles associated with malignant progression of glioma were successfully established, which provides a powerful tool for research on molecular mechanisms of malignant progression of gliomas.

  11. cDNA microarray reveals signaling pathways involved in hormones expression of human pituitary.

    Science.gov (United States)

    Ma, Yue-Yun; Qi, Xiao-Fei; Song, Shao-Jun; Zhao, Zhan-Yong; Zhu, Zhi-Dong; Qi, Jia; Zhang, Xin; Xiao, Hua-Sheng; Teng, Yun; Han, Ze-Guang

    2005-09-01

    Pituitary, a master gland of neuroendocrine system, secretes hormones that orchestrate many physiological processes, under the regulation of multiple signaling pathways. To investigate the genes involved in hormones expression of human pituitary, homemade cDNA microarray containing 14,800 human genes/ESTs were used to profile the gene expression in both fetal and adult pituitaries. Seven hundred and twelve known genes changed over 2-fold between the both tissues. Of which, 23 genes were changed with hormones expression in aging were confirmed by RT-PCR, not only the known regulators such as Pit1, GATA4, ESRRA, GABA-A, and EMK, but also LOC55884, DUSP3, PNN, and RCL, which had not been reported to be involved in the hormones expression. Correspondingly, the mRNAs of GH, PRL, POMC, TSH-beta, FSH-beta, and LH-beta, was increased as much as 6- to 20-fold in adult pituitary than those in fetal pituitary, by real-time quantitative RT-PCR assay. In addition, the mRNAs of signaling pathways, such as cAMP-PKA-CREB, PI3K-Akt, and PKA-ERK were further investigated. Of them, it was only cAMP-PKA-CREB pathway, but not PI3K-Akt and PKA-ERK have the same expressing pattern as hormones. It suggested that cDNA microarray is highly advantages to profile the differential expressed genes that were involved in hormones expression of human pituitary, but it might ignore some responding proteins regulated posttranscriptionally.

  12. Cloning and characterization of a cDNA clone encoding calreticulin from Haemaphysalis qinghaiensis (Acari: Ixodidae).

    Science.gov (United States)

    Gao, Jinliang; Luo, Jianxun; Fan, Ruiquan; Fingerle, Volker; Guan, Guiquan; Liu, Zhijie; Li, Youquan; Zhao, Haiping; Ma, Miling; Liu, Junlong; Liu, Aihong; Ren, Qiaoyun; Dang, Zhisheng; Sugimoto, Chihiro; Yin, Hong

    2008-03-01

    The application of anti-tick vaccine has been shown to be the most promising alternative strategy compared to the current use of acaricides that suffer from a number of serious limitations. The success of this method is dependent upon identification and cloning of potential tick vaccine antigens. Previously, we have cloned 21 positive clones (named from Hq02 to Hq22) by immunoscreening complimentary DNA (cDNA) libraries of Haemaphysalis qinghaiensis; however, some of those clones did not contain open reading frames (ORF). In this study, we amplified the entire sequence of Hq07 by using rapid amplification of the cDNA ends. Hq07 contains an ORF of 1,233 bp that encodes for 410 amino acid residues with a coding capacity of 47 kDa. Search of the cloned sequences against GenBank revealed that Hq07 is a calreticulin (CRT)-similar clone and designated HqCRT. Expression analysis by reverse transcription-polymerase chain reaction showed that this gene is ubiquitously expressed at different developmental stages and in different tissues of H. qinghaiensis. The gene was expressed as glutathione S-transferase-fused proteins in a prokaryotic system. Western blot analysis revealed that native HqCRT was secreted into their hosts by ticks during blood sucking. Vaccination of sheep with rHqCRT conferred protective immunity against ticks, resulting in 54.3% mortality in adult ticks, compared to the 38.7% death rate in the control group. These results demonstrated that rHqCRT might be a useful vaccine candidate antigen for biological control of H. qinghaiensis.

  13. Sequence characterization of a human embryonic craniofacial cDNA library

    Energy Technology Data Exchange (ETDEWEB)

    Padanilam, B.J.; Barsel, S.; Solursh, M. [and others

    1994-09-01

    Broad-based sequencing approaches for the characterization of human cDNA libraries have proven successful in identifying large numbers of novel genes of specific tissue or developmental stages. To pursue our interests in human craniofacial development, stages. To pursue our interests in human craniofacial development, we have made use of both subtracted and unsubtracted cDNA libraries constructed from embryonic craniofacial tissue obtained from pooled samples at 42-54 days gestation. Single-pass sequencing was carried out using an ABI automated sequencer and T3 or T7 primers. Sequences were characterized using BLAST and GRAIL, and the identified homologous sequences grouped according to gene class and family. Four genes have been mapped using repeat sequence elements identified in the clones. Using primers developed from sequence data, other genes are being mapped using a panel of somatic cell hybrids. To date, a total of 786 sequences have been returned with 35% identifying no homologies, and 35% with strong homologies to previously identified genes. A number of genes previously identified to play a role in human embryonic development have been returned from the sequence comparisons providing evidence that the library is representative of this tissue and stage of development. Previous characterization of the library has also identified a number of novel embryonically expressed human homeobox genes. Genes felt to be of special relevance based on their homology to characterized genes known to play a role in development or that are members of novel classes but with high scores on GRAIL searches are being characterized using whole mount in situ hybridization with mouse embryos. Characterization of the library with respect to chromosomal mapping, gene types and make-up, and embryonic expression patterns will be presented.

  14. 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.

  15. Molecular cloning and sequence analysis of hamster CENP-A cDNA

    Science.gov (United States)

    Figueroa, Javier; Pendón, Carlos; Valdivia, Manuel M

    2002-01-01

    Background The centromere is a specialized locus that mediates chromosome movement during mitosis and meiosis. This chromosomal domain comprises a uniquely packaged form of heterochromatin that acts as a nucleus for the assembly of the kinetochore a trilaminar proteinaceous structure on the surface of each chromatid at the primary constriction. Kinetochores mediate interactions with the spindle fibers of the mitotic apparatus. Centromere protein A (CENP-A) is a histone H3-like protein specifically located to the inner plate of kinetochore at active centromeres. CENP-A works as a component of specialized nucleosomes at centromeres bound to arrays of repeat satellite DNA. Results We have cloned the hamster homologue of human and mouse CENP-A. The cDNA isolated was found to contain an open reading frame encoding a polypeptide consisting of 129 amino acid residues with a C-terminal histone fold domain highly homologous to those of CENP-A and H3 sequences previously released. However, significant sequence divergence was found at the N-terminal region of hamster CENP-A that is five and eleven residues shorter than those of mouse and human respectively. Further, a human serine 7 residue, a target site for Aurora B kinase phosphorylation involved in the mechanism of cytokinesis, was not found in the hamster protein. A human autoepitope at the N-terminal region of CENP-A described in autoinmune diseases is not conserved in the hamster protein. Conclusions We have cloned the hamster cDNA for the centromeric protein CENP-A. Significant differences on protein sequence were found at the N-terminal tail of hamster CENP-A in comparison with that of human and mouse. Our results show a high degree of evolutionary divergence of kinetochore CENP-A proteins in mammals. This is related to the high diverse nucleotide repeat sequences found at the centromere DNA among species and support a current centromere model for kinetochore function and structural plasticity. PMID:12019018

  16. 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.

  17. 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...

  18. Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases

    NARCIS (Netherlands)

    Lücker, J.; El Tamer, M.K.; Schwab, W.; Verstappen, F.W.A.; Plas, van der L.H.W.; Bouwmeester, H.J.; Verhoeven, H.A.

    2002-01-01

    Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the pe

  19. The Arabian camel Camelus dromedarius heat shock protein 90α: cDNA cloning, characterization and expression.

    Science.gov (United States)

    Saeed, Hesham; Shalaby, Manal; Embaby, Amira; Ismael, Mohammad; Pathan, Akbar; Ataya, Farid; Alanazi, Mohammad; Bassiouny, Khalid

    2015-11-01

    Heat shock protein 90 (Hsp90) is a highly conserved ubiquitous molecular chaperone contributing to assisting folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. In the present study, a heat shock protein 90α full length coding cDNA was isolated and cloned from the Arabian one-humped camel by reverse transcription polymerase chain reaction (RT-PCR). The full length cDNA sequence was submitted to NCBI GeneBank under the accession number KF612338. The sequence analysis of the Arabian camel Hsp90α cDNA showed 2202bp encoding a protein of 733 amino acids with estimated molecular mass of 84.827kDa and theoretical isoelectric point (pI) of 5.31. Blast search analysis revealed that the C. dromedarius Hsp90α shared high similarity with other known Hsp90α. Comparative analyses of camel Hsp90α protein sequence with other mammalian Hsp90s showed high identity (85-94%). Heterologous expression of camel Hsp90α cDNA in E. coli JM109 (DE3) gave a fusion protein band of 86.0kDa after induction with IPTG for 4h.

  20. 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.

  1. Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi

    NARCIS (Netherlands)

    Gemeren, I.A. van; Musters, W.; Hondel, C.A.M.J.J. van den; Verrips, C.T.

    1995-01-01

    A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the subseq

  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. Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA mircoarray

    Institute of Scientific and Technical Information of China (English)

    Xiao-Jing Liu; Li Yang; Feng-Ming Luo; Hong-Bin Wu; Qu-Qiang

    2004-01-01

    AIM: To characterize the gene expression profiles associated with activation of mouse hepatic stellate cell (HSC) and provide novel insights into the pathogenesis of hepatic fibrosis.METHODS: Mice HSCs were isolated from BALB/c mice by in situ perfusion of collagenase and pronase and singlestep density Nycodenz gradient. Total RNA and mRNA of quiescent HSC and culture-activated HSC were extracted,quantified and reversely transcripted into cDNA. cDNAs from activated HSC were labeled with Cy5 and cDNAs from the quiescent HSC were labeled with Cy3, which were mixed with equal quantity, then hybridized with cDNA chips containing 4 000 genes. Chips were washed, scanned and analyzed. Increased expression of 4 genes and decreased expression of one gene in activated HSC were confirmed by reverse transcription- polymerase chain reaction (RT-PCR).RESULTS: A total of 835 differentially expressed genes were identified by cDNA chip between activated and quiescent HSC, and 465 genes were highly expressed in activated HSC. The differentially expressed genes included those involved in protein synthesis, cell-cycle regulation,apoptosis, and DNA damage response.CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis and proliferation were up-regulated in activated HSC. cDNA microarray is an effective technique in screening for differentially expressed genes between two different situations of the HSC. Further analysis of the obtained genes will help understand the molecular mechanism of activation of HSC and hepatic fibrosis.

  4. Cloning of a cDNA encoding a novel human nuclear phosphoprotein belonging to the WD-40 family

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder

    1994-01-01

    We have cloned and expressed in vaccinia virus a cDNA encoding an ubiquitous 501-amino-acid (aa) phosphoprotein that corresponds to protein IEF SSP 9502 (79,400 Da, pI 4.5) in the master 2-D-gel keratinocyte protein database [Celis et al., Electrophoresis 14 (1993) 1091-1198]. The deduced aa...

  5. Isolation and characterisation of the human lung NK-2 receptor gene using rapid amplification of cDNA ends.

    Science.gov (United States)

    Graham, A; Hopkins, B; Powell, S J; Danks, P; Briggs, I

    1991-05-31

    Functional cDNA clones for human NK-2 receptor were isolated from human lung RNA using a polymerase chain reaction (PCR) based method (RACE-PCR). In this method the cDNA was isolated as 5' end and 3'-end fragments; the entire cDNA was obtained by RNA-PCR. The sequence derived was 398 amino acids in length encoding an open-reading frame that was highly homologous to both the bovine and rat NK-2 receptor. The entire human cDNA sequence was cloned into a mammalian expression vector and mRNA was synthesised by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesised mRNA. The most potent of the three tachykinin peptides tested was neurokinin A. We have screened a human cosmid library and isolated a clone which contains the entire NK-2 receptor gene. The gene contains five exons and we have determined the complete sequence of the exons and the intron-exon junctions.

  6. Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, E; Spiering, M; Chow, KS; Mulder, PPMFA; Subroto, T; Beintema, JJ

    2001-01-01

    Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus whi

  7. Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase.

    Science.gov (United States)

    Giallongo, A; Feo, S; Moore, R; Croce, C M; Showe, L C

    1986-01-01

    We previously purified a 48-kDa protein (p48) that specifically reacts with an antiserum directed against the 12 carboxyl-terminal amino acids of the c-myc gene product. Using an antiserum directed against the purified p48, we have cloned a cDNA from a human expression library. This cDNA hybrid-selects an mRNA that translates to a 48-kDa protein that specifically reacts with anti-p48 serum. We have isolated a full-length cDNA that encodes p48 and spans 1755 bases. The coding region is 1299 bases long; 94 bases are 5' noncoding and 359 bases are 3' noncoding. The cDNA encodes a 433 amino acid protein that is 67% homologous to yeast enolase and 94% homologous to the rat non-neuronal enolase. The purified protein has been shown to have enolase activity and has been identified to be of the alpha type by isoenzyme analysis. The transcriptional regulation of enolase expression in response to mitogenic stimulation of peripheral blood lymphocytes and in response to heat shock is also discussed. Images PMID:3529090

  8. Massive Analysis of cDNA Ends (MACE for transcript-based marker design in pea (Pisum sativum L.

    Directory of Open Access Journals (Sweden)

    Aleksandr Zhernakov

    2017-03-01

    Full Text Available Aimed at gene-based markers design, we generated and analyzed transcriptome sequencing datasets for six pea (Pisum sativum L. genetic lines that have not previously been massively genotyped. Five cDNA libraries obtained from nodules or nodulated roots of genetic lines Finale, Frisson, Sparkle, Sprint-2 and NGB1238 were sequenced using a versatile 3′-RNA-seq protocol called MACE (Massive Analysis of cDNA Ends. MACE delivers a single next-generation sequence from the 3′-end of each individual cDNA molecule that precisely quantifies the respective transcripts. Since the contig generated from the 3′-end of the cDNA by assembling all sequences encompasses the highly polymorphic 3′-untranslated region (3′-UTR, MACE efficiently detects single nucleotide variants (SNVs. Mapping MACE reads to the reference nodule transcriptome assembly of the pea line SGE (Transcriptome Shotgun Assembly GDTM00000000.1 resulted in characterization of over 34,000 polymorphic sites in more than 9700 contigs. Several of these SNVs were located within recognition sequences of restriction endonucleases which allowed the design of co-dominant CAPS markers for the particular transcript. Cleaned reads of sequenced libraries are available from European Nucleotide Archive (http://www.ebi.ac.uk/ under accessions PRJEB18101, PRJEB18102, PRJEB18103, PRJEB18104, PRJEB17691.

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

    Science.gov (United States)

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

  10. Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labelled neurofilament cDNA probe.

    NARCIS (Netherlands)

    P. Liesi; J-P. Julien (Jean-Pierre); P. Vilja; F.G. Grosveld (Frank); L. Rechardt

    1986-01-01

    textabstractWe have used a biotinylated, 300-nucleotide cDNA probe which encodes the 68,000 MW neurofilament protein to detect neurofilament-specific mRNA in situ. The neurofilament message specifically demonstrates the neuronal cell bodies, in contrast to the usual antibody staining which detects t

  11. B-G cDNA clones have multiple small repeats and hybridize to both chicken MHC regions

    DEFF Research Database (Denmark)

    Kaufman, J; Salomonsen, J; Skjødt, K

    1989-01-01

    We used rabbit antisera to the chicken MHC erythrocyte molecule B-G and to the class I alpha chain (B-F) to screen lambda gt11 cDNA expression libraries made with RNA selected by oligo-dT from bone marrow cells of anemic B19 homozygous chickens. Eight clones were found to encode B-G molecules which...

  12. Construction of a metastasis-associated gene subtracted cDNA library of human colorectal carcinoma by suppression subtraction hybridization

    Institute of Scientific and Technical Information of China (English)

    Li Liang; Yan-Qing Ding; Xin Li; Guang-Zhi Yang; Jun Xiao; Li-Chun Lu; Jin-Hua Zhang

    2004-01-01

    AIM: To construct a differentially-expressed gene subtracted cDNA library from two colorectal carcinoma (CRC) cell lines with different metastatic phenotypes by suppression subtractive hybridization.METHODS: Two cell lines of human CRC from the same patient were used. SW620 cell line showing highly metastatic potential was regarded as tester in the forward subtractive hybridization, while SW480 cell line with lowly metastatic potential was treated as tester in the reverse hybridization. Suppression subtractive hybridization (SSH)was employed to obtain cDNA fragments of differentially expressed genes for the metastasis of CRC. These fragments were ligated with T vectors, screened through the bluewhite screening system to establish cDNA library.RESULTS: After the blue-white screening, 235 white clones were picked out from the positive-going hybridization and 232 from the reverse. PCR results showed that 200-700 bp inserts were seen in 98% and 91% clones from the forward and reverse hybridizations, respectively.CONCLUSIONS: A subtractive cDNA library of differentially expressed genes specific for metastasis of CRC can be constructed with SSH and T/A cloning techniques.

  13. 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.

  14. cDNA cloning and characterization of a gibberellin-responsive gene in hypocotyls of Cucumis sativus L.

    Science.gov (United States)

    Chono, M; Yamauchi, T; Yamaguchi, S; Yamane, H; Murofushi, N

    1996-07-01

    A cDNA clone corresponding to a gibberellin-responsive gene (CRG16) was isolated from cucumber hypocotyls. CRG16 was deduced to encode an extremely hydrophobic protein of 65 amino acids. The deduced sequence exhibited no significant homology to other proteins. Levels of CRG16 mRNA reflected the gibberellin-induced elongation of cucumber hypocotyls.

  15. Construction and characterization of a full-lengh cDNA library from non-fresh Giardia lamblia

    Institute of Scientific and Technical Information of China (English)

    Jun-Li Guo; Jie Jiang; Wen-Yu Zheng; Ming-Luan Li; Xi-Feng Tian; Xian-Min Feng; Yue-Hua Wang; Xiao-Hong Ju; Yue-Qiong Kong

    2012-01-01

    Objective: To construct rapidly a full-length cDNA library from nanogram amounts total RNA of Giardia lamblia (G. lamblia) trophozoites stocked in RNA stabilization reagent. Methods: Total RNA of Giardia was extracted using Trizol reagent. A full-length cDNA library of G. lamblia trophozoites was constructed by a long-distance PCR (LD-PCR) method. The recombinant rate and the coverage rate of full-length clones of the library were evaluated. The inserted fragments were identified and sequenced by PCR amplification. Results: The titer of cDNA library was 3.85 ×107 pfu/mL. The length of inserted fragments ranged from 0.4 to 2.5 kb, and the recombination efficiency accounted for 100% (20/20). The coverage rate of full-length clones is high (17/20). Conclusions: The RNA stabilization reagent may be used to fix the cells and prevent the RNA in cells even though delivered under normal atmospheric temperature. The long-distance PCR can be used to construct a full-length cDNA library rapidly and it needs less RNA than the traditional method from mRNA.

  16. Generation and characterisation of a full-length cDNA encoding murine myotonic dystrophy protein kinase from cardiac tissue

    Energy Technology Data Exchange (ETDEWEB)

    Carey, N.; Tongeren, T. van; Winchester, C. [Charing Cross & Wesminster Medical School, London (United Kingdom)] [and others

    1994-09-01

    The mutation underlying myotonic dystrophy (DM) is a CTG trinucleotide expansion in the 3{prime} untranslated region of a putative protein kinase gene (DMPK). We report the isolation of a full-length cDNA clone of the murine (DMPK) gene from a heart cDNA library. Sequence analysis shows that the clone is a splice isoform which has only previously been identified in brain, suggesting that there may be some flexibility of the splicing pattern in some tissues. We are currently analyzing the library for the presence of other isoforms. The full-length cDNA has been cloned into a bacterial expression system and the expressed protein is being used as an immunogen to generate both polyclonal and monoclonal antisera. These reagents will allow the analysis of the intracellular targets of the DMPK. Subclones of the cDNA have been generated for use as in situ hybridization probes, allowing investigation of the normal patterns of expression of the gene and the differential expression of the protein isoforms. These data will be essential for deciding on a rational use of rare patient material and will provide the necessary baseline for the analysis of transgenic and {open_quotes}knock-out{close_quotes} mice.

  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. 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.

  19. Terpenoids from a Chinese Gorgonian Anthogorgia sp. and Their Antifouling Activities

    Institute of Scientific and Technical Information of China (English)

    陈达伟; 刘东; 沈施; 程伟; 林文翰

    2012-01-01

    Chemical examination of a Chinese gorgonian Anthogorgia sp. resulted in the isolation of seven terpenoids, including two new compounds, an rearranged serrulatane-type diterpenoid anthogorgiene P (1) and a guaiazuenebased terpenoid anthogorgiene Q (2). Anthogorgiene P (1) contains an unprecedented cubebane nucleus which is rarely found from nature. The structures of new compounds were elucidated on the basis of extensive spectroscopic methods (IR, UV, MS, CD and NMR). Compound 7 showed potent antifouling activity against the larval settlement of Balanus amphitrite, while 1 and 4 possessed moderate inhibition.

  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. 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

  2. 诸葛菜基因EPSPS的cDNA核苷酸序列%The cDNA Nucleotide Sequence of EPSPS Gene from Orychophragmus violaceus

    Institute of Scientific and Technical Information of China (English)

    刘晓军; 邓运涛; 游大慧; 李旭锋

    2002-01-01

    @@1 Source The sequence was determined using the 3′ RACE(rapid amplification cDNA ends) RT-PCR and 5′RACE RT-PCR product, which was ligated to the pMD18-T vector, from the cDNA of Orychophragmus violaceus.

  3. Targeted rapid amplification of cDNA ends (T-RACE)--an improved RACE reaction through degradation of non-target sequences.

    Science.gov (United States)

    Bower, Neil I; Johnston, Ian A

    2010-11-01

    Amplification of the 5' ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3' end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-containing primers complimentary to the adapter sequences incorporated in the cDNA ends. The double-stranded cDNA-containing dUTP serves as a universal template for the specific amplification of the 3' or 5' end of any gene. To amplify the ends of cDNA, asymmetric PCR is performed using a single gene-specific primer and standard dNTPs. The asymmetric PCR product is purified and non-target transcripts containing dUTP degraded by Uracil DNA glycosylase, leaving only those transcripts produced during the asymmetric PCR. Subsequent PCR using a nested gene-specific primer and the 3' or 5' T-RACE primer results in specific amplification of cDNA ends. This method can be used to specifically amplify the 3' and 5' ends of numerous cDNAs from a single cDNA synthesis reaction.

  4. Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong Li; You-Yong Lu

    2002-01-01

    AIM: To develop and optimize cDNA representationaldifference analysis (cDNA RDA) method and to identify andclone garlic up-regulated genes in human gastric cancer(HGC) cells.METHODS: We performed cDNA RDA method by usingabundant double-stranded cDNA messages provided by twoself-constructed cDNA libraries (Allitridi-trested and paternalHGC cell line BGC823 cells cDNA libraries respectively).BamH Ⅰ and Xho I restriction sites harbored in the libraryvector were used to select representations. Northern andSlot blots analyses were employed to identify the obtaineddifference products.RESJLTS: Fragments released from the cDNA library vectorafter restriction endonuclease digestion acted as goodmarker indicating the appropriate digestion degree for libraryDNA. Two novel expressed sequence tags (ESTs) and arecombinant gene were obtained. Slot blots result showed a8-fold increase of gila-derived nexin/protease nexin 1 (GDN/PN1 ) gene expression level and 4-fold increase of hepatitis Bvirus x-interacting protein (XIP) mRNA level in BGC823 cellsafter Allitridi treatment for 72 h.CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAsinduced by Allitridi provide valuable molecular evidence forelucidating the garlic' s efficacies against neurodegenerativeand inflammatory diseases. Isolation of a recombinant geneand two novel ESTs further show cDNA RDA based on cDNAlibraries to be a powerful method with high specificity andreproducibility in cloning differentially expressed genes.

  5. 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.

  6. Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus.

    Science.gov (United States)

    Olsen, B S; Johansen, I E

    2001-01-01

    The complete nucleotide sequence of Pea seed-borne mosaic potyvirus isolate L1 has been determined from cloned virus cDNA. The PSbMV L1 genome is 9895 nucleotides in length excluding the poly(A) tail. Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9594 nucleotides. The ORF potentially encodes a polyprotein of 3198 amino acids with a deduced Mr of 363537. Nine putative proteolytic cleavage sites were identified by analogy to consensus sequences and genome arrangement in other potyviruses. Two full-length cDNA clones, p35S-L1-4 and p35S-L1-5, were assembled under control of an enhanced 35S promoter and nopaline synthase terminator. Clone p35S-L1-4 was constructed with four introns and p35S-L1-5 with five introns inserted in the cDNA. Clone p35S-L1-4 was unstable in Escherichia coli often resulting in amplification of plasmids with deletions. Clone p35S-L1-5 was stable and apparently less toxic to Escherichia coli resulting in larger bacterial colonies and higher plasmid yield. Both clones were infectious upon mechanical inoculation of plasmid DNA on susceptible pea cultivars Fjord, Scout, and Brutus. Eight pea genotypes resistant to L1 virus were also resistant to the cDNA derived L1 virus. Both native PSbMV L1 and the cDNA derived virus infected Chenopodium quinoa systemically giving rise to characteristic necrotic lesions on uninoculated leaves.

  7. Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiao-hong; CHEN Zhi; YAO Hang-ping; CHEN Feng; ZHU Hai-hong; ZHOU Hong-juan

    2005-01-01

    Objective: To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. Methods: The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5' end of the RNA transcript(SMART) technique and CDS Ⅲ/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction(LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to λTriplEx2 vector. Then λ phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. Results: The titers of unamplifed and amplified libraries were 1.94×106 pfu/ml and1.49×109 pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%.Conclusion: A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

  8. 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

  9. KARAKTERISTIK SEKUEN cDNA PENGKODE GEN ANTI VIRUS DARI UDANG WINDU, Penaeus monodon

    Directory of Open Access Journals (Sweden)

    Andi Parenrengi

    2016-11-01

    Full Text Available Transgenesis pada ikan merupakan sebuah teknik modern yang berpotensi besar dalam menghasilkan organisme yang memiliki karakter lebih baik melalui rekombinan DNA gen target termasuk gen anti virus dalam peningkatan resistensi pada udang. Gen anti virus PmAV (Penaeus monodon Anti Viral gene merupakan salah satu gen pengkode anti virus yang berasal dari spesies krustase. Penelitian ini dilakukan untuk mengetahui karakteristik gen anti virus yang diisolasi dari udang windu, Penaeus monodon. Isolasi gen anti virus menggunakan metode Polymerase Chain Reaction (PCR dan selanjutnya dipurifikasi untuk sekuensing. Data yang dihasilkan dianalisis dengan program Genetyx Versi 7 dan basic local alignment search tool (BLAST. Hasil penelitian menunjukkan bahwa gen anti virus PmAV yang berhasil diisolasi dari cDNA udang windu dengan panjang sekuen 520 bp yang mengkodekan 170 asam amino. BLAST-N menunjukkan tingkat similaritas yang sangat tinggi (100% dengan gen anti virus yang ada di GeneBank. Komposisi asam amino penyusun gen anti virus yang paling besar adalah serin (10,00%, sedangkan yang terkecil adalah asam amino prolin dan lisin masing-masing 1,76%. Analisis sekuen gen dan deduksi asam amino (BLAST-P memperlihatkan adanya C-type lectin-like domain (CTLD yang memiliki kemiripan dengan gen C-type lectin yang diisolasi dari beberapa spesies krustase. Transgenic fish technology is a potential modern technique in producing better character organism through DNA recombinant of target genes including anti viral gene for improvement of shrimp immunity. PmAV (Penaeus monodon Anti Viral gene is one of anti viral genes isolated from crustacean species. The research was conducted to analyze the characteristics anti viral gene isolated from tiger prawn, Penaeus monodon. Anti viral gene was isolated using Polymerase Chain Reaction (PCR technique and then purified for sequencing. Data obtained were analyzed using Genetyx Version 7 software and basic local alignment

  10. 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.

  11. 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.

  12. Identification of the differential expressive tumor associated genes in rectal cancers by cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    Xue-Qin Gao; Jin-Xiang Han; Zhong-Fa Xu; Wei-Dong Zhang; Hua-Ning Zhang; Hai-Yan Huang

    2007-01-01

    AIM: To identify tumor associated genes of rectal cancer and to probe the application possibility of gene expression profiles for the classification of tumors.METHODS: Rectal cancer tissues and their paired normal mucosa were obtained from patients undergoing surgical resection of rectal cancer. Total RNA was extracted using Trizol reagents. First strand cDNA synthesis was indirectly labeled with aminoallyl-dUTP and coupled with Cy3 or Cy5 dye NHS mono-functional ester. After normalization to total spots, the genes which background subtracted intensity did not exceed 2 SD above the mean blank were excluded. The data were then sorted to obtain genes differentially expressed by≥ 2 fold up or down in at least 5 of the 21 patients.RESULTS: In the 21 rectal cancer patients, 23 genes were up-regulated in at least 5 samples and 15 genes were down-regulated in at least 5 patients. Hierachical cluster analysis classified the patients into two groups according to the clinicopathological stage, with one group being all above stage Ⅱ and one group all below stage Ⅱ.CONCLUSION: The up-regulated genes and downregulated genes may be molecular markers of rectal cancer. The expression profiles can be used for classification of rectal cancer.

  13. Noise reduction of cDNA microarray images using complex wavelets.

    Science.gov (United States)

    Howlader, Tamanna; Chaubey, Yogendra P

    2010-08-01

    Noise reduction is an essential step of cDNA microarray image analysis for obtaining better-quality gene expression measurements. Wavelet-based denoising methods have shown significant success in traditional image processing. The complex wavelet transform (CWT) is preferred to the classical discrete wavelet transform for denoising of microarray images due to its improved directional selectivity for better representation of the circular edges of spots and near shift-invariance property. Existing CWT-based denoising methods are not efficient for microarray image processing because they fail to take into account the signal as well as noise correlations that exist between red and green channel images. In this paper, two bivariate estimators are developed for the CWT-based denoising of microarray images using the standard maximum a posteriori and linear minimum mean squared error estimation criteria. The proposed denoising methods are capable of taking into account both the interchannel signal and noise correlations. Significance of the proposed denoising methods is assessed by examining the effect of noise reduction on the estimation of the log-intensity ratio. Extensive experimentations are carried out to show that the proposed methods provide better noise reduction of microarray images leading to more accurate estimation of the log-intensity ratios as compared to the other CWT-based denoising methods.

  14. 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.

  15. Cloning and Expression Analysis of Downy Mildew Resistance-Related cDNA Sequences in Melon

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Melon downy mildew caused by Pseudoperonospora cubensis leads to significant losses in melon yields worldwide.Reverse-transcription Polymerase Chain Reaction (RT-PCR) was performed using cDNAs as templates from melonHuangdanzi induced with fungus Pseudoperonospora cubensis, and degenerate primers designed based on the conserved amino acid sequences of known plant disease-resistance genes. A polymorphic cDNA fragment which we named mp-19was cloned and sequenced. The Open Reading Frame (ORF) of this product comprised of 510 base pairs which encodes DNA or RNA-binding protein with 170 amino acids. The putative amino acid sequence of mp-19 appeared highly homologous with those of NBS-type resistant-genes isolated from other plants. Southern blot indicated that the melon genome contained more than 3 copies of mp-19. The obvious expression differences detected by semi-quantitative RTPCR could be observed between resistant-line Huangdanzi and susceptible-line Jiashi after Pseudoperonospora cubensis infection, which implied that mp-19 gene may be related to the resistance of downy mildew in melon.

  16. 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.

  17. 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.

  18. 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.

  19. Expression profiles of metastatic brain tumor from lung adenocarcinomas on cDNA microarray.

    Science.gov (United States)

    Kikuchi, Takefumi; Daigo, Yataro; Ishikawa, Nobuhisa; Katagiri, Toyomasa; Tsunoda, Tatsuhiko; Yoshida, Seiichi; Nakamura, Yusuke

    2006-04-01

    Distant metastasis is one of the crucial parameters determining the type of treatment and prognosis of patients. Previous studies discovered important factors involved in multiple steps of metastasis, the precise mechanisms of metastasis still remain to be clarified. To identify genes associated with this complicated biological feature of cancer, we analyzed expression profiles of 16 metastatic brain tumors derived from primary lung adenocarcinoma (ADC) using cDNA microarray representing 23,040 genes. We applied bioinformatic algorithm to compare the expression data of these 16 brain metastatic loci with those of 37 primary NSCLCs including 22 ADCs, and found that metastatic tumor cells has very different characteristics of gene expression patterns from primary ones. Two hundred and forty-four genes that showed significantly different expression levels between the two groups included plasma membrane bounding proteins, cellular antigens, and cytoskeletal proteins that might play important roles in altering cell-cell communication, attachment, and cell motility, and enhance the metastatic ability of cancer cells. Our results provide valuable information for development of predictive markers as well as novel therapeutic target molecules for metastatic brain tumor of ADC of the lung.

  20. Gene expression of panaxydol-treated human melanoma cells using radioactive cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Joong Youn; Yu, Su Jin; Soh, Jeong Won; Kim, Meyoung Kon [College of Medicine, Korea Univ., Seoul (Korea, Republic of)

    2001-07-01

    Polyacetylenic alcohols derived from Panax ginseng have been studied to be an anticancer reagent previously. One of the Panax ginseng polyacetylenic alcohols, i.e., panaxydol, has been studied to possess an antiproliferative effect on human melanoma cell line (SK-MEL-1). In ths study, radioactive cDNA microarrays enabled an efficient approach to analyze the pattern of gene expression (3.194 genes in a total) simultaneously. The bioinformatics selection of human cDNAs, which is specifically designed for immunology, apoptosis and signal transduction, were arrayed on nylon membranes. Using with {sup 33}P labeled probes, this method provided highly sensitive gene expression profiles of our interest including apoptosis, cell proliferation, cell cycle, and signal transduction. Gene expression profiles were also classified into several categories in accordance with the duration of panaxydol treatment. Consequently, the gene profiles of our interest were significantly up (199 genes, > 2.0 of Z-ratio) or down-(196 genes, < 2.0 of Z-ratio) regulated in panaxydol-treated human melanoma cells.

  1. 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.

  2. 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.

  3. Identification of some unknown transcripts from SSH cDNA library of buffalo follicular oocytes.

    Science.gov (United States)

    Rajput, S K; Kumar, P; Roy, B; Verma, A; Pandey, H P; Singh, D; De, S; Datta, T K

    2013-03-01

    A buffalo oocyte-specific subtracted cDNA library was constructed to identify exclusively or preferentially oocyte-expressed genes. The library represented an enriched population of transcripts obtained from oocytes of diverse ovarian follicular origin and at different stages of in vitro maturation. A total of 1173 high-quality sequences of oocyte-specific genes were clustered into 645 unique sequences, out of which 65.76% were represented as singlets and 34.26% as contig expressed sequence tags (ESTs; clusters). Analysis of sequences revealed that 498 of these sequences were identified as a known sequence in mammalian species including buffalo, 103 as uncharacterized ESTs and 44 unknown sequences including 1 novel EST, so far not reported in any species. Gene ontology annotation classified these sequences into functional categories of cellular events and biological processes associated with oocyte competence. Expression status of the isolated unknown ESTs confirmed that many of these are expressed in oocytes exclusively and in others preferentially, some in excess of 80-fold greater in comparison with a variety of somatic tissues. The isolated novel EST was detected to be expressed exclusively in oocytes and testicular cells only. To our knowledge, this is the first report giving a detailed transcriptome account of oocyte-expressed genes in buffalo. This study will provide important information on the physiological control of oocyte development, as well as many questions yet to be addressed on the reproductive process of buffalo.

  4. 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.

  5. Construction and analysis of a subtractive cDNA library of early embryonic development in duck.

    Science.gov (United States)

    Liu, Y L; Zhong, L X; Li, J J; Shen, J D; Wang, D Q; Tao, Z R; Shi, F X; Lu, L Z

    2013-07-08

    Several studies have documented the process of early embryonic development in poultry; however, the molecular mechanisms underlying its developmental regulation are poorly understood, particularly in ducks. In this study, we analyzed differential gene expression of embryos 6 and 25 h following oviposition to determine which genes regulate the early developmental stage in ducks. Among 216 randomly selected clones, 39 protein-encoding cDNAs that function in metabolism, transcription, transportation, proliferation/apoptosis, cell cycle, cell adhesion, and methylation were identified. Additionally, the full-length cDNA of the Nanog gene, encoding a 302-amino acid protein, was obtained. Quantitative real-time polymerase chain reaction analyses were performed to detect expression levels of the selected genes during early and late embryonic stages, which revealed that these genes are expressed in a particular spatial and temporal pattern. These results indicate that these genes may play pivotal roles in the process of area pellucida formation through a complex and precise regulatory network during development in duck embryos.

  6. cDNA cloning and sequence analysis of NIb gene of soybean mosaic virus

    Institute of Scientific and Technical Information of China (English)

    刘俊君; 彭学贤; 莽克强

    1995-01-01

    cDNA of soybean mosaic virus (Beijing isolate, SMV-BJ) has been synthesized, using viralgenomic RNA as template and random hexanucleotides as primers. Based on the sequences of SMV-BJ coat protein (CP) gene as well as SMV- and WMV-II-related regions, oligonucleotides were made as primers for polymerase chain reaction (PCR). NIb gene of SMV-BJ was amplified by PCR, and cloned into pBluescript SK. The complete sequence was determined. The comparison of NIb genes between SMV-BJ and WMV-II . (USA) shows that similarities for nucleotide sequence reach 80.3%, and the deduced amino acid sequence. 91 3%. In consideration of the high identities in between the CP gene and the 3’-non-coding region between them, WMV-II might be considered as a watermelon strain of SMV Besides, some unexpected sequences were found in the 3’-region of 2 NIb gene clones. Following modification and splicing, a binary vector of NIb gene has been constructed for its expression in higher plant for the purpose of studying the possible repl

  7. 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.

  8. Characterization and phylogenetic analysis of a cnidarian LMP X-like cDNA.

    Science.gov (United States)

    Dishaw, Larry J; Herrera, Manuel L; Bigger, Charles H

    2006-06-01

    Proteasomes are multisubunit protease complexes which are partly responsible for metabolism of intracellular, ubiquitinylated proteins. Vertebrates have adapted a second and specialized structure responsible for the generation of peptides presented to the adaptive immune system and is thus, commonly referred to as the immunoproteasome. This complex is assembled from paralogous copies of subunits belonging to the constitutive, housekeeping proteasome. The immunoproteasome is more efficient in the generation of peptides for display on major histocompatibility complex (MHC) molecules. Important components of this complex are the paralogous members, LMP X and 7; where the latter replaces the former in the assembly of the immunoproteasome of vertebrates. In this report, we describe an LMP X-like cDNA from an endosymbiont-free gorgonian coral, Swiftia exserta. Cnidarians predate the phylogenetic divergence of protostomes and deuterostomes (P-D split), and are becoming an essential model for our comprehension of immune system evolution. Phylogenetic analyses of available sequences indicates that invertebrate LMP X-like sequences are outgroups to vertebrate LMP X and LMP 7, and is in agreement with previous observations that the duplication event giving rise to the two rapidly diverging lineages of proteasomal subunits occurred before jawed fished divergence.

  9. 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.

  10. Cloning and Sequencing of Protein Kinase cDNA from Harbor Seal (Phoca vitulina Lymphocytes

    Directory of Open Access Journals (Sweden)

    Jennifer C. C. Neale

    2004-01-01

    Full Text Available Protein kinases (PKs play critical roles in signal transduction and activation of lymphocytes. The identification of PK genes provides a tool for understanding mechanisms of immunotoxic xenobiotics. As part of a larger study investigating persistent organic pollutants in the harbor seal and their possible immunomodulatory actions, we sequenced harbor seal cDNA fragments encoding PKs. The procedure, using degenerate primers based on conserved motifs of human protein tyrosine kinases (PTKs, successfully amplified nine phocid PK gene fragments with high homology to human and rodent orthologs. We identified eight PTKs and one dual (serine/threonine and tyrosine kinase. Among these were several PKs important in early signaling events through the B- and T-cell receptors (FYN, LYN, ITK and SYK and a MAP kinase involved in downstream signal transduction. V-FGR, RET and DDR2 were also expressed. Sequential activation of protein kinases ultimately induces gene transcription leading to the proliferation and differentiation of lymphocytes critical to adaptive immunity. PKs are potential targets of bioactive xenobiotics, including persistent organic pollutants of the marine environment; characterization of these molecules in the harbor seal provides a foundation for further research illuminating mechanisms of action of contaminants speculated to contribute to large-scale die-offs of marine mammals via immunosuppression.

  11. Characterization, cDNA cloning and expression pattern of relaxin gene during embryogenesis of Danio rerio.

    Science.gov (United States)

    Fiengo, Marcella; Donizetti, Aldo; del Gaudio, Rosanna; Minucci, Sergio; Aniello, Francesco

    2012-06-01

    We report the identification, the cDNA cloning, the temporal and spatial expression pattern analysis of the rln gene in the zebrafish Danio rerio. The deduced Rln B and A domains show different evolutionary conservation. Rln B domain shows higher similarity when compared to zebrafish and human RLN3 B domain than human RLN1 and RLN2 B domain. Differently, the zebrafish Rln A domain shows relatively low amino acid sequence similarity when compared with the same sequences. The rln gene is transcribed both during embryogenesis and in adult organism, where higher transcript level has been particularly evidenced in the brain. Moreover, we provide the first description of rln spatial expression pattern during embryonic development. In particular, we show restricted transcript localization starting at the pharyngula stage in olfactory placode, branchial arch region, and in a cell cluster near to otic vesicle. In larval stage, new transcription territories have been detected in both neural and non-neural regions. In particular, in the brain, rln expression has been revealed in telencephalic region around anterior commissure, in the preoptic area, and in restricted rombencephalic cell clusters. Expression of rln gene in extra-neural territories has been detected in the pancreatic and thyroid gland regions. Danio rerio rln expression pattern analysis reveals shared features with the mammalian RLN gene, particularly in the brain, where it might have a role in the neurophysiological processes. In addition, expression in the thyroid and pancreas region suggests a function as a paracrine and endocrine hormone.

  12. Characterization of cDNA for the large subunit of the transcription initiation factor TFIIF.

    Science.gov (United States)

    Aso, T; Vasavada, H A; Kawaguchi, T; Germino, F J; Ganguly, S; Kitajima, S; Weissman, S M; Yasukochi, Y

    1992-01-30

    At least six chromatographically resolvable general transcription factors may participate in accurate initiation by RNA polymerase II in HeLa cell-derived systems. TFIIF (also termed FC, RAP30/74 and beta/gamma) can bind directly to RNA polymerase II in solution and decrease the affinity of RNA polymerase II for nonspecific DNA. From studies on the kinetics of transcription initiation, on the composition of transcription initiation complexes fractionated by acrylamide gel electrophoresis, and on template competition experiments, TFIIF is known to act at an intermediate stage in initiation complex formation. It acts after TFIID firmly associates with DNA, but coincidentally with or immediately after RNA polymerase II binding to DNA, and before the recruitment of factor TFIIE. TFIIF may or may not have DNA helicase activity. The small subunit (RAP30) of TFIIF has been cloned and shows some amino-acid sequence homology to bacterial sigma factors. We have partially sequenced the RAP74 protein from purified HeLa cells, cloned its complementary DNA and shown that its translation product can interact with RAP30 in vitro as well as in vivo. The cDNA predicts an amino-acid sequence that lacks obvious DNA or RNA helicase motifs. It has regions rich in charged amino acids, including segments containing a higher content of acidic amino acids than are found in strong transcriptional activators such as VP16.

  13. Construction of Yeast Two-hybrid cDNA Library of Verticillium dahliae%大丽轮枝菌酵母双杂交 cDNA 文库的构建

    Institute of Scientific and Technical Information of China (English)

    康静敏; 刘坤; 刘严; 张怡; 李成伟; 谭光轩

    2015-01-01

    为了分离克隆与植物互作的大丽轮枝菌致病相关蛋白基因,利用 SMART 技术构建了大丽轮枝菌酵母双杂交 cDNA 文库。结果表明,构建的文库滴度为5.2×107 cfu/mL,库容为3.9×107 cfu;文库 cDNA 插入片段长度主要分布在0.5~2.0 kb,平均为1 kb;文库重组率为96%。表明文库质量较好,为筛选与植物互作的大丽轮枝菌致病蛋白基因奠定了基础。%To seek out the V. dahliae proteins involved in the interaction of plant with V. dahliae,a yeast two-hybrid library of V. dahliae was constructed using SMART technique. Results showed that the titer of cDNA library was 5. 2 × 107 cfu/mL,the library contained 3. 9 × 107 cfu independent clones,the size distribution of insert frag-ments ranged from 0. 5 -2. 0 kb,and the recombination rate was about 96%. These data demonstrated that the li-brary could meet the requirements of standard cDNA library,which laid a foundation for screening the interaction proteins from V. dahliae.

  14. 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.

  15. Fiscal 1998 achievement report. Industrial technology research and development project. (Strategic human cDNA genome application technology development); 1998 nendo senryakuteki hito cDNA genome oyo gijutsu kaihatsu seika hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    A human genome related project named above was started, and studies were conducted for base sequence determination and function analysis for approximately 10,000 kinds of full-length or long-chain human cDNA clones owned by research organizations in this country. The Institute of Medical Science of University of Tokyo and Helix Research Institute dealt with a full-length human cDNA library constructed by oligo-capping, and determined the base sequences of all specimens in the library. The Kazusa DNA Research Institute determined partial sequences for long-chain clones which are not shorter than 4-5kbp, and determined entire sequences for some bases. The obtained base sequence data were subjected to homology analysis, the base sequences were converted into amino acid sequences, and functions of proteins were predicted. In the analysis of gene functions, ATAC-PCR (adaptor tagged competitive-polymerase chain reaction) was applied to the clones covered by this project, and a database was prepared by use of the results of analyses of frequency-related information. For the preparation of a comprehensive gene expression profile, technologies for cDNA microarray construction were established. (NEDO)

  16. 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.

  17. 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.

  18. Gene discovery in Eimeria tenella by immunoscreening cDNA expression libraries of sporozoites and schizonts with chicken intestinal antibodies.

    Science.gov (United States)

    Réfega, Susana; Girard-Misguich, Fabienne; Bourdieu, Christiane; Péry, Pierre; Labbé, Marie

    2003-04-01

    Specific antibodies were produced ex vivo from intestinal culture of Eimeria tenella infected chickens. The specificity of these intestinal antibodies was tested against different parasite stages. These antibodies were used to immunoscreen first generation schizont and sporozoite cDNA libraries permitting the identification of new E. tenella antigens. We obtained a total of 119 cDNA clones which were subjected to sequence analysis. The sequences coding for the proteins inducing local immune responses were compared with nucleotide or protein databases and with expressed sequence tags (ESTs) databases. We identified new Eimeria genes coding for heat shock proteins, a ribosomal protein, a pyruvate kinase and a pyridoxine kinase. Specific features of other sequences are discussed.

  19. Isolation of cDNA encoding the catalytic site of phosphatidylinositol-specific phospholipase C from Coffea arabica L.

    Science.gov (United States)

    Sánchez-Cach, Lucila A; Ortiz-García, Matilde M; Minero-García, Yereni; Muñoz-Sánchez, J Armando; Hernández-Sotomayor, SM Teresa; Suárez-Solís, Víctor M

    2008-01-01

    A cDNA encoding the catalytic site of a phosphatidylinositol-specific phospholipase C (PI-PLC) was isolated from Coffea arabica suspension cells. The cDNA (designated CaPLC) encodes a polypeptide of 308 amino acids, containing the catalytic X and Y domains, and has 99% identity to the soybean gene. Recombinant CaPLC protein was expressed in Escherichia coli, purified, and used to produce a polyclonal antibody. The peptide has a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses. Immunoblots revealed the presence of PLC-like proteins in the tissues of different plant species. PMID:19513191

  20. Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas

    DEFF Research Database (Denmark)

    Wewer, U M; Gerecke, D R; Durkin, M E

    1994-01-01

    Overlapping cDNA clones that encode the full-length human laminin beta 2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature...... chain showed 86.1% sequence identity to the rat beta 2 chain, 50.0% to the human beta 1 chain, and 36.3% to the human beta 3 chain. The greatest sequence identity was in domains VI, V, and III. The sequence of a 24-amino-acid peptide fragment isolated from the beta 2 chain of laminin purified from human...

  1. Next-generation sequencing-based 5' rapid amplification of cDNA ends for alternative promoters.

    Science.gov (United States)

    Perera, Bambarendage P U; Kim, Joomyeong

    2016-02-01

    Mammalian genomes contain many unknown alternative first exons and promoters. Thus, we have modified the existing 5'RACE (5' rapid amplification of cDNA ends) approach into a next-generation sequencing (NGS)-based new protocol that can identify these alternative promoters. This protocol has incorporated two main ideas: (i) 5'RACE starting from the known second exons of genes and (ii) NGS-based sequencing of the subsequent cDNA products. This protocol also provides a bioinformatics strategy that processes the sequence reads from NGS runs. This protocol has successfully identified several alternative promoters for an imprinted gene, PEG3. Overall, this NGS-based 5'RACE protocol is a sensitive and reliable method for detecting low-abundant transcripts and promoters.

  2. Estabishment of A Human Liver Cancer Cell Line Transfected with IL-2 cDNA and Its Biologic Activity

    Institute of Scientific and Technical Information of China (English)

    孙跃明; 王学浩; 杜竞辉

    2001-01-01

    Objective To obtain IL-2 gene transfected human liver cancer cells and study IL-2 expression and its biologic activity in vivo. Methods Human liver cancer cells SMMC-7721 were cocultured with recombinant retroviral vector LNC-IL-2,and screening was performed in G418 medium.The exogenous IL-2 cDNA at the DNA,RNA,and protein levels were determined by using dot hybridization,PR-PCR and MTT methods respectively.The tumorigenesis and antitumorigenesis of the screened liver cancer cell with subcutaneous injection in nude mice were observed. Results and Conclusion The IL-2 cDNA was successfully integrated into SMMC-7721 cell genomic DNA and continuously expressed for more than 88 days.Subcutaneous vaccination of the nude mice with transfected cells revealed an obvious suppression of its tumorigenicity,and could induce antitumor activity in vivo.

  3. 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.

  4. Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes

    Energy Technology Data Exchange (ETDEWEB)

    Arino, J.; Woon, Chee Wai; Brautigan, D.L.; Miller, T.B. Jr.; Johnson, G.L. (Univ. of Massachusetts Medical School, Worcester (USA))

    1988-06-01

    Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. All of the amino acid substitutions were clustered in the amino-terminal domain of the protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity.

  5. Mass spectrometry-based cDNA profiling as a potential tool for human body fluid identification.

    Science.gov (United States)

    Donfack, Joseph; Wiley, Anissa

    2015-05-01

    Several mRNA markers have been exhaustively evaluated for the identification of human venous blood, saliva, and semen in forensic genetics. As new candidate human body fluid specific markers are discovered, evaluated, and reported in the scientific literature, there is an increasing trend toward determining the ideal markers for cDNA profiling of body fluids of forensic interest. However, it has not been determined which molecular genetics-based technique(s) should be utilized to assess the performance of these markers. In recent years, only a few confirmatory, mRNA/cDNA-based methods have been evaluated for applications in body fluid identification. The most frequently described methods tested to date include quantitative polymerase chain reaction (qPCR) and capillary electrophoresis (CE). However these methods, in particular qPCR, often favor narrow multiplex PCR due to the availability of a limited number of fluorescent dyes/tags. In an attempt to address this technological constraint, this study explored matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for human body fluid identification via cDNA profiling of venous blood, saliva, and semen. Using cDNA samples at 20pg input phosphoglycerate kinase 1 (PGK1) amounts, body fluid specific markers for the candidate genes were amplified in their corresponding body fluid (i.e., venous blood, saliva, or semen) and absent in the remaining two (100% specificity). The results of this study provide an initial indication that MALDI-TOF MS is a potential fluorescent dye-free alternative method for body fluid identification in forensic casework. However, the inherent issues of low amounts of mRNA, and the damage caused to mRNA by environmental exposures, extraction processes, and storage conditions are important factors that significantly hinder the implementation of cDNA profiling into forensic casework.

  6. 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.

  7. RNA transcripts of full-length cDNA clones of rabbit hepatitis E virus are infectious in rabbits.

    Science.gov (United States)

    Cossaboom, Caitlin M; Huang, Yao-Wei; Yugo, Danielle M; Kenney, Scott P; Piñeyro, Pablo; Matzinger, Shannon R; Heffron, C Lynn; Pierson, F William; Meng, Xiang-Jin

    2014-11-07

    Hepatitis E virus (HEV), the causative agent of hepatitis E, is a single-stranded positive-sense RNA virus belonging to the family Hepeviridae. At least four genotypes of the family infect humans: genotypes 1 and 2 are transmitted to humans through contaminated water, while genotypes 3 and 4 are zoonotic and have animal reservoirs. A novel strain of HEV recently identified in rabbits is a distant member of genotype 3, and thus poses a potential risk of zoonotic transmission to humans. The objective of this study was to construct and characterize an infectious cDNA clone of the rabbit HEV. Two full-length cDNA clones of rabbit HEV, pT7g-rabHEV and pT7-rabHEV, were constructed and their infectivity was tested by in vitro transfection of Huh7 human liver cells and by direct intrahepatic inoculation of rabbits with capped RNA transcripts. Results showed that positive signal for rabbit HEV protein was detected by an immunofluorescence assay with a HEV-specific antibody in Huh7 human liver cells transfected with capped RNA transcripts from the two full-length cDNA clones. Rabbits intrahepatically inoculated with capped RNA transcripts from each of the two clones developed active HEV infection as evidenced by seroconversion to anti-HEV antibodies, and detection of rabbit HEV RNA in sera and feces of inoculated animals. The availability of a rabbit HEV infectious cDNA clone now affords us the ability to delineate the mechanism of HEV replication and cross-species infection in a small animal model.

  8. 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.

  9. A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis.

    Science.gov (United States)

    Kim, Y S; Nosaka, K; Downs, D M; Kwak, J M; Park, D; Chung, I K; Nam, H G

    1998-08-01

    We report the characterization of a Brassica napus cDNA clone (pBTHI) encoding a protein (BTHI) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity. A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E. coli mutant strain. The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids. The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively. The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functionally complemented the S. typhimurium and E. coli thiD mutants deficient in the HMP-P kinase activity. These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus. This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus. Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned. The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene.

  10. 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.

  11. 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.

  12. Cross-species hybridisation of human and bovine orthologous genes on high density cDNA microarrays

    OpenAIRE

    2004-01-01

    Abstract Background Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a s...

  13. Mouse protein arrays from a TH1 cell cDNA library for antibody screening and serum profiling

    OpenAIRE

    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...

  14. Identification of Differentially Expressed IGFBP5-Related Genes in Breast Cancer Tumor Tissues Using cDNA Microarray Experiments

    OpenAIRE

    2015-01-01

    IGFBP5 is an important regulatory protein in breast cancer progression. We tried to identify differentially expressed genes (DEGs) between breast tumor tissues with IGFBP5 overexpression and their adjacent normal tissues. In this study, thirty-eight breast cancer and adjacent normal breast tissue samples were used to determine IGFBP5 expression by qPCR. cDNA microarrays were applied to the highest IGFBP5 overexpressed tumor samples compared to their adjacent normal breast tissue. Microarray a...

  15. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

    OpenAIRE

    1996-01-01

    A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model sys...

  16. Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library.

    OpenAIRE

    1988-01-01

    We have developed a subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in pi H3M, a vector containing the phage M13 origin of replication. We have used this strategy to isolate three transcripts whose abundance is increased in scrapie-infected brain. DNA sequence analysis showed that they represent glial fibrillary acidic protein, metallothionein II, and the B chain of alpha-crystallin; the latter two may represent a response to stress.

  17. Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis

    Directory of Open Access Journals (Sweden)

    Børresen-Dale Anne-Lise

    2002-10-01

    Full Text Available Abstract Background T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. Results Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation. Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3–3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. Conclusion T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays.

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

    Directory of Open Access Journals (Sweden)

    Wallis James G

    2007-07-01

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

  19. Novel human testis-specific cDNA: molecular cloning, expression and immunobiological effects of the recombinant protein.

    Science.gov (United States)

    Santhanam, R; Naz, R K

    2001-09-01

    A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-(lambda)gt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122-124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S-transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the approximately 17 kDa recombinant TSA-1, and a approximately 24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility.

  20. 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.

  1. 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.

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

    Science.gov (United States)

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

    2003-06-01

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

  3. cDNA sequence and protein bioinformatics analyses of MSTN in African catfish (Clarias gariepinus).

    Science.gov (United States)

    Kanjanaworakul, Poonmanee; Sawatdichaikul, Orathai; Poompuang, Supawadee

    2016-04-01

    Myostatin, also known as growth differentiation factor 8, has been identified as a potent negative regulator of skeletal muscle growth. The purpose of this study was to characterize and predict function of the myostatin gene of the African catfish (Cg-MSTN). Expression of Cg-MSTN was determined at three growth stages to establish the relationship between the levels of MSTN transcript and skeletal muscle growth. The partial cDNA sequence of Cg-MSTN was cloned by using published information from its congener walking catfish (Cm-MSTN). The Cg-MSTN was 1194 bp in length encoding a protein of 397 amino acids. The deduced MSTN sequence exhibited key functional sites similar to those of other members of the TGF-β superfamily, especially, the proteolytic processing site (RXXR motif) and nine conserved cysteines at the C-terminal. Expression of MSTN appeared to be correlated with muscle development and growth of African catfish. Protein bioinformatics revealed that the primary sequence of Cg-MSTN shared 98 % sequence identity with that of walking catfish Cm-MSTN with only two different residues, [Formula: see text]. and [Formula: see text]. The proposed model of Cg-MSTN revealed the key point mutation [Formula: see text] causing a 7.35 Å shorter distance between the N- and C-lobes and an approximately 11° narrow angle than those of Cm-MSTN. The substitution of a proline residue near the proteolytic processing site which altered the structure of myostatin may play a critical role in reducing proteolytic activity of this protein in African catfish.

  4. Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. (The Howard Florey Institute of Experimental Physiology and Medicine, Parkville, Victoria (Australia))

    1991-01-01

    Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CAVI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension. Overall the human CA VI protein has a sequence identity of 35 {percent} with human CA II, while residues involved in the active site of the enzymes have been conserved. The human and sheep secreted carbonic anhydrases have a sequence identity of 72 {percent}. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

  5. Factorial and time course designs for cDNA microarray experiments.

    Science.gov (United States)

    Glonek, G F V; Solomon, P J

    2004-01-01

    Microarrays are powerful tools for surveying the expression levels of many thousands of genes simultaneously. They belong to the new genomics technologies which have important applications in the biological, agricultural and pharmaceutical sciences. There are myriad sources of uncertainty in microarray experiments, and rigorous experimental design is essential for fully realizing the potential of these valuable resources. Two questions frequently asked by biologists on the brink of conducting cDNA or two-colour, spotted microarray experiments are 'Which mRNA samples should be competitively hybridized together on the same slide?' and 'How many times should each slide be replicated?' Early experience has shown that whilst the field of classical experimental design has much to offer this emerging multi-disciplinary area, new approaches which accommodate features specific to the microarray context are needed. In this paper, we propose optimal designs for factorial and time course experiments, which are special designs arising quite frequently in microarray experimentation. Our criterion for optimality is statistical efficiency based on a new notion of admissible designs; our approach enables efficient designs to be selected subject to the information available on the effects of most interest to biologists, the number of arrays available for the experiment, and other resource or practical constraints, including limitations on the amount of mRNA probe. We show that our designs are superior to both the popular reference designs, which are highly inefficient, and to designs incorporating all possible direct pairwise comparisons. Moreover, our proposed designs represent a substantial practical improvement over classical experimental designs which work in terms of standard interactions and main effects. The latter do not provide a basis for meaningful inference on the effects of most interest to biologists, nor make the most efficient use of valuable and limited resources.

  6. 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

  7. Isolation, characterization and cDNA sequencing of a Kazal family proteinase inhibitor from seminal plasma of turkey (Meleagris gallopavo).

    Science.gov (United States)

    Słowińska, Mariola; Olczak, Mariusz; Wojtczak, Mariola; Glogowski, Jan; Jankowski, Jan; Watorek, Wiesław; Amarowicz, Ryszard; Ciereszko, Andrzej

    2008-06-01

    The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.

  8. Analysis of gene expression patterns with cDNA micro-array during late stage of spermatogenesis in mice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The differentiation process of round spermatids to spermatozoa during the late stage of spermatogenesis is called spermiogenesis. To explore spermiogenesis-related genes, cDNA microarray was used to study expression patterns of 1176 genes in pachytene spermatocytes, round spermatids and elongating spermatids of Balb/c mice. The results showed that 208 genes were detected in all the three cell types. Most of them were down-regulated from pachytene spermatocytes to round spermatids and elongating spermatids. However, up-regulation of 7 genes expression in round spermatids and 3 genes in elongating spermatids were found. Expression of 7 differentially expressed genes in cDNA arrays was further confirmed by semi-quantitative RT-PCR study. The RT-PCR results indicated that the expression of 6 genes was consistent with that in cDNA arrays, only one gene did not show differential expression by RT-PCR. These results may provide important clues for studying of expression, regulation, and function of spermiogenesis-related genes.

  9. 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.

  10. Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee.

    Science.gov (United States)

    Whitfield, Charles W; Band, Mark R; Bonaldo, Maria F; Kumar, Charu G; Liu, Lei; Pardinas, Jose R; Robertson, Hugh M; Soares, M Bento; Robinson, Gene E

    2002-04-01

    To accelerate the molecular analysis of behavior in the honey bee (Apis mellifera), we created expressed sequence tag (EST) and cDNA microarray resources for the bee brain. Over 20,000 cDNA clones were partially sequenced from a normalized (and subsequently subtracted) library generated from adult A. mellifera brains. These sequences were processed to identify 15,311 high-quality ESTs representing 8912 putative transcripts. Putative transcripts were functionally annotated (using the Gene Ontology classification system) based on matching gene sequences in Drosophila melanogaster. The brain ESTs represent a broad range of molecular functions and biological processes, with neurobiological classifications particularly well represented. Roughly half of Drosophila genes currently implicated in synaptic transmission and/or behavior are represented in the Apis EST set. Of Apis sequences with open reading frames of at least 450 bp, 24% are highly diverged with no matches to known protein sequences. Additionally, over 100 Apis transcript sequences conserved with other organisms appear to have been lost from the Drosophila genome. DNA microarrays were fabricated with over 7000 EST cDNA clones putatively representing different transcripts. Using probe derived from single bee brain mRNA, microarrays detected gene expression for 90% of Apis cDNAs two standard deviations greater than exogenous control cDNAs. [The sequence data described in this paper have been submitted to Genbank data library under accession nos. BI502708-BI517278. The sequences are also available at http://titan.biotec.uiuc.edu/bee/honeybee_project.htm.

  11. An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones

    Science.gov (United States)

    Butterfield, Yaron S. N.; Marra, Marco A.; Asano, Jennifer K.; Chan, Susanna Y.; Guin, Ranabir; Krzywinski, Martin I.; Lee, Soo Sen; MacDonald, Kim W. K.; Mathewson, Carrie A.; Olson, Teika E.; Pandoh, Pawan K.; Prabhu, Anna-Liisa; Schnerch, Angelique; Skalska, Ursula; Smailus, Duane E.; Stott, Jeff M.; Tsai, Miranda I.; Yang, George S.; Zuyderduyn, Scott D.; Schein, Jacqueline E.; Jones, Steven J. M.

    2002-01-01

    We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites. PMID:12034834

  12. Construction and Characterization of a cDNA Expression Library for the North American Ginseng Root Tissues

    Institute of Scientific and Technical Information of China (English)

    WANG Ying; WANG Kun; BAO Yong-li; WU Yin; MENG Xiang-ying; LI Yu-xin

    2007-01-01

    The root of Panax ginseng plant undergoes a specific developmental process to become a biosynthesis and accumulation tissue for ginsenosides. To identify and analyze genes involved in the biosynthesis of ginsenoside, we constructed and characterized a full-length cDNA library for 6-year-old North American ginseng. The titer of primary cDNA library is 1.2×106 pfu/mL, the titer of amplified library is 2.6×1010 pfu/mL and the rate of recombinant is above 86%. The insert size ranges from 0.3 to 2.0 kb. Sequencing results show that 18 of 58 genes are high homologous to the genes(GBR5, GBR3 and GBR1) known in GenBank, which are involved in biosynthesis of ginsenoside in North American ginseng plant; 16 of 58 genes are novel genes. The full-length cDNA library of North American ginseng root tissues is essential for the cloning of genes known and it is also an initial key for the screening and cloning of new genes.

  13. Cloning a Chymotrypsin-Like 1 (CTRL-1 Protease cDNA from the Jellyfish Nemopilema nomurai

    Directory of Open Access Journals (Sweden)

    Yunwi Heo

    2016-07-01

    Full Text Available An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His69, Asp117, and Ser216. The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita or Hydrozoan (Hydra vulgaris. The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5′ donor splice (GT and 3′ acceptor splice sequences (AG are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.

  14. The mining of pearl formation genes in pearl oyster Pinctada fucata by cDNA suppression subtractive hybridization.

    Science.gov (United States)

    Wang, Ning; Kinoshita, Shigeharu; Nomura, Naoko; Riho, Chihiro; Maeyama, Kaoru; Nagai, Kiyohito; Watabe, Shugo

    2012-04-01

    Recent researches revealed the regional preference of biomineralization gene transcription in the pearl oyster Pinctada fucata: it transcribed mainly the genes responsible for nacre secretion in mantle pallial, whereas the ones regulating calcite shells expressed in mantle edge. This study took use of this character and constructed the forward and reverse suppression subtractive hybridization (SSH) cDNA libraries. A total of 669 cDNA clones were sequenced and 360 expressed sequence tags (ESTs) greater than 100 bp were generated. Functional annotation associated 95 ESTs with specific functions, and 79 among them were identified from P. fucata at the first time. In the forward SSH cDNA library, it recognized mass amount of nacre protein genes, biomineralization genes dominantly expressed in the mantle pallial, calcium-ion-binding genes, and other biomineralization-related genes important for pearl formation. Real-time PCR showed that all the examined genes were distributed in oyster mantle tissues with a consistence to the SSH design. The detection of their RNA transcripts in pearl sac confirmed that the identified genes were certainly involved in pearl formation. Therefore, the data from this work will initiate a new round of pearl formation gene study and shed new insights into molluscan biomineralization.

  15. Cloning and expression of core gene cDNA of Chinese hepatitis C virus in cosmid pTM3

    Institute of Scientific and Technical Information of China (English)

    Rong Long Jiang; Qiao Sheng Lu; Kang Xian Luo

    2000-01-01

    AIM To clone core gene cDNA of Chinese hepatitis C virus ( HCV ) into eukaryotic expression vector cosmid pTM3 and to express HCV core antigen in HepG2 cells. METHODS Core gene cDNA of HCV was introduced into eukaryotic expression vector cosmid pTM3. Using vaccinia virus/bacteriophage T7 hybrid expression system,HepG2 cells were transfected with the recombinant plasmid pTM3-Q534 by lipofectin. RESULTS From the transfected bacteria Top10F′, 2 pTM3-Q534 clones containing the recombinant plasmid were identified from randomly selected 10 ampicillin-resistant colonies. By reverse transcription PCR and indirect immunofluorescence technique, HCV RNA and core protein was identified in HepG2 cells transfected with the recombinant plasmid.CONCLUSION The construction of a recombinant plasmid and the expression of core gene cDNA of HCV in HepG2 was successful.

  16. cDNA of YP4, a follicular epithelium yolk protein subunit, in the moth, Plodia interpunctella.

    Science.gov (United States)

    Perera, O P; Shirk, P D

    1999-01-01

    YP4, a subunit of the follicular epithelium yolk protein in the moth, Plodia interpunctella, is produced in the follicle cells during vitellogenesis and after secretion is taken up into the oocyte and stored in the yolk spheres for utilization during embryogenesis. In order to identify the cDNA clones for YP4, a degenerate PCR primer was designed to six amino acid residues identified in the NH2-terminal sequence of mature YP4. The YP4 degenerate primer plus T7 reverse PCR primer produced a PCR product from a cDNA library for the majority of the YP4 coding sequence. Combined cDNA and 5' RACE sequencing showed the YP4 transcript to be 991 bp in length with a single open reading frame for a predicted polypeptide of 299 amino acids. Northern analysis showed a single YP4 transcript was present in ovarian RNA that was approximately 1 kb in length. The predicted amino acid sequence for YP4 from P. interpunctella was most closely related to the predicted YP4 protein from the moth, Galleria mellonella, and the spherulin 2a protein from the slime mold, Physarum polycephalum.

  17. Gene expression profiling in human peripheral blood mononuclear cells using high-density filter-based cDNA microarrays.

    Science.gov (United States)

    Walker, J; Rigley, K

    2000-05-26

    Microarray technology has provided the ability to analyse the expression profiles for thousands of genes in parallel. The need for highly specialised equipment to use certain types of microarrays has restricted the application of this technology to a small number of dedicated laboratories. High-density filter-based cDNA microarrays provide a low-cost option for performing high-throughput gene expression analysis. We have used a model system in which filter-based cDNA microarrays representing over 4000 known human genes were used to monitor the kinetics of gene expression in human peripheral blood mononuclear cells (PBMCs) stimulated with phytohaemagluttinin (PHA). Using software-based cluster analysis, we identified 104 genes that altered in expression levels in response to PHA stimulation of PBMCs and showed that there was a considerable overlap between genes with similar temporal expression profiles and similar functional roles. Comparison of microarray quantitation with quantitative PCR showed almost identical expression profiles for a number of genes. Coupled with the fact that our findings are in agreement with a large number of independent observations, we conclude that the use of filter-based cDNA microarrays is a valid and accurate method for high-throughput gene expression profiling.

  18. Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

    Science.gov (United States)

    Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

    1990-10-01

    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 extra-cellular 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).

  19. Tissue-specific expression and cDNA cloning of small nuclear ribonucleoprotein-associated polypeptide N

    Energy Technology Data Exchange (ETDEWEB)

    McAllister, G.; Amara, S.G.; Lerner, M.R. (Yale Univ. School of Medicine, New Haven, CT (USA))

    1988-07-01

    Sera from some patients with systemic lupus erythematosus and other autoimmune diseases have antibodies against nuclear antigens. An example is anti-Sm sera, which recognize proteins associated with small nuclear RNA molecules (small nuclear ribonucleoprotein (snRNP) particles). In this paper anti-Sm sera were used to probe immunoblots of various rat tissues. A previously unidentified M{sub r} 28,000 polypeptide was recognized by these anti-Sm sera. This polypeptide, referred to as N, is expressed in a tissue-specific manner, being most abundant in rat brain, less so in heart, and undetectable in the other tissues examined. Immunoprecipitation experiments using antibodies directed against the cap structure of small nuclear RNAs have demonstrated that N is a snRNP-associated polypeptide. Anti-Sm serum was also used to isolate a partial cDNA clone ({lambda}rb91) from a rat brain phage {lambda}gt11 cDNA expression library. A longer cDNA clone was obtained by rescreening the library with {lambda}rb91. In vitro transcription and subsequent translation of this subcloned, longer insert (pGMA2) resulted in a protein product with the same electrophoretic and immunological properties as N, confirming that pGMA2 encodes N. The tissue distribution of N and the involvement of snRNP particles in nuclear pre-mRNA processing may imply a role for N in tissue-specific pre-mRNA splicing.

  20. Molecular cloning of Ras cDNA from Penaeus japonicus (Crustacea, decapoda): geranylgeranylation and guanine nucleotide binding.

    Science.gov (United States)

    Huang, C F; Chuang, N N

    1998-12-11

    A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. The shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian K-Ras 4B protein and demonstrates identity in the guanine nucleotide binding domains. Expression of the shrimp cDNA in Escherichia coli yielded a 21-kDa polypeptide with a positive reactivity towards the monoclonal antibodies against mammalian Ras. The GTP binding of the shrimp ras-encoded fusion protein was approximated to be 30000units/mg of protein, whereas the binding for GDP was 5000units/mg of protein. Fluorography analysis demonstrated that the prenylation of both shrimp Ras GDP and shrimp Ras GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded the shrimp Ras nucleotide-free form by 10-fold, and fourfold, respectively; that is, the shrimp protein geranylgeranyltransferase I prefers to react with the shrimp ras-encoded p25 fusion protein in the GDP-bound form.

  1. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. (Vanderbilt Univ., Nashville, TN (United States))

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A{sub 2}, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human-hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and it gene regulation.

  2. Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification

    OpenAIRE

    2008-01-01

    Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synt...

  3. 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.

  4. 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

  5. cDNA sequence, mRNA expression and genomic DNA of trypsinogen from the indianmeal moth, Plodia interpunctella.

    Science.gov (United States)

    Zhu, Y C; Oppert, B; Kramer, K J; McGaughey, W H; Dowdy, A K

    2000-02-01

    Trypsin-like enzymes are major insect gut enzymes that digest dietary proteins and proteolytically activate insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). Resistance to Bt in a strain of the Indianmeal moth, Plodia interpunctella, was linked to the absence of a major trypsin-like proteinase (Oppert et al., 1997). In this study, trypsin-like proteinases, cDNA sequences, mRNA expression levels and genomic DNAs from Bt-susceptible and -resistant strains of the Indianmeal moth were compared. Proteinase activity blots of gut extracts indicated that the susceptible strain had two major trypsin-like proteinases, whereas the resistant strain had only one. Several trypsinogen-like cDNA clones were isolated and sequenced from cDNA libraries of both strains using a probe deduced from a conserved sequence for a serine proteinase active site. cDNAs of 852 nucleotides from the susceptible strain and 848 nucleotides from the resistant strain contained an open reading frame of 783 nucleotides which encoded a 261-amino acid trypsinogen-like protein. There was a single silent nucleotide difference between the two cDNAs in the open reading frame and the predicted amino acid sequence from the cDNA clones was most similar to sequences of trypsin-like proteinases from the spruce budworm, Choristoneura fumiferana, and the tobacco hornworm, Manduca sexta. The encoded protein included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Northern blotting analysis showed no major difference between the two strains in mRNA expression in fourth-instar larvae, indicating that transcription was similar in the strains. Southern blotting analysis revealed that the restriction sites for the trypsinogen genes from the susceptible and resistant strains were different. Based on an enzyme size comparison, the cDNA isolated in this study corresponded to the gene for the smaller of two

  6. 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

  7. Achieving high throughput sequencing of a cDNA library utilizing an alternative protocol for the bench top next-generation sequencing system.

    Science.gov (United States)

    Wan, Minxi; Faruq, Junaid; Rosenberg, Julian N; Xia, Jinlan; Oyler, George A; Betenbaugh, Michael J

    2013-02-15

    The development of next-generation sequencing (NGS) technologies has provided novel tools for genome analysis and expression profiling. A high throughput cDNA sequencing method using a bench top next-generation sequencing system, GS Junior, is now available. Here, we used an alternative protocol to the standard method for generating the cDNA library. This protocol can decrease the number of processing steps to manipulate RNA when constructing a cDNA library from an RNA sample, and does not require mRNA isolation from total RNA. Thus it can decrease the risk of RNA degradation and the cost for preparing a cDNA library. Also, the efficiency of sequencing data obtained with this approach is comparable to the standard method as verified by sequencing characteristics and expression levels of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

  8. 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.

  9. Cloning of a novel inhibin alpha cDNA from rhesus monkey testis

    Directory of Open Access Journals (Sweden)

    Woodruff Teresa K

    2004-10-01

    Full Text Available Abstract Background Inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion. Inhibin B is produced by testicular Sertoli cells and is the primary circulating form of inhibin in most adult male mammals. Inhibin B is comprised of the inhibin alpha subunit disulfide-linked to the inhibin/activin betaB subunit. Here we describe the cloning of the cDNAs encoding these subunits from adult rhesus monkey testis RNA. Methods The subunit cDNAs were cloned by a combination of reverse transcriptase polymerase chain reaction (RT-PCR and 5' rapid amplification of cDNA ends (RACE RT-PCR from adult rhesus monkey testis RNA. Results Both the inhibin alpha and betaB subunit nucleotide and predicted protein sequences are highly conserved with other mammalian species, particularly with humans. During the course of these investigations, a novel inhibin alpha mRNA isoform was also identified. This form, referred to as rhesus monkey inhibin alpha-variant 2, appears to derive from both alternative transcription initiation as well as alternative splicing. rmInhibin alpha-variant 2 is comprised of a novel 5' exon (exon 0, which is spliced in-frame with exon 2 of the conventional inhibin alpha isoforms (variant 1. Exon 1 is skipped in its entirety such that the pro-alpha and part of the alpha N regions are not included in the predicted protein. rmInhibin alpha -variant 2 is of relatively low abundance and its biological function has not yet been ascertained. Conclusion The data show that the predicted inhibin B protein is very similar between monkeys and humans. Therefore, studies in monkeys using recombinant human inhibins are likely to reflect actions of the homologous ligands. In addition, we have observed the first inhibin alpha subunit mRNA variant. It is possible that variants will be observed in other species as well and this may lead to novel insights into inhibin action.

  10. Folate biosynthesis in higher plants. cDNA cloning, heterologous expression, and characterization of dihydroneopterin aldolases.

    Science.gov (United States)

    Goyer, Aymeric; Illarionova, Victoria; Roje, Sanja; Fischer, Markus; Bacher, Adelbert; Hanson, Andrew D

    2004-05-01

    Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to be cloned and characterized from plants. This enzyme catalyzes conversion of 7,8-dihydroneopterin (DHN) to 6-hydroxymethyl-7,8-dihydropterin, and is encoded by the folB gene in Escherichia coli. The E. coli FolB protein also mediates epimerization of DHN to 7,8-dihydromonapterin. Searches of the Arabidopsis genome detected three genes encoding substantially diverged FolB homologs (AtFolB1-3, sharing 57%-73% identity), for which cDNAs were isolated. A fourth cDNA specifying a FolB-like protein (LeFolB1) was obtained from tomato (Lycopersicon esculentum) by reverse transcription-PCR. When overproduced in E. coli, recombinant AtFolB1, AtFolB2, and LeFolB1 proteins all had both dihydroneopterin aldolase and epimerase activities, and carried out the aldol cleavage reaction on the epimerization product, 7,8-dihydromonapterin, as well as on DHN. AtFolB3, however, could not be expressed in active form. Size exclusion chromatography indicated that the plant enzyme is an octamer, like the bacterial enzyme. Quantifying expression of the Arabidopsis genes by real-time reverse transcription-PCR showed that AtFolB1 and AtFolB2 messages occur at low levels throughout the plant, whereas the AtFolB3 mRNA was detected only in siliques and only with an extremely low abundance. Sequence comparisons and phylogenetic analysis of FolB homologs from 16 plants indicated that their N-terminal regions are highly variable, and that most species have a small number of FolB genes that diverged after separation of the lineages leading to families. The substantial divergence of FolB homologs in Arabidopsis and other plants suggests that some of them may act on substrates other than DHN.

  11. cDNA clone of hepatitis A virus encoding a virulent virus: induction of viral hepatitis by direct nucleic acid transfection of marmosets.

    OpenAIRE

    Emerson, S U; Lewis, M; Govindarajan, S. (Srinath); M. Shapiro(Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America); Moskal, T; Purcell, R. H.

    1992-01-01

    Direct inoculation of marmoset livers with an in vitro transcription mixture containing cDNA and full-length genomic RNA transcripts of hepatitis A virus resulted in acute viral hepatitis. Elevations in serum levels of liver enzymes were correlated with appearance of antibody to hepatitis A virus. Genomes of infectious hepatitis A virus isolated from the feces of transfected marmosets contained the same mutation as the cDNA template used for transfection. Liver biopsies confirmed that the vir...

  12. 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.

  13. Isolation and characterization of a cDNA clone of UDP-galactose: flavonoid 3-O-galactosyltransferase (UF3GaT) expressed in Vigna mungo seedlings.

    Science.gov (United States)

    Mato, M; Ozeki, Y; Itoh, Y; Higeta, D; Yoshitama, K; Teramoto, S; Aida, R; Ishikura, N; Shibata, M

    1998-11-01

    Four cDNA clones were isolated from Vigna mungo seedlings by the screening with cDNA encoding UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) of Antirrhinum majus as a probe; the product of the gene corresponding to one cDNA was more highly expressed in the first simple leaves than in stems. Nucleotide sequence analysis revealed 1,691 bp (including 326 bp non-reading) containing an open reading frame of 455 amino acids. The deduced amino acid sequence showed 42% and 23% identity with those of A. majus UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) and Petunia hybrida UDP-rhamnose:anthocyanidin 3-O-glucoside rhamnosyltransferase (RT), respectively. One region of the cDNA (amino acids 325 to 387) showed similarity to ceramide UDP-galactosyltransferases of mice, rats and humans. A crude extract from Escherichia coli, in which the protein was expressed from the cDNA, showed high UF3GaT activity but low UF3GT activity, and was similar in K(m), optimal pH and substrate specificity to UF3GaT from V. mungo. We conclude that we have obtained UDP-galactose:flavonoid 3-O-galactosyltransferase (UF3GaT) cDNA from V. mungo.

  14. 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.

  15. 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.

  16. ISOLATION OF HUMAN TRANSCRIPTS EXPRESSED IN 16HBE CELLS RELATED TO CHLOROPHYLLIN ANTITRANSFORMING ACTIVITY AGAINST ANTI-BPDE BY cDNA REPRESENTATIONAL DIFFERENCE ANALYSIS

    Institute of Scientific and Technical Information of China (English)

    ZHU Li-jin; JIANG Yi-guo

    2005-01-01

    Objective: To analyze the differentially expressed cDNA sequences related to chlorophyllin (CHL) mediated inhibition of malignant transformation of human bronchia1 epithelial cell line (16HBE). Methods: 16HBE cells treated with chlorophyllin and anti-BPDE were conducted as tester, 16HBE cells treated only with anti-BPDE were conducted as driver, and cDNA representational difference analysis (cDNA RDA) was used to compare the differential gene expression between the two kinds of cells. The cDNA fragments were ligated to pGEM-T vector and transformed into JM109 bacteria. The plasmid DNA was sequenced and compared with database in GenBank by BLASTN. Results: Among the 5 cloned cDNA sequences, three were novel and were registered in dbEST database, two showed sequence homology to alpha-enolase and a newly found gene ribosomal protein S18/S6-like. Conclusion: These 5 cDNA sequences might play important roles in antitransforming effect of chlorophyllin.

  17. Understanding the radiosensitivity of hematopoietic stem cells through CDNA micro-arrays profiling

    Energy Technology Data Exchange (ETDEWEB)

    Pawlik, A.; Cebo, Ch.; Vaigot, P.; Tronik-Le Roux, D. [Evry Univ., Lab. de Genomique et Radiobiologie de l' Hematopoiese, Service de Genomique Fonctionnelle, CEA, 91 (France)

    2006-07-01

    Eradication of circulating hematopoietic cells has been long known to be the first noticeable somatic effect following total body ionizing radiation (IR) exposure. Among these hematopoietic cells a marked differences in sensitivity to IR have been documented reflecting the remarkable degree of heterogeneity in cell type, proliferative capacity and cell cycle status within the bone marrow cells. From all the hematopoietic cells, the small lymphocyte has the greatest radiosensitivity. In fact, a decline in absolute lymphocyte count has been used to assess IR dose in the early phase of observation after IR exposure. At moderate doses, bone marrow recovery is triggered by the differentiation of stem/early progenitor cells, which confirms further their differential sensitivity to radiation exposure. Although differences in radiosensitivity of the stem cell pool have also been documented, little is known from a molecular viewpoint. To gain insight into the molecular programs underlying the response o f hematopoietic cells to radiation exposure, we have applied a genome wide analysis strategy based on cDNA micro arrays. This technology offers a unique opportunity to dissect complex biological process by assessing three types of questions, which are, in order of complexity: Which genes are differentially expressed among the samples studied:Which genes are expressed in a coordinated manner and what are the regulators involved,what are the global biological pathways mobilized. To answer these questions transcriptional changes occurring after exposure of mice to whole body irradiation (2 Gy) were monitored in bone marrow and spleen. The time course was established in vivo and encompassed the reversible eradication of cells. For each kinetic point RNA was collected from both, spleen or sorted B.M. populations from irradiated and sham irradiated mice. The sham irradiated mice were used to eliminate stress modifications due to handling.The results highlight numerous

  18. 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

  19. Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    Hai-Tao Wang; Jian-Ping Kong; Fang Ding; Xiu-Qin Wang; Ming-Rong Wang; Lian-Xin Liu; Min Wu; Zhi-Hua Liu

    2003-01-01

    AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1.METHODS: The authors first constructed pcDNA3.1/mychis expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes,classification was performed according to their function and cellular component.RESULTS: Human EMP-1 gene can be stably expressed in ECg706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion.CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved incell signaling, cell communication and adhesion regulators.

  20. 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.

  1. Construction of the Seed-Coat cDNA Microarray and Screening of Differentially Expressed Genes in Barley

    Institute of Scientific and Technical Information of China (English)

    Jin-Song PANG; Meng-Yuan HE; Bao LIU

    2004-01-01

    Some barley mutants can synthesize neither anthocyanins nor proanthocyanidins in the seed coat, which is related to several genes in locus Ant13, but the exact model of action remains unknown. We used the cDNA microarray technology with barley transcription-deficient mutant (ant13-152) that does not synthesize proanthocyanidins as the tester, and its wild type genotype (Triumph) as the driver, to study this question. Six-thousand and forty-eight clones from the wild type Morex testa+pericarp cDNA library were amplified using PCR, and the DNA fragments were spotted on commercial amino-modified glass slide as microarray. The mRNAs from the developing seed coat (8-15 days) of both the mutant and the wild-type barley plants were isolated, and labeled respectively with Cy3-dUTP and Cy5-dUTP when reversely transcribed to cDNAs. The labeled cDNAs were used as probes, mixed at the same molar concentration, and hybridized with the DNA fragments on the slide. Seventy clones exhibiting marked differential expression (ratio>4) were identified from the microarray. All the 25 cDNA clones that showed an over-expression in wild type in comparison to the mutant ant13-152 were sequenced. It was found that most of these overexpressing clones were transcription/translation and hordein-associated genes. These results have laid a solid material basis for further elucidation of the metabolic pathway in proanthocyanidin synthesis in barley and likely other plants.

  2. Molecular characterization of a cDNA encoding copper/zinc superoxide dismutase from cultured cells of Manihot esculenta.

    Science.gov (United States)

    Shin, Seung-Yong; Lee, Haeng-Soon; Kwon, Suk-Yoon; Kwon, Soon-Tae; Kwak, Sang-Soo

    2005-01-01

    Superoxide dismutase (SOD) cDNA, mSOD2, encoding cytosolic copper/zinc SOD (CuZnSOD) cDNA was isolated from suspension-cultured cells of cassava (Manihot esculenta Crantz) by cDNA library screening, and its expression was investigated in relation to environmental stress. mSOD2 is 774 bp in length with an open reading frame (ORF) of 152 amino acids, corresponding to a protein of predicted molecular mass 15 kDa and a pI of 5.22. One copy of the mSOD2 gene was found to be present in the cassava genome by Southern analysis using an mSOD2 cDNA-specific probe. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed diverse expression patterns for the mSOD2 gene in various tissues of intact cassava plants, at various stages of the growth in suspension cultures, and in the leaf tissues exposed to different stresses. The mSOD2 gene was highly expressed in suspension-cultured cells and in the stems of intact plants. However, it was expressed at low levels in leaves and roots. During suspension cell growth, the mSOD2 transcript progressively increased during culture. Moreover, the mSOD2 gene in excised cassava leaves responded to various stresses in different ways. In particular, it was highly induced in leaf tissue by several abiotic stresses, including high temperature (37 degrees C), chilling (4 degrees C), methyl viologen (MV) exposure, and wounding treatment. These results indicate that the mSOD2 gene is involved in the antioxidative process triggered by oxidative stress induced by environmental change.

  3. cDNA cloning and recombinant expression of the gen-eral odorant binding protein Ⅱ from Spodoptera litura

    Institute of Scientific and Technical Information of China (English)

    JIN FengLiang; DONG XiaoLin; XU XiaoXia; REN ShunXiang

    2009-01-01

    A cDNA encoding the general odorant binding protein Ⅱ (GOBP Ⅱ) was isolated from the antennae of Spodoptera fitura (SiGOBP Ⅱ, GenBank Accession No. EU086371) by homologous cloning and rapid amplification of cDNA ends (RACE). Sequencing and structural analyses revealed that the open reading frame (ORF) of SIGOBP Ⅱ was 489 bp, encoding 162 amino acids with a predicted MW of 18.2 kD and pl of 5.72. SIGOPB Ⅱ shared typical structural features of odorant binding proteins with other insects, including the six conservative cysteine residues. The deduced amino acid sequence of SIGOPB Ⅱ shared significant identity with the GOBP Ⅱ from S. frugiperda and S. exigua. RT-PCR and Northern blot analyses showed that SIGOBP Ⅱ was specifically expressed in the antennae, cDNA encoding SIGOBP Ⅱ was constructed into the pET-32a vector and the recombinant protein was highly expressed in Es-cherichia coil BL21 (DE3) after induction with IPTG. SDS electrophoresis and Western blot analysis confirmed the molecular weight of the recombinant SIGOBPⅡ i.e, 32 kD, which has a 6xHis tag at the N-terminus. The recombinant SIGOBP U was purified by single-step Ni-NTA affinity chromatography and used to raise antiserum in rabbits. ELISA showed that the titer of antiserum was 1 : 12800, while Western blot analysis showed that the recombinant SIGOBP Ⅱ was recognized as anti-SiGOBP Ⅱ an-tiserum.

  4. Construction and characterization of infectious cDNA clones of a chicken strain of hepatitis E virus (HEV), avian HEV.

    Science.gov (United States)

    Huang, F F; Pierson, F W; Toth, T E; Meng, X J

    2005-09-01

    Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important human pathogen. Increasing evidence indicates that hepatitis E is a zoonosis. Avian HEV was recently discovered in chickens with hepatitis-splenomegaly syndrome in the USA. Like swine HEV from pigs, avian HEV is also genetically and antigenically related to human HEV. The objective of this study was to construct and characterize an infectious cDNA clone of avian HEV for future studies of HEV replication and pathogenesis. Three full-length cDNA clones of avian HEV, pT7-aHEV-5, pT7G-aHEV-10 and pT7G-aHEV-6, were constructed and their infectivity was tested by in vitro transfection of leghorn male hepatoma (LMH) chicken liver cells and by direct intrahepatic inoculation of specific-pathogen-free (SPF) chickens with capped RNA transcripts from the three clones. The results showed that the capped RNA transcripts from each of the three clones were replication competent when transfected into LMH cells as demonstrated by detection of viral antigens with avian HEV-specific antibodies. SPF chickens intrahepatically inoculated with the capped RNA transcripts from each of the three clones developed active avian HEV infections as evidenced by seroconversion to avian HEV antibodies, viraemia and faecal virus shedding. The infectivity was further confirmed by successful infection of naïve chickens with the viruses recovered from chickens inoculated with the RNA transcripts. The results indicated that all three cDNA clones of avian HEV are infectious both in vitro and in vivo. The availability of these infectious clones for a chicken strain of HEV now affords an opportunity to study the mechanisms of HEV cross-species infection and tissue tropism by constructing chimeric viruses among human, swine and avian HEVs.

  5. Lung fibrosis: drug screening and disease biomarker identification with a lung slice culture model and subtracted cDNA Library.

    Science.gov (United States)

    Guo, Tong; Lok, Ka Yee; Yu, Changhe; Li, Zhuo

    2014-09-01

    Pulmonary fibrosis is a progressive and irreversible disorder with no appropriate cure. A practical and effective experimental model that recapitulates the disease will greatly benefit the research community and, ultimately, patients. In this study, we tested the lung slice culture (LSC) system for its potential use in drug screening and disease biomarker identification. Fibrosis was induced by treating rat lung slices with 1ng/ml TGF-β1 and 2.5μM CdCl2, quantified by measuring the content of hydroxyproline, and confirmed by detecting the expression of collagen type III alpha 1 (Col3α1) and connective tissue growth factor (CTGF) genes. The anti-fibrotic effects of pirfenidone, spironolactone and eplerenone were assessed by their capability to reduce hydroxyproline content. A subtractive hybridisation technique was used to create two cDNA libraries (subtracted and unsubtracted) from lung slices. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed to assess the subtraction efficiency of the subtracted cDNA library. Clones from the two libraries were sequenced and the genes were identified by performing a BLAST search on the NCBI GenBank database. Furthermore, the relevance of the genes to fibrosis formation was verified. The results presented here show that fibrosis was effectively induced in cultured lung slices, which exhibited significantly elevated levels of hydroxyproline and Col3α1/CTGF gene expression. Several inhibitors have demonstrated their anti-fibrotic effects by significantly reducing hydroxyproline content. The subtracted cDNA library, which was enriched for differentially expressed genes, was used to successfully identify genes associated with fibrosis. Collectively, the results indicate that our LSC system is an effective model for the screening of drug candidates and for disease biomarker identification.

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

    Science.gov (United States)

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

    2011-01-15

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

  7. Isolation and analysis of water stress induced genes in maize seedlings by subtractive PCR and cDNA macroarray.

    Science.gov (United States)

    Zheng, Jun; Zhao, Jinfeng; Tao, Yazhong; Wang, Jianhua; Liu, Yunjun; Fu, Junjie; Jin, Ying; Gao, Peng; Zhang, Jinpeng; Bai, Yunfeng; Wang, Guoying

    2004-08-01

    In order to identify genes induced during the water stress response in maize (Zea mays) seedlings, suppression subtractive hybridization (SSH) was performed using mixed cDNAs prepared from maize seedlings treated with 20% PEG as testers and cDNAs from unstressed maize seedlings as drivers. A forward subtractive cDNA library was constructed, from which 960 recombinant colonies were picked and amplified. Through differential screening of the subtractive cDNA library, 533 clones were identified as water stress induced. After sequencing, 190 unique expressed sequence tags (ESTs) were obtained by clustering and blast analysis, which included transcripts that had previously been reported as responsive to stress as well as some functionally unknown transcripts. The ESTs with significant protein homology were sorted into 13 functional categories. A cDNA marcoarray containing the 190 unique ESTs was used to analyze their expression profiles in maize seedling during both PEG treatment and natural drought. The results indicated that 67 ESTs in leaves and 113 ESTs in roots were significantly up-regulated by PEG-stress. 123 ESTs were found to be up-regulated for at least one time-course point in either maize leaves or roots. Correspondingly, 163 ESTs were significantly up-regulated by drought stress. Results from the hierarchical cluster analysis suggest that the leaves and roots of maize seedlings had different expression profiles after PEG treatment and that there was a lot of overlap between PEG- and drought-stress induced up-regulated transcripts. A set of transcripts has been identified, which have significantly increased expression and probably involved in water stress signaling pathway based on data analysis.

  8. Purification, characterization and cDNA cloning of a novel lipopolysaccharide-binding lectin from the shrimp Penaeus monodon.

    Science.gov (United States)

    Luo, Tian; Yang, Haijie; Li, Fang; Zhang, Xiaobo; Xu, Xun

    2006-01-01

    In invertebrates, C-type lectin plays an important role in innate immunity by mediating the recognition of pathogens to host cells and clearing microinvaders. A few C-type lectins have been identified from shrimps, but none of their gene or protein sequences is known to date. In this paper, a C-type lectin (named PmLec) specific for bacterial lipopolysaccharide was purified from the serum of the shrimp Penaeus monodon. The binding of PmLec to lipopolysaccharide was mainly mediated through the O-antigen. PmLec had a strong hemagglutinating and bacterial-agglutinating activity as well as an opsonic effect that enhances hemocyte phagocytosis. The PmLec cDNA sequence was obtained from the cDNA library of P. monodon by polymerase chain reaction with the degenerated primer designed according to the amino-terminal residue sequence of purified PmLec. A 546-bp open reading frame was found to encode a putative protein comprising 182 amino acids and containing a preceding signal peptide of 17 amino acids. A C-type lectin domain existed in PmLec, but no glycosylation site was found. The recombinant PmLec protein expressed in Escherichia coli also showed the same agglutinating activity and opsonic effect as that of the native protein. This is the first report of a lectin cDNA from the shrimp. PmLec functions as a pattern-recognition protein and an opsonin in the shrimp, and it provides a clue to elucidate the role of lectin in the innate immunity of aquatic invertebrates at the molecular level.

  9. 微量RNA的cDNA PCR文库的构建%The Construction of cDNA PCR Library from a Small Amount of RNA

    Institute of Scientific and Technical Information of China (English)

    李晶泉; 袁晓东; 汤敏谦

    2001-01-01

    By the method of PCR (Polymerase Chain Reaction),we have constructed the cDNA PCR library from mRNA.The cDNA PCR library can amplify the original cDNA up to hundreds of times.With the total RNA of human K562 cultured cell,the cDNA of β-Actin has been obtained by the methods of cDNA PCR library and reverse transcription respectively.As contrast,the amount of β-Actin′s cDNA from the cDNA PCR library is much higher than from reverse transcription.75pg total RNA of human K562 Cultured cell is employed to construct 50μl cDNA PCR library,and the cDNA of β-Actin can even be detected by using 1μl of the library as template to perform the PCR.Therefore cDNA PCR library can greatly enlarge the amount of information.%使用PCR(polymerase chain reaction)技术,调制了mRNA的cDNA PCR文库,实验证明,cDNA PCR文库能使原cDNA的量放大数百倍。同时,使用人体K562培养细胞的总RNA,对cDNA PCR文库法和反转录中的β-Actin的cDNA量进行了比较,cDNA PCR文库法中的β-Actin的cDNA量大大高于反转录中的β-Actin的cDNA量。使用75pg的人体K562培养细胞的总RNA,调制成50μl的cDNA PCR文库,使用1μl的cDNA PCR文库进行PCR反应时,可对文库中的β-Actin的cDNA进行PCR检测。因此,cDNA PCR文库显示了良好的信息放大性能。

  10. cDNA heterogeneity suggests structural variants related to the high-affinity IgE receptor.

    OpenAIRE

    Liu, F T; Albrandt, K; Robertson, M W

    1988-01-01

    The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, w...

  11. Identification and characterization of cDNA clones encoding hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase from tobacco.

    Science.gov (United States)

    Farmer, M J; Czernic, P; Michael, A; Negrel, J

    1999-08-01

    The sequences of three cDNA clones that include the complete coding region of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase (THT) from tobacco are reported. The three cDNAs were isolated by antibody screening of a cDNA expression library produced from poly(A)+RNA purified from tobacco leaves (Nicotiana tabacum cv. Bottom Special), previously infiltrated with an incompatible strain of Ralstonia solanacearum. The identity of these clones was confirmed by the detection of THT activity in extracts of transformed Escherichia coli and by matching the translated polypeptides with tryptic enzyme sequences. cDNA clones tht4 and tht11 differ only by their 5' leader and 3' UTRs and therefore encode the same protein, whereas tht10 and tht11 exhibit 95 and 99% sequence identity at the DNA and deduced amino acid levels, respectively. The three clones encode proteins of 226 amino acids with calculated molecular masses of 26 kDa. The deduced amino acid sequences show no similarity with the sequence of anthranilate hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus, the only enzyme exhibiting hydroxycinnamoyltransferase activity to be cloned so far in plants. In contrast, comparison of the THT amino acid sequence with protein sequence databases revealed substantial homology with mammalian diamine acetyltransferases. The THT clones hybridized to a 0.95-kb mRNA from elicited tobacco cell-suspension cultures and also to a mRNA of similar size from wound-healing potato tubers. The messengers for THT were also found to be expressed at relatively high levels in tobacco root tissues. Southern hybridization of tobacco genomic DNA with THT cDNA suggests that several copies of the THT gene occur in the tobacco genome. Inhibition experiments using amino-acid-specific reagents demonstrated that both histidyl and cysteyl residues are required for THT activity. In the course of these experiments THT was also found to be inhibited by (2-hydroxyphenyl) amino sulfinyl

  12. The Gene Expression Profile of D-galactose Induced Aging Model Rat Using cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    Li Min(李珉); Wang Gang; Zhang Wei; Wang Miqu; Zhang Yizheng

    2004-01-01

    In order to study the molecular mechanism of D-galactose induced aging model, cDNA microarray is used to analyze gene expression profiles of both normal and D-galactose induced aging model rats. D-galactose induced aging model rats are injected with D-galactose, while normal rats are injected with physiological saline as control. After 7 weeks, the two groups of rats are killed simultaneously. Their livers are harvested for genome-wide expression analysis. D-galactose treated rats showed changes in gene expression associated with increase or decrease in xenobiotic metabolism, protein metabolism and energy metabolism.

  13. 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,...... of RBP3 caused a 10-fold transactivation of the adenovirus E2 promoter, and this transactivation was dependent on the E2F recognition sequences. These properties suggest that RBP3 encodes E2F, or an E2F-like protein....

  14. Identification of two capa cDNA transcripts and detailed peptidomic characterization of their peptide products in Periplaneta americana

    Directory of Open Access Journals (Sweden)

    Susanne Neupert

    2014-06-01

    Full Text Available The first CAPA peptides of insects were identified from Periplaneta americana using extracts of 8000 abdominal perisympathetic organs. The corresponding gene of cockroaches and other basal insects, however, remained undiscovered. Here we identified two capa cDNA transcripts of P. americana. Single cell mass spectrometry of capa neurons in abdominal ganglia yielded a complete coverage of the precursor sequences of both transcripts; with intermediates of precursor processing more prominent in somata than in perisympathetic organs. The processing of the CAPA precursor in P. americana is compared with that in the beetle Tribolium castaneum which was re-evaluated in this study.

  15. Acetylcholinesterase of the Sand Fly, Phlebotomus papatasi (Scopoli): cDNA Sequence, Baculovirus Expression, and Biochemical Properties

    Science.gov (United States)

    2013-01-01

    temperature of 62.5°C and extension time of 3 min. at 72°C. The cDNA was cloned into pBlueBac4.5/V5-His TOPOW (Applied Biosystems/Life Technologies) and...Parasitol 2004, 20:328–332. 4. Kravchenko V, Wasserberg G, Warburg A: Bionomics of phlebotomine sandflies in the Galilee focus of cutaneous...phlebotomine sandflies . Med Vet Entomol 2011, 25:227–231. 7. Mirzaei A, Rouhani S, Taherkhani H, Farahmand M, Kazemi B, Hedayati M, Baghaei A, Davari B

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

    Directory of Open Access Journals (Sweden)

    Bendahmane Abdelhafid

    2011-05-01

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

  17. Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

    DEFF Research Database (Denmark)

    Ferreira, Elisa N; Pires, Lilian C; Parmigiani, Raphael B;

    2004-01-01

    The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification...... can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within...

  18. Genome-wide expression profiling of the response to terbinafine in Candida albicans using a cDNA microarray analysis

    Institute of Scientific and Technical Information of China (English)

    ZENG Yue-bin; QIAN Yuan-shu; MA Lian; GU Hong-ni

    2007-01-01

    Background Candida albicans is the most frequently seen opportunistic human fungal pathogen. Terbinafine is an allylamine antifungal agent that has been proven to have high clinical efficacy in the therapy of fungal infections, the mechanism of action of terbinafine involves the specific inhibition of fungal squalene epoxidase, resulting in ergosterol deficiency and accumulation of intracellular squalene. We used cDNA microarray analysis technology to monitor global expression profile changes of Candida albicans genes in response to terbinafine treatment, and we anticipated a panoramic view of the responses of Candida albicans cells to the representatives of allylamine antifungal agents at the molecular level in an effort to identify drug class-specific and mechanism-independent changes in gene expression.Methods Candida albicans strain ATCC 90028 was exposed to either medium alone or terbinafine at a concentration equivalent to the 1/2 minimal inhibitory concentrations (MICs, 4 mg/L) for 90 minutes. RNA was isolated and gene expression profiles were compared to identify the changes in the gene expression profile using a cDNA microarray analysis. Differential expression of 10 select genes detected by cDNA microarray analysis was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).Results A total of 222 genes were found to be responsive to terbinafine, including 121 up-regulated genes and 101 down-regulated genes. These included genes encoding membrane transport proteins belonging to the members of the ATP-binding cassette (ABC) or major facilitator superfamily (MFS; CDR1, AGP2, GAP6, PHO84, HOL3, FCY23, VCX1),genes involved in stress response and detoxification (CDR1, AGP2, HOL3), and gene involved in the ergosterol biosynthesis pathway (ERG12). The results of semi-quantitative RT-PCR were consistent with that of the cDNA microarray analysis.Conclusions The up-regulation of the gene encoding the multidrug resistance efflux pump

  19. 虹鳟 Ndufb2基因全长 cDNA 序列的克隆与分析%Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss

    Institute of Scientific and Technical Information of China (English)

    王家庆; 边佳; 李代宗; 马爽; 王亮; 那广宁

    2013-01-01

    Summary Rainbow trout belongs to Salmonidae aerobic fish,and it is necessary for high dissolved oxygen content of living water environment.If the dissolved oxygen content of living water is less than 5 mg/L,it will cause the increase of respiratory rate,which is the so-called“aquaculture floating head”phenomenon.Because the fish lives in hypoxia environment and the 90% oxygen consumption is in the mitochondria,the transmission mechanism in composition and electronic respiratory chain may be different from the terrestrial animal.At the mitochondrial inner membrane,electrons from NADH and succinate pass through the electron transport chain to oxygen,which is reduced to water.Complex I is one of the main sites at which premature electron leakage to oxygen occurs,thus being one of the main sites of production of harmful superoxide.The first isolation of mitochondrial complex I since 1 961,its composition and structure have had a primary understanding,but the specific mechanism of its participation in respiration,especially the function of each subunit is not clear.The protein encoded by Ndufb2 gene is a subunit of the multisubunit NADH:ubiquinone oxidoreductase(complex I). Mammalian complex I is composed of 45 different subunits.This protein has NADH dehydrogenase activity and oxidoreductase activity.It plays an important role in transferring electrons from NADH to the respiratory chain. Reverse transcription PCR(RT-PCR) and rapid amplification of cDNA ends(RACE)methods were used for the isolation of the whole cDNA of Ndufb2 gene from brain of Oncorhynchus mykiss .The assembly taskes of 3' and 5'-RACE sequence were completed by DNAman program.A pair of gene specific primers were designed to amplify the full-length cDNA sequence.ClustalX 1.81 and MEGA 3.0 software were used to calculate the amino acid sequence differences,and then the phylogenetic relationships of rainbow trout Ndufb2 gene sequence with other species were analyzed.Protein phosphorylation sites and

  20. Fiscal 1999 achievement report on the analysis of the complete sequencing of full-length cDNA; 1999 nendo dai 2 ji hosei yosan kanzencho cDNA kozo kaiseki seika hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    This report accommodates the results of study conducted during the period of April 1, 2000, through March 31, 2001. The study began with the partial sequence determination for cDNA (complementary deoxyribonucleic acid) terminals presented by the cDNA library, novel clones were then selected out of them, and efforts proceeded to sequence all the bases therein. In this study, partial sequences were determined for 519,000 clones, and entire sequences for 7252 clones. The obtained sequence data were subjected to a homological analysis and then converted into an amino acid sequence, and then protein function prediction and the like were performed using the SOSUI program or the like. A prototype system to isolate novel clones out of partial sequences and a system for the graphic display of cDNA-genome links were fabricated. As for expression profile databases, the iAFLP (introduced amplified fragment length polymorphism) method was used to construct a high-throughput system and a function analysis database. (NEDO)