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Sample records for synthase liver cdna

  1. cDNA sequence of the long mRNA for human glutamine synthase

    NARCIS (Netherlands)

    van den Hoff, M. J.; Geerts, W. J.; Das, A. T.; Moorman, A. F.; Lamers, W. H.

    1991-01-01

    Screening a human liver cDNA library in lambda ZAP revealed several clones for the mRNA of glutamine synthase. The longest clone was completely sequenced and consists of a 109 bp 5' untranslated region, a 1119 bp protein coding region, a 1498 bp 3' untranslated region and a poly(A) tract of 12 bp

  2. Human mitochondrial HMG CoA synthase: Liver cDNA and partial genomic cloning, chromosome mapping to 1p12-p13, and possible role in vertebrate evolution

    Energy Technology Data Exchange (ETDEWEB)

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Montreal (Canada)] [and others

    1994-10-01

    Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (mHS) is the first enzyme of ketogenesis, whereas the cytoplasmic HS isozyme (cHS) mediates an early step in cholersterol synthesis. We here report the sequence of human and mouse liver mHS cDNAs, the sequence of an HS-like cDNA from Caenorhabditis elegans, the structure of a partial human mHS genomic clone, and the mapping of the human mHS gene to chromosome 1p12-p13. the nucleotide sequence of the human mHS cDNA encodes a mature mHS peptide of 471 residues, with a mean amino acid identity of 66.5% with cHS from mammals and chicken. Comparative analysis of all known mHS and cHS protein and DNA sequences shows a high degree of conservation near the N-terminus that decreases progressively toward the C-terminus and suggests that the two isozymes arose from a common ancestor gene 400-900 million years ago. Comparison of the gene structure of mHS and cHS is also consistant with a recent duplication event. We hypothesize that the physiologic result of the HS gene duplication was the appearance of HS within the mitochondria around the time of emergence of early vertebrates, which linked preexisting pathways of beta oxidation and leucine catabolism and created the HMG CoA pathway of ketogenesis, thus providing a lipid-derived energy source for the vertebrate brain. 56 refs., 4 figs., 2 tabs.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  4. Isolation of an ATP synthase cDNA from Sinonovacula constricta ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... Complete cDNA sequence of ScATPase and its deduced amino acid sequence. Nucleotides were numbered from the first base at the 5'end. The canonical polyadenylation signal-sequence was italic and underlined. The asterisk indicated the stop codon. The domain for ATP synthase C was underlined.

  5. Isolation and sequence analysis of a chalcone synthase cDNA of Matthiola incana R. Br. (Brassicaceae).

    Science.gov (United States)

    Epping, B; Kittel, M; Ruhnau, B; Hemleben, V

    1990-06-01

    A cDNA clone (pcM12) of the chalcone synthase (CHS) of Matthiola incana R. Br. (Brassicaceae) was isolated from a cDNA library, sequenced and analysed. It comprises the complete coding sequence for the CHS and 5' and 3' untranslated regions. The deduced amino acid sequence shows that the Matthiola incana CHS consists of 394 amino acid residues. Comparison with CHS amino acid sequences of other plants indicates more than 82% homology.

  6. (+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.

    Science.gov (United States)

    Prosser, Ian; Phillips, Andy L; Gittings, Simon; Lewis, Mervyn J; Hooper, Antony M; Pickett, John A; Beale, Michael H

    2002-08-01

    Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.

  7. 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.

  8. cDNA isolation, functional expression, and characterization of (+)-alpha-pinene synthase and (-)-alpha-pinene synthase from loblolly pine (Pinus taeda): stereocontrol in pinene biosynthesis.

    Science.gov (United States)

    Phillips, Michael A; Wildung, Mark R; Williams, David C; Hyatt, David C; Croteau, Rodney

    2003-03-15

    The complex mixture of monoterpenes, sesquiterpenes, and diterpenes that comprises oleoresin provides the primary defense of conifers against bark beetles and their associated fungal pathogens. Monoterpene synthases produce the turpentine fraction of oleoresin, which allows mobilization of the diterpene resin acid component (rosin) and is also toxic toward invading insects; this is particularly the case for alpha-pinene, a prominent bicyclic monoterpene of pine turpentine. The stereochemistry of alpha-pinene is a critical determinant of host defense capability and has implications for host selection, insect pheromone biosynthesis, and tritrophic-level interactions. Pines produce both enantiomers of alpha-pinene, which appear to arise through antipodal reaction mechanisms by distinct enzymes. Using a cDNA library constructed with mRNA from flushing needles of loblolly pine (Pinus taeda), we employed a homology-based cloning strategy to isolate, and confirm by functional expression, the genes encoding (+)-(3R:5R)-alpha-pinene synthase, (-)-(3S:5S)-alpha-pinene synthase, and several other terpene synthases. The pinene synthases, which produce mirror-image products, share only 66% amino acid identity (72% similarity) but are similar in general properties to other monoterpene synthases of gymnosperms. The stereochemical control of monoterpene cyclization reactions, the evolution of "antipodal" enzymes, and the implications of turpentine composition in ecological interactions are discussed.

  9. [Construction of a cDNA library from liver tissue of rhesus monkey, Macaca mulatta].

    Science.gov (United States)

    Qin, Sheng-fang; Tan, Wei-dong; Chen, You-nan; Ding, Yang; Li, Sheng-fu; Li, Hong-xia; Wang, Li; Yang, Rong; Lu, Yan-rong

    2007-06-01

    To screen the target rhesus genes and give some basic genetic evidences to its value as one of the most important animal model in biomedical study, we constructed a cDNA expression library from liver tissue of a healthy rhesus monkey. With Trizol reagent, the total RNA was extracted from healthy rhesus liver tissue. By mutant Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT), the first-strand cDNA was synthesized from purified mRNA, and subsequently the second-strand cDNA was generated via E. coli DNA polymerase I . Then, the EcoR I adapter was added to the synthesized double-strand cDNA, which was subsequently digested by Xho I restriction enzyme and fractionated with CHROMA APIN-400 column. The fractionated cDNA fragments to be longer than 0. 5 kb were ligated into lambda ZAP express vector to form the phagemid cDNA recombinants, which were further packaged into the lambda ZAP cDNA library according to the standard protocol with phage lambda Gold packaging extract. In order to get more stable clones with larger quantity, the primary library was amplified through infecting the host strain XL1-Blue MRF'. Then, the library titre, recombinant rate and length of inserted cDNA were measured, respectively. The capacity of the primary stand or unamplified library was 1. 2X 10(6) pfu. The titers of the unamplified library or the amplified library was 1.1 X 10(6) mixture, pfu/mL or 7. 7 X 10(9) pfu/mL respectively, the percentages of recombinants were 99. 3% and 98. 2%, and the average lengths of the inserts were 2.0 kb and 2. 3 kb, respectively. An excellent cDNA expression library has been constructed successfully, which would lay solid foundation for transplantation study and pre-clinic evaluation of related drugs.

  10. [Construction and characterization of a cDNA library from human liver tissue of cirrhosis].

    Science.gov (United States)

    Chen, Xiao-hong; Chen, Zhi; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan; Yao, Hang-ping

    2005-03-01

    To construct a cDNA library from human liver tissue of cirrhosis. The total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb). A high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.

  11. Cloning, expression, and characterization of soluble starch synthase I cDNA from taro (Colocasia esculenta Var. esculenta).

    Science.gov (United States)

    Lin, Da-Gin; Jeang, Chii-Ling

    2005-10-05

    Soluble starch synthase I (SSSI) cDNA was isolated from taro (Colocasia esculenta var. esculenta) by RT-PCR and rapid amplification of cDNA ends reaction. The transcript of this single-copy gene is 2340 bp and encodes 642 amino acids protein containing a putative transit peptide of 54 residues. Recombinant SSSI protein displayed both primer-dependent and primer-independent activities of starch synthase. More SSSI transcript was expressed in taro leaves than in tubers, with no evident expression in petioles; and more transcript and protein were found in tubers of 597 +/- 37 g of fresh weight than in smaller or larger ones. Two forms of SSSI, i.e., 72 and 66 kDa, exist in leaves, and only the 66 kDa form was found in tubers. The taro SSSI, proposed as a novel member, was located only in the soluble fraction of tuber extract, while SSSI from other sources exist in both soluble and granule-bound forms.

  12. Germacrene C synthase from Lycopersicon esculentum cv. VFNT cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase.

    Science.gov (United States)

    Colby, S M; Crock, J; Dowdle-Rizzo, B; Lemaux, P G; Croteau, R

    1998-03-03

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, beta-caryophyllene, alpha-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton delta-cadinene synthase (50% identity).

  13. Isolation of an ATP synthase cDNA from Sinonovacula constricta ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... 2Ningbo City College of Vocational Technology, Ningbo, 315100 People's Republic of China. 3National Marine Environmental Monitoring Center. Dalian, 116023, People's ... The SMART cDNA library of S. constricta was constructed by our laboratory. Random sequencing of the library using T3 primer.

  14. Isolation and Characterization of Two Germacrene A Synthase cDNA Clones from Chicory1

    Science.gov (United States)

    Bouwmeester, Harro J.; Kodde, Jan; Verstappen, Francel W.A.; Altug, Iris G.; de Kraker, Jan-Willem; Wallaart, T. Eelco

    2002-01-01

    Chicory (Cichorium intybus) sesquiterpene lactones were recently shown to be derived from a common sesquiterpene intermediate, (+)-germacrene A. Germacrene A is of interest because of its key role in sesquiterpene lactone biosynthesis and because it is an enzyme-bound intermediate in the biosynthesis of a number of phytoalexins. Using polymerase chain reaction with degenerate primers, we have isolated two sesquiterpene synthases from chicory that exhibited 72% amino acid identity. Heterologous expression of the genes in Escherichia coli has shown that they both catalyze exclusively the formation of (+)-germacrene A, making this the first report, to our knowledge, on the isolation of (+)-germacrene A synthase (GAS)-encoding genes. Northern analysis demonstrated that both genes were expressed in all chicory tissues tested albeit at varying levels. Protein isolation and partial purification from chicory heads demonstrated the presence of two GAS proteins. On MonoQ, these proteins co-eluted with the two heterologously produced proteins. The Km value, pH optimum, and MonoQ elution volume of one of the proteins produced in E. coli were similar to the values reported for the GAS protein that was recently purified from chicory roots. Finally, the two deduced amino acid sequences were modeled, and the resulting protein models were compared with the crystal structure of tobacco (Nicotiana tabacum) 5-epi-aristolochene synthase, which forms germacrene A as an enzyme-bound intermediate en route to 5-epi-aristolochene. The possible involvement of a number of amino acids in sesquiterpene synthase product specificity is discussed. PMID:12011345

  15. Isolation and bacterial expression of a sesquiterpene synthase CDNA clone from peppermint(mentha .chi. piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Crock, John E. (Moscow, ID)

    1999-01-01

    A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-farnesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-farnesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

  16. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce; Crock, John E.

    2005-01-25

    A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-famesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-famesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

  17. Cloning and characterization of human liver cDNA encoding a protein S precursor

    International Nuclear Information System (INIS)

    Hoskins, J.; Norman, D.K.; Beckmann, R.J.; Long, G.L.

    1987-01-01

    Human liver cDNA encoding a protein S precursor was isolated from two cDNA libraries by two different techniques. Based upon the frequency of positive clones, the abundance of mRNA for protein S is ≅ 0.01%. Blot hybridization of electrophoretically fractionated poly(A) + RNA revealed a major mRNA ≅ 4 kilobases long and two minor forms of ≅ 3.1 and ≅ 2.6 kilobases. One of the cDNA clones contains a segment encoding a 676 amino acid protein S precursor, as well as 108 and 1132 nucleotides of 5' and 3' noncoding sequence, respectively, plus a poly(A) region at the 3' end. The cDNAs are adenosine plus thymidine-rich (60%) except for the 5' noncoding region, where 78% of the nucleotides are guanosine or cytosine. The protein precursor consists of a 41 amino acid leader peptide followed by 635 amino acids corresponding to mature protein S. Comparison of the mature protein region with homologous vitamin K-dependent plasma proteins shows that it is composed of the following domains: an amino-terminal γ-carboxyglutamic acid-rich region of 37 amino acids; a 36 amino acid linker region rich in hydroxy amino acids; four epidermal growth factor-like segments, each ≅ 45 amino acids long; and a 387 amino acid carboxyl-terminal domain of unrecognized structure and unknown function

  18. Germacrene C synthase from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

    Science.gov (United States)

    Colby, Sheila M.; Crock, John; Dowdle-Rizzo, Barbara; Lemaux, Peggy G.; Croteau, Rodney

    1998-01-01

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, β-caryophyllene, α-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton δ-cadinene synthase (50% identity). PMID:9482865

  19. Cloning and sequencing of a cDNA for the delta-subunit of photosynthetic ATP-synthase (EC 3.6.1.34) from pea (Pisum sativum).

    Science.gov (United States)

    Hoesche, J A; Berzborn, R J

    1992-12-29

    lambda gt10 cDNA clones for the nuclear encoded subunit delta of chloroplast ATP-synthase from Pisum sativum have been isolated. The 5' end was completed by PCR. The sequenced cDNA codes for the import precursor. N-Terminal sequencing of the mature protein isolated from chloroplasts revealed that the processing sites of the transit peptide from Pisum sativum and Spinacea oleracea are similar. The overall homology of the deduced amino acid sequences of the mature delta proteins from higher plants is about 40%. The conservation among hydrophilic residues is higher than for hydrophobic ones, indicating that the surface of delta is important for its function within the ATP-synthase.

  20. [Construction of a subtracted cDNA library of chronic intermittent hypoxia rabbit liver by suppression subtractive hybridization].

    Science.gov (United States)

    Wu, Yue-tao; Liu, Rui-hong; Yang, Yu; Luo, Ying-quan; Rong, Yao

    2007-12-01

    To construct a subtracted cDNA library of chronic intermittent hypoxia (CIH) rabbit liver by suppression subtractive hybridization (SSH). Twenty-four rabbits were divided into 4 groups: ordinary feeding group, full-fat food group, ordinary feeding in chronic intermittent hypoxia group, and full-fat food in chronic intermittent hypoxia group. The mRNAs were extracted from different rabbit livers and converted into double-strand cDNA. After digestion with restriction enzyme, the cDNA of hyperlipidemia-sensitive rabbit group was subdivided into 2 portions and each one was lighted with different adaptors. Two rounds of both hybridization and suppression PCR obtained the differentially expressed cDNA. The PCR products were inserted into T/A vector to set up the subtractive cDNA library. The clones were selected and amplified by PCR and identified. Based on the pathology of the abdominal aorta and liver, and the amplified library contained 500 positive bacteria clones, including 462 clones, which had inserts from 250 to 700 bp by PCR analysis. A novel rabbit gene, Cthrc1, involved in CHI had been cloned. The GenBank Accession Number is XM_418373. The molecular mechanism of CIH promoting atherogenesis formation is made clear.

  1. Cloning and characterization of an Armillaria gallica cDNA encoding protoilludene synthase, which catalyzes the first committed step in the synthesis of antimicrobial melleolides.

    Science.gov (United States)

    Engels, Benedikt; Heinig, Uwe; Grothe, Torsten; Stadler, Marc; Jennewein, Stefan

    2011-03-04

    Melleolides and related fungal sesquiterpenoid aryl esters are antimicrobial and cytotoxic natural products derived from cultures of the Homobasidiomycetes genus Armillaria. The initial step in the biosynthesis of all melleolides involves cyclization of the universal sesquiterpene precursor farnesyl diphosphate to produce protoilludene, a reaction catalyzed by protoilludene synthase. We achieved the partial purification of protoilludene synthase from a mycelial culture of Armillaria gallica and found that 6-protoilludene was its exclusive reaction product. Therefore, a further isomerization reaction is necessary to convert the 6-7 double bond into the 7-8 double bond found in melleolides. We expressed an A. gallica protoilludene synthase cDNA in Escherichia coli, and this also led to the exclusive production of 6-protoilludene. Sequence comparison of the isolated sesquiterpene synthase revealed a distant relationship to other fungal terpene synthases. The isolation of the genomic sequence identified the 6-protoilludene synthase to be present as a single copy gene in the genome of A. gallica, possessing an open reading frame interrupted with eight introns.

  2. Cloning and Characterization of an Armillaria gallica cDNA Encoding Protoilludene Synthase, Which Catalyzes the First Committed Step in the Synthesis of Antimicrobial Melleolides*

    Science.gov (United States)

    Engels, Benedikt; Heinig, Uwe; Grothe, Torsten; Stadler, Marc; Jennewein, Stefan

    2011-01-01

    Melleolides and related fungal sesquiterpenoid aryl esters are antimicrobial and cytotoxic natural products derived from cultures of the Homobasidiomycetes genus Armillaria. The initial step in the biosynthesis of all melleolides involves cyclization of the universal sesquiterpene precursor farnesyl diphosphate to produce protoilludene, a reaction catalyzed by protoilludene synthase. We achieved the partial purification of protoilludene synthase from a mycelial culture of Armillaria gallica and found that 6-protoilludene was its exclusive reaction product. Therefore, a further isomerization reaction is necessary to convert the 6–7 double bond into the 7–8 double bond found in melleolides. We expressed an A. gallica protoilludene synthase cDNA in Escherichia coli, and this also led to the exclusive production of 6-protoilludene. Sequence comparison of the isolated sesquiterpene synthase revealed a distant relationship to other fungal terpene synthases. The isolation of the genomic sequence identified the 6-protoilludene synthase to be present as a single copy gene in the genome of A. gallica, possessing an open reading frame interrupted with eight introns. PMID:21148562

  3. Construction and characterization of a cDNA library from liver tissue of Chinese Banna minipig inbred line.

    Science.gov (United States)

    Tan, W; Chen, Y; Zhang, L; Lu, Y; Li, S; Zeng, R; Zeng, Y; Li, Y; Cheng, J

    2006-09-01

    A xenograft that performs efficient functions is an essential premise for successful xenotransplantation. Our early study indicated that Chinese Banna minipig inbred line (BMI) was an ideal xenograft donor. However, the activities of some proteins synthesized by the BMI liver are different from the human, which could lead to functional disorders in coagulation, fibrinolysis, and anticoagulation after liver xenotransplantation. Therefore, it is important to investigate the genetic background of protein incompatibility and to provide new strategies for gene manipulation. In this study we constructed a cDNA expression library using BMI liver tissue to obtain an understanding of nucleic acid and protein differences between the two species. We extracted total RNA and purified mRNA of the liver tissue from one of the sixteenth inbred generation of BMI/JS 151 substrain. After double-strand cDNA synthesis, we fractionated it on a CHROMA APIN-400 column; ligated the longer than 500bp cDNA into a ZAP Express Vector; and performed a lambda: phage packaging reaction, library amplification, and titer. We randomly picked 12 plaques and tested the length of inserts. The titers of the primary and amplified libraries were 1.0 x 10(6) pfu/mL and 5.0 x 10(9) pfu/mL, respectively. The percentages of recombinants were 97.0% in the primary library and 98.0% in the amplified library. The lengths of most inserts were between 750 bp and 2.0 kb. Thus, we successfully constructed a cDNA expression library from BMI liver tissue. Using the library, we hope to get a full-length cDNA of some important genes and conduct further studies on porcine liver function in xenotransplantation.

  4. Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.).

    Science.gov (United States)

    Yamaguchi, S; Saito, T; Abe, H; Yamane, H; Murofushi, N; Kamiya, Y

    1996-08-01

    The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene. ent-Kaurene synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to ent-kaurene by ent-kaurene synthase B (KSB). A full-length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.). Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening. The isolated full-length cDNA was expressed in Escherichia coli as a fusion protein, which demonstrated the KSB activity to cyclize [3H]CDP to [3H]ent-kaurene. The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings. The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion-diphosphate complex binding site. A putative transit peptide sequence that may target the translated product into the plastids is present in the N-terminal region.

  5. Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis.

    Science.gov (United States)

    Lardizabal, K D; Metz, J G; Sakamoto, T; Hutton, W C; Pollard, M R; Lassner, M W

    2000-03-01

    Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.

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

    Science.gov (United States)

    Chen, Xiao-hong; Chen, Zhi; Yao, Hang-ping; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan

    2005-04-01

    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. 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 lambdaTriplEx2 vector. Then lambda 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. The titers of unamplifed and amplified libraries were 1.94 x 10(6) pfu/ml and 1.49 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% 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%. A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

  7. A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

    Science.gov (United States)

    Lassner, M W; Lardizabal, K; Metz, J G

    1996-02-01

    beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils.

  8. Cloning of a cDNA that encodes farnesyl diphosphate synthase and the blue-light-induced expression of the corresponding gene in the leaves of rice plants.

    Science.gov (United States)

    Sanmiya, K; Iwasaki, T; Matsuoka, M; Miyao, M; Yamamoto, N

    1997-02-28

    A cDNA encoding farnesyl diphosphate synthase (FPPS), a key enzyme in isoprenoid biosynthesis, was isolated from a cDNA library constructed from mRNA that had been prepared from etiolated rice (Oriza sativa L. variety Nipponbare) seedlings after three hours of illumination by a subtraction method. The putative polypeptide deduced from the 1289 bp nucleotide sequence consisted of 353 amino acids and had a molecular mass of 40 676 Da. The predicted amino acid sequence exhibited high homology to those of FPPS from Arabidopsis (73% to type 1, 72% to type 2) and white lupin (74%). Southern blot analysis showed that the rice genome might contain only one gene for FPPS. The highest level of expression of the gene was demonstrated in leaves by RNA blot analysis. Moreover, light, in particular blue light, effectively enhanced expression of the gene.

  9. Cloning and characterization of a female genital complex cDNA from the liver fluke Fasciola hepatica.

    Science.gov (United States)

    Zurita, M; Bieber, D; Ringold, G; Mansour, T E

    1987-01-01

    A cDNA clone whose RNA is abundant in the female genital complex of the liver fluke Fasciola hepatica has been isolated from a cDNA library in lambda gt10 by differential screening. The pattern of expression in different fluke tissues and at different stages of miracidium formation suggests that this gene is expressed in the F. hepatica vitelleria. The nucleotide sequence of the cloned cDNA was determined and the primary structure of the putative protein was deduced. The proposed protein is rich in glycine, lysine, and tyrosine and its overall amino acid composition agrees with that reported for the F. hepatica egg shell. The clone has homology with DNA from other trematodes; this homology is higher in organisms in which egg development is similar to that of F. hepatica and suggests that the protein is conserved in organisms in which miracidium formation occurs in fresh water. Images PMID:3470798

  10. Germacrene C synthase from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

    OpenAIRE

    Colby, Sheila M.; Crock, John; Dowdle-Rizzo, Barbara; Lemaux, Peggy G.; Croteau, Rodney

    1998-01-01

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, β-caryophyllene, α-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of...

  11. Mechanism of activation of liver glycogen synthase by swelling

    NARCIS (Netherlands)

    Meijer, A. J.; Baquet, A.; Gustafson, L.; van Woerkom, G. M.; Hue, L.

    1992-01-01

    The mechanism linking the stimulation of liver glycogen synthesis to swelling induced either by amino acids or hypotonicity was studied in hepatocytes, in gel-filtered liver extracts, and in purified preparations of particulate glycogen to which glycogen-metabolizing enzymes are bound. High

  12. Differential effects of nitric oxide synthase inhibitors on endotoxin-induced liver damage in rats

    NARCIS (Netherlands)

    Vos, TA; Gouw, ASH; Klok, PA; Havinga, R; vanGoor, H; Roelofsen, H; Kuipers, F; Jansen, PLM; Moshage, H

    1997-01-01

    Background & Aims: During endotoxemia, expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in the liver is increased, NO has been suggested to have a hepatoprotective function. The aim of this study was to investigate the distribution of iNOS and the effect of different

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

    NARCIS (Netherlands)

    Lücker, J.; Tamer, El 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

  14. Association of Endothelial Nitric Oxide Synthase Gene Polymorphisms With Acute Rejection in Liver Transplant Recipients.

    Science.gov (United States)

    Azarpira, Negar; Namazi, Soha; Malahi, Sayan; Kazemi, Kourosh

    2016-06-01

    Polymorphisms of the endothelial nitric oxide synthase gene have been associated with altered endothelial nitric oxide synthase activity. The purpose of this study was to investigate the relation between endothelial nitric oxide synthase -786T/C and 894G/T polymorphism and their haplotypes on the occurrence of acute rejection episodes in liver transplant recipients. We conducted a case control study in which 100 liver transplant recipients and 100 healthy controls were recruited from Shiraz Transplant Center. The patients used triple therapy including tacrolimus, mycophenolate mofetil, and prednisolone for immunosuppression maintenance. DNA was extracted from peripheral blood and endothelial nitric oxide synthase polymorphisms were determined by polymerase chain reaction and restriction fragment length polymorphism. Patients included 60 men and 40 women (mean age, 32.35 ± 10.2 y). There was a significant association of endothelial nitric oxide synthase 894G/T and acute rejection episode. The GT* gen-otype and acute rejection episodes had a significant association (odds ratio, 2.42; 95% confidence interval, 0.97-6.15; P = .03). The GG and GT* genotype and T* allele frequency were significantly different between patients and control subjects (P = .001). Haplotype TT* was higher in recipients than control subjects (odds ratio, 2.17; 95% confidence interval, 1.12-4.25; P = .01). Haplotype TG was higher in the control group (odds ratio, 0.62; 95% confidence interval, 0.40-0.96; P = .02). Our results suggest a relation between different endothelial nitric oxide synthase geno-types and risk of acute rejection episodes. However, further study is necessary to determine genetic susceptibility for transplant patients.

  15. [Construction and preliminary screening of a forward-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state].

    Science.gov (United States)

    Fang, Ding-Zhi; Liu, Bing-Wen; Shen, Tao; Bai, Huai

    2005-11-01

    To construct and preliminarily screen the forward-subtracted cDNA library of differentially expressed genes in rat liver of prothrombotic state (PTS). The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was constructed by suppression subtractive hybridization using cDNAs synthesized from mRNA of PTS rat as Tester and cDNAs from mRNA of control rat as Driver. The products from the last PCR amplification of suppression subtractive hybridization were inserted into a T/A plasmid vectors to transform the Escherichia coli JM109 cells. To produce the library, the transformed cells were incubated at 37 C overnight on a LB agar plate containing ampicillin (50 microg/ml), IPTG and X-gal. Forward-subtracted cDNA probes and reverse-subtracted cDNA probes were prepared by nested PCR amplification, which were labeled with HRP. Positive clones were selected by differential screening in which forward-subtracted and reverse-subtracted cDNA probes were separately hybridized with the membranes slot-blotted by plasmid DNAs amplified and isolated from the library. Inserts in the positive clones were submitted to DNA sequencing. Nucleic acid sequence homology search was performed against the GenBank DNA database (non-redundant, and non-mouse and non-human EST entries) using the Standard nucleotide-nucleotide BLAST [blastn] program via a network connection to the National Center for Biotechnology information. The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was successfully constructed. Two differentially expressed cDNA fragments were found after preliminary screening. The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was successfully constructed in the present study.

  16. [Construction of a subtracted cDNA library of differentially expressed genes in human normal liver tissue and primary hepatocellular carcinoma tissue].

    Science.gov (United States)

    Li, J; Xu, X; Han, B; Huang, G; Qian, G; Liang, P; Yang, T

    2001-12-01

    To construct a subtracted cDNA library of differentially expressed genes in human normal liver tissue and primary hepatocellular carcinoma (HCC) tissue. Using the suppression subtractive hybridization (SSH), a novel technique has been described recently. cDNA fragments of missing or low expressing tumor suppressor genes in HCC tissue were isolated using paracancerous normal liver tissue and HCC tissue as targets. Then these cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Amplification of the library was carried out with transformation of E.coli by high voltage electroperforation. One hundred positive bacteria clones were randomly picked and identified using enzyme restriction method. The amplified library contained more than 4,000 positive bacteria clones. Random analysis of 100 clones with enzyme restriction method showed that all clones contained 200-600 bp inserts. A subtracted cDNA library of differentially expressed genes in human normal liver tissue and HCC tissue is constructed successfully with SSH and T/A cloning techniques. The library is efficient and lays solid foundation for screening and cloning new and specific missing or low expressing tumor suppressor genes of HCC.

  17. Trehalose metabolism in the blue crab Callinectes sapidus: isolation of multiple structural cDNA isoforms of trehalose-6-phosphate synthase and their expression in muscles.

    Science.gov (United States)

    Shi, Q; Chung, J Sook

    2014-02-15

    Adult blue crab Callinectes sapidus exhibit behavioral and ecological dimorphisms: females migrating from the low salinity water to the high salinity area vs. males remaining in the same areas. The flesh basal muscle of the swimming paddle shows a dimorphic color pattern in that levator (Lev) and depressor (Dep) of females tend to be much darker than those of males, while both genders have the same light colored remoter (Rem) and promoter (Pro). The full-length cDNA sequence of four structural isoforms of trehalose-6-phosphate synthase (TPS) is isolated from chela muscles of an adult female, C. sapidus. Two isoforms of the C. sapidus TPS encode functional domains of TPS and trehalose-6-phosphorylase (TPP) in tandem as a fused gene product of Escherichia coli Ost A and Ost B. The other two isoforms contain only a single TPS domain. In both males and females, the darker (Lev+Dep) muscles exhibit greater amounts of trehalose, TPS and trehalase activities than the light colored (Rem+Pro). The fact that adult females show higher levels of trehalase activity in the basal muscles and of glucose in Lev+Dep than those of adult males suggests that there may be a metabolic dimorphism. Moreover, the involvement of trehalose in energy metabolism that was examined under the condition of strenuous swimming activity mimicked in adult females demonstrates the intrinsic trehalose metabolism in Lev+Dep, which subsequently results in hemolymphatic hyperglycemia and hyperlactemia. Our data support that trehalose serves as an additional carbohydrate source of hemolymphatic hyperglycemia in this species. Behavioral and ecological dimorphisms of C. sapidus adults may be supported by a functional dimorphism in energy metabolism. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Activity of glycogen synthase and glycogen phosphorylase in normal and cirrhotic rat liver during glycogen synthesis from glucose or fructose.

    Science.gov (United States)

    Bezborodkina, Natalia N; Chestnova, Anna Yu; Okovity, Sergey V; Kudryavtsev, Boris N

    2014-03-01

    Cirrhotic patients often demonstrate glucose intolerance, one of the possible causes being a decreased glycogen-synthesizing capacity of the liver. At the same time, information about the rates of glycogen synthesis in the cirrhotic liver is scanty and contradictory. We studied the dynamics of glycogen accumulation and the activity of glycogen synthase (GS) and glycogen phosphorylase (GP) in the course of 120min after per os administration of glucose or fructose to fasted rats with CCl4-cirrhosis or fasted normal rats. Blood serum and liver pieces were sampled for examinations. In the normal rat liver administration of glucose/fructose initiated a fast accumulation of glycogen, while in the cirrhotic liver glycogen was accumulated with a 20min delay and at a lower rate. In the normal liver GS activity rose sharply and GPa activity dropped in the beginning of glycogen synthesis, but 60min later a high synthesis rate was sustained at the background of a high GS and GPa activity. Contrariwise, in the cirrhotic liver glycogen was accumulated at the background of a decreased GS activity and a low GPa activity. Refeeding with fructose resulted in a faster increase in the GS activity in both the normal and the cirrhotic liver than refeeding with glucose. To conclude, the rate of glycogen synthesis in the cirrhotic liver is lower than in the normal one, the difference being probably associated with a low GS activity. Copyright © 2013 Elsevier GmbH. All rights reserved.

  19. Forest tent caterpillars (Malacosoma disstria) induce local and systemic diurnal emissions of terpenoid volatiles in hybrid poplar (Populus trichocarpa x deltoides): cDNA cloning, functional characterization, and patterns of gene expression of (-)-germacrene D synthase, PtdTPS1.

    Science.gov (United States)

    Arimura, Gen-Ichiro; Huber, Dezene P W; Bohlmann, Jörg

    2004-02-01

    Feeding forest tent caterpillars (FTCs) induced local and systemic diurnal emissions of (-)-germacrene D, along with (E)-beta-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), benzene cyanide, and (E,E)-alpha-farnesene, from leaves of hybrid poplar. FTC feeding induced substantially higher levels of volatiles in local and systemic leaves than did mechanical wounding. A full-length poplar sesquiterpene synthase cDNA (PtdTPS1) was isolated and functionally identified as (-)-germacrene D synthase. Expression of PtdTPS1, expression of genes of early, intermediate and late steps in terpenoid biosynthesis, and expression of a lipoxygenase gene (PtdLOX1) were analyzed in local FTC-infested and systemic leaves. Transcript levels of PtdTPS1 and PtdLOX1 were strongly increased in response to herbivory. PtdTPS1 was also induced by mechanical wounding or by methyl jasmonate (MeJA) treatment. FTC feeding did not affect transcript levels of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), and isoprene synthase (IPS). Two other TPS genes, PtdTPS2 and PtTPS3, and farnesyl diphosphate synthase were only very transiently induced. These results illustrate differential expression of terpenoid pathway genes in response to insect feeding and a key function of (-)-germacrene D synthase PtdTPS1 for herbivore-induced local and systemic volatile emissions in hybrid poplar. FTC-induced transcripts of PtdTPS1 followed diurnal rhythm. Spatial patterns of FTC-induced PtdTPS1 transcript accumulation revealed acropetal but not basipetal direction of the systemic response. Implications for tritrophic poplar-FTC-predator/parasitoid interactions are discussed.

  20. Endothelial Nitric Oxide Synthase Gene Variation Associated With Chronic Kidney Disease After Liver Transplant

    Science.gov (United States)

    Bambha, Kiran; Kim, W. Ray; Rosen, Charles B.; Pedersen, Rachel A.; Rys, Cynthia; Kolbert, Christopher P.; Cunningham, Julie M.; Therneau, Terry M.

    2010-01-01

    OBJECTIVE: To identify single nucleotide polymorphisms (SNPs) associated with risk of developing chronic kidney disease (CKD), a prevalent comorbidity, after liver transplant (LT). PATIENTS AND METHODS: This study consists of a cohort of adult (≥18 years) primary-LT recipients who had normal renal function before LT and who survived 1 year or more after LT at a high-volume US LT program between January 1, 1990, and December 31, 2000. Patients with adequate renal function (estimated glomerular filtration rate, ≥40 mL/min per 1.73 m2 during follow-up; n=308) and patients with incident CKD (estimated glomerular filtration rate, <40 mL/min per 1.73 m2 after LT; n=92) were identified. To investigate the association of 6 candidate genes with post-LT CKD, we selected SNPs that have been associated with renal function in the literature. Hazard ratios were estimated using Cox regression, adjusted for potential confounding variables. RESULTS: The variant allele (298Asp) of the Glu298Asp SNP in the endothelial nitric oxide synthase gene (NOS3) was significantly associated with CKD after LT (P=.05; adjusted for multiple comparisons). The 5-year incidence of CKD was 70% among patients homozygous for the NOS3 variant allele (298Asp) compared with 42% among those not homozygous for the NOS3 variant allele. Specifically, homozygosity for the NOS3 variant allele conferred a 2.5-fold increased risk of developing CKD after LT (P=.005, adjusted for confounding variables). CONCLUSION: Homozygosity for the variant allele of NOS3 (298Asp) is associated with CKD after LT and may be useful for identifying recipients at higher risk of post-LT CKD. PMID:20810793

  1. Metabolic impact of overexpression of liver glycogen synthase with serine-to-alanine substitutions in rat primary hepatocytes.

    Science.gov (United States)

    Kadotani, Akito; Fujimura, Maho; Nakamura, Takao; Ohyama, Sumika; Harada, Naomoto; Maruki, Hiroko; Tamai, Yoshitaka; Kanatani, Akio; Eiki, Jun-Ichi; Nagata, Yasufumi

    2007-10-15

    To investigate the effect of elevation of liver glycogen synthase (GYS2) activity on glucose and glycogen metabolism, we performed adenoviral overexpression of the mutant GYS2 with six serine-to-alanine substitutions in rat primary hepatocytes. Cell-free assays demonstrated that the serine-to-alanine substitutions caused constitutive activity and electrophoretic mobility shift. In rat primary hepatocytes, overexpression of the mutant GYS2 significantly reduced glucose production by 40% and dramatically induced glycogen synthesis via the indirect pathway rather than the direct pathway. Thus, we conclude that elevation of glycogen synthase activity has an inhibitory effect on glucose production in hepatocytes by shunting gluconeogenic precursors into glycogen. In addition, although intracellular compartmentation of glucose-6-phosphate (G6P) remains unclear in hepatocytes, our results imply that there are at least two G6P pools via gluconeogenesis and due to glucose phosphorylation, and that G6P via gluconeogenesis is preferentially used for glycogen synthesis in hepatocytes.

  2. Genome-Wide Screening of Genes Showing Altered Expression in Liver Metastases of Human Colorectal Cancers by cDNA Microarray

    Directory of Open Access Journals (Sweden)

    Rempei Yanagawa

    2001-01-01

    Full Text Available In spite of intensive and increasingly successful attempts to determine the multiple steps involved in colorectal carcinogenesis, the mechanisms responsible for metastasis of colorectal tumors to the liver remain to be clarified. To identify genes that are candidates for involvement in the metastatic process, we analyzed genome-wide expression profiles of 10 primary colorectal cancers and their corresponding metastatic lesions by means of a cDNA microarray consisting of 9121 human genes. This analysis identified 40 genes whose expression was commonly upregulated in metastatic lesions, and 7 that were commonly downregulated. The upregulated genes encoded proteins involved in cell adhesion, or remodeling of the actin cytoskeleton. Investigation of the functions of more of the altered genes should improve our understanding of metastasis and may identify diagnostic markers and/or novel molecular targets for prevention or therapy of metastatic lesions.

  3. Quantification of the glycogen cascade system: the ultrasensitive responses of liver glycogen synthase and muscle phosphorylase are due to distinctive regulatory designs

    Directory of Open Access Journals (Sweden)

    Venkatesh KV

    2005-05-01

    Full Text Available Abstract Background Signaling pathways include intricate networks of reversible covalent modification cycles. Such multicyclic enzyme cascades amplify the input stimulus, cause integration of multiple signals and exhibit sensitive output responses. Regulation of glycogen synthase and phosphorylase by reversible covalent modification cycles exemplifies signal transduction by enzyme cascades. Although this system for regulating glycogen synthesis and breakdown appears similar in all tissues, subtle differences have been identified. For example, phosphatase-1, a dephosphorylating enzyme of the system, is regulated quite differently in muscle and liver. Do these small differences in regulatory architecture affect the overall performance of the glycogen cascade in a specific tissue? We address this question by analyzing the regulatory structure of the glycogen cascade system in liver and muscle cells at steady state. Results The glycogen cascade system in liver and muscle cells was analyzed at steady state and the results were compared with literature data. We found that the cascade system exhibits highly sensitive switch-like responses to changes in cyclic AMP concentration and the outputs are surprisingly different in the two tissues. In muscle, glycogen phosphorylase is more sensitive than glycogen synthase to cyclic AMP, while the opposite is observed in liver. Furthermore, when the liver undergoes a transition from starved to fed-state, the futile cycle of simultaneous glycogen synthesis and degradation switches to reciprocal regulation. Under such a transition, different proportions of active glycogen synthase and phosphorylase can coexist due to the varying inhibition of glycogen-synthase phosphatase by active phosphorylase. Conclusion The highly sensitive responses of glycogen synthase in liver and phosphorylase in muscle to primary stimuli can be attributed to distinctive regulatory designs in the glycogen cascade system. The different

  4. Mitochondrial HMG to CoA synthase (mHS): cDNA cloning in human, mouse and C. elegans, mapping to human chromosome 1p12-13 and partial human genomic cloning

    Energy Technology Data Exchange (ETDEWEB)

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Montreal, Quebec (Canada)]|[Kingston General Hospital, Ontario (Canada)] [and others

    1994-09-01

    mHS catalyzes the rate-limiting first step of ketogenesis in the liver. A cytoplasmic HS isozyme, encoded by another gene, catalyzes an early step in cholesterol synthesis. Starting from a rat mHS cDNA obtained by RT-PCR from the published rat cDNA sequence, we obtained and sequenced human and mouse cDNAs spanning the entire coding sequence of natural human and mouse mHS, as well as sequencing C. elegans HS-like cDNA. Consensus sequences for 3 mitochondrial and 4 cytoplasmic HSs were created and compared to invertebrate HS sequences. We found high conversation in the active site and at other regions presumably important for HS function. We mapped the mHS locus, HMGCS2 by in situ hybridization to chromosome 1P12-13, in contrast to the human cHS locus (HMGCS1) known to be on chromosome 5p13. Comparative mapping results suggest that these two chromosomal regions may be contiguous in other species, constant with a recent gene duplication event. Furthermore, we have characterized a human genomic mHS subclone containing 4 mHS exons, and found the position of all splice junctions to be identical to that of the hamster cHS gene except for one site in the 3{prime} nontranslated region. We calculate that the mHS and cHS genes were derived from a common ancestor 400-700 Myrs ago, implying that ketogenesis from fat may have become possible around the time of emergence of vertebrates ({approximately}500 Myr ago). Ketogenesis has evolved into an important pathway of energy metabolism, and we predict the mHS deficiency may prove to be responsible for some as yet explained cases of Reye-like syndromes in humans. This hypothesis can now be tested at the molecular level without the necessity of obtaining hepatic tissue.

  5. The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys.

    Directory of Open Access Journals (Sweden)

    Sheo B Singh

    Full Text Available Platensimycin (PTM is a natural antibiotic produced by Streptomyces platensis that selectively inhibits bacterial and mammalian fatty acid synthase (FAS without affecting synthesis of other lipids. Recently, we reported that oral administration of PTM in mouse models (db/db and db/+ with high de novo lipogenesis (DNL tone inhibited DNL and enhanced glucose oxidation, which in turn led to net reduction of liver triglycerides (TG, reduced ambient glucose, and improved insulin sensitivity. The present study was conducted to explore translatability and the therapeutic potential of FAS inhibition for the treatment of diabetes in humans.We tested PTM in animal models with different DNL tones, i.e. intrinsic synthesis rates, which vary among species and are regulated by nutritional and disease states, and confirmed glucose-lowering efficacy of PTM in lean NHPs with quantitation of liver lipid by MRS imaging. To understand the direct effect of PTM on liver metabolism, we performed ex vivo liver perfusion study to compare FAS inhibitor and carnitine palmitoyltransferase 1 (CPT1 inhibitor.The efficacy of PTM is generally reproduced in preclinical models with DNL tones comparable to humans, including lean and established diet-induced obese (eDIO mice as well as non-human primates (NHPs. Similar effects of PTM on DNL reduction were observed in lean and type 2 diabetic rhesus and lean cynomolgus monkeys after acute and chronic treatment of PTM. Mechanistically, PTM lowers plasma glucose in part by enhancing hepatic glucose uptake and glycolysis. Teglicar, a CPT1 inhibitor, has similar effects on glucose uptake and glycolysis. In sharp contrast, Teglicar but not PTM significantly increased hepatic TG production, thus caused liver steatosis in eDIO mice.These findings demonstrate unique properties of PTM and provide proof-of-concept of FAS inhibition having potential utility for the treatment of diabetes and related metabolic disorders.

  6. The effects of tadalafil and pentoxifylline on apoptosis and nitric oxide synthase in liver ischemia/reperfusion injury

    Directory of Open Access Journals (Sweden)

    Sibel Bektas

    2016-07-01

    Full Text Available The aim of this study was to investigate the effects of tadalafil (TDF and pentoxifylline (PTX on hepatic apoptosis and the expressions of endothelial and inducible nitric oxide synthases (eNOS and iNOS after liver ischemia/reperfusion (IR. Forty Wistar albino rats were randomly divided into five groups (n=8 as follows: sham group; IR group with ischemia/reperfusion alone; low-dose and high-dose TDF groups received 2.5 mg/kg and 10 mg/kg TDF, respectively; and PTX group received 40 mg/kg PTX. Blood was collected for the analysis of serum alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, uric acid, malondialdehyde (MDA, and total antioxidant capacity (TAC. MDA and TAC also were measured in liver tissue. Histopathological examination was performed to assess the severity of hepatic injury. Apoptosis was evaluated using the apoptosis protease-activating factor 1 (APAF-1 antibody; the expressions of eNOS and iNOS were also assessed by immunohistochemistry in all groups. Serum alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, uric acid, MDA, and TAC, tissue MDA and TAC levels, hepatic injury, and score for extent and for intensity of eNOS, iNOS, and apoptosis protease-activating factor 1 were significantly different in TDF and PTX groups compared to the IR group. High dose-TDF and PTX have the best protective effect on IR-induced liver tissue damage. This study showed that TDF and PTX supplementation may be helpful in preventing free oxygen radical damage, lipid peroxidation, hepatocyte necrosis, and apoptosis in liver IR injury and minimizing liver damage.

  7. Ursolic acid and luteolin-7-glucoside improve lipid profiles and increase liver glycogen content through glycogen synthase kinase-3.

    Science.gov (United States)

    Azevedo, Marisa F; Camsari, Cagri; Sá, Carla M; Lima, Cristovao F; Fernandes-Ferreira, Manuel; Pereira-Wilson, Cristina

    2010-06-01

    In the present study, two phytochemicals - ursolic acid (UA) and luteolin-7-glucoside (L7G) - were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profile (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucose concentration. UA also significantly increased liver glycogen levels accompanied by phosphorylation of glycogen synthase kinase-3 (GSK3). The increase in glycogen deposition induced by UA (mediated by GSK3) could have contributed to the lower plasma glucose levels observed. Both compounds significantly lowered total plasma cholesterol and low-density lipoprotein levels, and, in addition, UA increased plasma high-density lipoprotein levels. Our results show that UA particularly may be useful in preventable strategies for people at risk of developing diabetes and associated cardiovascular complications by improving plasma glucose levels and lipid profile, as well as by promoting liver glycogen deposition.

  8. Homology analyses of the protein sequences of fatty acid synthases from chicken liver, rat mammary gland, and yeast

    International Nuclear Information System (INIS)

    Chang, Soo-Ik; Hammes, G.G.

    1989-01-01

    Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chicken and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the β-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution

  9. Ozagrel hydrochloride, a selective thromboxane A2 synthase inhibitor, alleviates liver injury induced by acetaminophen overdose in mice

    Directory of Open Access Journals (Sweden)

    Tomishima Yoshiro

    2013-01-01

    Full Text Available Abstract Background Overdosed acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP causes severe liver injury. We examined the effects of ozagrel, a selective thromboxane A2 (TXA2 synthase inhibitor, on liver injury induced by APAP overdose in mice. Methods Hepatotoxicity was induced to ICR male mice by an intraperitoneal injection with APAP (330 mg/kg. The effects of ozagrel (200 mg/kg treatment 30 min after the APAP injection were evaluated with mortality, serum alanine aminotransferase (ALT levels and hepatic changes, including histopathology, DNA fragmentation, mRNA expression and total glutathione contents. The impact of ozagrel (0.001-1 mg/mL on cytochrome P450 2E1 (CYP2E1 activity in mouse hepatic microsome was examined. RLC-16 cells, a rat hepatocytes cell line, were exposed to 0.25 mM N-acetyl-p-benzoquinone imine (NAPQI, a hepatotoxic metabolite of APAP. In this model, the cytoprotective effects of ozagrel (1–100 muM were evaluated by the WST-1 cell viability assay. Results Ozagel treatment significantly attenuated higher mortality, elevated serum alanine aminotransferase levels, excessive hepatic centrilobular necrosis, hemorrhaging and DNA fragmentation, as well as increase in plasma 2,3-dinor thromboxane B2 levels induced by APAP injection. Ozagrel also inhibited the hepatic expression of cell death-related mRNAs induced by APAP, such as jun oncogene, FBJ osteosarcoma oncogene (fos and C/EBP homologous protein (chop, but did not suppress B-cell lymphoma 2-like protein11 (bim expression and hepatic total glutathione depletion. These results show ozagrel can inhibit not all hepatic changes but can reduce the hepatic necrosis. Ozagrel had little impact on CYP2E1 activity involving the NAPQI production. In addition, ozagrel significantly attenuated cell injury induced by NAPQI in RLC-16. Conclusions We demonstrate that the TXA2 synthase inhibitor, ozagrel, dramatically alleviates liver injury induced by APAP in mice, and suggest

  10. Molecular cloning and characterization of a cDNA encoding ...

    African Journals Online (AJOL)

    enoh

    2012-03-29

    Nanjing) co., Ltd. The nucleotide sequences of these primers are as follows: ..... Ebizuka Y (2000). Molecular cloning and characterization of a cDNA for Glycyrrhiza glabra cycloartenol synthase. Biol. Pharm. Bull. 23(2):231-234.

  11. Diterpene resin acid biosynthesis in loblolly pine (Pinus taeda): functional characterization of abietadiene/levopimaradiene synthase (PtTPS-LAS) cDNA and subcellular targeting of PtTPS-LAS and abietadienol/abietadienal oxidase (PtAO, CYP720B1).

    Science.gov (United States)

    Ro, Dae-Kyun; Bohlmann, Jörg

    2006-08-01

    Diterpene resin acids are prominent defense compounds against insect pests and pathogens in conifers. Biochemical and molecular analyses in grand fir (Abies grandis), Norway spruce (Picea abies), and loblolly pine (Pinus taeda) have identified two classes of genes and enzymes that generate much of the structural diversity of terpenoid defense compounds: The terpenoid synthases (TPS) and cytochrome P450 monooxgenases (P450). Using a single substrate, geranylgeranyl diphosphate, families of single-product and multi-product diterpene synthases generate an array of cyclic diterpene olefins. These diterpenes are converted to diterpene resin acids by activity of one or more P450 enzymes. A few conifer diterpene synthases have previously been cloned and characterized in grand fir and in Norway spruce. We have also previously shown that the loblolly pine P450 abietadienol/abietadienal oxidase (PtAO) catalyzes multiple oxidations of several diterpene alcohols and aldehydes. Conifer diterpene synthases are thought to function in plastids while P450s can also be localized to plastids or to the endoplasmic reticulum (ER). Here, we show that a loblolly pine cDNA (PtTPS-LAS) encodes a typical multi-product conifer diterpene synthase that forms levopimaradiene, abietadiene, palustradiene, and neoabietadiene similar to the grand fir abietadiene synthase and Norway spruce levopimaradiene/abietadiene synthase. Subcellular targeting of PtTPS-LAS and PtAO to plastids and ER, respectively, was shown with green fluorescent fusion protein expression in tobacco cells. These data suggest that enzymes for conifer diterpene resin acid biosynthesis are localized to at least two different subcellular compartments, plastids and ER, requiring efficient transport of intermediates and secretion of diterpene resin acids into the extracelluar space.

  12. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  13. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  14. Type I Interferon Supports Inducible Nitric Oxide Synthase in Murine Hepatoma Cells and Hepatocytes and during Experimental Acetaminophen-Induced Liver Damage

    Directory of Open Access Journals (Sweden)

    Malte Bachmann

    2017-07-01

    Full Text Available Cytokine regulation of high-output nitric oxide (NO derived from inducible NO synthase (iNOS is critically involved in inflammation biology and host defense. Herein, we set out to characterize the role of type I interferon (IFN as potential regulator of hepatic iNOS in vitro and in vivo. In this regard, we identified in murine Hepa1-6 hepatoma cells a potent synergism between pro-inflammatory interleukin-β/tumor necrosis factor-α and immunoregulatory IFNβ as detected by analysis of iNOS expression and nitrite release. Upregulation of iNOS by IFNβ coincided with enhanced binding of signal transducer and activator of transcription-1 to a regulatory region at the murine iNOS promoter known to support target gene expression in response to this signaling pathway. Synergistic iNOS induction under the influence of IFNβ was confirmed in alternate murine Hepa56.1D hepatoma cells and primary hepatocytes. To assess iNOS regulation by type I IFN in vivo, murine acetaminophen (APAP-induced sterile liver inflammation was investigated. In this model of acute liver injury, excessive necroinflammation drives iNOS expression in diverse liver cell types, among others hepatocytes. Herein, we demonstrate impaired iNOS expression in type I IFN receptor-deficient mice which associated with diminished APAP-induced liver damage. Data presented indicate a vital role of type I IFN within the inflamed liver for fine-tuning pathological processes such as overt iNOS expression.

  15. Saturated free fatty acid sodium palmitate-induced lipoapoptosis by targeting glycogen synthase kinase-3β activation in human liver cells.

    Science.gov (United States)

    Cao, Jie; Feng, Xiao-Xia; Yao, Long; Ning, Bo; Yang, Zhao-Xia; Fang, Dian-Liang; Shen, Wei

    2014-02-01

    Elevated serum saturated fatty acid levels and hepatocyte lipoapoptosis are features of nonalcoholic fatty liver disease (NAFLD). The purpose of this study was to investigate saturated fatty acid induction of lipoapoptosis in human liver cells and the underlying mechanisms. Human liver L02 and HepG2 cells were treated with sodium palmitate, a saturated fatty acid, for up to 48 h with or without lithium chloride, a glycogen synthase kinase-3β (GSK-3β) inhibitor, or GSK-3β shRNA transfection. Transmission electron microscopy was used to detect morphological changes, flow cytometry was used to detect apoptosis, a colorimetric assay was used to detect caspase-3 activity, and western blot analysis was used to detect protein expression. The data showed that sodium palmitate was able to induce lipoapoptosis in L02 and HepG2 cells. Western blot analysis showed that sodium palmitate activated GSK-3β protein, which was indicated by dephosphorylation of GSK-3β at Ser-9. However, inhibition of GSK-3β activity with lithium chloride treatment or knockdown of GSK-3β expression with shRNA suppressed sodium palmitate-induced lipoapoptosis in L02 and HepG2 cells. On a molecular level, inhibition of GSK-3β expression or activity suppressed sodium palmitate-induced c-Jun-N-terminal kinase (JNK) phosphorylation and Bax upregulation, whereas GSK-3β inhibition did not affect endoplasmic reticulum stress-induced activation of unfolded protein response. The present data demonstrated that saturated fatty acid sodium palmitate-induced lipoapoptosis in human liver L02 and HepG2 cells was regulated by GSK-3β activation, which led to JNK activation and Bax upregulation. This finding indicates that GSK-3β inhibition may be a potential therapeutic target to control NAFLD.

  16. cDNA structure, genomic organization and expression patterns of ...

    African Journals Online (AJOL)

    Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin cDNA cloned from the liver was ...

  17. cDNA cloning and gene characterization of the mitochondrial large subunit (LSU) rRNA from the liver fluke Fasciola hepatica. Evidence of heterogeneity in the fluke mitochondrial genome.

    Science.gov (United States)

    Zurita, M; Bieber, D; Ringold, G; Mansour, T E

    1988-01-01

    A cDNA clone that encodes the large subunit of mitochondrial ribosomal RNA (LSU rRNA) from the liver fluke F. hepatica was isolated and characterized. This RNA molecule is polyadenylated at the 3' end and represents 10% of the poly A+RNA in adult F. hepatica. Fluke LSU rRNA has significant sequence homology to mosquito mitochondria LSU rRNA and is more closely related to the mitochondrial rRNA of hermaphroditic than dioecious trematodes. Mitochondrial DNA constitutes approximately 10% of the total cellular DNA of adult flukes. This percentage is lower in non-embryonated eggs as are the levels of LSU rRNA indicating eggs have lower metabolic activity. Analysis of transcription and the number of mitochondrial genomes in S. mansoni shows that the LSU rRNA is more abundant in females than in males. Restriction endonuclease analysis of the fluke mitochondrial LSU rRNA genes suggests the presence of heterogeneous repeated copies in the mitochondrial genome or heterogeneity among individual genomes of mitochondria. Images PMID:3405756

  18. 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 kb...

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

    International Nuclear Information System (INIS)

    Permutt, M.A.; Koranyi, L.; Keller, K.; Lacy, P.E.; Scharp, D.W.; Mueckler, M.

    1989-01-01

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

  20. ATP Synthase β-Chain Overexpression in SR-BI Knockout Mice Increases HDL Uptake and Reduces Plasma HDL Level

    Directory of Open Access Journals (Sweden)

    Kexiu Song

    2014-01-01

    Full Text Available HDL cholesterol is known to be inversely correlated with cardiovascular disease due to its diverse antiatherogenic functions. SR-BI mediates the selective uptake of HDL-C. SR-BI knockout diminishes but does not completely block the transport of HDL; other receptors may be involved. Ectopic ATP synthase β-chain in hepatocytes has been previously characterized as an apoA-I receptor, triggering HDL internalization. This study was undertaken to identify the overexpression of ectopic ATP synthase β-chain on DIL-HDL uptake in primary hepatocytes in vitro and on plasma HDL levels in SR-BI knockout mice. Human ATP synthase β-chain cDNA was delivered to the mouse liver by adenovirus and GFP adenovirus as control. The adenovirus-mediated overexpression of β-chain was identified at both mRNA and protein levels on mice liver and validated by its increasing of DiL-HDL uptake in primary hepatocytes. In response to hepatic overexpression of β-chain, plasma HDL-C levels and cholesterol were reduced in SR-BI knockout mice, compared with the control. The present data suggest that ATP synthase β-chain can serve as the endocytic receptor of HDL, and its overexpression can reduce plasma HDL-C.

  1. Normalizing cDNA libraries.

    Science.gov (United States)

    Bogdanov, Ekaterina A; Shagina, Irina; Barsova, Ekaterina V; Kelmanson, Ilya; Shagin, Dmitry A; Lukyanov, Sergey A

    2010-04-01

    The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex-specific nuclease (DSN)-based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full-length-enriched cDNA libraries, and enabling efficient RNA analyses. (c) 2010 by John Wiley & Sons, Inc.

  2. Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (-)-germacrene D synthase and expression of mono- and sesquiterpene synthases in grapevine flowers and berries.

    Science.gov (United States)

    Lücker, Joost; Bowen, Pat; Bohlmann, Jörg

    2004-10-01

    Valencene is a volatile sesquiterpene emitted from flowers of grapevine, Vitis vinifera L. A full-length cDNA from the cultivar Gewürztraminer was functionally expressed in Escherichia coli and found to encode valencene synthase (VvVal). The two major products formed by recombinant VvVal enzyme activity with farnesyl diphosphate (FPP) as substrate are (+)-valencene and (-)-7-epi-alpha-selinene. Grapevine valencene synthase is closely related to a second sesquiterpene synthase from this species, (-)-germacrene D synthase (VvGerD). VvVal and VvGerD cDNA probes revealed strong signals in Northern hybridizations with RNA isolated from grapevine flower buds. Transcript levels were lower in open pre-anthesis flowers, flowers after anthesis, or at early onset of fruit development. Similar results were obtained using a third probe, (-)-alpha-terpineol synthase, a monoterpenol synthase. Sesquiterpene synthase and monoterpene synthase transcripts were not detected in the mesocarp and exocarp during early stages of fruit development, but transcripts hybridizing with VvVal appeared during late ripening of the berries. Sesquiterpene synthase transcripts were also detected in young seeds.

  3. cDNA library preparation.

    Science.gov (United States)

    Kooiker, Maarten; Xue, Gang-Ping

    2014-01-01

    The construction of full-length cDNA libraries allows researchers to study gene expression and protein interactions and undertake gene discovery. Recent improvements allow the construction of high-quality cDNA libraries, with small amounts of mRNA. In parallel, these improvements allow for the incorporation of adapters into the cDNA, both at the 5' and 3' end of the cDNA. The 3' adapter is attached to the oligo-dT primer that is used by the reverse transcriptase, whereas the 5' adapter is incorporated by the template switching properties of the MMLV reverse transcriptase. This allows directional cloning and eliminates inefficient steps like adapter ligation, phosphorylation, and methylation. Another important step in the construction of high-quality cDNA libraries is the normalization. The difference in the levels of expression between genes might be several orders of magnitude. Therefore, it is essential that the cDNA library is normalized. With a recently discovered enzyme, duplex-specific nuclease, it is possible to normalize the cDNA library, based on the fact that more abundant molecules are more likely to reanneal after denaturation compared to rare molecules.

  4. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

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

    Science.gov (United States)

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

    1996-01-01

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

  6. Normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1997-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

  7. Nitric Oxide Synthase Type III Overexpression By Gene Therapy Exerts Antitumoral Activity In Mouse Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Raúl González

    2015-08-01

    Full Text Available Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO synthase type III (NOS-3 overexpression induces cell death in hepatoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. Hepa 1-6 cells were used for in vitro and in vivo experiments. The first generation adenovirus was designed to overexpress NOS-3 (or GFP and luciferase cDNA under the regulation of murine alpha-fetoprotein (AFP and Rous Sarcoma Virus (RSV promoters, respectively. Both adenoviruses were administered through the tail vein two weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-Luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8 activity in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by l-NAME or p53 siRNA. The tail vein infusion of AFP-NOS- 3/RSV-Luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.

  8. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  9. DHEA and non-alcoholic fat liver disease: increased gene expression of peroxisome proliferation-activated receptor γ (PPARγ and fatty acid synthase (FAS

    Directory of Open Access Journals (Sweden)

    Felipe Natali Almeida

    2014-05-01

    Full Text Available Dehydroespiandrosterone (DHEA is associated with improvements in chronic degenerative diseases, including obesity, insulin resistance, and cardiovascular diseases. Nevertheless, it is observed an increase in its concentration in individuals with liver lipid infiltration, but it is not precise if this condition emerges as a cause or a consequence. In this way, we aimed to identify gene expression alterations in lipid and glucose liver metabolism markers, as well as oxidative stress markers. For this purpose, male Wistar rats, 12-14 months old were treated with subcutaneous injections of DHEA (only dose of 10 mg kg-1; and after 7 days, hepatic gene expression by PCR real time were performed for the following genes:  G6Pase, PEPCK, FAS, PPARγ, malic enzyme, ChREBP, LXR, catalase, GPx, iNOS, NADPH oxidase subunits and PCNA. We observed a tendency of reduction in G6Pase gene expression in treated group (p = 0.08. In addition, it was identified an increase in liver PPARγ and FAS gene expressions, two markers of increased activity of lipogenic pathway. We also observed an increase in iNOS gene expression, a known inductor of systemic and hepatic insulin resistance. In conclusion, our data indicates that the treatment with DHEA can be associated with the development of liver lipid infiltration and hepatic insulin resistance.

  10. Cloning and expression analysis of chalcone synthase gene from ...

    Indian Academy of Sciences (India)

    Chalcone synthase (CHS) catalyses the first committed step of flavonoid biosynthetic pathway. Full-length cDNA, showing homology with plantCHS gene was isolated from leaves of C. forskohlii and named CfCHS (GenBank accession no. KF643243). Theoretical translation of CfCHS nucleotide sequence shows that it ...

  11. Molecular cloning and characterization of strictosidine synthase, a ...

    African Journals Online (AJOL)

    Mitragynine is one of the most dominant indole alkaloids present in the leaves of Mitragyna speciosa, a species of Rubiaceae. This alkaloid is believed to be synthesized via condensation of the amino acid derivative, tryptamine and secologanine by the action of strictosidine synthase (STR). The cDNA clone encoding STR ...

  12. Cloning and sequencing of cDNAs specifying a novel class of phosphoribosyl diphosphate synthase in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Krath, Britta N.; Eriksen, Tina A.; Poulsen, Tim S.

    1999-01-01

    cDNAs specifying four active phosphoribosyl diphosphate synthase isozymes were isolated from an Arabidopsis thaliana cDNA library. In contrast to other phosphoribosyl diphosphate synthases the activity of two of the A. thaliana isozymes are independent of Pi. Amino acid sequence comparison...

  13. Functional and evolutionary relationships between terpene synthases from Australian Myrtaceae.

    Science.gov (United States)

    Keszei, Andras; Brubaker, Curt L; Carter, Richard; Köllner, Tobias; Degenhardt, Jörg; Foley, William J

    2010-06-01

    Myrtaceae is one of the chemically most variable and most significant essential oil yielding plant families. Despite an abundance of chemical information, very little work has focussed on the biochemistry of terpene production in these plants. We describe 70 unique partial terpene synthase transcripts and eight full-length cDNA clones from 21 myrtaceous species, and compare phylogenetic relationships and leaf oil composition to reveal clades defined by common function. We provide further support for the correlation between function and phylogenetic relationships by the first functional characterisation of terpene synthases from Myrtaceae: a 1,8-cineole synthase from Eucalyptus sideroxylon and a caryophyllene synthase from Eucalyptusdives. Copyright 2010 Elsevier Ltd. All rights reserved.

  14. Studies on the chalcone synthase gene of two higher plants: petroselinum hortense and matthiola incana

    Energy Technology Data Exchange (ETDEWEB)

    Hemleben, V.; Frey, M.; Rall, S.; Koch, M.; Kittel, M.; Kreuzaler, F.; Ragg, H.; Fautz, E.; Hahlbrock, K.

    1982-01-01

    Two higher plant systems are presented which allow to study coordinated gene expression of the light-induced metabolic pathway of flavonoid biosynthesis: tissue culture cells of Petroselinum hortense (Apiaceae) and different developmental stages of various genotypes of Matthiola incana (Brassicaceae). The gene structure of the chalcone synthase is mainly studied. A cDNA clone (pLF56) of parsley has been constructed and characterized conferring the chalcone synthase gene sequence. Strong cross hybridization between the parsley cDNA and Matthiola DNA allowed to identify a HindIII fragment (6000 bp) identical in size for parsley and different Matthiola wild type lines and a mutant line.

  15. Studies on the chalcone synthase gene of two higher plants: petroselinum hortense and matthiola incana.

    Science.gov (United States)

    Hemleben, V; Frey, M; Rall, S; Koch, M; Kittel, M; Kreuzaler, F; Ragg, H; Fautz, E; Hahlbrock, K

    1982-01-01

    Two higher plant systems are presented which allow to study coordinated gene expression of the light-induced metabolic pathway of flavonoid biosynthesis: tissue culture cells of Petroselinum hortense (Apiaceae) and different developmental stages of various genotypes of Matthiola incana (Brassicaceae). The gene structure of the chalcone synthase is mainly studied. A cDNA clone (pLF56) of parsley has been constructed and characterized conferring the chalcone synthase gene sequence. Strong cross hybridization between the parsley cDNA and Matthiola DNA allowed to identify a HindIII fragment (6000 bp) identical in size for parsley and different Matthiola wild type lines and a mutant line.

  16. Molecular cloning and characterization of glycogen synthase in Eriocheir sinensis.

    Science.gov (United States)

    Li, Ran; Zhu, Li-Na; Ren, Li-Qi; Weng, Jie-Yang; Sun, Jin-Sheng

    2017-12-01

    Glycogen plays an important role in glucose and energy homeostasis at cellular and organismal levels. In glycogen synthesis, glycogen synthase (GS) is a rate-limiting enzyme catalysing the addition of α-1,4-linked glucose units from (UDP) 3 -glucose to a nascent glycogen chain using glycogenin (GN) as a primer. While studies on mammalian liver GS (GYS2) are numerous, enzymes from crustaceans, which also use glycogen and glucose as their main energy source, have received less attention. In the present study, we amplified full-length GS cDNA from Eriocheir sinensis. Tissue expression profiling revealed the highest expression of GS in the hepatopancreas. During moulting, GS expression and activity declined, and glycogen levels in the hepatopancreas were reduced. Recombinant GS was expressed in Escherichia coli Rosetta (DE3), and induction at 37°C or 16°C yielded EsGS in insoluble inclusion bodies (EsGS-I) or in soluble form (EsGS-S), respectively. Enzyme activity was measured in a cell-free system containing glucose-6-phosphate (G6P), and both forms possessed glycosyltransferase activity, but refolded EsGS-I was more active. Enzyme activity of both GS and EsGS-I in the hepatopancreas was optimum at 25°C, which is coincident with the optimum growth temperature of Chinese mitten crab, and higher (37°C) or lower (16°C) temperatures resulted in lower enzyme activity. Taken together, the results suggest that GS may be important for maintaining normal physiological functions such as growth and reproduction. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Cloning and sequencing of cDNAs specifying a novel class of phosphoribosyl diphosphate synthase in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Krath, Britta N.; Eriksen, Tina A.; Poulsen, Tim S.

    1999-01-01

    cDNAs specifying four active phosphoribosyl diphosphate synthase isozymes were isolated from an Arabidopsis thaliana cDNA library. In contrast to other phosphoribosyl diphosphate synthases the activity of two of the A. thaliana isozymes are independent of Pi. Amino acid sequence comparison...

  18. Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria ananassa)

    NARCIS (Netherlands)

    Lunkenbein, S.; Coiner, H.; Vos, de C.H.; Schaart, J.G.; Boone, M.J.; Krens, F.A.; Schwab, W.; Salentijn, E.M.J.

    2006-01-01

    An octaploid (Fragaria × ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria × ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens.

  19. Molecular cloning of goat 20alpha-hydroxysteroid dehydrogenase cDNA.

    Science.gov (United States)

    Jayasekara, Walimuni Samantha Nilanthi; Yonezawa, Tomohiro; Ishida, Maho; Yamanouchi, Keitaro; Nishihara, Masugi

    2004-06-01

    20Alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which catalyzes the conversion of progesterone to its inactive form 20alpha-dihydroprogesterone, is expressed in murine placenta and has been suggested to play roles in maintaining pregnancy. To understand the role of 20alpha-HSD during pregnancy in the goat, as a first step, cloning and sequencing of 20alpha-HSD cDNA were performed. The full nucleotide sequence of 20alpha-HSD cDNA was determined on samples obtained from the corpus luteum at the luteal phase of the estrous cycle and the placenta in late pregnancy by RT-PCR and 3' and 5' RACE systems. Cloned 20alpha-HSD cDNA consisted of 1124 bp and belonged to the aldo-keto reductase superfamily. From the start codon to stop codon there were 323 amino acids, the same as in other species. To verify whether the protein derived from goat 20alpha-HSD cDNA had 20alpha-HSD activity, the cDNA was expressed by bacteria. Bacterially expressed goat 20alpha-HSD protein showed 20alpha-HSD enzyme activity. A tissue distribution study demonstrated that 20alpha-HSD was expressed in the placenta, but not in the adrenal gland, liver and spleen during pregnancy. The present study suggests that goat 20alpha-HSD is another member of the aldo-keto reductase superfamily and that it plays a role in the placenta during pregnancy.

  20. Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.

    Science.gov (United States)

    Brodelius, Maria; Lundgren, Anneli; Mercke, Per; Brodelius, Peter E

    2002-07-01

    A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes.

  1. PCR-based cDNA library construction: general cDNA libraries at the level of a few cells.

    OpenAIRE

    Belyavsky, A; Vinogradova, T; Rajewsky, K

    1989-01-01

    A procedure for the construction of general cDNA libraries is described which is based on the amplification of total cDNA in vitro. The first cDNA strand is synthesized from total RNA using an oligo(dT)-containing primer. After oligo(dG) tailing the total cDNA is amplified by PCR using two primers complementary to oligo(dA) and oligo(dG) ends of the cDNA. For insertion of the cDNA into a vector a controlled trimming of the 3' ends of the cDNA by Klenow enzyme was used. Starting from 10 J558L ...

  2. Isolation and characterization of the murine alpha-L-iduronidase cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Clarke, L.A.; Zhang, H. Nasir, J. [Univ. of British Columbia, Vancouver (Canada)] [and others

    1994-09-01

    Mucopolysaccharidosis I (MPS I) are a group of disorders caused by deficiency of the lysosomal enzyme alpha-L-iduronidase. The characterization of the human gene and the identification of mutations underlying MPS I in humans has led to the delineation of the molecular basis of this disorder. Model systems are now needed for the evaluation and development of therapeutics for this disorder. Both canine and feline models for MPS type I have been described but only the canine gene has been isolated and characterized. We report here the cloning and expression of the murine alpha-L-iduronidase cDNA. The murine cDNA was obtained by screening a mouse liver cDNA library with a probe from the human cDNA. The full length murine cDNA is 3120 base pairs in length and thus is considerably larger than both the human and canine transcripts. The increase in size is due to a 1.2 kb 3{prime} untranslated region in the murine cDNA that contains a CA dinucleotide repeat. Within the coding region the murine cDNA shows sequences. At the protein level the murine protein shows 77% similarity with the human protein and 75% similarity with the canine protein. There are significant differences in both the start and stop sites with the murine protein 9 amino acids shorter at both the N terminal signal peptide region and the C terminus. Expression of the murine cDNA in COS-1 cells resulted in a 20 fold increase in intracellular alpha-L-iduronidase activity as well as the detection of considerable enzyme activity in the culture medium. Comparison of the reported missense mutations underlying MPS I in humans (A75T, H82P, R89Q, L218P, P533R, Q310X, T366P) has shown conservation of these amino acid residues in the murine protein. The isolation of the murine iduronidase cDNA will now allow for the development of a murine model for MPS I.

  3. Polyketide synthase from Fusarium

    DEFF Research Database (Denmark)

    Kvesel, Kasper; Wimmer, Reinhard; Sørensen, Jens Laurids

    Fungi produce a wide array of secondary metabolites, with interesting bioactivities by help of a number of enzyme complexes. Polyketide synthases (PKS) are a class of multidomain enzymes, producing a class of secondary metabolites called polyketides1,2. Only few structures of PKS’s have been...

  4. Molecular cloning and characterization of drimenol synthase from valerian plant (Valeriana officinalis).

    Science.gov (United States)

    Kwon, Moonhyuk; Cochrane, Stephen A; Vederas, John C; Ro, Dae-Kyun

    2014-12-20

    Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio-activities in nature. However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR analyses of the VoTPS3-produced terpene, and characterization of the VoTPS3 enzyme confirmed that VoTPS3 synthesizes (-)-drimenol. In feeding assays, possible reaction intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol, suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A mechanistic consideration of (-)-drimenol synthesis suggests that drimenol synthase is likely to use a protonation-initiated cyclization, which is rare for sesquiterpene synthases. VoTPS3 can be used to produce (-)-drimenol, from which useful drimane-type terpenes can be synthesized. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

    Directory of Open Access Journals (Sweden)

    Kawamukai Makoto

    2004-11-01

    Full Text Available Abstract Background Isopentenyl diphosphate (IPP, a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

  6. Expression in Arabidopsis of a strawberry linalool synthase gene under the control of the inducible potato P12 promoter

    NARCIS (Netherlands)

    Yang, L.; Mercke, P.; Loon, van J.J.A.; Fang, Zhiyuan; Dicke, M.; Jongsma, M.A.

    2008-01-01

    To investigate the role of inducible linalool in Arabidopsis-insect interactions, the FaNES1 linalool synthase (LIS) cDNA from strawberry with plastid targeting and a synthetic intron (LIS') was placed under the control of the wound inducible proteinase inhibitor 2 (PI2) promoter from potato. The

  7. Beta-Glucan Synthase Gene Expression in Pleurotus sp

    International Nuclear Information System (INIS)

    Azhar Mohamad; Nie, H.J.

    2016-01-01

    Pleurotus sp. is a popular edible mushroom, containing various functional component, in particular, Beta-glucan. Beta-glucans is a part of glucan family of polysaccharides and supposedly contribute to medicinal and nutritional value of Pleurotus.sp. In order to understand the distribution of Beta-glucan in Pleurotus.sp, the Beta-glucan synthase gene expression was determined and compared in different part of Pleurotus, namely mycelium, stripe and cap. The Pleurotus.sp RNA was extracted using commercial kit, employing Tissuelyser ll (Qiagen, USA) to disrupt the cell walls. Then the RNA was quantified by Nano drop (Thermo Fisher, USA) and visualized using denaturing agarose gel. RNA with good OD 260.280 reading (∼2.0) was chosen and converted to cDNA. Using Laccase synthase gene as home keeping gene, Beta-glucan synthase gene expression was quantified using CFX 96 Real Time PCR detection system (Biorad, USA). Preliminary result shows that Beta-glucan synthase was relatively expressed the most in stripe, followed by mycelium and barely in cap. (author)

  8. Characterization of cDNA clones encoding rabbit and human serum paraoxonase: The mature protein retains its signal sequence

    Energy Technology Data Exchange (ETDEWEB)

    Hassett, C.; Richter, R.J.; Humbert, R.; Omiecinski, C.J.; Furlong, C.E. (Univ. of Washington, Seattle (United States)); Chapline, C.; Crabb, J.W. (W.Alton Jones Cell Science Center, Lake Placid, NY (United States))

    1991-10-22

    Serum paraoxonase hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. High serum paraoxonase levels appear to protect against the neurotoxic effects of organophosphorus substrates of this enzyme. The amino acid sequence accounting for 42% of rabbit paraoxonase was determined. From these data, two oligonucleotide probes were synthesized and used to screen a rabbit liver cDNA library. Human paraoxonase clones were isolated from a liver cDNA library by using the rabbit cDNA as a hybridization probe. Inserts from three of the longest clones were sequenced, and one full-length clone contained an open reading frame encoding 355 amino acids, four less than the rabbit paraoxonase protein. Amino-terminal sequences derived from purified rabbit and human paraoxonase proteins suggested that the signal sequence is retained, with the exception of the initiator methionine residue. Characterization of the rabbit and human paraoxonase cDNA clones confirms that the signal sequences are not processed, except for the N-terminal methionine residue. The rabbit and human cDNA clones demonstrate striking nucleotide and deduced amino acid similarities (greater than 85%), suggesting an important metabolic role and constraints on the evolution of this protein.

  9. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    2000-10-03

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

  10. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    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 ...

  11. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Unknown

    317. 2.4 cDNA sequencing and analysis. The nucleotide sequence of the cloned H. fossilis GH. cDNA was determined by Sanger's dideoxy chain termi- nation method, using Perkin Elmer bigdye terminator kit in an ABI Prism 377 automated DNA sequencer. All other computational analysis of the GH cDNA was done using.

  12. Cleaning up polyketide synthases.

    Science.gov (United States)

    Kwan, Jason C; Schmidt, Eric W

    2012-03-23

    Complex biosynthetic enzymes such as polyketide synthases make mistakes. In this issue of Chemistry & Biology, Jensen et al. report that a discrete family of acyltransferases is responsible for error correction, hydrolyzing key biosynthetic intermediates from a multi-enzyme complex. This activity might find use in understanding polyketide biosynthesis, particularly in uncultivated organisms and in tailoring the synthesis of small molecules. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

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

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data DOI 10.18908/lsdba.nbdc00838-003 Description of data contents Phred's quality score. P...tion Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality

  15. Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA.

    Science.gov (United States)

    Sakajo, S; Nakamura, K; Asahi, T

    1987-06-01

    A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313-317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.

  16. The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum. Its gene structure relative to that of other polyketide synthases.

    Science.gov (United States)

    Beck, J; Ripka, S; Siegner, A; Schiltz, E; Schweizer, E

    1990-09-11

    6-Methylsalicylic acid synthase (MSAS) from Penicillium patulum is a homomultimer of a single, multifunctional protein subunit. The enzyme is induced, at the transcriptional level, during the end of the logarithmic growth phase. After approximately 150-fold purification, a homogeneous enzyme preparation was obtained exhibiting, upon SDS gel electrophoresis, a subunit molecular mass of 188 kDa. By immunological screening of a genomic P. patulum DNA expression library, the MSAS gene together with its flanking sequences was isolated; 7131 base pairs of the cloned genomic DNA were sequenced. Within this sequence the MSAS gene was identified as a 5322-bp-long open reading frame coding for a protein of 1774 amino acids and 190,731 Da molecular mass. Transcriptional initiation and termination sites were determined both by primer extension studies and from cDNA sequences specially prepared for the 5' and 3' portions of the gene. The same cDNA sequences revealed the presence of a 69-bp intron within the N-terminal part of the MSAS gene. The intron contains the canonical GT and AG dinucleotides at its 5'- and 3'-splice junctions. An internal TACTGAC sequence, resembling the TACTAAC consensus element of Saccharomyces cerevisiae introns is suggested to represent the branch point of the lariat splicing intermediate. When compared to other known polyketide synthases, distinct amino acid sequence similarities of limited lengths were observed with some, though not all, of them. A comparatively low degree of similarity was detected to the yeast and Penicillium FAS or to the plant chalcone and resveratrol synthases. In contrast, a significantly higher sequence similarity was found between MSAS and the rat fatty acid synthase, especially at their transacylase, 2-oxoacyl reductase, 2-oxoacyl synthase and acyl carrier protein domains. Besides several dissimilar, interspersed regions probably coding for MSAS- and FAS-specific functions, the sequential order of the similar domains was

  17. [Advances in isoprene synthase research].

    Science.gov (United States)

    Gou, Yan; Liu, Zhongchuan; Wang, Ganggang

    2017-11-25

    Isoprene emission can lead to significant consequence for atmospheric chemistry. In addition, isoprene is a chemical compound for various industrial applications. In the organisms, isoprene is produced by isoprene synthase that eliminates the pyrophosphate from the dimethylallyl diphosphate. As a key enzyme of isoprene formation, isoprene synthase plays an important role in the process of natural emission and artificial synthesis of isoprene. So far, isoprene synthase has been found in various plants. Isoprene synthases from different sources are of conservative structural and similar biochemical properties. In this review, the biochemical and structural characteristics of isoprene synthases from different sources were compared, the catalytic mechanism of isoprene synthase was discussed, and the perspective application of the enzyme in bioengineering was proposed.

  18. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  19. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  20. Cloning, expression, purification and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale.

    Science.gov (United States)

    Picaud, Sarah; Olsson, Mikael E; Brodelius, Maria; Brodelius, Peter E

    2006-08-01

    A cDNA clone encoding a sesquiterpene synthase, (+)-germacrene D synthase, has been isolated from ginger (Zingiber officinale). The full-length cDNA (AY860846) contains a 1650-bp open reading frame coding for 550 amino acids (63.8kDa) with a theoretical pI=5.59. The deduced amino acid sequence is 30-46% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a major product, (+)-germacrene D (50.2% of total sesquiterpenoids produced) and a co-product, germacrene B (17.1%) and a number of minor by-products. The optimal pH for the recombinant enzyme is around 7.5. Substantial (+)-germacrene D synthase activity is observed in the presence of Mg2+, Mn2+, Ni2+ or Co2+, while the enzyme is inactive when Cu2+ or Zn2+ is used. The Km- and kcat-values are 0.88 microM and 3.34 x 10(-3) s(-1), respectively. A reaction mechanism involving a double 1,2-hydride shift has been established using deuterium labeled substrates in combination with GC-MS analysis.

  1. Liver metastases

    Science.gov (United States)

    ... Esophageal cancer - liver metastases; Lung cancer - liver metastases; Melanoma - liver metastases ... through the probe that causes ice crystals to form around the probe. The cancer cells are frozen ...

  2. Expression of Clarkia S-linalool synthase in transgenic petunia plant results in the accumulation of S-linalyl-b-D-glucopyranoside

    NARCIS (Netherlands)

    Lücker, J.; Bouwmeester, H.J.; Schwab, W.; Blaas, J.; Plas, van der L.H.W.; Verhoeven, H.A.

    2001-01-01

    Petunia hybrida W115 was transformed with a Clarkia breweri S-linalool synthase cDNA (lis). Lis was expressed in all tissues analysed, and linalool was detected in leaves, sepals, corolla, stem and ovary, but not in nectaries, roots, pollen and style. However, the S-linalool produced by the plant in

  3. 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.

  4. Construction of Agrobacterium tumefaciens-mediated tomato black ring virus infectious cDNA clones.

    Science.gov (United States)

    Zarzyńska-Nowak, Aleksandra; Ferriol, Inmaculada; Falk, Bryce W; Borodynko-Filas, Natasza; Hasiów-Jaroszewska, Beata

    2017-02-15

    Tomato black ring virus (TBRV, genus Nepovirus) infects a wide range of economically important plants such as tomato, potato, tobacco and cucumber. Here, a successful construction of infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2) is reported for the first time. The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS). The symptoms induced on plants agroinoculated with both constructs were indistinguishable from those caused by the wild-type virus. The infectivity of obtained clones was verified by reinoculation to Nicotiana tabacum cv. Xanthi, Chenopodium quinoa and Cucumis sativus. The presence of viral particles and RNA was confirmed by electron microscopy and reverse transcription polymerase chain reaction, respectively. Constructed full-length infectious cDNA clones will serve as an excellent tool to study virus-host-vector interactions. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    1985-01-01

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

  6. Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jacob

    2007-01-01

    with the greatest number of different expressed genes, whereas tissues with more specialized function, such as developing liver, have fewer expressed genes. There are at least 65 high confidence housekeeping gene candidates and 876 cDNA library-specific gene candidates. We identified differential expression...

  7. Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum).

    Science.gov (United States)

    Yang, Chang-Qing; Wu, Xiu-Ming; Ruan, Ju-Xin; Hu, Wen-Li; Mao, Yin-Bo; Chen, Xiao-Ya; Wang, Ling-Jian

    2013-12-01

    Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. cDNA structure, genomic organization and expression patterns of ...

    African Journals Online (AJOL)

    use

    2011-11-23

    Nov 23, 2011 ... Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin. cDNA cloned ...

  9. Durable Expression of Minicircle DNA-Liposome-Delivered Androgen Receptor cDNA in Mice with Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Tian-You Chang

    2014-01-01

    Full Text Available Background. The most common gene-based cancer therapies involve the suppression of oncogenic molecules and enhancement of the expression of tumor-suppressor genes. Studies in noncancer disease animal models have shown that minicircle (MC DNA vectors are easy to deliver and that the proteins from said MC-carrying DNA vectors are expressed over a long period of time. However, delivery of therapeutic genes via a liposome-mediated, MC DNA complex has never been tested in vascular-rich hepatocellular carcinoma (HCC. Liposome-mediated DNA delivery exhibits high in vivo transfection efficiency and minimal systemic immune response, thereby allowing for repetitive interventions. In this study, we evaluated the efficacy of delivering an MC-liposome vector containing a 3.2 kb androgen receptor (AR; HCC metastasis suppressor cDNA into Hepatitis B Virus- (HBV- induced HCC mouse livers. Results. Protein expression and promoter luciferase assays revealed that liposome-encapsulated MC-AR resulted in abundant functional expression of AR protein (100 kD for up to two weeks. The AR cDNA was also successfully delivered into normal livers and diseased livers, where it was persistently expressed. In both normal livers and livers with tumors, the expression of AR was detectable for up to 60 days. Conclusion. Our results show that an MC/liposome delivery system might improve the efficacy of gene therapy in patients with HCC.

  10. Transcriptional Analysis of In vivo Plasmodium yoelii Liver Stage Gene Expression

    National Research Council Canada - National Science Library

    Sacci, Jr., John B; Ribeiro, Jose M; Huang, Fengying; Alama, Uzma; Russell, Joshua A; Blair, Peter L; Witney, Adam; Carucci, Daniel J; Azada, Abdu F; Aguiar, Joao C

    2005-01-01

    ... of parasites and large number of uninfected hepatocytes. We have overcome this obstruction by utilizing laser capture microdissection to provide a high quality source of parasite mRNA for the construction of a liver stage cDNA library...

  11. Three human alcohol dehydrogenase subunits: cDNA structure and molecular and evolutionary divergence

    International Nuclear Information System (INIS)

    Ikuta, T.; Szeto, S.; Yoshida, A.

    1986-01-01

    Class I human alcohol dehydrogenase (ADH; alcohol:NAD + oxidoreductase, EC 1.1.1.1) consists of several homo- and heterodimers of α, β, and γ subunits that are governed by the ADH1, ADH2, and ADH3 loci. The authors previously cloned a full length of cDNA for the β subunit, and the complete sequence of 374 amino acid residues was established. cDNAs for the α and γ subunits were cloned and characterized. A human liver cDNA library, constructed in phage λgt11, was screened by using a synthetic oligonucleotide probe that was matched to the γ but not to the β sequence. Clone pUCADHγ21 and clone pUCADHα15L differed from β cDNA with respect to restriction sites and hybridization with the nucleotide probe. Clone pUCADHγ21 contained an insertion of 1.5 kilobase pairs (kbp) and encodes 374 amino acid residues compatible with the reported amino acid sequence of the γ subunit. Clone pUCADHα15L contained an insertion of 2.4 kbp and included nucleotide sequences that encode 374 amino acid residues for another subunit, the γ subunit. In addition, this clone contained the sequences that encode the COOH-terminal part of the β subunit at its extended 5' region. The amino acid sequences and coding regions of the cDNAs of the three subunits are very similar. A high degree of resemblance is observed also in their 3' noncoding regions. However, distinctive differences exist in the vicinity of the Zn-binding cysteine residue at position 46. Based on the cDNA sequences and the deduced amino acid sequences of the three subunits, their structural and evolutionary relationships are discussed

  12. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

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

    1987-01-01

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

  13. [Construction of chicken embryo fibroblasts cDNA expression library].

    Science.gov (United States)

    Liu, Wei; Gao, Yu-long; Gao, Hong-lei; Wang, Xiao-mei; Xu, Xiu-hong

    2007-06-01

    Chicken embryo fibroblast (CEF) is a primary cellular material to research the infectious bursal disease virus (IBDV). Constructing the cDNA expression library of CEF is the foundation to research cell tropism and find cell receptors of IBDV from CEF. In order to achieve that purpose, a high-quality cDNA expression library of CEF was constructed by Gateway technology, which could avoid using the restriction enzyme for cloning to solve technical limitation of roution method. The mRNA was extracted from chicken embryonic fibroblast. Moreover, single-strand cDNA and double-strand cDNA were synthesized by using biotin-conjugated Oligo (dT) primer in turn. The double-strand cDNA was ligated Adapter and then purified by the cDNA Size Fractionation Columns. After BP recombination reaction, a cDNA entry library was constructed with a titer of 1 x 10(6) cfu/mL, total clones of 1.2 x 10(7) cfu and an average insertion size of about 2243 bp. After LR recombination reaction, the cDNA entry library was transformed into expression library which took on a titer of 5 x 10(5) cfu/mL, total clones of 5.5 x 10(6) cfu and an average insertion size of about 2411bp. The results indicate that the constructed cDNA expression library performs a remarkable high value in both recombination rate and library coverage. As a result, the cDNA expression library, with its good quality, may facilitate to identify the receptors associated with the resistance against IBDV in chicken embryonic fibroblast and to cast new light on the mechanism of cellular tropism. Moreover, it may also provide data of chicken embryonic fibroblast in transcription level and may be helpful to study its biological functions.

  14. [The construction of rapid amplification of cDNA ends cDNA libraries from human fetal bone and joint].

    Science.gov (United States)

    Liang, X; Gong, Y; Liu, Q; Li, J; Chen, B; Guo, C

    2001-02-01

    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. 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. 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. 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.

  15. Liver Hemangioma

    Science.gov (United States)

    Liver hemangioma Overview A liver hemangioma (he-man-jee-O-muh) is a noncancerous (benign) mass in the liver. A liver hemangioma is made up of a tangle of blood vessels. Other terms for a liver hemangioma are hepatic hemangioma and cavernous hemangioma. Most ...

  16. Cloning of the human androgen receptor cDNA

    International Nuclear Information System (INIS)

    Govindan, M.V.; Burelle, M.; Cantin, C.; Kabrie, C.; Labrie, F.; Lachance, Y.; Leblanc, G.; Lefebvre, C.; Patel, P.; Simard, J.

    1988-01-01

    The authors discuss how in order to define the functional domains of the human androgen receptor, complementary DNA (cDNA) clones encoding the human androgen receptor (hAR) have been isolated from a human testis λgtll cDNA library using synthetic oligonnucleotide probes, homologous to segments of the human glucocorticoid, estradiol and progesterone receptors. The cDNA clones corresponding to the human glucocorticoid, estradiol and progesterone receptors were eliminated after cross-hybridization with their respective cDNA probes and/or after restriction mapping of the cDNA clones. The remaining cDNA clones were classified into different groups after analysis by restriction digestion and cross-hybridization. Two of the largest cDNA clones from each group were inserted into an expression vector in both orientations. The linearized plasmids were used as templates in in vitro transcription with T7 RNA polymerase. Subsequent in vitro translation of the purified transcripts in rabbit reticulocyte lysate followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) permitted the characterization of the encoded polyeptides. The expressed proteins larger than 30,000 Da were analyzed for their ability to bind tritium-labelled dihydrotestosterone ([ 3 H] DHT) with high affinity and specificity

  17. 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...

  18. Liver biopsy

    Science.gov (United States)

    Biopsy - liver; Percutaneous biopsy ... the biopsy needle to be inserted into the liver. This is often done by using ultrasound. The ... the chance of damage to the lung or liver. The needle is removed quickly. Pressure will be ...

  19. Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues

    International Nuclear Information System (INIS)

    Prody, C.A.; Zevin-Sonkin, D.; Gnatt, A.; Goldberg, O.; Soreq, H.

    1987-01-01

    To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase and Torpedo electric organ true acetylcholinesterase. Using these probes, the authors isolated several cDNA clones from λgt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A) + RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These finding demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species

  20. Isolation and characterization of cDNA clones for the gamma subunit of Xenopus fibrinogen, the product of a coordinately regulated gene family.

    Science.gov (United States)

    Bhattacharya, A; Shepard, A R; Moser, D R; Holland, L J

    1990-09-10

    Fibrinogen, the major structural protein involved in blood coagulation, is synthesized and secreted by the liver. In the frog Xenopus laevis, fibrinogen production is dramatically induced by glucocorticoids. The hormonal stimulation requires synthesis of three separate subunits, designated A alpha, B beta, and gamma. For investigation of the molecular mechanisms underlying this coordinate induction, we have isolated cDNA clones for the subunits of Xenopus fibrinogen. In this communication we describe the identification of clones for the gamma chain. Initially, a Xenopus liver cDNA library in pBR322 was screened with a rat gamma chain cDNA and a clone representing half of the 1600-base frog gamma mRNA was identified. This clone was shown to be complementary to gamma mRNA by hybrid selection of mRNA that translated in vitro into the gamma polypeptide. A clone about 1460 base pairs in length was then isolated from a Xenopus liver lambda gt10 cDNA library and subcloned into Bluescript SK-. This clone, designated X1 gamma 3, contains the entire 3'-end and lacks 38 bases at the 5'-end of gamma mRNA. The deduced amino acid sequence at the N-terminal is compatible with a signal peptide of 20-23 amino acids, in agreement with the calculated size of the frog gamma chain signal peptide. Following the signal sequence is a region of highly conserved amino acids that participate in disulfide bond formation critical for the maintenance of tertiary structure in mammalian fibrinogen. The gamma cDNA clone was used to measure gamma mRNA in purified Xenopus liver cells treated with glucocorticoids in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Method for construction of normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1998-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

  2. Screening of cDNA libraries on glass slide microarrays.

    Science.gov (United States)

    Berger, Dave K; Crampton, Bridget G; Hein, Ingo; Vos, Wiesner

    2007-01-01

    A quantitative screening method was developed to evaluate the quality of cDNA libraries constructed by suppression subtraction hybridization (SSH) or other enrichment techniques. The SSH technique was adapted to facilitate screening of the resultant library on a small number of glass slide microarrays. A simple data analysis pipeline named SSHscreen using "linear models for microarray data" (limma) functions in the R computing environment was developed to identify clones in the cDNA libraries that are significantly differentially expressed, and to determine if they were rare or abundant in the original treated sample. This approach facilitates the choice of clones from the cDNA library for further analysis, such as DNA sequencing, Northern blotting, RT-PCR, or detailed expression profiling using a custom cDNA microarray. Furthermore, this strategy is particularly useful for studies of nonmodel organisms for which there is little genome sequence information.

  3. Constructing and detecting a cDNA library for mites.

    Science.gov (United States)

    Hu, Li; Zhao, YaE; Cheng, Juan; Yang, YuanJun; Li, Chen; Lu, ZhaoHui

    2015-10-01

    RNA extraction and construction of complementary DNA (cDNA) library for mites have been quite challenging due to difficulties in acquiring tiny living mites and breaking their hard chitin. The present study is to explore a better method to construct cDNA library for mites that will lay the foundation on transcriptome and molecular pathogenesis research. We selected Psoroptes cuniculi as an experimental subject and took the following steps to construct and verify cDNA library. First, we combined liquid nitrogen grinding with TRIzol for total RNA extraction. Then, switching mechanism at 5' end of the RNA transcript (SMART) technique was used to construct full-length cDNA library. To evaluate the quality of cDNA library, the library titer and recombination rate were calculated. The reliability of cDNA library was detected by sequencing and analyzing positive clones and genes amplified by specific primers. The results showed that the RNA concentration was 836 ng/μl and the absorbance ratio at 260/280 nm was 1.82. The library titer was 5.31 × 10(5) plaque-forming unit (PFU)/ml and the recombination rate was 98.21%, indicating that the library was of good quality. In the 33 expressed sequence tags (ESTs) of P. cuniculi, two clones of 1656 and 1658 bp were almost identical with only three variable sites detected, which had an identity of 99.63% with that of Psoroptes ovis, indicating that the cDNA library was reliable. Further detection by specific primers demonstrated that the 553-bp Pso c II gene sequences of P. cuniculi had an identity of 98.56% with those of P. ovis, confirming that the cDNA library was not only reliable but also feasible.

  4. Cloning and expression of trehalose-6-phosphate synthase 1 from Rhizopus oryzae.

    Science.gov (United States)

    Ozer Uyar, Ebru; Yücel, Meral; Hamamcı, Haluk

    2016-05-01

    Trehalose is a reducing disaccharide acting as a protectant against environmental stresses in many organisms. In fungi, Trehalose-6-phosphate synthase 1 (TPS1) plays a key role in the biosynthesis of trehalose. In this study, a full-length cDNA from Rhizopus oryzae encoding TPS1 (designated as RoTPS1) was isolated. The RoTPS1 cDNA is composed of 2505 nucleotides and encodes a protein of 834 amino acids with a molecular mass of 97.8 kDa. The amino acid sequence of RoTPS1 has a relatively high homology with the TPS1s in several other filamentous fungi. RoTPS1 was cloned into Saccharomyces cerevisiae and secretively expressed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. 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.

  6. Cloning of the cDNA for human 12-lipoxygenase

    International Nuclear Information System (INIS)

    Izumi, T.; Hoshiko, S.; Radmark, O.; Samuelsson, B.

    1990-01-01

    A full-length cDNA clone encoding 12-lipoxygenase was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme

  7. Bifunctional cis-Abienol Synthase from Abies balsamea Discovered by Transcriptome Sequencing and Its Implications for Diterpenoid Fragrance Production*

    Science.gov (United States)

    Zerbe, Philipp; Chiang, Angela; Yuen, Macaire; Hamberger, Björn; Hamberger, Britta; Draper, Jason A.; Britton, Robert; Bohlmann, Jörg

    2012-01-01

    The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of AbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production. PMID:22337889

  8. Purification, characterization and partial cDNA cloning of high-temperature stress-induced protein from French bean (Phaseolus vulgaris

    Directory of Open Access Journals (Sweden)

    Nagesh Babu, R.

    2013-02-01

    Full Text Available In order to identify the components of high temperature response in French bean, three heat shock proteins induced under high temperature were purified to homogeneity by Carboxy methyl cellulose and sephadex G-100 chromatography followed by preparative SDS-PAGE. Two of these, Hsp1 and Hsp3 were further characterized by immuno-detection with polyclonal antibodies. Hsp3 exhibited ATPase and chaperone activity with malate dehydrogenase and citrate synthase. Partial cDNA for Hsp3 synthesized using the primer derived from amino-terminal sequence was cloned and expressed in Escherichia coli. The recombinant protein possesses ATPase activity, and showed thermal protection at 50°C in Escherichia coli. The translated partial cDNA showed homology with stress induced proteins including ATPases from higher plants. These results supported the fact that French bean response to high temperature stress involves Hsps as one of the principal components.

  9. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...

  10. STRUCTURAL ENZYMOLOGY OF POLYKETIDE SYNTHASES

    OpenAIRE

    Tsai, Shiou-Chuan (Sheryl); Ames, Brian Douglas

    2009-01-01

    This chapter describes structural and associated enzymological studies of polyketide synthases, including isolated single domains and multidomain fragments. The sequence–structure–function relationship of polyketide biosynthesis, compared with homologous fatty acid synthesis, is discussed in detail. Structural enzymology sheds light on sequence and structural motifs that are important for the precise timing, substrate recognition, enzyme catalysis, and protein–protein interactions leading to ...

  11. cDNA library Table - KAIKOcDNA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data List Contact us KAIKOcDNA... cDNA library Table Data detail Data name cDNA library Table DOI 10.18908/lsdba.nbd...c00951-005 Description of data contents List of Bombyx mori cDNA libraries. Data file File name: kaiko_cdna_...iption Registered library name Registered name of the partial cDNA library Library synonym Another name for cDNA... Download License Update History of This Database Site Policy | Contact Us cDNA library Table - KAIKOcDNA | LSDB Archive ...

  12. Study on construction of cDNA library of the treated changliver cell and quality analysis

    OpenAIRE

    Juntang, Lin; Pramanik, Jogenananda; Congrui, Wang; Huiyong, Zhang; Huigen, Feng; Baosheng, Yang; Yuchang, Li; Cunshuan, Xu

    2004-01-01

    The study aims to construct cDNA library of Changliver cell by SMART (switching mechanism at 5′ end of RNA transcript) technique and analyze its quality. cDNA of Changliver cell was made with RT-PCR and LD-PCR (long-distance PCR), the cDNA library was constructed with SMART cDNA library construction kit. Through testing, the high quality cDNA library containing whole long cDNA of Changliver cell had been constructed. The titer of the amplified cDNA library was 4.5 × 1010 pfu/ml and the averag...

  13. Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

    International Nuclear Information System (INIS)

    Tian, Hongmei; Ma, Leyuan; Zhao, Cong; Hao, Hui; Gong, Biao; Yu, Xiyan; Wang, Xiufeng

    2010-01-01

    To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.

  14. cDNA library construction of two human Demodexspecies.

    Science.gov (United States)

    Niu, DongLing; Wang, RuiLing; Zhao, YaE; Yang, Rui; Hu, Li; Lei, YuYang; Dan, WeiChao

    2017-06-01

    The research of Demodex, a type of pathogen causing various dermatoses in animals and human beings, is lacking at RNA level. This study aims at extracting RNA and constructing cDNA library for Demodex. First, P. cuniculiand D. farinaewere mixed to establish homogenization method for RNA extraction. Second, D. folliculorumand D. breviswere collected and preserved in Trizol, which were mixed with D. farinaerespectively to extract RNA. Finally, cDNA library was constructed and its quality was assessed. The results indicated that for D. folliculorum& D. farinae, the recombination rate of cDNA library was 90.67% and the library titer was 7.50 × 104 pfu/ml. 17 of the 59 positive clones were predicted to be of D. folliculorum; For D. brevis& D. farinae, the recombination rate was 90.96% and the library titer was 7.85 x104 pfu/ml. 40 of the 59 positive clones were predicted to be of D. brevis. Further detection by specific primers demonstrated that mtDNA cox1, cox3and ATP6 detected from cDNA libraries had 96.52%-99.73% identities with the corresponding sequences in GenBank. In conclusion, the cDNA libraries constructed for Demodexmixed with D. farinaewere successful and could satisfy the requirements for functional genes detection.

  15. Second-strand cDNA synthesis: classical method

    International Nuclear Information System (INIS)

    Gubler, U.

    1987-01-01

    The classical scheme for the synthesis of double-stranded cDNA as it was reported in 1976 is described. Reverse transcription of mRNA with oligo(dT) as the primer generates first strands with a small loop at the 3' end of the cDNA (the end that corresponds to the 5' end of the mRNA). Subsequent removal of the mRNA by alkaline hydrolysis leaves single-stranded cDNA molecules again with a small 3' loop. This loop can be used by either reverse transcriptase or Klenow fragment of DNA polymerase I as a primer for second-strand synthesis. The resulting products are double-stranded cDNA molecules that are covalently closed at the end corresponding to the 5' end of the original mRNA. Subsequent cleavage of the short piece of single-stranded cDNA within the loop with the single-strand-specific S 1 nuclease generate open double-stranded molecules that can be used for molecular cloning in plasmids or in phage. Useful variations of this scheme have been described

  16. Gene expression profile analysis in human hepatocellular carcinoma by cDNA microarray.

    Science.gov (United States)

    Chung, Eun Jung; Sung, Young Kwan; Farooq, Mohammad; Kim, Younghee; Im, Sanguk; Tak, Won Young; Hwang, Yoon Jin; Kim, Yang Il; Han, Hyung Soo; Kim, Jung-Chul; Kim, Moon Kyu

    2002-12-31

    We performed gene expression profiling of normal and hepatocellular carcinoma (HCC) liver tissues using a high-density microarray that contained 3,063 human cDNA. The results of a microarray hybridization experiment from eight different HCC tissues were analyzed and classified by the Cluster program. Among these differentially-expressed genes, the galectin-3, serine/threonine kinase SGK, translation factor eIF-4A, -4B, -3, fibroblast growth factor receptor, and ribosomal protein L35A were up-regulated; the mRNAs of Nip3, decorin, and the insulin-like growth factor binding protein-3 were down-regulated in HCC. The differential expression of these genes was further confirmed by an RT-PCR analysis. In addition, our data suggest that the gene expression profile of HCC varies according to the histological types.

  17. Characterization of cDNA for human tripeptidyl peptidase II: The N-terminal part of the enzyme is similar to subtilisin

    International Nuclear Information System (INIS)

    Tomkinson, B.; Jonsson, A-K

    1991-01-01

    Tripeptidyl peptidase II is a high molecular weight serine exopeptidase, which has been purified from rat liver and human erythrocytes. Four clones, representing 4453 bp, or 90% of the mRNA of the human enzyme, have been isolated from two different cDNA libraries. One clone, designated A2, was obtained after screening a human B-lymphocyte cDNA library with a degenerated oligonucleotide mixture. The B-lymphocyte cDNA library, obtained from human fibroblasts, were rescreened with a 147 bp fragment from the 5' part of the A2 clone, whereby three different overlapping cDNA clones could be isolated. The deduced amino acid sequence, 1196 amino acid residues, corresponding to the longest open rading frame of the assembled nucleotide sequence, was compared to sequences of current databases. This revealed a 56% similarity between the bacterial enzyme subtilisin and the N-terminal part of tripeptidyl peptidase II. The enzyme was found to be represented by two different mRNAs of 4.2 and 5.0 kilobases, respectively, which probably result from the utilziation of two different polyadenylation sites. Futhermore, cDNA corresponding to both the N-terminal and C-terminal part of tripeptidyl peptidase II hybridized with genomic DNA from mouse, horse, calf, and hen, even under fairly high stringency conditions, indicating that tripeptidyl peptidase II is highly conserved

  18. Liver Immunology

    Science.gov (United States)

    Bogdanos, Dimitrios P.; Gao, Bin; Gershwin, M. Eric

    2014-01-01

    The liver is the largest organ in the body and is generally regarded by non-immunologists as not having lymphoid function. However, such is far from accurate. This review highlights the importance of the liver as a lymphoid organ. Firstly, we discuss experimental data surrounding the role of liver as a lymphoid organ. The liver facilitates a tolerance rather than immunoreactivity, which protects the host from antigenic overload of dietary components and drugs derived from the gut and is also instrumental to fetal immune tolerance. Loss of liver tolerance leads to autoaggressive phenomena which if are not controlled by regulatory lymphoid populations may lead to the induction of autoimmune liver diseases. Liver-related lymphoid subpopulations also act as critical antigen-presenting cells. The study of the immunological properties of liver and delineation of the microenvironment of the intrahepatic milieu in normal and diseased livers provides a platform to understand the hierarchy of a series of detrimental events which lead to immune-mediated destruction of the liver and the rejection of liver allografts. The majority of emphasis within this review will be on the normal mononuclear cell composition of the liver. However, within this context, we will discus select, but not all, immune mediated liver disease and attempt to place these data in the context of human autoimmunity. PMID:23720323

  19. cDNA libraries for virus-induced gene silencing.

    Science.gov (United States)

    Todd, Andrea T; Liu, Enwu; Page, Jonathan E

    2010-01-01

    Virus-induced gene silencing (VIGS) exploits endogenous plant antiviral defense mechanisms to posttranscriptionally silence the expression of targeted plant genes. VIGS is quick and relatively easy to perform and therefore serves as a powerful tool for high-throughput functional genomics in plants. Combined with the use of subtractive cDNA libraries for generating a collection of VIGS-ready cDNA inserts, VIGS can be utilized to screen a large number of genes to determine phenotypes resulting from the knockdown/knockout of gene function. Taking into account the optimal insert design for VIGS, we describe a methodology for producing VIGS-ready cDNA libraries enriched for inserts relevant to the biological process of interest.

  20. 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.

  1. Liver disease

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/000205.htm Liver disease To use the sharing features on this page, please enable JavaScript. The term "liver disease" applies to many conditions that stop the ...

  2. Liver Diseases

    Science.gov (United States)

    Your liver is the largest organ inside your body. It helps your body digest food, store energy, and remove poisons. There are many kinds of liver diseases: Diseases caused by viruses, such as hepatitis ...

  3. Fatty Liver

    Science.gov (United States)

    ... Drug Interactions Pill Identifier Commonly searched drugs Aspirin Metformin Warfarin Tramadol Lactulose Ranitidine News & Commentary Recent News ... Rarely, fat accumulates in the liver during late pregnancy. This disorder, called fatty liver of pregnancy or ...

  4. Liver Biopsy

    Science.gov (United States)

    ... instrument called a cannula to remove the liver tissue sample. What is the liver and what does it do? ... who specializes in diagnosing diseases—looks at the tissue with a microscope and sends a report to the person's health care provider. What are the risks of liver biopsy? The risks ...

  5. Isolation and Expression of a cDNA Encoding Methylmalonic Aciduria Type A Protein from Euglena gracilis Z

    Directory of Open Access Journals (Sweden)

    Fumio Watanabe

    2013-02-01

    Full Text Available In animals, cobalamin (Cbl is a cofactor for methionine synthase and methylmalonyl-CoA mutase (MCM, which utilizes methylcobalamin and 5′-deoxyadenosylcobalamin (AdoCbl, respectively. The cblA complementation class of inborn errors of Cbl metabolism in humans is one of three known disorders that affect AdoCbl synthesis. The gene responsible for cblA has been identified in humans (MMAA as well as its homolog (meaB in Methylobacterium extorquens. Recently, it has been reported that human MMAA plays an important role in the protection and reactivation of MCM in vitro. However, the physiological function of MMAA is largely unknown. In the present study, we isolated the cDNA encoding MMAA from Euglena gracilis Z, a photosynthetic flagellate. The deduced amino acid sequence of the cDNA shows 79%, 79%, 79% and 80% similarity to human, mouse, Danio rerio MMAAs and M. extorquens MeaB, respectively. The level of the MCM transcript was higher in Cbl-deficient cultures of E. gracilis than in those supplemented with Cbl. In contrast, no significant differences were observed in the levels of the MMAA transcript under the same two conditions. No significant difference in MCM activity was observed between Escherichia coli that expressed either MCM together with MMAA or expressed MCM alone.

  6. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    OpenAIRE

    Karuppiah, Vijaykumar; Ranaghan, Kara E.; Leferink, Nicole G. H.; Johannissen, Linus O.; Shanmugam, Muralidharan; Ní Cheallaigh, Aisling; Bennett, Nathan J.; Kearsey, Lewis J.; Takano, Eriko; Gardiner, John M.; van der Kamp, Marc W.; Hay, Sam; Mulholland, Adrian J.; Leys, David; Scrutton, Nigel S.

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS)...

  7. Genetic variants in promoters and coding regions of the muscle glycogen synthase and the insulin-responsive GLUT4 genes in NIDDM

    DEFF Research Database (Denmark)

    Bjørbaek, C; Echwald, Søren Morgenthaler; Hubricht, P

    1994-01-01

    To examine the hypothesis that variants in the regulatory or coding regions of the glycogen synthase (GS) and insulin-responsive glucose transporter (GLUT4) genes contribute to insulin-resistant glucose processing of muscle from non-insulin-dependent diabetes mellitus (NIDDM) patients, promoter r...... in the GLUT4 cDNA was a silent polymorphism at codon 130. Southern blotting of both gene loci did not detect any major abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)...

  8. Identification of a novel hedycaryol synthase gene isolated from Camellia brevistyla flowers and floral scent of Camellia cultivars.

    Science.gov (United States)

    Hattan, Jun-ichiro; Shindo, Kazutoshi; Ito, Tomoko; Shibuya, Yurica; Watanabe, Arisa; Tagaki, Chie; Ohno, Fumina; Sasaki, Tetsuya; Ishii, Jun; Kondo, Akihiko; Misawa, Norihiko

    2016-04-01

    A novel terpene synthase (Tps) gene isolated from Camellia brevistyla was identified as hedycaryol synthase, which was shown to be expressed specifically in flowers. Camellia plants are very popular because they bloom in winter when other plants seldom flower. Many ornamental cultivars of Camellia have been bred mainly in Japan, although the fragrance of their flowers has not been studied extensively. We analyzed floral scents of several Camellia cultivars by gas chromatography-mass spectrometry (GC-MS) and found that Camellia brevistyla produced various sesquiterpenes in addition to monoterpenes, whereas Camellia japonica and its cross-lines produced only monoterpenes, including linalool as the main product. From a flower of C. brevistyla, we isolated one cDNA encoding a terpene synthase (TPS) comprised of 554 amino acids, which was phylogenetically positioned to a sole gene clade. The cDNA, designated CbTps1, was expressed in mevalonate-pathway-engineered Escherichia coli, which carried the Streptomyces mevalonate-pathway gene cluster in addition to the acetoacetate-CoA ligase gene. A terpene product was purified from recombinant E. coli cultured with lithium acetoacetate, and analyzed by (1)H-nulcear magnetic resonance spectroscopy ((1)H-NMR) and GC-MS. It was shown that a sesquiterpene hedycaryol was produced, because (1)H-NMR signals of the purified product were very broad, and elemol, a thermal rearrangement product from hedycaryol, was identified by GC-MS analysis. Spectroscopic data of elemol were also determined. These results indicated that the CbTps1 gene encodes hedycaryol synthase. Expression analysis of CbTps1 showed that it was expressed specifically in flowers, and hedycaryol is likely to be one of the terpenes that attract insects for pollination of C. brevistyla. A linalool synthase gene, which was isolated from a flower of Camellia saluenensis, is also described.

  9. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Unknown

    cell embryo and the expression was monitored continuously. The expression shown here is in developing embryo and freshly hatched fish. The intensity of green colour indicate the strong expression of EGFP in all the tissues of the embryo/fry. The expression of EGPF indicates the co-expression of catfish GH cDNA and the ...

  10. cDNA cloning, structural analysis, SNP detection and tissue ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 96; Issue 2. cDNA cloning, structural analysis, SNP detection and tissue ... Abstract. Insulin-like growth factor 1 (IGF1) plays an important role in growth, reproduction, foetal development and cell proliferation. The present study was conducted to clone and sequence the ...

  11. Cloning and characterization of cDNA encoding xyloglucan ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... construction and restructuring of xyloglucan cross-links, thereby controlling the mechanical properties of cell wall. We cloned complete cDNA of an ..... are marked by horizontal lines. The conserved cysteine residues (amino acids 220, 229, 274 and 288 in P. glaucum) are marked by vertical blue arrows.

  12. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    RPS16 of eukaryote is a component of the 40S small ribosomal subunit encoded by RPS16 gene and is also a homolog of prokaryotic RPS9. The cDNA and genomic sequence of RPS16 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription-polymerase chain ...

  13. cDNA cloning, structural analysis, SNP detection and tissue ...

    Indian Academy of Sciences (India)

    THOMAS NAICY

    [Naicy T., Venkatachalapathy T., Aravindakshan T., Raghavan K. C., Mini M. and Shyama K. 2017 cDNA cloning, structural analysis, SNP detection and tissue expression profile of the IGF1 gene in Malabari and Attappady Black goats of India. J. Genet. 96, xx–xx]. Introduction. Insulin-like growth factor 1 (IGF1), an important ...

  14. Construction of yeast surface-displayed cDNA libraries.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2011-01-01

    Using yeast display, heterologous protein fragments can be efficiently displayed at high copy levels on the Saccharomyces cerevisiae cell wall. Yeast display can be used to screen large expressed protein libraries for proteins or protein fragments with specific binding properties. Recently, yeast surface-displayed cDNA libraries have been constructed and used to identify proteins that bind to various target molecules such as peptides, small molecules, and antibodies. Because yeast protein expression pathways are similar to those found in mammalian cells, human protein fragments displayed on the yeast cell wall are likely to be properly folded and functional. Coupled with fluorescence-activated cell sorting, yeast surface-displayed cDNA libraries potentially allow the selection of protein fragments or domains with affinity for any soluble molecule that can be fluorescently detected. In this report, we describe protocols for the construction and validation of yeast surface-displayed cDNA libraries using preexisting yeast two-hybrid cDNA libraries as a starting point.

  15. 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...

  16. Cloning and characterization of Ginkgo biloba levopimaradiene synthase which catalyzes the first committed step in ginkgolide biosynthesis.

    Science.gov (United States)

    Schepmann, H G; Pang, J; Matsuda, S P

    2001-08-15

    Levopimaradiene synthase, which catalyzes the initial cyclization step in ginkgolide biosynthesis, was cloned and functionally characterized. A Ginkgo biloba cDNA library was prepared from seedling roots and a probe was amplified using primers corresponding to conserved gymnosperm terpene synthase sequences. Colony hybridization and rapid amplification of cDNA ends yielded a full-length clone encoding a predicted protein (873 amino acids, 100,289 Da) similar to known gymnosperm diterpene synthases. The sequence includes a putative N-terminal plastid transit peptide and three aspartate-rich regions. The full-length protein expressed in Escherichia coli cyclized geranylgeranyl diphosphate to levopimaradiene, which was identical to a synthetic standard by GC/MS analysis. Removing 60 or 79 N-terminal residues increased levopimaradiene production, but a 128-residue N-terminal deletion lacked detectable activity. This is the first cloned ginkgolide biosynthetic gene and the first in vitro observation of an isolated ginkgolide biosynthetic enzyme. Copyright 2001 Academic Press.

  17. Isolation of 1-aminocyclopropane-1-carboxylate synthase gene from Oncidium Gower Ramsey.

    Science.gov (United States)

    Yang, G H; Liu, J P

    2014-10-20

    A full-length cDNA of a 1-aminocyclopropane-1-carboxylate synthase (ACS) family member from Oncidium, named OnACS1 (GenBank accession No. JQ822087) was cloned and characterized by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends technology. The full-length cDNA was 1586 bp, including a 1308-bp open reading frame, a 105-bp 5' untranslated region (UTR), and 173-bp 3' UTR, encoding 436 amino acids. The deduced amino acid sequence of OnACS1 shares 85, 84, and 83% homology with ACS proteins of Cattleya bicolor, Dendrobium crumenatum, and Phalaenopsis hybrid, respectively. Prokaryotic expression and sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that a specific band was produced and was consistent with the predicted protein size. A tissue-specific manner of OnACS1 expression was observed, and it was predominantly expressed in the gynostemium. The OnACS1 expression in the sepals and gynandria was upregulated by 1% ethephon treatment.

  18. Producing biofuels using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  19. Fatty Liver

    International Nuclear Information System (INIS)

    Filippone, A.; Digiovandomenico, V.; Digiovandomenico, E.; Genovesi, N.; Bonomo, L.

    1991-01-01

    The authors report their experience with the combined use of US and CT in the study of diffuse and subtotal fatty infiltration of the liver. An apparent disagreement was initially found between the two examinations in the study of fatty infiltration. Fifty-five patients were studied with US and CT of the upper abdomen, as suggested by clinics. US showed normal liver echogenicity in 30 patients and diffuse increased echogenicity (bright liver) in 25 cases. In 5 patients with bright liver, US demonstrated a solitary hypoechoic area, appearing as a 'skip area', in the quadrate lobe. In 2 patients with bright liver, the hypoechoic area was seen in the right lobe and exhibited no typical US features of 'Skip area'. Bright liver was quantified by measuring CT density of both liver and spleen. The relative attenuation values of spleen and liver were compared on plain and enhanced CT scans. In 5 cases with a hypoechoic area in the right lobe, CT findings were suggestive of hemangioma. A good correlation was found between broght liver and CT attenuation values, which decrease with increasing fat content of the liver. Moreover, CT attenuation values confirmed US findings in the study of typical 'skip area', by demonstrating normal density - which suggests that CT can characterize normal tissue in atypical 'skip area'

  20. 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.

  1. Molecular cloning and characterization of the sheep malic enzyme cDNA.

    Science.gov (United States)

    Stefos, Georgios C; Argyrokastritis, Alexandros; Bizelis, Iosif; Rogdakis, Emmanuel

    2008-10-15

    Malic enzyme catalyzes decarboxylation of malate to pyruvate and CO(2), providing de novo biosynthesis of fatty acids with NADPH. Since lipogenesis in ruminants, contrarily to some monogastric species like human and rodents, occurs predominantly in adipose tissue, the activity of many lipogenic enzymes is higher in adipose tissue compared to liver. Expression of malic enzyme is regulated by nutrition; refeeding after a period of starvation results to an induction of the enzyme. Here we present the nucleotide sequence of two transcripts of the ovine cytosolic malic enzyme gene that differ at the length of the 3' UTR. These are the first published cDNA sequences for ruminant species and share high similarity with the corresponding sequences of other species. Malic enzyme mRNA was present in every ovine tissue that was examined. In agreement with the fact that adipose tissue is the major lipogenic site for ruminants, mRNA levels in adipose tissue were higher than in liver. Refeeding after two weeks of caloric restriction resulted in a two-fold increase of the mRNA level of malic enzyme in adipose tissue.

  2. Full-length cDNA cloning of Toll-like receptor 4 in dogs and cats.

    Science.gov (United States)

    Asahina, Yuka; Yoshioka, Noriyuki; Kano, Rui; Moritomo, Tadaaki; Hasegawa, Atsuhiko

    2003-12-15

    In the present study, full length of canine and feline Toll-like receptor 4 (TLR4) cDNAs were sequenced, and the expression of canine and feline TLR4 mRNAs in dog and cat tissues were investigated. The full-length cDNA of TLR4 of dog and cat was 2709 bp encoding 637 amino acids and 3113 bp encoding 833 amino acids, respectively. The similarity of canine and feline TLR4 were 83.6% at the nucleotide sequence level and 77.6% at the amino acid sequence level. At the amino acid sequence level, canine and feline TLR4 showed sequence similarities of approximately 62-78% with those of Homo sapiens, Mus musculus, Bos taurus and Equus caballus, respectively. Southern hybridization analyses with TLR4 cDNA probes gave one distinct band in BamHI, EcoRI and HindIII digests of genomic DNA from dogs and cats, respectively, indicating the likely presence of a single TLR4 gene in each species. By RT-PCR analysis, mRNA of canine TLR4 was expressed highly in peripheral blood leukocytes (PBL), moderately in spleen, stomach and small intestine, at low levels in liver, with no expression in kidney, large intestine and skin. On the other hand, mRNA of feline TLR4 was expressed highly in lung, bladder and PBL, moderately in kidney, liver, spleen and large intestine and at low levels in pancreas and small intestine.

  3. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

    International Nuclear Information System (INIS)

    Gershenfeld, H.K.; Hershberger, R.J.; Shows, T.B.; Weissman, I.L.

    1988-01-01

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage λgt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16 + natural killer cells and CD3 + , CD16 - T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells

  4. Liver Transplant

    Science.gov (United States)

    ... Nutrition Clinical Trials Primary Biliary Cholangitis Definition & Facts Symptoms & Causes Diagnosis Treatment Eating, Diet, & Nutrition Clinical Trials Wilson Disease Liver Transplant View or Print All Sections Definition & ...

  5. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis.

    Science.gov (United States)

    Hegardt, F G

    1999-03-15

    Cytosolic and mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthases were first recognized as different chemical entities in 1975, when they were purified and characterized by Lane's group. Since then, the two enzymes have been studied extensively, one as a control site of the cholesterol biosynthetic pathway and the other as an important control site of ketogenesis. This review describes some key developments over the last 25 years that have led to our current understanding of the physiology of mitochondrial HMG-CoA synthase in the HMG-CoA pathway and in ketogenesis in the liver and small intestine of suckling animals. The enzyme is regulated by two systems: succinylation and desuccinylation in the short term, and transcriptional regulation in the long term. Both control mechanisms are influenced by nutritional and hormonal factors, which explains the incidence of ketogenesis in diabetes and starvation, during intense lipolysis, and in the foetal-neonatal and suckling-weaning transitions. The DNA-binding properties of the peroxisome-proliferator-activated receptor and other transcription factors on the nuclear-receptor-responsive element of the mitochondrial HMG-CoA synthase promoter have revealed how ketogenesis can be regulated by fatty acids. Finally, the expression of mitochondrial HMG-CoA synthase in the gonads and the correction of auxotrophy for mevalonate in cells deficient in cytosolic HMG-CoA synthase suggest that the mitochondrial enzyme may play a role in cholesterogenesis in gonadal and other tissues.

  6. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  7. Expression, purification and characterization of recombinant (E)-beta-farnesene synthase from Artemisia annua.

    Science.gov (United States)

    Picaud, Sarah; Brodelius, Maria; Brodelius, Peter E

    2005-05-01

    A cDNA clone (GenBank Accession No. AY835398) encoding a sesquiterpene synthase, (E)-beta-farnesene synthase, has been isolated from Artemisia annua L. It contains a 1746-bp open reading frame coding for 574 amino acids (66.9 kDa) with a calculated pI=5.03. The deduced amino acid sequence is 30-50% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, beta-farnesene, from farnesyl diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the K(m)- and k(cat)-values for farnesyl diphosphate, is 2.1 microM and 9.5 x 10(-3) s(-1), respectively resulting in the efficiency 4.5 x 10(-3) M(-1)s(-1). The enzyme exhibits substantial activity in the presence of Mg(2+), Mn(2+) or Co(2+) but essentially no activity when Zn(2+), Ni(2+) or Cu(2+) is used as cofactor. The concentration required for maximum activity are estimated to 5 mM, 0.5 mM and <10 microM for Mg(2+), Co(2+) or Mn(2+), respectively. Geranyl diphosphate is not a substrate for the recombinant enzyme.

  8. Molecular cloning and characterization of a cDNA encoding ...

    African Journals Online (AJOL)

    Fritillaria thunbergii Miq., known as the bulbous plants of the genus fritillaria, produces a large amount of sterols. Homology based polymerase chain reactions (PCRs) with degenerate primers designed from the conserved sequences among the known cycloartenol synthase (CAS) resulted in cloning of a CAS from the ...

  9. Molecular cloning of growth hormone encoding cDNA of Indian ...

    Indian Academy of Sciences (India)

    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 ...

  10. cDNA cloning and characterization of a mannose-binding lectin from ...

    Indian Academy of Sciences (India)

    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 ...

  11. Isolation and characterization of a Coffea canephora ERF-like c-DNA

    African Journals Online (AJOL)

    A cDNA corresponding to an ERF gene has been isolated from a Coffea canephora fruit cDNA library. The cDNA was 1,317 nucleotides long and has an open reading frame of 987 bp. The predicted polypeptide showed a great similitude with equivalent proteins from others plant species. The binding domain shows 98.3% ...

  12. Molecular cloning of growth hormone encoding cDNA of Indian

    Indian Academy of Sciences (India)

    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 ...

  13. cDNA cloning and characterization of a mannose-binding lectin

    Indian Academy of Sciences (India)

    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 ...

  14. cDNA library information - Dicty_cDB | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available (C) 5) sexually fusion-competent KAX3 cells (Gamete phase) (F) cDNA library construction method How to const...dir) 2) Full-length cDNA libraries (oligocapped method)(fl) 3) Gamete-specific subtraction library (sub) cDNA library construction

  15. [CDNA cloning of human leptin and its expression].

    Science.gov (United States)

    Jia, Zhen-Yu; Fu, Xiao-Min; Jin, Ai-Hua; Cao, Jiang

    2003-07-01

    To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins.

  16. Construction of cDNA library of Pyrocystis lunula (Pyrophyta)

    Science.gov (United States)

    Sui, Zhenghong; Kowallik, Klaus V.

    2004-10-01

    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 20µgg-1 net cells, and the band intensity ratio of 28S/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.

  17. Construction of cDNA libraries in vaccinia virus.

    Science.gov (United States)

    Smith, Ernest S; Shi, Shuying; Zauderer, Maurice

    2004-01-01

    Poxvirus expression vectors have gained widespread use for expression of foreign proteins and as delivery vehicles for vaccine antigens. We have developed a novel method using the poxvirus as a library vector for functional selection of specific cDNA. Poxviruses have several unique and useful properties as a library vector. Most importantly, because poxviruses are packaged into fully infectious particles in the cell cytoplasm, specific recombinants can be readily recovered even from a very small number of selected cells. Moreover, in contrast to libraries constructed in retrovirus or plasmid-based vectors, recombinant vaccinia virus can be efficiently recovered even from cells that have been induced to undergo apoptosis or cessation of cell growth. In the past, the major obstacle in this application to poxviruses has been the low frequency with which recombinants can be generated. The most commonly used method to construct recombinant poxvirus is homologous recombination. The frequency of recombinants derived in this manner is of the order of 0.1%, sufficient to recover a recombinant of a purified DNA clone in a transfer plasmid, but far too low to permit construction of a representative cDNA library. We have developed a method that generates nearly 100% recombinant vaccinia viruses at good titer. We have termed this method trimolecular recombination. cDNA libraries of as many as 107 or more independent viral recombinants can be constructed by trimolecular recombination. For the first time, large, diverse, and representative cDNA libraries can be screened in a vaccinia virus-based expression vector.

  18. cDNA cloning, structural analysis, SNP detection and tissue ...

    Indian Academy of Sciences (India)

    THOMAS NAICY

    E-mail: naicy@kvasu.ac.in. and conception rate ... transformed into DH5α strain of Escherichia coli and the clones harbouring ... Primer pairs for caprine IGF1 and GAPDH were designed using Primer3 software (table 1). RTq-PCR was conducted in a 25 μL reaction volume containing 50 ng of cDNA and 2× Max- ima SYBR ...

  19. Ketogenesis prevents diet-induced fatty liver injury and hyperglycemia

    OpenAIRE

    Cotter, David G.; Ercal, Baris; Huang, Xiaojing; Leid, Jamison M.; d’Avignon, D. André; Graham, Mark J.; Dietzen, Dennis J.; Brunt, Elizabeth M.; Patti, Gary J.; Crawford, Peter A.

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) spectrum disorders affect approximately 1 billion individuals worldwide. However, the drivers of progressive steatohepatitis remain incompletely defined. Ketogenesis can dispose of much of the fat that enters the liver, and dysfunction in this pathway could promote the development of NAFLD. Here, we evaluated mice lacking mitochondrial 3-hydroxymethylglutaryl CoA synthase (HMGCS2) to determine the role of ketogenesis in preventing diet-induced steatohe...

  20. CDNA encoding a polypeptide including a hevein sequence

    Science.gov (United States)

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  1. cDNA library generation from ribonucleoprotein particles.

    Science.gov (United States)

    Rederstorff, Mathieu; Hüttenhofer, Alexander

    2011-02-01

    Most, if not all, known noncoding RNAs (ncRNAs) are associated with RNA binding proteins, thus forming ribonucleoprotein particles or RNPs. Here we describe a protocol for the generation of a specialized cDNA library from RNPs, thereby increasing the proportion of functional ncRNA species in the library. To that end, cellular extracts are fractionated on 10-30% glycerol gradients. Subsequently, RNP-derived ncRNAs are isolated and 3'-tailed by cytidine triphosphate and poly(A) polymerase; this is followed by 5' adapter ligation by T4 RNA ligase. Reverse transcription of ncRNAs into cDNAs is carried out with an oligo-d(G) anchor primer. The generated cDNA libraries are subsequently submitted to high-throughput sequencing. This RNP selection procedure increases the probability of the presence of biologically relevant ncRNA species in the library compared with libraries generation methods that use size-selected, protein-devoid ncRNAs. The protocol enables the generation of deep-sequencing-compatible cDNA libraries that code for functional ncRNAs within 1 week.

  2. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  3. Liver regeneration

    NARCIS (Netherlands)

    Chamuleau, R. A.; Bosman, D. K.

    1988-01-01

    Despite great advances in analysing hemodynamic, morphological and biochemical changes during the process of liver regeneration, the exact (patho)physiological mechanism is still unknown. A short survey of literature is given of the kinetics of liver regeneration and the significance of different

  4. [Cloning and characterization of a novel mouse short-chain dehydrogenase/reductases cDNA mHsdl2#, encoding a protein with a SDR domaid and a SCP2 domain].

    Science.gov (United States)

    Dai, J; Li, P; Ji, Ch; Feng, C; Gui, M; Sun, Y; Zhang, J; Zhu, J; Dou, Ch; Gu, Sh

    2005-01-01

    The short-chain dehydrogenases/reductases (SDRs) play important roles in body's metabolism. We cloned a novel mouse SDR cDNA which encodes a deduced HSD-like protein with a conserved SDR domain and a SCP2 domain. The 1.8 kb cDNA consists of 11 exons and is mapped to mouse chromosome 4B3. The corresponding gene is widely expressed in normal mouse tissues and its expression level in liver increases after inducement with cholesterol food. The predicted mouse HSDL2 protein, which has a peroxisomal target signal, is localized in the cytoplasm of NIH 3T3 cells.

  5. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B. (DuPont); (NWU)

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  6. [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.

  7. [Construction and identification of the expression library of album pollen allergens cDNA].

    Science.gov (United States)

    Zhang, Jie; Sun, Xiu-zhen; Yan, Hong; Zhang, Ni; Feng, Xiang-li

    2011-05-01

    To construct and identify the express library of album pollen allergens cDNA. Total RNA were extracted from the album pollen with TRIzol reagent and the mRNA was isolate for the amplify followed. A double stranded cDNA (ds cDNA) was synthesized using primers containing Xho I and Poly(dT) sequence by ZAP Express®cDNA synthesis kit. The ds cDNA was modified and purified by gel chromatography, and then the cDNA fragment with the length of more than 400 bp containing sticky ends was obtained. The cDNA fragment was ligated with Uni-ZAP XR vector and subsequently treated with in vitro packaging using phage by ZAP-cDNA express GigapackIII Gold cloning kit. The express library of album pollen cDNA was constructed by in vitro packaging. The recombination rate and the lengths of fragments inserted of the cDNA library were detected by polymerase chain reaction. The titer and the recombination rate of cDNA expression library constructed were 9.7×10(5) and 100%, respectively. The capacity of the library was 4.85 Pfu. The average length of cDNA fragments inserted was about 1.0 kb. Based on the capacity of cDNA expression library constructed and the length of cDNA insertion fragments, the cDNA expression library constructed is qualified to screening target cDNA clone, laying the foundation for preparation of gene recombinant allergen pollen vaccine.

  8. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    Science.gov (United States)

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids. PMID:28966840

  9. Liver transplant

    Science.gov (United States)

    ... a liver becomes available. Make sure that, no matter where you are going, you can be contacted ... ADAM Health Solutions. About MedlinePlus Site Map FAQs Customer Support Get email updates Subscribe to RSS Follow ...

  10. Liver Transplant

    Science.gov (United States)

    ... Legacy Society Make Gifts of Stock Donate Your Car Personal Fundraising Partnership & Support Share Your Story Spread the Word Give While You Shop Contact Us Donate Now Liver Transplant Back In ...

  11. Enlarged Liver

    Science.gov (United States)

    ... of liver damage. Medicinal herbs. Certain herbs, including comfrey, ma huang and mistletoe, can increase your risk ... herbs to avoid include germander, chaparral, senna, mistletoe, comfrey, ma huang, valerian root, kava, celandine and green ...

  12. An unusual plant triterpene synthase with predominant α-amyrin-producing activity identified by characterizing oxidosqualene cyclases from Malus × domestica.

    Science.gov (United States)

    Brendolise, Cyril; Yauk, Yar-Khing; Eberhard, Ellen D; Wang, Mindy; Chagne, David; Andre, Christelle; Greenwood, David R; Beuning, Lesley L

    2011-07-01

    The pentacyclic triterpenes, in particular ursolic acid and oleanolic acid and their derivatives, exist abundantly in the plant kingdom, where they are well known for their anti-inflammatory, antitumour and antimicrobial properties. α-Amyrin and β-amyrin are the precursors of ursolic and oleanolic acids, respectively, formed by concerted cyclization of squalene epoxide by a complex synthase reaction. We identified three full-length expressed sequence tag sequences in cDNA libraries constructed from apple (Malus × domestica 'Royal Gala') that were likely to encode triterpene synthases. Two of these expressed sequence tag sequences were essentially identical (> 99% amino acid similarity; MdOSC1 and MdOSC3). MdOSC1 and MdOSC2 were expressed by transient expression in Nicotiana benthamiana leaves and by expression in the yeast Pichia methanolica. The resulting products were analysed by GC and GC-MS. MdOSC1 was shown to be a mixed amyrin synthase (a 5 : 1 ratio of α-amyrin to β-amyrin). MdOSC1 is the only triterpene synthase so far identified in which the level of α-amyrin produced is > 80% of the total product and is, therefore, primarily an α-amyrin synthase. No product was evident for MdOSC2 when expressed either transiently or in yeast, suggesting that this putative triterpene synthase is either encoded by a pseudogene or does not express well in these systems. Transcript expression analysis in Royal Gala indicated that the genes are mostly expressed in apple peel, and that the MdOSC2 expression level was much lower than that of MdOSC1 and MdOSC3 in all the tissues tested. Amyrin content analysis was undertaken by LC-MS, and demonstrated that levels and ratios differ between tissues, but that the true consequence of synthase activity is reflected in the ursolic/oleanolic acid content and in further triterpenoids derived from them. Phylogenetic analysis placed the three triterpene synthase sequences with other triterpene synthases that encoded either

  13. Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments

    NARCIS (Netherlands)

    Hakvoort, T. B.; Spijkers, J. A.; Vermeulen, J. L.; Lamers, W. H.

    1996-01-01

    We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes

  14. Succinyl-CoA:3-oxoacid transferase (SCOT): Human cDNA and genomic cloning and chromosomal mapping of the human gene

    Energy Technology Data Exchange (ETDEWEB)

    Kassovska-Bratinova, S.; Mitchell, G.A. [Hopital Ste-Justine, Montreal, Quebec (Canada); Duncan, A. [Kingston General Hospital, Kingston, Ontario (Canada)] [and others

    1994-09-01

    SCOT (EC 2.8.3.5) mediates the activation and utilization of ketone bodies in extrahepatic tissues, especially brain, heart and kidney. Children with hereditary SCOT deficiency have episodes of severe ketoacidosis. Using a partial human heart SCOT cDNA, hSCOT-G, we detected a single {approximately}3 Kb mRNA in human heart and leukocytes, but not in liver. The length of the mouse SCOT mRNA detected with hSCOT-G in muscle, heart, kidney and brain is {approximately}3 Kb. We mapped the human SCOT gene to chromosome 5p13 by in situ hybridization. To date we have isolated human heart cDNAs spanning 2.9 Kb and including a 1248 bp open reading frame. The 3{prime} nontranslated region of the human SCOT mRNA extends at least 1712 bp, in contrast to the 209 bp sequence reported for pig SCOT cDNA. In one heart cDNA clone we detected a 58 bp insertion 258 bp downstream from the stop codon. We performed RT-PCR using a 5{prime} degenerate-sequence primer designed from the pig SCOT leader peptide sequence and 3{prime} human-specific primers. We obtained a fragment of the expected 320 bp length which strongly hybridizes to an internal oligonucleotide and which we are now characterizing. Human genomic Southern blot analysis with a partial human cDNA as probe suggests that the length of the human SCOT gene is about 40 K. Using hSCOT-G as a probe, we screened a human leukocyte genomic library in EMBL-3 phage and isolated two genomic clones. One of them contains a processed pseudogene. The other contains at least two exons of the human SCOT gene spanning cDNA residues 431 to 734. These findings will be useful for mutation analysis in SCOT-deficient patients.

  15. 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.

  16. Construction and characterization of a normalized cDNA library.

    OpenAIRE

    Soares, M B; Bonaldo, M F; Jelene, P; Su, L; Lawton, L; Efstratiadis, A

    1994-01-01

    We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each...

  17. Liver Transplantation for Acute Intermittent Porphyria: Biochemical and Pathologic Studies of the Explanted Liver.

    Science.gov (United States)

    Yasuda, Makiko; Erwin, Angelika L; Liu, Lawrence U; Balwani, Manisha; Chen, Brenden; Kadirvel, Senkottuvelan; Gan, Lin; Fiel, M Isabel; Gordon, Ronald E; Yu, Chunli; Clavero, Sonia; Arvelakis, Antonios; Naik, Hetanshi; Martin, L David; Phillips, John D; Anderson, Karl E; Sadagoparamanujam, Vaithamanithi M; Florman, Sander S; Desnick, Robert J

    2015-06-05

    Acute intermittent porphyria (AIP) is an autosomal-dominant hepatic disorder caused by the half-normal activity of hydroxymethylbilane (HMB) synthase. Symptomatic individuals experience life-threatening acute neurovisceral attacks that are precipitated by factors that induce the hepatic expression of 5-aminolevulinic acid synthase 1 (ALAS1), resulting in the marked accumulation of the putative neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Here, we provide the first detailed description of the biochemical and pathologic alterations in the explanted liver of an AIP patient who underwent orthotopic liver transplantation (OLT) due to untreatable and debilitating chronic attacks. After OLT, the recipient's plasma and urinary ALA and PBG rapidly normalized, and her attacks immediately stopped. In the explanted liver, (a) ALAS1 mRNA and activity were elevated approximately ~3- and 5-fold, and ALA and PBG concentrations were increased ~3- and 1,760-fold, respectively; (b) uroporphyrin III concentration was elevated; (c) microsomal heme content was sufficient, and representative cytochrome P450 activities were essentially normal; (d) HMB synthase activity was approximately half-normal (~42%); (e) iron concentration was slightly elevated; and (f) heme oxygenase I mRNA was increased approximately three-fold. Notable pathologic findings included nodular regenerative hyperplasia, previously not reported in AIP livers, and minimal iron deposition, despite the large number of hemin infusions received before OLT. These findings suggest that the neurovisceral symptoms of AIP are not associated with generalized hepatic heme deficiency and support the neurotoxicity of ALA and/or PBG. Additionally, they indicate that substrate inhibition of hepatic HMB synthase activity by PBG is not a pathogenic mechanism in acute attacks.

  18. Characterization of ovine hepatic gene expression profiles in response to Escherichia coli lipopolysaccharide using a bovine cDNA microarray

    Directory of Open Access Journals (Sweden)

    Boermans Herman J

    2006-11-01

    Full Text Available Abstract Background During systemic gram-negative bacterial infections, lipopolysaccharide (LPS ligation to the hepatic Toll-like receptor-4 complex induces the production of hepatic acute phase proteins that are involved in the host response to infection and limit the associated inflammatory process. Identifying the genes that regulate this hepatic response to LPS in ruminants may provide insight into the pathogenesis of bacterial diseases and eventually facilitate breeding of more disease resistant animals. The objective of this research was to profile the expression of ovine hepatic genes in response to Escherichia coli LPS challenge (0, 200, 400 ng/kg using a bovine cDNA microarray and quantitative real-time PCR (qRT-PCR. Results Twelve yearling ewes were challenged iv with E. coli LPS (0, 200, 400 ng/kg and liver biopsies were collected 4–5 hours post-challenge to assess hepatic gene expression profiles by bovine cDNA microarray and qRT-PCR analyses. The expression of CD14, C3, IL12R, NRAMP1, SOD and IGFBP3 genes was down regulated, whereas the expression of ACTHR, IFNαR, CD1, MCP-1 and GH was increased during LPS challenge. With the exception of C3, qRT-PCR analysis of 7 of these genes confirmed the microarray results and demonstrated that GAPDH is not a suitable housekeeping gene in LPS challenged sheep. Conclusion We have identified several potentially important genes by bovine cDNA microarray and qRT-PCR analyses that are differentially expressed during the ovine hepatic response to systemic LPS challenge. Their potential role in regulating the inflammatory response to LPS warrants further investigation.

  19. Fungal type III polyketide synthases.

    Science.gov (United States)

    Hashimoto, Makoto; Nonaka, Takamasa; Fujii, Isao

    2014-10-01

    This article covers the literature on fungal type III polyketide synthases (PKSs) published from 2005 to 2014. Since the first discovery of fungal type III PKS genes in Aspergillus oryzae, reported in 2005, putative genes for type III PKSs have been discovered in fungal genomes. Compared with type I PKSs, type III PKSs are much less abundant in fungi. However, type III PKSs could have some critical roles in fungi. This article summarizes the studies on fungal type III PKS functional analysis, including Neurospora crassa ORAS, Aspergillus niger AnPKS, Botrytis cinerea BPKS and Aspergillus oryzae CsyA and CsyB. It is mostly in vitro analysis using their recombinant enzymes that has revealed their starter and product specificities. Of these, CsyB was found to be a new kind of type III PKS that catalyses the coupling of two β-keto fatty acyl CoAs. Homology modelling reported in this article supports the importance of the capacity of the acyl binding tunnel and active site cavity in fungal type III PKSs.

  20. Terpene synthases from Cannabis sativa.

    Directory of Open Access Journals (Sweden)

    Judith K Booth

    Full Text Available Cannabis (Cannabis sativa plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E-β-ocimene, (--limonene, (+-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  1. [Construction and Identification of the cDNA Expression Library for Human Esophageal Cancer Cells].

    Science.gov (United States)

    Zhang, Zhe; Wu, Xiang-Yu; Feng, Lu; Huang, Shang-Ke; Luo, Min-Na; Shao, Shan; Zhao, Xin-Han

    2016-09-01

    To construct a cDNA phage expression library for human esophageal cancer cells. After the total RNA were obtained from esophageal cancer cells, the mRNA were separated with magnetic beads adsorption method, and the single-strand and double-strand cDNA were synthesized through reverse transcription. With the undesirable cDNA fragments removed, the remaining cDNA (linked with Eco R1 aptamer and phosphorylated its 5'end) combined with the carrier of T7 Select10-3b. The recombinant phage were packaged in vitro for preliminary cDNA library. PCR was used to identify the size of inserted cDNA. The constructed original cDNA phage expression library for human esophageal cancer cells was consisted of 2.01×10⁶ pfu/mL bacteriophages with a recombination rate of 100%. The length of the inserted cDNA fragments were range from 300 bp to 1 500 bp. The cDNA phage expression library of human esophageal cell is successfully constructed to meet the currently recognized standards, and can be well used to screen cDNA-cloned genes of human esophageal cancer antigens by serological analysis of recombinantly expressed cDNA clone (SEREX).

  2. Characterization of a cDNA encoding cottonseed catalase.

    Science.gov (United States)

    Ni, W; Turley, R B; Trelease, R N

    1990-06-21

    A 1.7 kb cDNA clone was isolated from our lambda gt11 library constructed from poly(A) RNA of 24-h-old cotyledons. The cDNA encodes a full-length catalase peptide (492 amino acid residues). The calculated molecular mass is 56,800, similar to that determined for purified enzyme (57,000 SDS-PAGE). Among higher plant catalases, this cotton catalase shows the highest amino acid sequence identity (85%) to the subunit of homotetrameric maize CAT 1, a developmental counterpart to the homotetrameric CAT A isoform of cotton seeds. Comparison of sequences from cotton, sweet potato, maize CAT 1, and yeast with bovine catalase revealed that the amino acid residues and regions that are involved in catalytic activity and/or required to maintain basic catalase structure, are highly conserved. The C-terminus region, which has the lowest nucleotide sequence identity between plant and mammalian catalases, does not terminate with a tripeptide, S-K/R/H-L, a putative targeting signal for peroxisomal proteins.

  3. Infectious Maize rayado fino virus from Cloned cDNA.

    Science.gov (United States)

    Edwards, Michael C; Weiland, John J; Todd, Jane; Stewart, Lucy R

    2015-06-01

    A full-length cDNA clone was produced from a U.S. isolate of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus within the family Tymoviridae. Infectivity of transcripts derived from cDNA clones was demonstrated by infection of maize plants and protoplasts, as well as by transmission via the known leafhopper vectors Dalbulus maidis and Graminella nigrifrons that transmit the virus in a persistent-propagative manner. Infection of maize plants through vascular puncture inoculation of seed with transcript RNA resulted in the induction of fine stipple stripe symptoms typical of those produced by wild-type MRFV and a frequency of infection comparable with that of the wild type. Northern and Western blotting confirmed the production of MRFV-specific RNAs and proteins in infected plants and protoplasts. An unanticipated increase in subgenomic RNA synthesis over levels in infected plants was observed in protoplasts infected with either wild-type or cloned virus. A conserved cleavage site motif previously demonstrated to function in both Oat blue dwarf virus capsid protein and tymoviral nonstructural protein processing was identified near the amino terminus of the MRFV replicase polyprotein, suggesting that cleavage at this site also may occur.

  4. Molecular Cloning and Functional Analysis of Squalene Synthase 2(SQS2 in Salvia miltiorrhiza Bunge

    Directory of Open Access Journals (Sweden)

    Qixian Rong

    2016-08-01

    Full Text Available Salvia miltiorrhiza Bunge,which is also known as a traditional Chinese herbal medicine,is widely studied for its ability to accumulate the diterpene quinone Tanshinones. In addition to producing a variety of diterpene quinone, S. miltiorrhiza Bunge also accumulates sterol, brassinosteroid and triterpenoids. During their biosynthesis, squalene synthase (SQS, EC 2.5.1.21 converts two molecules of the hydrophilic substrate farnesyl diphosphate into a hydrophobic product, squalene. In the present study, cloning and characterization of S. miltiorrhiza Bunge squalene synthase 2 (SmSQS2, Genbank Accession Number: KM408605 cDNA was investigated subsequently followed by its recombinant expression and preliminary enzyme activity. The full-length cDNA of SmSQS2 was 1 597 bp in length, with an open reading frame (ORF of 1 245 bp encoding 414 amino acids. The deduced amino acid sequence of SmSQS2 shared high similarity with those of SQSs from other plants. To obtain soluble recombinant enzymes, the truncated SmSQS2 in which 28 amino acids were deleted from the carboxy terminus was expressed as GST-Tag fusion protein in Escherichia coli BL21 (DE3 and confirmed by SDS-PAGE and Western Blot analysis, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. The gene expression level was analyzed through Quantitative real-time PCR, and was found to be higher in roots as compared to the leaves, and was up-regulated upon YE+ Ag+ treatment. These results could serve as an important to understand the function of the SQS family. In addition, the identification of SmSQS2 is important for further studies of terpenoid and sterol biosynthesis in S. miltiorrhiza Bunge.

  5. Molecular Cloning and Functional Analysis of Squalene Synthase 2(SQS2) in Salvia miltiorrhiza Bunge.

    Science.gov (United States)

    Rong, Qixian; Jiang, Dan; Chen, Yijun; Shen, Ye; Yuan, Qingjun; Lin, Huixin; Zha, Liangping; Zhang, Yan; Huang, Luqi

    2016-01-01

    Salvia miltiorrhiza Bunge, which is also known as a traditional Chinese herbal medicine, is widely studied for its ability to accumulate the diterpene quinone Tanshinones. In addition to producing a variety of diterpene quinone, S. miltiorrhiza Bunge also accumulates sterol, brassinosteroid and triterpenoids. During their biosynthesis, squalene synthase (SQS, EC 2.5.1.21) converts two molecules of the hydrophilic substrate farnesyl diphosphate (FPP) into a hydrophobic product, squalene. In the present study, cloning and characterization of S. miltiorrhiza Bunge squalene synthase 2 (SmSQS2, Genbank Accession Number: KM408605) cDNA was investigated subsequently followed by its recombinant expression and preliminary enzyme activity. The full-length cDNA of SmSQS2 was 1 597 bp in length, with an open reading frame of 1 245 bp encoding 414 amino acids. The deduced amino acid sequence of SmSQS2 shared high similarity with those of SQSs from other plants. To obtain soluble recombinant enzymes, the truncated SmSQS2 in which 28 amino acids were deleted from the carboxy terminus was expressed as GST-Tag fusion protein in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western Blot analysis, and the resultant bacterial crude extract was incubated with FPP and NADPH. Gas chromatograph-mass spectrometer analysis showed that squalene was detected in the in vitro reaction mixture. The gene expression level was analyzed through Quantitative real-time PCR, and was found to be higher in roots as compared to the leaves, and was up-regulated upon YE+ Ag(+) treatment. These results could serve as an important to understand the function of the SQS family. In addition, the identification of SmSQS2 is important for further studies of terpenoid and sterol biosynthesis in S. miltiorrhiza Bunge.

  6. Preparation of full-length cDNA libraries: focus on metazoans.

    Science.gov (United States)

    Harada, Masako; Hayashizaki, Yoshihide

    2009-01-01

    Critical steps in a cDNA library preparation include efficient cDNA synthesis, selection of full-length cDNAs, normalizing their abundance, and the subtraction of redundant transcripts. The use of trehalose and sorbiol stabilizes the activity of the reverse transcriptase leading to efficient cDNA synthesis and the cap-trapping method is used for efficient full-length cDNA selection. Through the incorporation of additional normalization and subtraction steps that eliminate the size bias and expressed gene frequency, it is possible to attain cDNA libraries that include larger or rarely expressed genes. This chapter describes an efficient method to construct a full-length cDNA library, with a focus on metazoan samples.

  7. Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis)

    Science.gov (United States)

    Zak, Megan A.; Regish, Amy M.; McCormick, Stephen; Manzon, Richard G.

    2017-01-01

    Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19 °C) or below (8 °C) the thermal optimum (13 °C) and exposure to exogenous thyroid hormone (60 µg T4/g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation.

  8. Solid-phase cDNA library construction, a versatile approach.

    OpenAIRE

    Roeder, T

    1998-01-01

    A rapid and versatile method for cDNA library construction was developed. It is based on conventional cDNA library synthesis including all enzymatic steps usually required, but is performed on a solid support. The cDNA is immobilised via a biotin residue to streptavidin coupled magnetic beads, which allows rapid and easy to perform changes of buffers and enzymes. Therefore, it combines speed (library construction within a single day) with high quality libraries, making it ideally suited for m...

  9. [Software development in data analysis and mining for cDNA microarray].

    Science.gov (United States)

    Wu, Bin; Wang, Jianguo; Wang, Miqu

    2007-12-01

    Data analysis and mining is a key issue to microarray technology and is usually implemented through software development. This paper summarizes the state-of-art software development in cDNA microarray data analysis and mining. The updated software developments are discussed in three stages: data inquisition from cDNA microarray tests, statistical treatment of cDNA data and data mining from gene network.

  10. Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits).

    Science.gov (United States)

    Borejsza-Wysocki, W.; Hrazdina, G.

    1996-03-01

    p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.

  11. Functional cloning using pFB retroviral cDNA expression libraries.

    Science.gov (United States)

    Felts, Katherine A; Chen, Keith; Zaharee, Kim; Sundar, Latha; Limjoco, Jamie; Miller, Anna; Vaillancourt, Peter

    2002-09-01

    Retroviral cDNA expression libraries allow the efficient introduction of complex cDNA libraries into virtually any mitotic cell type for screening based on gene function. The cDNA copy number per cell can be easily controlled by adjusting the multiplicity of infection, thus cell populations may be generated in which >90% of infected cells contain one to three cDNAs. We describe the isolation of two known oncogenes and one cell-surface receptor from a human Burkitt's lymphoma (Daudi) cDNA library inserted into the high-titer retroviral vector pFB.

  12. Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge

    Directory of Open Access Journals (Sweden)

    Wei Lei

    2010-01-01

    Full Text Available A cDNA encoding chalcone synthase (CHS, the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE. The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no. EU386767, was 1649 bp with a 1170 bp open reading frame (ORF that corresponded to a deduced protein of 390 amino acid residues, a calculated molecular mass of 42.56 kDa and a theoretical isoelectric point (pI of 5.79. Multiple sequence alignments showed that SvCHS shared high homology with CHS from other plants. Functional analysis in silico indicated that SvCHS was a hydrophilic protein most likely associated with intermediate metabolism. The active sites of the malonyl-CoA binding motif, coumaroyl pocket and cyclization pocket in CHS of Medicago sativa were also found in SvCHS. Molecular modeling indicated that the secondary structure of SvCHS contained mainly α-helixes and random coils. Phylogenetic analysis showed that SvCHS was most closely related to CHS from Scutellaria baicalensis. In agreement with its function as an elicitor-responsive gene, the expression of Svchs was induced and coordinated by methyl jasmonate. To our knowledge, this is the first report to describe the isolation and expression of a gene from S. viscidula.

  13. Saponin biosynthesis in Saponaria vaccaria. cDNAs encoding beta-amyrin synthase and a triterpene carboxylic acid glucosyltransferase.

    Science.gov (United States)

    Meesapyodsuk, Dauenpen; Balsevich, John; Reed, Darwin W; Covello, Patrick S

    2007-02-01

    Saponaria vaccaria (Caryophyllaceae), a soapwort, known in western Canada as cowcockle, contains bioactive oleanane-type saponins similar to those found in soapbark tree (Quillaja saponaria; Rosaceae). To improve our understanding of the biosynthesis of these saponins, a combined polymerase chain reaction and expressed sequence tag approach was taken to identify the genes involved. A cDNA encoding a beta-amyrin synthase (SvBS) was isolated by reverse transcription-polymerase chain reaction and characterized by expression in yeast (Saccharomyces cerevisiae). The SvBS gene is predominantly expressed in leaves. A S. vaccaria developing seed expressed sequence tag collection was developed and used for the isolation of a full-length cDNA bearing sequence similarity to ester-forming glycosyltransferases. The gene product of the cDNA, classified as UGT74M1, was expressed in Escherichia coli, purified, and identified as a triterpene carboxylic acid glucosyltransferase. UGT74M1 is expressed in roots and leaves and appears to be involved in monodesmoside biosynthesis in S. vaccaria.

  14. Heme synthesis in normal mouse liver and mouse liver tumors

    International Nuclear Information System (INIS)

    Stout, D.L.; Becker, F.F.

    1990-01-01

    Hepatic cancers from mice and rats demonstrate decreased levels of delta-aminolevulinic acid synthase, the rate-limiting enzyme in the heme synthetic pathway, and increased heme oxygenase, the heme-catabolizing enzyme. These findings suggest that diminution of P-450, b5, and catalase in these lesions may result from a heme supply that is limited by decreased heme synthesis and increased heme catabolism. Heme synthesis was measured in mouse liver tumors (MLT) and adjacent tumor-free lobes (BKG) by administering the radiolabeled heme precursors 55 FeCl3 and [2- 14 C]glycine and subsequently extracting the heme for determination of specific activity. Despite reduced delta-aminolevulinic acid synthase activity in MLT, both tissues incorporated [2-14C]glycine into heme at similar rates. At early time points, heme extracted from MLT contained less 55Fe than that from BKG. This was attributed to the findings that MLT took up 55Fe at a slower rate than BKG and had larger iron stores than BKG. The amount of heme per milligram of protein was also similar in both tissues. These findings militate against the hypothesis that diminished hemoprotein levels in MLT result from limited availability of heme. It is probable, therefore, that decreased hemoprotein levels in hepatic tumors are linked to a general program of dedifferentiation associated with the cancer phenotype. Diminution of hemoprotein in MLT may result in a relatively increased intracellular heme pool. delta-Aminolevulinic acid synthase and heme oxygenase are, respectively, negatively and positively regulated by heme. Thus, their alteration in MLT may be due to the regulatory influences of the heme pool

  15. Functional identification of a Lippia dulcis bornyl diphosphate synthase that contains a duplicated, inhibitory arginine-rich motif.

    Science.gov (United States)

    Hurd, Matthew C; Kwon, Moonhyuk; Ro, Dae-Kyun

    2017-08-26

    Lippia dulcis (Aztec sweet herb) contains the potent natural sweetener hernandulcin, a sesquiterpene ketone found in the leaves and flowers. Utilizing the leaves for agricultural application is challenging due to the presence of the bitter-tasting and toxic monoterpene, camphor. To unlock the commercial potential of L. dulcis leaves, the first step of camphor biosynthesis by a bornyl diphosphate synthase needs to be elucidated. Two putative monoterpene synthases (LdTPS3 and LdTPS9) were isolated from L. dulcis leaf cDNA. To elucidate their catalytic functions, E. coli-produced recombinant enzymes with truncations of their chloroplast transit peptides were assayed with geranyl diphosphate (GPP). In vitro enzyme assays showed that LdTPS3 encodes bornyl diphosphate synthase (thus named LdBPPS) while LdTPS9 encodes linalool synthase. Interestingly, the N-terminus of LdBPPS possesses two arginine-rich (RRX 8 W) motifs, and enzyme assays showed that the presence of both RRX 8 W motifs completely inhibits the catalytic activity of LdBPPS. Only after the removal of the putative chloroplast transit peptide and the first RRX 8 W, LdBPPS could react with GPP to produce bornyl diphosphate. LdBPPS is distantly related to the known bornyl diphosphate synthase from sage in a phylogenetic analysis, indicating a converged evolution of camphor biosynthesis in sage and L. dulcis. The discovery of LdBPPS opens up the possibility of engineering L. dulcis to remove the undesirable product, camphor. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Molecular characterization of a transient expression gene encoding for 1-aminocyclopropane-1-carboxylate synthase in cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    Wang, Xia; Zhang, Ying; Zhang, Jiedao; Cheng, Cheng; Guo, Xingqi

    2007-09-30

    Ethylene performs an important function in plant growth and development. 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), the key enzyme involved in ethylene biosynthesis, has been the focus of most ethylene studies. Here, a cotton ACS gene referred to as Gossypium hirsutum ACS1 (GhACS1), was isolated. The full-length cDNA of GhACS1 encodes for a 476-amino acid protein which harbors seven conserved regions, 11 invariant amino acid residues, and the PLP binding active site, all of which characterize ACC synthases. Alignment analysis showed that GhACS1 shared a high degree of identity with other known ACC synthases from different species. Two introns were detected in the genomic DNA sequence, and the results of Southern blot analysis suggested that there might be a multi-gene family encoding for ACC synthase in cotton. From the phylogenetic tree constructed with 24 different kinds of ACC synthases, we determined that GhACS1 falls into group II, and was closely associated with the wound-inducible ACS of citrus. The analysis of the 5' flanking region of GhACS1 revealed a group of putative cis-acting elements. The results of expression analysis showed that GhACS1 displayed its transient expression nature after wounding, abscisic acid (ABA), and CuCl(2) treatments. These results indicate that GhACS1, which was transiently expressed in response to certain stimuli, may be involved in the production of ethylene for the transmission of stress signals.

  17. Alleviation of Plasma Homocysteine Level by Phytoestrogen α-Zearalanol Might Be Related to the Reduction of Cystathionine β-Synthase Nitration

    Directory of Open Access Journals (Sweden)

    Hui Zhang

    2014-01-01

    Full Text Available Hyperhomocysteinemia is strongly associated with cardiovascular diseases. Previous studies have shown that phytoestrogen α-zearalanol can protect cardiovascular system from hyperhomocysteinemia and ameliorate the level of plasma total homocysteine; however, the underlying mechanisms remain to be clarified. The aim of this research is to investigate the possible molecular mechanisms involved in ameliorating the level of plasma homocysteine by α-zearalanol. By the successfully established diet-induced hyperhomocysteinemia rat models, we found that, after α-zearalanol treatment, the activity of cystathionine β-synthase, the key enzyme in homocysteine metabolism, was significantly elevated and level of nitrative stress in liver was significantly reduced. In correlation with this, results also showed a decreased nitration level of cystathionine β-synthase in liver. Together data implied that alleviation of plasma homocysteine level by phytoestrogen α-zearalanol might be related to the reduction of cystathionine β-synthase nitration.

  18. Expression analysis of a ''Cucurbita'' cDNA encoding endonuclease

    International Nuclear Information System (INIS)

    Szopa, J.

    1995-01-01

    The nuclear matrices of plant cell nuclei display intrinsic nuclease activity which consists in nicking supercoiled DNA. A cDNA encoding a 32 kDa endonuclease has been cloned and sequenced. The nucleotide and deduced amino-acid sequences show high homology to known 14-3-3-protein sequences from other sources. The amino-acid sequence shows agreement with consensus sequences for potential phosphorylation by protein kinase A and C and for calcium, lipid and membrane-binding sites. The nucleotide-binding site is also present within the conserved part of the sequence. By Northern blot analysis, the differential expression of the corresponding mRNA was detected; it was the strongest in sink tissues. The endonuclease activity found on DNA-polyacrylamide gel electrophoresis coincided with mRNA content and was the highest in tuber. (author). 22 refs, 6 figs

  19. Elevated Liver Enzymes

    Science.gov (United States)

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  20. Liver cancer - hepatocellular carcinoma

    Science.gov (United States)

    Primary liver cell carcinoma; Tumor - liver; Cancer - liver; Hepatoma ... Hepatocellular carcinoma accounts for most liver cancers. This type of cancer occurs more often in men than women. It is usually diagnosed in people age 50 or ...

  1. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...

  2. Activation of anthocyanin synthesis genes by white light in eggplant hypocotyl tissues, and identification of an inducible P-450 cDNA

    International Nuclear Information System (INIS)

    Toguri, T.; Umemoto, N.; Kobayashi, O.; Ohtani, T.

    1993-01-01

    Eggplant seedlings (Solanum melongena) grown under red light irradiation showed a normal morphology with green, fully expanded cotyledons. When the seedlings grown under red light were irradiated with ultraviolet-containing white light, anthocyanin synthesis was induced in the hypocotyl tissues, especially when a UV light supplement was added. The accumulation of pigments was closely associated with the expression of genes involved in flavonoid synthesis. These genes include chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR). Using subtracted probes, which had been enriched for the accumulated mRNA, one white light-responsive cDNA was identified as being a P450 gene by comparison with database sequences. The maximal amino acid homology this cDNA had with other P450s was 36%. This was with CYP71 from avocado (Persea americana). Thus it represents a new P-450 family, which has been named CYP75. The mRNA of this gene was localized in the hypocotyl tissues of eggplant seedlings, which had been white light-irradiated. The transcript was accumulated by changing the light source, as in the case of other flavonoid biosynthesis genes. In delphinidin producing petunia plants, the mRNAs corresponding to the eggplant P-450 and flavonoid biosynthesis genes such as CHS and DFR were most abundant during the mid stage of flower bud development, but could not be detected in leaf tissues. These results suggest that this P-450 gene encodes a hydroxylating enzyme involved in flavonoid biosynthesis. (author)

  3. Characterization of Squalene synthase gene from Chlorophytum borivilianum (Sant. and Fernand.).

    Science.gov (United States)

    Kalra, Shikha; Kumar, Sunil; Lakhanpal, Neha; Kaur, Jagdeep; Singh, Kashmir

    2013-07-01

    Saponins are important group of secondary metabolites known for their pharmacological properties. Chlorophytum borivilianum contains high amount of saponins and is thus, recognized as an important medicinal plant with aphrodisiac properties. Though the plant is well known for its pharmaceutical properties, there is meager information available about the genes and enzymes responsible for biosynthesis of saponins from this plant. Squalene synthase (SqS) is the key enzyme of saponin biosynthesis pathway and here, we report cloning and characterization of SqS gene from C. borivilianum. A full-length CbSqS cDNA consisting of 1,760 bp was cloned which contained an open reading frame (ORF) of 1,233 bp, encoding a protein of 411 amino acids. Analysis of deduced amino acid sequence of CbSqS predicted the presence of conserved isoprenoid family domain and catalytic sites. Phylogenetic analysis revealed that CbSqS is closer to Glycine max and monocotyledonous plants. 3D structure prediction using various programs showed CbSqS structure to be similar to SqS from other species. C-terminus truncated recombinant squalene synthase (TruncCbSqS) was expressed in E. coli M15 cells with optimum expression induced with 1 mM IPTG at 37 °C. The gene expression level was analyzed through semi-quantitative RT-PCR and was found to be higher in leaves as compared to the roots.

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

    International Nuclear Information System (INIS)

    Han, J.H.; Stratowa, C.; Rutter, W.J.

    1987-01-01

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

  5. Molecular cloning of a cDNA and chromosomal localization of a human theta-class glutathione S-transferase gene (GSTT2) to chromosome 22

    Energy Technology Data Exchange (ETDEWEB)

    Tan, K.L.; Baker, R.T.; Board, P.G. [Australian National Univ., Canberra (Australia)] [and others

    1995-01-20

    Until recently the Theta-class glutathione S-transferases (GSTs) were largely overlooked due to their low activity with the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) and their failure to bind to immobilized glutathione affinity matrices. Little is known about the number of genes in this class. Recently, Pemble et al. reported the cDNA cloning of a human Theta-class GST, termed GSTT1. In this study, we describe the molecular cloning of a cDNA encoding a second human Theta-class GST (GSTT2) from a {lambda}gt11 human liver 5{prime}-stretch cDNA library. The encoded protein contains 244 amino acids and has 78.3% sequence identity with the rat subunit 12 and only 55.0% identity with human GSTT1. GSTT2 has been mapped to chromosome 22 by somatic cell hybrid analysis. The precise position of the gene was localized to subband 22q11.2 by in situ hybridization. The absence of other regions of hybridization suggests that there are no closely related sequences (e.g., reverse transcribed pseudogenes) scattered throughout the genome and that if there are closely related genes, they must be clustered near GSTT2. Southern blot analysis of human DNA digested with BamHI shows that the size of the GSTT2 gene is relatively small, as the coding sequence falls within a 3.6-kb BamHI fragment. 35 refs., 6 figs.

  6. Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments

    Directory of Open Access Journals (Sweden)

    Cushman Mark

    2003-12-01

    Full Text Available Abstract Background Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs. Results A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20°C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg-1 min-1 with an apparent KM of 5.7 microM. 19F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. 19F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins. Conclusion Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H-pyrimidinedione. The 19F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important

  7. Acute Liver Failure

    Science.gov (United States)

    ... begins in or spreads to your liver can cause your liver to fail. Shock. Overwhelming infection (sepsis) and shock can severely impair blood flow to the liver, causing liver failure. Many cases of acute liver failure have no apparent ... liver failure often causes complications, including: ...

  8. Expression, purification, and characterization of recombinant amorpha-4,11-diene synthase from Artemisia annua L.

    Science.gov (United States)

    Picaud, Sarah; Olofsson, Linda; Brodelius, Maria; Brodelius, Peter E

    2005-04-15

    A cDNA clone encoding amorpha-4,11-diene synthase from Artemisia annua was subcloned into a bacterial expression vector in frame with a His6-tag. Recombinant amorpha-4,11-diene synthase was produced in Escherichia coli and purified to apparent homogeneity. The enzyme showed pH optimum at pH 6.5, and a minimum at pH 7.5. Substantial activity was observed in the presence of Mg2+, Mn2+ or Co2+ as cofactor. The enzyme exhibits a low activity in the presence of Ni2+ and essentially no activity with Cu2+ or Zn2+. The sesquiterpenoids produced from farnesyl diphosphate in the presence of Mg2+ were analyzed by GC-MS. In addition to amorpha-4,11-diene, 15 sesquiterpenoids were produced. Only small quantitative differences in product pattern were observed at pH 6.5, 7.5, or 9.5. Amorpha-4,11-diene synthase showed significant increased product selectivity in the presence of Mn2+ or Co2+. Km for farnesyl diphosphate was 3.3, 8.0, and 0.7 microM in the presence of Mg2+, Mn2+ or Co2+, respectively. The corresponding kcat-values were 6.8, 15.0, and 1.3 x 10(-3) s(-1), respectively. Km and kcat for geranyl diphosphate were 16.9 microM and 7.0 x 10(-4) s(-1), respectively, at pH 6.5, in the presence of Mn2+.

  9. A Copal-8-ol Diphosphate Synthase from the Angiosperm Cistus creticus subsp. creticus Is a Putative Key Enzyme for the Formation of Pharmacologically Active, Oxygen-Containing Labdane-Type Diterpenes1[OA

    Science.gov (United States)

    Falara, Vasiliki; Pichersky, Eran; Kanellis, Angelos K.

    2010-01-01

    The resin of Cistus creticus subsp. creticus, a plant native to Crete, is rich in labdane-type diterpenes with significant antimicrobial and cytotoxic activities. The full-length cDNA of a putative diterpene synthase was isolated from a C. creticus trichome cDNA library. The deduced amino acid sequence of this protein is highly similar (59%–70% identical) to type B diterpene synthases from other angiosperm species that catalyze a protonation-initiated cyclization. The affinity-purified recombinant Escherichia coli-expressed protein used geranylgeranyl diphosphate as substrate and catalyzed the formation of copal-8-ol diphosphate. This diterpene synthase, therefore, was named CcCLS (for C. creticus copal-8-ol diphosphate synthase). Copal-8-ol diphosphate is likely to be an intermediate in the biosynthesis of the oxygen-containing labdane-type diterpenes that are abundant in the resin of this plant. RNA gel-blot analysis revealed that CcCLS is preferentially expressed in the trichomes, with higher transcript levels found in glands on young leaves than on fully expanded leaves, while CcCLS transcript levels increased after mechanical wounding. Chemical analyses revealed that labdane-type diterpene production followed a similar pattern, with higher concentrations in trichomes of young leaves and increased accumulation upon wounding. PMID:20595348

  10. Construction and analysis of a cDNA library from yellow-fruit ginseng

    African Journals Online (AJOL)

    The total RNA was isolated from yellow-fruit ginseng (Panax ginseng C.A. Meyer) leaf tissue. A cDNA library of panax ginseng leaves was constructed by using pDNR-LIB vector according to the SMART cDNA library construction kit protocol. We obtained 378 high quality sequences (GenBank accession number: ...

  11. cDNA table - RPD | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data ...ile URL: ftp://ftp.biosciencedbc.jp/archive/rpd/LATEST/rpd_cdna.zip File size: 15 KB Simple search URL http:...age About This Database Database Description Download License Update History of This Database Site Policy | Contact Us cDNA table - RPD | LSDB Archive ...

  12. [Construction of cDNA library of Magnaporthe grisea with magnetic bead].

    Science.gov (United States)

    Feng, Xu; Xiaoli, Wu; Dewen, Qiu

    2008-06-01

    We constructed cDNA library of Magnaporthe grisea. The good quality cDNA library could facilitate finding proteinaceous elicitors of M. grisea, and elucidating the mechanisms of the M. grisea--rice interaction. The Oligo(dT) combined with the magnetic bead was used to extract mRNA from total RNA of Magnaporthe grisea and as primers to synthesize the first-strand cDNA. Terminal transferase introduced PolyA into 3'terminal of the first cDNA strand, then the PolyA was used for amplifying the second-strand cDNA. Restriction enzyme and adapter were avoided in this research, which could solve technical limitation of the traditional method. Because all reactions were done in one centrifuge tube, this process could reduce the risk of cDNA loss and cross-contamination. The primers designed in this research could clone the amplified cDNAs into expression vector in a desirable orientation. The cDNA library constructed had a high titer of 8.9 x 10(6) cfu/mL, and contained a total clones of 8.9 x 10(7) cfu, with an average inserts size of about 1380 bp. Constructing cDNA library with magnetic bead was a highly efficient method using only small amount of experimental materials within a short period.

  13. Development of a simple and powerful method, cDNA AFLP-SSPAG ...

    African Journals Online (AJOL)

    Differential cDNAs were easily obtained from silver stained cDNA-AFLP separated on polyacylamide gels. The cDNA was then reamplified, cloned and fragments were sequenced. Sequenced clones were used as probes in northern dot blot analyses and library screening. Full-length cDNA was cloned from a library ...

  14. Genomic and cDNA cloning of a novel mouse lipoxygenase gene

    NARCIS (Netherlands)

    Willems van Dijk, K.; Steketee, K.; Havekes, L.; Frants, R.; Hofker, M.

    1995-01-01

    A novel 12- and 15-lipoxygenase related gene was isolated from a mouse strain 129 genomic phage library in a screen with a human 15-lipoxygenase cDNA probe. The complete genomic sequence revealed 14 exons and 13 introns covering 7.3 kb of DNA. The splice junctions were verified from the cDNA

  15. Construction of full-length cDNA library of white flower Salvia ...

    African Journals Online (AJOL)

    In order to screen and isolate secondary metabolite biosynthesis related gene, we construct a cDNA library of white flower Salvia miltiorrhiza bge. f.alba. High quality of total RNA was successfully isolated from roots of white flower S. miltiorrhiza using modified CTAB method. Double strand cDNA was cloned into pDNR-LIB ...

  16. Cloning and sequence analysis of H. contortus HC58cDNA gene ...

    African Journals Online (AJOL)

    Phylogenetic analysis revealed close evolutionary proximity of the protein sequence to counterpart sequences in the cathepsin B like proteases, suggesting that HC58cDNA was a member of the papain family. Keywords:Haemonchus contortus, HC58cDNA, cathepsin B like protease, papain family. Kenya Veterinarian Vol.

  17. [A novel vector for construction of a cDNA library].

    Science.gov (United States)

    Fedchenko, V I; Kaloshin, A A; Medvedev, A E

    2010-01-01

    A new original vector pEM-(dT)40(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The pGEM-(dT)40f(+) is initially transformed into single stranded and then into a linear form and its (dT)40 tail at 3'-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector cyclization and synthesis of the second strand cDNA. This approach significantly simplifies cDNA library construction, it does not require PCR reaction (which can induce artifact mutations in cDNA sequences) and restrictase treatment.

  18. Class II recombinant phosphoribosyl diphosphate synthase from spinach

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows...

  19. Evolutionary and mechanistic insights from the reconstruction of (+)-humulene synthases from a modern (+)-Germacrene A Synthase

    OpenAIRE

    Gonzalez Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J.; Faraldos, Juan A.; Allemann, Rudolf Konrad

    2014-01-01

    Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and

  20. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    Endothelial nitric oxide synthase (NOS3) is involved in key steps of immune response. Genetic factors predispose individuals to periodontal disease. This study's aim was to explore the association between NOS3 gene polymorphisms and clinical parameters in patients with periodontal disease. Genomic DNA was obtained ...

  1. Glutamate synthase: An archaeal horizontal gene transfer?

    Indian Academy of Sciences (India)

    (GOGAT) which is a key enzyme in ammonia assimilation in bacteria, algae and plants. It catalyzes the reductive transamidation of amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate (Temple et al 1998). Glutamate synthases differ according to their molecular weights, subunit compositions, ...

  2. Relationship between endothelial nitric oxide synthase gene ...

    African Journals Online (AJOL)

    Introduction: Endothelial nitric oxide synthase (eNOS), the enzyme in charge of nitric oxide production, plays a crucial role in vascular biology. However, the impact of single nucleotide polymorphisms (SNPs) affecting the gene encoding for eNOS (eNOS) on coronary artery diseases remains under debate and no data were ...

  3. Producing alpha-olefins using polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Katz, Leonard; Steen, Eric J.; Keasling, Jay D.

    2018-01-02

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an .alpha.-olefin, such as 1-hexene or butadiene. The present invention also provides for a host cell comprising the PKS and when cultured produces the .alpha.-olefin.

  4. Expression of Deinococcus geothermalis trehalose synthase gene ...

    African Journals Online (AJOL)

    A novel trehalose synthase gene from Deinococcus geothermalis (DSMZ 11300) containing 1692 bp reading-frame encoding 564 amino acids was amplified using polymerase chain reaction (PCR). The gene was ligated into pET30Ek/LIC vector and expressed after isopropyl β-D-thiogalactopyranoside induction in ...

  5. Cloning and expression of pineapple sucrosephosphate synthase ...

    African Journals Online (AJOL)

    A 1132-base pairs (bp) polymerase-chain-reaction product of sucrose-phosphate synthase (SPS) (EC 2.3.1.14) from pineapple (Ananas comosus cv. Comte de paris) fruit was cloned and nominated as Ac- SPS1. The sequence encodes a putative 377 amino acids protein containing two serine conserved features that had ...

  6. High-efficiency system for the construction of adenovirus vectors and its application to the generation of representative adenovirus-based cDNA expression libraries.

    Science.gov (United States)

    Hillgenberg, Moritz; Hofmann, Christian; Stadler, Herbert; Löser, Peter

    2006-06-01

    We here describe a convenient system for the production of recombinant adenovirus vectors and its use for the construction of a representative adenovirus-based cDNA expression library. The system is based on direct site-specific insertion of transgene cassettes into a replicating donor virus. The transgene is inserted into a donor plasmid containing the viral 5' inverted terminal repeat, the complete viral packaging signal, and a single loxP site. The plasmid is then transfected into a Cre recombinase-expressing packaging cell line that has been infected with a donor virus containing a partially deleted packaging signal flanked by loxP sites. Cre recombinase, by two steps of action, sequentially catalyzes the generation of a nonpackageable donor virus acceptor substrate and the generation of the desired recombinant adenovirus vector. Due to its growth impairment, residual donor virus can efficiently be counterselected during amplification of the recombinant adenovirus vector. By using this adenovirus construction system, a plasmid-based human liver cDNA library was converted by a single step into an adenovirus-based cDNA expression library with about 10(6) independent adenovirus clones. The high-titer purified library was shown to contain about 44% of full-length cDNAs with an average insert size of 1.3 kb. cDNAs of a gene expressed at a high level (human alpha(1)-antitrypsin) and a gene expressed at a relatively low level (human coagulation factor IX) in human liver were isolated from the adenovirus-based library using an enzyme-linked immunosorbent assay-based screening procedure.

  7. Construction and characterization of a normalized cDNA library.

    Science.gov (United States)

    Soares, M B; Bonaldo, M F; Jelene, P; Su, L; Lawton, L; Efstratiadis, A

    1994-09-27

    We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low C0t, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.

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

    OpenAIRE

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

    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.

  9. Influence of DNA-Microbubble Coupling on Contrast Ultrasound-Mediated Gene Transfection in Muscle and Liver.

    Science.gov (United States)

    Xie, Aris; Wu, Melinda D; Cigarroa, Gabriella; Belcik, J Todd; Ammi, Azzdine; Moccetti, Federico; Lindner, Jonathan R

    2016-08-01

    Contrast ultrasound-mediated gene delivery (CUMGD) is a promising approach for enhancing gene therapy that relies on microbubble (MB) cavitation to augment complementary deoxyribonucleic acid (cDNA) transfection. The aims of this study were to determine optimal conditions for charge-coupling cDNA to MBs and to evaluate the advantages of surface loading for gene transfection in muscle and liver. Charge coupling of fluorescently labeled cDNA to either neutral MBs (MBN) or cationic MBs (MB+) in low- to high-ionic conditions (0.3%-1.8% NaCl) was assessed by flow cytometry. MB aggregation from cDNA coupling was determined by electrozone sensing. Tissue transfection of luciferase in murine hindlimb skeletal muscle and liver was made by CUMGD with MBN or MB+ combined with subsaturated, saturated, or supersaturated cDNA concentrations (2.5, 50, and 200 μg/10(8) MBs). Charge-coupling of cDNA was detected for MB+ but not MBN. Coupling occurred over almost the entire range of ionic conditions, with a peak at 1.2% NaCl, although electrostatic interference occurred at >1.5% NaCl. DNA-mediated aggregation of MB+ was observed at ≤0.6% NaCl but did not reduce the ability to produce inertial cavitation. Transfection with CUMGD in muscle and liver was low for both MBs at subsaturation concentrations. In muscle, higher cDNA concentrations produced a 10-fold higher degree of transfection with MB+, which was approximately fivefold higher (P transfection with MBN equal to that of MB+. Efficient charge-coupling of cDNA to MB+ but not MBN occurs over a relatively wide range of ionic conditions without aggregation. Transfection with CUMGD is much more efficient with charge-coupling of cDNA to MBs and is not affected by supersaturation except in the liver, which is specialized for macromolecular and cDNA uptake. Copyright © 2016 American Society of Echocardiography. Published by Elsevier Inc. All rights reserved.

  10. 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.

  11. Suppressive Role of PPARγ-Regulated Endothelial Nitric Oxide Synthase in Adipocyte Lipolysis.

    Directory of Open Access Journals (Sweden)

    Yoko Yamada

    Full Text Available Metabolic syndrome causes insulin resistance and is associated with risk factor clustering, thereby increasing the risk of atherosclerosis. Recently, endothelial nitric oxide synthase deficient (eNOS-/- mice have been reported to show metabolic disorders. Interestingly, eNOS has also been reported to be expressed in non-endothelial cells including adipocytes, but the functions of eNOS in adipocytes remain unclear.The eNOS expression was induced with adipocyte differentiation and inhibition of eNOS/NO enhanced lipolysis in vitro and in vivo. Furthermore, the administration of a high fat diet (HFD was able to induce non-alcoholic steatohepatitis (NASH in eNOS-/- mice but not in wild type mice. A PPARγ antagonist increased eNOS expression in adipocytes and suppressed HFD-induced fatty liver changes.eNOS-/- mice induce NASH development, and these findings provide new insights into the therapeutic approach for fatty liver disease and related disorders.

  12. [cDNA libraries construction and screening in gene expression profiling of disease resistance in wheat].

    Science.gov (United States)

    Luo, Meng; Kong, Xiu-Ying; Liu, Yue; Zhou, Rong-Hua; Jia, Ji-Zeng

    2002-09-01

    A wheat line, Bai Nong 3217/Mardler BC5F4 with resistance to powdery mildew, was used to construct a conventional cDNA library and a suppression subtractive hybridization (SSH) cDNA library from wheat leaves inoculated by Erysiphe graminis DC. Three hundred and eighty-seven non-redundant ESTs from the conventional cDNA library and 760 ESTs from the SSH cDNA library were obtained, and the ESTs similarity analysis using BLASTn and BLASTx were conducted by comparing these ESTs with sequences in GenBank. The results showed that the redundancy of some kinds of genes such as photosynthesis related genes and ribosome related genes was higher in the conventional cDNA library but the varieties and quantities of disease resistance genes were less than those in the SSH cDNA library. The SSH cDNA library was found to have obvious advantages in gene expression profiling of disease resistance such as simple library construction procedure, rich specific DRR (disease-resistance-related) genes and decreased sequencing amount. To acquire genes that were involved in the powdery mildew resistance of wheat, hybridization with high-density dots membranes was used to screen the two libraries. The result showed that the method was relatively simple in operation, and the membranes could be used for many times. But some problems also existed with this screening method. For instance, a large amount of mRNA and radioactive isotope were needed and the hybridization procedure must be repeated several times to obtain stable hybridization results. About 54.1% function-known ESTs in the SSH cDNA library were identified to be DRR genes by screening. There were 247 clones of the SSH cDNA library that had positive signal in the repeated hybridizations with the pathogen uninfected probe. The identified DRR genes distributed in the whole procedure of powdery mildew resistance, but mainly focused on the SAR (systemic of acquired resistance).

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

    International Nuclear Information System (INIS)

    Spicer, E.K.; Horton, R.; Bloem, L.

    1987-01-01

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

  14. Cloning of human purine-nucleoside phosphorylase cDNA sequences by complementation in Escherichia coli.

    OpenAIRE

    Goddard, J M; Caput, D; Williams, S R; Martin, D W

    1983-01-01

    We have obtained cDNA clones that contain the entire coding region of the human purine-nucleoside phosphorylase (PNP; EC 2.4.2.1) mRNA. The cDNA sequences were generated by reverse transcription of PNP-enriched mRNA obtained by immunoadsorption of HeLa cell polyribosomes with monospecific antibody to human PNP. cDNA molecules that were close in length to PNP mRNA were separated by agarose gel electrophoresis and inserted into the Pst I site of the plasmid pBR322. Plasmid DNA from the pooled c...

  15. Generation of cDNA expression libraries enriched for in-frame sequences

    OpenAIRE

    Davis, Claytus A.; Benzer, Seymour

    1997-01-01

    Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore deve...

  16. Generation of full-length cDNA libraries: focus on plants.

    Science.gov (United States)

    Seki, Motoaki; Kamiya, Asako; Carninci, Piero; Hayashizaki, Yoshihide; Shinozaki, Kazuo

    2009-01-01

    Full-length cDNAs are essential for the correct annotation of transcriptional units and gene products from genomic sequence data and for functional analysis of the genes. Full-length cDNA libraries are very important resources for isolation of the full-length cDNAs. The biotinylated cap trapper method using the trehalose-thermostabilized reverse transcriptase has been developed and has become an efficient method for construction of high-content full-length cDNA libraries. We have constructed full-length cDNA libraries from various plants and animals using this method. The protocol of the method is described in this chapter.

  17. 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.

  18. 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 List Contact us 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...or-capping method, the sequence quality score generated by the Phred software, and links to SGD, dbEST and U...es. FASTA format. Quality Phred's quality score About This Database Database Desc...g yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive ...

  19. Cloning, expression, and characterization of epi-cedrol synthase, a sesquiterpene cyclase from Artemisia annua L.

    Science.gov (United States)

    Mercke, P; Crock, J; Croteau, R; Brodelius, P E

    1999-09-15

    Sesquiterpene cyclases (synthases) catalyze the conversion of the isoprenoid intermediate farnesyl diphosphate to various sesquiterpene structural types. In plants, many sesquiterpenes are produced as defensive chemicals (phytoalexins) or mediators of chemical communication (i.e., pollinator attractants). A number of sesquiterpene synthases are present in Artemisia annua L. (annual wormwood). We have isolated a cDNA clone encoding one of these, epi-cedrol synthase. This clone contains a 1641-bp open reading frame coding for 547 amino acids (63.5 kDa), a 38-bp 5'-untranslated end, and a 272-bp 3'-untranslated sequence. The deduced amino acid sequence was 32 to 43% identical with the sequences of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (3%) and oxygenated (97%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as alpha-cedrene (57% of the olefins), beta-cedrene (13%), (E)-beta-farnesene (5%), alpha-acoradiene (1%), (E)-alpha-bisabolene (8%), and three unknown olefins (16%) and the oxygenated sesquiterpenes (97% of total sesquiterpene generated, exclusive of farnesol and nerolidol) as cedrol (4%) and epi-cedrol (96%). epi-Cedrol synthase was not active with geranylgeranyl diphosphate as substrate, whereas geranyl diphosphate was converted to monoterpenes by the recombinant enzyme at a rate of about 15% of that observed with farnesyl diphosphate as substrate. The monoterpene olefin products are limonene (45%), terpinolene (42%), gamma-terpinene (8%), myrcene (5%), and alpha-terpinene (2%); a small amount of the monoterpene alcohol terpinen-4-ol is also produced. The pH optimum for the recombinant enzyme is 8.5-9.0 (with farnesyl diphosphate as substrate) and the K(m) values for farnesyl diphosphate are 0.4 and 1.3 microM at pH 7. 0 and 9.0, respectively. The K(m) for Mg(2+) is 80 microM at pH 7.0 and 9.0.

  20. Effect of tenoxicam on rat liver tissue.

    Science.gov (United States)

    Karatopuk, D Ulusoy; Gökçımen, Alpaslan

    2010-06-01

    Tenoxicam is a non-steroidal antiinflammatory drug, which has antipyretic and antiinflammatory effects. Though it is known that the major side effect of non-steroidal antiinflammatory drugs is on the gastrointestinal tract and liver, there have been few studies regarding the effects of tenoxicam. In this study, we aimed to investigate whether tenoxicam has a deleterious effect on liver tissue using immunohistochemical staining and biochemical analysis. A total of 30 male Wistar albino rats were included in this study. Animals were equally and randomly divided into three groups as follows: Group I (Controls), Group II (Injection with 10 mg/kg/day of tenoxicam) and Group III (Injection with 20 mg/kg/day of tenoxicam). At the end of the study, some liver tissue samples were taken and kept in neutral formalin for histological and immunohistochemical evaluation. Liver tissue samples were embedded in paraffin blocks after routine tissue preparation procedures, and were stained with hematoxylin-eosin and immunohistochemical stain. Liver samples taken for biochemical analysis were washed with physiological saline. Thiobarbituric acid reactive substances and superoxide dismutase activity were measured in the obtained supernatants. There were significant structural changes in liver tissues of the tenoxicam-administered groups when compared with the controls. We observed that hepatic (inducible nitric oxide synthase) receptors were increased in the study groups. Furthermore, hepatic superoxide dismutase and malondialdehyde levels were prominently higher in the tenoxicam-administered groups when compared to levels of the control group. Nitric oxide may exert an antioxidative effect against lipid peroxidation to one point at low levels; however, it may also have the opposite effect at higher levels in tenoxicam induced liver injury.

  1. Engineering of plant type III polyketide synthases.

    Science.gov (United States)

    Wakimoto, Toshiyuki; Morita, Hiroyuki; Abe, Ikuro

    2012-01-01

    Members of the chalcone synthase superfamily of type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkable structural diversity and biological activities. The homodimeric type III PKSs share a common three-dimensional overall fold with a conserved Cys-His-Asn catalytic triad; notably, only a slight modification of the active site dramatically expands the catalytic repertoire of the enzymes. In addition, the enzymes exhibit extremely promiscuous substrate specificities, and accept a variety of nonphysiological substrates, making the type III PKSs an excellent platform for the further production of unnatural, novel polyketide scaffolds with promising biological activities. This chapter summarizes recent advances in the engineering of plant type III PKS enzymes in our laboratories, using approaches combining structure-based enzyme engineering and precursor-directed biosynthesis with rationally designed substrate analogs. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Liver transplant - slideshow

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/presentations/100090.htm Liver transplant - series—Normal anatomy To use the sharing ... to slide 5 out of 5 Overview The liver is in the right upper abdomen. The liver ...

  3. Pyogenic liver abscess

    Science.gov (United States)

    Liver abscess; Bacterial liver abscess ... There are many possible causes of liver abscesses, including: Abdominal infection, such as appendicitis , diverticulitis , or a perforated bowel Infection in the blood Infection of the bile draining tubes ...

  4. Fatty Liver Disease

    Science.gov (United States)

    What is fatty liver disease? Your liver is the largest organ inside your body. It helps your body digest food, store energy, and remove poisons. Fatty liver disease is a condition in which fat builds up ...

  5. Engineering cotton (+)-delta-cadinene synthase to an altered function: germacrene D-4-ol synthase.

    Science.gov (United States)

    Yoshikuni, Yasuo; Martin, Vincent J J; Ferrin, Thomas E; Keasling, Jay D

    2006-01-01

    The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-delta-cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-delta-cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-delta-cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-delta-cadinene synthase suggested that the G helix plays a very important role in (+)-delta-cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).

  6. Chalcone synthase genes from milk thistle (Silybum marianum ...

    Indian Academy of Sciences (India)

    Analysis of the resultant nucleotide and deduced amino acid sequences led to the identification of two different members of gene family, 1 and 2. Third member, full-length cDNA (3) was isolated by rapid amplification of cDNA ends (RACE), whose open reading frame contained 1239 bp ...

  7. Cloning and expression pattern of chitin synthase (CHS) gene in ...

    African Journals Online (AJOL)

    USER

    2010-08-16

    Aug 16, 2010 ... This study provided an important information for further research on development of RNA interference. (RNAi) technology to control E. ... transcriptional gene silencing; RNAi, RNA interference; RACE, rapid amplification of cDNA .... Alignment showed that the alternately spliced. cDNA sequence of CHS-A in ...

  8. Abietadiene synthase from grand fir (Abies grandis): characterization and mechanism of action of the "pseudomature" recombinant enzyme.

    Science.gov (United States)

    Peters, R J; Flory, J E; Jetter, R; Ravn, M M; Lee, H J; Coates, R M; Croteau, R B

    2000-12-19

    The oleoresin secreted by grand fir (Abies grandis) is composed of resin acids derived largely from the abietane family of diterpene olefins as precursors which undergo subsequent oxidation of the C18-methyl group to a carboxyl function, for example, in the conversion of abieta-7,13-diene to abietic acid. A cDNA encoding abietadiene synthase has been isolated from grand fir and the heterologously expressed bifunctional enzyme shown to catalyze both the protonation-initiated cyclization of geranylgeranyl diphosphate to the intermediate (+)-copalyl diphosphate and the ionization-dependent cyclization of (+)-copalyl diphosphate, via a pimarenyl intermediate, to the olefin end products. Abietadiene synthase is translated as a preprotein bearing an N-terminal plastidial targeting sequence, and this form of the recombinant protein expressed in Escherichia coli proved to be unsuitable for detailed structure-function studies. Since the transit peptide-mature protein cleavage site could not be determined directly, a truncation series was constructed to delete the targeting sequence and prepare a "pseudomature" form of the enzyme that resembled the native abietadiene synthase in kinetic properties. Both the native synthase and the pseudomature synthase having 84 residues deleted from the preprotein converted geranylgeranyl diphosphate and the intermediate (+)-copalyl diphosphate to a nearly equal mixture of abietadiene, levopimaradiene, and neoabietadiene, as well as to three minor products, indicating that this single enzyme accounts for production of all of the resin acid precursors of grand fir. Kinetic evaluation of abietadiene synthase with geranylgeranyl diphosphate and (+)-copalyl diphosphate provided evidence for two functionally distinct active sites, the first for the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate and the second for the cyclization of (+)-copalyl diphosphate to diterpene end products, and demonstrated that the rate

  9. Amebic liver abscess

    Science.gov (United States)

    Hepatic amebiasis; Extraintestinal amebiasis; Abscess - amebic liver ... Amebic liver abscess is caused by Entamoeba histolytica. This parasite causes amebiasis , an intestinal infection that is also called ...

  10. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  11. 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...... in human. Although additional minor bands of 1.5 and 3.3 kb were found in Northern blots, RT-PCR (reverse transcription polymerase chain reaction) analysis failed to provide evidence that these minor bands are products of the tetranectin gene. Finally, the genetic map location for this gene, Tna...

  12. Cloning and sequencing of complete τ-crystallin cDNA from ...

    Indian Academy of Sciences (India)

    Unknown

    length τ-crystallin cDNA from crocodilian lens and α-enolase from other tissues. ... human (Acc. No. NM_001428). The sequences were used to construct a phylogenetic tree depicting gene lineage, using the clustering program DNAML.

  13. Construction and characterization of a cDNA expression library from the endangered Hu sheep.

    Science.gov (United States)

    Hu, P-F; Li, X-C; Liu, H-K; Guan, W-J; Ma, Y-H

    2014-10-31

    Hu sheep is one of the most important species in China; it is also listed as one of the 78 nationally protected domestic animals by the Chinese government in 2000. The construction of cDNA expression library of Hu sheep is of great significance for protecting individual genomes, generating transgenic sheep, and conducting clinical research using cDNA from Hu sheep. In this study, the total RNA from the ear tissue of Hu sheep was extracted, and a cDNA expression library was constructed using the SMART(TM) technique. The titer of amplified cDNA library was 1.09 x 10(10) PFU/mL, the rate of recombination was above 91.6%, and the average size of fragments was 1.1 kb. This study has an important significance for the preservation of Hu sheep resources at the genome level.

  14. 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...

  15. cDNA - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available ing/transcriptional initiation regionss About This Database Database Description Download License Update History of This Database Site Policy | Contact Us cDNA - ASTRA | LSDB Archive ...

  16. Construction of primary and subtracted cDNA libraries from early embryos.

    Science.gov (United States)

    Rothstein, J L; Johnson, D; Jessee, J; Skowronski, J; DeLoia, J A; Solter, D; Knowles, B B

    1993-01-01

    By modifying current cDNA cloning and electroporation methods, large and representative murine cDNA libraries were synthesized from 10 to 100 ng mRNA isolated from unfertilized egg and preimplantation mouse embryos. High cloning efficiency is essential for complete representation of genes expressed in egg and preimplantation embryos and for the isolation of stage-specific genes using subtractive hybridization. Because the mouse embryo contains no more than 50 pg of poly(A)+ mRNA at any stage of preimplantation development, approximately 5000-10,000 embryos are required to obtain enough mRNA to synthesize libraries using current methods. To obtain a representative library that also includes rare transcripts, the size of the library should be at least 10(6) clones. The average percent conversion of mRNA to single-stranded cDNA was 20-40%, so that a cloning efficiency of nearly 2 x 10(8) cfu/microgram cDNA is required for such a cDNA library. No previous methods have provided directional cloning of cDNA into plasmids with these high efficiencies. The advent of electroporation methods for the introduction of nucleic acids into bacteria has made possible the use of standard plasmid vectors for high-efficiency cDNA cloning. Plasmid vectors are currently available that can accommodate the directional cloning of cDNA such that T7 and T3 RNA polymerase promoter sequences can be used to generate sense and anti-sense transcripts for subtractive hybridization and riboprobe synthesis. The cDNA libraries we derived using this methodology are a reusable and abundant source of genetic information about the control of preimplantation development. Specialized subtractive cDNA libraries enriched for genes expressed exclusively at a predetermined time in development give access to genes expressed in a stage-specific manner. The ability to construct new cDNA libraries from limited amounts of starting material ensures the provision of new and important resources for the identification

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

    International Nuclear Information System (INIS)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon

    2003-01-01

    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

  18. 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.

  19. Enhancement of liver regeneration and liver surgery

    NARCIS (Netherlands)

    Olthof, P.B.

    2017-01-01

    Liver regeneration allows surgical resection of up to 75% of the liver and enables curative treatment potential for patients with primary or secondary hepatic malignancies. Liver surgery is associated with substantial risks, reflected by considerable morbidity and mortality rates. Optimization of

  20. [Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

    Science.gov (United States)

    Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

    2017-04-12

    Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

  1. cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes.

    OpenAIRE

    Candeliere, G A; Rao, Y; Floh, A; Sandler, S D; Aubin, J E

    1999-01-01

    A cDNA fingerprinting strategy was developed to identify genes based on their differential expression pattern during osteoblast development. Preliminary biological and molecular staging of cDNA pools prepared by global amplification PCR allowed discrim-inating choices to be made in selection of expressed sequence tags (ESTs) to be isolated. Sequencing of selected ESTs confirmed that both known and novel genes can be isolated from any developmental stage of interest, e.g. from primitive progen...

  2. Construction and characteristics of 3-end enriched cDNA library from individual embryos of cattle.

    Science.gov (United States)

    Long, Jian-Er; He, Li-Qiang; Cai, Xia; Ren, Zhao-Rui; Huang, Shu-Zhen; Zeng, Yi-Tao

    2006-11-01

    To analyze stage-specific gene expression profiles of pre-implantation embryos and evaluate potential viability, techniques were adapted to generate 3-end enriched cDNA libraries from individual embryos of cattle based on RT-PCR methodology. The reproducibility of constructing a cDNA library was tested by five independent PCR experiments with specific primers for the presence of several rare genes such as DNMT1 (DNA methylation transferase 1), DNMT2, DNMT3A, Oct-4/3 (octmer-binding transcription factor), IFN-iota, IGF-2r (insulin like growth factor 2 receptor), and the housekeeping genes, H2A and beta-actin. Results indicated repeatability and that a proportion of expressed genes in the cDNA library from an individual embryo was not affected by limited PCR amplification. From the cDNA library, 134 clones were randomly selected for sequencing and showed that structure related elements accounted for 33.5% of transcripts and the energy- and metabolism-related genes were also an important component being 11.9% in the cDNA library. Approximately 14% of genes in the library were functionally unknown including greater than 5% of genes that were likely novel because there was no identity in Genbank. The frequency of structure-related genes such as beta-actin and ribosomal proteins in the cDNA library corresponded to other reports and suggested that the cDNA library constructed by RT-PCR might be proportional to the mRNA populations. The cDNA libraries constructed from different stage embryos will provide a powerful tool to explore novel genes relevant to embryogenesis, determine the profiling of stage-specific gene expression, and evaluate the potential viability of embryos.

  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...

  4. Construction of an infectious cDNA clone of foot-and-mouth disease ...

    Indian Academy of Sciences (India)

    A stable, full-length cDNA clone of FMDV type O1BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEMR-7Zf(–). An ∼8.2 kb PCR product was amplified from the cDNA clone and a full-length RNA was generated from it by in vitro transcription. Transfection of BHK-21 ...

  5. cDNA cloning and mRNA expression of heat shock protein 70 gene ...

    African Journals Online (AJOL)

    In this study, the full-length heat shock protein 70 of Tegillarca granosa was cloned from cDNA library by rapid amplification of cDNA end (RACE). The open reading frame (ORF) of heat shock protein 70 was 1968 bp, and it encoded a protein of 655 amino acids with a predicted molecular weight of 71.48 kDa and an ...

  6. Cloning of cDNA encoding steroid 11β-hydroxylase (P450c11)

    International Nuclear Information System (INIS)

    Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

    1987-01-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11β-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage λ vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11β-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia

  7. Trehalose as a good candidate for enriching full-length cDNAs in cDNA library construction.

    Science.gov (United States)

    Chen, Lei; Cao, Lixue; Zhou, Longhai; Jing, Yudong; Chen, Zuozhou; Deng, Cheng; Shen, Yu; Chen, Liangbiao

    2007-01-10

    It has been reported that the disaccharide trehalose is capable of increasing the thermostability and thermoactivity of reverse transcriptase, and therefore improving the length of cDNA synthesis. However, no test has been done on how the disaccharide trehalose performs in the context of the entire cDNA synthesis processes, or whether it can seamlessly integrate into the commercially available cDNA synthesis kit. In this report, we optimized a protocol to incorporate trehalose in the Stratagene's cDNA library construction kit in order to demonstrate great improvement in cDNA's length (average length of 1.8 kb in the trehalose group versus 1.0 kb in the control). Sequence analysis of the cDNA clones showed that the addition of trehalose did not increase the error rate of the RT products but greatly increase the quantity of full-length in cDNA library.

  8. Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate

    Science.gov (United States)

    Yoon, Ju-Yeon; Cho, In-Sook; Choi, Gug-Seoun; Choi, Seung-Kook

    2014-01-01

    Chrysanthemum stunt viroid (CSVd), a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1) were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants. PMID:25288987

  9. Construction of a T7 Human Lung Cancer cDNA Library

    Directory of Open Access Journals (Sweden)

    Wentao YUE

    2008-10-01

    Full Text Available Background and objective Currently, only a limited numbers of tumor markers for non small lung cancer (NSCLC diagnosis, new biomarker, such as serum autoantibody may improve the early detection of lung cancer. Our objective is construction human lung squamous carcinoma and adenocarcinoma T7 phage display cDNA library from the tissues of NSCLC patients. Methods mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas, and then mRNA was reverse transcribed into double stranded cDNA. After digestion, the cDNA was inserted into T7Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library.Results Two T7 phage display cDNA library were established. Plaque assay show the titer of lung squamas carcinoma library was 1.8×106 pfu, and the adenocarcinoma library was 5×106 pfu. The phage titer of the amplified library were 3.2×1010 pfu/mL and 2.5×1010 pfu/mL. PCR amplification of random plaque show insert ratio were 100% (24/24 in adenocarcinoma library and 95.8% in human lung squamas carcinoma library (23/24. Insert range from 300 bp to 1 500 bp. Conclusion Two phage display cDNA library from NSCLC were constructed.

  10. [Construction of suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus].

    Science.gov (United States)

    Wu, Jia-hong; Zhao, Tong-yan; Dong, Yan-de

    2006-08-01

    To construct the suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus. Total RNA was extracted from the deltamethrin-resistant (R-lab) and -sensitive (S-lab) isolates, mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Two subtractions were performed by suppression subtractive hybridization with S-lab as tester and R-lab as driver or S-lab as driver and R-lab as tester. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. The subtracted cDNA libraries contained 580 and 477 positive clones respectively. The PCR results of 150 clones picked randomly from each library showed that the positive ratio of constructed cDNA libraries was 93%, with a length of cDNA fragments ranged from 150bp to 750bp. The suppression subtracted cDNA library of deltamethrin-resistant Ae. albopictus is constructed.

  11. [Construction of the subtracted cDNA libraries related to artemisinin-resistance of Plasmodium berghei].

    Science.gov (United States)

    Bei, Zhu-chun; Wang, Jing-yan

    2004-06-01

    To construct the subtracted cDNA libraries related to artemisinin-resistance of Plasmodium berghei using suppression subtractive hybridization PCR (SSH PCR). Total RNA was extracted from the artemisinin-sensitive (NS) and artemisinin-resistant (AR) strains of Plasmodium berghei K173. The cDNA synthesis followed the protocol of super SMART cDNA synthesis kit. Taking the NS as driver, AR as tester and reverse, two subtractions were performed by SSH PCR. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. The subtracted cDNA libraries of NS-AR and AR-NS contained 395 and 506 positive clones respectively. The PCR results of 108 clones picked randomly from each library showed 100 and 104 positive inserts contained in the plasmids respectively, and distributing in 250-2000 bp. The successful construction of the subtracted cDNA libraries related to artemisinin-resistance of P. berghei enable us to identify the different expressed genes involved in the resistance mechanism.

  12. [cDNA library constructing and specific antigen expression of Streptomyces thermohydroscopicus].

    Science.gov (United States)

    Xu, Lei; Wang, Ling-ling; Liu, Shuo; Ling, Yuan; Ma, Lie; Wang, Qun; Zhang, Li-jiao; He, Xiao-yu; Zhao, Ming-jing; Wang, Xiao-ge

    2012-03-01

    To construct a cDNA library from Streptomyces thermohydroscopicus and screen genes with virulence, obtain the recombinant fusion virulence proteins by prokaryotic expression system. The Streptomyces thermohydroscopicus cDNA library was constructed by switching mechanism at 5'end of RNA transcript approach. A total of 1020 clones randomly selected from the cDNA library were sequenced and these expressed sequence tags (EST) were further analyzed for the screen of antigen-specific genes. The two candidate genes were subcloned into expression vector pET-28a. The recombinants were transformed into BL2 and proteins were expressed by the induction of isopropyl-β-D-1-thiogalactopyranoside (IPTG). A high-quality cDNA library from Streptomyces thermohydroscopicus was constructed and a set of 978 valid sequences were obtained. Clustering and assembly of these cDNA sequences resulted in 347 unique genes, among which 2 potential antigen-specific genes were highly allied with outer membrane lipoprotein (51%) and transferring-binding protein B (42%) from Actinobacillus pleuropneumoniae serotype (APP). The open reading frame (ORF) of the two candidate genes are 1554 bp and 726 bp, which coded two peptides with 517 and 241 amino acids, respectively. The molecular weights of the recombinant fusion proteins were 63 000 and 30 000. The cDNA library of Streptomyces thermohydroscopicus reached the quality requirement of gene library. EST database in the library would greatly facilitate further screening of virulence genes.

  13. Molecular Cloning, Characterization and mRNA Expression of a Chitin Synthase 2 Gene from the Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae

    Directory of Open Access Journals (Sweden)

    Kang-Kang Xu

    2013-08-01

    Full Text Available Chitin synthase (CHS, a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2 was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis.

  14. Cloning and functional characterization of a gene for capsanthin-capsorubin synthase from tiger lily (Lilium lancifolium Thunb. 'Splendens').

    Science.gov (United States)

    Jeknić, Zoran; Morré, Jeffrey T; Jeknić, Stevan; Jevremović, Sladana; Subotić, Angelina; Chen, Tony H H

    2012-11-01

    The orange color of tiger lily (Lolium lancifolium 'Splendens') flowers is due, primarily, to the accumulation of two κ-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-degenerate primer that was based on the sequence of a gene for lycopene β-cyclase (lcyB). The full-length cDNA of Llccs was 1,785 bp long and contained an open reading frame of 1,425 bp that encoded a polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence. Analysis by reverse transcription-PCR (RT-PCR) revealed that expression of Llccs was spatially and temporally regulated, with expression in flower buds and flowers of L. lancifolium but not in vegetative tissues. Stable overexpression of the Llccs gene in callus tissue of Iris germanica, which accumulates several xanthophylls including violaxanthin, the precursor of capsorubin, resulted in transgenic callus whose color had changed from its normal yellow to red-orange. This novel red-orange coloration was due to the accumulation of two non-native κ-xanthophylls, capsanthin and capsorubin, as confirmed by HPLC and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis with authentic standards. Cloning of the Llccs gene should advance our understanding of the molecular and genetic mechanisms of the biosynthesis of κ-carotenoids in general and in the genus Lilium in particular, and will facilitate transgenic alterations of the colors of flowers and fruits of many plant species.

  15. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...... in the sarcolemma as well as the cytoplasm of type I muscle fibres. NADPH diaphorase activity confirmed a higher level of NO synthase activity in the sarcolemma as well as the cytoplasm of type I muscle fibers. Histochemical staining for cytochrome oxidase showed a staining pattern similar to that observed for type...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...

  16. Functional plasticity of paralogous diterpene synthases involved in conifer defense

    OpenAIRE

    Keeling, Christopher I.; Weisshaar, Sabrina; Lin, Roy P. C.; Bohlmann, Jörg

    2008-01-01

    The diversity of terpenoid compounds produced by plants plays an important role in mediating various plant–herbivore, plant–pollinator, and plant–pathogen interactions. This diversity has resulted from gene duplication and neofunctionalization of the enzymes that synthesize and subsequently modify terpenes. Two diterpene synthases in Norway spruce (Picea abies), isopimaradiene synthase and levopimaradiene/abietadiene synthase, provide the hydrocarbon precursors for most of the diterpene resin...

  17. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney Bruce [Pullman, WA; Burke, Charles Cullen [Moscow, ID

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  18. Efficacy Testing of H56 cDNA Tattoo Immunization against Tuberculosis in a Mouse Model.

    Science.gov (United States)

    Platteel, Anouk C M; Nieuwenhuizen, Natalie E; Domaszewska, Teresa; Schürer, Stefanie; Zedler, Ulrike; Brinkmann, Volker; Sijts, Alice J A M; Kaufmann, Stefan H E

    2017-01-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), remains a global threat. The only approved vaccine against TB, Mycobacterium bovis bacillus Calmette-Guérin (BCG), provides insufficient protection and, being a live vaccine, can cause disseminated disease in immunocompromised individuals. Previously, we found that intradermal cDNA tattoo immunization with cDNA of tetanus toxoid fragment C domain 1 fused to cDNA of the fusion protein H56, comprising the Mtb antigens Ag85B, ESAT-6, and Rv2660c, induced antigen-specific CD8 + T cell responses in vivo . As cDNA tattoo immunization would be safer than a live vaccine in immunocompromised patients, we tested the protective efficacy of intradermal tattoo immunization against TB with H56 cDNA, as well as with H56_E, a construct optimized for epitope processing in a mouse model. As Mtb antigens can be used in combination with BCG to boost immune responses, we also tested the protective efficacy of heterologous prime-boost, using dermal tattoo immunization with H56_E cDNA to boost BCG immunization in mice. Dermal H56 and H56_E cDNA immunization induced H56-specific CD4 + and CD8 + T cell responses and Ag85B-specific IgG antibodies, but did not reduce bacterial loads, although immunization with H56_E ameliorated lung pathology. Both subcutaneous and intradermal immunization with BCG resulted in broad cellular immune responses, with increased frequencies of CD4 + T effector memory cells, T follicular helper cells, and germinal center B cells, and resulted in reduced bacterial loads and lung pathology. Heterologous vaccination with BCG/H56_E cDNA induced increased H56-specific CD4 + and CD8 + T cell cytokine responses compared to vaccination with BCG alone, and lung pathology was significantly decreased in BCG/H56_E cDNA immunized mice compared to unvaccinated controls. However, bacterial loads were not decreased after heterologous vaccination compared to BCG alone. CD4 + T cells responding to Ag85B- and ESAT-6

  19. Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer

    International Nuclear Information System (INIS)

    Kaur, Jatinder; Sawhney, Meenakshi; DattaGupta, Siddartha; Shukla, Nootan K; Srivastava, Anurag; Ralhan, Ranju

    2010-01-01

    We reported increased levels of Phosphatidyl Inositol synthase (PI synthase), (enzyme that catalyses phosphatidyl inositol (PI) synthesis-implicated in intracellular signaling and regulation of cell growth) in smokeless tobacco (ST) exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC) and premalignant lesions (leukoplakia), and identified the downstream signaling proteins in PI synthase pathway that are perturbed by smokeless tobacco (ST) exposure. Tissue microarray (TMA) Immunohistochemistry, Western blotting, Confocal laser scan microscopy, RT-PCR were performed to define the expression of PI synthase in clinical samples and in oral cell culture systems. Significant increase in PI synthase immunoreactivity was observed in premalignant lesions and OSCCs as compared to oral normal tissues (p = 0.000). Further, PI synthase expression was significantly associated with de-differentiation of OSCCs, (p = 0.005) and tobacco consumption (p = 0.03, OR = 9.0). Exposure of oral cell systems to smokeless tobacco (ST) in vitro confirmed increase in PI synthase, Phosphatidylinositol 3-kinase (PI3K) and cyclin D1 levels. Collectively, increased PI synthase expression was found to be an early event in oral cancer and a target for smokeless tobacco

  20. Divinyl ether synthase gene and protein, and uses thereof

    Science.gov (United States)

    Howe, Gregg A [East Lansing, MI; Itoh, Aya [Tsuruoka, JP

    2011-09-13

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  1. Molecular cloning and expression of squalene synthase and 2,3-oxidosqualene cyclase genes in persimmon (Diospyros kaki L.) fruits.

    Science.gov (United States)

    Zhou, Chunhua; Zhao, Daqiu; Sheng, Yanle; Liang, Guohua; Tao, Jun

    2012-02-01

    Oleanolic acid (OA) and ursolic acid (UA) are the main triterpene acids in persimmon fruit, and squalene synthase and 2,3-oxidosqualene cyclases are important enzymes in pentacyclic triterpene biosynthesis. In order to study their relationship, DkSQS and DkOSC were cloned from persimmon fruits in the present study. The full-length cDNA of DkSQS was 1647 bp, containing an open reading frame (ORF) of 1245 bp that encoded a peptide of 415 amino acids (AA). The 3'-end of DkOSC cDNA fragment contained 522 bp, including a partial ORF of 298 bp, a full poly A tail that encoded 98 AA. Two cultivars of persimmon, i.e. cv. Nishimurawase and cv. Niuxinshi, were used to study the content of OA and UA and the related gene expression. Results showed that OA and UA contents changed in both cultivars during fruit development, the difference in cv. Nishimurawase was greater than that in cv. Niuxinshi. The expression of DkSQS and DkOSC had no obvious correlation with the biosynthesis of OA and UA in the flesh. There may be two main reasons. Firstly, different enzymes involved in the biosynthesis of triterpenes and mutual adjustment were existed in different gene expressions. Secondly, it was not clear that the DkOSC cloned in this research belonged to which subfamily. Therefore, the real relationship between triterpenes and DkSQS and DkOSC in persimmon fruits is still to be revealed.

  2. Pediatric Liver Transplantation.

    Science.gov (United States)

    Rawal, Nidhi; Yazigi, Nada

    2017-06-01

    Excellent outcomes over the last 3 decades have made liver transplantation the treatment of choice for many advanced liver disorders. This success also opened liver transplantation to new indications such as liver tumors and metabolic disorders. The emergence of such new indications for liver transplantation is bringing a new stream of patients along with disease-specific challenges. The cumulative number of liver transplant recipients is peaking, requiring novel systems of health care delivery that meet the needs of this special patient population. This article reviews updates and new development in pediatric liver transplantation. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Saponin Biosynthesis in Saponaria vaccaria. cDNAs Encoding β-Amyrin Synthase and a Triterpene Carboxylic Acid Glucosyltransferase1[OA

    Science.gov (United States)

    Meesapyodsuk, Dauenpen; Balsevich, John; Reed, Darwin W.; Covello, Patrick S.

    2007-01-01

    Saponaria vaccaria (Caryophyllaceae), a soapwort, known in western Canada as cowcockle, contains bioactive oleanane-type saponins similar to those found in soapbark tree (Quillaja saponaria; Rosaceae). To improve our understanding of the biosynthesis of these saponins, a combined polymerase chain reaction and expressed sequence tag approach was taken to identify the genes involved. A cDNA encoding a β-amyrin synthase (SvBS) was isolated by reverse transcription-polymerase chain reaction and characterized by expression in yeast (Saccharomyces cerevisiae). The SvBS gene is predominantly expressed in leaves. A S. vaccaria developing seed expressed sequence tag collection was developed and used for the isolation of a full-length cDNA bearing sequence similarity to ester-forming glycosyltransferases. The gene product of the cDNA, classified as UGT74M1, was expressed in Escherichia coli, purified, and identified as a triterpene carboxylic acid glucosyltransferase. UGT74M1 is expressed in roots and leaves and appears to be involved in monodesmoside biosynthesis in S. vaccaria. PMID:17172290

  4. Liver disease in menopause.

    Science.gov (United States)

    Brady, Carla W

    2015-07-07

    There are numerous physiologic and biochemical changes in menopause that can affect the function of the liver and mediate the development of liver disease. Menopause represents a state of growing estrogen deficiency, and this loss of estrogen in the setting of physiologic aging increases the likelihood of mitochondrial dysfunction, cellular senescence, declining immune responses to injury, and disarray in the balance between antioxidant formation and oxidative stress. The sum effect of these changes can contribute to increased susceptibility to development of significant liver pathology, particularly nonalcoholic fatty liver disease and hepatocellular carcinoma, as well as accelerated progression of fibrosis in liver diseases, as has been particularly demonstrated in hepatitis C virus liver disease. Recognition of the unique nature of these mediating factors should raise suspicion for liver disease in perimenopausal and menopausal women and offer an opportunity for implementation of aggressive treatment measures so as to avoid progression of liver disease to cirrhosis, liver cancer and liver failure.

  5. Presence of monoterpene synthase in four Labiatae species and Solid-Phase Microextraction- Gas chromatography-Mass Spectroscopy analysis of their aroma profiles.

    Science.gov (United States)

    Saeidnia, Soodabeh; Gohari, Ahmad Reza; Haddadi, Azita; Amin, Gholamreza; Nikan, Marjan; Hadjiakhoondi, Abbass

    2014-04-01

    The family Lamiaceae (Labiatae) has included some medicinal plants. some monoterpene synthases, including linalool and limonene synthases, have been cloned and functionally characterized from several plants of Labiatae family. In this study, presence of linalool and limonene synthases, in four species of Labiatae family including Nepeta cataria, Lavandula angustifolia, Hyssopus officinalis and Salvia sclarea has been determined by molecular biological techniques together with the Head space SPME - GC-MS analysis of the aroma profile of these species. Indicated that none of the plant species produced distinguishable bands with primer pairs related to d-limonene synthase. Distinguishable bands around 1800 bp in cDNA samples of L. angustifolia, H. officinalis and S. sclarea were observed regarding to the presence of linalool synthase. Head space SPME-GC-MS analysis of the aroma profiles of the above-mentioned plants showed that linalool (31.0%), linalyl acetate (18.2%), were found as the major compounds of L. angustifolia, while geraniol (5.5%), nerol (34.0%) and α- citral (52.0%) were identified as the main compounds of the N. cataria. The major components of H. officinalis and S. sclarea oils were determined as cis-pinocamphone (57.3%), and linalool (19.0%), linalyl acetate (51.5%), respectively. H. officinalis was rich of cyclic monoterpenes, L. angustifolia, N. cataria and S. sclarea showed considerable amount of linear monoterpenes. The aroma profile of the above-mentioned plants contained low concentration of sesquiterpenes except N. cataria, which indicated no sesquiterpene. The profiles of the main components of these plants are in agreement with molecular assays.

  6. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity

    DEFF Research Database (Denmark)

    Yang, Ting; Gao, Liping; Hu, Hao

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first path-way-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate...

  7. Impaired ATP synthase assembly associated with a mutation in the human ATP synthase subunit 6 gene.

    NARCIS (Netherlands)

    Nijtmans, L.G.J.; Henderson, N.S.; Attardi, G.; Holt, L.J.

    2001-01-01

    Mutations in human mitochondrial DNA are a well recognized cause of disease. A mutation at nucleotide position 8993 of human mitochondrial DNA, located within the gene for ATP synthase subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome.

  8. Immune mediated liver failure

    OpenAIRE

    Wang, Xiaojing; Ning, Qin

    2014-01-01

    Liver failure is a clinical syndrome of various etiologies, manifesting as jaundice, encephalopathy, coagulopathy and circulatory dysfunction, which result in subsequent multiorgan failure. Clinically, liver failure is classified into four categories: acute, subacute, acute-on-chronic and chronic liver failure. Massive hepatocyte death is considered to be the core event in the development of liver failure, which occurs when the extent of hepatocyte death is beyond the liver regenerative capac...

  9. The effect of dexamethasone treatment on the expression of the regulatory genes of ketogenesis in intestine and liver of suckling rats.

    Science.gov (United States)

    Arias, G; Asins, G; Hegardt, F G; Serra, D

    1998-01-01

    The influence of the injection of dexamethasone on ketogenesis in 12 day old suckling rats was studied in intestine and liver by determining mRNA levels and enzyme activity of the two genes responsible for regulation of ketogenesis: carnitine palmitoyl transferase I (CPT I) and mitochondrial HMG-CoA synthase. Dexamethasone produced a 2 fold increase in mRNA and activity of CPT I in intestine, but led to a decrease in mit. HMG-CoA synthase. In liver the mRNA levels and activity of both CPT I and mit. HMG-CoA synthase decreased. Comparison of these values with the ketogenic rate of both tissues following dexamethasone treatment suggests that mit. HMG-CoA synthase could be the main gene responsible for the regulation of ketogenesis in suckling rats. The changes produced in serum ketone bodies by dexamethasone, with a profile that is more similar to the ketogenic rate in the liver than that in the intestine, indicate that liver contributes more to ketone body synthesis in suckling rats. Two day treatment with dexamethasone produced no change in mRNA or activity levels for CPT I in liver or intestine. While mRNA levels for mit. HMG-CoA synthase changed little, the enzyme activity is decreased in both tissues.

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

    International Nuclear Information System (INIS)

    Szybka, Malgorzata; Kordek, Radzislaw; Zakrzewska, Magdalena; Rieske, Piotr; Pasz-Walczak, Grazyna; Kulczycka-Wojdala, Dominika; Zawlik, Izabela; Stawski, Robert; Jesionek-Kupnicka, Dorota; Liberski, Pawel P

    2009-01-01

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

  11. [Construction of phage display cDNA library from adult worms of Schistosoma japonicum].

    Science.gov (United States)

    Sun, Yi; Jia, Ren-chu; Liu, Jin-ming; Yuan, Chun-xiu; Shi, Yao-jun; Lu, Ke; Fu, Zhi-qiang; Sun, Huan; Cai, You-min; Lin, Jiao-jiao

    2007-10-01

    To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum. Total RNA was extracted from adult worms of S. japonicum by Trizol reagent anti mRNA was isolated from the total RNA. The ds cDNA was synthesized by reverse transcription using random primer. Directional EcoR I/ Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoR I anti Hind III, which resulted in ds cDNA with EcoR I and Hind III adhering ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector. After packaging in citro, the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library. Plaque assay and PCR were used to evaluate the library. Seven known objective genes of S. japonicum were screened by PCR to detect the representation of the library. Primary library capacity was 4.98 x 10(6) pfu, and the titer of amplified library was 3.85 x 10(11) pfu/mL. The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%, in which 95.6% inserted cDNA fragments were longer than 300 bp in length. All the seven known objective genes of S. japonicum were amplified from the library. The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

  12. [Construction of cDNA expression library of unfed female Haemaphysalis longicornis and immuno-screening].

    Science.gov (United States)

    Chai, Hui-ping; Liu, Guang-yuan; Zhang, Lin; Gong, Zhen-li; Xie, Jun-ren; Tian, Zhan-cheng; Wang, Lu; Jia, Ning

    2009-02-28

    To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes. Total RNA was isolated from unfed female ticks, mRNA was purified and a library of oligo (dT) -primed cDNA with added directional EcoR I /Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I /Hind III arms of the lambda SCREEN vector. Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8. Recombinant plasmids that were subcloned to E. coli BM25.8 were isolated and transformed into E. coli JM109. Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing. The recombinant phage DNA was packaged by using phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.8 x 10(6) pfu. Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4 x 10(9) pfu/ml. Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library. Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H. longicornis tropomyosin mRNA, Rhipicephalus annulatus unknown larval protein mRNA, chromosome 2R of Drosophila melanogaster, mitochondrial DNA of H. flava, clones HqL09 unkown mRNA and Hq05 mRNA of H. qinghaiensis, and myosin alkali light chain protein mRNA. The cDNA expression library from unfed female H. longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.

  13. Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vassinated mice

    International Nuclear Information System (INIS)

    Dalton, J.P.; Tom, T.D.; Strand, M.

    1987-01-01

    Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. Binding was measured by radioimmunoassay. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in λgt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein

  14. Human placental Na+, K+-ATPase α subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

    International Nuclear Information System (INIS)

    Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.

    1987-01-01

    A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na + , K + -ATPase α subunit was cloned from human placenta and its sequence was identical to that encoding the α subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na + , K + -ATPase gene from various human tissues and cell lines revealed only one band (≅ 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) λgt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the α subunit is on the short is on the short arm (band p11-p13) of chromosome 1

  15. Mouse microsomal triglyceride transfer protein large subunit: cDNA cloning, tissue-specific expression, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Nakamuta, Makoto; Chang, Benny Hung-Junn; Hoogeveen, R. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1996-04-15

    Microsomal triglyceride transfer protein (MTP) catalyzes the transfer of triglyceride, cholesteryl ester, and phospholipid between membranes. It is essential for the secretion of apolipoprotein B from the cell. Mutations in MTP are a major cause of abetalipoproteinemia. The mouse is a popular animal model for lipoprotein metabolism. We have cloned and sequenced mouse MTP cDNA. The DNA-deduced amino acid sequence indicates that mouse protein shows 93, 86, and 83% sequence indicates that mouse MTP contains 894 amino acids; the mouse protein shows 93, 86, and 83% sequence identity to the hamster, human, and bovine sequences, respectively. Northern blot analysis indicates that mouse MTP mRNA is expressed at high levels in the small intestine and at substantially lower levels in the liver and that it is not detectable in six other tissues examined. The mouse MTP gene has been localized to the distal region of chromosome 3 by Southern blots of interspecific backcross panels using progeny derived from matings of (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei. Comparison of MTP sequences from human, bovine, hamster, and mouse indicates that the C-terminal region of MTP is better conserved than its N-terminal region. 21 refs., 2 figs.

  16. α/sub i/-3 cDNA encodes the α subunit of G/sub k/, the stimulatory G protein of receptor-regulated K+ channels

    International Nuclear Information System (INIS)

    Codina, J.; Olate, J.; Abramowitz, J.; Mattera, R.; Cook, R.G.; Birnbaumer, L.

    1988-01-01

    cDNA cloning has identified the presence in the human genome of three genes encoding α subunits of pertussis toxin substrates, generically called G/sub i/. They are named α/sub i/-1, α/sub i/-2 and α/sub i/-3. However, none of these genes has been functionally identified with any of the α subunits of several possible G proteins, including pertussis toxin-sensitive G/sub p/'s, stimulatory to phospholipase C or A 2 , G/sub i/, inhibitory to adenylyl cyclase, or G/sub k/, stimulatory to a type of K + channels. The authors now report the nucleotide sequence and the complete predicted amino acid sequence of human liver α/sub i/-3 and the partial amino acid sequence of proteolytic fragments of the α subunit of human erythrocyte G/sub k/. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of α/sub i/-3, thus identifying it as α/sub k/. The probable identity of α/sub i/-1 with α/sub p/ and possible roles for α/sub i/-2, as well as additional roles for α/sub i/-1 and α/sub i/-3 (α/sub k/) are discussed

  17. Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate.

    Science.gov (United States)

    Burke, Charles; Croteau, Rodney

    2002-02-01

    Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C(5) units, of the respective C(10), C(15), and C(20) precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56-75%) with geranylgeranyl diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case. These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C(10) chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase.

  18. Evolution of floral scent in Clarkia: novel patterns of S-linalool synthase gene expression in the C. breweri flower.

    Science.gov (United States)

    Dudareva, N; Cseke, L; Blanc, V M; Pichersky, E

    1996-01-01

    Flowers of Clarkia breweri, an annual plant from the coastal range of California, emit a strong sweet scent of which S-linalool, an acyclic monoterpene, is a major component. Chromosomal, chemical, and morphological data, and the species' geographic distribution, suggest that C. breweri evolved from an extant nonscented species, C. concinna. A cDNA of Lis, the gene encoding S-linalool synthase, was isolated from C. breweri. We show that in C. breweri, Lis is highly expressed in cells of the transmitting tract of the stigma and style and in the epidermal cells of petals, as well as in stamens, whereas in the nonscented C. concinna, Lis is expressed only in the stigma and at a relatively low level. In both species, changes in protein levels parallel changes in mRNA levels, and changes in enzyme activity levels parallel changes in protein levels. The results indicate that in C. breweri, the expression of Lis has been upregulated and its range enlarged to include cells not expressing this gene in C. concinna. These results show how scent can evolve in a relatively simple way without the evolution of highly specialized "scent glands" and other specialized structures. Lis encodes a protein that is structurally related to the family of proteins termed terpene synthases. The protein encoded by Lis is the first member of this family found to catalyze the formation of an acyclic monoterpene. PMID:8768373

  19. Liver Transplants for Alcoholic Liver Disease

    Directory of Open Access Journals (Sweden)

    RJ Fingerote

    1991-01-01

    Full Text Available Alcohol related end-stage liver disease is a principal cause of liver failure. The scarcity of donor livers and the predominance of alcohol related end-stage liver disease has raised the issue of including alcoholics as candidates for liver transplantation. In rationalizing the arguments for and against the treatment of alcoholic end-stage liver disease with transplantation, factors such as recidivism, resource allocation and principles of medical practice must be considered. Public confidence in organ transplantation depends on the scientific validity and moral integrity of the policies adopted. Sound policies will prove defensible while policies based on perceptions or prejudices will, in the long run, harm the process.

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

    Directory of Open Access Journals (Sweden)

    Regenbogen Johannes

    2002-03-01

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

  1. Uncovering the structures of modular polyketide synthases.

    Science.gov (United States)

    Weissman, Kira J

    2015-03-01

    The modular polyketide synthases (PKSs) are multienzyme proteins responsible for the assembly of diverse secondary metabolites of high economic and therapeutic importance. These molecular 'assembly lines' consist of repeated functional units called 'modules' organized into gigantic polypeptides. For several decades, concerted efforts have been made to understand in detail the structure and function of PKSs in order to facilitate genetic engineering of the systems towards the production of polyketide analogues for evaluation as drug leads. Despite this intense activity, it has not yet been possible to solve the crystal structure of a single module, let alone a multimodular subunit. Nonetheless, on the basis of analysis of the structures of modular fragments and the study of the related multienzyme of animal fatty acid synthase (FAS), several models of modular PKS architecture have been proposed. This year, however, the situation has changed - three modular structures have been characterized, not by X-ray crystallography, but by the complementary methods of single-particle cryo-electron microscopy and small-angle X-ray scattering. This review aims to compare the cryo-EM structures and SAXS-derived structural models, and to interpret them in the context of previously obtained data and existing architectural proposals. The consequences for genetic engineering of the systems will also be discussed, as well as unresolved questions and future directions.

  2. Generation of cDNA expression libraries enriched for in-frame sequences.

    Science.gov (United States)

    Davis, C A; Benzer, S

    1997-03-18

    Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60-80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.

  3. [Construction of a yeast two-hybrid cDNA library from the human testis].

    Science.gov (United States)

    Zheng, Ying; Zhang, Lu-Ping; Jia, Xiao-Qin; Wang, Hai-Yan

    2012-04-01

    To construct a human testis cDNA library for yeast two-hybrid screening. Human normal testis mRNA was purified from total RNA, and ds cDNA was synthesized and amplified using primers SMART III and CDS III oligo (dT) as the base of recombination. The purified PCR products and linearized plasmid pGADT7-Rec were co-transformed into the competent yeast Y187 and recombined by yeast homologous recombinase in the yeast cells to form an active cyclic plasmid. All the clones growing on the SD/-Leu plates were harvested to constitute a human testis cDNA library. We constructed a human testis cDNA library with high multiplication and adequate capacity, from which 2.0 x 10(6) recombinants were obtained. The amplified PCR fragments were between 0.3 kb and 4.0 kb in length. The yeast two-hybrid cDNA library of human testis was successfully constructed by the Clontech SMART method, which has prepared a ground for further studies on the molecular mechanism of spermatogenesis.

  4. [Construction of cDNA expression library of salivary gland from Boophilus microplus].

    Science.gov (United States)

    Tian, Zhan-Cheng; Liu, Guang-Yuan; Xie, Jun-Ren; Gong, Zhen-Li

    2008-10-30

    Total RNA were isolated from salivary gland dissected from partially engorged Boophilus microplus. The mRNA was purified. A library of oligo (dT)-primed cDNA with added directional EcoR I/Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I/Hind III arms of the lambda SCREEN vector. The recombinant phage DNA was packaged by phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.38x10(6) PFU. Data showed 100% of the library were recombinant and the titer of the amplified library was 2x10(9) PFU/ml. A partial cDNA encoding cytochrome oxidase C subunit II of B. microplus was screened from the expression library with rabbit serum against B. microplus salivary gland proteins. The results is suggested that the cDNA expression library has been constructed.

  5. Construction of equalized short hairpin RNA library from human brain cDNA.

    Science.gov (United States)

    Xu, Lei; Li, Jingqi; Liu, Li; Lu, Lixia; Gao, Jingxia; Li, Xueli

    2007-02-20

    Short hairpin RNA (shRNA) library is a powerful new tool for high-throughput loss-of-function genetic screens in mammalian cells. An shRNA library can be constructed from synthetic oligonucleotides or enzymatically cleaved natural cDNA. Here, we describe a new method for constructing equalized shRNA libraries from cDNA. First, enzymatically digested cDNA fragments are equalized by a suppression PCR-based method modified from suppression subtractive hybridization. The efficiency of equalization was confirmed by quantitative real-time PCR. The fragments are then converted into an shRNA library by a series of enzymatic treatments. With this new technology, we constructed a library from human brain cDNA. Sequence analysis showed that most of the randomly selected clones had inverted repeat sequences converted from different cDNA. After transfecting HEK 293T cells and detecting gene expression, three out of eight clones were demonstrated to significantly inhibit their target genes.

  6. [Combining SSH and cDNA microarray for identification of lung cancer related genes].

    Science.gov (United States)

    Fan, Baoxing; Zhang, Kaitai; Da, Jiping; Xie, Ling; Wang, Shengqi; Wu, Dechang

    2003-04-20

    To screen and identify differentially expressed genes among lung cancer tissues, paracancerous pulmonary tissues and some other kinds of tumor tissues using suppression subtractive hybridization (SSH) and cDNA Microarray. One cDNA chip was made by gathering clones of three differentially expressed cDNA libraries which came from BEP2D cell lines during three different malignant transformed phases. Then the clones were hybridizated with cDNA probes which extracted from 15 cases of lung cancer tissues, 5 cases of paracancerous pulmonary tissues and 24 cases of other 8 kinds of tumor tissues respectively. Twenty-six cDNAs were obtained which expressed higher in lung cancer tissues than that in paracancerous pulmonary tissues. Thirty-one cDNAs expressed remarkably higher in paracancerous tissues than those in cancer tissues. Compared with other 8 kinds of tumors, paracancerous tissues had 63 overexpressed cDNAs and lung cancer tissues had 87 overexpressed cDNAs. The combination of SSH and cDNA microarray is rapid and effective for screening and identification of differentially expressed genes in different samples. It may be potentially useful for diagnosis of lung cancer to further study the differentially expressed genes among lung cancer tissues, paracancerous pulmonary tissues and other tumor tissues.

  7. Sequence of a cDNA encoding turtle high mobility group 1 protein.

    Science.gov (United States)

    Zheng, Jifang; Hu, Bi; Wu, Duansheng

    2005-07-01

    In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.

  8. Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Rumessen, J J; Qvortrup, K

    1991-01-01

    Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase...

  9. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed ...

  10. Sequence analysis of cereal sucrose synthase genes and isolation ...

    African Journals Online (AJOL)

    SERVER

    2007-10-18

    Oct 18, 2007 ... sequencing of sucrose synthase gene fragment from sor- ghum using primers designed at their conserved exons. MATERIALS AND METHODS. Multiple sequence alignment. Sucrose synthase gene sequences of various cereals like rice, maize, and barley were accessed from NCBI Genbank database.

  11. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i...

  12. Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase.

    Directory of Open Access Journals (Sweden)

    Catalina Sanz

    Full Text Available Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.

  13. Biomarkers for liver fibrosis

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, Jon M.; Burnum-Johnson, Kristin E.; Baker, Erin M.; Smith, Richard D.; Gritsenko, Marina A.; Orton, Daniel

    2017-05-16

    Methods and systems for diagnosing or prognosing liver fibrosis in a subject are provided. In some examples, such methods and systems can include detecting liver fibrosis-related molecules in a sample obtained from the subject, comparing expression of the molecules in the sample to controls representing expression values expected in a subject who does not have liver fibrosis or who has non-progressing fibrosis, and diagnosing or prognosing liver fibrosis in the subject when differential expression of the molecules between the sample and the controls is detected. Kits for the diagnosis or prognosis of liver fibrosis in a subject are also provided which include reagents for detecting liver fibrosis related molecules.

  14. Expressed sequence tags: normalization and subtraction of cDNA libraries expressed sequence tags\\ normalization and subtraction of cDNA libraries.

    Science.gov (United States)

    Soares, Marcelo Bento; de Fatima Bonaldo, Maria; Hackett, Jeremiah D; Bhattacharya, Debashish

    2009-01-01

    Expressed Sequence Tags (ESTs) provide a rapid and efficient approach for gene discovery and analysis of gene expression in eukaryotes. ESTs have also become particularly important with recent expanded efforts in complete genome sequencing of understudied, nonmodel eukaryotes such as protists and algae. For these projects, ESTs provide an invaluable source of data for gene identification and prediction of exon-intron boundaries. The generation of EST data, although straightforward in concept, requires nonetheless great care to ensure the highest efficiency and return for the investment in time and funds. To this end, key steps in the process include generation of a normalized cDNA library to facilitate a high gene discovery rate followed by serial subtraction of normalized libraries to maintain the discovery rate. Here we describe in detail, protocols for normalization and subtraction of cDNA libraries followed by an example using the toxic dinoflagellate Alexandrium tamarense.

  15. Cloning and Comparative Studies of Seaweed Trehalose-6-Phosphate Synthase Genes

    Science.gov (United States)

    Wang, Guoliang; Zhao, Ge; Feng, Yanbin; Xuan, Jinsong; Sun, Jianwei; Guo, Baotai; Jiang, Guoyong; Weng, Manli; Yao, Jianting; Wang, Bin; Duan, Delin; Liu, Tao

    2010-01-01

    The full-length cDNA sequence (3219 base pairs) of the trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS) was isolated by RACE-PCR and deposited in GenBank (NCBI) with the accession number AY729671. PyTPS encodes a protein of 908 amino acids before a stop codon, and has a calculated molecular mass of 101,591 Daltons. The PyTPS protein consists of a TPS domain in the N-terminus and a putative TPP domain at the C-terminus. Homology alignment for PyTPS and the TPS proteins from bacteria, yeast and higher plants indicated that the most closely related sequences to PyTPS were those from higher plants (OsTPS and AtTPS5), whereas the most distant sequence to PyTPS was from bacteria (EcOtsAB). Based on the identified sequence of the PyTPS gene, PCR primers were designed and used to amplify the TPS genes from nine other seaweed species. Sequences of the nine obtained TPS genes were deposited in GenBank (NCBI). All 10 TPS genes encoded peptides of 908 amino acids and the sequences were highly conserved both in nucleotide composition (>94%) and in amino acid composition (>96%). Unlike the TPS genes from some other plants, there was no intron in any of the 10 isolated seaweed TPS genes. PMID:20714424

  16. Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway.

    Directory of Open Access Journals (Sweden)

    Wei Sun

    Full Text Available Chalcone synthase (CHS catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1 encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

  17. Suppression of allene oxide synthase 3 in potato increases degree of arbuscular mycorrhizal fungal colonization.

    Science.gov (United States)

    Morcillo, Rafael Jorge León; Navarrete, María Isabel Tamayo; Bote, Juan Antonio Ocampo; Monguio, Salomé Prat; García-Garrido, José Manuel

    2016-01-15

    Arbuscular mycorrhizal (AM) is a mutually beneficial interaction among higher plants and soil fungi of the phylum Glomeromycota. Numerous studies have pointed that jasmonic acid plays an important role in the development of the intraradical fungus. This compound belongs to a group of biologically active compounds known as oxylipins which are derived from the oxidative metabolism of polyunsaturated fatty acids. Studies of the regulatory role played by oxylipins in AM colonization have generally focused on jasmonates, while few studies exist on the 9-LOX pathway of oxylipins during AM formation. Here, the cDNA of Allene oxide synthase 3 (AOS3), a key enzyme in the 9-LOX pathway, was used in the RNA interference (RNAi) system to transform potato plants in order to suppress its expression. Results show increases in AOS3 gene expression and 9-LOX products in roots of wild type potato mycorrhizal plants. The suppression of AOS3 gene expression increases the percentage of root with mycorrhizal colonization at early stages of AM formation. AOS3 RNA interference lead to an induction of LOXA and 13-LOX genes, a reduction in AOS3 derived 9-LOX oxylipin compounds and an increase in jasmonic acid content, suggesting compensation between 9 and 13-LOX pathways. The results in a whole support the hypothesis of a regulatory role for the 9-LOX oxylipin pathway during mycorrhization. Copyright © 2015 Elsevier GmbH. All rights reserved.

  18. Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway.

    Science.gov (United States)

    Sun, Wei; Meng, Xiangyu; Liang, Lingjie; Jiang, Wangshu; Huang, Yafei; He, Jing; Hu, Haiyan; Almqvist, Jonas; Gao, Xiang; Wang, Li

    2015-01-01

    Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

  19. Cloning and Comparative Studies of Seaweed Trehalose-6-Phosphate Synthase Genes

    Directory of Open Access Journals (Sweden)

    Bin Wang

    2010-07-01

    Full Text Available The full-length cDNA sequence (3219 base pairs of the trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS was isolated byRACE-PCR and deposited in GenBank (NCBI with the accession number AY729671. PyTPS encodes a protein of 908 amino acids before a stop codon, and has a calculated molecular mass of 101,591 Daltons. The PyTPS protein consists of a TPS domain in the N-terminus and a putative TPP domain at the C-terminus. Homology alignment for PyTPS and the TPS proteins from bacteria, yeast and higher plants indicated that the most closely related sequences to PyTPS were those from higher plants (OsTPS and AtTPS5, whereas the most distant sequence to PyTPS was from bacteria (EcOtsAB. Based on the identified sequence of the PyTPS gene, PCR primers were designed and used to amplify the TPS genes from nine other seaweed species. Sequences of the nine obtained TPS genes were deposited in GenBank (NCBI. All 10 TPS genes encoded peptides of 908 amino acids and the sequences were highly conserved both in nucleotide composition (>94% and in amino acid composition (>96%. Unlike the TPS genes from some other plants, there was no intron in any of the 10 isolated seaweed TPS genes.

  20. ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development.

    Science.gov (United States)

    Alba, Rob; Fei, Zhangjun; Payton, Paxton; Liu, Yang; Moore, Shanna L; Debbie, Paul; Cohn, Jonathan; D'Ascenzo, Mark; Gordon, Jeffrey S; Rose, Jocelyn K C; Martin, Gregory; Tanksley, Steven D; Bouzayen, Mondher; Jahn, Molly M; Giovannoni, Jim

    2004-09-01

    Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit.

  1. Construction and analysis of full-length and normalized cDNA libraries from citrus.

    Science.gov (United States)

    Marques, M Carmen; Perez-Amador, Miguel A

    2012-01-01

    We have developed an integrated method to generate a normalized cDNA collection enriched in full-length and rare transcripts from citrus, using different species and multiple tissues and developmental stages. Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. In this regard, the availability of full-length cDNA clones facilitates functional analysis of the corresponding genes enabling manipulation of their expression and the generation of a variety of tagged versions of the native protein. The development of full-length cDNA sequences has the power to improve the quality of genome annotation, as well as provide tools for functional characterization of genes.

  2. 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.

  3. [Screening of specifically expressed genes in amphioxus neurula by construction of a subtractive cDNA library].

    Science.gov (United States)

    Zhang, Lei; Yang, Yong-Jie; Zhang, Yan-Jun

    2010-12-01

    To screen specifically expressed genes in the development of nerve, muscle, and body axis of amphioxus, Branchiostoma belcheri tsingtauenese. A subtractive cDNA library was constructed from the 12-hour amphioxus neurula cDNA after subtractively hybridized with the 6-hour amphioxus gastrula cDNA. The total RNA was extracted from the 12-hour neurula and 6-hour gastrula, then reverse transcribed into cDNA. The 12-hour neurula cDNA was designated as the experimental group (the tester) and the 6-hour gastrula cDNA as the control group (the driver). The differentially expressed sequences were exponentially amplified using suppression PCR. Background was subtracted and differentially expressed sequences were further enriched. The PCR products were ligated to the T Vector. After transformation of the recombinant plasmid carrying inserted amphioxus cDNA into E.coli host cells, the cDNA library was constructed successfully. Two hundred randomly chosen positive clones were sequenced and some of neurula-specifically expressed genes were obtained. SSH is an effective method for searching differentially expressed genes. The subtractive cDNA library we generated provides a tool for further study of regulatory mechanisms of amphioxus early embryonic development.

  4. Gene therapy for bladder pain with gene gun particle encoding pro-opiomelanocortin cDNA.

    Science.gov (United States)

    Chuang, Yao-Chi; Chou, A-K; Wu, P-C; Chiang, Po-Hui; Yu, T-J; Yang, L-C; Yoshimura, Naoki; Chancellor, Michael B

    2003-11-01

    Interstitial cystitis is a bladder hypersensitivity disease associated with bladder pain that has been a major challenge to understand and treat. We hypothesized that targeted and localized expression of endogenous opioid peptide in the bladder could be useful for the treatment of bladder pain. Pro-opiomelanocortin (POMC) is one of such precursor molecules. In this study we developed a gene gun method for the transfer of POMC cDNA in vivo and investigated its therapeutic effect on acetic acid induced bladder hyperactivity in rats. Human POMC cDNA was cloned into a modified pCMV plasmid and delivered into the bladder wall of adult female rats by direct injection or the gene gun. Three days after gene therapy continuous cystometrograms were performed using urethane anesthesia by filling the bladder (0.08 ml per minute) with saline, followed by 0.3% acetic acid. Bladder immunohistochemical testing was used to detect endorphin after POMC cDNA transfer. The intercontraction interval was decreased after intravesical instillation of acetic acid (73.1% or 68.1% decrease) in 2 control groups treated with saline or the gene gun without POMC cDNA, respectively. However, rats that received POMC cDNA via the gene gun showed a significantly decreased response (intercontraction interval 35% decreased) to acetic acid instillation, whereas this antinociceptive effect was not detected in the plasmid POMC cDNA direct injection group. This effect induced by POMC gene gun treatment was reversed by intramuscular naloxone (1 mg/kg), an opioid antagonist. Increased endorphin immunoreactivity with anti-endorphin antibodies was observed in the bladder of gene gun treated animals. The POMC gene can be transferred in the bladder using the gene gun and increased bladder expression of endorphin can suppress nociceptive responses induced by bladder irritation. Thus, POMC gene gun delivery may be useful for the treatment of interstitial cystitis and other types of visceral pain.

  5. Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA

    Directory of Open Access Journals (Sweden)

    Karlovsky Petr

    2008-10-01

    Full Text Available Abstract Background cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to generate cDNA fragments negatively affects the performance of cDNA-AFLP. Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively. Results With the goal of improving the coverage of cDNA-AFLP without increasing its redundancy, we designed a modified cDNA-AFLP protocol. Immobilized cDNA is sequentially digested with several restriction endonucleases and the released DNA fragments are collected in mutually exclusive pools. To investigate the performance of the protocol, software tool MECS (Multiple Enzyme cDNA-AFLP Simulation was written in Perl. cDNA-AFLP protocols described in the literatur and the new sequential digestion protocol were simulated on sets of cDNA sequences from mouse, human and Arabidopsis thaliana. The redundancy and coverage, the total number of PCR reactions, and the average fragment length were calculated for each protocol and cDNA set. Conclusion Simulation revealed that sequential digestion of immobilized cDNA followed by the partitioning of released fragments into mutually exclusive pools outperformed other cDNA-AFLP protocols in terms of coverage, redundancy, fragment length, and the total number of PCRs. Primers generating 30 to 70 amplicons per PCR provided the highest fraction of electrophoretically distinguishable fragments suitable for normalization. For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each

  6. Display of a maize cDNA library on baculovirus infected insect cells.

    Science.gov (United States)

    Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

    2008-08-12

    Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

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

    International Nuclear Information System (INIS)

    Oshima, A.; Kyle, J.W.; Miller, R.D.

    1987-01-01

    The authors report here the cDNA sequence for human placental β-glucuronidase (β-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH 2 -terminal amino acid sequence determined for human spleen β-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human β-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human β-glucuronidase, demonstrate the existence of two populations of mRNA for β-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length

  8. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    Directory of Open Access Journals (Sweden)

    Nevzat Selim Gokay

    2016-01-01

    Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  9. Identification of differential expression of genes in hepatocellular carcinoma by suppression subtractive hybridization combined cDNA microarray.

    Science.gov (United States)

    Liu, Yuefang; Zhu, Xiaojing; Zhu, Jin; Liao, Shibing; Tang, Qi; Liu, Kaikun; Guan, Xiaohong; Zhang, Jianping; Feng, Zhenqing

    2007-10-01

    The genetic background of hepatocellular carcinoma (HCC) has yet to be completely understood. Here, we describe the application of suppression subtractive hybridization (SSH) coupled with cDNA microarray analysis for the isolation and identification of differential expression of genes in HCC. Twenty-six known genes were validated as up-regulated and 19 known genes as down-regulated in HCC. The known genes identified were found to have diverse functions. In addition to the overexpression of AFP, these genes (increased in the presence of HCC) are involved in many processes, such as transcription and protein biosynthesis (HNRPDL, PABPC1, POLR2K, SRP9, SNRPA, and six ribosomal protein genes including RPL8, RPL14, RPL41, RPS5, RPS17, RPS24), the metabolism of lipids and proteins (FADS1, ApoA-II, ApoM, FTL), cell proliferation (Syndecan-2, and Annexin A2), and signal transduction (LRRC28 and FMR1). Additionally, a glutathione-binding protein involved in the detoxification of methylglyoxal known as GLO1 and an enzyme which increases the formation of prostaglandin E(2) known as PLA2G10 were up-regulated in HCC. Among the underexpressed genes discovered in HCC, most were responsible for liver-synthesized proteins (fibrinogen, complement species, amyloid, albumin, haptoglobin, hemopexin and orosomucoid). The enzyme implicated in the biotransformation of CYP family members (LOC644587) was decreased. The genes coding enzymes ADH1C, ALDH6A1, ALDOB, Arginase and CES1 were also found. Additionally, we isolated a zinc transporter (Zip14) and a function-unknown gene named ZBTB11 (Zinc finger and BTB domain containing 11) which were underexpressed, and seven expression sequence tags deregulated in HCC without significant homology reported in the public database. Essentially, by using SSH combined with a cDNA microarray we have identified a number of genes associated with HCC, most of which have not been previously reported. Further characterization of these differentially expressed

  10. Deficiency of a glycogen synthase-associated protein, Epm2aip1, causes decreased glycogen synthesis and hepatic insulin resistance.

    Science.gov (United States)

    Turnbull, Julie; Tiberia, Erica; Pereira, Sandra; Zhao, Xiaochu; Pencea, Nela; Wheeler, Anne L; Yu, Wen Qin; Ivovic, Alexander; Naranian, Taline; Israelian, Nyrie; Draginov, Arman; Piliguian, Mark; Frankland, Paul W; Wang, Peixiang; Ackerley, Cameron A; Giacca, Adria; Minassian, Berge A

    2013-11-29

    Glycogen synthesis is a major component of the insulin response, and defective glycogen synthesis is a major portion of insulin resistance. Insulin regulates glycogen synthase (GS) through incompletely defined pathways that activate the enzyme through dephosphorylation and, more potently, allosteric activation. We identify Epm2aip1 as a GS-associated protein. We show that the absence of Epm2aip1 in mice impairs allosteric activation of GS by glucose 6-phosphate, decreases hepatic glycogen synthesis, increases liver fat, causes hepatic insulin resistance, and protects against age-related obesity. Our work identifies a novel GS-associated GS activity-modulating component of insulin resistance.

  11. Optimized cDNA libraries for virus-induced gene silencing (VIGS using tobacco rattle virus

    Directory of Open Access Journals (Sweden)

    Page Jonathan E

    2008-01-01

    Full Text Available Abstract Background Virus-induced gene silencing (VIGS has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV. Results NbPDS inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the NbPDS cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A or poly(G homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated N. benthamiana roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401–500 bp in length and 99.5% lacked poly(A tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine N-methyltransferase (PMT, with ten different VIGS-NbPMT constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs. Conclusion Based on the silencing of NbPDS and NbPMT, we suggest the following design guidelines for constructs in TRV vectors: (1 Insert lengths should be in the range of ~200 bp to ~1300 bp, (2 they should be positioned in

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

    Science.gov (United States)

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

    2009-01-01

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

  13. Development of an HIV-based cDNA expression cloning system.

    Science.gov (United States)

    van Maanen, Marc; Tidwell, Jennie K; Donehower, Lawrence A; Sutton, Richard E

    2003-07-01

    Expression cloning of cDNAs is a powerful tool with which to identify genes based on their specific functional properties. Here we describe the development of a cDNA library transfer system based on the human immunodeficiency virus type-1 (HIV). This system represents an improvement over current oncoretroviral cDNA expression systems in terms of target cell range and the inclusion of a selectable marker. By use of a simple packaging system, we were able to produce high-titer vector stocks from HIV vector-based cDNA libraries and demonstrate highly efficient cDNA expression cloning in three model experiments. First, HOS TK(-) cells, which are null for thymidine kinase (TK) expression, were transduced with an HIV-based cDNA library derived from primary human foreskin fibroblasts (HFFs) and functionally selected for TK expression. In a second experiment, hypoxanthine guanine phosphoribosyltransferase-1-deficient (HPRT(-)) fibroblasts were transduced with a T cell (PM1) line-derived cDNA library and selected for HPRT expression. Both TK (frequency 1 in 5.0 x 10(4)) and HPRT (frequency 1 in 2.0 x 10(4)) cDNAs were readily isolated from these HIV-based cDNA libraries. As a third example, we demonstrated the ability of this vector system to allow functional cDNA library screens to be performed in primary, mitotically inactive cell types. Using senescent HFFs as a target cell population, we were able to isolate SV40 large T antigen cDNA-containing clones (frequency 1 in 2.5 x 10(4)) based on their ability to overcome the senescence-induced block to cell proliferation. Thus, this system can be used to clone relatively low-abundance cDNAs based upon their expression. Because of the ability of HIV-based vectors to transduce primary and nondividing cells efficiently, this vector system will further broaden the range of cell types in which expression cloning studies can be performed.

  14. Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase

    Science.gov (United States)

    Ober, Dietrich; Hartmann, Thomas

    1999-01-01

    Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids. PMID:10611289

  15. American Liver Foundation

    Science.gov (United States)

    ... Progression of Liver Disease Reye Syndrome Type I Glycogen Storage Disease Wilson Disease Find Your Local Division ... about liver cancer HERE . Thanks to Hep B Free San Francisco, our campaign partner in Northern California, ...

  16. Antioxidants in liver health

    Science.gov (United States)

    Casas-Grajales, Sael; Muriel, Pablo

    2015-01-01

    Liver diseases are a worldwide medical problem because the liver is the principal detoxifying organ and maintains metabolic homeostasis. The liver metabolizes various compounds that produce free radicals (FR). However, antioxidants scavenge FR and maintain the oxidative/antioxidative balance in the liver. When the liver oxidative/antioxidative balance is disrupted, the state is termed oxidative stress. Oxidative stress leads to deleterious processes in the liver and produces liver diseases. Therefore, restoring antioxidants is essential to maintain homeostasis. One method of restoring antioxidants is to consume natural compounds with antioxidant capacity. The objective of this review is to provide information pertaining to various antioxidants found in food that have demonstrated utility in improving liver diseases. PMID:26261734

  17. Diet - liver disease

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/002441.htm Diet - liver disease To use the sharing features on this page, please enable JavaScript. Some people with liver disease must eat a special diet. This diet ...

  18. Autoimmune liver disease panel

    Science.gov (United States)

    Liver disease test panel - autoimmune ... Autoimmune disorders are a possible cause of liver disease. The most common of these diseases are autoimmune hepatitis and primary biliary cholangitis (formerly called primary biliary cirrhosis). This group of tests ...

  19. Hepatic (Liver) Function Panel

    Science.gov (United States)

    ... or kidney disease, or nutritional problems. Liver enzymes: Alkaline phosphatase (ALP), alanine aminotransferase (ALT) , and aspartate aminotransferase (AST) . These enzymes help the liver convert food into energy. When their levels are high, it ...

  20. Liver Function Tests

    Science.gov (United States)

    ... Legacy Society Make Gifts of Stock Donate Your Car Personal Fundraising Partnership & Support Share Your Story Spread the Word Give While You Shop Contact Us Donate Now Diagnosing Liver Disease – Liver ...

  1. Novel applications of plant polyketide synthases.

    Science.gov (United States)

    Abe, Ikuro

    2012-04-01

    The structurally and mechanistically simple type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkably diverse structures and biological activities. By exploiting the enzymes, we combined precursor-directed biosynthesis with nitrogen-containing substrates and structure-based enzyme engineering and generated unnatural, novel polyketide-alkaloid scaffolds with promising biological activities. The nucleophilic nitrogen atom and the engineered enzymes thus facilitated the formation of additional CC and CN bonds during the enzymatic transformations. The methodology will contribute to the further production of chemically and structurally divergent, unnatural natural products, as well as the rational design of novel biocatalysts with unprecedented catalytic functions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Tyrosine nitration affects thymidylate synthase properties.

    Science.gov (United States)

    Dąbrowska-Maś, Elżbieta; Frączyk, Tomasz; Ruman, Tomasz; Radziszewska, Karolina; Wilk, Piotr; Cieśla, Joanna; Zieliński, Zbigniew; Jurkiewicz, Agata; Gołos, Barbara; Wińska, Patrycja; Wałajtys-Rode, Elżbieta; Leś, Andrzej; Nizioł, Joanna; Jarmuła, Adam; Stefanowicz, Piotr; Szewczuk, Zbigniew; Rode, Wojciech

    2012-01-14

    Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.

  3. CLYBL is a polymorphic human enzyme with malate synthase and β-methylmalate synthase activity

    Science.gov (United States)

    Strittmatter, Laura; Li, Yang; Nakatsuka, Nathan J.; Calvo, Sarah E.; Grabarek, Zenon; Mootha, Vamsi K.

    2014-01-01

    CLYBL is a human mitochondrial enzyme of unknown function that is found in multiple eukaryotic taxa and conserved to bacteria. The protein is expressed in the mitochondria of all mammalian organs, with highest expression in brown fat and kidney. Approximately 5% of all humans harbor a premature stop polymorphism in CLYBL that has been associated with reduced levels of circulating vitamin B12. Using comparative genomics, we now show that CLYBL is strongly co-expressed with and co-evolved specifically with other components of the mitochondrial B12 pathway. We confirm that the premature stop polymorphism in CLYBL leads to a loss of protein expression. To elucidate the molecular function of CLYBL, we used comparative operon analysis, structural modeling and enzyme kinetics. We report that CLYBL encodes a malate/β-methylmalate synthase, converting glyoxylate and acetyl-CoA to malate, or glyoxylate and propionyl-CoA to β-methylmalate. Malate synthases are best known for their established role in the glyoxylate shunt of plants and lower organisms and are traditionally described as not occurring in humans. The broader role of a malate/β-methylmalate synthase in human physiology and its mechanistic link to vitamin B12 metabolism remain unknown. PMID:24334609

  4. Molecular Pathogenesis of Liver Steatosis Induced by Hepatitis C Virus

    Directory of Open Access Journals (Sweden)

    Cheng Jun

    2012-09-01

    Full Text Available Liver steatosis is a pathological hallmark in patients with chronic hepatitis C (CHC. Increased lipid uptake, decreased lipid secretion, increased lipid synthesis and decreased lipid degradation are all involved in pathogenesis of steatosis induced by hepatitic C virus (HCV infection. Level of low density lipoprotein receptor (LDL-R and activity of peroxisome proliferator-activated receptor (PPAR α is related to liver uptake of lipid from circulation, and affected by HCV. Secretion via microsomal triglyceride transfer protein (MTTP, and formation of very low density lipoprotein (VLDL have been hampered by HCV infection. Up-regulation of lipid synthesis related genes, such as sterol regulatory element-binding protein (SREBP-1, SREBP-2, SREBP-1c, fatty acid synthase (FASN, HMG CoA reductase (HMGCR, liver X receptor (LXR, acetyl-CoA carboxylase 1 (ACC1, hepatic CB (1 receptors, retinoid X receptor (RXR α, were the main stay of liver steatosis pathogenesis. Degradation of lipid in liver is decreased in patients with CHC. There is strong evidence that heterogeneity of HCV core genes of different genotypes affect their effects of liver steatosis induction. A mechanism in which steatosis is involved in HCV life cycle is emerging.

  5. Immune mediated liver failure.

    Science.gov (United States)

    Wang, Xiaojing; Ning, Qin

    2014-01-01

    Liver failure is a clinical syndrome of various etiologies, manifesting as jaundice, encephalopathy, coagulopathy and circulatory dysfunction, which result in subsequent multiorgan failure. Clinically, liver failure is classified into four categories: acute, subacute, acute-on-chronic and chronic liver failure. Massive hepatocyte death is considered to be the core event in the development of liver failure, which occurs when the extent of hepatocyte death is beyond the liver regenerative capacity. Direct damage and immune-mediated liver injury are two major factors involved in this process. Increasing evidence has suggested the essential role of immune-mediated liver injury in the pathogenesis of liver failure. Here, we review the evolved concepts concerning the mechanisms of immune-mediated liver injury in liver failure from human and animal studies. Both innate and adaptive immunity, especially the interaction of various immune cells and molecules as well as death receptor signaling system are discussed. In addition, we highlight the concept of "immune coagulation", which has been shown to be related to the disease progression and liver injury exacerbation in HBV related acute-on-chronic liver failure.

  6. Liver transplantation : an update

    NARCIS (Netherlands)

    Verdonk, R. C.; Van den Berg, A. P.; Slooff, M. J. H.; Porte, R. J.; Haagsma, E. B.

    2007-01-01

    Liver transplantation has been an accepted treatment for end-stage liver disease since the 1980s. Currently it is a highly successful treatment for this indication. The aim of this review is to give a general update on recent developments in the field of liver transplantation. In the last decades

  7. Liver Tumors (For Parents)

    Science.gov (United States)

    ... Staying Safe Videos for Educators Search English Español Liver Tumors KidsHealth / For Parents / Liver Tumors What's in this article? Types of Tumors ... Cancerous) Tumors Symptoms Diagnosis Treatment Coping Print The liver is the body's largest solid organ. Lying next ...

  8. 492 Review Liver

    African Journals Online (AJOL)

    Marinda

    2009-11-26

    Nov 26, 2009 ... Severe bleeding still occurs in a minority of cases and efforts to define clinical and blood test predictors for major bleeding during liver surgery remain elusive. Excessive bleeding during liver surgery can be due to surgical factors, haemostatic problems due to liver disease or other causes and poor ...

  9. Acetolactate Synthase Activity in Developing Maize (Zea mays L.) Kernels

    Science.gov (United States)

    Muhitch, Michael J.

    1988-01-01

    Acetolactate synthase (EC 4.1.3.18) activity was examined in maize (Zea mays L.) endosperm and embryos as a function of kernel development. When assayed using unpurified homogenates, embryo acetolactate synthase activity appeared less sensitive to inhibition by leucine + valine and by the imidazolinone herbicide imazapyr than endosperm acetolactate synthase activity. Evidence is presented to show that pyruvate decarboxylase contributes to apparent acetolactate synthase activity in crude embryo extracts and a modification of the acetolactate synthase assay is proposed to correct for the presence of pyruvate decarboxylase in unpurified plant homogenates. Endosperm acetolactate synthase activity increased rapidly during early kernel development, reaching a maximum of 3 micromoles acetoin per hour per endosperm at 25 days after pollination. In contrast, embryo activity was low in young kernels and steadily increased throughout development to a maximum activity of 0.24 micromole per hour per embryo by 45 days after pollination. The sensitivity of both endosperm and embryo acetolactate synthase activities to feedback inhibition by leucine + valine did not change during kernel development. The results are compared to those found for other enzymes of nitrogen metabolism and discussed with respect to the potential roles of the embryo and endosperm in providing amino acids for storage protein synthesis. PMID:16665871

  10. Liver metabolism traits in two rabbit lines divergently selected for intramuscular fat.

    Science.gov (United States)

    Martínez-Álvaro, M; Paucar, Y; Satué, K; Blasco, A; Hernández, P

    2017-10-26

    Intramuscular fat (IMF) has a large effect in the sensory properties of meat because it affects tenderness, juiciness and flavour. A divergent selection experiment for IMF in longissimus dorsi (LD) muscle was performed in rabbits. Since liver is the major site of lipogenesis in rabbits, the objective of this work is to study the liver metabolism in the lines of the divergent selection experiment. Intramuscular fat content, perirenal fat weight, liver weight, liver lipogenic activities and plasma metabolites related to liver metabolism were measured in the eighth generation of selection. Direct response on IMF was 0.34 g/100 g of LD, which represented 2.7 SD of the trait, and selection showed a positive correlated response in the perirenal fat weight. High-IMF line showed greater liver size and greater liver lipogenic activities of enzymes glucose-6-phosphate dehydrogenase and malic enzyme. We did not find differences between lines for fatty acid synthase lipogenic activity. With regard to plasma metabolites, low-IMF line showed greater plasma concentration of triglycerides, cholesterol, bilirubin and alkaline phosphatase than high-IMF line, whereas high-IMF line showed greater albumin and alanine transaminase concentrations than low-IMF line. We did not observe differences between lines for glucose, total protein and plasma concentrations. Phenotypic correlations between fat (IMF and perirenal fat weight) and liver traits showed that liver lipogenesis affects fat deposition in both, muscle and carcass. However, the mechanisms whereby liver lipogenesis affected IMF content remain to be clarified.

  11. Hypervascular liver lesions in radiologically normal liver

    Energy Technology Data Exchange (ETDEWEB)

    Amico, Enio Campos; Alves, Jose Roberto; Souza, Dyego Leandro Bezerra de; Salviano, Fellipe Alexandre Macena; Joao, Samir Assi; Liguori, Adriano de Araujo Lima, E-mail: ecamic@uol.com.br [Hospital Universitario Onofre Lopes (HUOL/UFRN), Natal, RN (Brazil). Clinica Gastrocentro e Ambulatorios de Cirurgia do Aparelho Digestivo e de Cirurgia Hepatobiliopancreatica

    2017-09-01

    Background: The hypervascular liver lesions represent a diagnostic challenge. Aim: To identify risk factors for cancer in patients with non-hemangiomatous hypervascular hepatic lesions in radiologically normal liver. Method: This prospective study included patients with hypervascular liver lesions in radiologically normal liver. The diagnosis was made by biopsy or was presumed on the basis of radiologic stability in follow-up period of one year. Cirrhosis or patients with typical imaging characteristics of haemangioma were excluded. Results: Eighty eight patients were included. The average age was 42.4. The lesions were unique and were between 2-5 cm in size in most cases. Liver biopsy was performed in approximately 1/3 of cases. The lesions were benign or most likely benign in 81.8%, while cancer was diagnosed in 12.5% of cases. Univariate analysis showed that age >45 years (p< 0.001), personal history of cancer (p=0.020), presence of >3 nodules (p=0.003) and elevated alkaline phosphatase (p=0.013) were significant risk factors for cancer. Conclusion: It is safe to observe hypervascular liver lesions in normal liver in patients up to 45 years, normal alanine amino transaminase, up to three nodules and no personal history of cancer. Lesion biopsies are safe in patients with atypical lesions and define the treatment to be established for most of these patients. (author)

  12. Hypervascular liver lesions in radiologically normal liver

    International Nuclear Information System (INIS)

    Amico, Enio Campos; Alves, Jose Roberto; Souza, Dyego Leandro Bezerra de; Salviano, Fellipe Alexandre Macena; Joao, Samir Assi; Liguori, Adriano de Araujo Lima

    2017-01-01

    Background: The hypervascular liver lesions represent a diagnostic challenge. Aim: To identify risk factors for cancer in patients with non-hemangiomatous hypervascular hepatic lesions in radiologically normal liver. Method: This prospective study included patients with hypervascular liver lesions in radiologically normal liver. The diagnosis was made by biopsy or was presumed on the basis of radiologic stability in follow-up period of one year. Cirrhosis or patients with typical imaging characteristics of haemangioma were excluded. Results: Eighty eight patients were included. The average age was 42.4. The lesions were unique and were between 2-5 cm in size in most cases. Liver biopsy was performed in approximately 1/3 of cases. The lesions were benign or most likely benign in 81.8%, while cancer was diagnosed in 12.5% of cases. Univariate analysis showed that age >45 years (p< 0.001), personal history of cancer (p=0.020), presence of >3 nodules (p=0.003) and elevated alkaline phosphatase (p=0.013) were significant risk factors for cancer. Conclusion: It is safe to observe hypervascular liver lesions in normal liver in patients up to 45 years, normal alanine amino transaminase, up to three nodules and no personal history of cancer. Lesion biopsies are safe in patients with atypical lesions and define the treatment to be established for most of these patients. (author)

  13. N-acetylglutamate synthase deficiency: an insight into the genetics, epidemiology, pathophysiology, and treatment

    Directory of Open Access Journals (Sweden)

    Caldovic L

    2011-08-01

    Full Text Available Nicholas Ah Mew, Ljubica CaldovicCenter for Genetic Medicine Research, Children’s Research Institute, Children’s National Medical Center, Washington DC, USAAbstract: The conversion of ammonia into urea by the human liver requires the coordinated function of the 6 enzymes and 2 transporters of the urea cycle. The initial and rate-limiting enzyme of the urea cycle, carbamylphosphate synthetase 1 (CPS1, requires an allosteric activator, N-acetylglutamate (NAG. The formation of this unique cofactor from glutamate and acetyl Coenzyme-A is catalyzed by N-acetylglutamate synthase (NAGS. An absence of NAG as a consequence of NAGS deficiency may compromise flux through CPS1 and result in hyperammonemia. The NAGS gene encodes a 528-amino acid protein, consisting of a C-terminal catalytic domain, a variable segment, and an N-terminal mitochondrial targeting signal. Only 22 mutations in the NAGS gene have been reported to date, mostly in the catalytic domain. NAGS is primarily expressed in the liver and intestine. However, it is also surprisingly expressed in testis, stomach and spleen, and during early embryonic development at levels not concordant with the expression of other urea cycle enzymes, CPS1, or ornithine transcarbamylase. The purpose of NAGS expression in these tissues, and its significance to NAGS deficiency is as yet unknown. Inherited NAGS deficiency is the rarest of the urea cycle disorders, and we review the currently reported 34 cases. Treatment of NAGS deficiency with N-carbamyglutamate, a stable analog of NAG, can restore deficient urea cycle function and normalize blood ammonia in affected patients.Keywords: urea cycle, urea cycle disorder, N-acetyl-L-glutamate, N-acetylglutamate synthase, hyperammonemia, N-carbamyl-L-glutamate

  14. Liver and gastrointestinal tract

    International Nuclear Information System (INIS)

    Shahid, M.A.

    1992-01-01

    Liver is often a site of a variety of diseases. A palpable liver during a routine clinical examination is an important finding and requires further investigations. The availability of non-invasive liver imaging procedures using nuclear, ultrasound, CT (and now MRI) techniques have immensely enhanced diagnostic accuracy in liver diseases. In this Chapter, a detailed description of routinely practised nuclear medicine procedures related to liver is given. Brief reference is also made to other imaging techniques, particularly ultrasonography, only for the purposes of comparison. Most of the information is based on our own clinical experience of past 30 years

  15. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown-PCR technology ...

  16. Cloning and sequencing of complete τ-crystallin cDNA from ...

    Indian Academy of Sciences (India)

    Unknown

    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 τ-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 ...

  17. 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

  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. 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.

  20. cDNA, genomic sequence cloning and analysis of the ribosomal ...

    African Journals Online (AJOL)

    Ribosomal protein L37A (RPL37A) is a component of 60S large ribosomal subunit encoded by the RPL37A gene, which belongs to the family of ribosomal L37AE proteins, located in the cytoplasm. The complementary deoxyribonucleic acid (cDNA) and the genomic sequence of RPL37A were cloned successfully from giant ...

  1. Construction and primary characterization of Echinococcus multilocularis protoscolex cDNA expression library.

    Science.gov (United States)

    Li, S; Chen, Y

    2001-02-01

    To construct a lambda gt11 cDNA expression library of Echinococcus multilocularis protoscolex isolated in China. Echinococcus multilocularis protoscolex mRNA was extracted using a Quickprep MicromRNA purification kit based on combining of the disruptive and protective properties of guanidinium thiocyanate (GTC) with the speed and selectivity of oligo (dT)-cellulose chromatography in a spum-column with some modification. Purified mRNA (1.8 micrograms) was submitted to reverse transcription using random hexamers [pd(N6)]. The double-strand blunt-ended cDNAs were ligated with an EcoRI/Notl adaptor to form a cohesive EcoRI end. Subsequently the synthesized cDNA was inserted into vector lambda gt11 EcoRI arms. After being packaged in vitro, lambda gt11 was put to an infectious bacteria Echinococcus coli (E. coli) strain Y1090; the recombinants were screened by color selection. PCR amplification was performed to evaluate the size of insertion DNA fragments. The recombinant ratio was nearly 100% and approximately 1 x 10(6) clones could be derived from this lambda gt11 cDNA library. PCR results indicated that the insertion DNAs were about 1.48 kb. A lambda gt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multicularis protoscolex mRNA. Further studies on this library are deserved.

  2. 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...

  3. Cloning a cDNA for the lysosomal alpha-glucosidase

    NARCIS (Netherlands)

    KONINGS, A.; HUPKES, P.; Versteeg, R.; Grosveld, G.; Reuser, A.; Galjaard, H.

    1984-01-01

    Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most

  4. Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library

    NARCIS (Netherlands)

    Akkerdaas, Jaap H.; Schocker, Frauke; Vieths, Stefan; Versteeg, Serge; Zuidmeer, Laurian; Hefle, Sue L.; Aalberse, Rob C.; Richter, Klaus; Ferreira, Fatima; van Ree, Ronald

    2006-01-01

    The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA

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

    Science.gov (United States)

    Cartolano, Maria; Huettel, Bruno; Hartwig, Benjamin; Reinhardt, Richard; Schneeberger, Korbinian

    2016-01-01

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

  6. 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...

  7. Primary structure of a lipoxygenase from barley grain as deduced from its cDNA sequence

    NARCIS (Netherlands)

    Mechelen, J.R. van; Smits, M.; Douma, A.C.; Rouster, J.; Cameron-Mills, V.; Heidekamp, F.; Valk, B.E.

    1995-01-01

    A full length cDNA sequence for a barley grain lipoxygenase was obtained. It includes a 5' untranslated region of 69 nucleotides, an open reading frame of 2586 nucleotides encoding a protein of 862 amino acid residues and a 3' untranslated region of 142 nucleotides. The molecular mass of the encoded

  8. cDNA cloning and expression analysis of two distinct Sox8 genes in ...

    Indian Academy of Sciences (India)

    2010-08-06

    Aug 6, 2010 ... cDNA cloning and expression analysis of two distinct Sox8 genes in. Paramisgurnus dabryanus (Cypriniformes). XIAOHUA XIA, JIE ZHAO, QIYAN DU and ZHONGJIE CHANG. ∗. Molecular and Genetic Laboratory, College of Life Sciences, Henan Normal University, 46 East of Construction Road,. Xinxiang ...

  9. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    enoh

    2012-03-13

    Mar 13, 2012 ... Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The. cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown- ...

  10. Construction and characterization of a cDNA library from human ...

    African Journals Online (AJOL)

    The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network, and the challenge to understand the network is to identify p53 downstream genes. In order to isolate and identify new p53 regulated genes, we constructed and characterized a normalized cDNA library from human brain ...

  11. First-strand cDNA synthesis primed with oligo(dT)

    International Nuclear Information System (INIS)

    Krug, M.S.; Berger, S.L.

    1987-01-01

    The quality of a cDNA library depends on the integrity of the messenger RNA and the fidelity with which it can be reverse transcribed. RNA cannot be cloned directly; in a reaction catalyzed by reverse transcriptase, the RNA, together with a suitable primer and a supply of deoxyribonucleoside triphosphates (dNTPs), must be converted to a double-stranded molecule. The product contains a complementary strand (first, antisense, or minus-strand cDNA) that is hybridized to what remains of the original RNA template. Such DNA-RNA hybrids can be cloned albeit often with lower efficiency than their double-stranded DNA counterparts. Usually the hybrid molecules are treated as intermediates in a scheme aimed at replacing the fragmented RNA with continuous DNA to form a double-stranded cDNA molecule. From this brief summary of cDNA cloning, it should be obvious that, regardless of the strategy, reverse transcriptase does and how it does it in vitro is discussed

  12. 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...

  13. cDNA cloning and primary structure analysis of invariant chain in ...

    African Journals Online (AJOL)

    cDNA cloning and primary structure analysis of invariant chain in Chinese Pengze crucian carp. X Liu, W Yu, J Li, F Chen, S Liu, C Wu, J Xu. Abstract. Invariant chain (Ii) plays an important role in MHC class II molecules assembly and exogenous peptide presentation in vertebrates. Although mammalian Ii has been ...

  14. Molecular cloning of growth hormone encoding cDNA of Indian ...

    Indian Academy of Sciences (India)

    Unknown

    Evans and Long 1921) and the human growth hormone (GH) encoding cDNA was per- haps the first to be isolated and characterized (Li and. Evans 1944). GH, chorionic somatomamotropin (placental lactogen) and prolactin (PRL) are all a family of ...

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

    Science.gov (United States)

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

    2003-11-01

    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. 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 SMART 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. 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. 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 gastrocarcinoma.

  16. New Approaches to Attenuated Hepatitis a Vaccine Development: Cloning and Sequencing of Cell-Culture Adapted Viral cDNA.

    Science.gov (United States)

    1987-10-13

    Insert frag- ments from p16 cDNA clones were subcloned into the phage vector Ml3mp8 or Ml3mpl9 qnd subjected to rapid sequencing using the...2), and selected cDNA insert fragments were subcloned into M13 vectors for sequencing. The sequence of the complete genome was determined, with over

  17. 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...

  18. Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L.

    Science.gov (United States)

    Mercke, P; Bengtsson, M; Bouwmeester, H J; Posthumus, M A; Brodelius, P E

    2000-09-15

    In plants, sesquiterpenes of different structural types are biosynthesized from the isoprenoid intermediate farnesyl diphosphate. The initial reaction of the biosynthesis is catalyzed by sesquiterpene cyclases (synthases). In Artemisia annua L. (annual wormwood), a number of such sesquiterpene cyclases are active. We have isolated a cDNA clone encoding one of these, amorpha-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis. This clone contains a 1641-bp open reading frame coding for 546 amino acids (63.9 kDa), a 12-bp 5'-untranslated end, and a 427-bp 3'-untranslated sequence. The deduced amino acid sequence is 32 to 51% identical with the sequence of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5%) and oxygenated (2.5%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as (E)-beta-farnesene (0.8%), amorpha-4,11diene (91.2%), amorpha-4,7(11)-diene (3.7%), gamma-humulene (1.0%), beta-sesquiphellandrene (0.5%), and an unknown olefin (0.2%) and the oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorpha-4-en-7-ol (2.1%), and alpha-bisabolol (0.3%) (tentatively). Using geranyl diphosphate as substrate, amorpha-4,11-diene synthase did not produce any monoterpenes. The recombinant enzyme has a broad pH optimum between 7.5 and 9.0 and the Km values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 70, and 13 microM, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested.

  19. Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A

    International Nuclear Information System (INIS)

    Miziorko, H.M.; Behnke, C.E.

    1985-01-01

    3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1- 14 C]-3-Chloropropionyl-CoA binds covalently to the enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of the enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [ 14 C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue

  20. 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

  1. [Construction and immunoscreening of cDNA library of Babesia orientalis].

    Science.gov (United States)

    Liu, Qin; Zhou, Dan-Na; Zhou, Yan-Qin; Zhang, Ying; He, Lan; Yao, Bao-An; Zhao, Jun-Long

    2009-06-01

    To construct a cDNA library for Babesia orientalis and screen immunologically positive clones. Total RNA of B. orientalis in red blood cells from an infected calf was isolated. cDNA was synthesized by reverse transcriptase, amplified by PCR and ligated into lambdaTriplEx2 vector. The recombined vectors were packaged and the unamplified cDNA library was constructed. The cDNA library was then amplified and immunologically screened with rabbit anti-B. orientalis serum. The recombinant lambdaTriplEx2 of positive clones were converted to the corresponding recombinant pTriplEx2. The inserted fragments were identified by PCR amplification. The plasmids were sequenced and compared against GenBank database by Blast. The titer of the unamplified library was 2.0 x 10(6) pfu/ml. The inserted fragment length of the library ranged from 500 to 3,000 bp, and the recombination efficiency accounted for 98.8%. The titer of the amplified library was 5.8 x 10(8) pfu/ml. Three positive clones were selected by serum immunological screening and named B04, B05, and B41, respectively. The inserted fragments of the B04, B05 and B41 were about 1,300 bp, 1,000 bp, and 2,400 bp, respectively. Sequence analysis revealed that the 3 clones contained open reading frames. Blast results showed that they were highly homologous to the nuclear movement protein gene, the hypothetical protein gene and the heat shock protein 70 (HSP70) gene, respectively. The deduced amino acid sequences of B04, B05 and B41 contained 310, 192 and 647 amino acid residues, with Mr of 34,000, 21,000, and 70,700, respectively. A qualified cDNA library of B. orientalis has been constructed and three positive clones of B. orientalis discovered.

  2. Screening a cDNA Library for Protein–Protein Interactions Directly in Planta[W

    Science.gov (United States)

    Lee, Lan-Ying; Wu, Fu-Hui; Hsu, Chen-Tran; Shen, Shu-Chen; Yeh, Hsuan-Yu; Liao, De-Chih; Fang, Mei-Jane; Liu, Nien-Tze; Yen, Yu-Chen; Dokládal, Ladislav; Sýkorová, Eva; Gelvin, Stanton B.; Lin, Choun-Sea

    2012-01-01

    Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein– or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ∼2 × 105 cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species. PMID:22623495

  3. Chalcone synthase genes from milk thistle (Silybum marianum ...

    Indian Academy of Sciences (India)

    Leyva et al. 1995), UV treatments and blue light (Hartmann et al. 1998; Wade et al. 2001; Zhou et al. 2007), elicitor treatments such as salicylic acid and. Keywords. chalcone synthase; real-time PCR; silymarin; anthocyanin; Silybum marianum.

  4. Identification and expression analysis of peroxisome proliferator-activated receptors cDNA in a reptile, the leopard gecko (Eublepharis macularius).

    Science.gov (United States)

    Kato, Keisuke; Oka, Yoshitaka; Park, Min Kyun

    2008-05-01

    Despite the physiological and evolutionary significance of lipid metabolism in amniotes, the molecular mechanisms involved have been unclear in reptiles. To elucidate this, we investigated peroxisome proliferators-activated receptors (PPARs) in the leopard gecko (Eublepharis macularius). PPARs belong to a nuclear hormone-receptor family mainly involved in lipid metabolism. Although PPARs have been widely studied in mammals, little information about them is yet available from reptiles. We identified in the leopard gecko partial cDNA sequences of PPARalpha and beta, and full sequences of two isoforms of PPARgamma. This is the first report of reptilian PPARgamma mRNA isoforms. We also evaluated the organ distribution of expression of these genes by using RT-PCR and competitive PCR. The expression level of PPARalpha mRNA was highest in the large intestine, and moderate in the liver and kidney. The expression level of PPARbeta mRNA was highest in the kidney and large intestine, and moderate in the liver. Similarly to the expression of human PPARgamma isoforms, PPARgammaa was expressed ubiquitously, whereas the expression of PPARgammab was restricted. The highest levels of their expression, however, were observed in the large intestine, rather than in the adipose tissue as in mammals. Taken together, these results showed that the profile of PPARbeta mRNA expression in the leopard gecko is similar to that in mammals, and that those of PPAR alpha and gamma are species specific. This may reflect adaptation to annual changes in lipid storage due to seasonal food availability.

  5. Hydrodynamic delivery of human IL-15 cDNA increases murine natural killer cell recovery after syngeneic bone marrow transplantation.

    Science.gov (United States)

    Barao, Isabel; Alvarez, Maite; Redelman, Doug; Weiss, Jonathan M; Ortaldo, John R; Wiltrout, Robert H; Murphy, William J

    2011-12-01

    Immune deficiency immediately following bone marrow transplantation (BMT) increases susceptibility to opportunistic infections as well as tumor relapse. Natural Killer (NK) cells play important roles in the resistance to virally infected and transformed cells. Interleukin (IL)-15 has been shown to be essential for NK cell development and survival. We administered human (h) IL-15 cDNA (pIL-15) via hydrodynamic delivery to murine recipients undergoing congenic BMT to determine its effects on NK cell reconstitution. Hydrodynamic pIL-15 delivery resulted in high levels of hIL-15 protein in the serum that lasted for several days and then quickly declined. The appearance of hIL-15 was followed by a significant increase of mature donor-derived NK cells within the bone marrow, spleens, and livers of the treated recipients. No accumulation of immature NK cell progenitors was observed. The NK cells from IL-15-treated recipients displayed an activated phenotype and were lytically active toward tumor targets in vitro to a similar degree as did those cells from recipients treated with control plasmid. This suggests that the predominant effect of IL-15 was a quantitative increase in total NK cell numbers and not qualitative changes in NK cell functions. No toxicities or adverse effects were observed. Studies performed in transplanted mice bearing renal carcinoma tumors demonstrated that this mode of hIL-15 gene delivery resulted in increased antitumor responses. These results support the use of cytokine gene transfer-based regimens as a platform to augment NK cell recovery after BMT. Copyright © 2011 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  6. A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase.

    Science.gov (United States)

    Ahmad, Zulfiqar; Hassan, Sherif S; Azim, Sofiya

    2017-11-20

    For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phytochemicals is based on tradition or word of mouth with few evidence-based studies. Moreover, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become pertinent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of dietary phytochemicals are known to inhibit ATP synthase. Structural modifications of phytochemicals have been shown to increase the inhibitory potency and extent of inhibition. Sitedirected mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can result in selective binding and inhibition of microbial ATP synthase. In this review, the therapeutic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective targeting of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  7. Engineering fatty acid synthases for directed polyketide production.

    Science.gov (United States)

    Gajewski, Jan; Buelens, Floris; Serdjukow, Sascha; Janßen, Melanie; Cortina, Niña; Grubmüller, Helmut; Grininger, Martin

    2017-04-01

    In this study, we engineered fatty acid synthases (FAS) for the biosynthesis of short-chain fatty acids and polyketides, guided by a combined in vitro and in silico approach. Along with exploring the synthetic capability of FAS, we aim to build a foundation for efficient protein engineering, with the specific goal of harnessing evolutionarily related megadalton-scale polyketide synthases (PKS) for the tailored production of bioactive natural compounds.

  8. Regulation of CDP-diacylglycerol synthase activity in Saccharomyces cerevisiae.

    OpenAIRE

    Homann, M J; Henry, S A; Carman, G M

    1985-01-01

    The addition of ethanolamine or choline to inositol-containing growth medium resulted in a reduction of CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) activity in Saccharomyces cerevisiae. The reduction of activity did not occur in the absence of inositol. CDP-diacylglycerol synthase activity was not regulated in a S. cerevisiae mutant strain (opi1; an inositol biosynthesis regulatory mutant) by the addition of phospholipid precursors to the growth medium.

  9. Understanding plant cellulose synthases through a comprehensive investigation of the cellulose synthase family sequences.

    Directory of Open Access Journals (Sweden)

    Andrew eCarroll

    2011-03-01

    Full Text Available The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized subfamilies and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair cellulose synthase function, and compare these sites to those observed in the closest cellulose synthase-like (Csl families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments.

  10. Homocystinuria due to cystathionine beta synthase deficiency

    Directory of Open Access Journals (Sweden)

    Rao T

    2008-01-01

    Full Text Available A two year-old male child presented with cutis marmorata congenita universalis, brittle hair, mild mental retardation, and finger spasms. Biochemical findings include increased levels of homocysteine in the blood-106.62 µmol/L (normal levels: 5.90-16µmol/L. Biochemical tests such as the silver nitroprusside and nitroprusside tests were positive suggesting homocystinuria. The patient was treated with oral pyridoxine therapy for three months. The child responded well to this therapy and the muscle spasms as well as skin manifestations such as cutis marmorata subsided. The treatment is being continued; the case is reported here because of its rarity. Homocysteinuria arising due to cystathionine beta-synthase (CBS deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine It manifests itself in vascular, central nervous system, cutaneous, and connective tissue disturbances and phenotypically resembles Marfan′s syndrome. Skin manifestations include malar flush, thin hair, and cutis reticulata / marmorata.

  11. Type III polyketide synthases in microorganisms.

    Science.gov (United States)

    Katsuyama, Yohei; Ohnishi, Yasuo

    2012-01-01

    Type III polyketide synthases (PKSs) are simple homodimers of ketosynthases which catalyze the condensation of one to several molecules of extender substrate onto a starter substrate through iterative decarboxylative Claisen condensation reactions. Type III PKSs have been found in bacteria and fungi, as well as plants. Microbial type III PKSs, which are involved in the biosynthesis of some lipidic compounds and various secondary metabolites, have several interesting characteristics that are not shared by plant type III PKSs. Further, many compounds produced by microbial type III PKSs have significant biological functions and/or important pharmaceutical activities. Thus, studies on this class of enzymes will expand our knowledge of the biosynthetic machineries that generate natural products and generate new findings about microbial physiology. The recent development of next-generation DNA sequencing has allowed for an increase in the number of microbial genomes sequenced and the discovery of many microbial type III PKS genes. Here, we describe basic methods to study microbial type III PKSs whose genes are easy to clone. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.

    Science.gov (United States)

    Sinko, William; Wang, Yang; Zhu, Wei; Zhang, Yonghui; Feixas, Ferran; Cox, Courtney L; Mitchell, Douglas A; Oldfield, Eric; McCammon, J Andrew

    2014-07-10

    There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ∼2 μM, Ki ∼300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25-4 μg/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism.

  13. Nitric oxide synthase in ferret brain: localization and characterization.

    Science.gov (United States)

    Matsumoto, T.; Mitchell, J. A.; Schmidt, H. H.; Kohlhaas, K. L.; Warner, T. D.; Förstermann, U.; Murad, F.

    1992-01-01

    1. In the present study, we have investigated the distribution of nitric oxide synthase in the ferret brain. Nitric oxide synthase was determined biochemically and immunochemically. 2. In the rat brain, the highest nitric oxide synthase activity has been detected in the cerebellum. However, in the ferret brain, the highest activity was found in the striatum and the lowest in the cerebellum and cerebral cortex. The enzymatic activity was localized predominantly in the cytosolic fractions, it was dependent on NADPH and Ca2+, and inhibited by NG-nitro-L-arginine or NG-methyl-L-arginine. 3. Western blot analysis revealed that all regions of the ferret brain contained a 160 kD protein crossreacting with an antibody to nitric oxide synthase purified from the rat cerebellum, and the levels of relative intensity of staining by the antibody correlated with the distribution of nitric oxide synthase activity. 4. These results indicate that the ferret brain contains a nitric oxide synthase similar to the rat brain, but the distribution of enzymatic activity in the ferret brain differs markedly from the rat brain. Images Figure 1 PMID:1282076

  14. 25 Ways to Love Your Liver

    Science.gov (United States)

    ... Related Liver Disease Alpha 1 Antitrypsin Deficiency Autoimmune Hepatitis Benign Liver Tumors Biliary Atresia Cirrhosis of the Liver Galactosemia ... Related Liver Disease Alpha 1 Antitrypsin Deficiency Autoimmune Hepatitis Benign Liver Tumors Biliary Atresia Cirrhosis of the Liver Galactosemia ...

  15. Construction of C35 gene bait recombinants and T47D cell cDNA library.

    Science.gov (United States)

    Yin, Kun; Xu, Chao; Zhao, Gui-Hua; Liu, Ye; Xiao, Ting; Zhu, Song; Yan, Ge

    2017-11-20

    C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C35 1-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMART TM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 10 7 pfu/mL with high transformation efficiency of 1.4 × 10 6 , and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

  16. Diagnosis of fatty liver

    International Nuclear Information System (INIS)

    Saitoh, Shuichi; Nagamine, Takeaki; Takagi, Hitoshi

    1988-01-01

    Diagnostic values of various ultrasonographic findings were evaluated from fatty infiltration ratio calculated by liver specimens in 42 patients. The ratio of the CT number of liver to those of spleen were also compared with fatty infiltration ratio in 11 patients. Fatty bandless sign one plus (perirenal bright echo between the liver and the right kidney is masked partially) or more and the fatty score 3 (it is calculated by several ultrasonographic findings) and the less than 0.90 of the ratio of CT number of liver to those of spleen were useful for diagnosis of fatty liver, the sensitivity was 100%, 87.5%, 85.7% and the accuracy was 78.1%, 81.8%, 81.8% respectively. It was considered that these criteria were suitable in screening study of fatty liver. (author)

  17. Liver disease in pregnancy

    Directory of Open Access Journals (Sweden)

    Shashank Shekhar

    2015-10-01

    Full Text Available Deranged liver function tests are encountered in 3% of pregnancies. The potential causes are classified as those unique to and those just incidental to pregnancy. Pregnancy-related diseases are the most frequent causes of liver dysfunction during pregnancy and exhibit a trimester-specific occurrence during pregnancy. Differentiation of liver dysfunction as that related to and just incidental to pregnancy is the key to management, especially when liver dysfunction is encountered after 28 weeks of pregnancy. It can be judged from the fact that delivery remains the cornerstone of management of pregnancy-related diseases except hyperemesis gravidarum. This is an overview of the causes of liver dysfunction during pregnancy; an update on the underlying mechanisms of their occurrence, especially liver diseases unique to pregnancy; and a methodological approach to their diagnosis and management.

  18. The effect of fasting/refeeding and insulin treatment on the expression of the regulatory genes of ketogenesis in intestine and liver of suckling rats.

    Science.gov (United States)

    Arias, G; Asins, G; Hegardt, F G; Serra, D

    1997-04-15

    The influence of fasting/refeeding and insulin treatment on ketogenesis in 12-day-old suckling rats was studied in intestine and liver by determining mRNA levels and enzyme activity of the two genes responsible for regulation of ketogenesis: carnitine palmitoyl transferase I (CPT I) and mitochondrial HMG-CoA synthase. Fasting produced hardly any change in mRNA or activity of CPT 1 in intestine, but led to a decrease in mitochondrial (mit.) HMG-CoA synthase. In liver, while mRNA levels and activity for CPT I increased, neither parameter was changed in HMG-CoA synthase. The comparison of these values with the ketogenic rate of both tissues under the fasting/refeeding treatment shows that HMG-CoA synthase could be the main gene responsible for regulation of ketogenesis in suckling rats. The small changes produced in serum ketone bodies in fasting/refeeding, with a profile similar to the ketogenic rate of the liver, indicate that liver contributes most to ketone body synthesis in suckling rats under these experimental conditions. Short-term insulin treatment produced increases in mRNA levels and activity in CPT I in intestine, but it also decreased both parameters in mit. HMG-CoA synthase. In liver, graphs of mRNA and activity were nearly identical in both genes. There was a marked decrease in mRNA levels and activity, resembling those values observed in adult rats. As in fasting/refeeding, the ketogenic rate correlated better to mit. HMG-CoA synthase than CPT I, and liver was the main organ regulating ketogenesis after insulin treatment. Serum ketone body concentrations were decreased by insulin but recovered after the second hour. Long-term insulin treatment had little effect on the mRNA levels for CPT I or mit. HMG-CoA synthase, but both the expressed and total activities of mit. HMG-CoA synthase were reduced by half in both intestine and liver. The ketogenic rate of both organs was decreased to 40% by long-term insulin treatment. The different effects of refeeding

  19. Robotic liver surgery

    Science.gov (United States)

    Leung, Universe

    2014-01-01

    Robotic surgery is an evolving technology that has been successfully applied to a number of surgical specialties, but its use in liver surgery has so far been limited. In this review article we discuss the challenges of minimally invasive liver surgery, the pros and cons of robotics, the evolution of medical robots, and the potentials in applying this technology to liver surgery. The current data in the literature are also presented. PMID:25392840

  20. Converting S-limonene synthase to pinene or phellandrene synthases reveals the plasticity of the active site.

    Science.gov (United States)

    Xu, Jinkun; Ai, Ying; Wang, Jianhui; Xu, Jingwei; Zhang, Yongkang; Yang, Dong

    2017-05-01

    S-limonene synthase is a model monoterpene synthase that cyclizes geranyl pyrophosphate (GPP) to form S-limonene. It is a relatively specific enzyme as the majority of its products are composed of limonene. In this study, we converted it to pinene or phellandrene synthases after introducing N345A/L423A/S454A or N345I mutations. Further studies on N345 suggest the polarity of this residue plays a critical role in limonene production by stabilizing the terpinyl cation intermediate. If it is mutated to a non-polar residue, further cyclization or hydride shifts occurs so the carbocation migrates towards the pyrophosphate, leading to the production of pinene or phellandrene. On the other hand, mutant enzymes that still possess a polar residue at this position produce limonene as the major product. N345 is not the only polar residue that may stabilize the terpinyl cation because it is not strictly conserved among limonene synthases across species and there are also several other polar residues in this area. These residues could form a "polar pocket" that may collectively play this stabilizing role. Our study provides important insights into the catalytic mechanism of limonene synthases. Furthermore, it also has wider implications on the evolution of terpene synthases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Adipokines in Liver Cirrhosis.

    Science.gov (United States)

    Buechler, Christa; Haberl, Elisabeth M; Rein-Fischboeck, Lisa; Aslanidis, Charalampos

    2017-06-29

    Liver fibrosis can progress to cirrhosis, which is considered a serious disease. The Child-Pugh score and the model of end-stage liver disease score have been established to assess residual liver function in patients with liver cirrhosis. The development of portal hypertension contributes to ascites, variceal bleeding and further complications in these patients. A transjugular intrahepatic portosystemic shunt (TIPS) is used to lower portal pressure, which represents a major improvement in the treatment of patients. Adipokines are proteins released from adipose tissue and modulate hepatic fibrogenesis. These proteins affect various biological processes that are involved in liver function, including angiogenesis, vasodilation, inflammation and deposition of extracellular matrix proteins. The best studied adipokines are adiponectin and leptin. Adiponectin protects against hepatic inflammation and fibrogenesis, and leptin functions as a profibrogenic factor. These and other adipokines are supposed to modulate disease severity in patients with liver cirrhosis. Consequently, circulating levels of these proteins have been analyzed to identify associations with parameters of hepatic function, portal hypertension and its associated complications in patients with liver cirrhosis. This review article briefly addresses the role of adipokines in hepatitis and liver fibrosis. Here, studies having analyzed these proteins in systemic blood in cirrhotic patients are listed to identify adipokines that are comparably changed in the different cohorts of patients with liver cirrhosis. Some studies measured these proteins in systemic, hepatic and portal vein blood or after TIPS to specify the tissues contributing to circulating levels of these proteins and the effect of portal hypertension, respectively.

  2. Acute Liver Failure.

    Science.gov (United States)

    Newland, Catherine D

    2016-12-01

    Pediatric acute liver failure (ALF) is a complex and rapidly progressive syndrome that results from a variety of age-dependent etiologies. It is defined by the acute onset of liver disease with no evidence of chronic liver disease. There must be biochemical or clinical evidence of severe liver dysfunction as defined by an international normalized ratio (INR) ≥2. If hepatic encephalopathy is present, INR should be ≥1.5. Unfortunately, due to the rarity of ALF in pediatric patients, there is a paucity of diagnostic and management algorithms and each patient must have an individualized approach. [Pediatr Ann. 2016;45(12):e433-e438.]. Copyright 2016, SLACK Incorporated.

  3. Nonalcoholic fatty liver disease

    DEFF Research Database (Denmark)

    Patrick-Melin, A J; Kalinski, M I; Kelly, K R

    2009-01-01

    Nonalcoholic fatty liver disease (NAFLD) is a rapidly emerging chronic liver disease and is reported to affect up to 70-80% of overweight and obese individuals. NAFLD represents a spectrum of liver diseases that range from simple hepatic steatosis, to a more severe and treatment resistant stage...... that features steatosis plus inflammation, termed nonalcoholic steatohepatitis (NASH), which may in turn progress to hepatic fibrosis, cirrhosis, and sub-acute liver failure. Thus, NAFLD and its subsequent complications create a significant health burden, and currently there is no effective treatment strategy...

  4. Construction and characterization of a cDNA library from head kidney of Japanese sea bass (Lateolabrax japonicus).

    Science.gov (United States)

    Shao, Zhan-tao; Cong, Xiao; Yuan, Jin-duo; Yang, Gui-wen; Chen, Ying; Pan, Jie; An, Li-guo

    2009-09-01

    In this paper, a cDNA expression library from head kidney of Japanese sea bass (Lateolabrax japonicus) was constructed for the first time. The first-strand cDNA was synthesized with Moloney Murine Leukaemia virus reverse transcriptase and the double-stranded cDNA was digested by Xho I enzyme. Size fractionation was performed on CHROMA SPIN-400 columns. cDNA fragments longer than 500 bps were ligated into the lambdaZAPExpress vector. The recombinant DNA was packaged in vitro with Gigapack III gold packaging extract. The titers of the primary and amplified library were 1.0 x 10(5) and 5.0 x 10(9) pfu/ml, respectively. To characterize the constructed cDNA library, 15 phage plaques were selected randomly to test the inserted fragments. The results showed that the inserts were mostly longer than 500 bps. To test the utility, the library was screened with primers designed for three immune-related genes of, Myxovirus resistant (Mx), tumor necrosis factor-alpha (TNF-alpha) and Toll-like receptor (TLR). Results of Blastn and alignment showed that they are members of Mx, TNF-alpha and TLR gene families, respectively, which meets our anticipates for this cDNA library as an immune-related one. These results confirmed that the cDNA library constructed will provide a useful tool for gene cloning and expression analysis in immune system of Japanese sea bass.

  5. [Construction and characterization of normalized cDNA library of maize inbred Mo17 from multiple tissues and developmental stages].

    Science.gov (United States)

    Zhang, Z X; Zhang, F D; Tang, W H; Pi, Y J; Zheng, Y L

    2005-01-01

    Comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10.830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarry was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate gene, and developing microarrays in maize genomics research.

  6. [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.

  7. [Cloning and sequence analysis of Eg95 cDNA from different stages of Echinococcus granulosus in Xinjiang].

    Science.gov (United States)

    Lin, Ren-yong; Ding, Jian-bing; Wen, Hao; Zhang, Wen-bao; Li, Jun; Lu, Xiao-mei

    2003-01-01

    To study expression and sequence differences of Echinococcus granulosus 95(Eg95) antigen cDNA from different stages of protoscolex, oncosphere and adult worm of E. granulosus from Xinjiang Uighur Aut. Reg. In accordance with the sequence of Eg95 antigen cDNA, the primers of Eg95 were designed. Eg95 antigen cDNAs were amplified by PCR from protoscolex, oncosphere and adult worm cDNA libraries of E. granulosus, respectively and were cloned into pUCm-T plasmid, and sequenced. The sequences were analyzed by DNAman and GenBank/BLAST biosoftware. PCR results showed that Eg95 antigen cDNA was amplified from three stages of E. granulosus cDNA libraries. Sequencing analysis indicated that the Eg95 cDNA length was 402 bp, same as the reported data in GenBank. The Eg95 antigen cDNA was expressed in the different life-cycle stages of E. granulosus in Xinjiang and there was no nucleic acid sequence difference of Eg95 antigen among the protoscolex, oncosphere and adult worm of E. granulosus.

  8. Phenotype of transgenic mice overexpressed with inducible nitric oxide synthase in the retina.

    Directory of Open Access Journals (Sweden)

    Guey Shuang Wu

    Full Text Available Unlike its constitutive isoforms, including neuronal and endothelial nitric oxide synthase, inducible nitric oxide synthase (iNOS along with a series of cytokines are generated in inflammatory pathologic conditions in retinal photoreceptors. In this study, we constructed transgenic mice overexpressing iNOS in the retina to evaluate the effect of sustained, intense iNOS generation in the photoreceptor damage.For construction of opsin/iNOS transgene in the CMVSport 6 expression vector, the 4.4 kb Acc65I/Xhol mouse rod opsin promoter was ligated upstream to a 4.1 kb fragment encoding the complete mouse cDNA of iNOS. From the four founders identified, two heterozygote lines and one homozygote line were established. The presence of iNOS in the retina was confirmed and the pathologic role of iNOS was assessed by detecting nitrotyrosine products and apoptosis. Commercial TUNEL kit was used to detect DNA strand breaks, a later step in a sequence of morphologic changes of apoptosis process.The insertion and translation of iNOS gene were demonstrated by an intense single 130 kDa band in Western blot and specific immunolocalization at the photoreceptors of the retina. Cellular toxicity in the retinas of transgenic animals was detected by a post-translational modification product, tyrosine-nitrated protein, the most significant one of which was nitrated cytochrome c. Following the accumulation of nitrated mitochondrial proteins and cytochrome c release, marked apoptosis was detected in the photoreceptor cell nuclei of the retina.We have generated a pathologic phenotype with sustained iNOS overexpression and, therefore, high output of nitric oxide. Under basal conditions, such overexpression of iNOS causes marked mitochondrial cytochrome c nitration and release and subsequent photoreceptor apoptosis in the retina. Therefore, the modulation of pathways leading to iNOS generation or its effective neutralization can be of significant therapeutic benefit in the

  9. Characterization of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS) gene from Ginkgo biloba.

    Science.gov (United States)

    Kim, Sang-Min; Kim, Soo-Un

    2010-02-01

    Diterpene trilactone ginkgolides, one of the major constituents of Ginkgo biloba extract, have shown interesting bioactivities including platelet-activating factor antagonistic activity. 1-Hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), converting 2-C-methyl-d-erythritol-2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate, is the penultimate enzyme of the seven-step 2-C-methyl-d-erythritol 4-phosphate pathway that supplies building blocks for plant isoprenoids of plastid origin such as ginkgolides and carotenoids. Here, we report on the isolation and characterization of the full-length cDNA encoding HDS (GbHDS, GenBank accession number: DQ251630) from G. biloba. Full-length cDNA of GbHDS, 2,763 bp long, contained an ORF of 2,226 bp encoding a protein composed of 741 amino acids. The theoretical molecular weight and pI of the deduced mature GbHDS of 679 amino acid residues are 75.6 kDa and 5.5, respectively. From 2 weeks after initiation of the culture onward, transcription level of this gene in the ginkgo embryo roots increased to about two times higher than that in the leaves. GbHDS was predicted to possess chloroplast transit peptide of 62 amino acid residues, suggesting its putative localization in the plastids. The transient gene expression in Arabidopsis protoplasts confirmed that the transit peptide was capable of delivering the GbHDS protein from the cytosol into the chloroplasts. The isolation and characterization of GbHDS gene enabled us to further understand the role of GbHDS in the terpenoid biosynthesis in G. biloba.

  10. Arginase activity in mitochondria - An interfering factor in nitric oxide synthase activity assays

    Energy Technology Data Exchange (ETDEWEB)

    Venkatakrishnan, Priya; Nakayasu, Ernesto S.; Almeida, Igor C. [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968 (United States); Miller, R.T., E-mail: tmiller2@utep.edu [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968 (United States)

    2010-04-09

    Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [{sup 14}C]-L-arginine to [{sup 14}C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent . Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [{sup 14}C]-L-arginine to [{sup 14}C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

  11. Cloning and Functional Characterization of a Gene for Capsanthin-Capsorubin Synthase from Tiger Lily (Lilium lancifolium Thunb. ‘Splendens’)

    Science.gov (United States)

    Chen, Tony H.H.

    2012-01-01

    The orange color of tiger lily (Lolium lancifolium ‘Splendens’) flowers is due, primarily, to the accumulation of two κ-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-degenerate primer that was based on the sequence of a gene for lycopene β-cyclase (lcyB). The full-length cDNA of Llccs was 1,785 bp long and contained an open reading frame of 1,425 bp that encoded a polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence. Analysis by reverse transcription–PCR (RT–PCR) revealed that expression of Llccs was spatially and temporally regulated, with expression in flower buds and flowers of L. lancifolium but not in vegetative tissues. Stable overexpression of the Llccs gene in callus tissue of Iris germanica, which accumulates several xanthophylls including violaxanthin, the precursor of capsorubin, resulted in transgenic callus whose color had changed from its normal yellow to red-orange. This novel red-orange coloration was due to the accumulation of two non-native κ-xanthophylls, capsanthin and capsorubin, as confirmed by HPLC and ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) analysis with authentic standards. Cloning of the Llccs gene should advance our understanding of the molecular and genetic mechanisms of the biosynthesis of κ-carotenoids in general and in the genus Lilium in particular, and will facilitate transgenic alterations of the colors of flowers and fruits of many plant species. PMID:23008421

  12. Identification of breast cancer-restricted antigens by antibody screening of SKBR3 cDNA library using a preselected patient's serum.

    Science.gov (United States)

    Forti, Stefania; Scanlan, Matthew J; Invernizzi, Annamaria; Castiglioni, Fabio; Pupa, Sandro; Agresti, Roberto; Fontanelli, Rosanna; Morelli, Daniele; Old, Lloyd J; Pupa, Serenella M; Ménard, Sylvie

    2002-06-01

    Screening of a breast cancer cDNA library from SKBR3 human breast cancer cells by SEREX (serological analysis of cDNA expression library) using a preselected serum from a breast cancer patient revealed 13 genes, two of which, INT-MI-1 and INT-MI-2, encode novel gene products, while the remaining 11 genes and their products are identical with or highly homologous to known GenBank entries. Immunoscreening of the 13 clones using 20 allogeneic sera from breast cancer patients and 20 samples from age- and gender-matched healthy donors showed that lactate dehydrogenase-A (LDH-A), lactate dehydrogenase-B (LDH-B), fibulin-1, and thyroid hormone-binding protein (THBP) were recognized principally by the breast cancer patient sera, indicating the immunogenicity of these molecules in vivo. The other antigens were similarly recognized by normal and patients sera, and thus not tumor-restricted immunologically. RT-PCR analysis revealed strong expression of fibulin-1 in tumor cell lines and surgical specimen whereas in the same experimental conditions, normal tissues scored negative. Also THBP expression was found in various tumors whereas in normal tissues, its expression is restricted to the testis and, at lower levels, in ovary, liver, and spleen. In contrast, LDH-A and LDH-B were ubiquitously expressed in normal and tumor tissues, with LDH-B levels considerably lower and heterogeneous in normal samples compared to those expressed in tumor cell lines. The differential expression of fibulin-1 between the normal tissues and breast carcinoma cell lines (5/6) and surgical specimens (5/6) suggests the possible involvement of the overexpression of this extracellular matrix-associated glycoprotein in the pathogenesis of this neoplasm.

  13. Plasma succinylacetone is persistently raised after liver transplantation in tyrosinaemia type 1.

    Science.gov (United States)

    Bartlett, David C; Preece, Mary Anne; Holme, Elisabeth; Lloyd, Carla; Newsome, Phil N; McKiernan, Patrick J

    2013-01-01

    Tyrosinaemia type 1 (HT1) is a rare disorder leading to accumulation of toxic metabolites such as succinylacetone (SA) and a high risk of hepatocellular carcinoma. Children with HT1 traditionally required liver transplantation (OLT) and while the need for this has been reduced by the introduction of nitisinone some still require OLT. SA inhibits the enzyme porphobilinogen (PBG) synthase and its activity can be used as a marker of active SA. Elevated urinary SA post OLT has been reported previously. This study describes a novel finding of elevated plasma SA following OLT for HT1. A retrospective analysis was performed of patients treated for HT1 at our institution from 1989-2010. Thirteen patients had an OLT for HT1. In patients who received nitisinone prior to OLT, mean urinary and plasma SA were elevated prior to treatment but normalised by the time of OLT (p ≤ 0.01). Mean PBG synthase activity increased from 0.032 to 0.99 nkat/gHb (ref range 0.58-1.25) at the time of OLT (p PBG synthase activity were not available prior to OLT for this group. Following OLT, mean urinary and plasma SA were elevated in all for the duration of follow-up and associated with low-normal PBG synthase activity. Urinary and plasma SA levels are elevated following OLT for HT1. Low-normal PBG synthase activity suggests the plasma SA may be active. The clinical significance of this is unclear.

  14. 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

  15. cDNA cloning of a major allergen from timothy grass (Phleum pratense) pollen; characterization of the recombinant Phl pV allergen

    NARCIS (Netherlands)

    Vrtala, S.; Sperr, W. R.; Reimitzer, I.; van Ree, R.; Laffer, S.; Müller, W. D.; Valent, P.; Lechner, K.; Rumpold, H.; Kraft, D.

    1993-01-01

    We isolated a cDNA encoding a major grass pollen allergen from a timothy grass (Phleum pratense) pollen expression cDNA library using allergic patients' IgE. The complete cDNA encoded an allergen that binds IgE from about 80% of grass pollen-allergic patients. Significant sequence homology was found

  16. 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.

  17. 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.

  18. [Construction and identification of a full-length cDNA library from Spirometra erinaceieuropaei].

    Science.gov (United States)

    Lv, Gang; Lu, Ya-Jun; Fan, Zhi-Gang; Shi, Da-Zhong; Gan, Xiu-Feng; Zhong, Sai-Feng

    2010-10-30

    The full-length pBluescript II SK cDNA library of adult Spirometra erinaceieuropaei was constructed by using the SMART method. Data showed that 95.5% of the library was recombinant and the titer of the library was 1.06 x 10(6). The average insert size of the library was about 1.4 kb. Forty-eight randomly selected clones were sequenced. A set of 36 effective expressed sequence tags (ESTs) with the average size of 674 bp was obtained after excluding clones shorter than 450 bp. The unigenes occupied 58.3% of the 36 ESTs. The rate of full-length cDNAs were 57.7% (15/26). The high-quality of full-length cDNA library could be used for large scale EST sequencing.

  19. Construction and analysis of full-length cDNA library of Cryptosporidium parvum.

    Science.gov (United States)

    Yamagishi, Junya; Wakaguri, Hiroyuki; Sugano, Sumio; Kawano, Suguru; Fujisaki, Kozo; Sugimoto, Chihiro; Watanabe, Junichi; Suzuki, Yutaka; Kimata, Isao; Xuan, Xuenan

    2011-06-01

    A full-length cDNA library was constructed from the sporozoite of Cryptosporidium parvum. Normalized clones were subjected to Solexa shotgun sequencing, and then complete sequences for 1066 clones were reconfigured. Detailed analyses of the sequences revealed that 13.5% of the transcripts were spliced; the average and median 5' UTR lengths were 213.5 and 122 nucleotides, respectively. There were 148 inconsistencies out of 562 examined genes between the experimentally described cDNA sequence and the predicted sequence from its genome. In addition, we identified 118 sequences that had little homology against annotated genes of C. parvum as prospective candidates for addable genes. These observations should improve the reliability of C. parvum transcriptome and provide a versatile resource for further studies. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

    Rong, Mingqiang; Yang, Shilong; Wen, Bo

    2015-01-01

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

  1. Molecular cloning and characterization of cDNA encoding fibrinolytic enzyme-3 from earthworm Eisenia foetida.

    Science.gov (United States)

    Dong, Guo-Qing; Yuan, Xiao-Ling; Shan, Ya-Jun; Zhao, Zhen-Hu; Chen, Jia-Pei; Cong, Yu-Wen

    2004-04-01

    The earthworm fibrinolytic enzyme-3 (EFE-3, GenBank accession No: AY438622), from the earthworm Eisenia foetida, is a component of earthworm fibrinolytic enzymes. In this study, cDNA encoding the EFE-3 was cloned by RT-PCR. The cDNA 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.

  2. 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.

  3. 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.

  4. 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...... that random pentadecamers can replace random hexamers in reverse transcription reactions on both poly(A) RNA and amplified RNA, resulting in higher cDNA yields and quality....... 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...

  5. TIMP-1 attenuates blood–brain barrier permeability in mice with acute liver failure

    Science.gov (United States)

    Chen, Feng; Radisky, Evette S; Das, Pritam; Batra, Jyotica; Hata, Toshiyuki; Hori, Tomohide; Baine, Ann-Marie T; Gardner, Lindsay; Yue, Mei Y; Bu, Guojun; del Zoppo, Gregory; Patel, Tushar C; Nguyen, Justin H

    2013-01-01

    Blood–brain barrier (BBB) dysfunction in acute liver failure (ALF) results in increased BBB permeability that often precludes the patients from obtaining a life-saving liver transplantation. It remains controversial whether matrix metalloproteinase-9 (MMP-9) from the injured liver contributes to the deregulation of BBB function in ALF. We selectively upregulated a physiologic inhibitor of MMP-9 (TIMP-1) with a single intracerebroventricular injection of TIMP-1 cDNA plasmids at 48 and 72 hours, or with pegylated-TIMP-1 protein. Acute liver failure was induced with tumor necrosis factor-α and 𝒟-(+)-galactosamine in mice. Permeability of BBB was assessed with sodium fluorescein (NaF) extravasation. We found a significant increase in TIMP-1 within the central nervous system (CNS) after the administration of TIMP-1 cDNA plasmids and that increased TIMP-1 within the CNS resulted in an attenuation of BBB permeability, a reduction in activation of epidermal growth factor receptor and p38 mitogen-activated protein kinase signals, and a restoration of the tight junction protein occludin in mice with experimental ALF. Pegylated TIMP-1 provided similar protection against BBB permeability in mice with ALF. Our results provided a proof of principle that MMP-9 contributes to the BBB dysfunction in ALF and suggests a potential therapeutic role of TIMP-1 in ALF. PMID:23532086

  6. Cloning and characterization of cellulose synthase-like gene, PtrCSLD2 from developing xylem of aspen trees.

    Science.gov (United States)

    Samuga, Anita; Joshi, Chandrashekhar P.

    2004-04-01

    Genetic improvement of cell wall polymer synthesis in forest trees is one of the major goals of forest biotechnology that could possibly impact their end product utilization. Identification of genes involved in cell wall polymer biogenesis is essential for achieving this goal. Among various candidate cell wall-related genes, cellulose synthase-like D (CSLD) genes are intriguing due to their hitherto unknown functions in cell wall polymer synthesis but strong structural similarity with cellulose synthases (CesAs) involved in cellulose deposition. Little is known about CSLD genes from trees. In the present article PtrCSLD2, a first CSLD gene from an economically important tree, aspen (Populus tremuloides) is reported. PtrCSLD2 cDNA was isolated from an aspen xylem cDNA library and encodes a protein that shares 90% similarity with Arabidopsis AtCSLD3 protein involved in root hair tip growth. It is possible that xylem fibers that also grow by intrusive tip growth may need expression of PtrCSLD2 for controlling the length of xylem fibers, a wood quality trait of great economical importance. PtrCSLD2 protein has a N-terminal cysteine-rich putative zinc-binding domain; eight transmembrane domains; alternating conserved and hypervariable domains; and a processive glycosyltransferases signature, D, D, D, QXXRW; all similar to aspen CesA proteins. However, PtrCSLD2 shares only 43-48% overall identity with the known aspen CesAs suggesting its distinct functional role in cell wall polymer synthesis perhaps other than cellulose biosynthesis. Based on Southern analysis, the aspen CSLD gene family consists of at least three genes and this gene copy estimate is supported by phylogenetic analysis of available CSLDs from plants. Moreover, gene expression studies using RT-PCR and in situ mRNA hybridization showed that PtrCSLD2 is expressed at a low level in all aspen tissues examined with a slightly higher expression level in secondary cell wall-enriched aspen xylem as compared to

  7. Attenuation and cell culture adaptation of hepatitis A virus (HAV): a genetic analysis with HAV cDNA.

    OpenAIRE

    Cohen, J I; Rosenblum, B; Feinstone, S M; Ticehurst, J; Purcell, R H

    1989-01-01

    RNA transcripts of hepatitis A virus (HAV) HM-175 cDNA from attenuated, cell culture-adapted HAV were infectious in cell culture. A full-length HAV cDNA from wild-type HAV (propagated in marmosets in vivo) was constructed. Chimeric cDNAs that contained portions of both wild-type and attenuated genomes were produced. Oligonucleotide-directed mutagenesis was used to engineer a point mutation into the VP1 gene of attenuated HAV cDNA, so that the sequence of this capsid protein would be identical...

  8. Identification of differential genes by suppression subtractive hybridization: I. Preparation of subtracted cDNA or genomic DNA library.

    Science.gov (United States)

    Rebrikov, Denis V

    2008-07-01

    INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes the preparation of a subtracted cDNA or genomic DNA library, and includes methods for cDNA synthesis, tester and driver DNA digestion, and adapter ligation.

  9. Construction of an infectious cDNA clone of foot-and-mouth disease ...

    Indian Academy of Sciences (India)

    Prakash

    Foot-and-mouth disease virus (FMDV) serotype O is the most predominant among the endemic serotypes in India. A stable, full-length cDNA clone of FMDV type O1BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEMR-7Zf(–). An ~8.2 kb PCR product was amplified ...

  10. Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA

    OpenAIRE

    Yadav, Rajiv Kumar; Barbi, Florian; Ziller, Antoine; Luis, Patricia; Marmeisse, Roland; Reddy, M Sudhakara; Fraissinet-Tachet, Laurence

    2014-01-01

    Background: Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. Results: We report here a ...

  11. cDNA cloning and expression analysis of a mannose-binding lectin ...

    Indian Academy of Sciences (India)

    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 ...

  12. Liver transplantation in polycystic liver disease

    DEFF Research Database (Denmark)

    Krohn, Paul S; Hillingsø, Jens; Kirkegaard, Preben

    2008-01-01

    OBJECTIVE: Polycystic liver disease (PLD) is a rare, hereditary, benign disorder. Hepatic failure is uncommon and symptoms are caused by mass effects leading to abdominal distension and pain. Liver transplantation (LTX) offers fully curative treatment, but there is still some controversy about...... whether it is a relevant modality considering the absence of liver failure, relative organ shortage, perioperative risks and lifelong immunosuppression. The purpose of this study was to review our experience of LTX for PLD and to compare the survival with the overall survival of patients who underwent LTX....../kidney transplantation. One patient had undergone kidney transplantation 10 years earlier. RESULTS: Median follow-up was 55 months. One patient who underwent combined transplantation died after 5.4 months because of multiorgan failure after re-LTX, and one patient, with well-functioning grafts, died of lymphoma after 7...

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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

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

    Directory of Open Access Journals (Sweden)

    Yu-Juan Xiang

    2015-06-01

    Full Text Available 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 quantitatively 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. Keywords: Breast neoplasms, Candidate genes, Microarray

  15. A novel method using edge detection for signal extraction from cDNA microarray image analysis.

    Science.gov (United States)

    Kim, J H; Kim, H Y; Lee, Y S

    2001-06-30

    Gene expression analyses by probes of hybridization from mRNA to cDNA targets arrayed on membranes or activated glass surfaces have revolutionized the way of profiling mega level gene expression. The main remaining problems however are sensitivity of detection, reproducibility and data processing. During processing of microarray images, especially irregularities of spot position and shape could generate significant errors: small regions of signal spots can be mis-included into background area and vice versa. Here we report a novel method to eliminate such obstacles by sensing their edges. Application of edge detection technology on separating spots from the background decreases the probability of the errors and gives more accurate information about the states of spots such as the pixel number, degree of fragmentation, width and height of spot, and circumference of spot. Such information can be used for the quality control of cDNA microarray experiments and filtering of low quality spots. We analyzed the cDNA microarray image that contains 10,368 genes using edge detection and compared the result with that of conventional method which draws circle around the spot.

  16. Growth hormone and prolactin in Andrias davidianus: cDNA cloning, tissue distribution and phylogenetic analysis.

    Science.gov (United States)

    Yang, Liping; Meng, Zining; Liu, Yun; Zhang, Yong; Liu, Xiaochun; Lu, Danqi; Huang, Junhai; Lin, Haoran

    2010-01-15

    The Chinese giant salamander (Andrias davidianus) is one of the largest and 'living fossil' species of amphibian. To obtain genetic information for this species, the cDNAs encoding growth hormone (adGH) and prolactin (adPRL) were cloned from a pituitary cDNA library. The isolated adGH cDNA consisted of 864 bp and encoded a propeptide of 215 amino acids, while the cDNA of adPRL was 1106 bp in length and encoded a putative peptide of 229 amino acids. Expression of the GH and PRL mRNA was only detected in the pituitary. Phylogenetic analyses were performed based on the isolated pituitary hormone sequences using maximum parsimony and neighbor-joining algorithms. The clustering results are similar to that based on the morphological characteristics or the rRNA genes, which indicate that the two orders (Anura and Caudata) of amphibian were monophyletic, and that A. davidianus was diverged early in the Caudate clade. These results indicated that both the GH and PRL sequence might be useful to study the phylogenies of relatively moderate evolved groups.

  17. Generation of longer 3' cDNA fragments from massively parallel signature sequencing tags.

    Science.gov (United States)

    Silva, Ana Paula M; Chen, Jianjun; Carraro, Dirce M; Wang, San Ming; Camargo, Anamaria A

    2004-07-06

    Massively Parallel Signature Sequencing (MPSS) is a powerful technique for genome-wide gene expression analysis, which, similar to SAGE, relies on the production of short tags proximal to the 3'end of transcripts. A single MPSS experiment can generate over 10(7) tags, providing a 10-fold coverage of the transcripts expressed in a human cell. A significant fraction of MPSS tags cannot be assigned to known transcripts (orphan tags) and are likely to be derived from transcripts expressed at very low levels (approximately 1 copy per cell). In order to explore the potential of MPSS for the characterization of the human transcriptome, we have adapted the GLGI protocol (Generation of Longer cDNA fragments from SAGE tags for Gene Identification) to convert MPSS tags into their corresponding 3' cDNA fragments. GLGI-MPSS was applied to 83 orphan tags and 41 cDNA fragments were obtained. The analysis of these 41 fragments allowed the identification of novel transcripts, alternative tags generated from polymorphic and alternatively spliced transcripts, as well as the detection of artefactual MPSS tags. A systematic large-scale analysis of the genome by MPSS, in combination with the use of GLGI-MPSS protocol, will certainly provide a complementary approach to generate the complete catalog of human transcripts.

  18. Generation of longer 3′ cDNA fragments from massively parallel signature sequencing tags

    Science.gov (United States)

    Silva, Ana Paula M.; Chen, Jianjun; Carraro, Dirce M.; Wang, San Ming; Camargo, Anamaria A.

    2004-01-01

    Massively Parallel Signature Sequencing (MPSS) is a powerful technique for genome-wide gene expression analysis, which, similar to SAGE, relies on the production of short tags proximal to the 3′end of transcripts. A single MPSS experiment can generate over 107 tags, providing a 10-fold coverage of the transcripts expressed in a human cell. A significant fraction of MPSS tags cannot be assigned to known transcripts (orphan tags) and are likely to be derived from transcripts expressed at very low levels (∼1 copy per cell). In order to explore the potential of MPSS for the characterization of the human transcriptome, we have adapted the GLGI protocol (Generation of Longer cDNA fragments from SAGE tags for Gene Identification) to convert MPSS tags into their corresponding 3′ cDNA fragments. GLGI-MPSS was applied to 83 orphan tags and 41 cDNA fragments were obtained. The analysis of these 41 fragments allowed the identification of novel transcripts, alternative tags generated from polymorphic and alternatively spliced transcripts, as well as the detection of artefactual MPSS tags. A systematic large-scale analysis of the genome by MPSS, in combination with the use of GLGI-MPSS protocol, will certainly provide a complementary approach to generate the complete catalog of human transcripts. PMID:15247327

  19. Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA.

    Science.gov (United States)

    Yadav, Rajiv Kumar; Barbi, Florian; Ziller, Antoine; Luis, Patricia; Marmeisse, Roland; Reddy, M Sudhakara; Fraissinet-Tachet, Laurence

    2014-09-03

    Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.

  20. [Construction and analysis of suppression subtractive cDNA libraries of continuous monoculture Rehmannia glutinosa].

    Science.gov (United States)

    Zhang, Zhongyi; Fan, Huamin; Yang, Yanhui; Li, Mingjie; Li, Juan; Xu, Haixia; Chen, Junying; Chen, Xinjian

    2011-02-01

    To explore the molecular mechanism of continuous monoculture problem by constructing the cDNA libraries of continuous monoculture Rehmannia glutinosa. To use the suppression subtractive hybridization (SSH) technique to construct the forward and reverse subtractive cDNA libraries of continuous monoculture R. glutinosa to adopt blue-white colony screening and PCR to detect the positive clones which would be sequenced and analyzed by bioinformatics. The subtracted cDNA libraries of continuous monoculture R. glutinosa. were successfully constructed, and the result showed that the forward and reverse subtracted libraries obtained 300 positive clones, respectively. The forward and reverse libraries got different ESTs, and produced 232 (forward library) and 214 (reverse library) unique ESTs by sequencing. Based on homology search of BLASTX and BLASTN in NCBI, 200 and 195 of unique ESTs were homologous to known genes in the forward and reverse libraries, respectively. Categories of orthologous group (COG) showed that the forward and reverse libraries got 60 and 61 ESTs with the corresponding gene annotation, involving 21 metabolic pathways. The information of differential expression genes in continuous monoculture R. glutinosa, and their functional annotation of differentially expressed genes indicate that continuous monoculture has a profound effect on expression of the genes in R. glutinosa. Furthermore, the research analyzed several key genes in response to replant problem, which provided a foundation for revealing the molecular mechanism of continuous monoculture R. glutinosa.

  1. CONSTRUCTION AND APPLICATIONS OF A MYCORRHIZAL ARBUSCULAR SPECIFIC cDNA LIBRARY.

    Science.gov (United States)

    Isayenkov, S; Maathuis, F J M

    2016-01-01

    To exploit the potential benefits of mycorrhizas, we need to investigate the processes that occur in these symbiotic interactions, particularly in the arbuscular compartment where nutrients are exchanged between the plant and the fungus. Progress in this area is restricted due to the intricacy and complexity of this plant-fungus interface and many techniques that have been employed successfully in other plants and animal systems cannot be used. An effective approach to study processes in arbuscules is to examine transcript composition and dynamics. We applied laser capture microdissection (LCM) to isolate approximately 3000 arbuscules from Glomus intraradices colonised Me- dicago truncatula roots. Total RNA was extracted from microdissected arbuscules and subjected to T7 RNA polymerase-based linear amplification. Amplified RNA was then usedfor construction of a cDNA library. The presence and level of enrichment of mycorrhiza-specific transcripts was determined by quantitative Real-time and conventional PCR. To improve enrichment a cDNA library subtraction was performed. Complementation of yeast mutants deficient in the uptake of.potassium, phosphate, sulphate, amino acids, ammonium and of a Mn²⁺sensitive strain, demonstrates the functionality of our cDNA library.

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

    Science.gov (United States)

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

    2010-10-01

    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 1.30×10(6) pfu/ml and 1.62×10(9) 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 1,142 bps 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.

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

    Science.gov (United States)

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

    2014-02-01

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

  4. Effects of heavy metals on Drosophila larvae and a metallothionein cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Maroni, G.; Lastowski-Perry, D.; Otto, E.; Watson, D.

    1986-03-01

    Drosophila melanogaster larvae reared on food containing radioactive cadmium retained over 80% of it, mostly in the intestinal epithelium. The majority of this radioactivity was associated with a soluble protein of less than 10,000 molecular weight. Synthesis of this cadmium-binding protein was induced by the metal as demonstrated by incorporation of radioactive cysteine. Most copper ingested by larvae was also found to associate with a low molecular weight, inducible protein, but some of it was found in an insoluble fraction. A D. melanogaster cDNA clone was isolated based on its more intense hybridization to copies of RNA sequences from copper-fed larvae than from control larvae. This clone showed strong hybridization to mouse metallothionein-I cDNA at reduced stringency. Its nucleotide sequence includes an open-reading segment which codes for a 40 amino acid protein; this protein was identified as metallothionein based on its similarity to the amino-terminal portion of mammalian and crab metalloproteins. The ten cysteine residues present occur in five pairs of near-vicinal cysteines (Cys-X-Cys). This cDNA sequence hybridized to a 400-nucleotide polyadenylated RNA whose presence in the cells of the alimentary canal of larvae was stimulated by ingestion of cadmium or copper; in other tissues this RNA was present at much lower levels.

  5. Sequencing of first-strand cDNA library reveals full-length transcriptomes.

    Science.gov (United States)

    Agarwal, Saurabh; Macfarlan, Todd S; Sartor, Maureen A; Iwase, Shigeki

    2015-01-21

    Massively parallel strand-specific sequencing of RNA (ssRNA-seq) has emerged as a powerful tool for profiling complex transcriptomes. However, many current methods for ssRNA-seq suffer from the underrepresentation of both the 5' and 3' ends of RNAs, which can be attributed to second-strand cDNA synthesis. The 5' and 3' ends of RNA harbour crucial information for gene regulation; namely, transcription start sites (TSSs) and polyadenylation sites. Here we report a novel ssRNA-seq method that does not involve second-strand cDNA synthesis, as we Directly Ligate sequencing Adaptors to the First-strand cDNA (DLAF). This novel method with fewer enzymatic reactions results in a higher quality of the libraries than the conventional method. Sequencing of DLAF libraries followed by a novel analysis pipeline enables the profiling of both 5' ends and polyadenylation sites at near-base resolution. Therefore, DLAF offers the first genomics tool to obtain the 'full-length' transcriptome with a single library.

  6. An efficient and sensitive method for preparing cDNA libraries from scarce biological samples.

    Science.gov (United States)

    Sterling, Catherine H; Veksler-Lublinsky, Isana; Ambros, Victor

    2015-01-01

    The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. 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.

  8. Isolation and characterization of cDNA clones for human erythrocyte β-spectrin

    International Nuclear Information System (INIS)

    Prchal, J.T.; Morley, B.J.; Yoon, S.H.; Coetzer, T.L.; Palek, J.; Conboy, J.G.; Kan, Y.W.

    1987-01-01

    Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical α (M/sub r/ 240,000) and β (M/sub r/ 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. The authors report here the isolation and characterization of a human erythroid-specific β-spectrin cDNA clone that encodes parts of the β-9 through β-12 repeat segments. This cDNA was used as a hybridization probe to assign the β-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte β-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the β-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities

  9. Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

    International Nuclear Information System (INIS)

    Chao, S.; Chao, L.; Chao, J.

    1989-01-01

    A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins. Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available

  10. cDNA encoding a polypeptide including a hev ein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, Natasha V. (Okemos, MI); Broekaert, Willem F. (Dilbeek, BE); Chua, Nam-Hai (Scarsdale, NY); Kush, Anil (New York, NY)

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

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

    Science.gov (United States)

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

    2010-01-01

    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. PMID:20941376

  12. Rat liver arginase system under acetaminophen-induced toxic injury and protein deprivation

    Directory of Open Access Journals (Sweden)

    H. P. Kopylchuk

    2017-04-01

    Full Text Available Arginase activity and L-arginine content in both cytosolic and mitochondrial fractions of rat liver cells under the conditions of toxic injury on the background of protein deprivation was studied. The most significant reduction of arginase activity in liver cells and depletion of L-arginine pool was found in rats with toxic acetaminophen-induced liver injury maintained on the ration balanced by all nutrients as well as in protein deficiency rats. It was concluded that reduction of the arginase activity in the cytosolic fraction of rat liver cells, combined with simultaneous decrease of L-arginine content, may be considered as one of the mechanisms of ornithine cycle disturbance. The decline of activity of mitochondrial isoform of arginase II, for certain, is related with activation of NO-synthase system.

  13. Liver regeneration and restoration of liver function after partial hepatectomy in patients with liver tumors

    NARCIS (Netherlands)

    Jansen, P. L.; Chamuleau, R. A.; van Leeuwen, D. J.; Schipper, H. G.; Busemann-Sokole, E.; van der Heyde, M. N.

    1990-01-01

    Liver regeneration and restoration of liver function were studied in six patients who underwent partial hepatectomy with removal of 30-70% of the liver. Liver volume and liver regeneration were studied by single-photon computed tomography (SPECT), using 99mTc-colloid as tracer. The method was

  14. Effect of acute beer ingestion on the liver: studies in female mice.

    Science.gov (United States)

    Kanuri, Giridhar; Wagnerberger, Sabine; Landmann, Marianne; Prigl, Eva; Hellerbrand, Claus; Bischoff, Stephan C; Bergheim, Ina

    2015-04-01

    The aim of the present study was to assess whether the effects of acute consumption of stout or pilsner beer on the liver differ from those of plain ethanol in a mouse model. Seven-week-old female C57BL/6J mice received either ethanol, stout or pilsner beer (ethanol content: 6 g/kg body weight) or isocaloric maltodextrin solution. Plasma alanine transaminase, markers of steatosis, lipogenesis, activation of the toll-like receptor-4 signaling cascade as well as lipid peroxidation and fibrogenesis in the liver were measured 12 h after acute ethanol or beer intake. Acute alcohol ingestion caused a marked ~11-fold increase in hepatic triglyceride accumulation in comparison to controls, whereas in mice exposed to stout and pilsner beer, hepatic triglyceride levels were increased only by ~6.5- and ~4-fold, respectively. mRNA expression of sterol regulatory element-binding protein 1c and fatty acid synthase in the liver did not differ between alcohol and beer groups. In contrast, expression of myeloid differentiation primary response gene 88, inducible nitric oxide synthases, but also the concentrations of 4-hydroxynonenal protein adducts, nuclear factor κB and plasminogen activator inhibitor-1 were induced in livers of ethanol treated mice but not in those exposed to the two beers. Taken together, our results suggest that acute ingestion of beer and herein especially of pilsner beer is less harmful to the liver than the ingestion of plain ethanol.

  15. Cloning, Expression Profiling and Functional Analysis of CnHMGS, a Gene Encoding 3-hydroxy-3-Methylglutaryl Coenzyme A Synthase from Chamaemelum nobile.

    Science.gov (United States)

    Cheng, Shuiyuan; Wang, Xiaohui; Xu, Feng; Chen, Qiangwen; Tao, Tingting; Lei, Jing; Zhang, Weiwei; Liao, Yongling; Chang, Jie; Li, Xingxiang

    2016-03-08

    Roman chamomile (Chamaemelum nobile L.) is renowned for its production of essential oils, which major components are sesquiterpenoids. As the important enzyme in the sesquiterpenoid biosynthesis pathway, 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) catalyze the crucial step in the mevalonate pathway in plants. To isolate and identify the functional genes involved in the sesquiterpene biosynthesis of C. nobile L., a HMGS gene designated as CnHMGS (GenBank Accession No. KU529969) was cloned from C. nobile. The cDNA sequence of CnHMGS contained a 1377 bp open reading frame encoding a 458-amino-acid protein. The sequence of the CnHMGS protein was highly homologous to those of HMGS proteins from other plant species. Phylogenetic tree analysis revealed that CnHMGS clustered with the HMGS of Asteraceae in the dicotyledon clade. Further functional complementation of CnHMGS in the mutant yeast strain YSC6274 lacking HMGS activity demonstrated that the cloned CnHMGS cDNA encodes a functional HMGS. Transcript profile analysis indicated that CnHMGS was preferentially expressed in flowers and roots of C. nobile. The expression of CnHMGS could be upregulated by exogenous elicitors, including methyl jasmonate and salicylic acid, suggesting that CnHMGS was elicitor-responsive. The characterization and expression analysis of CnHMGS is helpful to understand the biosynthesis of sesquiterpenoid in C. nobile at the molecular level and also provides molecular wealth for the biotechnological improvement of this important medicinal plant.

  16. Characterisation and vascular expression of nitric oxide synthase 3 in amphibians.

    Science.gov (United States)

    Cameron, Melissa S; Trajanovska, Sofie; Forgan, Leonard G; Donald, John A

    2016-12-01

    In mammals, nitric oxide (NO) produced by nitric oxide synthase 3 (NOS3) localised in vascular endothelial cells is an important vasodilator but the presence of NOS3 in the endothelium of amphibians has been concluded to be absent, based on physiological studies. In this study, a nos3 cDNA was sequenced from the toad, Rhinella marina. The open reading frame of R. marina nos3 encoded an 1170 amino acid protein that showed 81 % sequence identity to the recently cloned Xenopus tropicalis nos3. Rhinella marina nos3 mRNA was expressed in a range of tissues and in the dorsal aorta and pulmonary, mesenteric, iliac and gastrocnemius arteries. Furthermore, nos3 mRNA was expressed in the aorta of Xenopus laevis and X. tropicalis. Quantitative real-time PCR showed that removal of the endothelium of the lateral aorta of R. marina significantly reduced the expression of nos3 mRNA compared to control aorta with the endothelium intact. However, in situ hybridisation was not able to detect any nos3 mRNA in the dorsal aorta of R. marina. Immunohistochemistry using a homologous R. marina NOS3 antibody showed immunoreactivity (IR) within the basal region of many endothelial cells of the dorsal aorta and iliac artery. NOS3-IR was also observed in the proximal tubules and collecting ducts of the kidney but not within the capillaries of the glomeruli. This is the first study to demonstrate that vascular endothelial cells of an amphibian express NOS3.

  17. Malate synthase gene expression during fruit ripening of Cavendish banana (Musa acuminata cv. Williams).

    Science.gov (United States)

    Pua, Eng-Chong; Chandramouli, Sumana; Han, Ping; Liu, Pei

    2003-01-01

    Malate synthase (MS) is a key enzyme responsible for malic acid synthesis in the glyoxylate cycle, which functions to convert stored lipids to carbohydrates, by catalysing the glyoxylate condensation reaction with acetyl-CoA in the peroxisome. In this study, the cloning of an MS cDNA, designated MaMS-1, from the banana fruit is reported. MaMS-1 was 1801 bp in length encoding a single polypeptide of 556 amino acid residues. Sequence analysis revealed that MaMS-1 possessed the conserved catalytic domain and a putative peroxisomal targeting signal SK(I/L) at the carboxyl terminal. MaMS-1 also shared an extensive sequence homology (79-81.3%) with other plant MS homologues. Southern analysis indicated that MS might be present as multiple members in the banana genome. In Northern analysis, MaMS-1 was expressed specifically in ripening fruit tissue and transcripts were not detected in other organs such as roots, pseudostem, leaves, ovary, male flower, and in fruit at different stages of development. However, the transcript abundance in fruit was affected by stage of ripening, during which transcript was barely detectable at the early stage of ripening (FG and TY), but the level increased markedly in MG and in other fruits at advanced ripening stages. Furthermore, MaMS-1 expression in FG fruit could be stimulated by treatment with 1 microl l(-1) exogenous ethylene, but the stimulatory effect was abolished by the application of an ethylene inhibitor, norbornadiene. Results of this study clearly show that MS expression in banana fruit is temporally regulated during ripening and is ethylene-inducible.

  18. Analyses of the sucrose synthase gene family in cotton: structure, phylogeny and expression patterns

    Directory of Open Access Journals (Sweden)

    Chen Aiqun

    2012-06-01

    Full Text Available Abstract Background In plants, sucrose synthase (Sus is widely considered as a key enzyme involved in sucrose metabolism. Several paralogous genes encoding different isozymes of Sus have been identified and characterized in multiple plant genomes, while limited information of Sus genes is available to date for cotton. Results Here, we report the molecular cloning, structural organization, phylogenetic evolution and expression profiles of seven Sus genes (GaSus1 to 7 identified from diploid fiber cotton (Gossypium arboreum. Comparisons between cDNA and genomic sequences revealed that the cotton GaSus genes were interrupted by multiple introns. Comparative screening of introns in homologous genes demonstrated that the number and position of Sus introns are highly conserved among Sus genes in cotton and other more distantly related plant species. Phylogenetic analysis showed that GaSus1, GaSus2, GaSus3, GaSus4 and GaSus5 could be clustered together into a dicot Sus group, while GaSus6 and GaSus7 were separated evenly into other two groups, with members from both dicot and monocot species. Expression profiles analyses of the seven Sus genes indicated that except GaSus2, of which the transcripts was undetectable in all tissues examined, and GaSus7, which was only expressed in stem and petal, the other five paralogues were differentially expressed in a wide ranges of tissues, and showed development-dependent expression profiles in cotton fiber cells. Conclusions This is a comprehensive study of the Sus gene family in cotton plant. The results presented in this work provide new insights into the evolutionary conservation and sub-functional divergence of the cotton Sus gene family in response to cotton fiber growth and development.

  19. Prolactin and liver disease

    NARCIS (Netherlands)

    A.G.C. Bauer (Alexander)

    1982-01-01

    textabstractCirrhosis of the liver is associated with profound endocrinological disturbances. Until recently it was thought that these disturbances were caused mainly by ineffective elimination of hormones by the diseased liver. It is now known that the pathogenesis of disturbed hormonal function in

  20. Acute liver failure

    DEFF Research Database (Denmark)

    Bernal, William; Lee, William M; Wendon, Julia

    2015-01-01

    Over the last three decades acute liver failure (ALF) has been transformed from a rare and poorly understood condition with a near universally fatal outcome, to one with a well characterized phenotype and disease course. Complex critical care protocols are now applied and emergency liver...

  1. Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila.

    Directory of Open Access Journals (Sweden)

    Praveen Balabaskaran Nina

    2010-07-01

    Full Text Available The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1 sector catalyzes ATP synthesis, whereas the F(o sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1 and F(o sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a

  2. Plasticity and evolution of (+)-3-carene synthase and (-)-sabinene synthase functions of a sitka spruce monoterpene synthase gene family associated with weevil resistance.

    Science.gov (United States)

    Roach, Christopher R; Hall, Dawn E; Zerbe, Philipp; Bohlmann, Jörg

    2014-08-22

    The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (-)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3-carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (-)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene, whereas phenylalanine promotes (-)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Liver cancer oncogenomics

    DEFF Research Database (Denmark)

    Marquardt, Jens U; Andersen, Jesper B

    2015-01-01

    Primary liver cancers are among the most rapidly evolving malignant tumors worldwide. An underlying chronic inflammatory liver disease, which precedes liver cancer development for several decades and frequently creates a pro-oncogenic microenvironment, impairs progress in therapeutic approaches....... Molecular heterogeneity of liver cancer is potentiated by a crosstalk between epithelial tumor and stromal cells that complicate translational efforts to unravel molecular mechanisms of hepatocarcinogenesis with a drugable intend. Next-generation sequencing has greatly advanced our understanding of cancer...... development. With regards to liver cancer, the unprecedented coverage of next-generation sequencing has created a detailed map of genetic alterations and identified key somatic changes such as CTNNB1 and TP53 as well as several previously unrecognized recurrent disease-causing alterations that could...

  4. Sucrose Phosphate Synthase and Sucrose Accumulation at Low Temperature 1

    Science.gov (United States)

    Guy, Charles L.; Huber, Joan L. A.; Huber, Steven C.

    1992-01-01

    The influence of growth temperature on the free sugar and sucrose phosphate synthase content and activity of spinach (Spinacia oleracea) leaf tissue was studied. When plants were grown at 25°C for 3 weeks and then transferred to a constant 5°C, sucrose, glucose, and fructose accumulated to high levels during a 14-d period. Predawn sugar levels increased from 14- to 20-fold over the levels present at the outset of the low-temperature treatment. Sucrose was the most abundant free sugar before, during, and after exposure to 5°C. Leaf sucrose phosphate synthase activity was significantly increased by the low-temperature treatment, whereas sucrose synthase and invertases were not. Synthesis of the sucrose phosphate synthase subunit was increased during and after low-temperature exposure and paralleled an increase in the steady-state level of the subunit. The increases in sucrose and its primary biosynthetic enzyme, sucrose phosphate synthase, are discussed in relation to adjustment of metabolism to low nonfreezing temperature and freezing stress tolerance. Images Figure 1 Figure 2 Figure 3 PMID:16652990

  5. A Comparative Analysis of Acyl-Homoserine Lactone Synthase Assays.

    Science.gov (United States)

    Shin, Daniel; Frane, Nicole D; Brecht, Ryan M; Keeler, Jesse; Nagarajan, Rajesh

    2015-12-01

    Quorum sensing is cell-to-cell communication that allows bacteria to coordinate attacks on their hosts by inducing virulent gene expression, biofilm production, and other cellular functions, including antibiotic resistance. AHL synthase enzymes synthesize N-acyl-l-homoserine lactones, commonly referred to as autoinducers, to facilitate quorum sensing in Gram-negative bacteria. Studying the synthases, however, has proven to be a difficult road. Two assays, including a radiolabeled assay and a colorimetric (DCPIP) assay are well-documented in literature to study AHL synthases. In this paper, we describe additional methods that include an HPLC-based, C-S bond cleavage and coupled assays to investigate this class of enzymes. In addition, we compare and contrast each assay for both acyl-CoA- and acyl-ACP-utilizing synthases. The expanded toolkit described in this study should facilitate mechanistic studies on quorum sensing signal synthases and expedite discovery of antivirulent compounds. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Heterologous expression of an active chitin synthase from Rhizopus oryzae.

    Science.gov (United States)

    Salgado-Lugo, Holjes; Sánchez-Arreguín, Alejandro; Ruiz-Herrera, José

    2016-12-01

    Chitin synthases are highly important enzymes in nature, where they synthesize structural components in species belonging to different eukaryotic kingdoms, including kingdom Fungi. Unfortunately, their structure and the molecular mechanism of synthesis of their microfibrilar product remain largely unknown, probably because no fungal active chitin synthases have been isolated, possibly due to their extreme hydrophobicity. In this study we have turned to the heterologous expression of the transcript from a small chitin synthase of Rhizopus oryzae (RO3G_00942, Chs1) in Escherichia coli. The enzyme was active, but accumulated mostly in inclusion bodies. High concentrations of arginine or urea solubilized the enzyme, but their dilution led to its denaturation and precipitation. Nevertheless, use of urea permitted the purification of small amounts of the enzyme. The properties of Chs1 (Km, optimum temperature and pH, effect of GlcNAc) were abnormal, probably because it lacks the hydrophobic transmembrane regions characteristic of chitin synthases. The product of the enzyme showed that, contrasting with chitin made by membrane-bound Chs's and chitosomes, was only partially in the form of short microfibrils of low crystallinity. This approach may lead to future developments to obtain active chitin synthases that permit understanding their molecular mechanism of activity, and microfibril assembly. Copyright © 2016. Published by Elsevier Inc.

  7. cDNA sequences reveal considerable gene prediction inaccuracy in the Plasmodium falciparum genome

    Directory of Open Access Journals (Sweden)

    Valenzuela Jesus G

    2007-07-01

    Full Text Available Abstract Background The completion of the Plasmodium falciparum genome represents a milestone in malaria research. The genome sequence allows for the development of genome-wide approaches such as microarray and proteomics that will greatly facilitate our understanding of the parasite biology and accelerate new drug and vaccine development. Designing and application of these genome-wide assays, however, requires accurate information on gene prediction and genome annotation. Unfortunately, the genes in the parasite genome databases were mostly identified using computer software that could make some erroneous predictions. Results We aimed to obtain cDNA sequences to examine the accuracy of gene prediction in silico. We constructed cDNA libraries from mixed blood stages of P. falciparum parasite using the SMART cDNA library construction technique and generated 17332 high-quality expressed sequence tags (EST, including 2198 from primer-walking experiments. Assembly of our sequence tags produced 2548 contigs and 2671 singletons versus 5220 contigs and 5910 singletons when our EST were assembled with EST in public databases. Comparison of all the assembled EST/contigs with predicted CDS and genomic sequences in the PlasmoDB database identified 356 genes with predicted coding sequences fully covered by EST, including 85 genes (23.6% with introns incorrectly predicted. Careful automatic software and manual alignments found an additional 308 genes that have introns different from those predicted, with 152 new introns discovered and 182 introns with sizes or locations different from those predicted. Alternative spliced and antisense transcripts were also detected. Matching cDNA to predicted genes also revealed silent chromosomal regions, mostly at subtelomere regions. Conclusion Our data indicated that approximately 24% of the genes in the current databases were predicted incorrectly, although some of these inaccuracies could represent alternatively

  8. Identification of a cryptic prokaryotic promoter within the cDNA encoding the 5' end of dengue virus RNA genome.

    Directory of Open Access Journals (Sweden)

    Dongsheng Li

    Full Text Available Infectious cDNA clones of RNA viruses are important research tools, but flavivirus cDNA clones have proven difficult to assemble and propagate in bacteria. This has been attributed to genetic instability and/or host cell toxicity, however the mechanism leading to these difficulties has not been fully elucidated. Here we identify and characterize an efficient cryptic bacterial promoter in the cDNA encoding the dengue virus (DENV 5' UTR. Following cryptic transcription in E. coli, protein expression initiated at a conserved in-frame AUG that is downstream from the authentic DENV initiation codon, yielding a DENV polyprotein fragment that was truncated at the N-terminus. A more complete understanding of constitutive viral protein expression in E. coli might help explain the cloning and propagation difficulties generally observed with flavivirus cDNA.

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

    International Nuclear Information System (INIS)

    Taira, M.; Yoshida, T.; Miyagawa, K.; Sakamoto, H.; Terada, M.; Sugimura, T.

    1987-01-01

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

  10. Liver biopsy in liver patients with coagulopathy

    DEFF Research Database (Denmark)

    Ott, P.; Gronbaek, H.; Clausen, M.R.

    2008-01-01

    The risk of severe bleeding after liver biopsy is estimated to be 1:12,000 in patients with near normal coagulation (INR 60 billion /l). Beyond these limits, the risk is higher, but still uncertain. The Danish guidelines require INR > 1.5, platelet count

  11. Bornyl-diphosphate synthase from Lavandula angustifolia: A major monoterpene synthase involved in essential oil quality.

    Science.gov (United States)

    Despinasse, Yolande; Fiorucci, Sébastien; Antonczak, Serge; Moja, Sandrine; Bony, Aurélie; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis; Jullien, Frédéric

    2017-05-01

    Lavender essential oils (EOs) of higher quality are produced by a few Lavandula angustifolia cultivars and mainly used in the perfume industry. Undesirable compounds such as camphor and borneol are also synthesized by lavender leading to a depreciated EO. Here, we report the cloning of bornyl diphosphate synthase of lavender (LaBPPS), an enzyme that catalyzes the production of bornyl diphosphate (BPP) and then by-products such as borneol or camphor, from an EST library. Compared to the BPPS of Salvia officinalis, the functional characterization of LaBPPS showed several differences in amino acid sequence, and the distribution of catalyzed products. Molecular modeling of the enzyme's active site suggests that the carbocation intermediates are more stable in LaBPPS than in SoBPPS leading probably to a lower efficiency of LaBPPS to convert GPP into BPP. Quantitative RT-PCR performed from leaves and flowers at different development stages of L. angustifolia samples show a clear correlation between transcript level of LaBPPS and accumulation of borneol/camphor, suggesting that LaBPPS is mainly responsible of in vivo biosynthesis of borneol/camphor in fine lavender. A phylogenetic analysis of terpene synthases (TPS) pointed out the basal position of LaBPPS in the TPSb clade, suggesting that LaBPPS could be an ancestor of others lavender TPSb. Finally, borneol could be one of the first monoterpenes to be synthesized in the Lavandula subgenus. Knowledge gained from these experiments will facilitate future studies to improve the lavender oils through metabolic engineering or plant breeding. Accession numbers: LaBPPS: KM015221. Copyright © 2017. Published by Elsevier Ltd.

  12. Understanding Plant Cellulose Synthases through a Comprehensive Investigation of the Cellulose Synthase Family Sequences.

    Science.gov (United States)

    Carroll, Andrew; Specht, Chelsea D

    2011-01-01

    The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA) family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized clades and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair CesA function, and compare these sites to those observed in the closest cellulose synthase-like (Csl) families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments.

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

    Science.gov (United States)

    Soares, Marcelo Bento; Bonaldo, Maria de Fatima

    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.

  14. Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incana R. Br. (Brassicaceae).

    Science.gov (United States)

    Hemleben, Vera; Dressel, Angela; Epping, Bernhard; Lukacin, Richard; Martens, Stefan; Austin, Michael

    2004-05-01

    For Matthiola incana (Brassicaceae), used as a model system to study biochemical and genetical aspects of anthocyanin biosynthesis, several nearly isogenic colored wild type lines and white-flowering mutant lines are available, each with a specific defect in the genes responsible for anthocyanin production (genes e, f, and g). For gene f supposed to code for chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the flavonoid/anthocyanin biosynthesis pathway belonging to the group of type III polyketide synthases (PKS), the wild type genomic sequence of M. incana line 04 was determined in comparison to the white-flowering CHS mutant line 18. The type of mutation in the chs gene was characterized as a single nucleotide substitution in a triplet AGG coding for an evolutionary conserved arginine into AGT coding for serine (R72S). Northern blots and RT-PCR demonstrated that the mutated gene is expressed in flower petals. Heterologous expression of the wild type and mutated CHS cDNA in E. Scherichia coli, verified by Western blotting and enzyme assays with various starter molecules, revealed that the mutant protein had no detectable activity, indicating that the strictly conserved arginine residue is essential for the enzymatic reaction. This mutation, which previously was not detected by mutagenic screening, is discussed in the light of structural and functional information on alfalfa CHS and related type III PKS enzymes.

  15. Enzymatic Assays to Investigate Acyl-Homoserine Lactone Autoinducer Synthases.

    Science.gov (United States)

    Shin, Daniel; Nagarajan, Rajesh

    2018-01-01

    Bacteria use chemical molecules called autoinducers as votes to poll their numerical strength in a colony. This polling mechanism, commonly referred to as quorum sensing, enables bacteria to build a social network and provide a collective response for fighting off common threats. In Gram-negative bacteria, AHL synthases synthesize acyl-homoserine lactone (AHL) autoinducers to turn on the expression of several virulent genes including biofilm formation, protease secretion, and toxin production. Therefore, inhibiting AHL signal synthase would limit quorum sensing and virulence. In this chapter, we describe four enzymatic methods that could be adopted to investigate a broad array of AHL synthases. The enzymatic assays described here should accelerate our mechanistic understanding of quorum-sensing signal synthesis that could pave the way for discovery of potent antivirulence compounds.

  16. Impaired glycogen synthase activity and mitochondrial dysfunction in skeletal muscle

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    Insulin resistance in skeletal muscle is a major hallmark of type 2 diabetes and an early detectable abnormality in the development of this disease. The cellular mechanisms of insulin resistance include impaired insulin-mediated muscle glycogen synthesis and increased intramyocellular lipid content......, whereas impaired insulin activation of muscle glycogen synthase represents a consistent, molecular defect found in both type 2 diabetic and high-risk individuals. Despite several studies of the insulin signaling pathway believed to mediate dephosphorylation and hence activation of glycogen synthase......, the molecular mechanisms responsible for this defect remain unknown. Recently, the use of phospho-specific antibodies in human diabetic muscle has revealed hyperphosphorylation of glycogen synthase at sites not regulated by the classical insulin signaling pathway. In addition, novel approaches such as gene...

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

    International Nuclear Information System (INIS)

    Li Jianxiang; Nie Jihua; Tong Jian; Fu Chunling; Zhou Jianwei

    2008-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kristen M Pluchino

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

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

    Directory of Open Access Journals (Sweden)

    Poustka Annemarie

    2004-06-01

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

  20. [Construction of forward and reverse subtracted cDNA libraries between muscle tissue of Meishan and Landrace pigs].

    Science.gov (United States)

    Xu, De-Quan; Zhang, Yi-Bing; Xiong, Yuan-Zhu; Gui, Jian-Fang; Jiang, Si-Wen; Su, Yu-Hong

    2003-07-01

    Using suppression subtractive hybridization (SSH) technique, forward and reverse subtracted cDNA libraries were constructed between Longissimus muscles from Meishan and Landrace pigs. A housekeeping gene, G3PDH, was used to estimate the efficiency of subtractive cDNA. In two cDNA libraries, G3PDH was subtracted very efficiently at appropriate 2(10) and 2(5) folds, respectively, indicating that some differentially expressed genes were also enriched at the same folds and the two subtractive cDNA libraries were very successful. A total of 709 and 673 positive clones were isolated from forward and reverse subtracted cDNA libraries, respectively. Analysis of PCR showed that most of all plasmids in the clones contained 150-750 bp inserts. The construction of subtractive cDNA libraries between muscle tissue from different pig breeds laid solid foundations for isolating and identifying the genes determining muscle growth and meat quality, which will be important to understand the mechanism of muscle growth, determination of meat quality and practice of molecular breeding.