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Sample records for human cdna encoding

  1. Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor.

    PubMed Central

    Weiser, W Y; Temple, P A; Witek-Giannotti, J S; Remold, H G; Clark, S C; David, J R

    1989-01-01

    A cDNA encoding a human macrophage migration inhibitory factor (MIF) was isolated, through functional expression cloning in COS-1 cells, from a cDNA library prepared from a lectin-stimulated T-cell hybridoma, T-CEMB. The 115-amino acid polypeptide encoded by the MIF cDNA (p7-1) was effectively released from the transfected COS-1 cells and yielded readily detectable MIF activity in the culture supernatant despite the apparent lack of a classical protein secretory sequence. Insertional mutational analysis and elution of MIF activity from polyacrylamide gel slices demonstrated that the Mr 12,000 protein with MIF activity released by the COS-1 cells is encoded by p7-1. The p7-1 cDNA hybridized with a 700-base mRNA expressed by Con-A-stimulated lymphocytes but not unstimulated lymphocytes. The availability of the MIF cDNA clone and recombinant MIF will facilitate the analysis of the role of this lymphokine in cell-mediated immunity, immunoregulation, and inflammation. Images PMID:2552447

  2. Molecular cloning, sequencing and expression of cDNA encoding human trehalase.

    PubMed

    Ishihara, R; Taketani, S; Sasai-Takedatsu, M; Kino, M; Tokunaga, R; Kobayashi, Y

    1997-11-20

    A complete cDNA clone encoding human trehalase, a glycoprotein of brush-border membranes, has been isolated from a human kidney library. The cDNA encodes a protein of 583 amino acids with a calculated molecular weight of 66,595. Human enzyme contains a typical cleavable signal peptide at amino terminus, five potential glycosylation sites, and a hydrophobic region at carboxyl terminus where the protein is anchored to plasma membranes via glycosylphosphatidylinositol. The deduced amino acid sequence of the human enzyme showed similarity to sequences of the enzyme from rabbit, silk worm, Tenebrio molitor, Escherichia coli and yeast. Northern blots revealed that human trehalase mRNA of approx. 2.0 kb was found mainly in the kidney, liver and small intestine. Expression of the recombinant trehalase in E. coli provided a high level of the enzyme activity. The isolation and expression of cDNA for human trehalase should facilitate studies of the structure of the gene, as well as a basis for a better understanding of the catalytic mechanism.

  3. Aspartylglucosaminuria: cDNA encoding human aspartylglucosaminidase and the missense mutation causing the disease.

    PubMed Central

    Ikonen, E; Baumann, M; Grön, K; Syvänen, A C; Enomaa, N; Halila, R; Aula, P; Peltonen, L

    1991-01-01

    We have isolated a 2.1 kb cDNA which encodes human aspartylglucosaminidase (AGA, E.C. 3.5.1.26). The activity of this lysosomal enzyme is deficient in aspartylglucosaminuria (AGU), a recessively inherited lysosomal accumulation disease resulting in severe mental retardation. The polypeptide chain deduced from the AGA cDNA consists of 346 amino acids, has two potential N-glycosylation sites and 11 cysteine residues. Transient expression of this cDNA in COS-1 cells resulted in increased expression of immunoprecipitable AGA protein. Direct sequencing of amplified AGA cDNA from an AGU patient revealed a G----C transition resulting in the substitution of cysteine 163 with serine. This mutation was subsequently found in all the 20 analyzed Finnish AGU patients, in the heterozygous form in all 53 carriers and in none of 67 control individuals, suggesting that it represents the major AGU causing mutation enriched in this isolated population. Since the mutation produces a change in the predicted flexibility of the AGA polypeptide chain and removes an intramolecular S-S bridge, it most probably explains the deficient enzyme activity found in cells and tissues of AGU patients. Images PMID:1703489

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

    SciTech Connect

    Rokeach, L.A.; Haselby, J.A.; Hoch, S.O. )

    1988-07-01

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

  5. Expression cloning of a human cDNA encoding folylpoly(gamma-glutamate) synthetase and determination of its primary structure.

    PubMed Central

    Garrow, T A; Admon, A; Shane, B

    1992-01-01

    A human cDNA for folypoly(gamma-glutamate) synthetase [FPGS; tetrahydrofolate:L-glutamate gamma-ligase (ADP forming), EC 6.3.2.17] has been cloned by functional complementation of an Escherichia coli folC mutant. The cDNA encodes a 545-residue protein of M(r) 60,128. The deduced sequence has regions that are highly homologous to peptide sequences obtained from purified pig liver FPGS and shows limited homology to the E. coli and Lactobacillus casei FPGSs. Expression of the cDNA in E. coli results in elevated expression of an enzyme with characteristics of mammalian FPGS. Expression of the cDNA in AUXB1, a mammalian cell lacking FPGS activity, overcomes the cell's requirement for thymidine and purines but does not overcome the cell's glycine auxotrophy, consistent with expression of the protein in the cytosol but not the mitochondria. PMID:1409616

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

    SciTech Connect

    Hassett, C.; Richter, R.J.; Humbert, R.; Omiecinski, C.J.; Furlong, C.E. ); Chapline, C.; Crabb, J.W. )

    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.

  7. Sanfilippo syndrome type B: cDNA and gene encoding human {alpha}-N-acetylglucosaminidase

    SciTech Connect

    Zhao, H.G.; Lopez, R.; Rennecker, J.

    1994-09-01

    Deficiency of the lysosomal enzyme {alpha}-N-acetlyglucosaminidase underlies the type B Sanfilippo syndrome (MPS III B), a mucopolysaccharide storage disease with profound neurologic deterioration. We are acquiring tools to study the molecular basis of the disorder. The enzyme was purified from bovine testis; after ConA-, DEAE- and phenyl-Sepharose chromatography, it was subjected to SDS-PAGE without preheating. Of two bands of activity detected on the gel, 170 kDa and 87 kDa, the larger one, which coincided with a well-defined Coomassie blue band, was selected for sequence analysis. Degenerate 17-base oligonucleotides, corresponding to the ends of an internal 23 amino acid sequence, were used for RT-PCR of RNA from human fibroblasts. A 41-mer was synthesized from the sequence of the RT-PCR product and used to screen a human testis cDNA library. A number of cDNA inserts were isolated, all lacking the 5{prime} end and none longer than 1.7 kb. An additional 300 bp segment has been obtained by RACE. The cDNA sequence accounts for 9 of 11 peptides, allowing for species difference. Northern analysis of fibroblast RNA with a 1.5 kb cDNA probe showed the presence of a 3 kb mRNA; marked deficiency of this mRNA in two MPS III B fibroblast lines confirmed the authenticity of the cloned cDNA. While no homologous amino acid sequence has been found in a search of GenBank, the nucleotide sequence (interrupted by 4 introns) is present in a flanking region upstream of an unrelated gene on chromosome 17q11-21 (human 17{beta}-hydroxysteroid dehydrogenase). This must therefore be the chromosomal locus of the {alpha}-N-acetylglucosaminidase gene and of MPS III B.

  8. Cloning and characterization of a cDNA encoding transformation-sensitive tropomyosin isoform 3 from tumorigenic human fibroblasts

    SciTech Connect

    Lin, C.S.; Leavitt, J.

    1988-01-01

    The authors isolated a cDNA clone from the tumorigenic human fibroblast cell line HuT-14 that contains the entire protein coding region of tropomyosin isoform 3 (Tm3) and 781 base pairs of 5'- and 3'-untranslated sequences. Tm3, despite its apparent smaller molecular weight than Tm1 in two-dimensional gels, has the same peptide length as Tm1 (284 amino acids) and shares 83% homology with Tm1. Tm3 cDNA hybridized to an abundant mRNA of 1.3 kilobases in fetal muscle and cardiac muscle, suggesting that Tm3 is related to an ..cap alpha../sub fast/-tropomyosin. The first 188 amino acids of Tm3 are identical to those of rat or rabbit skeletal muscle ..cap alpha..-tropomyosin, and the last 71 amino acids differ from those of rat smooth muscle ..cap alpha..-tropomyosin by only 1 residue. Tm3 therefore appears to be encoded by the same gene that encodes the fast skeletal muscle ..cap alpha..-tropomyosin and the smooth muscle ..cap alpha..-tropomyosin via an alternative RNA-splicing mechanism. In contrast to Tm4 and Tm5, Tm3 has a small gene family, with, at best, only one pseudogene.

  9. Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase

    PubMed Central

    Roldán-Arjona, Teresa; Wei, Ying-Fei; Carter, Kenneth C.; Klungland, Arne; Anselmino, Catherine; Wang, Rui-Ping; Augustus, Meena; Lindahl, Tomas

    1997-01-01

    The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine (8-oxo-7,8-dihydroguanine). In bacteria and Saccharomyces cerevisiae, this damaged base is excised by a DNA glycosylase with an associated lyase activity for chain cleavage. We have cloned, sequenced, and expressed a human cDNA with partial sequence homology to the relevant yeast gene. The encoded 47-kDa human enzyme releases free 8-hydroxyguanine from oxidized DNA and introduces a chain break in a double-stranded oligonucleotide specifically at an 8-hydroxyguanine residue base paired with cytosine. Expression of the human protein in a DNA repair-deficient E. coli mutM mutY strain partly suppresses its spontaneous mutator phenotype. The gene encoding the human enzyme maps to chromosome 3p25. These results show that human cells have an enzyme that can initiate base excision repair at mutagenic DNA lesions caused by active oxygen. PMID:9223306

  10. Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

    PubMed

    Sawada, R; Ohashi, K; Anaguchi, H; Okazaki, H; Hattori, M; Minato, N; Naruto, M

    1990-04-01

    To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.

  11. Molecular cloning and characterization of a human cDNA and gene encoding a novel acid ceramidase-like protein.

    PubMed

    Hong, S B; Li, C M; Rhee, H J; Park, J H; He, X; Levy, B; Yoo, O J; Schuchman, E H

    1999-12-01

    Computer-assisted database analysis of sequences homologous to human acid ceramidase (ASAH) revealed a 1233-bp cDNA (previously designated cPj-LTR) whose 266-amino-acid open reading frame had approximately 36% identity with the ASAH polypeptide. Based on this high degree of homology, we undertook further molecular characterization of cPj-LTR and now report the full-length cDNA sequence, complete gene structure (renamed human ASAHL since it is a human acid ceramidase-like sequence), chromosomal location, primer extension and promoter analysis, and transient expression results. The full-length human ASAHL cDNA was 1825 bp and contained an open-reading frame encoding a 359-amino-acid polypeptide that was 33% identical and 69% similar to the ASAH polypeptide over its entire length. Numerous short regions of complete identity were observed between these two sequences and two sequences obtained from the Caenorhabditis elegans genome database. The 30-kb human ASAHL genomic sequence contained 11 exons, which ranged in size from 26 to 671 bp, and 10 introns, which ranged from 150 bp to 6.4 kb. The gene was localized to the chromosomal region 4q21.1 by fluorescence in situ hybridization analysis. Northern blotting experiments revealed a major 2.0-kb ASAHL transcript that was expressed at high levels in the liver and kidney, but at relatively low levels in other tissues such as the lung, heart, and brain. Sequence analysis of the 5'-flanking region of the human ASAHL gene revealed a putative promoter region that lacked a TATA box and was GC rich, typical features of a housekeeping gene promoter, as well as several tissue-specific and/or hormone-induced transcription regulatory sites. 5'-Deletion analysis localized the promoter activity to a 1. 1-kb fragment within this region. A major transcription start site also was located 72 bp upstream from the ATG translation initiation site by primer extension analysis. Expression analysis of a green fluorescence protein/ASAHL fusion

  12. Cloning and expression of the cDNA encoding human fumarylacetoacetate hydrolase, the enzyme deficient in hereditary tyrosinemia: assignment of the gene to chromosome 15.

    PubMed Central

    Phaneuf, D; Labelle, Y; Bérubé, D; Arden, K; Cavenee, W; Gagné, R; Tanguay, R M

    1991-01-01

    Type 1 hereditary tyrosinemia (HT) is an autosomal recessive disease characterized by a deficiency of the enzyme fumarylacetoacetate hydrolase (FAH; E.C.3.7.1.2). We have isolated human FAH cDNA clones by screening a liver cDNA expression library using specific antibodies and plaque hybridization with a rat FAH cDNA probe. A 1,477-bp cDNA was sequenced and shown to code for FAH by an in vitro transcription-translation assay and sequence homology with tryptic fragments of purified FAH. Transient expression of this FAH cDNA in transfected CV-1 mammalian cells resulted in the synthesis of an immunoreactive protein comigrating with purified human liver FAH on SDS-PAGE and having enzymatic activity as shown by the hydrolysis of the natural substrate fumarylacetoacetate. This indicates that the single polypeptide chain encoded by the FAH gene contains all the genetic information required for functional activity, suggesting that the dimer found in vivo is a homodimer. The human FAH cDNA was used as a probe to determine the gene's chromosomal localization using somatic cell hybrids and in situ hybridization. The human FAH gene maps to the long arm of chromosome 15 in the region q23-q25. Images Figure 1 Figure 3 Figure 4 Figure 6 Figure 8 PMID:1998338

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

    SciTech Connect

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

    1988-02-01

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage lambdagt10 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/sup +/ natural killer cells and CD3/sup +/, CD16/sup -/ 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.

  14. Isolation and characterization of a cDNA clone encoding the 60-kD component of the human SS-A/Ro ribonucleoprotein autoantigen.

    PubMed Central

    Ben-Chetrit, E; Gandy, B J; Tan, E M; Sullivan, K F

    1989-01-01

    SS-A/Ro is a nucleocytoplasmic ribonucleoprotein (RNP) particle that is a common target of autoimmune response in Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Previously, SS-A/Ro has been shown to be composed of at least two polypeptide antigens of 60 and 52 kD noncovalently associated with a set of small RNAs, designated Y1-Y5. A serum from an SS patient was selected to screen a lambda gt11 cDNA library constructed from human T cell lymphoblastic leukemia (MOLT-4) mRNA. An immunoreactive clone was isolated that possessed a 1.8-kb cDNA insert. In vitro transcription and translation of the cDNA resulted in the synthesis of a 57.5-kD polypeptide which was specifically immunoprecipitated by SS-A/Ro antisera. The identity of the cDNA encoded protein as the 60-kD SS-A/Ro antigen was established by proteolytic peptide mapping of the cDNA-encoded protein and the 60-kD HeLa cell antigen. The sequence of the cDNA shows that the 60-kD SS-A/Ro protein possesses both RNA binding protein consensus sequences and a single zinc-finger motif. Recombinant SS-A/Ro antigen produced in bacteria proved to be a sensitive and specific reagent for detection of anti-SS-A/Ro antibodies in patient sera. The availability of the 60-kD SS-A/Ro cDNA will enable detailed analysis of the molecular structure and function of the SS-A/Ro RNP particle and its role in autoimmune pathology. Images PMID:2649513

  15. Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA.

    PubMed Central

    Segel, G B; Boal, T R; Cardillo, T S; Murant, F G; Lichtman, M A; Sherman, F

    1992-01-01

    A human cDNA library in lambda-yes plasmid was used to transform a strain of Saccharomyces cerevisiae with defects in histidine biosynthesis (his4-401) and histidine permease (hip1-614) and with the general amino acid permease (GAP) repressed by excess ammonium. We investigated three plasmids complementing the transport defect on a medium with a low concentration of histidine. Inserts in these plasmids hybridized with human genomic but not yeast genomic DNA, indicating their human origin. mRNA corresponding to the human DNA insert was produced by each yeast transformant. Complementation of the histidine transport defect was confirmed by direct measurement of histidine uptake, which was increased 15- to 65-fold in the transformants as compared with the parental strain. Competitive inhibition studies, measurement of citrulline uptake, and lack of complementation in gap1- strains indicated that the human cDNA genes code for proteins that prevent GAP repression by ammonium. The amino acid sequence encoded by one of the cDNA clones is related to T-complex proteins, which suggests a "chaperonin"-like function. We suggest that the human chaperonin-like protein stabilizes the NPR1 gene product and prevents inactivation of GAP. Images PMID:1352881

  16. cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family

    SciTech Connect

    Cool, D.E.; Tonks, N.K.; Charbonneau, H.; Walsh, K.A.; Fischer, E.H.; Krebs, E.G. )

    1989-07-01

    A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase. One positive clone was isolated and the nucleotide sequence was determined. It contained 1,305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3{prime} untranslated end, although a poly(A){sup +} tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues. This was supported by the synthesis of a M{sub r} 48,000 protein in an in vitro reticulocyte lysate translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low M{sub r} PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes.

  17. Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumor associated with humoral hypercalcemia of malignancy

    SciTech Connect

    Mangin, M.; Webb, A.C.; Dreyer, B.E.; Posillico, J.T.; Ikeda, K.; Weir, E.C.; Stewart, A.F.; Bander, N.H.; Milstone, L.; Barton, D.E.

    1988-01-01

    Humoral hypercalcemia of malignancy is a common paraneoplastic syndrome that appears to be mediated in many instances by a parathyroid hormone-like peptide. Poly(A)/sup +/ RNA from a human renal carcinoma associated with this syndrome was enriched by preparative electrophoresis and used to construct an enriched cDNA library in phage lambdagt10. The library was screened with a codon-preference oligonucleotide synthesized on the basis of a partial N-terminal amino acid sequence from a human tumor-derived peptide, and a 2.0 kilo-base cDNA was identified. The cDNA encodes a 177 amino acid protein consisting of a 36 amino acid leader sequence and a 141 amino acid mature peptide. The first 13 amino acids of the deduced sequence of the mature peptide display strong homology to human PTH, with complete divergence thereafter. RNA blot-hybridization analysis revealed multiple transcripts in mRNA from tumors associated with the humor syndrome and also in mRNA from normal human keratinocytes. Southern blot analysis of genomic DNA from humans and rodents revealed a simple pattern compatible with a single-copy gene. The gene has been mapped to chromosome 12.

  18. Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated fatty acids.

    PubMed Central

    Leonard, A E; Bobik, E G; Dorado, J; Kroeger, P E; Chuang, L T; Thurmond, J M; Parker-Barnes, J M; Das, T; Huang, Y S; Mukerji, P

    2000-01-01

    The Saccharomyces cerevisiae protein ELO2p is involved in the elongation of saturated and monounsaturated fatty acids. Among several sequences with limited identity with the S. cerevisiae ELO2 gene, a consensus cDNA sequence was identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. Human liver cDNA was amplified by PCR using oligonucleotides complementary to the 5' and 3' ends of the putative human cDNA sequence. The resulting full-length sequence, termed HELO1, consisted of 897 bp, which encoded 299 amino acids. However, in contrast with the ELO2 gene, expression of this open reading frame in S. cerevisiae demonstrated that the encoded protein was involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of gamma-linolenic acid (C(18:3, n-6)) into dihomo-gamma-linolenic acid (C(20:3, n-6)), arachidonic acid (C(20:4, n-6)) into adrenic acid (C(22:4, n-6)), stearidonic acid (C(18:4, n-3)) into eicosatetraenoic acid (C(20:4, n-3)), eicosapentaenoic acid (C(20:5, n-3)) into omega3-docosapentaenoic acid (C(22:5, n-3)) and alpha-linolenic acid (C(18:3, n-3)) into omega3-eicosatrienoic acid (C(20:3, n-3)). The predicted amino acid sequence of the open reading frame had only 29% identity with the yeast ELO2 sequence, contained a single histidine-rich domain and had six transmembrane-spanning regions, as suggested by hydropathy analysis. The tissue expression profile revealed that the HELO1 gene is highly expressed in the adrenal gland and testis. Furthermore, the HELO1 gene is located on chromosome 6, best known for encoding the major histocompatibility complex, which is essential to the human immune response. PMID:10970790

  19. Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: definition of a family of DNA repair enzymes.

    PubMed Central

    Demple, B; Herman, T; Chen, D S

    1991-01-01

    Abasic (AP) sites are common, potentially mutagenic DNA damages that are attacked by AP endonucleases. The biological roles of these enzymes in metazoans have not been tested. We have cloned the human cDNA (APE) that encodes the main nuclear AP endonuclease. The predicted Ape protein, which contains likely nuclear transport signals, is a member of a family of DNA repair enzymes that includes two bacterial AP endonucleases (ExoA protein of Streptococcus pneumoniae and exonuclease III of Escherichia coli) and Rrp1 protein of Drosophila melanogaster. Purified Ape protein lacks the 3'-exonuclease activity against undamaged DNA that is found in the bacterial and Drosophila enzymes, but the lack of obvious amino acid changes to account for this difference suggests that the various enzyme functions evolved by fine tuning a conserved active site. Expression of the active human enzyme in AP endonuclease-deficient E. coli conferred significant resistance to killing by the DNA-alkylating agent methyl methanesulfonate. The APE cDNA provides a molecular tool for analyzing the role of this central enzyme in maintaining genetic stability in humans. Images PMID:1722334

  20. Expression of the developmental I antigen by a cloned human cDNA encoding a member of a beta-1,6-N-acetylglucosaminyltransferase gene family.

    PubMed

    Bierhuizen, M F; Mattei, M G; Fukuda, M

    1993-03-01

    The blood group i/I antigens were the first identified alloantigens that display a dramatic change during human development. The i and I antigens are determined by linear and branched poly-N-acetyllactosaminoglycans, respectively. In human erythrocytes during embryonic development, the fetal (i) antigen is replaced by the adult (I) antigen as a result of the appearance of a beta-1,6-N-acetylglucosaminyltransferase, the I-branching enzyme. Here, we report the cDNA cloning and expression of this branching enzyme that converts linear into branched poly-N-acetyllactosaminoglycans, thus introducing the I antigen in transfected cells. The cDNA sequence predicts a protein with type II membrane topology as has been found for all other mammalian glycosyltransferases cloned to date. The Chinese hamster ovary cells that stably express the isolated cDNA acquire I-branched structures as evidenced by the structural analysis of glycopeptides from these cells. Comparison of the amino acid sequence with those of other glycosyltransferases revealed that this I-branching enzyme and another beta-1,6-N-acetylglucosaminyltransferase that forms a branch in O-glycans are strongly homologous in the center of their putative catalytic domains. Moreover, the genes encoding these two beta-1,6-N-acetylglucosaminyltransferases were found to be located at the same locus on chromosome 9, band q21. These results indicate that the I-branching enzyme represents a member of a beta-1,6-N-acetylglucosaminyltransferase gene family of which expression is controlled by developmental programs.

  1. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

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

    1993-02-16

    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 1,018 nucleotides long and includes an open reading frame of 204 amino acids.

  2. Antibody to a human DNA repair protein allows for cloning of a Drosophila cDNA that encodes an apurinic endonuclease

    SciTech Connect

    Kelley, M.R. ); Venugopal, S.; Harless, J.; Deutsch, W.A. . Dept. of Biochemistry)

    1989-03-01

    The cDNA of a Drosophila DNA repair gene, AP3, was cloned by screening an embryonic lambda gt11 expression library with an antibody that was originally prepared against a purified human apurinicapyrimidine (AP) endonuclease. The 1.2-kilobase (kb) AP3 cDNA mapped to a region on the third chromosome where a number of mutagen-sensitive alleles were located. The cDNA clone yielded an in vitro translation product of 35,000 daltons, in agreement with the predicted size of the translation product of the only open reading frame of AP3, and identical to the molecular size of an AP endonuclease activity recovered following sodium dodecyl sulfate-polyacrymalide gel electrophoresis of Drosophilia extracts. The C-terminal portion of the predicted protein contained regions of presumptive DNA-binding domains, while the DNA sequence at the amino end of AP3 showed similarity to the Escherichia coli recA gene. AP3 is expressed as an abundant 1.3-kb mRNA that is detected throughout the life cycle of Drosophila melanogaster. Another 3.5-klb mRNA also hybridized to the AP3 cDNA, but species was restricted to the early stages of development.

  3. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

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

    1993-02-16

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

  4. Molecular characterization of an autoantigen of PM-Scl in the polymyositis/scleroderma overlap syndrome: a unique and complete human cDNA encoding an apparent 75-kD acidic protein of the nucleolar complex

    PubMed Central

    1991-01-01

    About 50% of patients with the polymyositis/scleroderma (PM-Scl) overlap syndrome are reported to have autoantibodies to a nuclear/nucleolar particle termed PM-Scl. The particle is composed of several polypeptides of which two have been identified as autoantigens. In this report, human cDNA clone coding for the entire 75-kD autoantigen of the PM-Scl particle (PM-Scl 75) was isolated from a MOLT- 4 lambda gt-11 library. The deduced amino acid sequence of the cDNA clone represented a protein of 355 amino acids and 39.2 kD; the in vitro translation product of this cDNA migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at approximately 70 kD. The aberrant migration of the polypeptide in SDS-PAGE was shown to be related to the COOH half that was rich in acidic residues. Authenticity of the cDNA coding for PM-Scl 75 was shown by immunoreactivity of PM-Scl sera with in vitro translation products and recombinant fusion proteins encoded by the cDNA. In addition, rabbit antibodies raised to recombinant fusion protein reacted in immunofluorescence, immunoblotting, and immunoprecipitation with the characteristic features displayed by human anti-PM-Scl sera. PMID:2007859

  5. Identification of cDNA encoding an additional. alpha. subunit of a human GTP-binding protein: Expression of three. alpha. sub i subtypes in human tissues and cell lines

    SciTech Connect

    Kim, S.; Ang, S.L.; Bloch, D.B.; Bloch, K.D.; Kawahara, Y.; Tolman, C.; Lee, R.; Seidman, J.G.; Neer, E.J. )

    1988-06-01

    The guanine nucleotide-binding proteins (G proteins), which mediate hormonal regulation of many membrane functions, are composed of {alpha}, {beta}, and {gamma} subunits. The authors have cloned and characterized cDNA from a human T-cell library encoding a form of {alpha}{sub i} that is different from the human {alpha}{sub i} subtypes previously reported. {alpha}{sub i} is the {alpha} subunit of a class of G proteins that inhibits adenylate cyclase and regulates other enzymes and ion channels. This cDNA encodes a polypeptide of 354 amino acids and is assigned to encode the {alpha}{sub i-3} subtype of G proteins on the basis of its similarity to other {alpha}{sub i}-like cDNAs and the presence of a predicted site for ADP ribosylation by pertussis toxin. They have determined the expression of mRNA for this and two other subtypes of human {alpha}{sub i} ({alpha}{sub i-1} and {alpha}{sub i-2}) in a variety of human fetal tissues and in human cell lines. All three {alpha}{sub i} subtypes were present in the tissues tested. However, analysis of individual cell types reveals specificity of {alpha}{sub i-1} expression. mRNA for {alpha}{i-1} is absent in T cells, B cells, and monocytes but is present in other cell lines. The finding of differential expression of {alpha}{sub i-1} genes may permit characterization of distinct physiological roles for this {alpha}{sub i} subunit. mRNA for {alpha}{sub i-2} and {alpha}{sub i-3} was found in all the primary and transformed cell lines tested. Thus, some cells contain all three {alpha}{sub i} subtypes. This observation raises the question of how cells prevent cross talk among receptors that are coupled to effectors through such similar {alpha} proteins.

  6. Characterization and chromosomal assignment of a human cDNA encoding a protein related to the murine 102-kDa cadherin-associated protein ([alpha]-catenin)

    SciTech Connect

    Claverie, J.M. ); Hardelin, J.P.; Legouis, R.; Levilliers, J.; Petit, C. ); Bougueleret, L. ); Mattei, M.G. )

    1993-01-01

    We report the characterization of a human cDNA encompassing the complete coding region of a 945-residue putative protein (CAP-R) 80% identical to the recently described murine 102-kDa [alpha]-catenin (CAP102). The CAP-R protein mostly differs from CAP102 by the presence of a 48-residue insert. This insert exhibits similarity with a segment of the type 1 neurofibromatosis gene product. The analysis of a publicly available human [open quote]expressed sequence tag[close quotes] collection revealed the existence of another human cDNA more closely related (89% identical) to CAP 102. This strongly suggests that CAP-R is not the human homologue of the murine 102- kDa [alpha]-catenin but a new closely related gene of the vinculin family. This is further supported by the computed mutation rates falling outside the range observed for mammalian orthologous genes. Using in situ hybridization, the CAP-R gene could be mapped to the pll.l-pl2 region of human chromosome 2 and to the homologous B3-D region of mouse chromosome 6. 32 refs., 4 fig.

  7. Human cyclooxygenase-2 cDNA.

    PubMed Central

    Hla, T; Neilson, K

    1992-01-01

    Cyclooxygenase (Cox), also known as prostaglandin (PG) H synthase (EC 1.14.99.1), catalyzes the rate-limiting step in the formation of inflammatory PGs. A major regulatory step in PG biosynthesis is at the level of Cox: growth factors, cytokines, and tumor promoters induce Cox activity. We have cloned the second form of the Cox gene (Cox-2) from human umbilical vein endothelial cells (HUVEC). The cDNA encodes a polypeptide of 604 amino acids that is 61% identical to the previously isolated human Cox-1 polypeptide. In vitro translation of the human (h)Cox-2 transcript in rabbit reticulocyte lysates resulted in the synthesis of a 70-kDa protein that is immunoprecipitated by antiserum to ovine Cox. Expression of the hCox-2 open reading frame in Cos-7 monkey kidney cells results in the elaboration of cyclooxygenase activity. hCox-2 cDNA hybridizes to a 4.5-kilobase mRNA species in HUVEC, whereas the hCox-1 cDNA hybridizes to 3- and 5.3-kilobase species. Both Cox-1 and Cox-2 mRNAs are expressed in HUVEC, vascular smooth muscle cells, monocytes, and fibroblasts. Cox-2 mRNA was preferentially induced by phorbol 12-myristate 13-acetate and lipopolysaccharide in human endothelial cells and monocytes. Together, these data demonstrate that the Cox enzyme is encoded by at least two genes that are expressed and differentially regulated in a variety of cell types. High-level induction of the hCox-2 transcript in mesenchymal-derived inflammatory cells suggests a role in inflammatory conditions. Images PMID:1380156

  8. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

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

    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 1,018 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 figures.

  9. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

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

    1999-05-04

    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. 12 figs.

  10. CDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    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.

  11. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

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

    1999-05-04

    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.

  12. cDNA encoding a polypeptide including a hevein sequence

    SciTech Connect

    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.

  13. Sequence of the cDNA encoding an actin homolog in the crayfish Procambarus clarkii.

    PubMed

    Kang, W K; Naya, Y

    1993-11-15

    A cDNA library was constructed by using mRNAs purified from crayfish (Procambarus clarkii) muscle. Using a homology search of the nucleotide (nt) sequences, a clone of the library was found to encode a protein homologous to actin (Act). The insert fragment of this cDNA clone was 1072 nt in length. The amino acid sequence deduced from the nt sequence showed significant similarity to Act of various organisms as follows: 88.1% to Drosophila melanogaster, 88.2% to silk worm, 87.3% to brine shrimp, 86.3% to rat, and 86.3% to human (% identity).

  14. Horse cDNA clones encoding two MHC class I genes

    SciTech Connect

    Barbis, D.P.; Maher, J.K.; Stanek, J.; Klaunberg, B.A.; Antczak, D.F.

    1994-12-31

    Two full-length clones encoding MHC class I genes were isolated by screening a horse cDNA library, using a probe encoding in human HLA-A2.2Y allele. The library was made in the pcDNA1 vector (Invitrogen, San Diego, CA), using mRNA from peripheral blood lymphocytes obtained from a Thoroughbred stallion (No. 0834) homozygous for a common horse MHC haplotype (ELA-A2, -B2, -D2; Antczak et al. 1984; Donaldson et al. 1988). The clones were sequenced, using SP6 and T7 universal primers and horse-specific oligonucleotides designed to extend previously determined sequences.

  15. Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec complex recognized by autoantibodies from patients with type-1 autoimmune hepatitis

    PubMed Central

    Costa, M; Rodríguez-Sánchez, J L; Czaja, A J; Gelpí, C

    2000-01-01

    We previously described autoantibodies against a UGA serine tRNA–protein complex (tRNP(Ser)Sec) in patients with type-1 autoimmune hepatitis [1] and now define the specificity and frequency of this autoantibody and the DNA sequence encoding the tRNA(Ser)Sec-associated antigenic protein. The presence of anti‐tRNP(Ser)Sec antibodies was highly specific for type-1 autoimmune hepatitis, as 47·5% of patients were positive compared with none of the control subjects. To characterize the antigenic protein(s), we immunoscreened a human cDNA library with anti-tRNP(Ser)Sec-positive sera. Two clones (19 and 13) were isolated. Clone 19 encodes a protein with a predicted molecular mass of 48·8 kD. Clone 13 is a shorter cDNA, almost identical to clone 19, which encodes a 35·9-kD protein. Expression of both cDNAs was accomplished in Escherichia coli as His-tagged recombinant proteins. Antibodies eluted from both purified recombinant proteins were able to immunoprecipitate the tRNA(Ser)Sec from a HeLa S3 cell extract, demonstrating their cross-reactivity with the mammalian antigenic complex. Recent cloning data relating to the target antigen(s) of autoantibodies in autoimmune hepatitis patients that react with a soluble liver antigen (SLA) and a liver-pancreas antigen (LP) have revealed that these two autoantibodies are identical and that the cloned antigen shows 99% amino acid sequence homology with tRNP(Ser)Sec. PMID:10931155

  16. Cloning of a cDNA encoding a putative human very low density lipoprotein/Apolipoprotein E receptor and assignment of the gene to chromosome 9pter-p23[sup 6

    SciTech Connect

    Gafvels, M.E.; Strauss, J.F. III ); Caird, M.; Patterson, D. ); Britt, D.; Jackson, C.L. )

    1993-11-01

    The authors report the cloning of a 3656-bp cDNA encoding a putative human very low density lipoprotein (VLDL)/apolipoprotein E (ApoE) receptor. The gene encoding this protein was mapped to chromosome 9pter-p23. Northern analysis of human RNA identified cognate mRNAs of 6.0 and 3.8 kb with most abundant expression in heart and skeletal muscle, followed by kidney, placenta, pancreas, and brain. The pattern of expression generally paralleled that of lipoprotein lipase mRNA but differed from that of the low density lipoprotein (LDL) receptor and the low density lipoprotein receptor-related protein/[alpha][sub 2]-macroglobulin receptor (LRP), which are members of the same gene family. VLDL/ApoE receptor message was not detected in liver, whereas mRNAs for both LDL receptor and LRP were found in hepatic tissue. In mouse 3T3-L1 cells, VLDL/ApoE receptor mRNA was induced during the transformation of the cells into adipocytes. Expression was also detected in human choriocarcinoma cells, suggesting that at least part of the expression observed in placenta may be in trophoblasts, cells which would be exposed to maternal blood. Expression in brain may be related to high levels of ApoE expression in that organ, an observation of potential relevance to the recently hypothesized role for ApoE in late onset Alzheimer disease. The results suggest that the putative VLDL/ApoE receptor could play a role in the uptake of triglyceride-rich lipoprotein particles by specific organs including striated and cardiac muscle and adipose tissue and in the transport of maternal lipids across the placenta. The findings presented here, together with recent observations from other laboratories, bring up the possibility that a single gene, the VLDL/ApoE receptor, may play a role in the pathogenesis of certain forms of atherosclerosis, Alzheimer disease, and obesity.

  17. Cloning and expression of human tyrosine aminotransferase cDNA.

    PubMed

    Séralini, G E; Luu-Thé, V; Labrie, F

    1995-01-02

    Complementary DNA clones encoding human tyrosine aminotransferase (TAT) were isolated by screening a normal adult woman liver lambda gt11 library with rat TAT cDNA. The largest isolated cDNA is 2051 bp long (EMBL accession number X55675). This cDNA was subcloned downstream of the cytomegalovirus promoter in the pCMV vector for transfection into human cervical carcinoma HeLa cells. Expression of the TAT cDNA resulted in the synthesis of a protein with a molecular mass of approximately 50 kDa, as assessed by Western analysis, a value which is in close agreement with the predicted molecular weight of 50,399, for a deduced sequence of 454 amino acids. The expressed protein catalyzed specifically the conversion of L-[14C]tyrosine into p-[14C]hydroxyphenylpyruvate. The availability of a functional TAT cDNA provides a useful tool for detailed study of the structure-function relationship of the enzyme and its mutated derivatives.

  18. Isolation and sequencing of a cDNA clone encoding the 85 kDa human lysosomal sialoglycoprotein (hLGP85) in human metastatic pancreas islet tumor cells.

    PubMed

    Fujita, H; Takata, Y; Kono, A; Tanaka, Y; Takahashi, T; Himeno, M; Kato, K

    1992-04-30

    A full length cDNA for a human lysosomal membrane sialoglycoprotein (hLGP85) was isolated as a probe of the cDNA of rat LGP85 (rLGP85) from the cDNA library prepared from total mRNA of QGP-1NL cells, a human pancreatic islet tumor cell with a high metastatic activity. The deduced amino acid sequence shows that hLGP85 consists of 478 amino acid residues (MW. 54,289). The protein has 10 putative N-glycosylation sites and 2 hydrophobic regions at the NH2- and near the COOH-termini, respectively. Thus, both domains probably constitute putative transmembrane domains. It exhibits 86% and 79% sequence similarities in amino acids and nucleic acids to rat lysosomal membrane sialoglycoprotein (rLGP85), respectively. The protein contained the short cytoplasmic tail at the COOH-terminus which does not form the glycine-tyrosine sequence (GY motif), the so-called lysosomal targetting signal.

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

    PubMed

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

    1984-12-10

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

  20. Molecular cloning of cDNA encoding the Xenopus homolog of mammalian RelB.

    PubMed Central

    Suzuki, K; Yamamoto, T; Inoue, J

    1995-01-01

    We have molecularly cloned cDNA encoding a new Rel-related protein in Xenopus laevis. Nucleotide sequencing revealed that the product is most homologous to mammalian RelB in its N-terminal region. Furthermore, the putative protein kinase A phosphorylation site (RRPS), found in most of the Rel family proteins, but replaced by QRLT in mammalian RelB, is replaced by QRIT, indicating that our cDNA most likely encodes the Xenopus homolog of mammalian RelB (XrelB). As in the case of mouse RelB, XrelB alone does not bind to DNA efficiently, while XrelB/human p50 heterodimers bind to kappa B sites and activate transcription. XrelB transcripts are present at all stages of oocyte maturation and in adult tissues examined. However, in staged embryos XrelB is undetectable from neurula to stage 28 and resumes expression at stage 47, while Xrel1/XrelA, the Xenopus homolog of p65, has been demonstrated to be expressed throughout embryogenesis. These results raise the possibility that XrelB and Xrel1/XrelA play different roles in the development of X.laevis. Images PMID:8524658

  1. Cloning and sequencing of a cDNA encoding a taste-modifying protein, miraculin.

    PubMed

    Masuda, Y; Nirasawa, S; Nakaya, K; Kurihara, Y

    1995-08-19

    A cDNA clone encoding a taste-modifying protein, miraculin (MIR), was isolated and sequenced. The encoded precursor to MIR was composed of 220 amino acid (aa) residues, including a possible signal sequence of 29 aa. Northern blot analysis showed that the mRNA encoding MIR was already expressed in fruits of Richadella dulcifica at 3 weeks after pollination and was present specifically in the pulp.

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

    PubMed

    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.

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

    SciTech Connect

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

    1991-01-15

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

  4. Cloning and sequencing of a dextranase-encoding cDNA from Penicillium minioluteum.

    PubMed

    Garcia, B; Margolles, E; Roca, H; Mateu, D; Raices, M; Gonzales, M E; Herrera, L; Delgado, J

    1996-10-01

    A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-alpha-glucan hydrolase, EC 3.2.1.11) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.

  5. Analysis of cDNA encoding the hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) of Schistosoma mansoni; a putative target for chemotherapy.

    PubMed Central

    Craig, S P; McKerrow, J H; Newport, G R; Wang, C C

    1988-01-01

    Because of the lack of de novo purine biosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is a critical enzyme in the purine metabolic pathway of the human parasite, Schistosoma mansoni. Using a cDNA clone encoding mouse HGPRTase and subsequently a synthetic oligonucleotide derived from sequencing a clone of genomic DNA, two clones were isolated from an adult schistosome cDNA library. One clone is 1.374 Kilobases (Kb) long and has an open reading frame of 693 bases. The deduced 231 amino acid sequence has 47.9% identity in a 217 amino acid overlap with human HGPRTase. Northern blot analysis indicates that the full length of mRNA for the S. mansoni HGPRTase is 1.45-1.6 Kb. Analysis of the primary structures of the putative active site for human and parasite enzymes reveal specific differences which may eventually be exploitable in the design of drugs for the treatment of schistosomiasis. Images PMID:3136439

  6. Human somatostatin I: sequence of the cDNA.

    PubMed Central

    Shen, L P; Pictet, R L; Rutter, W J

    1982-01-01

    RNA has been isolated from a human pancreatic somatostatinoma and used to prepare a cDNA library. After prescreening, clones containing somatostatin I sequences were identified by hybridization with an anglerfish somatostatin I-cloned cDNA probe. From the nucleotide sequence of two of these clones, we have deduced an essentially full-length mRNA sequence, including the preprosomatostatin coding region, 105 nucleotides from the 5' untranslated region and the complete 150-nucleotide 3' untranslated region. The coding region predicts a 116-amino acid precursor protein (Mr, 12.727) that contains somatostatin-14 and -28 at its COOH terminus. The predicted amino acid sequence of human somatostatin-28 is identical to that of somatostatin-28 isolated from the porcine and ovine species. A comparison of the amino acid sequences of human and anglerfish preprosomatostatin I indicated that the COOH-terminal region encoding somatostatin-14 and the adjacent 6 amino acids are highly conserved, whereas the remainder of the molecule, including the signal peptide region, is more divergent. However, many of the amino acid differences found in the pro region of the human and anglerfish proteins are conservative changes. This suggests that the propeptides have a similar secondary structure, which in turn may imply a biological function for this region of the molecule. Images PMID:6126875

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

    SciTech Connect

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

    1989-11-01

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-K{sub 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.

  8. Isolation and characterization of a Paracentrotus lividus cDNA encoding a stress-inducible chaperonin

    PubMed Central

    Gianguzza, Fabrizio; Antonietta Ragusa, Maria; Roccheri, Maria Carmela; Liegro, Italia Di; Rinaldi, Anna Maria

    2000-01-01

    Chaperonins are ubiquitous proteins that facilitate protein folding in an adenosine triphosphate–dependent manner. Here we report the isolation of a sea urchin cDNA (Plhsp60) coding for mitochondrial chaperonin (Cpn60), whose basal expression is further enhanced by heat shock. The described cDNA corresponds to a full-length mRNA encoding a protein of 582 amino acids, the first 32 of which constitute a putative mitochondrial targeting leader sequence. Comparative analysis has demonstrated that this protein is highly conserved in evolution. PMID:11147969

  9. Isolation and nucleotide sequence of a cDNA clone encoding rat mitochondrial malate dehydrogenase.

    PubMed Central

    Grant, P M; Tellam, J; May, V L; Strauss, A W

    1986-01-01

    We have determined the complete sequence of the rat mitochondrial malate dehydrogenase (mMDH) precursor derived from nucleotide sequence of the cDNA. A single synthetic oligodeoxynucleotide probe was used to screen a rat atrial cDNA library constructed in lambda gt10. A 1.2 kb full-length cDNA clone provided the first complete amino acid sequence of pre-mMDH. The 1014 nucleotide-long open reading frame encodes the 314 residue long mature mMDH protein and a 24 amino acid NH2-terminal extension which directs mitochondrial import and is cleaved from the precursor after import to generate mature mMDH. The amino acid composition of the transit peptide is polar and basic. The pre-mMDH transit peptide shows marked homology with those of two other enzymes targeted to the rat mitochondrial matrix. Images PMID:3755817

  10. Isolation and characterization of a cDNA clone encoding wheat germ agglutinin

    SciTech Connect

    Raikhel, N.V.; Wilkins, T.A.

    1987-10-01

    Two sets of synthetic oligonucleotides coding for amino acids in the amino- and carboxyl-terminal portions of wheat germ agglutinin were synthesized and used as hybridization probes to screen cDNA libraries derived from developing embryos of tetraploid wheat. The nucleotide sequence for a cDNA clone recovered from the cDNA library was determined by dideoxynucleotide chain-termination sequencing in vector M13. The amino acid sequence deduced from the DNA sequence indicated that this cDNA clone (pNVR1) encodes isolectin 3 of wheat germ agglutinin. Comparison of the deduced amino acid sequence of clone pNVR1 with published sequences indicates isolectin 3 differs from isolectins 1 and 2 by 10 and 8 amino acid changes, respectively. In addition, the protein encoded by pNVR1 extends 15 amino acids beyond the carboxyl terminus of the published amino acid sequence for isolectins 1 and 2 and includes a potential site for N-linked glycosylation. Utilizing the insert of pNVR1 as a hybridization probe, the authors have demonstrated that the expression of genes for wheat germ agglutinin is modulated by exogenous abscisic acid. Striking homology is observed between wheat germ agglutinin and chitinase, both of which are proteins that bind chitin.

  11. Isolation and characterization of human defensin cDNA clones

    SciTech Connect

    Daher, K.A.; Lehrer, R.I.; Ganz, T.; Kronenberg, M. )

    1988-10-01

    Four clones that encode defensins, a group of microbicidal and cytotoxic peptides made by neutrophils, were isolated from an HL-60 human promyelocytic leukemia cDNA library. Analysis of these clones indicated that the defensins are made as precursor proteins, which must be cleaved to yield the mature peptides. Defensin mRNA was detected in normal bone marrow cells, but not in normal peripheral blood leukocytes. Defensin transcripts were also found in the peripheral leukocytes of some leukemia patients and in some lung and intestine tissues. Defensin mRNA content was augmented by treatment of HL-60 cells with dimethyl sulfoxide. These results define important aspects of the mechanism of synthesis and the tissue-specific expression of a major group of neutrophil granule proteins.

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

    PubMed

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

    2002-06-01

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

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

    NASA Astrophysics Data System (ADS)

    Wang, Zai-Zhao; Xiang, Jian-Hai

    2002-09-01

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

  14. Isolation and characterization of cDNA clones encoding pig gastric mucin.

    PubMed Central

    Turner, B S; Bhaskar, K R; Hadzopoulou-Cladaras, M; Specian, R D; LaMont, J T

    1995-01-01

    Polyclonal antibodies raised to deglycosylated pig gastric mucin were used to screen a cDNA library constructed with pig stomach mucosal mRNA. Immunocytochemistry indicated that the antibody recognizes intracellular and secreted mucin in surface mucous cells of pig gastric epithelium. A total of 70 clones producing proteins immunoreactive to this antibody were identified, two of which (PGM-2A,9B) were fully sequenced from both ends. Clone PGM-9B hybridized to a polydisperse mRNA (3-9 kb) from pig stomach, but not liver, intestine or spleen, nor to mRNA from human, mouse, rabbit or rat stomach. Sequence analysis indicated that PGM-9B encodes 33 tandem repeats of a 16-amino-acid consensus sequence rich in serine (46%) and threonine (17%). Using the restriction enzyme MwoI, which has a single target site in the repeat, it was demonstrated that PGM-9B consists entirely of this tandem repeat. Southern-blot analysis indicated that the repeat region is contained in a 20 kb HindIII-EcoRI fragment, and BamHI digestion suggested that most of the repeats are contained in a 10 kb fragment. In situ hybridization with an antisense probe to PGM-9B showed an intense signal in the entire gastric gland. Clone PGM-2A also contains the same repeat sequence as 9B, but, in addition, has a 64-amino-acid-long non-repeat region at its 5' end. Interestingly the non-repeat region of PGM-2A has five cysteine residues, the arrangement of which is identical with that reported for human intestinal mucin gene MUC2. Images Figure 1 Figure 2 Figure 5 Figure 6 Figure 7 Figure 8 PMID:7755593

  15. Isolation of cDNA and genomic DNA clones encoding type II collagen.

    PubMed Central

    Young, M F; Vogeli, G; Nunez, A M; Fernandez, M P; Sullivan, M; Sobel, M E

    1984-01-01

    A cDNA library constructed from total chick embryo RNA was screened with an enriched fraction of type II collagen mRNA. Two overlapping cDNA clones were characterized and shown to encode the COOH propeptide of type II collagen. In addition, a type II collagen clone was isolated from a Charon 4A library of chick genomic fragments. Definitive identification of the clones was based on DNA sequence analysis. The 3' end of the type II collagen gene appears to be similar to that of other interstitial collagen genes. Northern hybridization data indicates that there is a marked decrease in type II collagen mRNA levels in chondrocytes treated with the dedifferentiating agent 5-bromodeoxyuridine. The major type II collagen mRNA species is 5300 bases long, similar to that of other interstitial collagen RNAs. Images PMID:6203098

  16. Cloning and functional expression of a cDNA encoding coffee bean alpha-galactosidase.

    PubMed

    Zhu, A; Goldstein, J

    1994-03-25

    Purified coffee bean alpha-galactosidase (alpha Gal) has been used for removing terminal alpha-galactose residues from the glyco-conjugates at the red cell surface, in studies of blood group conversion. Here, we report the isolation and sequence of the full-length clone for coffee bean alpha Gal by using the polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques. The cDNA clone (1.4 kb) contains a single open reading frame which encodes a protein of 378 amino acids (aa). Its authenticity is confirmed by perfect alignment of aa sequences obtained from purified coffee bean alpha Gal, and by immune reaction with the antibody raised against the enzyme. Furthermore, the protein produced in insect cells shows enzymatic activity towards a synthetic alpha Gal substrate, p-nitro-phenyl-alpha-galactopyranoside.

  17. cDNA encoding a polypeptide including a hev ein sequence

    DOEpatents

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

    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.

  18. Cloning and expression of cDNA encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation.

    PubMed Central

    White, P C; New, M I; Dupont, B

    1984-01-01

    We isolated a cDNA clone encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation (P-450C21). Serum from rabbits immunized with purified P-450C21 precipitated a single protein from the products of an in vitro translation reaction using bovine adrenal mRNA. This protein migrated with P-450C21 on NaDodSO4/polyacrylamide gel electrophoresis. After sucrose gradient sedimentation, mRNA encoding P-450C21 was found in the 19S fraction. This fraction was reverse transcribed into double-stranded cDNA and inserted into the Pst I site of pBR322 by the dC X dG tailing procedure. Escherichia coli cells transformed with recombinant plasmids were screened with an in situ immunoassay using anti-P-450C21 serum and 125I-labeled staphylococcal protein A. Two colonies consistently bound anti-P-450C21 serum. They were identified as carrying the same plasmid by restriction mapping. This plasmid, pC21a, contains an insert of 520 base pairs. It hybridizes with mRNA encoding P-450C21. The peptide encoded by the insert in pC21a is highly homologous to two peptides isolated from porcine P-450C21 and shows limited homology to the P-450 induced by phenobarbital in rat liver. This clone may be useful in studying the molecular genetics of human congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Images PMID:6609358

  19. Isolation and analysis of a cDNA clone encoding an S. guttatum alternataive oxidase protein

    SciTech Connect

    Rhoads, D.M.; McIntosh, L. Michigan State Univ., East Lansing )

    1990-05-01

    Antibodies that recognize the 35, 36, and 37 kilodalton (kDa) alternative oxidase proteins were used to isolate a cDNA proteins were used to isolate a cDNA clone of a nuclearly encoded protein of Sauromatum guttatum. The amino acid sequence deduced from clone pAOSG81 revealed a protein with a predicted molecular mass of 44 kDa, while a 42 kDa protein is immunoprecipitated from in vitro translation products made using S. guttatum poly A+ RNA. The protein contains a 60-65 amino acid transit peptide which is predicted to form amphiphilic helices. We have also identified regions of the mature 42 kDa protein which are likely to be membrane associated. Clone pAOSG81 is being used to screen a genomic library. The genomic clone encoding the 42 kDa protein will be used to investigate the salicylic-acid-controlled transcriptional regulation of the S. guttatum alternative oxidase proteins.

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

    PubMed

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

    2004-06-01

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

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

    SciTech Connect

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

    1987-01-01

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

  2. Cloning of the cDNA for the human. beta. /sub 1/-adrenergic receptor

    SciTech Connect

    Frielle, T.; Collins, S.; Daniel, K.W.; Caron, M.G.; Lefkowitz, R.J.; Kobilka, B.K.

    1987-11-01

    Screening of a human placenta lambdagt11 library has led to the isolation of the cDNA for the human ..beta../sub 1/-adrenergic receptor (..beta../sub 1/AR). Used as the probe was the human genomic clone termed G-21. This clone, which contains an intronless gene for a putative receptor, was previously isolated by virtue of its cross hybridization with the human ..beta../sub 2/-adrenergic receptor (..beta../sub 2/AR). The 2.4-kilobase cDNA for the human ..beta../sub 1/AR encodes a protein of 477 amino acid residues that is 69% homologous with the avian ..beta..AR but only 54% homologous with the human ..beta../sub 2/AR. This suggests that the avian gene encoding ..beta..AR and the human gene encoding ..beta../sub 1/AR evolved from a common ancestral gene. RNA blot analysis indicates a message of 2.5 kilobases in rat tissues, with a pattern of tissue distribution consistent with ..beta../sub 1/AR binding. This pattern is quite distinct from the pattern obtained when the ..beta../sub 2/AR cDNA is used as a probe. Expression of receptor protein in Xenopus laevis oocytes conveys adenylate cyclase responsiveness to catecholamines with a typical ..beta../sub 1/AR specificity. This contrasts with the typical ..beta../sub 2/ subtype specificity observed when the human ..beta../sub 2/AR cDNA is expressed in this system. Mammalian ..beta../sub 1/AR and ..beta../sub 2/AR are thus products of distinct genes, both of which are apparently related to the putative G-21 receptor.

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

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

  4. Cloning and molecular characterization of cDNA encoding a mouse male-enhanced antigen-2 (Mea-2): a putative family of the Golgi autoantigen.

    PubMed

    Kondo, M; Sutou, S

    1997-01-01

    The male-enhanced antigen-2 (Mea-2) gene was originally identified with a monoclonal histocompatibility Y (H-Y) antibody (mAb4VII). There is no report of the full length cDNA encode for Mea-2 product until this report. In this study, we isolated the full length mouse Mea-2 cDNA by screening a testis cDNA library with a PCR-amplified Mea-2 product, and direct PCR amplification of its upstream sequences from the cDNA library. The primary structure of the Mea-2 peptide, deduced from this nucleotide sequence, shows that it encode a 150 kDa protein, of 1325 amino acid residues, which contained five putative N-glycosylation sites and four leucine zipper motifs. A data bank search indicated that it has high homology with a human Golgi autoantigen (golgin-160) both in its nucleotides (78%) and amino acids sequence (83%). This suggests that Mea-2 gene product may encode a golgi structural protein. In situ hybridization analysis suggested that the Mea-2 gene is expressed in spermatids during spermatogenesis as already shown by Mea-1, suggesting that Mea-2 gene product as well as Mea-1 have also some role for spermatogenesis.

  5. cDNA sequence and chromosomal localization of human enterokinase, the proteolytic activator of trypsinogen.

    PubMed

    Kitamoto, Y; Veile, R A; Donis-Keller, H; Sadler, J E

    1995-04-11

    Enterokinase is a serine protease of the duodenal brush border membrane that cleaves trypsinogen and produces active trypsin, thereby leading to the activation of many pancreatic digestive enzymes. Overlapping cDNA clones that encode the complete human enterokinase amino acid sequence were isolated from a human intestine cDNA library. Starting from the first ATG codon, the composite 3696 nt cDNA sequence contains an open reading frame of 3057 nt that encodes a 784 amino acid heavy chain followed by a 235 amino acid light chain; the two chains are linked by at least one disulfide bond. The heavy chain contains a potential N-terminal myristoylation site, a potential signal anchor sequence near the amino terminus, and six structural motifs that are found in otherwise unrelated proteins. These domains resemble motifs of the LDL receptor (two copies), complement component Clr (two copies), the metalloprotease meprin (one copy), and the macrophage scavenger receptor (one copy). The enterokinase light chain is homologous to the trypsin-like serine proteinases. These structural features are conserved among human, bovine, and porcine enterokinase. By Northern blotting, a 4.4 kb enterokinase mRNA was detected only in small intestine. The enterokinase gene was localized to human chromosome 21q21 by fluorescence in situ hybridization.

  6. Identification of a human cDNA with high homology to yeast omnipotent suppressor 45.

    PubMed

    Grenett, H E; Bounelis, P; Fuller, G M

    1992-01-15

    Omnipotent suppression is a well-established phenomenon in yeast and bacteria in which nonsense mutations are misread. Wild-type (wt) suppressors are presumed to be involved in ensuring the fidelity of translation. We report a human homolog to wt yeast omnipotent suppressor 45 which shares 63% identity at the nucleotide level in the area of open reading frame (ORF) and 73% similarity at the amino acid (aa) level. The aa sequence of the human protein was deduced from a 2.3-kb cDNA (TB3-1) isolated from an adenocarcinoma T84 cell line cDNA library. The cDNA contains an ORF of 1284 bp which encodes a 47.8-kDa protein. Two transcripts for the clone were identified (2.6 and 4.0 kb) in a variety of human cell types. The strong structural similarity to yeast omnipotent suppressor 45, and its widespread expression suggest that this cDNA may play a role in the accurate recognition of nonsense codons in mammalian cells.

  7. Molecular cloning and characterization of a cDNA encoding the cerebrovascular and the neuritic plaque amyloid peptides

    SciTech Connect

    Robakis, N.K.; Ramakrishna, N.; Wolfe, G.; Wisniewski, H.M.

    1987-06-01

    Deposits of amyloid fibers are found in large numbers in the walls of blood vessels and in neuritic plaques in the brains of patients with Alzheimer disease and adults with Down syndrome. The authors used the amino acid sequence of the amyloid peptide to synthesize oligonucleotide probes specific for the gene encoding this peptide. When a human brain cDNA library was screened with this probe, a clone was found with a 1.7-kilobase insert that contains a long open reading frame coding for 412 amino acid residues including the 28 amino acids of the amyloid peptide. RNA gel blots revealed that a 3.3-kilobase mRNA species was present in the brains of individuals with Alzheimer disease, with Down syndrome, or with not apparent neurological disorders. Southern blots showed that homologous genes are present in the genomic DNA of humans, rabbits, sheep, hamsters, and mice, suggesting that this gene has been conserved through mammalian evolution. Localization of the corresponding genomic sequences on human chromosome 21 suggest a genetic relationship between Alzheimer disease and Down syndrome, and it may explain the early appearance of large numbers of neuritic plaques in adult Down syndrome patients.

  8. Molecular cloning and expression of a cDNA encoding an olfactory-specific mouse phenol sulphotransferase.

    PubMed Central

    Tamura, H O; Harada, Y; Miyawaki, A; Mikoshiba, K; Matsui, M

    1998-01-01

    Previously we demonstrated the presence of phenol sulphotransferase (P-ST) in mouse nasal cytosols and identified its zonal location in mouse nasal cavity by staining with an antiserum raised against a rat liver P-ST isoenzyme, PSTg. In the present study a cDNA was isolated from a mouse olfactory cDNA library by immunological screening with the antiserum. The isolated cDNA consisted of 1347 bp with a 912 bp open reading frame encoding a 304-residue polypeptide. Both the nucleotide and deduced amino acid sequences of the cDNA were 94% identical with those of a rat liver P-ST isoenzyme, ST1C1. The expressed enzyme in Escherichia coli displayed high P-ST activity towards phenolic odorants such as eugenol and guaiacol, and it showed a high N-hydroxy-2-acetylaminofluorene sulphation activity in comparison with the rat ST1C1 enzyme. These results indicate that the olfactory P-ST encoded by the cDNA is a mouse orthologue of rat ST1C1; however, expression of the olfactory P-ST mRNA is specific for nasal tissues as revealed by reverse transcriptase-mediated PCR (RT-PCR). PMID:9560327

  9. Cloning and characterization of cDNA encoding an elicitor of Phytophthora colocasiae.

    PubMed

    Mishra, Ajay Kumar; Sharma, Kamal; Misra, Raj Shekhar

    2010-02-28

    The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. A cDNA encoding elicitor, the major secreted extracellular glycoprotein of Phytophthora colocasiae, a pathogen of taro (Colocasia esculenta) plants, was isolated, sequenced and characterized. The expression of the corresponding elicitor gene during the disease cycle of P. colocasiae was analyzed. Elicitor was shown to be expressed in mycelium grown in culture media, whereas it was not expressed in sporangiospores and zoospores. In planta, during infection of taro, particularly during the biotrophic stage, expression of elicitor was down-regulated compared to in vitro. The highest levels of expression of elicitor were observed in in vitro grown mycelium and in late stages of infection when profuse sporulation and leaf necrosis occur. The elicitation of the suspension-cultured taro cells was effective in the induction of the enzyme activity of l-phenylalanine-ammonia lyase, peroxidase and lipoxygenase as well as the expression of defense-related endochitinase gene. All these biological activities were exerted within a low concentration range. The glycoprotein represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance. It may also be useful to engineer broad disease protection in taro plant against Phytophthora leaf blight.

  10. Toward a cDNA map of the human genome

    SciTech Connect

    Korenberg, J.R.; Chen, X.N.; Adams, M.D.; Venter, J.C.

    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.

  11. Cloning and characterization of the cDNA encoding guinea-pig properdin: a comparison of properdin from three species.

    PubMed Central

    Maves, K K; Guenthner, S T; Densen, P; Moser, D R; Weiler, J M

    1995-01-01

    The cDNA sequence encoding properdin was generated from guinea-pig spleen RNA by the reverse transcription-polymerase chain reaction. This sequence was approximately 75% homologous with human and 71% homologous with murine properdin at the nucleic acid level. Guinea-pig properdin had six thrombospondin repeat sequences consisting of about 60 amino acids, each with six cysteine and three tryptophan residues. Additionally, the Valine-Threonine-Cysteine-Glycine sequence, reported to have important cell adhesive properties in malarial circumsporozoite proteins and thrombospondin, was conserved in the properdin sequence of guinea-pigs. Finally, mouse spleen was also examined to complete the sequence determination of the leader peptide and the initial four residues of murine properdin. This allowed a thorough comparison of the primary structure of properdin from all three species. Like human and murine properdin cDNAs, the guinea pig sequence contained a region of unique, non-homologous sequence (18 base pairs in length) within the fifth thrombospondin repeat, the significance of which remains unclear. PMID:8550088

  12. Molecular cloning and characterization of cDNA encoding a putative stress-induced heat-shock protein from Camelus dromedarius.

    PubMed

    Elrobh, Mohamed S; Alanazi, Mohammad S; Khan, Wajahatullah; Abduljaleel, Zainularifeen; Al-Amri, Abdullah; Bazzi, Mohammad D

    2011-01-01

    Heat shock proteins are ubiquitous, induced under a number of environmental and metabolic stresses, with highly conserved DNA sequences among mammalian species. Camelus dromedaries (the Arabian camel) domesticated under semi-desert environments, is well adapted to tolerate and survive against severe drought and high temperatures for extended periods. This is the first report of molecular cloning and characterization of full length cDNA of encoding a putative stress-induced heat shock HSPA6 protein (also called HSP70B') from Arabian camel. A full-length cDNA (2417 bp) was obtained by rapid amplification of cDNA ends (RACE) and cloned in pET-b expression vector. The sequence analysis of HSPA6 gene showed 1932 bp-long open reading frame encoding 643 amino acids. The complete cDNA sequence of the Arabian camel HSPA6 gene was submitted to NCBI GeneBank (accession number HQ214118.1). The BLAST analysis indicated that C. dromedaries HSPA6 gene nucleotides shared high similarity (77-91%) with heat shock gene nucleotide of other mammals. The deduced 643 amino acid sequences (accession number ADO12067.1) showed that the predicted protein has an estimated molecular weight of 70.5 kDa with a predicted isoelectric point (pI) of 6.0. The comparative analyses of camel HSPA6 protein sequences with other mammalian heat shock proteins (HSPs) showed high identity (80-94%). Predicted camel HSPA6 protein structure using Protein 3D structural analysis high similarities with human and mouse HSPs. Taken together, this study indicates that the cDNA sequences of HSPA6 gene and its amino acid and protein structure from the Arabian camel are highly conserved and have similarities with other mammalian species.

  13. Cloning and sequence analysis of an Ophiophagus hannah cDNA encoding a precursor of two natriuretic peptide domains.

    PubMed

    Lei, Weiwei; Zhang, Yong; Yu, Guoyu; Jiang, Ping; He, Yingying; Lee, Wenhui; Zhang, Yun

    2011-04-01

    The king cobra (Ophiophagus hannah) is the largest venomous snake. Despite the components are mainly neurotoxins, the venom contains several proteins affecting blood system. Natriuretic peptide (NP), one of the important components of snake venoms, could cause local vasodilatation and a promoted capillary permeability facilitating a rapid diffusion of other toxins into the prey tissues. Due to the low abundance, it is hard to purify the snake venom NPs. The cDNA cloning of the NPs become a useful approach. In this study, a 957 bp natriuretic peptide-encoding cDNA clone was isolated from an O. hannah venom gland cDNA library. The open-reading frame of the cDNA encodes a 210-amino acid residues precursor protein named Oh-NP. Oh-NP has a typical signal peptide sequence of 26 amino acid residues. Surprisingly, Oh-NP has two typical NP domains which consist of the typical sequence of 17-residue loop of CFGXXDRIGC, so it is an unusual NP precursor. These two NP domains share high amino acid sequence identity. In addition, there are two homologous peptides of unknown function within the Oh-NP precursor. To our knowledge, Oh-NP is the first protein precursor containing two NP domains. It might belong to another subclass of snake venom NPs.

  14. Cloning and sequencing of a cDNA encoding a heat-stable sweet protein, mabinlin II.

    PubMed

    Nirasawa, S; Masuda, Y; Nakaya, K; Kurihara, Y

    1996-11-28

    A cDNA clone encoding a heat-stable sweet protein, mabinlin II (MAB), was isolated and sequenced. The encoded precursor to MAB was composed of 155 amino acid (aa) residues, including a signal sequence of 20 aa, an N-terminal extension peptide of 15 aa, a linker peptide of 14 aa and one residue of C-terminal extension. Comparison of the proteolytic cleavage sites during post-translational processing of MAB precursor with those of like 2S seed-storage proteins of Arabidopsis thaliana, Brassica napus and Bertholletia excelsa shows that the three individual cleavage sites between respective species are conserved.

  15. Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae.

    PubMed

    Leustek, T; Murillo, M; Cervantes, M

    1994-07-01

    ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana. APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast). Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein. The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S. cerevisiae, with which it is 25% identical. Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase. Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts. Several genomic DNA fragments that hybridize with the APS1 probe were identified. The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb).

  16. Complete cDNA and derived amino acid sequence of human factor V

    SciTech Connect

    Jenny, R.J.; Pittman, D.D.; Toole, J.J.; Kriz, R.W.; Aldape, R.A.; Hewick, R.M.; Kaufman, R.J.; Mann, K.G.

    1987-07-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A)tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approx. 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approx. 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approx. 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

  17. Molecular cloning and characterization of a novel human protein phosphatase 2C cDNA (PP2C epsilon*).

    PubMed

    Jin, Feng; Ji, Chaoneng; Liu, Lingfeng; Dai, Jianfeng; Gu, Shaohua; Sun, Xianfei; Xie, Yi; Mao, Yumin

    2004-09-01

    We have isolated a novel cDNA from the human fetal brain cDNA library with homology to the Mg2+ -dependent serine/threonine protein phosphatase 2C (PP2C) family. The cDNA is 3055 bp in length, and the predicted coding region encodes a 360-amino-acid protein, which shows 99% identity to the PP2C epsilon from rat and mouse. Then we term it human PP2C epsilon gene. The gene is mapped to chromosome 3q26.1 and contains 4 exons. RT-PCR analysis shows that the PP2C epsilon is widely expressed in human tissues and the expression levels in heart, placenta, lung, liver, kidney, and pancreas are relatively high.

  18. Chromosomal localization of a human cDNA containing a DIDS binding domain and demonstrating high homology to yeast omnipotent suppressor 45.

    PubMed

    Grenett, H E; Eipers, P G; Kidd, V J; Bounelis, P; Fuller, G M

    1992-01-01

    We recently have identified a full-length cDNA (TB3-1) from a human adenocarcinoma cell line T84 cDNA library that encodes a 47.8-kDa protein. TB3-1 shares identity with the putative yeast translation termination factor omnipotent suppressor 45. Using human-mouse somatic cell panel analysis, a family of sequences with high homology to the TB3-1 cDNA clone were localized to human chromosomes 5, 6, 7, and X. Southern analysis of a panel of mammalian and chicken genomic DNA demonstrates that TB3-1 is well conserved in higher vertebrates.

  19. Sequence of a cDNA encoding nitrite reductase from the tree Betula pendula and identification of conserved protein regions.

    PubMed

    Friemann, A; Brinkmann, K; Hachtel, W

    1992-02-01

    The sequence of an mRNA encoding nitrite reductase (NiR, EC 1.7.7.1.) from the tree Betula pendula was determined. A cDNA library constructed from leaf poly(A)+ mRNA was screened with an oligonucleotide probe deduced from NiR sequences from spinach and maize. A 2.5 kb cDNA was isolated that hybridized to an mRNA, the steady-state level of which increased markedly upon induction with nitrate. The nucleotide sequence of the cDNA contains a reading frame encoding a protein of 583 amino acids that reveals 79% identity with NiR from spinach. The transit peptide of the NiR precursor from birch was determined to be 22 amino acids in size by sequence comparison with NiR from spinach and maize and is the shortest transit peptide reported so far. A graphical evaluation of identities found in the NiR sequence alignment revealed nine well conserved sections each exceeding ten amino acids in size. Sequence comparisons with related redox proteins identified essential residues involved in cofactor binding. A putative binding site for ferredoxin was found in the N-terminal half of the protein.

  20. Human TOP3: a single-copy gene encoding DNA topoisomerase III.

    PubMed Central

    Hanai, R; Caron, P R; Wang, J C

    1996-01-01

    A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12. Images Fig. 2 PMID:8622991

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Complete sequence analysis of cDNA clones encoding rat whey phosphoprotein: homology to a protease inhibitor.

    PubMed

    Dandekar, A M; Robinson, E A; Appella, E; Qasba, P K

    1982-07-01

    Lactoprotein clones have been isolated from a rat mammary gland recombinant library of cDNA plasmids. Clones p-Wp 52 and p-Wp 47 were shown by hybrid selection, in vitro translation, and immunoprecipitation to represent a cloned DNA sequence encoding rat whey phosphoprotein. We report here the nucleotide sequence of the cDNA insert of p-Wp 52 and shows that it encodes the complete whey phosphoprotein sequence. The encoded sequence shows a high content of half-cystine, glutamic acid, aspartic acid, and serine but an absence of tyrosine. The half-cystines appear in unique arrangements and are repeated in two domains of the protein. The second domain has striking similarities with the second domain of the red sea turtle protease inhibitor. Clone p-Wp 52 has allowed the study of expression of whey phosphoprotein mRNA during functional differentiation of rat mammary gland and in mammary tumors. The whey phosphoprotein mRNA is detected during midpregnancy and lactation in the rat mammary gland but is barely detected in mammary tumors in which other milk protein mRNAs are expressed. The whey phosphoprotein gene in these tumors is hypermethylated, correlating with the reduced expression of this gene.

  3. Cloning and sequencing of cDNA and genomic DNA encoding PDM phosphatase of Fusarium moniliforme.

    PubMed

    Yoshida, Hiroshi; Iizuka, Mari; Narita, Takao; Norioka, Naoko; Norioka, Shigemi

    2006-12-01

    PDM phosphatase was purified approximately 500-fold through six steps from the extract of dried powder of the culture filtrate of Fusarium moniliforme. The purified preparation appeared homogeneous on SDS-PAGE although the protein band was broad. Amino acid sequence information was collected on tryptic peptides from this preparation. cDNA cloning was carried out based on the information. A full-length cDNA was obtained and sequenced. The sequence had an open reading frame of 651 amino acid residues with a molecular mass of 69,988 Da. Cloning and sequencing of the genomic DNA corresponding to the cDNA was also conducted. The deduced amino acid sequence could account for many but not all of the tryptic peptides, suggesting presence of contaminant protein(s). SDS-PAGE analysis after chemical deglycosylation showed two proteins with molecular masses of 58 and 68 kDa. This implied that the 58 kDa protein had been copurified with PDM phosphatase. Homology search showed that PDM phosphatase belongs to the purple acid phosphatase family, which is widely distributed in the biosphere. Sequence data of fungal purple acid phosphatases were collected from the database. Processing of the data revealed presence of two types, whose evolutionary relationships were discussed.

  4. Cloning and mapping of a novel human cDNA homologous to DROER, the enhancer of the Drosophila melanogaster rudimentary gene

    SciTech Connect

    Isomura, Minoru; Okui, Keiko; Nakamura, Yusuke

    1996-02-15

    This article reports on the isolation and localization to human chromosome 7q34 of a human cDNA clone that encodes a protein which is homologous to DROER, the enhancer of the Drosophila melanogaster rudimentary gene. The structure and expression of this gene is also discussed. 12 refs., 3 figs.

  5. Cloning and characterization of a cDNA encoding type 1 diacylglycerol acyltransferase from sunflower (Helianthus annuus L.).

    PubMed

    Sun, Li; Ouyang, Chao; Kou, Shanglong; Wang, Shenghua; Yao, Yunyi; Peng, Tong; Xu, Ying; Tang, Lin; Chen, Fang

    2011-01-01

    A full-length cDNA encoding a putative diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) was obtained from sunflower (Helianthus annuus L.) seeds. The 1524-bp open reading frame of this cDNA, designated as HaDGAT1, encodes a protein of 507 amino acids with a molecular mass of 58.5 kDa showing high homology to DGAT1 enzymes of other plants. The protein characters, such as a predicted structure with a long N-terminal hydrophilic domain followed by 9 transmembrane domains, acyl-CoA-binding signature, diacylglycerol (DAG)-binding and putative endoplasmic reticulum retrieval motifs (ER-DIR), also indicated that HaDGAT belongs to the DGAT1 family. HaDGAT1 is expressed in all plant tissues especially in developing seeds. Expression of recombinant HaDGAT1 in yeast showed an 1.76-fold increase of total fatty acids, especially unsaturated fatty acids such as palmitoleic acid (enhanced by 86.6%) and oleic acid (enhanced by 81.6%).

  6. Characterization and expression of a cDNA encoding a tubuliform silk protein of the golden web spider Nephila antipodiana.

    PubMed

    Huang, W; Lin, Z; Sin, Y M; Li, D; Gong, Z; Yang, D

    2006-07-01

    Spider silks are renowned for their excellent mechanical properties. Although several spider fibroin genes, mainly from dragline and capture silks, have been identified, there are still many members in the spider fibroin gene family remain uncharacterized. In this study, a novel silk cDNA clone from the golden web spider Nephila antipodiana was isolated. It is serine rich and contains two almost identical fragments with one varied gap region and one conserved spider fibroin-like C-terminal domain. Both in situ hybridization and immunoblot analyses have shown that it is specifically expressed in the tubuliform gland. Thus, it likely encodes the silk fibroin from the tubuliform gland, which supplies the main component of the inner egg case. Unlike other silk proteins, the protein encoded by the novel cDNA in water solution exhibits the characteristic of an alpha-helical protein, which implies the distinct property of the egg case silk, though the fiber of tubuliform silk is mainly composed of beta-sheet structure. Its sequence information facilitates elucidation of the evolutionary history of the araneoid fibroin genes.

  7. Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger.

    PubMed Central

    Tse, C M; Ma, A I; Yang, V W; Watson, A J; Levine, S; Montrose, M H; Potter, J; Sardet, C; Pouyssegur, J; Donowitz, M

    1991-01-01

    A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5'-untranslated region, 2451 bp of open reading frame and 1163 bp of 3'-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI-AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger. Images PMID:1712287

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

    PubMed

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

    1991-05-31

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

  9. Glia of the cholinergic electromotor nucleus of Torpedo are the source of the cDNA encoding a GAT-1-like GABA transporter.

    PubMed

    Swanson, G T; Umbach, J A; Gundersen, C B

    1994-07-01

    A PCR-based strategy was used to clone DNAs encoding Na(+)- and Cl(-)-dependent cotransport proteins using DNA from the cholinergic electromotor nucleus of Torpedo californica. This cloning strategy resulted in the isolation of a cDNA clone that shows strong nucleotide sequence homology to the GABA transporter-1 (GAT-1) types of rat and human brain. When expressed in frog oocytes, this transporter mediates the uptake of GABA. Moreover, physiologically and pharmacologically, the Torpedo protein behaves very similarly to the rat and human GAT-1 proteins. However, in contrast to the predominantly neuronal localization of the mammalian GAT-1 proteins, the mRNA for the fish protein is found almost exclusively in glial elements of the electromotor nucleus. This unexpected discovery of a GABA transporter cDNA in a nucleus that has no previously characterized GABAergic innervation raises questions about the role of GABA and this transporter in the electromotor system. Several speculative models for GABA function are proposed.

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

    PubMed

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

    1992-09-01

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

  11. Characterization of a cDNA encoding a 34-kDa Purkinje neuron protein recognized by sera from patients with paraneoplastic cerebellar degeneration

    SciTech Connect

    Furneaux, H.M.; Dropcho, E.J.; Barbut, D.; Chen, Yaotseng; Rosenblum, M.K.; Old, L.J.; Posner, J.B. )

    1989-04-01

    Paraneoplastic cerebellar degeneration is a neurological disorder of unknown cause occurring in patients with an identified or occult cancer. An autoimmune etiology is likely since autoantibodies directed against the Purkinje cells of the cerebellum have been found in the serum and cerebrospinal fluid of some patients. Two Purkinje cell-specific antigens are recognized by these autoantibodies, a major antigen of 62 kDa (CDR 62, cerebellar degeneration-related 62-kDa protein) and a minor antigen of 34 kDa (CDR 34). Previous studies have described the isolation and characterization of a human cerebellar cDNA that encodes an epitope recognized by sera from patients with paraneoplastic cerebellar degeneration. The authors have now established by two independent methods that this gene is uniquely expressed in Purkinje cells of the cerebellum and corresponds to the minor antigen CDR 34. This antigen is also expressed in tumor tissue from a patient with paraneoplastic cerebellar degeneration.

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

    PubMed Central

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

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene. Images PMID:3039499

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

    PubMed

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

    1987-08-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene.

  14. Isolation and characterization of another cDNA encoding a chorismate mutase from the phytoparasitic nematode Meloidogyne arenaria.

    PubMed

    Long, Hai; Wang, Xuan; Xu, Jian Hua; Hu, Yong Jian

    2006-06-01

    A new cDNA, named Ma-cm-2, encoding a chorismate mutase (CM), has been isolated from Meloidogyne arenaria. The full-length cDNA, carrying the trans-spliced SL1 leader sequence, was 753-bp long with an open reading frame of 576 bp. The deduced protein MA-CM-2 including amino-terminal signal peptide shows significant similarity to CMs of Meloidogyne incognita, Meloidogyne javanica, and also bacteria. Secondary structure prediction of MA-CM-2 indicates the presence of the three conserved alpha-helix domains present in the Escherichia coli CMs. Reverse transcription and polymerase chain reaction analysis showed that its transcript abundance is high in the early developmental stages and low in later ones. In situ mRNA hybridization revealed that the transcripts of Ma-cm-2 accumulated specifically in the two subventral oesophageal gland cells of M. arenaria. The widespread existence of CMs in the sedentary endoparasitic nematodes implicates that this enzyme plays an important role in the host-parasite interaction.

  15. Isolation and characterization of a cDNA encoding a mammalian cathepsin L-like cysteine proteinase from Acanthamoeba healyi

    PubMed Central

    Hong, Yeon-Chul; Hwang, Mi-Yul; Yun, Ho-Cheol; Yu, Hak-Sun; Kong, Hyun-Hee; Yong, Tai-Soon

    2002-01-01

    We have cloned a cDNA encoding a cysteine proteinase of the Acanthamoeba healyi OC-3A strain isolated from the brain of a granulomatous amoebic encephalitis patient. A DNA probe for an A. healyi cDNA library screening was amplified by PCR using degenerate oligonucleotide primers designed on the basis of conserved amino acids franking the active sites of cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteinases. Cysteine proteinase gene of A. healyi (AhCP1) was composed of 330 amino acids with signal sequence, a proposed pro-domain and a predicted active site made up of the catalytic residues, Cys25, His159, and Asn175. Deduced amino acid sequence analysis indicates that AhCP1 belong to ERFNIN subfamily of C1 peptidases. By Northern blot analysis, no direct correlation was observed between AhCP1 mRNA expression and virulence of Acanthamoeba, but the gene was expressed at higher level in amoebae isolated from soil than amoeba from clinical samples. These findings raise the possibility that Ahcp1 protein may play a role in protein metabolism and digestion of phagocytosed bacteria or host tissue debris rather than in invasion of amoebae into host tissue. PMID:11949209

  16. Cloning of human PEX cDNA. Expression, subcellular localization, and endopeptidase activity.

    PubMed

    Lipman, M L; Panda, D; Bennett, H P; Henderson, J E; Shane, E; Shen, Y; Goltzman, D; Karaplis, A C

    1998-05-29

    Mutations in the PEX gene are responsible for X-linked hypophosphatemic rickets. To gain insight into the role of PEX in normal physiology we have cloned the human full-length cDNA and studied its tissue expression, subcellular localization, and peptidase activity. We show that the cDNA encodes a 749-amino acid protein structurally related to a family of neutral endopeptidases that include neprilysin as prototype. By Northern blot analysis, the size of the full-length PEX transcript is 6.5 kilobases. PEX expression, as determined by semi-quantitative polymerase chain reaction, is high in bone and in tumor tissue associated with the paraneoplastic syndrome of renal phosphate wasting. PEX is glycosylated in the presence of canine microsomal membranes and partitions exclusively in the detergent phase from Triton X-114 extractions of transiently transfected COS cells. Immunofluorescence studies in A293 cells expressing PEX tagged with a c-myc epitope show a predominant cell-surface location for the protein with its COOH-terminal domain in the extracellular compartment, substantiating the assumption that PEX, like other members of the neutral endopeptidase family, is a type II integral membrane glycoprotein. Cell membranes from cultured COS cells transiently expressing PEX efficiently degrade exogenously added parathyroid hormone-derived peptides, demonstrating for the first time that recombinant PEX can function as an endopeptidase. PEX peptidase activity may provide a convenient target for pharmacological intervention in states of altered phosphate homeostasis and in metabolic bone diseases.

  17. Differential processing of colony-stimulating factor 1 precursors encoded by two human cDNAs.

    PubMed Central

    Rettenmier, C W; Roussel, M F

    1988-01-01

    The biosynthesis of macrophage colony-stimulating factor 1 (CSF-1) was examined in mouse NIH-3T3 fibroblasts transfected with a retroviral vector expressing the 554-amino-acid product of a human 4-kilobase (kb) CSF-1 cDNA. Similar to results previously obtained with a 1.6-kb human cDNA that codes for a 256-amino-acid CSF-1 precursor, the results of the present study showed that NIH-3T3 cells expressing the product of the 4-kb clone produced biologically active human CSF-1 and were transformed by an autocrine mechanism when cotransfected with a vector containing a human c-fms (CSF-1 receptor) cDNA. The 4-kb CSF-1 cDNA product was synthesized as an integral transmembrane glycoprotein that was assembled into disulfide-linked dimers and rapidly underwent proteolytic cleavage to generate a soluble growth factor. Although the smaller CSF-1 precursor specified by the 1.6-kb human cDNA was stably expressed as a membrane-bound glycoprotein at the cell surface and was slowly cleaved to release the extracellular growth factor, the cell-associated product of the 4-kb clone was efficiently processed to the secreted form and was not detected on the plasma membrane. Digestion with glycosidic enzymes indicated that soluble CSF-1 encoded by the 4-kb cDNA contained both asparagine(N)-linked and O-linked carbohydrate chains, whereas the product of the 1.6-kb clone had only N-linked oligosaccharides. Removal of the carbohydrate indicated that the polypeptide chain of the secreted 4-kb cDNA product was longer than that of the corresponding form encoded by the smaller clone. These differences in posttranslational processing may reflect diverse physiological roles for the products of the two CSF-1 precursors in vivo. Images PMID:3264877

  18. Cloning of a full-length cDNA encoding ent-kaurene synthase from Gibberella fujikuroi: functional analysis of a bifunctional diterpene cyclase.

    PubMed

    Toyomasu, T; Kawaide, H; Ishizaki, A; Shinoda, S; Otsuka, M; Mitsuhashi, W; Sassa, T

    2000-03-01

    We report here the nucleotide sequence of a full-length cDNA encoding ent-kaurene synthase that was isolated by a reverse-transcription polymerase chain reaction from Gibberella fujikuroi (Gcps/ks). This cDNA encodes 952 amino acid residues with a relative molecular mass of 107 kDa. The sequence similarity between Gcps/ks and ent-kaurene synthase of the gibberellin A1-producing fungus, Phaeosphaeria sp. L487, is very high, suggesting that Gcps/ks is also a bifunctional diterpene cyclase. Its recombinant protein expressed in Escherichia coli converted geranylgeranyl diphosphate to copalyl diphosphate and ent-kaurene.

  19. Molecular cloning of a cDNA encoding interleukin 11, a stromal cell-derived lymphopoietic and hematopoietic cytokine.

    PubMed Central

    Paul, S R; Bennett, F; Calvetti, J A; Kelleher, K; Wood, C R; O'Hara, R M; Leary, A C; Sibley, B; Clark, S C; Williams, D A

    1990-01-01

    Hematopoiesis occurs in close association with a complex network of cells loosely termed the hematopoietic microenvironment. Analysis of the mechanisms of microenvironmental regulation of hematopoiesis has been hindered by the complexity of the microenvironment as well as the heterogeneity of hematopoietic stem cells and early progenitor cells. We have established immortalized primate bone marrow-derived stromal cell lines to facilitate analysis of the interactions of hematopoietic cells with the microenvironment in a large animal species. One such line, PU-34, was found to produce a variety of growth factors, including an activity that stimulates the proliferation of an interleukin 6-dependent murine plasmacytoma cell line. A cDNA encoding the plasmacytoma stimulatory activity was isolated through functional expression cloning in mammalian cells. The nucleotide sequence contained a single long reading frame of 597 nucleotides encoding a predicted 199-amino acid polypeptide. The amino acid sequence of this cytokine, designated interleukin 11 (IL-11), did not display significant similarity with any other sequence in the GenBank data base. Preliminary biological characterization indicates that in addition to stimulating plasmacytoma proliferation, IL-11 stimulates the T-cell-dependent development of immunoglobulin-producing B cells and synergizes with IL-3 in supporting murine megakaryocyte colony formation. These properties implicate IL-11 as an additional multifunctional regulator in the hematopoietic microenvironment. Images PMID:2145578

  20. Cloning and over-expression of a cDNA encoding a polyketide synthase from Cannabis sativa.

    PubMed

    Raharjo, Tri J; Chang, Wen-Te; Verberne, Marianne C; Peltenburg-Looman, Anja M G; Linthorst, Huub J M; Verpoorte, Robert

    2004-04-01

    A polyketide synthase has been suggested to play an important role in cannabinoid biosynthesis in Cannabis sativa L. This enzyme catalyzes the biosynthesis of olivetolic acid, one of the precursors for cannabinoid biosynthesis. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) based on the DNA homology of chalcone synthase (EC 2.3.1.156) and valerophenone synthase (EC 2.3.1.156) of hop (Humulus lupulus), a cDNA encoding a polyketide synthase in C. sativa was identified. The coding region of the gene is 1170 bp long encoding a 389 amino acid protein of a predicted 42.7 kDa molecular mass and with a pI of 6.04. The gene shares a high homology with a chalcone synthase gene of H. lupulus, 85% and 94% homology on the level of DNA and protein, respectively. Over-expression of the construct in Escherichia coli M15 resulted in a 45 kDa protein. The protein has chalcone synthase activity as well as valerophenone synthase activity, a chalcone synthase-like activity. Using n-hexanoyl-CoA and malonyl-CoA as substrates did not give olivetol or olivetolic acid as a product.

  1. Molecular cloning and characterization of a new cDNA sequence encoding a venom peptide from the centipede Scolopendra subspinipes mutilans.

    PubMed

    Liu, Wanhong; Luo, Feng; He, Jing; Cao, Zhijian; Miao, Lixia

    2012-01-01

    Many studies have been performed on venomous peptides derived from animals. However, little of this research has focused on peptides from centipede venoms. Here, a venom gland cDNA library was successfully constructed for the centipede Scolopendra subspinipes mutilans. A new cDNA encoding the precursor of a venom peptide, named SsmTx, was cloned from the venomous gland cDNA library of the centipede S. subspinipes mutilans. The full-length SsmTx cDNA sequence is 465 nt, including a 249 nt ORF, a 45 nt 5' UTR and a 171 nt 3' UTR. There is a signal tail AATAAA 31 nt upstream of the poly (A) tail. The precursor nucleotide sequence of SsmTx encodes a signal peptide of 25 residues and a mature peptide of 57 residues, which is bridged by two pairs of disulfide bonds. SsmTx displays a unique cysteine motif that is completely different from that of other venomous animal toxins. This is the first reported cDNA sequence encoding a venom peptide from the centipede S. subspinipes mutilans.

  2. Herbicide safener-binding protein of maize. Purification, cloning, and expression of an encoding cDNA.

    PubMed

    Scott-Craig, J S; Casida, J E; Poduje, L; Walton, J D

    1998-03-01

    Dichloroacetamide safeners protect maize (Zea mays L.) against injury from chloroacetanilide and thiocarbamate herbicides. Etiolated maize seedlings have a high-affinity cytosolic-binding site for the safener [3H](R,S)-3-dichloroacetyl-2,2,5-trimethyl-1, 3-oxazol-idine ([3H]Saf), and this safener-binding activity (SafBA) is competitively inhibited by the herbicides. The safener-binding protein (SafBP), purified to homogeneity, has a relative molecular weight of 39,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 5.5. Antiserum raised against purified SafBP specifically recognizes a 39-kD protein in etiolated maize and sorghum (Sorghum bicolor L.), which have SafBA, but not in etiolated wheat (Triticum aestivum L.), oat (Avena sativa L.), barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), or Arabidopsis, which lack SafBA. SafBP is most abundant in the coleoptile and scarcest in the leaves, consistent with the distribution of SafBA. SBP1, a cDNA encoding SafBP, was cloned using polymerase chain reaction primers based on purified proteolytic peptides. Extracts of Escherichia coli cells expressing SBP1 have strong [3H]Saf binding, which, like binding to the native maize protein, is competitively inhibited by the safener dichlormid and the herbicides S-ethyl dipropylthiocarbamate, alachlor, and metolachlor. SBP1 is predicted to encode a phenolic O-methyltransferase, but SafBP does not O-methylate catechol or caffeic acid. The acquisition of its encoding gene opens experimental approaches for the evaluation of the role of SafBP in response to the relevant safeners and herbicides.

  3. Human liver apolipoprotein B-100 cDNA: complete nucleic acid and derived amino acid sequence.

    PubMed Central

    Law, S W; Grant, S M; Higuchi, K; Hospattankar, A; Lackner, K; Lee, N; Brewer, H B

    1986-01-01

    Human apolipoprotein B-100 (apoB-100), the ligand on low density lipoproteins that interacts with the low density lipoprotein receptor and initiates receptor-mediated endocytosis and low density lipoprotein catabolism, has been cloned, and the complete nucleic acid and derived amino acid sequences have been determined. ApoB-100 cDNAs were isolated from normal human liver cDNA libraries utilizing immunoscreening as well as filter hybridization with radiolabeled apoB-100 oligodeoxynucleotides. The apoB-100 mRNA is 14.1 kilobases long encoding a mature apoB-100 protein of 4536 amino acids with a calculated amino acid molecular weight of 512,723. ApoB-100 contains 20 potential glycosylation sites, and 12 of a total of 25 cysteine residues are located in the amino-terminal region of the apolipoprotein providing a potential globular structure of the amino terminus of the protein. ApoB-100 contains relatively few regions of amphipathic helices, but compared to other human apolipoproteins it is enriched in beta-structure. The delineation of the entire human apoB-100 sequence will now permit a detailed analysis of the conformation of the protein, the low density lipoprotein receptor binding domain(s), and the structural relationship between apoB-100 and apoB-48 and will provide the basis for the study of genetic defects in apoB-100 in patients with dyslipoproteinemias. PMID:3464946

  4. Isolation and characterization of a cDNA clone encoding an alternative oxidase protein of Sauromatum guttatum (Schott).

    PubMed Central

    Rhoads, D M; McIntosh, L

    1991-01-01

    Polyclonal and monoclonal antibodies that recognize the 35-, 36-, and 37-kDa alternative oxidase proteins of Sauromatum guttatum (Schott) were used to isolate a cDNA clone, pAOSG81, from an S. guttatum cDNA expression library. A fusion protein with an apparent molecular mass of 48 kDa was expressed from a pUC119 derivative of pAOSG81 (pAOSG81-119) in Escherichia coli cells and was recognized by the monoclonal antibodies. When the in vitro translated and immunoprecipitated products made from mRNA hybrid-selected by pAOSG81 were analyzed, a single band corresponding to a protein with an apparent molecular mass of 42 kDa was observed. DNA sequence characterization showed that pAOSG81 contains the entire coding region of a protein with a calculated molecular mass of 38.9 kDa, a putative 63-amino acid transit peptide, and a 9-amino acid match to the authentic N-terminal sequence of the 36-kDa alternative oxidase protein. Analyses of the deduced amino acid sequence indicate: (i) that the transit peptide is predicted to form amphiphilic helices, and (ii) that three regions of the processed protein are likely to form transmembrane alpha-helices. We conclude from these data that pAOSG81 represents a nuclear gene, aox1, encoding a precursor protein of one or more of the alternative oxidase proteins of S. guttatum. Images PMID:1706518

  5. Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds.

    PubMed

    Games, Patrícia D; Dos Santos, Izabela S; Mello, Erica O; Diz, Mariângela S S; Carvalho, André O; de Souza-Filho, Gonçalo A; Da Cunha, Maura; Vasconcelos, Ilka M; Ferreira, Beatriz Dos S; Gomes, Valdirene M

    2008-12-01

    The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301-9], with some modifications. A DEAE-Sepharose, equilibrated with 20mM Tris-HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS-Tricine gel electrophoresis with a molecular mass of approximately 6kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.

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

    SciTech Connect

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

    1988-10-01

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

  7. cDNA cloning and sequence of MAL, a hydrophobic protein associated with human T-cell differentiation.

    PubMed Central

    Alonso, M A; Weissman, S M

    1987-01-01

    We have isolated a human cDNA that is expressed in the intermediate and late stages of T-cell differentiation. The cDNA encodes a highly hydrophobic protein, termed MAL, that lacks a hydrophobic leader peptide sequence and contains four potential transmembrane domains separated by short hydrophilic segments. The predicted configuration of the MAL protein resembles the structure of integral proteins that form pores or channels in the plasma membrane and that are believed to act as transporters of water-soluble molecules and ions across the lipid bilayer. The presence of MAL mRNA in a panel of T-cell lines that express both the T-cell receptor and the T11 antigen suggests that MAL may be involved in membrane signaling in T cells activated via either T11 or T-cell receptor pathways. Images PMID:3494249

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

    SciTech Connect

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. )

    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.

  9. Cloning and characterization of a cDNA encoding a cobalamin-independent methionine synthase from potato (Solanum tuberosum L.).

    PubMed

    Zeh, Michaela; Leggewie, Georg; Hoefgen, Rainer; Hesse, Holger

    2002-02-01

    A potato cDNA clone, StMS1, that encodes a methionine synthase was isolated. This protein was identified on the basis of both structural and functional evidence. The predicted sequence of the protein encoded by StMS1 shows a high degree of similarity to methionine synthases from other organisms and the expression of StMS1 in bacterial mutant strains restored the mutant's ability to synthesize methionine. Genomic organization and expression analyses suggest that StMS1 is a low-copy gene and is differentially expressed in potato organs. StMS1 expression was found in all tissues, but at elevated levels in flowers, basal levels in sink and source leaves, roots and stolons, and low levels in stems and tubers. RNA expression data were confirmed by western blot analysis except that the protein content in leaves was less than expected from the RNA data. Western blot analysis of subcellular fractions revealed that the protein is located in the cytosol. However, the changing pattern of gene expression during the day/night period implied a light-dependent control of MS transcription normally seen for enzymes localized in plastids. The expression of MS was shown to be light-inducible with its highest expression at midday. These RNA data were not confirmed at the protein level since protein content levels remained constant over the whole day. Feeding experiments of detached leaves revealed that sucrose or sucrose-derived products are responsible for StMS1 induction. This induction can be blocked by treatment with DCMU during the light period. Western analysis revealed that the amount of StMS1 is not affected by either treatment. This experiment confirmed the presence of a day/night rhythm. Methionine synthase expression is regulated by photoassimilates but this seems not to detectably alter protein levels.

  10. Cloning and expression of a cDNA encoding a maize glutathione-S-transferase in E. coli.

    PubMed

    Moore, R E; Davies, M S; O'Connell, K M; Harding, E I; Wiegand, R C; Tiemeier, D C

    1986-09-25

    The isolation and characterization of a family of maize glutathione-S-transferases (GST's) has been described previously. These enzymes are designated GSTs I, II and III based on size, substrate specificity and responsiveness to safeners. GST III has been shown to act on the herbicide alachlor as well as the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Clones were isolated from a maize cDNA library in lambda gt10. Three clones contained the entire coding region for GST III. The sequences of these clones were consistent with the known amino terminal GST III protein sequence. Moreover, expression of one of these clones in E. coli resulted in a GST activity as measured with both CDNB and alachlor, proving that at least one of the clones encodes an active GST III species. With the enzyme expressed in E. coli it will become possible to study enzyme structure-function relationships ex planta. While a number of different GST proteins are present in maize tissue the GST III gene is present in single or low copy in the genome.

  11. Cloning and expression of a cDNA encoding a maize glutathione-S-transferase in E. coli.

    PubMed Central

    Moore, R E; Davies, M S; O'Connell, K M; Harding, E I; Wiegand, R C; Tiemeier, D C

    1986-01-01

    The isolation and characterization of a family of maize glutathione-S-transferases (GST's) has been described previously. These enzymes are designated GSTs I, II and III based on size, substrate specificity and responsiveness to safeners. GST III has been shown to act on the herbicide alachlor as well as the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Clones were isolated from a maize cDNA library in lambda gt10. Three clones contained the entire coding region for GST III. The sequences of these clones were consistent with the known amino terminal GST III protein sequence. Moreover, expression of one of these clones in E. coli resulted in a GST activity as measured with both CDNB and alachlor, proving that at least one of the clones encodes an active GST III species. With the enzyme expressed in E. coli it will become possible to study enzyme structure-function relationships ex planta. While a number of different GST proteins are present in maize tissue the GST III gene is present in single or low copy in the genome. Images PMID:3532034

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

    NASA Astrophysics Data System (ADS)

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

    1990-10-01

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

  13. A hot pepper cDNA encoding ascorbate peroxidase is induced during the incompatible interaction with virus and bacteria.

    PubMed

    Yoo, Tae Hyoung; Park, Chang-Jin; Lee, Gil-Je; Shin, Ryoung; Yun, Ji-Hyun; Kim, Ki-Jeong; Rhee, Ki-Hyeong; Paek, Kyung-Hee

    2002-08-31

    Capsicum annuum L. is infected by a number of viruses, including the tobacco mosaic virus (TMV). To study the defense-related genes that are induced by TMV in hot peppers, the pepper plant, which is susceptible to P1.2 but resistant to the P0 pathotype of TMV, was inoculated with TMV-P0. Differential screening isolated the genes that were specifically up- or down-regulated during the hypersensitive response (HR). The CaAPX1 cDNA clone that putatively encodes a polypeptide of cytosolic ascorbate peroxidase was selected as an up-regulated gene. It was isolated for further study. The full-length cDNA for CaAPX1, which is 972 bp long, contained the open-reading frame of 250-amino acid residues. A genomic Southern blot analysis showed that there were only limited copies of the CaAPX1 gene in the hot pepper genome. In hot pepper cv. Bugang, which is resistant to TMV-P0 and susceptible to TMV-P1.2, the CaAPX1 gene transcript was accumulated by TMV-P0, but not by TMV-P1.2 inoculation. CaAPX1 transcripts began to accumulate 24 h post-inoculation of TMV-P0, and increased gradually until 96 h. To investigate whether each transcript is induced by other stimuli, the plants were treated with various chemicals and wounding. A striking induction of the CaAPX1 transcript was observed at 2 h. It subsided 12 h after salicylic acid (SA), ethephon, and methyl jasmonate (MeJA) treatments. The response of the gene upon other pathogen infection was also examined by a bacterial pathogen (Xanthomonas campestris pv. vesicatoria race 3) inoculation. The CaAPX1 gene was induced in a hot pepper (C. annuum cv. ECW 20R) that was resistant to this bacterial pathogen, but not in a susceptible hot pepper (C. annuum cv. ECW). These results suggest the possible role(s) for the CaAPX1 gene in plant defense against viral and bacterial pathogen.

  14. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  15. Nucleotide sequence and the encoded amino acids of human apolipoprotein A-I mRNA.

    PubMed Central

    Law, S W; Brewer, H B

    1984-01-01

    The cDNA clones encoding the precursor form of human liver apolipoprotein A-I (apoA-I), preproapoA-I, have been isolated from a cDNA library. A 17-base synthetic oligonucleotide based on residues 108-113 of apoA-I and a 26-base primer-extended, dideoxynucleotide-terminated cDNA were used as hybridization probes to select for recombinant plasmids bearing the apoA-I sequence. The complete nucleic acid sequence of human liver preproapoA-I has been determined by analysis of the cloned cDNA. The sequence is composed of 801 nucleotides encoding 267 amino acid residues. PreproapoA-I contains an 18-amino-acid prepeptide and a 6-amino-acid propeptide connected to the amino terminus of the 243-amino acid mature apoA-I. Southern blotting analysis of chromosomal DNA obtained from peripheral blood indicated the apoA-I gene is contained in a 2.1-kilobase-pair Pst I fragment and there is no gross difference in structural organization between the normal apoA-I gene and the Tangier disease apoA-I gene. Images PMID:6198645

  16. Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine.

    PubMed

    Altenbach, S B; Pearson, K W; Leung, F W; Sun, S S

    1987-05-01

    The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.

  17. Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

    PubMed Central

    Zhu, Ge-Jian; Yu, Ying-Nian; Li, Xin; Qian, Yu-Li

    2002-01-01

    AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells. METHODS: After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography (HPLC). RESULTS: The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild typeCYP2C9. However, there were two base differences, i.e. 21T > C, 1146C > T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 ± 0.109 μmol•min-1 ·g-1 S9 protein or 8.62 ± 2.02 mol•min-1 ·mol-1 CYP, but was undetectable in parental CHL cell. CONCLUSION: The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL-CYP2C9, efficiently expressing the protein of CYP2C9, was established. PMID:11925616

  18. A cDNA encoding a cold-induced glycine-rich RNA binding protein from Prunus avium expressed in embryonic axes.

    PubMed

    Stephen, John R; Dent, Katherine C; Finch-Savage, William E

    2003-11-27

    A cDNA clone encoding a presumed full-length glycine-rich ribonucleic acid (RNA) binding protein was isolated from a lambda-ZAP Express cDNA library generated from primarily nondormant Prunus avium (wild cherry) embryonic axes. The cDNA, designated Pa-RRM-GRP1 (Prunus avium RNA recognition motif glycine-rich protein 1), contains a single N-terminal RNA recognition motif (RRM) and single C-terminal glycine-rich domain. The glycine-rich domain is unusually long at 91 amino acids, 58 of which are glycines. The 534-base pair (bp) open reading frame (ORF) of this clone encodes a 178-amino-acid polypeptide with a predicted molecular weight of 17.33 kDa and pI of 7.84. Comparative sequence alignment of Pa-RRM-GRP1 reveals extensive homology to known and presumed glycine-rich RNA binding proteins from angiosperms and gymnosperms. Genomic Southern blot analysis suggests that this gene exists as a single copy in P. avium. Expression of this gene in P. avium embryonic axes during low-temperature dormancy-breaking treatments was studied and found to be induced by cold (3 degrees C) using real-time PCR of total cDNA supported by Northern blot analysis of total RNA. Expression dropped during prolonged storage at 3 degrees C and was reduced to control levels by interruption of cold treatment by warming to 20 degrees C.

  19. Molecular cloning of the cDNA for the human U2 snRNA-specific A' protein.

    PubMed Central

    Sillekens, P T; Beijer, R P; Habets, W J; van Verooij, W J

    1989-01-01

    The A' polypeptide is one of the protein constituents of the U2 snRNP particle. A potentially full-length cDNA clone containing the complete coding sequence for this U2 snRNP-specific protein was isolated by screening of a human lambda gt11 expression vector library with an autoimmune anti-(U1,U2)RNP serum. Monospecific antibodies, eluted from the 140-150 kD fusion protein of this cDNA recombinant, specifically recognized the A' protein on immunoblots and immunoprecipitated U2 snRNP particles from nuclear extracts. The identity of the clone was confirmed by in vitro translation of hybrid-selected mRNA or an RNA transcript synthesized from the cDNA insert. RNA blot analysis showed strong hybridization to a single polyadenylated transcript of 1.3 kb in human cells. The nucleotide sequence of the 1054 bp cDNA contains an open reading frame of 756 bp encoding a polypeptide of 255 amino acids with a predicted molecular weight of 28,444 D. The coding sequence is preceded by a 49 bp 5'-untranslated region and followed by a 226 bp 3'-untranslated region containing a single polyadenylation signal. Most striking feature of the deduced primary structure for the A' protein is a leucine-rich region in the amino-terminal half of the polypeptide. In contrast to the other U2 snRNP-specific protein B", the A' protein does not contain segments homologous to the RNP consensus sequences RNP1 and RNP2, common amino acid motifs found in several RNA-binding proteins. In the A' protein, however, the extremely hydrophilic carboxy terminus may constitute an RNA-binding moiety. Images PMID:2928112

  20. [Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper].

    PubMed

    Ramazanova, A S; Fil'kin, S Iu; Starkov, V G; Utkin, Iu N

    2011-01-01

    Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.

  1. Alternative splicing of the mRNA encoding the human cholesteryl ester transfer protein

    SciTech Connect

    Inazu, Akihiro; Quinet, E.M.; Suke Wang; Brown, M.L.; Stevenson, S.; Barr, M.L.; Moulin, P.; Tall, A.R. )

    1992-03-03

    The plasma cholesteryl ester transfer protein (CETP) is known to facilitate the transfer of lipids between plasma lipoproteins. The human CETP gene is a complex locus encompassing 16 exons. The CETP mRNA is found in liver and small intestine as well as in a variety of peripheral tissues. While the CETP cDNA from human adipose tissue was being cloned, a variant CETP cDNA was discovered which excluded the complete sequence encoded by exon 9, but which was otherwise identical to the full-length CETP cDNA, suggesting modification of the CETP gene transcript by an alternative RNA splicing mechanism. RNase protection analysis of tissue RNA confirmed the presence of exon 9 deleted transcripts and showed that they represented a variable proportion of the total CETP mRNA in various human tissues including adipose tissue (25%), liver (33%), and spleen (46%). Transient expression of the exon 9 deleted cDNA in COS cells or stable expression in CHO cells showed that the protein encoded by the alternatively spliced transcript was inactive in neutral lipid transfer, smaller, and poorly secreted compared to the protein derived from the full-length cDNA. Endo H digestion suggested that the inactive, cell-associated protein was present within the endoplasmic reticulum. The experiments show that the expression of the human CETP gene is modified by alternative splicing of the ninth exon, in a tissue-specific fashion. The function of alternative splicing is unknown but could serve to produce a protein with a function other than plasma neutral lipid transfer, or as an on-off switch to regulate the local concentration of biologically active protein.

  2. Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein.

    PubMed Central

    Taylor, M E; Brickell, P M; Craig, R K; Summerfield, J A

    1989-01-01

    The N-terminal sequence of the major human serum mannose-binding protein (MBP1) was shown to be identical at all positions determined with the amino acid sequence predicted from a cDNA clone of a human liver MBP mRNA. An oligonucleotide corresponding to part of the sequence of this cDNA clone was used to isolate a cosmid genomic clone containing a homologous gene. The intron/exon structure of this gene was found to closely resemble that of the gene encoding a rat liver MBP (MBP A). The nucleotide sequence of the exons differed in several places from that of the human cDNA clone published by Ezekowitz, Day & Herman [(1988) J. Exp. Med. 167, 1034-1046]. The MBP molecule comprises a signal peptide, a cysteine-rich domain, a collagen-like domain, a 'neck' region and a carbohydrate-binding domain. Each domain is encoded by a separate exon. This genomic organization lends support to the hypothesis that the gene arose during evolution by a process of exon shuffling. Several consensus sequences that may be involved in controlling the expression of human serum MBP have been identified in the promoter region of the gene. The consensus sequences are consistent with the suggestion that this mammalian serum lectin is regulated as an acute-phase protein synthesized by the liver. PMID:2590164

  3. Human and Tree Shrew Alpha-synuclein: Comparative cDNA Sequence and Protein Structure Analysis.

    PubMed

    Wu, Zheng-Cun; Huang, Zhang-Qiong; Jiang, Qin-Fang; Dai, Jie-Jie; Zhang, Ying; Gao, Jia-Hong; Sun, Xiao-Mei; Chen, Nai-Hong; Yuan, Yu-He; Li, Cong; Han, Yuan-Yuan; Li, Yun; Ma, Kai-Li

    2015-10-01

    The synaptic protein alpha-synuclein (α-syn) is associated with a number of neurodegenerative diseases, and homology analyses among many species have been reported. Nevertheless, little is known about the cDNA sequence and protein structure of α-syn in tree shrews, and this information might contribute to our understanding of its role in both health and disease. We designed primers to the human α-syn cDNA sequence; then, tree shrew α-syn cDNA was obtained by RT-PCR and sequenced. Based on the acquired tree shrew α-syn cDNA sequence, both the amino acid sequence and the spatial structure of α-syn were predicted and analyzed. The homology analysis results showed that the tree shrew cDNA sequence matches the human cDNA sequence exactly except at nucleotide positions 45, 60, 65, 69, 93, 114, 147, 150, 157, 204, 252, 270, 284, 298, 308, and 324. Further protein sequence analysis revealed that the tree shrew α-syn protein sequence is 97.1 % identical to that of human α-syn. The secondary protein structure of tree shrew α-syn based on random coils and α-helices is the same as that of the human structure. The phosphorylation sites are highly conserved, except the site at position 103 of tree shrew α-syn. The predicted spatial structure of tree shrew α-syn is identical to that of human α-syn. Thus, α-syn might have a similar function in tree shrew and in human, and tree shrew might be a potential animal model for studying the pathogenesis of α-synucleinopathies.

  4. A majority of Ig H chain cDNA of normal human adult blood lymphocytes resembles cDNA for fetal Ig and natural autoantibodies

    SciTech Connect

    Huang, C.; Stollar, B.D. )

    1993-11-15

    Certain Ig V[sub H] gene segments, with few or no mutations, recur frequently in natural autoantibodies, fetal antibodies, and products of B cell tumors. The goal of this study was to determine whether similar Ig gene segment usage occurs in normal human adult PBL. Extending previous analyses, 105 randomly picked H chain V region clones of representative cDNA libraries from PBL were sequenced. Clones were from: IgM and IgG libraries from one RNA sample of a normal adult; a second IgM library from the same subject 11 mo later; and one IgM library from a second subject. Although some clones had clear evidence of mutation, 48 of 77 IgM clones (62%) shared 99% or more identity with known germline V[sub H] segments, and most of these had no mutations in the CDR3 portion of the J[sub H] segment. Certain V[sub H] gene segments, expressed in autoantibodies and fetal antibodies, occurred at high frequency in these libraries. Fourteen of the clones with 99% identity to known V[sub H] segments had CDR3 segments identical to portions of known germline D[sub H] gene sequences; two such clones had no N nucleotides at the V[sub H] D[sub H] or D[sub H]J[sub H] junctions. IgG-encoding sequences had more mutations than IgM-encoding sequences. J[sub H] and D[sub H] usage was not random. The circulating B cell population may represent a distinct compartment, with a large proportion of cells similar to those of the fetal and natural autoantibody repertoire. Polyreactive Ig products of these circulating cells may serve a screening function, binding and delivering diverse Ag to secondary lymphoid tissues where more highly selective antibodies are formed to foreign or Self-Ag. 40 refs., 2 figs., 6 tabs.

  5. cDNA sequence of a human skeletal muscle ADP/ATP translocator: lack of a leader peptide, divergence from a fibroblast translocator cDNA, and coevolution with mitochondrial DNA genes

    SciTech Connect

    Neckelmann, N.; Li, K.; Wade, R.P.; Shuster, R.; Wallace, D.C.

    1987-11-01

    The authors have characterized a 1400-nucleotide cDNA for the human skeletal muscle ADP/ATP translocator. The deduced amino acid sequence is 94% homologous to the beef heart ADP/ATP translocator protein and contains only a single additional amino-terminal methionine. This implies that the human translocator lacks an amino-terminal targeting peptide, a conclusion substantiated by measuring the molecular weight of the protein synthesized in vitro. A 1400-nucleotide transcript encoding the skeletal muscle translocator was detected on blots of total RNA from human heart, kidney, skeletal muscle, and HeLa cells by hybridization with oligonucleotide probes homologous to the coding region and 3' noncoding region of the cDNA. However, the level of this mRNA varied substantially among tissues. Comparison of our skeletal muscle translocator sequence with that of a recently published human fibroblast translocator cognate revealed that the two proteins are 88% identical and diverged about 275 million years ago. Hence, tissues vary both in the level of expression of individual translocator genes and in differential expression of cognate translocator genes. Comparison of the base substitution rates of the ADP/ATP translocator and the oxidative phosphorylation genes encoded by mitochondrial DNA revealed that the mitochondrial DNA genes fix 10 times more synonymous substitutions and 12 times more replacement substitutions; yet, these nuclear and cytoplasmic respiration genes experience comparable evolutionary constraints. This suggest that the mitochondrial DNA genes are highly prone to deleterious mutations.

  6. Characterization and distribution of a maize cDNA encoding a peptide similar to the catalytic region of second messenger dependent protein kinases

    NASA Technical Reports Server (NTRS)

    Biermann, B.; Johnson, E. M.; Feldman, L. J.

    1990-01-01

    Maize (Zea mays) roots respond to a variety of environmental stimuli which are perceived by a specialized group of cells, the root cap. We are studying the transduction of extracellular signals by roots, particularly the role of protein kinases. Protein phosphorylation by kinases is an important step in many eukaryotic signal transduction pathways. As a first phase of this research we have isolated a cDNA encoding a maize protein similar to fungal and animal protein kinases known to be involved in the transduction of extracellular signals. The deduced sequence of this cDNA encodes a polypeptide containing amino acids corresponding to 33 out of 34 invariant or nearly invariant sequence features characteristic of protein kinase catalytic domains. The maize cDNA gene product is more closely related to the branch of serine/threonine protein kinase catalytic domains composed of the cyclic-nucleotide- and calcium-phospholipid-dependent subfamilies than to other protein kinases. Sequence identity is 35% or more between the deduced maize polypeptide and all members of this branch. The high structural similarity strongly suggests that catalytic activity of the encoded maize protein kinase may be regulated by second messengers, like that of all members of this branch whose regulation has been characterized. Northern hybridization with the maize cDNA clone shows a single 2400 base transcript at roughly similar levels in maize coleoptiles, root meristems, and the zone of root elongation, but the transcript is less abundant in mature leaves. In situ hybridization confirms the presence of the transcript in all regions of primary maize root tissue.

  7. cap alpha. /sub i/-3 cDNA encodes the. cap alpha. subunit of G/sub k/, the stimulatory G protein of receptor-regulated K/sup +/ channels

    SciTech Connect

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

    1988-05-15

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

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

    EPA Science Inventory

    GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN cDNA MICROARRAY ANALYSES

    B.S. Pukazhenthi1, J. C. Rockett2, M. Ouyang3, D.J. Dix2, J.G. Howard1, P. Georgopoulos4, W.J. J. Welsh3 and D. E. Wildt1

    1Department of Reproductiv...

  9. Cloning and characterization of a cDNA encoding beta-amyrin synthase from petroleum plant Euphorbia tirucalli L.

    PubMed

    Kajikawa, Masataka; Yamato, Katsuyuki T; Fukuzawa, Hideya; Sakai, Yasuyoshi; Uchida, Hidenobu; Ohyama, Kanji

    2005-08-01

    Euphorbia tirucalli L., known as the petroleum plant, produces a large amount of triterpenes, such as beta-amyrin. Degenerate RT-PCR based on the sequences conserved among known beta-amyrin synthases led to cloning of a putative triterpene synthase cDNA, EtAS, from leaves of E. tirucalli. The deduced amino acid sequence of the EtAS cDNA showed the highest identity of 82% to the Panax ginseng beta-amyrin synthase. Heterologous expression of the EtAS ORF in the methylotrophic yeast, Pichia pastoris, resulted in production of beta-amyrin, revealing that the EtAS cDNA codes for a beta-amyrin synthase. This is the first report of a gene involved in the triterpene synthetic pathway from Euphorbiaceae plants.

  10. Molecular cloning and expression of cDNAs encoding human alpha-mannosidase II and a previously unrecognized alpha-mannosidase IIx isozyme.

    PubMed Central

    Misago, M; Liao, Y F; Kudo, S; Eto, S; Mattei, M G; Moremen, K W; Fukuda, M N

    1995-01-01

    Golgi alpha-mannosidase II (alpha-MII) is an enzyme involved in the processing of N-linked glycans. Using a previously isolated murine cDNA clone as a probe, we have isolated cDNA clones encompassing the human alpha-MII cDNA open reading frame and initiated isolation of human genomic clones. During the isolation of genomic clones, genes related to that encoding alpha-MII were isolated. One such gene was found to encode an isozyme, designated alpha-MIIx. A 5-kb cDNA clone encoding alpha-MIIx was then isolated from a human melanoma cDNA library. However, comparison between alpha-MIIx and alpha-MII cDNAs suggested that the cloned cDNA encodes a truncated polypeptide with 796 amino acid residues, while alpha-MII consists of 1144 amino acid residues. To reevaluate the sequence of alpha-MIIx cDNA, polymerase chain reaction (PCR) was performed with lymphocyte mRNAs. Comparison of the sequence of PCR products with the alpha-MIIx genomic sequence revealed that alternative splicing of the alpha-MIIx transcript can result in an additional transcript encoding a 1139-amino acid polypeptide. Northern analysis showed transcription of alpha-MIIx in various tissues, suggesting that the alpha-MIIx gene is a housekeeping gene. COS cells transfected with alpha-MIIx cDNA containing the full-length open reading frame showed an increase of alpha-mannosidase activity. The alpha-MIIx gene was mapped to human chromosome 15q25, whereas the alpha-MII gene was mapped to 5q21-22. Images Fig. 5 PMID:8524845

  11. 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.

    PubMed

    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.

  12. Isolation and characterization of a cDNA encoding a membrane bound acyl-CoA binding protein from Agave americana L. epidermis.

    PubMed

    Guerrero, Consuelo; Martín-Rufián, M; Reina, José J; Heredia, Antonio

    2006-01-01

    A cDNA encoding an acyl-CoA binding protein (ACBP) homologue has been cloned from a cDNA library made from mRNA isolated from epidermis of young leaves of Agave americana L. The derived amino acid sequence reveals a protein corresponding to the membrane-associated form of ACBPs only previously described in Arabidopsis and rice. Northern blot analysis showed that the A. americana ACBP gene is mainly expressed in the epidermis of mature zone of the leaves. The epidermis of A. americana leaves have a well developed cuticle with the highest amounts of the cuticular components waxes, cutin and cutan suggesting a potential role of the protein in cuticle formation.

  13. Isolation of cDNA encoding a binding protein specific to 5'-phosphorylated single-stranded DNA with G-rich sequences.

    PubMed Central

    Mizuta, T R; Fukita, Y; Miyoshi, T; Shimizu, A; Honjo, T

    1993-01-01

    We have isolated the cDNA encoding a binding protein to the sequence motif of the immunoglobulin S mu region by the southwestern method. The binding protein designated S mu bp-2 specifically binds to 5'-phosphorylated single-stranded DNA containing 5'-G and GGGG stretches. The amino acid sequence deduced from the cDNA sequence showed that the S mu bp-2 belongs to the putative helicase superfamily which is involved in replication, recombination and repair. Expression of S mu bp-2 mRNA is ubiquitous and augmented in spleen cells stimulated with lipopolysaccharide and interleukin 4 which also induce class switching. The S mu bp-2 gene is conserved among vertebrates. Possible involvement of S mu bp-2 in class switching is discussed. Images PMID:8493094

  14. Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: Evidence for more than one receptor class

    SciTech Connect

    Gronwald, R.G.K.; Grant, F.J.; Haldeman, B.A.; Hart, C.E.; O'Hara, P.J.; Hagen, F.S.; Ross, R.; Bowen-Pope, D.F.; Murray, M.J. )

    1988-05-01

    The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A){sup +} RNA from human dermal fibroblasts detects a major and a minor transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an {approx} 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF. Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class.

  15. Identification of cDNA clones encoding secretory isoenzyme forms: sequence determination of canine pancreatic prechymotrypsinogen 2 mRNA.

    PubMed Central

    Pinsky, S D; LaForge, K S; Luc, V; Scheele, G

    1983-01-01

    A cDNA library has been constructed from canine poly(A)+ mRNA. Clones containing cDNA inserts coding for prechymotrypsinogen 2 (isoelectric point = 7.1; Mr = 27,500), one of three canine pancreatic isoenzyme forms, were selected by colony hybridization using a cDNA probe synthesized from immunoselected prechymotrypsinogen 2 mRNA. To verify that cDNA clones code for prechymotrypsinogen 2 forms that translocate across rough endoplasmic reticulum membranes and fold into stable and identifiable secretory proteins, we conducted in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and optimal concentrations of glutathione and analyzed nascent translation products in their nonreduced state by two-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and fluorography. A near full-length chymotrypsinogen 2 cDNA and its primed extension were used to determine the nucleotide sequence for the entire coding region of prechymotrypsinogen 2 mRNA and 87 residues, including a poly(A) addition signal, in the 3' nontranslated region. The deduced amino acid sequence shows a 263-residue presecretory protein containing an 18-residue amino-terminal transport peptide (Met-Ala-Phe-Leu-Trp-Leu-Leu-Ser-Cys-Phe-Ala-Leu-Leu-Gly-Thr-Ala-Phe-Gly ), which we have previously shown to mediate the translocation of chymotrypsinogen 2 across the rough endoplasmic reticulum membrane. Following the transport peptide is a 245-residue proenzyme, which shows 82% and 80% sequence identity with bovine chymotrypsinogens A and B, respectively. Conserved among the three zymogens are 10 Cys residues that form five disulfide bonds in bovine chymotrypsinogens A and B and the residues that are required for zymogen activation, substrate binding, and catalytic activity. Images PMID:6584866

  16. Human cDNA mapping using fluorescence in situ hybridization

    SciTech Connect

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  17. Cloning of the genes encoding two murine and human cochlear unconventional type I myosins

    SciTech Connect

    Crozet, F.; El Amraoui, Z.; Blanchard, S.

    1997-03-01

    Several lines of evidence indicate a crucial role for unconventional myosins in the function of the sensory hair cells of the inner ear. We report here the characterization of the cDNAs encoding two unconventional type I myosins from a mouse cochlear cDNA library. The first cDNA encodes a putative protein named Myo1c, which is likely to be the murine orthologue of the bullfrog myosin I{beta} and which may be involved in the gating of the mechanotransduction channel of the sensory hair cells. This myosin belongs to the group of short-tailed myosins I, with its tail ending shortly after a polybasic, TH-1-like domain. The second cDNA encodes a novel type I myosin Myo1f which displays three regions: a head domain with the conserved ATP- and actin-binding sites, a neck domain with a single IQ motif, and a tail domain with the tripartite structure initially described in protozoan myosins I. The tail of Myo1f includes (1) a TH-1 region rich in basic residues, which may interact with anionic membrane phospholipids; (2) a TH-2 proline-rich region, expected to contain an ATP-insensitive actin-binding site; and (3) an SH-3 domain found in a variety of cytoskeletal and signaling proteins. Northern blot analysis indicated that the genes encoding Myo1c and Myo1f display a widespread tissue expression in the adult mouse. Myo1c and Myo1f were mapped by in situ hybridization to the chromosomal regions 11D-11E and 17B-17C, respectively. The human orthologuous genes MYO1C and MYO1F were also characterized, and mapped to the human chromosomal regions 17p13 and 19p13.2- 19p1.3.3, respectively. 45 refs., 5 figs., 2 tabs.

  18. Molecular cloning and characterization of a cDNA encoding a laminin-binding protein (AhLBP) from Acanthamoeba healyi.

    PubMed

    Hong, Yeon-Chul; Lee, Won-Myung; Kong, Hyun-Hee; Jeong, Hae-Jin; Chung, Dong-Il

    2004-01-01

    Adherence of Acanthamoeba to host tissue is believed to be crucial in the establishment of amoebic keratitis or GAE. We have isolated a cDNA from a GAE-causing gymnoamoeba, Acanthamoeba healyi, encoding a protein that binds laminin by screening with a peptide G-specific DNA probe. The cDNA clone (AhLBP) was identified on the basis of sequence homology to the nonintegrin mammalian metastasis-associated 67-kDa laminin receptor (67-LR). The predicted amino acid sequence is 256 residues long with a calculated molecular mass of 28.2kDa and a theoretical pI of 5.48. Southern and Northern blot analyses suggested the gene as a single copy in A. healyi genome and expressed as a single transcript of approximately 1.0kb. Virulent strains of Acanthamoeba revealed higher level of the AhLBP mRNA expression than soil isolates. Specific binding of the purified recombinant protein to laminin was confirmed by sandwich Western blot. The polypeptide encoded by AhLBP shared substantial identity with the acidic class ribosomal proteins involved in protein synthesis. Therefore, the AhLBP may be multifunctional in A. healyi, acting as a laminin-binding molecule but also playing a role in cell division and growth. AhLBP-EGFP fusion protein expressed in A. healyi was localized mainly at the cell membrane and nucleus and at cytoplasm with lesser degree. N-terminal 64 amino acids were important for the localization at the cell membrane. This is the first description of a cDNA encoding a laminin-binding protein from protozoan parasites.

  19. Sequence of a cDNA encoding the bi-specific NAD(P)H-nitrate reductase from the tree Betula pendula and identification of conserved protein regions.

    PubMed

    Friemann, A; Brinkmann, K; Hachtel, W

    1991-05-01

    Nitrate reductase (NR) assays revealed a bispecific NAD(P)H-NR (EC 1.6.6.2.) to be the only nitrate-reducing enzyme in leaves of hydroponically grown birches. To obtain the primary structure of the NAD(P)H-NR, leaf poly(A)+ mRNA was used to construct a cDNA library in the lambda gt11 phage. Recombinant clones were screened with heterologous gene probes encoding NADH-NR from tobacco and squash. A 3.0 kb cDNA was isolated which hybridized to a 3.2 kb mRNA whose level was significantly higher in plants grown on nitrate than in those grown on ammonia. The nucleotide sequence of the cDNA comprises a reading frame encoding a protein of 898 amino acids which reveals 67%-77% identity with NADH-nitrate reductase sequences from higher plants. To identify conserved and variable regions of the multicentre electron-transfer protein a graphical evaluation of identities found in NR sequence alignments was carried out. Thirteen well-conserved sections exceeding a size of 10 amino acids were found in higher plant nitrate reductases. Sequence comparisons with related redox proteins indicate that about half of the conserved NR regions are involved in cofactor binding. The most striking difference in the birch NAD(P)H-NR sequence in comparison to NADH-NR sequences was found at the putative pyridine nucleotide binding site. Southern analysis indicates that the bi-specific NR is encoded by a single copy gene in birch.

  20. Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

    PubMed Central

    Ridley, R G; Patel, H V; Gerber, G E; Morton, R C; Freeman, K B

    1986-01-01

    A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein. Images PMID:3012461

  1. cDNA cloning and sequence analysis of human pancreatic procarboxypeptidase A1.

    PubMed Central

    Catasús, L; Villegas, V; Pascual, R; Avilés, F X; Wicker-Planquart, C; Puigserver, A

    1992-01-01

    Using polyclonal antibodies raised against human pancreatic procarboxypeptidases, a full-length cDNA coding for an A-type proenzyme was isolated from a lambda gt11 human pancreatic library. This cDNA contains standard 3' and 5' flanking regions, a poly(A)+ tail and a central region of 1260 nucleotides coding for a protein of 419 amino acids. On the basis of sequence comparisons, the human protein was classified as a procarboxypeptidase A1 which is very similar to the previously described A1 forms from rat and bovine pancreatic glands. The presence of the amino acid sequences assumed to be of importance for the zymogen inhibition by its activation segment, primarily on the basis of the recently reported crystal structure of the B form, further supports the proposed classification. PMID:1417781

  2. Molecular cloning and nucleotide sequencing of human immunoglobulin epsilon chain cDNA.

    PubMed Central

    Seno, M; Kurokawa, T; Ono, Y; Onda, H; Sasada, R; Igarashi, K; Kikuchi, M; Sugino, Y; Nishida, Y; Honjo, T

    1983-01-01

    DNA complementary to mRNA of human immunoglobulin E heavy chain (epsilon chain) isolated and purified from U266 cells has been synthesized and inserted into the PstI site of pBR322 by G-C tailing. This recombinant plasmid was used to transform E. coli chi 1776 to screen 1445 tetracycline resistant colonies. Nine clones (pGETI - 9) containing cDNA coding for the human epsilon chain were recognized by colony hybridization and Southern blotting analysis with a nick-translated human IgE genome fragment. The nucleotide sequence of the longest cDNA contained in pGET2 was determined. The results indicate that the sequence of 1657 nucleotides codes for 494 amino acids covering a part of the variable region and all of the constant region of the human epsilon chain. Most of the amino acid sequence deduced from the nucleotide sequence is in substantial agreement with that reported. Furthermore a termination codon after the -COOH terminal amino acid marks the beginning of a 3' untranslated region of 125 nucleotides with a poly A tail. Taking this into account, the structure of the human epsilon chain mRNA, except a part of the 5' end, is conserved fairly well in the cDNA insert in pGET2. Images PMID:6300763

  3. Cloning and characterization of a cDNA encoding a cellobiose dehydrogenase from the white rot fungus Phanerochaete chrysosporium.

    PubMed

    Raices, M; Paifer, E; Cremata, J; Montesino, R; Ståhlberg, J; Divne, C; Szabó, I J; Henriksson, G; Johansson, G; Pettersson, G

    1995-08-07

    The cDNA of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium has been cloned and sequenced. The 5' end was obtained by PCR amplification. The cDNA contains 2310 translated bases excluding the poly(A) tail. The deduced mature protein contains 770 amino acid residues and is preceded by a 18 residue long signal peptide. The regions of the amino acid sequence corresponding to the heme and FAD domains of CDH were identified as well as the nucleotide-binding motif, the disulfide pairing and a methionine residue chelating the heme iron. No homologous sequences were found for the heme domain, however, the FAD domain appears to be distantly related to the GMC oxidoreductase family.

  4. Molecular characterization and expression of a cDNA encoding fructan:fructan 6G-fructosyltransferase from asparagus (Asparagus officinalis).

    PubMed

    Ueno, Keiji; Onodera, Shuichi; Kawakami, Akira; Yoshida, Midori; Shiomi, Norio

    2005-03-01

    * Fructan:fructan 6G-fructosyltransferase (6G-FFT) catalyses a transfructosylation from fructooligosaccharides to C6 of the glucose residue of sucrose or fructooligosacchrides. In asparagus (Asparagus officinalis), 6G-FFT is important for the synthesis of inulin neoseries fructan. Here, we report the isolation and functional analysis of the gene encoding asparagus 6G-FFT. * A cDNA clone was isolated from asparagus cDNA library. Recombinant protein was produced by expression system of Pichia pastoris. To measure enzymatic activity, recombinant protein was incubated with sucrose, 1-kestose, 1-kestose and sucrose, or neokestose. The reaction products were detected by high performance anion-exchange chromatography. * The deduced amino acid sequence of isolated cDNA was similar to that of fructosyltransferases and vacuolar type invertases from plants. Recombinant protein mainly produced inulin neoseries fructan, such as 1F, 6G-di-beta-D-fructofuranosylsucrose and neokestose. * Recombinant protein demonstrates 6G-FFT activity, and slight fructan:fructan 1-fructosyltransferase (1-FFT) activity. The ratio of 6G-FFT activity to 1-FFT activity was calculated to be 13. The characteristics of the recombinant protein closely resemble those of the 6G-FFT from asparagus roots, except for a difference in accompanying 1-FFT activity.

  5. Isolation of cDNA clones for differentially expressed genes of the human parasite Schistosoma mansoni.

    PubMed Central

    Davis, A H; Blanton, R; Rottman, F; Maurer, R; Mahmoud, A

    1986-01-01

    Little is known about the mechanisms that control transformations during the life cycle of Schistosoma mansoni. To enable isolation of DNA sequences encoding developmentally regulated antigens a cDNA expression library in the vector lambda gt11 amp3 was constructed from adult mRNA and immunologically screened with sera from infected individuals. We report here on the properties of three recombinant clones that derive from developmentally regulated genes. Clone 10-3 encoded a beta-galactosidase fusion protein present in high abundance in infected Escherichia coli. Clones 7-2 and 8-2 also produced immunologically recognized proteins; however, the peptides did not appear to be beta-galactosidase fusion proteins. The expression of mRNAs hybridizing to these cDNAs was examined in the different stages of the parasite life cycle. Messenger RNA corresponding to clone 10-3, approximately equal to 1000 bases in length, was present in higher abundance in male worms than in females but was not detected in schistosome eggs. A 900-base mRNA hybridizing to clone 7-2 was observed in adult worms and eggs. Both clone 10-3 and clone 7-2 hybridized to smaller mRNAs in cercariae and freshly transformed schistosomula than in adult worms. Clone 8-2 contained tandem cDNA inserts. One cDNA hybridized to a 1700-base mRNA present in all stages, while the second hybridized to an 800-base mRNA specific to adult female worms. Images PMID:3461448

  6. cDNA and deduced amino acid sequence of human pulmonary surfactant-associated proteolipid SPL(Phe)

    SciTech Connect

    Glasser, S.W.; Korfhagen, T.R.; Weaver, T.; Pilot-Matias, T.; Fox, J.L.; Whitsett, J.A.

    1987-06-01

    Hydrophobic surfactant-associated protein of M/sub r/ 6000-14,000 was isolated from either/ethanol or chloroform/methanol extracts of mammalian pulmonary surfactant. Automated Edman degradation in a gas-phase sequencer showed the major N-terminus of the human low molecular weight protein to be Phe-Pro-Ile-Pro-Leu-Pro-Try-Cys-Trp-Leu-Cys-Arg-Ala-Leu-. Because of the N-terminal phenylalanine, the surfactant protein was designated SPL(Phe). Antiserum generated against hydrophobic surfactant protein(s) from bovine pulmonary surfactant recognized protein of M/sub r/ 6000-14,000 in immunoblot analysis and was used to screen a lambdagt11 expression library constructed from adult human lung poly(A)/sup +/ RNA. This resulted in identification of a 1.4-kilobase cDNA clone that was shown to encode the N-terminus of the surfactant polypeptide SPL(Phe) (Phe-Pro-Ile-Pro-Leu-Pro-) within an open reading frame for a larger protein. Expression of a fused ..beta..-galactosidase-SPL (Phe) gene in Escherichia coli yielded an immunoreactive M/sub r/ 34,000 fusion peptide. Hybrid-arrested translation with the cDNA and immunoprecipitation of (/sup 35/S)methionine-labeled in vitro translation products of human poly(A)/sup +/ RNA with a surfactant polyclonal antibody resulted in identification of a M/sub r/ 40,000 precursor protein. Blot hybridization analysis of electrophoretically fractionated RNA from human lung detected a 2.0-kilobase RNA that was more abundant in adult lung than in fetal lung. These proteins, and specifically SPL(Phe), may therefore be useful for synthesis of replacement surfactants for treatment of hyaline membrane disease in newborn infants or of other surfactant-deficient states.

  7. Molecular cloning and characterization of a tomato cDNA encoding a systemically wound-inducible bZIP DNA-binding protein

    NASA Technical Reports Server (NTRS)

    Stankovic, B.; Vian, A.; Henry-Vian, C.; Davies, E.

    2000-01-01

    Localized wounding of one leaf in intact tomato (Lycopersicon esculentum Mill.) plants triggers rapid systemic transcriptional responses that might be involved in defense. To better understand the mechanism(s) of intercellular signal transmission in wounded tomatoes, and to identify the array of genes systemically up-regulated by wounding, a subtractive cDNA library for wounded tomato leaves was constructed. A novel cDNA clone (designated LebZIP1) encoding a DNA-binding protein was isolated and identified. This clone appears to be encoded by a single gene, and belongs to the family of basic leucine zipper domain (bZIP) transcription factors shown to be up-regulated by cold and dark treatments. Analysis of the mRNA levels suggests that the transcript for LebZIP1 is both organ-specific and up-regulated by wounding. In wounded wild-type tomatoes, the LebZIP1 mRNA levels in distant tissue were maximally up-regulated within only 5 min following localized wounding. Exogenous abscisic acid (ABA) prevented the rapid wound-induced increase in LebZIP1 mRNA levels, while the basal levels of LebZIP1 transcripts were higher in the ABA mutants notabilis (not), sitiens (sit), and flacca (flc), and wound-induced increases were greater in the ABA-deficient mutants. Together, these results suggest that ABA acts to curtail the wound-induced synthesis of LebZIP1 mRNA.

  8. Molecular cloning and characterization of a cDNA encoding the N-acetyl-beta-D-glucosaminidase homologue of Paracoccidioides brasiliensis.

    PubMed

    Santos, Mônica O; Pereira, Maristela; Felipe, Maria Sueli S; Jesuino, Rosalia Santos A; Ulhoa, Cirano J; Soares, Renata de Bastos A; Soares, Celia Maria de A

    2004-06-01

    A cDNA encoding the N-acetyl-beta-D-glucosaminidase (NAG) protein of Paracoccidioides brasiliensis, Pb NAG1, was cloned and characterized. The 2663-nucleotide sequence of the cDNA consisted of a single open reading frame encoding a protein with a predicted molecular mass of 64.73 kDa and an isoeletric point of 6.35. The predicted protein includes a putative 30-amino-acid signal peptide. The protein as a whole shares considerable sequence similarity with 'classic' NAG. The primary sequence of Pb NAG1 was used to infer phylogenetic relationships. The amino acid sequence of Pb NAG1 has 45, 31 and 30% identity, respectively, with homologous sequences from Trichoderma harzianum, Aspergillus nidulans and Candida albicans. In particular, striking homology was observed with the active site regions of the glycosyl hydrolase group of proteins (family 20). The expected active site consensus motif G X D E and catalytic Asp and Glu residues at positions 373 and 374 were found, reinforcing that Pb NAG1 belongs to glycosyl hydrolase family 20. The nucleotide sequence of Pb nag1 and its flanking regions have been deposited, along with the amino acid sequence of the deduced protein, in GenBank under accession number AF419158.

  9. Sequence analysis and molecular characterization of larval midgut cDNA transcripts encoding peptidases from the yellow mealworm, Tenebrio molitor L.

    PubMed

    Prabhakar, S; Chen, M-S; Elpidina, E N; Vinokurov, K S; Smith, C M; Marshall, J; Oppert, B

    2007-08-01

    Peptidase sequences were analysed in randomly picked clones from cDNA libraries of the anterior or posterior midgut or whole larvae of the yellow mealworm, Tenebrio molitor Linnaeus. Of a total of 1528 sequences, 92 encoded potential peptidases, from which 50 full-length cDNA sequences were obtained, including serine and cysteine proteinases and metallopeptidases. Serine proteinase transcripts were predominant in the posterior midgut, whereas transcripts encoding cysteine and metallopeptidases were mainly found in the anterior midgut. Alignments with other proteinases indicated that 40% of the serine proteinase sequences were serine proteinase homologues, and the remaining ones were identified as either trypsin, chymotrypsin or other serine proteinases. Cysteine proteinase sequences included cathepsin B- and L-like proteinases, and metallopeptidase transcripts were similar to carboxypeptidase A. Northern blot analysis of representative sequences demonstrated the differential expression profile of selected transcripts across five developmental stages of Te. molitor. These sequences provide insights into peptidases in coleopteran insects as a basis to study the response of coleopteran larvae to external stimuli and to evaluate regulatory features of the response.

  10. Structure of LEP100, a glycoprotein that shuttles between lysosomes and the plasma membrane, deduced from the nucleotide sequence of the encoding cDNA

    PubMed Central

    1988-01-01

    LEP100, a membrane glycoprotein that has the unique property of shuttling from lysosomes to endosomes to plasma membrane and back, was purified from chicken brain. Its NH2-terminal amino acid sequence was determined, and an oligonucleotide encoding part of this sequence was used to clone the encoding cDNA. The deduced amino acid sequence consists of 414 residues of which the NH2-terminal 18 constitute a signal peptide. The sequence includes 17 sites for N-glycosylation in the NH2-terminal 75% of the polypeptide chain followed by a region lacking N-linked oligosaccharides, a single possible membrane-spanning segment, and a cytoplasmic domain of 11 residues, including three potential phosphorylation sites. Eight cysteine residues are spaced in a regular pattern through the lumenal (extracellular) domain, while a 32-residue sequence rich in proline, serine, and threonine occurs at its midpoint. Expression of the cDNA in mouse L cells resulted in targeting of LEP100 primarily to the mouse lysosomes. PMID:3339090

  11. [Identification and expression analysis of a full-length cDNA encoding Brassica napus small nuclear ribonucleoprotein BnSmD1].

    PubMed

    Yuan, Xiao-Meng; Zhou, Yun-Tao; Zhang, Hong-Yan; Xue, Hua; Zhou, Lin; Zhao, Yun

    2007-12-01

    By using substractive hybridization (SSH) and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR), a full-length cDNA encoding Brassica napus small nuclear ribonucleoprotein, named BnSmD1, was obtained. It had 484 base pairs in length containing an open reading frame (ORF) of 354 bp and encoding a predicted protein of 118 amino acids with a molecular weight of 13 kDa. The BnSmD1 protein shares two highly conserved Sm folds (Sm-1 and Sm-2) and a C-terminal RG dipeptide repeat. Northern blot analysis revealed that BnSmD1 was expressed in all tested organs in B. napus, but its transcript level in early floral buds was much higher than that in leaf and stem tissues. No obvious expression difference was observed in leaf and stem tissues between the apetalous line Apet33-10 petalled near-isogenic line Pet33-10. Compared with wild type, the expression of BnSmD1 in the early floral buds of apetalous mutant Apet33-10 was significantly reduced. Taken together, our results suggest that BnSmD1 may play an important role in early floral petal development in B. napus.

  12. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    NASA Astrophysics Data System (ADS)

    Qi, Fei; Guo, Huarong; Wang, Jian

    2008-02-01

    Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  13. cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera

    SciTech Connect

    Aris, J.P.; Blobel, G. )

    1991-02-01

    The authors have isolated a 1.1-kilobase cDNA clone that encodes human fibrillarin by screening a hepatoma library in parallel with DNA probes derived from the fibrillarin genes of Saccharomyces cerevisiae (NOP1) and Xenopus laevis. RNA blot analysis indicates that the corresponding mRNA is {approximately}1,300 nucleotides in length. Human fibrillarin expressed in vitro migrates on SDS gels as a 36-kDa protein that is specifically immunoprecipitated by antisera from humans with scleroderma autoimmune disease. Human fibrillarin contains an amino-terminal repetitive domain {approximately}75-80 amino acids in length that is rich in glycine and arginine residues and is similar to amino-terminal domains in the yeast and Xenopus fibrillarins. The occurrence of a putative RNA-binding domain and an RNP consensus sequence within the protein is consistent with the association of fibrillarin with small nucleolar RNAs. Protein sequence alignments show that 67% of amino acids from human fibrillarin are identical to those in yeast fibrillarin and that 81% are identical to those in Xenopus fibrillarin. This identity suggests the evolutionary conservation of an important function early in the pathway for ribosome biosynthesis.

  14. Cloning of cDNA for proteinase 3: a serine protease, antibiotic, and autoantigen from human neutrophils

    PubMed Central

    1990-01-01

    Closely similar but nonidentical NH2-terminal amino acid sequences have been reported for a protein or proteins in human neutrophils whose bioactivities is/are diverse (as a serine protease, antibiotic, and Wegener's granulomatosis autoantigen) but that share(s) several features: localization in the azurophil granules, a molecular mass of approximately 29 kD, reactivity with diisopropylfluorophosphate, and the ability to degrade elastin. We previously purified one such entity, termed p29b. Using a monospecific antibody, we have cloned from human bone marrow a cDNA encoding the complete p29b protein in its mature form, along with pre- and pro-sequences. The predicted amino acid sequence agrees closely with the NH2-terminal sequence obtained previously from purified p29b, as well as with sequences newly obtained from CNBr fragments. The primary structure is highly homologous to elastase, cathepsin G, T cell granzymes, and other serine proteases, and shares both the catalytic triad and substrate binding pocket of elastase. Hybridization of the full-length cDNA with restriction enzyme digests of human genomic DNA revealed only one fragment. This suggests that the closely related species described previously are the same, and can be subsumed by the term used for the first-described activity, proteinase 3. Proteinase 3 is more abundant in neutrophils than elastase and has a similar proteolytic profile and specific activity. Thus, proteinase 3 may share the role previously attributed to neutrophil elastase in tissue damage, and has the potential to function as an antimicrobial agent. PMID:2258701

  15. Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

    PubMed Central

    Imanishi, Tadashi; Itoh, Takeshi; Suzuki, Yutaka; O'Donovan, Claire; Fukuchi, Satoshi; Koyanagi, Kanako O; Barrero, Roberto A; Tamura, Takuro; Yamaguchi-Kabata, Yumi; Tanino, Motohiko; Yura, Kei; Miyazaki, Satoru; Ikeo, Kazuho; Homma, Keiichi; Kasprzyk, Arek; Nishikawa, Tetsuo; Hirakawa, Mika; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Ashurst, Jennifer; Jia, Libin; Nakao, Mitsuteru; Thomas, Michael A; Mulder, Nicola; Karavidopoulou, Youla; Jin, Lihua; Kim, Sangsoo; Yasuda, Tomohiro; Lenhard, Boris; Eveno, Eric; Suzuki, Yoshiyuki; Yamasaki, Chisato; Takeda, Jun-ichi; Gough, Craig; Hilton, Phillip; Fujii, Yasuyuki; Sakai, Hiroaki; Tanaka, Susumu; Amid, Clara; Bellgard, Matthew; Bonaldo, Maria de Fatima; Bono, Hidemasa; Bromberg, Susan K; Brookes, Anthony J; Bruford, Elspeth; Carninci, Piero; Chelala, Claude; Couillault, Christine; de Souza, Sandro J.; Debily, Marie-Anne; Devignes, Marie-Dominique; Dubchak, Inna; Endo, Toshinori; Estreicher, Anne; Eyras, Eduardo; Fukami-Kobayashi, Kaoru; R. Gopinath, Gopal; Graudens, Esther; Hahn, Yoonsoo; Han, Michael; Han, Ze-Guang; Hanada, Kousuke; Hanaoka, Hideki; Harada, Erimi; Hashimoto, Katsuyuki; Hinz, Ursula; Hirai, Momoki; Hishiki, Teruyoshi; Hopkinson, Ian; Imbeaud, Sandrine; Inoko, Hidetoshi; Kanapin, Alexander; Kaneko, Yayoi; Kasukawa, Takeya; Kelso, Janet; Kersey, Paul; Kikuno, Reiko; Kimura, Kouichi; Korn, Bernhard; Kuryshev, Vladimir; Makalowska, Izabela; Makino, Takashi; Mano, Shuhei; Mariage-Samson, Regine; Mashima, Jun; Matsuda, Hideo; Mewes, Hans-Werner; Minoshima, Shinsei; Nagai, Keiichi; Nagasaki, Hideki; Nagata, Naoki; Nigam, Rajni; Ogasawara, Osamu; Ohara, Osamu; Ohtsubo, Masafumi; Okada, Norihiro; Okido, Toshihisa; Oota, Satoshi; Ota, Motonori; Ota, Toshio; Otsuki, Tetsuji; Piatier-Tonneau, Dominique; Poustka, Annemarie; Ren, Shuang-Xi; Saitou, Naruya; Sakai, Katsunaga; Sakamoto, Shigetaka; Sakate, Ryuichi; Schupp, Ingo; Servant, Florence; Sherry, Stephen; Shiba, Rie; Shimizu, Nobuyoshi; Shimoyama, Mary; Simpson, Andrew J; Soares, Bento; Steward, Charles; Suwa, Makiko; Suzuki, Mami; Takahashi, Aiko; Tamiya, Gen; Tanaka, Hiroshi; Taylor, Todd; Terwilliger, Joseph D; Unneberg, Per; Veeramachaneni, Vamsi; Watanabe, Shinya; Wilming, Laurens; Yasuda, Norikazu; Yoo, Hyang-Sook; Stodolsky, Marvin; Makalowski, Wojciech; Go, Mitiko; Nakai, Kenta; Takagi, Toshihisa; Kanehisa, Minoru; Sakaki, Yoshiyuki; Quackenbush, John; Okazaki, Yasushi; Hayashizaki, Yoshihide; Hide, Winston; Chakraborty, Ranajit; Nishikawa, Ken; Sugawara, Hideaki; Tateno, Yoshio; Chen, Zhu; Oishi, Michio; Tonellato, Peter; Apweiler, Rolf; Okubo, Kousaku; Wagner, Lukas; Wiemann, Stefan; Strausberg, Robert L; Isogai, Takao; Auffray, Charles; Nomura, Nobuo; Sugano, Sumio

    2004-01-01

    The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In

  16. Identification and isolation of cDNA clones encoding the abundant secreted proteins in the saliva proteome of Culicoides nubeculosus.

    PubMed

    Russell, C L; Heesom, K J; Arthur, C J; Helps, C R; Mellor, P S; Day, M J; Torsteinsdottir, S; Björnsdóttir, T S; Wilson, A D

    2009-06-01

    Culicoides spp. are vectors of several infectious diseases of veterinary importance and a major cause of allergy in horses and other livestock. Their saliva contains a number of proteins which enable blood feeding, enhance disease transmission and act as allergens. We report the construction of a novel cDNA library from Culicoides nubeculosus linked to the analysis of abundant salivary gland proteins by mass spectrometry. Fifty-four novel proteins sequences are described including those of the enzymes maltase, hyaluronidase and two serine proteases demonstrated to be present in Culicoides salivary glands, as well as several members of the D7 family and protease inhibitors with putative anticoagulant activity. In addition, several families of abundant proteins with unknown function were identified including some of the major candidate allergens that cause insect bite hypersensitivity in horses.

  17. Cloning of a cDNA encoding cathepsin D from salamander, Hynobius leechii, and its expression in the limb regenerates.

    PubMed

    Ju, B G; Kim, W S

    2000-01-01

    Cathepsin D is a major lysosomal aspartic proteinase participating in the degradation or modification of intra- and extracellular matrix molecules, and its activity is known to increase in the process of tissue reorganization during the early phase of salamander limb regeneration. Here, we report the cloning of a salamander cathepsin D cDNA from Hynobius leechii and its expression profile in normal and retinoic acid (RA) treated limb regenerates. The gene expression of cathepsin D increased notably during the dedifferentiation stage and decreased gradually thereafter. Furthermore, RA that enhances dedifferentiation of regenerating salamander limb caused the elevation of cathepsin D expression both in terms of level and duration. These results suggest that cathepsin D plays important role(s) in the dedifferentiation process, and enhancement of cathepsin D expression might be closely related to RA-evoked pattern duplication.

  18. Cloning of a cDNA encoding the Saussurea medusa chalcone isomerase and its expression in transgenic tobacco.

    PubMed

    Li, F; Jin, Z; Qu, W; Zhao, D; Ma, F

    2006-01-01

    Chalcone isomerase (CHI; EC 5.5.1.6) is a key enzyme in the flavonoid biosynthesis pathway. We isolated a CHI gene (SmCHI) from a cDNA library derived from Saussurea medusa (Asteraceae) cell cultures. The cDNA and genomic sequences of SmCHI are the same; in other words, this gene is intronless. The coding region of the gene is 699 bp long, and its deduced protein consists of 232 amino acids with a predicted molecular mass of 24 kDa and a pI of 4.7. The deduced amino acid sequence of SmCHI shares 79.3% identity with CHI from Callistephus chinensis, a familial relative to S. medusa; this homology is higher than those with CHI's from any other plant species. A functional bioassay for SmCHI was performed by transforming Nicotiana tabacum plants in the sense or antisense orientation under the regulation of the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic tobacco plants overexpressing sense SmCHI produced up to fivefold total flavonoids over wild-type tobacco plants, mainly due to an enhanced accumulation of rutin. Transgenic tobacco plants with antisense SmCHI accumulated smaller amounts of flavonoids; this is apparently brought about by suppressed expression of the endogenous CHI gene. CHI activities also positively correlated with the amounts of total flavonoids accumulated in the transgenic plants. It is concluded that overexpression of SmCHI can be used as a useful approach to increase flavonoid production in transgenic plants.

  19. Molecular cloning of cDNAs encoding human carnitine acetyltransferase and mapping of the corresponding gene to chromosome 9q34.1

    SciTech Connect

    Corti, O.; Finocchiaro, G.; DiDonato, S.

    1994-09-01

    Using a combination of PCR screening of cDNA libraries and reverse transcription PCR, we have cloned three overlapping DNA fragments that encode human carnitine acetyltransferase (CAT), a key enzyme for metabolic pathways involved with the control of the acyl-Co/CoA ratio in mitochondria, peroxisomes, and endoplasmic reticulum. The resulting cDNA (2436 bp) hybridizes to a mRNA species of {approximately}2.9 kb that is particularly abundant in skeletal muscle and encodes a 68-kDa protein containing a peroxisomal targeting signal. The sequence matches those of several tryptic peptides obtained from purified human liver CAT and shows striking similarities with other members of the carnitine/choline acetyltransferase family very distant throughout evolution. CAT cDNA has also been used for fluorescence in situ hybridization on metaphase spreads of human chromosomes, and the corresponding gene, CAT1, has been mapped to chromosome 9q34.1. 29 refs., 4 figs.

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

    PubMed

    Fukushima, D; Kitamura, N; Nakanishi, S

    1985-12-31

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

  1. Human beta-mannosidase cDNA characterization and first identification of a mutation associated with human beta-mannosidosis.

    PubMed

    Alkhayat, A H; Kraemer, S A; Leipprandt, J R; Macek, M; Kleijer, W J; Friderici, K H

    1998-01-01

    Human beta-mannosidosis is an autosomal recessive, lysosomal storage disease caused by a deficiency of the enzyme beta-mannosidase. Unlike the severe clinical manifestation of the disease in ruminants, in which it leads to neonatal death, the human disease phenotype is generally milder. In addition, the phenotypic manifestation among the reported cases of human beta-mannosidosis is variable, even among members of the same family. To understand the molecular basis of the human disease and the mechanisms for such clinical variability, we sequenced the entire coding region of the human beta-mannosidase gene using a combination of cDNA library screening, RT-PCR and 5' rapid amplification of cDNA ends (RACE). The composite cDNA is 3293 nt, consisting of an 87 nt 5'-untranslated region, 2640 nt coding region and 566 nt 3'-untranslated region. The gene was localized to human chromosome 4q22-25. Analysis of a multiple tissue northern blot demonstrated a single 3.7 kb transcript. Mutation analysis of a Czech gypsy family with two siblings differently affected with beta-mannosidosis demonstrated a homozygous A-->G transition 2 bp upstream of a splice acceptor site. The associated cryptic splice site activation and exon skipping caused by this mutation resulted in two abnormally spliced mutant mRNA species in both siblings.

  2. Cloning and functional expression of a cDNA encoding stearoyl-ACP Δ9-desaturase from the endosperm of coconut (Cocos nucifera L.).

    PubMed

    Gao, Lingchao; Sun, Ruhao; Liang, Yuanxue; Zhang, Mengdan; Zheng, Yusheng; Li, Dongdong

    2014-10-01

    Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176 bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC-MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future.

  3. Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues.

    PubMed

    Ando-Akatsuka, Y; Saitou, M; Hirase, T; Kishi, M; Sakakibara, A; Itoh, M; Yonemura, S; Furuse, M; Tsukita, S

    1996-04-01

    Occludin has been identified from chick liver as a novel integral membrane protein localizing at tight junctions (Furuse, M., T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 123:1777-1788). To analyze and modulate the functions of tight junctions, it would be advantageous to know the mammalian homologues of occludin and their genes. Here we describe the nucleotide sequences of full length cDNAs encoding occludin of rat-kangaroo (potoroo), human, mouse, and dog. Rat-kangaroo occludin cDNA was prepared from RNA isolated from PtK2 cell culture, using a mAb against chicken occludin, whereas the others were amplified by polymerase chain reaction based on the sequence found around the human neuronal apoptosis inhibitory protein gene. The amino acid sequences of the three mammalian (human, murine, and canine) occludins were very closely related to each other (approximately 90% identity), whereas they diverged considerably from those of chicken and rat-kangaroo (approximately 50% identity). Implications of these data and novel experimental options in cell biological research are discussed.

  4. Transgenic Tobacco Overexpressing Tea cDNA Encoding Dihydroflavonol 4-Reductase and Anthocyanidin Reductase Induces Early Flowering and Provides Biotic Stress Tolerance

    PubMed Central

    Kumar, Vinay; Nadda, Gireesh; Kumar, Sanjay; Yadav, Sudesh Kumar

    2013-01-01

    Flavan-3-ols contribute significantly to flavonoid content of tea (Camellia sinensis L.). Dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR) are known to be key regulatory enzymes of flavan-3-ols biosynthesis. In this study, we have generated the transgenic tobacco overexpressing individually tea cDNA CsDFR and CsANR encoding for DFR and ANR to evaluate their influence on developmental and protective abilities of plant against biotic stress. The transgenic lines of CsDFR and CsANR produced early flowering and better seed yield. Both types of transgenic tobacco showed higher content of flavonoids than control. Flavan-3-ols such as catechin, epicatechin and epicatechingallate were found to be increased in transgenic lines. The free radical scavenging activity of CsDFR and CsANR transgenic lines was improved. Oxidative stress was observed to induce lesser cell death in transgenic lines compared to control tobacco plants. Transgenic tobacco overexpressing CsDFR and CsANR also showed resistance against infestation by a tobacco leaf cutworm Spodoptera litura. Results suggested that the overexpression of CsDFR and CsANR cDNA in tobacco has improved flavonoids content and antioxidant potential. These attributes in transgenic tobacco have ultimately improved their growth and development, and biotic stress tolerance. PMID:23823500

  5. Isolation and mapping of human chromosome 21 cDNA: Progress in constructing a chromosome 21 expression map

    SciTech Connect

    Jan-Fang Cheng; Boyartchuk, V.; Zhu Y.

    1994-09-01

    We have isolated 175 cDNA clones from a fetal brain library by direct cDNA selection using genomic DNA isolated from pools of human chromosome 21 (HC21) cosmids. DNA sequences have revealed that 16 of these cDNA clones contain overlapping sequences. Of the other 159 cDNA sequences, 10 match previously identified HC21 genes, and 9 match previously determined cDNA sequences, including the Wilms tumor related transcript (QM), the human testican cDNA, the mammalian calponin cDNA, and 6 anonymous expressed sequence tags. All isolated cDNAs were hybridized to their corresponding cosmids, which suggests that they originated from HC21. We have localized 92 cDNA clones to previously reported HC21q YACs. The remaining unmapped cDNAs contain either sequences not included in the isolated HC21q YACs or sequences that hybridize to yeast DNA. The cDNAs not included in the YACs should be useful in isolating new YACs to bridge the gaps. PCR primers were derived from 4 novel cDNA sequences that had been mapped to the YACs in the suspected Down syndrome region and used in RT-PCR analysis. All 4 primer sequences amplified RNA fragments with the expected sizes, suggesting that these sequences could be used for expression analysis. The construction of a chromosome 21 cDNA map not only is important in the refinement of physical maps, but also will identify a set of genes in the disease regions for detailed characterization. 30 refs., 2 figs., 2 tabs.

  6. cDNA immunization of mice with human thyroglobulin generates both humoral and T cell responses: a novel model of thyroid autoimmunity.

    PubMed

    Jacobson, Eric M; Concepcion, Erlinda; Ho, Kenneth; Kopp, Peter; Vono Toniolo, Jussara; Tomer, Yaron

    2011-04-29

    Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis.

  7. cDNA Immunization of Mice with Human Thyroglobulin Generates Both Humoral and T Cell Responses: A Novel Model of Thyroid Autoimmunity

    PubMed Central

    Jacobson, Eric M.; Concepcion, Erlinda; Ho, Kenneth; Kopp, Peter; Vono Toniolo, Jussara; Tomer, Yaron

    2011-01-01

    Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis. PMID:21559421

  8. Characterization of a human glycoprotein with a potential role in sperm-egg fusion: cDNA cloning, immunohistochemical localization, and chromosomal assignment of the gene (AEGL1)

    SciTech Connect

    Hayashi, Masaru; Fujimoto, Seiichiro; Takano, Hiroko

    1996-03-05

    Acidic epididymal glycoprotein (AEG), thus far identified only in rodents, is one of the sperm surface proteins involved in the fusion of the sperm and egg plasma membranes. In the present study, we describe the isolation and characterization of cDNA encoding a human glycoprotein related to AEG. Although this protein, designated ARP (AEG-related protein), is not the ortholog of rodent AEG, it resembles AEG in that it is an epididymal secretory glycoprotein that binds to the postacrosomal region of the sperm head. The fact that no AEG mRNA can be detected in the human epididymis suggests that ARP might be the functional counterpart of rodent AEG. The gene encoding ARP (AEGL1) was mapped by fluorescence in situ hybridization to 6p21.1-p21.2. This result indicates that AEGL1 and the mouse gene for AEG are located in the chromosomal segments with conserved syntenies. 43 refs., 6 figs.

  9. Evaluation of the immune response of male and female rats vaccinated with cDNA encoding a cysteine proteinase of Fasciola hepatica (FhPcW1).

    PubMed

    Wesołowska, Agnieszka; Norbury, Luke J; Januszkiewicz, Kamil; Jedlina, Luiza; Jaros, Sławomir; Zawistowska-Deniziak, Anna; Zygner, Wojciech; Wędrychowicz, Halina

    2013-06-01

    Not only do males and females of many species vary in their responses to certain parasitic infections, but also to treatments such as vaccines. However, there are very few studies investigating differences among sexes following vaccination and infection. Here we demonstrate that female Sprague-Dawley rats vaccinated with cDNA encoding a recently discovered cysteine proteinase of Fasciola hepatica (FhPcW1) develop considerably lower liver fluke burdens after F. hepatica infection than their male counterparts. This is accompanied by differences in the course of their immune responses which involve different eosinophil and monocyte responses throughout the study as well as humoral responses. It is evident that host gender influences the outcome of parasitic infections after vaccination and research on both sexes should be considered when developing new treatments against parasites.

  10. Evidence that a human soluble beta-galactoside-binding lectin is encoded by a family of genes.

    PubMed Central

    Gitt, M A; Barondes, S H

    1986-01-01

    Two cDNA clones were isolated by immunoscreening a human hepatoma cDNA library with an antiserum that bound specifically to a human soluble beta-galactoside-binding lectin with Mr of approximately 14,000. The deduced amino acid sequences of the inserts of these two clones show considerable homology with each other, the sequence of chicken skin beta-galactoside-binding lectin, and eight peptides derived from purified human lung lectin of Mr approximately 14,000. However, the sequence differences between the two hepatoma clones as well as among each clone and the lung peptides suggest that at least three variants of the gene encoding this lectin are expressed in human tissue. Images PMID:3020551

  11. Methods to optimize the generation of cDNA from postmortem human brain tissue.

    PubMed

    Miller, Christine L; Yolken, Robert H

    2003-02-01

    The analysis of gene transcript levels in postmortem human brain is a valuable tool for the study of neurological and psychiatric diseases. Optimization of the methods of RNA extraction and cDNA generation is particularly important in this application because postmortem human brain tissue is in limited supply and generally yields less RNA than many other human tissues. We compared column extraction and solvent extraction for total RNA, reverse transcription (RT) with random hexamers versus oligo-dT priming, and incubation of the RNA with or without DNase for effect on the cDNA product derived from the same homogenized pool of postmortem human frontal cortex. The total RNA obtained from the solvent method was found to be less stable at room temperature and to contain a higher proportion of non-messenger RNA than that obtained from the column method. Evaluating the RT-PCR results per wet weight of tissue extracted, we found that the signal strength was increased >20-fold by a protocol of Qiagen RNeasy column extraction, random hexamer RT priming and omitting DNase treatment of the RNA.

  12. Molecular cloning and sequencing of a cDNA encoding the thioesterase domain of the rat fatty acid synthetase.

    PubMed

    Naggert, J; Witkowski, A; Mikkelsen, J; Smith, S

    1988-01-25

    A cloned cDNA containing the entire coding sequence for the long-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase I) component as well as the 3'-noncoding region of the fatty acid synthetase has been isolated using an expression vector and domain-specific antibodies. The coding region was assigned to the thioesterase I domain by identification of sequences coding for characterized peptide fragments, amino-terminal analysis of the isolated thioesterase I domain and the presence of the serine esterase active-site sequence motif. The thioesterase I domain is 306 amino acids long with a calculated molecular mass of 33,476 daltons; its DNA is flanked at the 5'-end by a region coding for the acyl carrier protein domain and at the 3'-end by a 1,537-base pairs-long noncoding sequence with a poly(A) tail. The thioesterase I domain exhibits a low, albeit discernible, homology with the discrete medium-chain S-acyl fatty acid synthetase thioester hydrolases (thioesterase II) from rat mammary gland and duck uropygial gland, suggesting a distant but common evolutionary ancestry for these proteins.

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

    SciTech Connect

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

    1987-06-01

    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 lambdagt11. 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. 2058 Expressed sequence tags (ESTs) from a human fetal lung cDNA library

    SciTech Connect

    Kazunori, Sudo |; Katsuya Chinen; Yusuke Nakamura

    1994-11-15

    ESTs (expressed sequence tags) provide complementary resources for structural and functional analyses of the human genome. The authors have performed single-pass sequencing of 2058 randomly selected, directionally cloned cDNAs isolated from a fetal-lung cDNA library constructed with oligo (dT) primers. Computer analyses of the 5{prime}-end sequences revealed that 60.4% of the clones were considered to be identical to previously reported human genes or ESTs; 9.0% of them showed significant homology to known genes in human, other mammals, or lower organisms; 30.6% showed no homology to any genes or DNA sequences in the public database. These data and reagents will be useful for future investigations of gene expression during prenatal development of human lung. 11 refs., 1 fig., 2 tabs.

  15. cDNA cloning, tissue distribution, and chromosomal localization of Ocp2, a gene encoding a putative transcription-associated factor predominantly expressed in the auditory organs

    SciTech Connect

    Chen, Hong; Thalmann, I.; Thalmann, R.

    1995-06-10

    We report the cloning of the Ocp2 gene encoding OCP-II from a guinea pig organ-of-Corti cDNA library. The predicted open reading frame encodes a protein of 163 amino acids with an estimated molecular mass of 18.6 kDa. A homology search revealed that Ocp2 shares significant sequence similarity with p15, a sub-unit of transcription factor SIII that regulates the activity of the RNA polymerase II elongation complex. The Ocp2 messenger RNA is expressed abundantly in the cochlea while not significantly in any other tissues examined, including brain, eye, heart, intestine, kidney, liver, lung, thigh muscle, and testis, demonstrating that the expression of this gene may be restricted to auditory organs. A polyclonal antiserum was raised against the N-terminal region of OCP-II. Immunohistochemical staining of paraffin-embedded sections of the cochlea showed that OCP-II is localized abundantly in nonsensory cells in the organ of Corti; in addition, it was also detected, at a lower concentration, in vestibular sensory organs, as well as auditory and vestibular brain stem nuclei. The Ocp2 gene was mapped to mouse chromosome 4 as well as 11. Our results suggest that OCP-II may be involved in transcription regulation for the development or maintenance of specialized functions of the inner ear. 40 refs., 5 figs.

  16. Face Encoding and Recognition in the Human Brain

    NASA Astrophysics Data System (ADS)

    Haxby, James V.; Ungerleider, Leslie G.; Horwitz, Barry; Maisog, Jose Ma.; Rapoport, Stanley I.; Grady, Cheryl L.

    1996-01-01

    A dissociation between human neural systems that participate in the encoding and later recognition of new memories for faces was demonstrated by measuring memory task-related changes in regional cerebral blood flow with positron emission tomography. There was almost no overlap between the brain structures associated with these memory functions. A region in the right hippocampus and adjacent cortex was activated during memory encoding but not during recognition. The most striking finding in neocortex was the lateralization of prefrontal participation. Encoding activated left prefrontal cortex, whereas recognition activated right prefrontal cortex. These results indicate that the hippocampus and adjacent cortex participate in memory function primarily at the time of new memory encoding. Moreover, face recognition is not mediated simply by recapitulation of operations performed at the time of encoding but, rather, involves anatomically dissociable operations.

  17. Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFI

    SciTech Connect

    Vieira, P.; De Waal-Malefyt, R.; Dang, M.N.; Johnson, K.E.; Kastelein, R.; Fiorentino, D.F.; DeVries, J.E.; Roncarolo, M.G.; Mosmann, T.R.; Moore, K.W. )

    1991-02-15

    The authors demonstrated the existence of human cytokine synthesis inhibitory factor (DSIF) (interleukin 10 (IL-10)). cDNA clones encoding human IL-10 (hIL-10) were isolated from a tetanus toxin-specific human T-cell clone. Like mouse IL-10, hIL-10 exhibits strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus, BDRFL. hIL-10 and the BCRFI product inhibit cytokine synthesis by activated human peripheral blood mononuclear cells and by a mouse Th1 clone. Both hIL-10 and mouse IL-10 sustain the viability of a mouse mast cell line in culture, but BCRFI lacks comparable activity in this way, suggesting that BCRFI may have conserved only a subset of hIL-10 activities.

  18. Isolation of a human cDNA for heme A:farnesyltransferase by functional complementation of a yeast cox10 mutant.

    PubMed Central

    Glerum, D M; Tzagoloff, A

    1994-01-01

    We have cloned the human homolog of the Saccharomyces cerevisiae COX10 gene by functional complementation of a yeast cox10 null mutant. The 2.8-kb cDNA encoding the human heme A:farnesyltransferase codes for a 443-aa protein with high homology to the yeast and bacterial farnesylases. The human COX10 homolog, however, does not complement the mutation as efficiently as the yeast COX10 protein, likely due to the heterologous environment. PCR amplification and Southern analysis confirm the existence of a large mRNA for the human protein, with an unusually long 3' untranslated region. This clone can now be used to screen patients with inherited deficiencies in cytochrome oxidase in which the mutations remain unidentified and are likely to reside in a protein influencing the assembly of the enzyme. Images PMID:8078902

  19. Exons I and VII of the gene (Ker10) encoding human keratin 10 undergo structural rearrangements within repeats.

    PubMed

    Tkachenko, A V; Buchman, V L; Bliskovsky, V V; Shvets YuP; Kisselev, L L

    1992-07-15

    A genomic fragment containing the K51 gene previously isolated from a rat genomic library by hybridization with the v-mos probe in nonstringent conditions [Chumakov et al., Dokl. Akad. Nauk SSSR 290 (1986) 1252-1254], resembles a human keratin type-I-encoding gene [Shvets et al., Mol. Biol. 24 (1990) 663-677]. This genomic clone, K51, has been used as a probe to search for related human genes. A recombinant clone, HK51, with a 1.5-kb insert, was isolated from a human embryonic skin cDNA library, and its nucleotide (nt) sequence was determined. Analysis has shown that the cloned cDNA encodes human keratin 10 (Ker10). All presently known nt sequences of the human Ker10-encoding gene (Ker10) are not identical. Differences are concentrated in the 5'-end of the first exon and in the middle of the seventh exon within repeats. In spite of structural rearrangements in two of eight exons, the reading frame and position of the stop codon are preserved. The genetic rearrangements cause changes in hydrophobicity profiles of the N and C termini of Ker10. It was also noticed that insertion of one nt leads to the formation of an unusual 3'-end of the transcript.

  20. The KUP gene, located on human chromosome 14, encodes a protein with two distant zinc fingers.

    PubMed

    Chardin, P; Courtois, G; Mattei, M G; Gisselbrecht, S

    1991-04-11

    We have isolated a human cDNA (kup), encoding a new protein with two distantly spaced zinc fingers of the C2H2 type. This gene is highly conserved in mammals and is expressed mainly in hematopoietic cells and testis. Its expression was not higher in the various transformed cells tested than in the normal corresponding tissues. The kup gene is located in region q23-q24 of the long arm of human chromosome 14. The kup protein is 433 a.a. long, has a M.W. close to 50 kD and binds to DNA. Although the structure of the kup protein is unusual, the isolated fingers resemble closely those of the Krüppel family, suggesting that this protein is also a transcription factor. The precise function and DNA motif recognized by the kup protein remain to be determined.

  1. Human synaptonemal complex protein 1 (SCP1): Isolation and characterization of the cDNA and chromosomal localization of the gene

    SciTech Connect

    Meuwissen, R.L.J.; Meerts, I.; Heyting, C.

    1997-02-01

    Synaptonemal complexes (SCs) are structures that are formed between homologous chromosomes (homologs) during meiotic prophase. They consist of two proteinaceous axes, one along each homolog, that are connected along their length by numerous transverse filaments (TFs). The cDNA encoding one major component of TFs of SCs of the rat, rnSCP1, has recently been isolated and characterized. In this paper we describe the isolation and characterization of the cDNA encoding the human protein homologous to rnSCP1, hsSCP1. hsSCP1 and rnSCP1 have 75% amino acid identity. The most prominent structural features and amino acid sequence motifs of rnSCP1 have been conserved in hsSCP1. Most probably, hsSCP1 is functionally homologous to rnSCP1. The hsSCP1 gene was assigned to human chromosome 1p12-p13 by fluorescence in situ hybridization. 44 refs., 4 figs.

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

    PubMed

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

    1998-03-13

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

  3. Localization of the human fibromodulin gene (FMOD) to chromosome 1q32 and completion of the cDNA sequence

    SciTech Connect

    Sztrolovics, R.; Grover, J.; Roughley, P.J.

    1994-10-01

    This report describes the cloning of the 3{prime}-untranslated region of the human fibromodulin cDNA and its use to map the gene. For somatic cell hybrids, the generation of the PCR product was concordant with the presence of chromosome 1 and discordant with the presence of all other chromosomes, confirming that the fibromodulin gene is located within region q32 of chromosome 1. The physical mapping of genes is a critical step in the process of identifying which genes may be responsible for various inherited disorders. Specifically, the mapping of the fibromodulin gene now provides the information necessary to evaluate its potential role in genetic disorders of connective tissues. The analysis of previously reported diseases mapped to chromosome 1 reveals two genes located in the proximity of the fibromodulin locus. These are Usher syndrome type II, a recessive disorder characterized by hearing loss and retinitis pigmentosa, and Van der Woude syndrome, a dominant condition associated with abnormalities such as cleft lip and palate and hyperdontia. The genes for both of these disorders have been projected to be localized to 1q32 of a physical map that integrates available genetic linkage and physical data. However, it seems improbable that either of these disorders, exhibiting restricted tissue involvement, could be linked to the fibromodulin gene, given the wide tissue distribution of the encoded proteoglycan, although it remains possible that the relative importance of the quantity and function of the proteoglycan may avry between tissues. 11 refs., 1 fig.

  4. Human Genomic Signatures of Brain Oscillations During Memory Encoding.

    PubMed

    Berto, Stefano; Wang, Guang-Zhong; Germi, James; Lega, Bradley C; Konopka, Genevieve

    2017-04-05

    Memory encoding is an essential step for all learning. However, the genetic and molecular mechanisms underlying human memory encoding remain poorly understood, and how this molecular framework permits the emergence of specific patterns of brain oscillations observed during mnemonic processing is unknown. Here, we directly compare intracranial electroencephalography recordings from the neocortex in individuals performing an episodic memory task with human gene expression from the same areas. We identify genes correlated with oscillatory memory effects across 6 frequency bands. These genes are enriched for autism-related genes and have preferential expression in neurons, in particular genes encoding synaptic proteins and ion channels, supporting the idea that the genes regulating voltage gradients are involved in the modulation of oscillatory patterns during successful memory encoding across brain areas. Memory-related genes are distinct from those correlated with other forms of cognitive processing and resting state fMRI. These data are the first to identify correlations between gene expression and active human brain states as well as provide a molecular window into memory encoding oscillations in the human brain.

  5. cDNA cloning and chromosomal mapping of a novel human GAP (GAP1M), GTPase-activating protein of Ras

    SciTech Connect

    Li, Shaowei; Nakamura, Shun; Hattori, Seisuke

    1996-08-01

    We have previously isolated a novel Ras GTPase-activating protein (Ras GAP), Gapl{sup m}, from rat brain. Gap1{sup m} is considered to be a negative regulator of the Ras signaling pathways, like other Ras GAPs, neurofibromin, which is a gene product of the neurofibromatosis type I gene, and p120GAP. In this study we have isolated a human cDNA of this Gap and mapped the gene. The gene encodes a protein of 853 amino acids that shows 89% sequence identity to rat Gapl{sup m}. The human gene was mapped to chromosome 3 by PCR analysis on a panel of human-mouse hybrid cells. FISH analysis refined the location of the gene further to 3q22-q23. 11 refs., 2 figs.

  6. cDNA sequence and gene locus of the human retinal phosphoinositide-specific phospholipase-C{beta}4 (PLCB4)

    SciTech Connect

    Alvarez, R.A.; Ghalayini, A.J.; Anderson, R.E.

    1995-09-01

    Defects in the Drosophila norpA (no receptor potential A) gene encoding a phosphoinositide-specific phospholipase C (PLC) block invertebrate phototransduction and lead to retinal degeneration. The mammalian homolog, PLCB4, is expressed in rat brain, bovine cerebellum, and the bovine retina in several splice variants. To determine a possible role of PLCB4 gene defects in human disease, we isolated several overlapping cDNA clones from a human retina library. The composite cDNA sequence predicts a human PLC{beta}4 polypeptide of 1022 amino acid residues (MW 117,000). This PLC{beta}4 variant lacks a 165-amino-acid N-terminal domain characteristic for the rat brain isoforms, but has a distinct putative exon 1 unique for human and bovine retina isoforms. A PLC{beta}4 monospecific antibody detected a major (130 kDa) and a minor (160 kDa) isoform in retina homogenates. Somatic cell hybrids and deletion panels were used to localize the PCLB4 gene to the short arm of chromosome 20. The gene was further sublocalized to 20p12 by florescence in situ hybridization. 4 refs., 5 figs.

  7. cDNA sequences of variant forms of human placenta diamine oxidase

    SciTech Connect

    Zhang, X.; Kim, J.; McIntire, S.

    1995-08-01

    Genes for two forms of human placenta diamine oxidase (dao) were cloned from a cDNA library and sequenced. One gene, pdao1, is identical in length to human kidney dao but differs from it by two bases in the coding region and differs slightly in the 3{prime} - and 5{prime}-noncoding regions. The second gene, pdao2, is nearly identical to these genes in the coding region, except that it has an extra 57-nucleotide coding segment near the 3{prime} end of this region. This segment corresponds to the contiguous sequence of the 3{prime} end of intron 3 of human kidney dao. pdao2 also differs significantly from pdao1 and human kidney dao in a 13-base sequence in the t{prime}-noncoding region. It is proposed that pdao1 and human kidney dao are polymorphic forms of the same allele. Whether pdao2 is a polymorph of these two is not certain, because of the significant differences in the coding and noncoding regions. pdao2 may represent a different allele. 21 refs., 2 figs.

  8. Molecular cloning, sequence, and expression of a human GDP-L-fucose:. beta. -D-galactoside 2-. alpha. -L-fucosyltransferase cDNA that can form the H blood group antigen

    SciTech Connect

    Larsen, R.D.; Ernst, L.K.; Nair, R.P.; Lowe, J.B. )

    1990-09-01

    The authors have previously used a gene-transfer scheme to isolate a human genomic DNA fragment that determines expression of a GDP-L-fucose:{beta}D-galactoside 2-{alpha}-L-fucosyltransferase. Although this fragment determined expression of an {alpha}(1,2)FT whose kinetic properties mirror those of the human H blood group {alpha}(1,2)FT, their precise nature remained undefined. They describe here the molecular cloning, sequence, and expression of a human of cDNA corresponding to these human genomic sequences. When expressed in COS-1 cells, the cDNA directs expression of cell surface H structures and a cognate {alpha}(1,2)FT activity with properties analogous to the human H blood group {alpha}(1,2)FT. The cDNA sequence predicts a 365-amino acid polypeptide characteristic of a type II transmembrane glycoprotein with a domain structure analogous to that of other glycosyltransferases but without significant primary sequence similarity to these or other known proteins. To directly demonstrate that the cDNA encodes an {alpha}(1,2)FT, the COOH-terminal domain predicted to be Golgi-resident was expressed in COS-1 cells as a catalytically active, secreted, and soluble protein A fusion peptide. Southern blot analysis showed that this cDNA identified DNA sequences syntenic to the human H locus on chromosome 19. These results strongly suggest that this cloned {alpha}(1,2)FT cDNA represents the product of the human H blood group locus.

  9. Twenty Drosophila visual system cDNA clones: one is a homolog of human arrestin.

    PubMed Central

    Hyde, D R; Mecklenburg, K L; Pollock, J A; Vihtelic, T S; Benzer, S

    1990-01-01

    From a group of 436 Drosophila melanogaster cDNA clones, we selected 39 that are expressed exclusively or predominantly in the adult visual system. By sequence analysis, 20 of the clones appear to represent previously unreported distinct cDNAs. The corresponding transcripts are detected in the retina and optic lobes. The genes are scattered throughout the genome, some near mutations known to affect eye function. One of these clones has been identified, by sequence analysis, as the structural gene (Arr) for a Drosophila homolog of human arrestin. Vertebrate arrestin interacts with rhodopsin in phototransduction and has been associated with an autoimmune form of uveitis in primates. The presence of an arrestin homolog in Drosophila suggests that both the vertebrate and invertebrate phototransduction cascades are regulated in a similar manner. Images PMID:2105491

  10. Sequence of a cDNA clone encoding the polysialic acid-rich and cytoplasmic domains of the neural cell adhesion molecule N-CAM.

    PubMed Central

    Hemperly, J J; Murray, B A; Edelman, G M; Cunningham, B A

    1986-01-01

    Purified fractions of the neural cell-adhesion molecule N-CAM from embryonic chicken brain contain two similar polypeptides (Mr, 160,000 and 130,000), each containing an amino-terminal external binding region, a carbohydrate-rich central region, and a carboxyl-terminal region that is associated with the cell. Previous studies indicate that the two polypeptides arise by alternative splicing of mRNAs transcribed from a single gene. We report here the 3556-nucleotide sequence of a cDNA clone (pEC208) that encodes 964 amino acids from the carbohydrate and cell-associated domains of the larger N-CAM polypeptide followed by 664 nucleotides of 3' untranslated sequence. The predicted protein sequence contains attachment sites for polysialic acid-containing oligosaccharides, four tandem homologous regions of polypeptide resembling those seen in the immunoglobulin superfamily, and a single hydrophobic sequence that appears to be the membrane-spanning segment. The cytoplasmic domain carboxyl terminal to this segment includes a block of approximately equal to 250 amino acids present in the larger but not in the smaller N-CAM polypeptide. We designate these the ld (large domain) polypeptide and the sd (small domain) polypeptide. The intracellular domains of the ld and sd polypeptides are likely to be critical for cell-surface modulation of N-CAM by interacting in a differential fashion with other intrinsic proteins or with the cytoskeleton. PMID:3458261

  11. Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of Mycobacterium bovis BCG induce interferons and activate natural killer cells.

    PubMed

    Tokunaga, T; Yano, O; Kuramoto, E; Kimura, Y; Yamamoto, T; Kataoka, T; Yamamoto, S

    1992-01-01

    Thirteen kinds of 45-mer single-stranded oligonucleotide, having sequence randomly selected from the known cDNA encoding BCG proteins, were tested for their capability to augment natural killer (NK) cell activity of mouse spleen cells in vitro. Six out of the 13 oligonucleotides showed the activity, while the others did not. In order to know the minimal and essential sequence(s) responsible for the biological activity, 2 kinds of 30-mer and 5 kinds of 15-mer oligonucleotide fragments of an active 45-mer nucleotide were tested for their activity. One of the 30-mer oligonucleotides, designated BCG-A4a, was active, but the other 30-mer was inactive. All of the 15-mer oligonucleotide fragments were inactive. The BCG-A4a also stimulated the spleen cells to produce interferon (IFN)-alpha and -gamma. An experiment using anti-IFN antisera showed that the NK cell activation by the oligonucleotide was ascribed to the IFN-alpha produced. It was noticed that all of the biologically active oligonucleotides possessed one or more palindrome sequence(s), and the inactive ones did not, with an exception of a 45-mer inactive oligonucleotide containing overlapping palindrome sequences (GGGCCCGGG). These findings strongly suggest that certain palindrome sequences, like GACGTC, GGCGCC and TGCGCA, are essential for 30-mer oligonucleotides, like BCG-A4a, to induce IFNs.

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

    PubMed

    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.

  13. The human homolog of the JE gene encodes a monocyte secretory protein.

    PubMed Central

    Rollins, B J; Stier, P; Ernst, T; Wong, G G

    1989-01-01

    The mouse fibroblast gene, JE, was one of the first platelet-derived growth factor-inducible genes to be described as such. The protein encoded by JE (mJE) is the prototype of a large family of secreted, cytokinelike glycoproteins, all of whose members are induced by a mitogenic or activation signal in monocytes macrophages, and T lymphocytes; JE is the only member to have been identified in fibroblasts. We report the identification of a human homolog for murine JE, cloned from human fibroblasts. The protein predicted by the coding sequence of human JE (hJE) is 55 amino acids shorter than mJE, and its sequence is identical to that of a recently purified monocyte chemoattractant. When expressed in COS cells, the human JE cDNA directed the secretion of N-glycosylated proteins of Mr 16,000 to 18,000 as well as proteins of Mr 15,500, 15,000, and 13,000. Antibodies raised against mJE recognized these hJE species, all of which were secreted by human fibroblasts. hJE expression was stimulated in HL60 cells during phorbol myristate acetate-induced monocytoid differentiation. However, resting human monocytes constitutively secreted hJE; treatment with gamma interferon did not enhance hJE expression in monocytes, and treatment with phorbol myristate acetate or lipopolysaccharide inhibited its expression. Thus, human JE encodes yet another member of the large family of JE-related cytokinelike proteins, in this case a novel human monocyte and fibroblast secretory protein. Images PMID:2513477

  14. Cloning of cDNAs that encode human mast cell carboxypeptidase A, and comparison of the protein with mouse mast cell carboxypeptidase A and rat pancreatic carboxypeptidases

    SciTech Connect

    Reynolds, D.S.; Gurley, D.S.; Stevens, R.L.; Austen, K.F.; Serafin, W.E. Brigham and Women's Hospital, Boston, MA ); Sugarbaker, D.J. )

    1989-12-01

    Human skin and lung mast cells and rodent peritoneal cells contain a carboxypeptidase in their secretory granules. The authors have screened human lung cDNA libraries with a mouse mast cell carboxypeptidase A (MC-CPA) cDNA probe to isolate a near-full-length cDNA that encodes human MC-CPA. The 5{prime} end of the human MC-CPA transcript was defined by direct mRNA sequencing and by isolation and partial sequencing of the human MC-CPA gene. Human MC-CPA is predicted to be translated as a 417 amino acid preproenzyme which includes a 15 amino acid signal peptide and a 94-amino acid activation peptide. The mature human MC-CPA enzyme has a predicted size of 36.1 kDa, a net positive charge of 16 at neutral pH, and 86% amino acid sequence identity with mouse MC-CPA. DNA blot analyses showed that human MC-CPA mRNA is transcribed from a single locus in the human genome. Comparison of the human MC-CPA with mouse MC-CPA and with three rat pancreatic carboxypeptidases shows that these enzymes are encoded by distinct but homologous genes.

  15. Robust encoding of scene anticipation during human spatial navigation

    PubMed Central

    Shikauchi, Yumi; Ishii, Shin

    2016-01-01

    In a familiar city, people can recall scene views (e.g., a particular street corner scene) they could encounter again in the future. Complex objects with multiple features are represented by multiple neural units (channels) in the brain, but when anticipating a scene view, the kind of feature that is assigned to a specific channel is unknown. Here, we studied neural encoding of scene view anticipation during spatial navigation, using a novel data-driven analysis to evaluate encoding channels. Our encoding models, based on functional magnetic resonance imaging (fMRI) activity, provided channel error correction via redundant channel assignments that reflected the navigation environment. We also found that our encoding models strongly reflected brain activity in the inferior parietal gyrus and precuneus, and that details of future scenes were locally represented in the superior prefrontal gyrus and temporal pole. Furthermore, a decoder associated with the encoding models accurately predicted future scene views in both passive and active navigation. These results suggest that the human brain uses scene anticipation, mediated especially by parietal and medial prefrontal cortical areas, as a robust and effective navigation processing. PMID:27874089

  16. The human U1-70K snRNP protein: cDNA cloning, chromosomal localization, expression, alternative splicing and RNA-binding.

    PubMed Central

    Spritz, R A; Strunk, K; Surowy, C S; Hoch, S O; Barton, D E; Francke, U

    1987-01-01

    We have isolated and sequenced cDNA clones encoding the human U1-70K snRNP protein, and have mapped this locus (U1AP1) to human chromosome 19. The gene produces two size classes of RNA, a major 1.7-kb RNA and a minor 3.9-kb RNA. The 1.7-kb species appears to be the functional mRNA; the role of the 3.9-kb RNA, which extends further in the 5' direction, is unclear. The actual size of the hU1-70K protein is probably 52 kd, rather than 70 kd. The protein contains three regions similar to known nucleic acid-binding proteins, and it binds RNA in an in vitro assay. Comparison of the cDNA sequences indicates that there are multiple subclasses of mRNA that arise by alternative pre-mRNA splicing of at least four alternative exon segments. This suggests that multiple forms of the hU1-70K protein may exist, possibly with different functions in vivo. Images PMID:2447561

  17. Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway.

    PubMed Central

    Teutsch, H G; Hasenfratz, M P; Lesot, A; Stoltz, C; Garnier, J M; Jeltsch, J M; Durst, F; Werck-Reichhart, D

    1993-01-01

    Cinnamate 4-hydroxylase [CA4H; trans-cinnamate,NADPH:oxygen oxidoreductase (4-hydroxylating), EC 1.14.13.11] is a cytochrome P450 that catalyzes the first oxygenation step of the general phenylpropanoid metabolism in higher plants. The compounds formed are essential for lignification and defense against predators and pathogens. We recently reported the purification of this enzyme from Mn(2+)-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. Highly selective polyclonal antibodies raised against the purified protein were used to screen a lambda gt11 cDNA expression library from wound-induced Jerusalem artichoke, allowing isolation of a 1130-base-pair insert. Typical P450 domains were identified in this incomplete sequence, which was used as a probe for the isolation of a 1.7-kilobase clone in a lambda gt10 library. A full-length open reading frame of 1515 base pairs, encoding a P450 protein of 505 residues (M(r) = 57,927), was sequenced. The N terminus, essentially composed of hydrophobic residues, matches perfectly the microsequenced N terminus of the purified protein. The calculated pI is 9.78, in agreement with the chromatographic behavior and two-dimensional electrophoretic analysis of CA4H. Synthesis of the corresponding mRNA is induced in wounded plant tissues, in correlation with CA4H enzymatic activity. This P450 protein exhibits the most similarity (28% amino acid identity) with avocado CYP71, but also good similarity with CYP17 and CYP21, or with CYP1 and CYP2 families. According to current criteria, it qualifies as a member of a new P450 family. Images Fig. 4 PMID:8097885

  18. Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans.

    PubMed Central

    Smeekens, S P; Avruch, A S; LaMendola, J; Chan, S J; Steiner, D F

    1991-01-01

    PC2 and furin are two recently identified members of a class of mammalian proteins homologous to the yeast precursor processing protease kex2 and the bacterial subtillisins. We have used the polymerase chain reaction to identify and clone a cDNA (PC3) from the mouse AtT20 anterior pituitary cell line that represents an additional member of this growing family of mammalian proteases. PC3 encodes a 753-residue protein that begins with a signal peptide and contains a 292-residue domain closely related to the catalytic modules of PC2, furin, and kex2. Within this region 58%, 65%, and 50% of the amino acids of PC3 are identical to those of the aligned PC2, furin, and kex2 sequences, respectively, and the catalytically important Asp, His, and Ser residues are all conserved. On Northern blots, PC3 hybridizes to two transcripts of 3 and 5 kilobases. Tissue distribution studies indicate that both PC2 and PC3 are expressed in a variety of neuroendocrine tissues, including pancreatic islets and brain, but are not expressed in liver, kidney, skeletal muscle, and spleen. The high degree of similarity of PC3, PC2, and furin suggests that they are all members of a superfamily of mammalian proteases that are involved in the processing of prohormones and/or other protein precursors. In contrast to furin, PC3, like PC2, lacks a hydrophobic transmembrane anchor, but it has a potential C-terminal amphipathic helical segment similar to the putative membrane anchor of carboxypeptidase H. These and other differences suggest that these proteins carry out compartmentalized proteolysis within cells, such as processing within regulated versus constitutive secretory pathways. Images PMID:1988934

  19. Characterization of a novel cDNA encoding a short venom peptide derived from venom gland of scorpion Buthus martensii Karsch: trans-splicing may play an important role in the diversification of scorpion venom peptides.

    PubMed

    Zeng, Xian-Chun; Luo, Feng; Li, Wen-Xin

    2006-04-01

    A novel cDNA clone (named BmKT-u) which is a hybrid molecule of the 5'-terminal region of BmKT' cDNA and the 3'-terminal region of an undocumented cDNA (named BmKu), was isolated from a cDNA library made from the venom gland of scorpion Buthus martensii Karsch. BmKT-u codes for a 30 amino acid residue precursor peptide composed of a 20-residue signal sequence, and a putative 10-residue novel mature peptide. Northern blot hybridization showed BmKT-u cDNA is generated from a transcript. RT-PCR experiments excluded the possibility that BmKT-u cDNA is an artifact generated during reverse transcription. Genomic amplifications performed with three pairs of BmKT-u gene-specific primers showed the BmKT-u gene does not exist in the genome of the scorpion as a single transcriptional unit. Genomic cloning for BmKT' showed that the BmKT' gene contains an intron of 509 bp inserted into the region encoding the C-terminal region of the signal peptide. A sequence alignment comparison of the cDNA of BmKT-u with genomic BmKT' revealed that the junction site of the hybrid molecule is located at the 5'-splicing site of the intron. The data suggest that the BmKT-u transcript is a naturally occurring mature mRNA that is generated by trans-splicing. Trans-splicing may contribute to the diversity of venom peptides from venomous animals.

  20. ERCC2: cDNA cloning and molecular characterization of a human nucleotide excision repair gene with high homology to yeast RAD3.

    PubMed Central

    Weber, C A; Salazar, E P; Stewart, S A; Thompson, L H

    1990-01-01

    Human ERCC2 genomic clones give efficient, stable correction of the nucleotide excision repair defect in UV5 Chinese hamster ovary cells. One clone having a breakpoint just 5' of classical promoter elements corrects only transiently, implicating further flanking sequences in stable gene expression. The nucleotide sequences of a cDNA clone and genomic flanking regions were determined. The ERCC2 translated amino acid sequence has 52% identity (73% homology) with the yeast nucleotide excision repair protein RAD3. RAD3 is essential for cell viability and encodes a protein that is a single-stranded DNA dependent ATPase and an ATP dependent helicase. The similarity of ERCC2 and RAD3 suggests a role for ERCC2 in both cell viability and DNA repair and provides the first insight into the biochemical function of a mammalian nucleotide excision repair gene. Images Fig. 5. PMID:2184031

  1. Sequence, tissue distribution, and chromosomal localization of mRNA encoding a human glucose transporter-like protein

    SciTech Connect

    Fukumoto, Hirofumi; Seino, Susumu; Imura, Hiroo; Seino, Yutaka; Eddy, R.L.; Fukushima, Yoshimitsu; Byers, M.G.; Shows, T.B.; Bell, G.I. )

    1988-08-01

    Recombinant DNA clones encoding a glucose transporter-like protein have been isolated from adult human liver and kidney cDNA libraries by cross-hybridization with the human HepG2/erythrocyte glucose transporter cDNA. Analysis of the sequence of this 524-amino acid glucose transporter-like protein indicates that is has 55.5% identity with the HepG2/erythrocyte glucose transporter as well as a similar structural organization. Studies of the tissue distribution of the mRNA coding for this glucose transporter-like protein in adult human tissues indicate that the highest amounts are present in liver with lower amounts in kidney and small intestine. The amounts of glucose transporter-like mRNA in other tissues, including colon, stomach, cerebrum, skeletal muscle, and adipose tissue, were below the level of sensitivity of our assay. The single-copy gene encoding this glucose transporter-like protein has been localized to the q26.1{yields}q26.3 region of chromosome 3.

  2. Human ficolin: cDNA cloning, demonstration of peripheral blood leucocytes as the major site of synthesis and assignment of the gene to chromosome 9.

    PubMed Central

    Lu, J; Tay, P N; Kon, O L; Reid, K B

    1996-01-01

    Pig ficolins and a number of other proteins contain sequences that are homologous to the C-terminal halves of fibrinogen beta- and gamma-chains. To clone the cDNA for human ficolin, two degenerate oligonucleotide primers were synthesized, based on two stretches of protein sequence that were highly conserved among those proteins, and used for PCR with cDNA from a human uterus lambda gt11 library as a template. A PCR product with a predicted size of 300 bp was obtained and this was used to screen a uterus cDNA library. Of the positive clones isolated, two (U1 and U2), containing inserts of 1.7 and 1.1 kb respectively, were found to encode human ficolin. The cDNA-derived amino acid sequence of human ficolin has approx. 75% identity with, and a similar domain organization to, the two pig ficolin sequences, which are characterized by the presence of a leader peptide, a short N-terminal segment followed by a collagen-like region and then by a C-terminal fibrinogen-like domain. The 1.1 kb insert of clone U2 was used in Northern-blot analysis, and a very strong signal for a 1.4 kb mRNA species was detected in mRNA from human peripheral blood leucocytes. This showed that, despite the initial characterization of pig ficolin as a putative receptor on uterine cells for transforming growth factor beta 1, blood leucocytes are probably the major site of human ficolin synthesis. Much weaker signals of the same size were also detected in spleen, lung and thymus and may be due to the presence of tissue macrophages or trapped blood in these tissues. An mRNA species of approx. 1.3 kb in human liver also weakly hybridized to the U2 probe, indicating the presence of a sequence that was distinct from, but related to, ficolin. The gene for human ficolin has been mapped to chromosome 9. PMID:8573080

  3. Human renal carcinoma expresses two messages encoding a parathyroid hormone-like peptide: evidence for the alternative splicing of a single-copy gene.

    PubMed Central

    Thiede, M A; Strewler, G J; Nissenson, R A; Rosenblatt, M; Rodan, G A

    1988-01-01

    A peptide secreted by tumors associated with the clinical syndrome of humoral hypercalcemia of malignancy was recently purified from human renal carcinoma cell line 786-0. The N-terminal amino acid sequence of this peptide has considerable similarity with those of parathyroid hormone (PTH) and of peptides isolated from human breast and lung carcinoma (cell line BEN). In this study we obtained the nucleotide sequence of a 1595-base cDNA complementary to mRNA encoding the PTH-like peptide produced by 786-0 cells. The cDNA contains an open reading frame encoding a leader sequence of 36 amino acids and a 139-residue peptide, in which 8 of the first 13 residues are identical to the N terminus of PTH. Through the first 828 bases the sequence of this cDNA is identical with one recently isolated from a BEN cell cDNA library; however, beginning with base 829 the sequences diverge, shortening the open reading frame by 2 amino acids. Differential RNA blot analysis revealed that 786-0 cells express two major PTH-like peptide mRNAs with different 3' untranslated sequences, one of which hybridizes with the presently described sequence and the other one with that reported for the BEN cell PTH-like peptide cDNA. Primer-extension analysis of 786-0 poly(A)+ RNA together with Southern blot analysis of human DNA confirmed the presence of a single-copy gene coding for multiple mRNAs through alternate splicing. In addition, the 3' untranslated sequence of the cDNA described here has significant similarity to the c-myc protooncogene. Images PMID:3290897

  4. [ENCODE apophenia or a panglossian analysis of the human genome].

    PubMed

    Casane, Didier; Fumey, Julien; Laurenti, Patrick

    2015-01-01

    In September 2012, a batch of more than 30 articles presenting the results of the ENCODE (Encyclopaedia of DNA Elements) project was released. Many of these articles appeared in Nature and Science, the two most prestigious interdisciplinary scientific journals. Since that time, hundreds of other articles dedicated to the further analyses of the Encode data have been published. The time of hundreds of scientists and hundreds of millions of dollars were not invested in vain since this project had led to an apparent paradigm shift: contrary to the classical view, 80% of the human genome is not junk DNA, but is functional. This hypothesis has been criticized by evolutionary biologists, sometimes eagerly, and detailed refutations have been published in specialized journals with impact factors far below those that published the main contribution of the Encode project to our understanding of genome architecture. In 2014, the Encode consortium released a new batch of articles that neither suggested that 80% of the genome is functional nor commented on the disappearance of their 2012 scientific breakthrough. Unfortunately, by that time many biologists had accepted the idea that 80% of the genome is functional, or at least, that this idea is a valid alternative to the long held evolutionary genetic view that it is not. In order to understand the dynamics of the genome, it is necessary to re-examine the basics of evolutionary genetics because, not only are they well established, they also will allow us to avoid the pitfall of a panglossian interpretation of Encode. Actually, the architecture of the genome and its dynamics are the product of trade-offs between various evolutionary forces, and many structural features are not related to functional properties. In other words, evolution does not produce the best of all worlds, not even the best of all possible worlds, but only one possible world.

  5. Adenovirus-mediated transfer of a recombinant human alpha 1-antitrypsin cDNA to human endothelial cells.

    PubMed Central

    Lemarchand, P; Jaffe, H A; Danel, C; Cid, M C; Kleinman, H K; Stratford-Perricaudet, L D; Perricaudet, M; Pavirani, A; Lecocq, J P; Crystal, R G

    1992-01-01

    To evaluate the feasibility of using a replication-deficient recombinant adenovirus to transfer human genes to the human endothelium, human umbilical vein endothelial cells were infected in vitro with adenovirus vectors containing the lacZ gene or a human alpha 1-antitrypsin (alpha 1AT) cDNA. After in vitro infection with the lacZ adenovirus vector, cultured endothelial cells expressed beta-galactosidase. In parallel studies with the alpha 1AT adenovirus vector, infected cells expressed human alpha 1AT transcripts, as evidenced by in situ hybridization and Northern analysis, and de novo synthesized and secreted glycosylated, functional alpha 1AT within 6 hr of infection, as shown by [35S]methionine labeling and immunoprecipitation. Quantification of the culture supernatants demonstrated 0.3-0.6 micrograms of human alpha 1AT secreted per 10(6) cells in 24 hr, for at least 14 days after adenovirus vector infection. To demonstrate the feasibility of direct transfer of genes into endothelial cells in human blood vessels, lacZ or alpha 1AT adenovirus vectors were placed in the lumen of intact human umbilical veins ex vivo. Histologic evaluation of the veins after 24 hr demonstrated transfer and expression of the lacZ gene specifically to the endothelium. alpha 1AT adenovirus infection resulted both in expression of alpha 1AT transcripts in the endothelium and in de novo synthesis and secretion of alpha 1AT. Quantification of alpha 1AT in the vein perfusates showed average levels of 13 micrograms/ml after 24 hr. These observations strongly support the feasibility of in vivo human gene transfer to the endothelium mediated by replication-deficient adenovirus vectors. Images PMID:1631146

  6. Differential Encoding of Losses and Gains in the Human Striatum

    PubMed Central

    Seymour, Ben; Daw, Nathaniel; Dayan, Peter; Singer, Tania; Dolan, Ray

    2009-01-01

    Studies on human monetary prediction and decision making emphasize the role of the striatum in encoding prediction errors for financial reward. However, less is known about how the brain encodes financial loss. Using Pavlovian conditioning of visual cues to outcomes that simultaneously incorporate the chance of financial reward and loss, we show that striatal activation reflects positively signed prediction errors for both. Furthermore, we show functional segregation within the striatum, with more anterior regions showing relative selectivity for rewards and more posterior regions for losses. These findings mirror the anteroposterior valence-specific gradient reported in rodents and endorse the role of the striatum in aversive motivational learning about financial losses, illustrating functional and anatomical consistencies with primary aversive outcomes such as pain. PMID:17475790

  7. Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin

    PubMed Central

    1991-01-01

    Antibodies producing an unusual immunofluorescent pattern were identified in the sera of patients with diverse autoimmune features. This pattern was characterized by the presence of up to six round discrete nuclear bodies in interphase cell nuclei. Immunoblotting analysis showed that these sera recognized an 80-kD nuclear protein, and affinity-purified anti-p80 antibody from the protein band reproduced the fluorescent staining of nuclear bodies. Colloidal gold immunoelectron microscopy showed that the affinity-purified anti-p80 antibody recognized the coiled body, an ultramicroscopic nuclear structure probably first described by the Spanish cytologist Ramon y Cajal. Five cDNA clones were isolated from a MOLT-4 cell lambda gt-11 expression library using human antibody and oligonucleotide probes. The longest cDNA insert was 2.1 kb and had an open reading frame of 405 amino acids. A clone encoding a 14-kD COOH-terminal region of the protein was used for expression of a beta-galactosidase fusion protein. An epitope was present in this COOH-terminal 14-kD region, which was recognized by 18 of 20 sera with anti-p80 reactivity, and affinity- purified antibody from the recombinant protein also reacted in immunofluorescence to show specific staining of the coiled body. This is the first demonstration and molecular cloning of a protein that appears to have particular identification with the coiled body, and it was designated p80-coilin. Autoantibody to p80-coilin may be useful for the elucidation of the structure and function of the coiled body, and the availability of a cDNA sequence could be helpful in further studies to clarify the clinical significance of this autoantibody response. PMID:2033369

  8. ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

    PubMed Central

    Brookman, K W; Lamerdin, J E; Thelen, M P; Hwang, M; Reardon, J T; Sancar, A; Zhou, Z Q; Walter, C A; Parris, C N; Thompson, L H

    1996-01-01

    ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues. PMID:8887684

  9. Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.

    PubMed

    Ding, Ling; Azam, Mark; Lin, Yu-Huei; Sheridan, James; Wei, Shuanghong; Gupta, Gigi; Singh, Rakesh K; Pauling, Michelle H; Chu, Waihei; Tran, Antares; Yu, Nai-Xuan; Hu, Jiefeng; Wang, Wei; Long, Hao; Xiang, Dong; Zhu, Li; Hua, Shao-Bing

    2010-10-01

    We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single-chain variable fragment (scFv) antibody library was constructed in a yeast two-hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin-8 (hIL8) into the yeast two-hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error-prone PCR of the scFv sequence followed by additional rounds of yeast two-hybrid screening. The scFv antibodies of both primary and affinity-matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.

  10. cDNA sequence encoding metallothionein protein from Aegiceras corniculatum and its gene expression induced by Pb²⁺ and Cd²⁺ stresses.

    PubMed

    Yuhong, Li; Atagana, Harrison I; Jingchun, Liu; Wenlin, Wu; Shijun, Wu

    2013-12-01

    Constructing various green wetland examples for mangrove wetland systems is a useful way to use natural power to remediate the polluted wetlands at intertidal zones. Metallothioneins (MT) are involved in heavy metal tolerance, homeostasis, and detoxification of intracellular metal ions in plants. In order to understand the mechanism of heavy metal uptake in Aegiceras corniculatum, we isolated its metallothionein gene and studied the MT gene expression in response to heavy metals contamination. Here, we report the isolation and characterization of MT2 genes from young stem tissues of A. corniculatum growing in the cadmium (Cd) and lead (Pb) polluted wetlands of Quanzhou Bay, southeast of China. The obtained cDNA sequence of MT is 512 bp in length, and it has an open reading frame encoding 79 amino acid residues with a molecular weight of 7.92 kDa and the theoretical isoelectric point of 4.55. The amino acids include 14 cysteine residues and 14 glycine residues. It is a non-transmembrane hydrophilic protein. Sequence and homology analysis showed the MT protein sequence shared more than 60% homology with other plant type 2 MT-like protein genes. The results suggested that the expression level of MT gene of A. corniculatum young stems induced by a certain range concentration of Cd(2+) and Pb(2+) stresses (0.2 mmol L(-1) Pb(2+), 1 mmol L(-1) Pb(2+), 0.2 mmol L(-1) Pb(2+), and 40 μmmol L(-1) Cd(2+); 1 mmol L(-1) Pb(2+) and 40 μmol L(-1) Cd(2+)) compared with control might show an adaptive protection. The expression levels of MT gene at 20 h stress treatment were higher than those at 480 h stress treatment. The expression levels of MT gene with 0.2 mmol L(-1) Pb(2+) stress treatment were higher than those with 0.2 mmol L(-1) Pb(2+) and 40 μmol L(-1) Cd(2+) stress treatment, and the MT gene expression levels with 1 mmol L(-1) Pb(2+) treatment were higher than those with 1 mmol L(-1) Pb(2+) and 40 μmol L(-1) Cd(2+) treatment. There exists an antagonistic action between

  11. Isolation of human hexosaminidase. cap alpha. cDNA and expression of. cap alpha. chains in E. coli

    SciTech Connect

    Wiktorowicz, J.E.; Whitman, J.M.

    1986-05-01

    Pooled antisera against homogeneous, glutaraldehyde cross-linked hexosaminidase (hex) A was adsorbed with E. coli lysate insolubilized on Sepharose 4B. Aliquots of a human liver lambdagtll cDNA library (50,000-100,000 pfu) were plated on E. coli Y1090. Expression of cloned cDNA, after sufficient plaque growth at 42/sup 0/, was accomplished by induction with isopropylthiogalactoside soaked nitrocellulose filters. Identification of hex cDNA clones was performed by incubation of the filters with purified antisera. Protein A labelled with I-125 was used to develop the reactive plaques. Positive plaques, identified by autoradiography, were picked, replated at a lower density, and rescreened. This was repeated several more times until all plaques yielded positive signals. Identification of the clones as containing ..cap alpha.. or ..beta.. cDNA was accomplished by replating the purified phage and rescreening the plaques with anti-hex B antiserum preadsorbed with E. coli lysate. According to this protocol several hex ..cap alpha.. clones have been identified. While these clones generate ..beta..-galactosidase: hex ..cap alpha.. fusion proteins, these findings suggest that in the future it may be possible to obtain large quantities of unmodified hex ..cap alpha.. and ..beta.. polypeptides from E. coli for the study of the structural and enzymatic properties of these polypeptides and for diagnostic purposes in the GM2 gangliosidoses.

  12. Stable expression of human H1-histamine-receptor cDNA in Chinese hamster ovary cells. Pharmacological characterisation of the protein, tissue distribution of messenger RNA and chromosomal localisation of the gene.

    PubMed

    Moguilevsky, N; Varsalona, F; Noyer, M; Gillard, M; Guillaume, J P; Garcia, L; Szpirer, C; Szpirer, J; Bollen, A

    1994-09-01

    A cDNA clone for the histamine H1 receptor was isolated from a human lung cDNA library; it encoded a protein of 487 amino acids which showed characteristic features of G-protein-coupled receptors. The percentages of identity of the deduced amino acid sequence with bovine, rat and guinea pig H1 histamine receptors were 82.6%, 79.4% and 73.3%, respectively, whereas these percentages decreased to 74.6%, 66% and 56.7% for the amino acid sequence of the third intracellular loop. The human H1-receptor cDNA was transfected into Chinese hamster ovary cells (CHO) via an eukaryotic expression vector; the receptor protein present on cell membranes specifically bound [3H]mepyramine with a Kd of 3.7 nM. The binding was displaced by H1-histamine-receptor antagonists and histamine. Northern blot analysis indicated the presence of two histamine H1 receptor mRNAs of 3.5 kb and 4.1 kb in various human tissues and an additional mRNA of 4.8 kb restricted to the human brain. Finally, by means of somatic cell hybrids segregating either human or rat chromosomes, the gene for histamine H1 receptor was found to reside on human chromosome 3 and rat chromosome 4.

  13. Dynamic Encoding of Speech Sequence Probability in Human Temporal Cortex

    PubMed Central

    Leonard, Matthew K.; Bouchard, Kristofer E.; Tang, Claire

    2015-01-01

    Sensory processing involves identification of stimulus features, but also integration with the surrounding sensory and cognitive context. Previous work in animals and humans has shown fine-scale sensitivity to context in the form of learned knowledge about the statistics of the sensory environment, including relative probabilities of discrete units in a stream of sequential auditory input. These statistics are a defining characteristic of one of the most important sequential signals humans encounter: speech. For speech, extensive exposure to a language tunes listeners to the statistics of sound sequences. To address how speech sequence statistics are neurally encoded, we used high-resolution direct cortical recordings from human lateral superior temporal cortex as subjects listened to words and nonwords with varying transition probabilities between sound segments. In addition to their sensitivity to acoustic features (including contextual features, such as coarticulation), we found that neural responses dynamically encoded the language-level probability of both preceding and upcoming speech sounds. Transition probability first negatively modulated neural responses, followed by positive modulation of neural responses, consistent with coordinated predictive and retrospective recognition processes, respectively. Furthermore, transition probability encoding was different for real English words compared with nonwords, providing evidence for online interactions with high-order linguistic knowledge. These results demonstrate that sensory processing of deeply learned stimuli involves integrating physical stimulus features with their contextual sequential structure. Despite not being consciously aware of phoneme sequence statistics, listeners use this information to process spoken input and to link low-level acoustic representations with linguistic information about word identity and meaning. PMID:25948269

  14. Dynamic encoding of speech sequence probability in human temporal cortex.

    PubMed

    Leonard, Matthew K; Bouchard, Kristofer E; Tang, Claire; Chang, Edward F

    2015-05-06

    Sensory processing involves identification of stimulus features, but also integration with the surrounding sensory and cognitive context. Previous work in animals and humans has shown fine-scale sensitivity to context in the form of learned knowledge about the statistics of the sensory environment, including relative probabilities of discrete units in a stream of sequential auditory input. These statistics are a defining characteristic of one of the most important sequential signals humans encounter: speech. For speech, extensive exposure to a language tunes listeners to the statistics of sound sequences. To address how speech sequence statistics are neurally encoded, we used high-resolution direct cortical recordings from human lateral superior temporal cortex as subjects listened to words and nonwords with varying transition probabilities between sound segments. In addition to their sensitivity to acoustic features (including contextual features, such as coarticulation), we found that neural responses dynamically encoded the language-level probability of both preceding and upcoming speech sounds. Transition probability first negatively modulated neural responses, followed by positive modulation of neural responses, consistent with coordinated predictive and retrospective recognition processes, respectively. Furthermore, transition probability encoding was different for real English words compared with nonwords, providing evidence for online interactions with high-order linguistic knowledge. These results demonstrate that sensory processing of deeply learned stimuli involves integrating physical stimulus features with their contextual sequential structure. Despite not being consciously aware of phoneme sequence statistics, listeners use this information to process spoken input and to link low-level acoustic representations with linguistic information about word identity and meaning.

  15. Human cytomegalovirus UL49 encodes an early, virion-associated protein essential for virus growth in human foreskin fibroblasts.

    PubMed

    Zhu, Feng; Yuan, Jian; Li, Hong-Jian; Zeng, Zhi-Feng; Luo, Zhi-Wen; Li, Shi-Qian; He, Chi-Qiang; Jia, Xue-Fang; Zhang, Xin; Zuo, Hui; Liu, Yi-Min; Chang, Martin; Li, Yue-Qin; Zhou, Tian-Hong

    2016-05-01

    Despite recent results of deletion experiments showing that open reading frame (ORF) UL49 of human cytomegalovirus (HCMV) is essential, the expression, function and functional location of its encoded protein remain unknown. We generated an antibody specific for pUL49 to investigate the protein product encoded by the UL49 ORF and identified its function in HCMV-infected host foreskin fibroblasts. A bacterial artificial chromosome (BAC) of HCMV strain Towne (pRV-Towne) and the UL49-deleted mutant pRV-delUL49Towne were used to observe virus growth by plaque assay. Using a UL49-protein-binding antibody, we located pUL49 in the fibroblast cytoplasm. pUL49 exhibited expression kinetics resembling those of the class β-2 proteins and was detected in the virion tegument. Following deletion of UL49 ORF, the virus failed to replicate, but it could be recovered by addition of pUL49 from pCDNA3.1 (+)-UL49. Our findings indicate that UL49 ORF is essential for HCMV replication in host foreskin fibroblasts.

  16. Cloning and sequencing of human intestinal alkaline phosphatase cDNA

    SciTech Connect

    Berger, J.; Garattini, E.; Hua, J.C.; Udenfriend, S.

    1987-02-01

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

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

    PubMed

    Donfack, Joseph; Wiley, Anissa

    2015-05-01

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

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

    PubMed

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

    1995-04-03

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

  19. cDNA cloning, molecular characterization, and chromosomal localization of NET(EPHT2), a human EPH-related receptor protein-tyrosine kinase gene preferentially expressed in brain

    SciTech Connect

    Tang, X.X.; Yoshioka, A.; Pleasure, D.E.

    1995-09-20

    By screening a human fetal brain cDNA expression library using a monoclonal anti-phosphotyrosine antibody , we have isolated a cDNA clone encoding a receptor type protein-tyrosine kinase belonging to the EPH family, NET (neuronally expressed EPH-related tyrosine kinase). NET shows 87% homology in nucleotide sequence and 99% homology in the deduced amino acid sequence to rat elk, suggesting that NET is the human homologue of elk. The NET gene is mapped to human chromosome 3q21-q23 by PCR screening of a human rodent somatic cell hybrid panel and by fluorescence in situ hybridization. Examination of NET mRNA expression in several human tissues has shown that the NET gene is expressed preferentially in brain as a 5-kb transcript. Steady-state levels of NET mRNA in human brain are greater in the midterm fetus than in the adult. Lower levels of NET mRNA are found in fetal kidney and adult skeletal muscle. The expression pattern of NET mRNA thus differs from that of elk, suggesting that these two gene products may preform distinct roles in human and rat. NET transcripts are detected in human acid-induced neuronal differentiation. Several human tumor cell lines derived from neuroectoderm including primitive neuroblastoma also express NET transcripts. Since the NET mRNA expression in human brain is developmentally regulated and is induced during neuronal differentiation, NET potentially plays important roles in human neurogenesis. 89 refs., 7 figs.

  20. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes

    PubMed Central

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene. PMID:26529408

  1. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

    PubMed

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

  2. Human placental Na/sup +/, K/sup +/-ATPase. cap alpha. subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

    SciTech Connect

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

    1987-11-01

    A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na/sup +/, K/sup +/-ATPase ..cap alpha.. subunit was cloned from human placenta and its sequence was identical to that encoding the ..cap alpha.. subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na/sup +/, K/sup +/-ATPase gene from various human tissues and cell lines revealed only one band (approx. = 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) lambdagt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the ..cap alpha.. subunit is on the short is on the short arm (band p11-p13) of chromosome 1.

  3. Production of glycosylated physiologically "normal" human alpha 1-antitrypsin by mouse fibroblasts modified by insertion of a human alpha 1-antitrypsin cDNA using a retroviral vector.

    PubMed Central

    Garver, R I; Chytil, A; Karlsson, S; Fells, G A; Brantly, M L; Courtney, M; Kantoff, P W; Nienhuis, A W; Anderson, W F; Crystal, R G

    1987-01-01

    Alpha 1-Antitrypsin (alpha 1AT) deficiency is a hereditary disorder characterized by reduced serum levels of alpha 1AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment alpha 1AT levels in this disorder with physiologically normal human alpha 1AT, we have integrated a full-length normal human alpha 1AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the alpha 1AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi 2 and infected NIH 3T3. The clones produced three mRNA transcripts (5.8, 4.8, and 2.4 kilobases) containing human alpha 1AT sequences, secreted an alpha 1AT molecule recognized by an anti-human alpha 1AT antibody, with the same molecular mass (52 kDa) as normal human alpha 1AT and that complexed with and inhibited human neutrophil elastase. The psi 2 produced alpha 1AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal alpha 1AT purified from human plasma and markedly longer than that of nonglycosylated human alpha 1AT cDNA-directed yeast-produced alpha 1AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human alpha 1AT cDNA into non-alpha 1AT-producing cells, resulting in the synthesis and secretion of physiologically "normal" human alpha 1AT. Images PMID:3029759

  4. The human heat-shock protein family. Expression of a novel heat-inducible HSP70 (HSP70B') and isolation of its cDNA and genomic DNA.

    PubMed Central

    Leung, T K; Rajendran, M Y; Monfries, C; Hall, C; Lim, L

    1990-01-01

    The human heat-shock protein multigene family comprises several highly conserved proteins with structural and functional properties in common, but which vary in the extent of their inducibility in response to metabolic stress. We have isolated and characterized a novel human HSP70 cDNA, HSP70B' cDNA, and its corresponding gene sequence. HSP70B' cDNA hybrid-selected an mRNA encoding a more basic 70 kDa heat-shock protein that both the major stress-inducible HSP70 and constitutively expressed HSC70 heat-shock proteins, which in common with other heat-shock 70 kDa proteins bound ATP. The complete HSP70B' gene was sequenced and, like the major inducible HSP70 gene, is devoid of introns. The HSP70B' gene has 77% sequence similarity to the HSP70 gene and 70% similarity to HSC70 cDNA, with greatest sequence divergence towards the 3'-terminus. The HSP70B' gene represents a functional gene, as indicated by Northern-blot analysis with specific oligonucleotides, hybrid-selected translation with a specific 3' cDNA sequence and S1 nuclease protection experiments. In contrast with HSP70 mRNA, which is present at low concentrations in HeLa cells and readily induced by heat or CdCl2 treatment in both fibroblasts and HeLa cells, HSP70B' mRNA was induced only at higher temperature and showed no basal expression. The differences in patterns of induction may be due to the special features of the promoter region of the HSP70B' gene. Images Fig. 1. Fig. 4. Fig. 6. PMID:2327978

  5. Ipsilateral directional encoding of joystick movements in human cortex.

    PubMed

    Sharma, Mohit; Gaona, Charles; Roland, Jarod; Anderson, Nick; Freudenberg, Zachary; Leuthardt, Eric C

    2009-01-01

    The majority of Brain Computer Interfaces have relied on signals related to primary motor cortex and the operation of the contralateral limb. Recently, the physiology associated with same-sided (ipsilateral) motor movements has been found to have a unique cortical physiology. This study sets out to assess whether more complex motor movements can be discerned utilizing ipsilateral cortical signals. In this study, three invasively monitored human subjects were recorded while performing a center out joystick task with the hand ipsilateral to the hemispheric subdural grid array. It was found that directional tuning was present in ipsilateral cortex. This information was encoded in both distinct anatomic populations and spectral distributions. These findings support the notion that ipsilateral signals may provide added information for BCI operation in the future.

  6. Tenascin-X: a novel extracellular matrix protein encoded by the human XB gene overlapping P450c21B

    PubMed Central

    1993-01-01

    A human gene termed XB overlaps the P450c21B gene encoding steroid 21- hydroxylase and encodes a protein that closely resembles extracellular matrix proteins. Sequencing of phage and cosmid clones and of cDNA fragments shows that the XB gene spans 65 kb of DNA, consisting of 39 exons that encode a 12-kb mRNA. The predicted protein of over 400 kD consists of five distinct domains: a signal peptide, a hydrophobic domain containing three heptad repeats, a series of 18.5 EGF-like repeats, 29 fibronectin type III repeats, and a carboxy-terminal fibrinogen-like domain. Because the structure of the protein encoded by the XB gene closely resembles tenascin, we term this protein tenascin-X (TN-X), and propose a simplified nomenclature system for the family of tenascins. RNase protection experiments show that the TN-X transcript is expressed ubiquitously in human fetal tissues, with the greatest expression in the fetal testis and in fetal skeletal, cardiac, and smooth muscle. Two adrenal-specific transcripts, P450c21B (steroid 21- hydroxylase) and Y (an untranslated transcript) overlap the XB gene on the complementary strand of DNA, yielding a unique array of overlapping transcripts: a "polygene." In situ hybridization histochemistry experiments show that the TN-X transcript and the P450c21 and Y transcripts encoded on the complementary DNA strand are all expressed in the same cells of the human adrenal cortex. Genetic data suggest that TN-X may be essential for life. PMID:7686164

  7. Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor.

    PubMed Central

    Elhag, G A; Thomas, F J; McCreery, T P; Bourque, D P

    1992-01-01

    Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco. Images PMID:1542565

  8. Cloning and expression of a cDNA encoding a Vorticella convallaria spasmin: an EF-hand calcium-binding protein.

    PubMed

    Maciejewski, J J; Vacchiano, E J; McCutcheon, S M; Buhse, H E

    1999-01-01

    The stalked, ciliated protozoan Vorticella convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.

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

    PubMed

    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.

  10. Csa-19, a radiation-responsive human gene, identified by an unbiased two-gel cDNA library screening method in human cancer cells

    NASA Technical Reports Server (NTRS)

    Balcer-Kubiczek, E. K.; Meltzer, S. J.; Han, L. H.; Zhang, X. F.; Shi, Z. M.; Harrison, G. H.; Abraham, J. M.

    1997-01-01

    A novel polymerase chain reaction (PCR)-based method was used to identify candidate genes whose expression is altered in cancer cells by ionizing radiation. Transcriptional induction of randomly selected genes in control versus irradiated human HL60 cells was compared. Among several complementary DNA (cDNA) clones recovered by this approach, one cDNA clone (CL68-5) was downregulated in X-irradiated HL60 cells but unaffected by 12-O-tetradecanoyl phorbol-13-acetate, forskolin, or cyclosporin-A. DNA sequencing of the CL68-5 cDNA revealed 100% nucleotide sequence homology to the reported human Csa-19 gene. Northern blot analysis of RNA from control and irradiated cells revealed the expression of a single 0.7-kilobase (kb) messenger RNA (mRNA) transcript. This 0.7-kb Csa-19 mRNA transcript was also expressed in a variety of human adult and corresponding fetal normal tissues. Moreover, when the effect of X- or fission neutron-irradiation on Csa-19 mRNA was compared in cultured human cells differing in p53 gene status (p53-/- versus p53+/+), downregulation of Csa-19 by X-rays or fission neutrons was similar in p53-wild type and p53-null cell lines. Our results provide the first known example of a radiation-responsive gene in human cancer cells whose expression is not associated with p53, adenylate cyclase or protein kinase C.

  11. Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase.

    PubMed Central

    Thelander, L; Berg, P

    1986-01-01

    Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2, which are differentially regulated during the cell cycle. We have isolated expressible cDNA clones of both subunits from an Okayama-Berg cDNA library made with mRNA from hydroxyurea-resistant, M2 protein-overproducing mouse TA3 cells. Expression of M2 protein could be demonstrated by electron paramagnetic resonance spectroscopy after transfection of COS-7 monkey cells with the plasmid. Electrophoresis and blot analyses of the parent and hydroxyurea-resistant TA3 mRNA revealed two M2 transcripts, a major one of 2.1 kilobases and a minor one of about 1.6 kilobases. Restriction endonuclease mapping of the corresponding cDNAs indicated that the two mRNAs differed only in the length of the 3' untranslated ends. By contrast, there was only one mRNA corresponding to the M1 protein, and its mobility corresponded to about 3.1 kilobases. The hydroxyurea-resistant TA3 cells contained a 50- to 100-fold excess of the M2 mRNAs over that of the parent cells and a 10-fold excess of the M1 mRNA. However, a Southern blot analysis of the corresponding genomic DNA sequences showed that the M2 gene was amplified fivefold but the M1 gene was still single copy. The complete nucleotide sequence of the 2,111-base-pair-long M2 cDNA revealed an open reading frame coding for 390 amino acids, which corresponds to a molecular weight of 45,100. The mouse M2 protein sequence was quite homologous to the equivalent protein in the clam Spisula solidissima, while the homology to the smaller subunits of Epstein-Barr virus, herpes simplex virus type 2, and Escherichia coli ribonucleotide reductases were less pronounced. Images PMID:3025593

  12. Nucleotide sequence of a tobacco cDNA encoding plastidic glutamine synthetase and light inducibility, organ specificity and diurnal rhythmicity in the expression of the corresponding genes of tobacco and tomato.

    PubMed

    Becker, T W; Caboche, M; Carrayol, E; Hirel, B

    1992-06-01

    A full-length cDNA encoding glutamine synthetase (GS) was cloned from a lambda gt10 library of tobacco leaf RNA, and the nucleotide sequence was determined. An open reading frame accounting for a primary translation product consisting of 432 amino acids has been localized on the cDNA. The calculated molecular mass of the encoded protein is 47.2 kDa. The predicted amino acid sequence of this precursor shows higher homology to GS-2 protein sequences from other species than to a leaf GS-1 polypeptide sequence, indicating that the cDNA isolated encodes the chloroplastic isoform (GS-2) of tobacco GS. The presence of C- and N-terminal extensions which are characteristic of GS-2 proteins supports this conclusion. Genomic Southern blot analysis indicated that GS-2 is encoded by a single gene in the diploid genomes of both tomato and Nicotiana sylvestris, while two GS-2 genes are very likely present in the amphidiploid tobacco genome. Western blot analysis indicated that in etiolated and in green tomato cotyledons GS-2 subunits are represented by polypeptides of similar size, while in green tomato leaves an additional GS-2 polypeptide of higher apparent molecular weight is detectable. In contrast, tobacco GS-2 is composed of subunits of identical size in all organs examined. GS-2 transcripts and GS-2 proteins could be detected at high levels in the leaves of both tobacco or tomato. Lower amounts of GS-2 mRNA were detected in stems, corolla, and roots of tomato, but not in non-green organs of tobacco. The GS-2 transcript abundance exhibited a diurnal fluctuation in tomato leaves but not in tobacco leaves. White or red light stimulated the accumulation of GS-2 transcripts and GS-2 protein in etiolated tomato cotyledons. Far-red light cancelled this stimulation. The red light response of the GS-2 gene was reduced in etiolated seedlings of the phytochrome-deficient aurea mutant of tomato. These results indicate a phytochrome-mediated light stimulation of GS-2 gene expression

  13. Molecular characterization of a cDNA encoding Cu/Zn superoxide dismutase from Deschampsia antarctica and its expression regulated by cold and UV stresses

    PubMed Central

    Sánchez-Venegas, Jaime R; Dinamarca, Jorge; Moraga, Ana Gutiérrez; Gidekel, Manuel

    2009-01-01

    Background The Copper/Zinc superoxide dismutase (Cu/ZnSOD) gene, SOD gene, was isolated from a Deschampsia antarctica Desv. by cDNA library screening. The expression of SOD gene in the leaves of D. antarctica was determined by RT-PCR and its differential expression of gene transcripts in conditions of cold and UV radiation stresses was revealed by northern blot. Findings The molecular characterization shows that SOD cDNA is 709 bp in length, which translates an ORF of 152 amino acids that correspond to a protein of predicted molecular mass of 15 kDa. The assay shows that the expression of SOD gene increases when D. antarctica is acclimatised to 4°C and exposed to UV radiation. These results indicate that the SOD gene of D. antarctica is involved in the antioxidative process triggered by oxidative stress induced by the conditions of environmental change in which they live. Conclusion The present results allow us to know the characteristics of Cu/ZnSOD gene from D. antarctica and understand that its expression is regulated by cold and UV radiation. PMID:19785762

  14. Characterization of a cDNA clone encoding the carboxy-terminal domain of a 90-kilodalton surface antigen of Trypanosoma cruzi metacyclic trypomastigotes.

    PubMed Central

    Franco, F R; Paranhos-Bacallà, G S; Yamauchi, L M; Yoshida, N; da Silveira, J F

    1993-01-01

    We have cloned and sequenced a cDNA for a metacyclic trypomastigote-specific glycoprotein with a molecular mass of 90 kDa, termed MTS-gp90. By immunoblotting, antibodies to the MTS-gp90 recombinant protein reacted exclusively with a 90-kDa antigen of metacyclic trypomastigotes. The insert of the MTS-gp90 cDNA clone strongly hybridized with a single 3.0-kb mRNA of metacyclic forms, whereas the hybridization signal with epimastigote mRNA was weak and those with RNAs from other developmental stages were negative, indicating that transcription of the MTS-gp90 gene is developmentally regulated. A series of experiments showed that the MTS-gp90 gene is present in multiple copies in the Trypanosoma cruzi genome, arranged in a nontandem manner, and that there are at least 40 copies of the gene per haploid genome. Sequence analysis of recombinant MTS-gp90 revealed 40 to 60% identity at the amino acid level with members of a family of mammalian stage-specific, 85-kDa surface antigens of T. cruzi. However, there are considerable differences in the amino acid compositions outside the homology region. Images PMID:8406808

  15. Encoding of marginal utility across time in the human brain

    PubMed Central

    Pine, Alex; Seymour, Ben; Roiser, Jonathan P; Bossaerts, Peter; Friston, Karl J.; Curran, H. Valerie; Dolan, Raymond J.

    2010-01-01

    Marginal utility theory prescribes the relationship between the objective property of the magnitude of rewards and their subjective value. Despite its pervasive influence, however, there is remarkably little direct empirical evidence for such a theory of value, let alone of its neurobiological basis. We show that human preferences in an inter-temporal choice task are best described by a model that integrates marginally diminishing utility with temporal discounting. Using functional magnetic resonance imaging (fMRI), we show that activity in the dorsal striatum encodes both the marginal utility of rewards, over and above that which can be described by their magnitude alone, and the discounting associated with increasing time. In addition, our data show that dorsal striatum may be involved in integrating subjective valuation systems inherent to time and magnitude, thereby providing an overall metric of value used to guide choice behaviour. Furthermore, during choice we show that anterior cingulate activity correlates with the degree of difficulty associated with dissonance between value and time. Our data support an integrative architecture for decision-making, revealing the neural representation of distinct subcomponents of value that may contribute to impulsivity and decisiveness. PMID:19641120

  16. Functional Genomics of 5- to 8-Cell Stage Human Embryos by Blastomere Single-Cell cDNA Analysis

    PubMed Central

    Galán, Amparo; Montaner, David; Póo, M. Eugenia; Valbuena, Diana; Ruiz, Verónica; Aguilar, Cristóbal; Dopazo, Joaquín; Simón, Carlos

    2010-01-01

    Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented. PMID:21049019

  17. Epstein-barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells

    SciTech Connect

    Margolskee, R.F.; Kavathas, P.; Berg, P.

    1988-07-01

    Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromcying B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastodi cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10/sup 6/ to 10/sup 7/ independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBP-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthing-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2. Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10/sup 6/ total clones or one clone of EBO-pcD-Leu-2 per 2 x 10/sup 4/ total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.

  18. Genetic mapping in human and mouse of the locus encoding TRBP, a protein that binds the TAR region of the human immunodeficiency virus (HIV-1)

    SciTech Connect

    Kozak, C.A.; Gatignol, A.; Graham, K.

    1995-01-01

    Productive infection with HIV-1, the virus responsible for AIDS, requires the involvement of host cell factors for completion of the replicative cycle, but the identification of these factors and elucidation of their specific functions has been difficult. A human cDNA, TRBP, was recently cloned and characterized as a positive regulator of gene expression that binds to the TAR region of the HIV-1 genome. Here we demonstrate that this factor is encoded by a gene, TARBP2, that maps to human chromosome 12 and mouse chromosome 15, and we also identify and map one human pseudogene (TARBP2P) and two mouse TRBP-related sequences. The map location of the expressed gene identifies it as a candidate for the previously identified factor encoded on human chromosome 12 that has been shown to be important for expression of HIV-1 genes. Western blotting indicates that despite high sequence conservation in human and mouse, the TARBP2 protein differs in apparent size in primate and rodent cells. 41 refs., 5 figs., 1 tab.

  19. Structure of the human gene encoding the associated microfibrillar protein (MFAP1) and localization to chromosome 15q15-q21

    SciTech Connect

    Yeh, H.; Chow, M.; Abrams, W.R.

    1994-09-15

    Microfibrils with a diameter of 10-12 nm, found either in assocation with elastin or independently, are an important component of the extracellular matrix of many tissues. To extend understanding of the proteins composing these microfibrils, the cDNA and gene encoding the human associated microfibril protein (MRAP1) have been cloned and characterized. The coding portion is contained in 9 exons, and the sequence is very homologous to the previously described chick cDNA, but does not appear to share homology or domain motifs with any other known protein. Interestingly, the gene has been localized to chromosome 15q15-q21 by somatic hybrid cell and chromosome in situ analyses. This is the same chromosomal region to which the fibrillin gene, FBN1, known to be defective in the Marfan syndrome, has been mapped. MFAP1 is a candidate gene for heritable diseases affecting microfibrils. 38 refs., 6 figs.

  20. Partial purification of the chloroplast ATP synthase from Chlamydomonas reinhardtii and the cloning and sequencing of a cDNA encoding the gamma subunit

    SciTech Connect

    Yu, L.M.

    1988-01-01

    The chloroplast ATP synthase was partially purified from the green alga Chlamydomonas reinhardtii by extracting membranes with deoxycholate and KCl, followed by centrifugation and ammonium sulfate fractionation of the supernatant. The enzyme assay involved the reconstitution of such fractions with bacteriorhodopsin and soybean phospholipids to form vesicles capable of light-dependent ({sup 32}P)-phosphate esterification. A cDNA for the gamma subunit from Chlamydomonas was isolated, expressed in vitro and sequenced. It contains the entire coding region for the gamma subunit precursor. A 35 amino acid long transit peptide resides at the NH{sub 2}-terminus of a 323 amino acid long mature peptide that is 77% similar to the spinach gamma subunit. Six cysteines were found; three were conserved in Chlamydomonas and spinach.

  1. The human sorbitol dehydrogenase gene: cDNA cloning, sequence determination, and mapping by fluorescence in situ hybridization

    SciTech Connect

    Lee, F.K.; Chung, S. ); Cheung, M.C. )

    1994-05-15

    The cDNA for human sorbitol dehydrogenase (SORD) has been cloned and sequenced. It translates into a peptide of 356 amino acid residues, one more than the sequence previously reported from peptide analysis. An extra alanine was found at the acetyl-blocked N-terminal, between positions 1 and 4. This matches the rat cDNA, which also has 356 amino acids, with an extra proline at position 3. Four other mismatches were also observed, but these are all amino acid substitutions that occur outside proposed functionally important regions. Further work must be performed to determine whether these discrepancies represent polymorphic forms of the enzyme. The SORD gene was mapped by fluorescence in situ hybridization and found to occupy a single site on chromosome 15q15, indicating that it is a single-copy gene. This was confirmed by Southern blot hybridization. SORD is thought to be involved in the etiology of diabetic complications, and its deficiency has been linked to congenital cataracts. The cloned gene could be used as a probe to study the role of this enzyme in the pathogenesis of these diseases. 24 refs., 4 figs.

  2. Nucleotide sequence of alkyl-dihydroxyacetonephosphate synthase cDNA from Dictyostelium discoideum.

    PubMed

    de Vet, E C; van den Bosch, H

    1998-11-27

    The nucleotide sequence is reported of alkyl-dihydroxyacetonephosphate synthase cDNA from the cellular slime mold Dictyostelium discoideum. The open reading frame encodes a protein of 611 amino acids which shows a 33% amino acid identity to the human enzyme. This D. discoideum homolog carries a variant of the peroxisomal targeting signal type 1 at its C-terminus (PKL). Expression of the cDNA in Escherichia coli yielded an enzymatically active protein.

  3. Cloning of a chicken liver cDNA encoding 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase by functional complementation of Escherichia coli pur mutants.

    PubMed Central

    Chen, Z D; Dixon, J E; Zalkin, H

    1990-01-01

    We have used functional complementation of Escherichia coli pur mutants to clone avian cDNA encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase-5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase, the bifunctional enzyme catalyzing steps 6 and 7 in the pathway for de novo purine nucleotide synthesis. Mutational analyses have been used to establish the structure-function relationship: NH2-SAICAR synthetase-AIR carboxylase-COOH. The amino acid sequence of the SAICAR synthetase domain is homologous to that of bacterial purC-encoded enzymes, and the sequence of the following AIR carboxylase domain is homologous to that of bacterial purE-encoded enzymes. In E. coli, AIR carboxylase is the product of genes purEK with the purK subunit postulated to have a role in CO2 binding. The avian enzyme lacks sequences corresponding to purK yet functions in E. coli. Functional complementation of E. coli pur mutants can be used to clone additional avian cDNAs for de novo purine nucleotide synthesis. Images PMID:1691501

  4. Subunit organization of the abalone Haliotis tuberculata hemocyanin type 2 (HtH2), and the cDNA sequence encoding its functional units d, e, f, g and h.

    PubMed

    Lieb, B; Altenhein, B; Lehnert, R; Gebauer, W; Markl, J

    1999-10-01

    We have developed a HPLC procedure to isolate the two different hemocyanin types (HtH1 and HtH2) of the European abalone Haliotis tuberculata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, PAGE, N-terminal protein sequencing and cDNA sequencing, we have identified eight different 40-60-kDa functional units (FUs) in HtH2, termed HtH2-a to HtH2-h, and determined their linear arrangement within the elongated 400-kDa subunit. From a Haliotis cDNA library, we have isolated and sequenced a cDNA clone which encodes the five C-terminal FUs d, e, f, g and h of HtH2. As shown by multiple sequence alignments, defg of HtH2 correspond structurally to defg from Octopus dofleini hemocyanin. HtH2-e is the first FU of a gastropod hemocyanin to be sequenced. The new Haliotis hemocyanin sequences are compared to their counterparts in Octopus, Helix pomatia and HtH1 (from the latter, the sequences of FU-f, FU-g and FU-h have recently been determined) and discussed in relation to the recent 2.3 A X-ray structure of FU-g from Octopus hemocyanin and the 15 A three-dimensional reconstruction of the Megathura crenulata hemocyanin didecamer from electron micrographs. This data allows, for the first time, an insight into the evolution of the two functionally different hemocyanin isoforms found in marine gastropods. It appears that they evolved several hundred million years ago within the Prosobranchia, after separation of the latter from the branch leading to the Pulmonata. Moreover, as a structural explanation for the inefficiency of the type 1 hemocyanin to form multidecamers in vivo, the additional N-glycosylation sites in HtH1 compared to HtH2 are discussed.

  5. Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7

    SciTech Connect

    Flores, J.; Sears, J.; Schael, I.P.; White, L.; Garcia, D.; Lanata, C.; Kapikian, A.Z. )

    1990-08-01

    We have synthesized {sup 32}P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies.

  6. Human substance P receptor (NK-1): Organization of the gene, chromosome localization, and functional expression of cDNA clones

    SciTech Connect

    Gerard, N.P.; Paquet, J.L. Children's Hospital, Boston, MA Harvard Medical School, Boston, MA ); Garraway, L.A. Harvard Medical School, Boston, MA ); Eddy, R.L. Jr.; Shows, T.B. ); Iijima, Hideya Harvard School of Public Health, Boston, MA ); Gerard, C. )

    1991-11-05

    The gene for the human substance P receptor (NK-1) was cloned using cDNA probes made by the polymerase chain reaction from primers based on the rat sequence. The gene spans 45-60 kb and is contained in five exons, with introns interrupting at sites homologous to those in the NK-2 receptor gene. Analysis of restriction digests of genomic DNA from mouse/human cell hybrids indicates the NK-1 receptor is a single-copy gene located on human chromosome 2. Polymerase chain reaction using primers based on the 5{prime} and 3{prime} ends of the coding sequence was used to generate full-length cDNAs from human lung and from IM9 lymphoblast cells. When transfected into COS-7 cells, the NK-1 receptor binds {sup 125}I-BHSP with a K{sub d} of 0.35 {plus minus} 0.07 nM and mediates substance P induced phosphatidylinositol metabolism. The receptor is selective for substance P; the relative affinity for neurokinin A and neurokinin B is 100- and 500-fold lower, respectively. Human IM9 lymphoblast cells express relatively high levels of the NK-1 receptor, and Northern blot analysis indicates modulation of mRNA levels by glucocorticoids and growth factors, suggesting that this cell line may be useful as a model for studying the control of NK-1 receptor gene expression.

  7. Isolation of an insulin-like growth factor II cDNA with a unique 5 prime untranslated region from human placenta

    SciTech Connect

    Shen, Shujane; Daimon, Makoto; Wang, Chunyeh; Ilan, J. ); Jansen, M. )

    1988-03-01

    Human insulin-like growth factor II (IGF-II) cDNA from a placental library was isolated and sequenced. The 5{prime} untranslated region (5{prime}-UTR) sequence of this cDNA differs completely from that of adult human liver and has considerable base sequence identity to the same region of an IGF-II cDNA of a rat liver cell line, BRL-3A. Human placental poly(A){sup +} RNA was probed with either the 5{prime}-UTR of the isolated human placental IGF-II cDNA or the 5{prime}-UTR of the IGF-II cDNA obtained from adult human liver. No transcripts were detected by using the 5{prime}-UTR of the adult liver IGF-II as the probe. In contrast, three transcripts of 6.0, 3.2, and 2.2 kilobases were detected by using the 5{prime}-UTR of the placental IGF-II cDNA as the probe or the probe from the coding sequence. A fourth IGF-II transcript of 4.9 kilobases presumably containing a 5{prime}-UTR consisting of a base sequence dissimilar to that of either IGF-II 5{prime}-UTR was apparent. Therefore, IGF-II transcripts detected may be products of alternative splicing as their 5{prime}-UTR sequence is contained within the human IGF-II gene or they may be a consequence of alternative promoter utilization in placenta.

  8. The complete sequence of a full length cDNA for human liver glyceraldehyde-3-phosphate dehydrogenase: evidence for multiple mRNA species.

    PubMed Central

    Arcari, P; Martinelli, R; Salvatore, F

    1984-01-01

    A recombinant M13 clone (O42) containing a 65 b.p. cDNA fragment from human fetal liver mRNA coding for glyceraldehyde-3-phosphate dehydrogenase has been identified and it has been used to isolate from a full-length human adult liver cDNA library a recombinant clone, pG1, which has been subcloned in M13 phage and completely sequenced with the chain terminator method. Besides the coding region of 1008 b.p., the cDNA sequence includes 60 nucleotides at the 5'-end and 204 nucleotides at the 3'-end up to the polyA tail. Hybridization of pG1 to human liver total RNA shows only one band about the size of pG1 cDNA. A much stronger hybridization signal was observed using RNA derived from human hepatocarcinoma and kidney carcinoma cell lines. Sequence homology between clone 042 and the homologous region of clone pG1 is 86%. On the other hand, homology among the translated sequences and the known human muscle protein sequence ranges between 77 and 90%; these data demonstrate the existence of more than one gene coding for G3PD. Southern blot of human DNA, digested with several restriction enzymes, also indicate that several homologous sequences are present in the human genome. Images PMID:6096821

  9. Encoding of configural regularity in the human visual system.

    PubMed

    Kubilius, Jonas; Wagemans, Johan; Op de Beeck, Hans P

    2014-08-13

    The visual system is very efficient in encoding stimulus properties by utilizing available regularities in the inputs. To explore the underlying encoding strategies during visual information processing, we presented participants with two-line configurations that varied in the amount of configural regularity (or degrees of freedom in the relative positioning of the two lines) in a fMRI experiment. Configural regularity ranged from a generic configuration to stimuli resembling an "L" (i.e., a right-angle L-junction), a "T" (i.e., a right-angle midpoint T-junction), or a "+",-the latter being the most regular stimulus. We found that the response strength in the shape-selective lateral occipital area was consistently lower for a higher degree of regularity in the stimuli. In the second experiment, using multivoxel pattern analysis, we further show that regularity is encoded in terms of the fMRI signal strength but not in the distributed pattern of responses. Finally, we found that the results of these experiments could not be accounted for by low-level stimulus properties and are distinct from norm-based encoding. Our results suggest that regularity plays an important role in stimulus encoding in the ventral visual processing stream.

  10. Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis.

    PubMed

    Beaudoin, Guillaume A W; Facchini, Peter J

    2013-02-15

    Sanguinarine is a benzo[c]phenenthridine alkaloid with potent antimicrobial properties found commonly in plants of the Papaveraceae, including the roots of opium poppy (Papaver somniferum). Sanguinarine is formed from the central 1-benzylisoquinoline intermediate (S)-reticuline via the protoberberine alkaloid (S)-scoulerine, which undergoes five enzymatic oxidations and an N-methylation. The first four oxidations from (S)-scoulerine are catalyzed by cytochromes P450, whereas the final conversion involves a flavoprotein oxidase. All but one gene in the biosynthetic pathway from (S)-reticuline to sanguinarine has been identified. In this communication, we report the isolation and characterization of (S)-cis-N-methylstylopine 14-hydroxylase (MSH) from opium poppy based on the transcriptional induction in elicitor-treated cell suspension cultures and root-specific expression of the corresponding gene. Along with protopine 6-hydroxylase, which catalyzes the subsequent and penultimate step in sanguinarine biosynthesis, MSH is a member of the CYP82N subfamily of cytochromes P450. The full-length MSH cDNA was expressed in Saccharomyces cerevisiae and the recombinant microsomal protein was tested for enzymatic activity using 25 benzylisoquinoline alkaloids representing a wide range of structural subgroups. The only enzymatic substrates were the N-methylated protoberberine alkaloids N-methylstylopine and N-methylcanadine, which were converted to protopine and allocryptopine, respectively.

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

    PubMed Central

    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

  12. Lectin cDNA and transgenic plants derived therefrom

    DOEpatents

    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.

  13. hSmad5 gene, a human hSmad family member: its full length cDNA, genomic structure, promoter region and mutation analysis in human tumors.

    PubMed

    Gemma, A; Hagiwara, K; Vincent, F; Ke, Y; Hancock, A R; Nagashima, M; Bennett, W P; Harris, C C

    1998-02-19

    hSmad (mothers against decapentaplegic)-related proteins are important messengers within the Transforming Growth Factor-beta1 (TGF-beta1) superfamily signal transduction pathways. To further characterize a member of this family, we obtained a full length cDNA of the human hSmad5 (hSmad5) gene by rapid amplification of cDNA ends (RACE) and then determined the genomic structure of the gene. There are eight exons and two alternative transcripts; the shorter transcript lacks exon 2. We identified the hSmad5 promoter region from a human genomic YAC clone by obtaining the nucleotide sequence extending 1235 base pairs upstream of the 5' end of the cDNA. We found a CpG island consistent with a promoter region, and we demonstrated promoter activity in a 1232 bp fragment located upstream of the transcription initiation site. To investigate the frequency of somatic hSmad5 mutations in human cancers, we designed intron-based primers to examine coding regions by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Neither homozygous deletions or point mutations were found in 40 primary gastric tumors and 51 cell lines derived from diverse types of human cancer including 20 cell lines resistant to the growth inhibitory effects of TGF-beta1. These results suggest that the hSmad5 gene is not commonly mutated and that other genetic alterations mediate the loss of TGF-beta1 responsiveness in human cancers.

  14. Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen p53.

    PubMed Central

    Harlow, E; Williamson, N M; Ralston, R; Helfman, D M; Adams, T E

    1985-01-01

    Three clones for the human tumor antigen p53 were isolated from a cDNA library prepared from A431 cells. One of these clones, pR4-2, contains the entire coding region for human p53. This clone directs the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of an authentic p53 molecule in an in vitro transcription-translation reaction. Although the pR4-2 clone contains the coding region for p53, it is not a full-length copy of the human p53 mRNA. Northern analysis showed that the p53 mRNA is approximately 2,500 nucleotides long, whereas the pR4-2 insert is only 1,760 base pairs in length. Analysis of the DNA sequence of this clone suggests that the human p53 polypeptide has 393 amino acids. We compared the predicted amino acid sequence of the pR4-2 clone with similar clones for the mouse p53 and found long regions of amino acid homology between these two molecules. Images PMID:3894933

  15. The human gene CGT encoding the UDP-galactose ceramide galactosyl transferase (cerebroside synthase): Cloning, characterization, and assignment to human chromosome 4, band q26

    SciTech Connect

    Bosio, A.; Binczek, E.; Stoffel, W.

    1996-05-15

    We have previously cloned the human UDP-galactose ceramide galactosyltransferase (CGT, E.C. 2.4.1.45) cDNA. Its open reading frame encodes the key enzyme in the biosynthesis of the glycosphingolipids, cerebrosides and sulfatides, essential constituents of the myelin membrane of the central nervous system (CNS) and PNS. Expression of the CGT gene and of the myelin-specific proteins in the terminal differentiated oligodendrocyte of CNS and in Schwann cells of PNS is cell-specific and highly time-regulated. The CGT gene therefore is important in the differentiation program of the oligodendrocyte lineage. Here we report the structural organization and the chromosomal localization of the human CGT gene. The coding sequence is separated into five exons, which are distributed over >40 kb. The CGT locus was mapped to the distal region of human chromosome 4, band q26. The organization of the CGT gene and of the UGT (uridylglucuronosyl-transferases) gene family suggests a correlation to functional domains of the encoded proteins. 19 refs., 4 figs., 1 tab.

  16. Molecular cloning of cDNA for the zeta isoform of the 14-3-3 protein: homologous sequences in the 3'-untranslated region of frog and human zeta isoforms.

    PubMed

    Miura, I; Nakajima, T; Ohtani, H; Kashiwagi, A; Nakamura, M

    1997-10-01

    14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that possess diverse biochemical activities such as regulation of gene transcription, cell proliferation and activation of protein kinase C. At least 7 subtypes (alpha to theta) of 14-3-3 protein are known, but the zeta subtype of this protein has been cloned only in mammals. We cloned the zeta subtype of 14-3-3 protein (14-3-3 zeta) from the frog, Rana rugosa. The sequence encoded 245 amino acids that share 92% identity with rat and bovine 14-3-3 zeta s, and 92% with human phospholipase A2 (PLA2; 14-3-3 zeta). Northern blot analysis revealed a single band of about 1.8 kb in tadpoles at stage 25. The 14-3-3 zeta mRNA level was high in the brain, lung, spleen and kidney, and low in the heart and testis, as opposed to the mRNA level, which was only faintly detected in the liver, pancreas, ovary and muscle. Furthermore, high similarity in the 3'-untranslated region (3'-UTR) was observed between frog and human 14-3-3 zeta cDNA. The results suggest that 14-3-3 zeta is highly conserved throughout eukaryotic evolution, and that the homologous sequence in the 3'-UTR of 14-3-3 zeta cDNA may be conserved in frogs and humans.

  17. Human germline antibody gene segments encode polyspecific antibodies.

    PubMed

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens

    2013-04-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  18. Molecular cloning of a novel human gene encoding a 63-kDa protein and its sublocalization within the 11q13 locus

    SciTech Connect

    Perelman, B.; Dafni, N.; Naiman, T.

    1997-05-01

    A human cDNA previously isolated by virtue of its ability to complement partially the ultraviolet sensitivity of a xeroderma pigmentosum cell line was further characterized. The transcription unit is expressed as a single 4.0-kb mRNA that encodes a novel 63-kDa cytoplasmic protein, possibly initiating from an internal AUG codon. The gene encoding this protein, named UVRAG, has been extremely well conserved during evolution, implying an important role for this gene product in cell metabolism. The transcribed mRNA is constitutively expressed in a wide variety of human tissues. The protein encoded by this gene is predicted to contain a coiled-coil structure and is likely to be metabolically unstable based on the occurrence of a strong PEST domain. UVRAG was assigned to human chromosome 11 by Southern hybridization to a somatic cell hybrid panel. Fluorescence in situ hybridization coupled with PCR analysis of human/rodent somatic cell hybrids containing segments of human chromosome 11 has localized this gene to a subregion of 11q13 in between the D11S916 and the D11S906 loci. Importantly, this region has been shown to be amplified in a variety of human malignancies, including breast cancer. 28 refs., 7 figs.

  19. Human jagged polypeptide, encoding nucleic acids and methods of use

    DOEpatents

    Li, Linheng; Hood, Leroy

    2000-01-01

    The present invention provides an isolated polypeptide exhibiting substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the polypeptide does not have the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. The invention further provides an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the nucleotide sequence does not encode the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Also provided herein is a method of inhibiting differentiation of hematopoietic progenitor cells by contacting the progenitor cells with an isolated JAGGED polypeptide, or active fragment thereof. The invention additionally provides a method of diagnosing Alagille Syndrome in an individual. The method consists of detecting an Alagille Syndrome disease-associated mutation linked to a JAGGED locus.

  20. Measuring human ventilation for apnoea detection using an optical encoder.

    PubMed

    Weinberg, G M; Webster, J G

    1998-08-01

    We have designed, built and tested a proof-of-concept system based on optical encoder technology for measuring adult or infant ventilation. It uses change in chest circumference to provide an indirect measure of ventilation. The Hewlett-Packard HEDS-9720 optical encoder senses displacement of its matching codestrip. It yields a resolution of 0.17 mm and is accurate to 0.008 mm over a 10 mm test distance. The encoder is mounted on a nylon web belt wrapped around the torso and responds to changes in circumference. Motion of the code strip during respiration is converted to direction of movement (inhalation or exhalation) as well as magnitude of circumference change. Use of two sensor bands, one on the chest and one on the abdomen, may allow detection of obstructive apnoea in which there is no air flow out of or into the subject despite respiratory movement. Applications of this technology include infant apnoea monitoring as well as long-term adult monitoring.

  1. Construction of a transcription map of a 300 kb region around the human G6PD locus by direct cDNA selection.

    PubMed

    Sedlacek, Z; Korn, B; Konecki, D S; Siebenhaar, R; Coy, J F; Kioschis, P; Poustka, A

    1993-11-01

    A transcription map covering a 300 kb region around the G6PD gene in the human Xq28 region was constructed by the direct cDNA selection method and the analysis of the resulting region-specific enriched cDNA sublibrary. Seven new genes and two loci of endogenous retrovirus HERV-K were identified. The distribution of the genes across the region is strongly non-uniform and follows the non-uniform distribution of GpG islands in the area. While one of the novel genes was found to be highly homologous to bovine smg p25A GDP-dissociation inhibitor, the remaining genes did not detect any homology to known genes. The analysis of region-specific cDNA sublibraries represents a simple, rapid and efficient tool for the generation of a regional transcription map.

  2. UMD‐Predictor: A High‐Throughput Sequencing Compliant System for Pathogenicity Prediction of any Human cDNA Substitution

    PubMed Central

    Salgado, David; Desvignes, Jean‐Pierre; Rai, Ghadi; Blanchard, Arnaud; Miltgen, Morgane; Pinard, Amélie; Lévy, Nicolas; Collod‐Béroud, Gwenaëlle

    2016-01-01

    ABSTRACT Whole‐exome sequencing (WES) is increasingly applied to research and clinical diagnosis of human diseases. It typically results in large amounts of genetic variations. Depending on the mode of inheritance, only one or two correspond to pathogenic mutations responsible for the disease and present in affected individuals. Therefore, it is crucial to filter out nonpathogenic variants and limit downstream analysis to a handful of candidate mutations. We have developed a new computational combinatorial system UMD‐Predictor (http://umd‐predictor.eu) to efficiently annotate cDNA substitutions of all human transcripts for their potential pathogenicity. It combines biochemical properties, impact on splicing signals, localization in protein domains, variation frequency in the global population, and conservation through the BLOSUM62 global substitution matrix and a protein‐specific conservation among 100 species. We compared its accuracy with the seven most used and reliable prediction tools, using the largest reference variation datasets including more than 140,000 annotated variations. This system consistently demonstrated a better accuracy, specificity, Matthews correlation coefficient, diagnostic odds ratio, speed, and provided the shortest list of candidate mutations for WES. Webservices allow its implementation in any bioinformatics pipeline for next‐generation sequencing analysis. It could benefit to a wide range of users and applications varying from gene discovery to clinical diagnosis. PMID:26842889

  3. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1--December 31, 1992

    SciTech Connect

    Korenberg, J.R.

    1993-12-31

    The ultimate goal of this proposal is to create a cDNA map of the human genome. Mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach will generate 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  4. Efficient expression of codon-adapted human acetaldehyde dehydrogenase 2 cDNA with 6xHis tag in Pichia pastoris.

    PubMed

    Zhao, YuFeng; Lei, MingKe; Wu, YuanXin; Zhang, ZiSheng; Wang, CunWen

    2009-10-01

    Human mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) catalyzes the oxidation of acetaldehyde to acetic acid. Therefore, ALDH2 has therapeutic potential in detoxification of acetaldehyde. Furthermore, ALDH2 catalyzes nitroglycerin to nitrate and 1, 2-glyceryldinitrate during therapy for angina pectoris, myocardial infarction, and heart failure. Large quantities of ALDH2 will be needed for potential clinical practice. In this study, Pichia pastoris was used as a platform for expression of human ALDH2. Based on the ALDH2*1 cDNA sequence, we designed ALDH2 cDNA by choosing the P. pastoris preferred codons and by decreasing the G + C content level. The sequence was synthesized using the overlap extension PCR method. The cDNA and 6xHis tags were subcloned into the plasmid pPIC9K. The recombinant protein was expressed in P. pastoris GS115 and purified using Ni(2+)-Sepharose affinity chromatography. The amount of secreted protein in the culture was 80 mg/L in shake-flask cultivation and 260 mg/L in high-density bioreactor fermentation. Secreted ALDH2 was easily purified from the culture supernatant by using Ni(2+)-Sepharose affinity chromatography. After purification of the fermentation supernatant, the enzyme had a specific activity of 1.2 U/mg protein. The yield was about 16 mg/L in a shake flask culture of P. pastoris GS115 which contained the original human ALDH2*1 cDNA.

  5. Cloning, characterization and subcellular localization of a gene encoding a human Ubiquitin-conjugating enzyme (E2) homologous to the Arabidopsis thaliana UBC-16 gene product.

    PubMed

    Yin, Gang; Ji, Chaoneng; Wu, Tong; Shen, Zhouliang; Xu, Xin; Xie, Yi; Mao, Yumin

    2006-05-01

    Ubiquitin charging and activation of class III E2 enzymes has been directly linked to their nuclear import. It has not been published whether other classes E2s also abide by this mechanism. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone that is 2252 base pair in length, encoding a putative 162 amino acid protein, which shares high homology to Arabidopsis thaliana ubiquitin-conjugating enzyme 16 (Accession number NP_565110, 51% identity and 71% similarity) at protein level. Bioinformatics analysis revealed that the gene is composed of 7 exons, located on human chromosome 8q13-8q21.1, and that the predicted protein of the gene is a class I E2, for only composed of a conserved approximately 150-amino acid catalytic core, ubiquitin-conjugating enzyme E2 domain (UBC domain). In the C-terminal of the UBC domain sequence, there are two nuclear localization signals (NLSs). RT-PCR showed that this gene is ubiquitously expressed in 16 kinds of normal human tissues, but expression level is very low, unless in human heart, brain, liver, and pancreas. The subcellular localizations of the new human Ubiquitin conjugating enzyme E2 and its mutation were also examined, which showed that the nuclear localization of hUBC16 depended on two conditions: It has NLS, and at the same time, has enzyme active site, too, at least in HEK293 cells.

  6. Molecular cloning and characterization of the human ASB-8 gene encoding a novel member of ankyrin repeat and SOCS box containing protein family.

    PubMed

    Liu, Yongzhong; Li, Jinjun; Zhang, Fengrui; Qin, Wenxin; Yao, Genfu; He, Xianghuo; Xue, Peng; Ge, Chao; Wan, Dafang; Gu, Jianren

    2003-01-24

    We have cloned a new member of human ankyrin repeat and SOCS box containing protein family (ASB), designed as hASB-8, from a human placental cDNA library and further extended by 5(') and 3(')-RACE. The full-length cDNA was 2545bp in length, with a predicted open reading frame encoding a protein of 288 amino acids, which was 96% identical to mouse ASB-8 protein. Computer analysis revealed that the deduced amino acid sequence of the human ASB-8 contained four Ankyrin repeats and one SOCS box. The gene had four exons separated by three introns and was mapped to human chromosome 12q13. Human ASB-8 mRNA was expressed at the highest level of expression in skeletal muscle and at a varied level of expression in heart, brain, placenta, liver, kidney, and pancreas. The transcript of hASB-8 was not detected in adult normal lung tissue, but found in lung carcinoma cell lines SPC-A1, A549, and NCI-H446. Subcellular localization analysis showed that the EGFP-tagged hASB-8 protein was localized at cytoplasm in human hepatocellular carcinoma cell line BEL-7402. We also provided evidence that hASB-8 could interact with Elongin B-C complex in vitro. Furthermore, transfection with the truncated mutant of hASB-8 cDNA lacking SOCS box could suppress cell growth of lung adenocarcinoma SPC-A1 cells in vitro, which suggests that this gene might be related to the development of lung cancer.

  7. A cDNA clone for human glucosamine-6-sulphatase reveals differences between arylsulphatases and non-arylsulphatases.

    PubMed Central

    Robertson, D A; Freeman, C; Morris, C P; Hopwood, J J

    1992-01-01

    Glucosamine-6-sulphatase is an exo-hydrolase required for the lysosomal degradation of heparan sulphate and keratan sulphate. Deficiency of glucosamine-6-sulphatase activity leads to the lysosomal storage of the glycosaminoglycan, heparan sulphate and the monosaccharide sulphate N-acetylglucosamine 6-sulphate and the autosomal recessive genetic disorder mucopolysaccharidosis type IIID. Glucosamine-6-sulphatase can be classified as a non-arylsulphatase since, relative to arylsulphatase B, it shows negligible activity toward 4-methylumbelliferyl sulphate. We have isolated human cDNA clones and derived amino acid sequence coding for the entire glucosamine-6-sulphatase protein. The predicted sequence has 552 amino acids with a leader peptide of 36 amino acids and contains 13 potential N-glycosylation sites, of which it is likely that 10 are used. Glucosamine-6-sulphatase shows strong sequence similarity to other sulphatases such as the family of arylsulphatases, although the degree of similarity is not as high as that between members of the arylsulphatase family. This pattern of inter- and intra-family similarity delineates regions and amino acid residues that may be critical for sulphatase function and substrate specificity. PMID:1463457

  8. apl-1, a Caenorhabditis elegans gene encoding a protein related to the human beta-amyloid protein precursor.

    PubMed Central

    Daigle, I; Li, C

    1993-01-01

    The major component of senile plaques found in the brains of Alzheimer disease patients is the beta-amyloid peptide, which is derived from a larger amyloid precursor protein (APP). Recently, a number of APP and APP-related proteins have been identified in different organisms and constitute the family of APP proteins. We have isolated several cDNAs encoding an APP-related protein in the nematode Caenorhabditis elegans and have designated the corresponding gene as apl-1. The apl-1 transcripts undergo two forms of posttranscriptional modification: trans-splicing and alternative polyadenylylation. In vitro translation of an apl-1 cDNA results in a protein of approximately the expected size. Similar to the Drosophila, human, and mouse APP-related proteins, APL-1 does not appear to contain the beta-amyloid peptide. Because APP-related proteins seem to be conserved through evolution, the apl-1 gene from C. elegans should be important for determining the normal function of human APP. Images Fig. 2 Fig. 3 PMID:8265668

  9. Cloning of human Ca2+ homoeostasis endoplasmic reticulum protein (CHERP): regulated expression of antisense cDNA depletes CHERP, inhibits intracellular Ca2+ mobilization and decreases cell proliferation.

    PubMed Central

    Laplante, J M; O'Rourke, F; Lu, X; Fein, A; Olsen, A; Feinstein, M B

    2000-01-01

    A monoclonal antibody which blocks InsP(3)-induced Ca(2+) release from isolated endoplasmic reticulum was used to isolate a novel 4.0 kb cDNA from a human erythroleukaemia (HEL) cell cDNA expression library. A corresponding mRNA transcript of approx. 4.2 kb was present in all human cell lines and tissues examined, but cardiac and skeletal muscle had an additional transcript of 6.4 kb. The identification in GenBank(R) of homologous expressed sequence tags from many tissues and organisms suggests that the gene is ubiquitously expressed in higher eukaryotes. The gene was mapped to human chromosome 19p13.1. The cDNA predicts a 100 kDa protein, designated Ca(2+) homoeostasis endoplasmic reticulum protein (CHERP), with two putative transmembrane domains, multiple consensus phosphorylation sites, a polyglutamine tract of 12 repeats and regions of imperfect tryptophan and histadine octa- and nona-peptide repeats. In vitro translation of the full-length cDNA produced proteins of M(r) 128000 and 100000, corresponding to protein bands detected by Western blotting of many cell types. CHERP was co-localized in HEL cells with the InsP(3) receptor by two-colour immunofluorescence. Transfection of HEL cells with antisense cDNA led to an 80% decline in CHERP within 5 days of antisense induction, with markedly decreased intracellular Ca(2+) mobilization by thrombin, decreased DNA synthesis and growth arrest, indicating that the protein has an important function in Ca(2+) homoeostasis, growth and proliferation. PMID:10794731

  10. Isolation and characterization of cDNA clones encoding the Drosophila homolog of the HMG-box SSRP family that recognizes specific DNA structures.

    PubMed Central

    Bruhn, S L; Housman, D E; Lippard, S J

    1993-01-01

    Recently an HMG-box protein denoted SSRP1, for structure-specific recognition protein 1, has been discovered which binds to specific DNA structural elements such as the bent, unwound conformations that occur upon the formation of intrastrand crosslinks by the anticancer drug cisplatin. The SSRP family includes the mouse protein T160, which recognizes recombination signal sequences. In order to delineate functional domains more clearly, a homolog of SSRP1 was cloned from Drosophila melanogaster. This homolog maps to polytene region 60A (1-4) and shares 54% identity with human SSRP1. Comparison of the predicted amino acid sequences among SSRP family members reveals 48% identity, with structural conservation in the carboxy terminus of the HMG box as well as domains of highly charged residues. Interestingly, however, the most highly conserved regions of the protein are in the less well understood amino terminus, strongly suggesting that this portion of the protein is critical for its function. Images PMID:8479916

  11. Characterization of Leukemia-Inducing Genes Using a Proto-Oncogene/Homeobox Gene Retroviral Human cDNA Library in a Mouse In Vivo Model.

    PubMed

    Jang, Su Hwa; Lee, Sohyun; Chung, Hee Yong

    2015-01-01

    The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML) using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM) cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30%) had MYC as the only transgene, and seven mice (70%) had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy.

  12. A Drosophila gene encoding a protein resembling the human. beta. -amyloid protein precursor

    SciTech Connect

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K. )

    1989-04-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human {beta}-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human {beta}-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.

  13. A Drosophila gene encoding a protein resembling the human beta-amyloid protein precursor.

    PubMed Central

    Rosen, D R; Martin-Morris, L; Luo, L Q; White, K

    1989-01-01

    We have isolated genomic and cDNA clones for a Drosophila gene resembling the human beta-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human beta-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development. Images PMID:2494667

  14. Isolation and characterization of a cDNA clone encoding an auxin influx carrier in carnation cuttings. Expression in different organs and cultivars and its relationship with cold storage.

    PubMed

    Oliveros-Valenzuela, María Del Rocío; Reyes, David; Sánchez-Bravo, José; Acosta, Manuel; Nicolás, Carlos

    2008-12-01

    Polar auxin transport (PAT) is necessary for the formation of adventitious roots in the base of leafy stem cuttings, as has been demonstrated in several studies in which the application of PAT inhibitors strongly inhibited the rooting of cuttings. However, unlike in the case of lateral roots, there is almost no information on the molecular mechanism that controls PAT in the formation of adventitious roots. A novel cDNA encoding an auxin influx carrier has been isolated and characterized from carnation (Dianthus caryophyllus) cuttings. The full length of DcAUX1 was obtained and the deduced aminoacid sequence revealed a high degree of identity with the corresponding auxin carrier proteins from several species. The expression of this gene depended on the organ, the carnation cultivar and the length of time cuttings had been stored in a cold chamber. As a rule, expression was higher in stem than in leaves, in the basal than in the first internode and in mature than in young leaves irrespective of the cultivar and the duration of the storage. This pattern of expression agrees with the results of a previous study showing that auxin from mature leaves was essential for rooting, while exogenous auxin applied to mature leaves was polarly transported in the stem and accumulated in the basal internode (the rooting zone). Variations in the expression observed during storage (depending of the cultivar) might be related to the variation in PAT and rooting reported in previous studies.

  15. cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules

    SciTech Connect

    Claesson-Welsh, L.; Eriksson, A.; Moren, A.; Severinsson, L.; Ek, B.; Ostman, A.; Betsholtz, C.; Heldin, C.H.

    1988-08-01

    The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGFR receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGR receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different /sup 125/I-labeled dimeric forms of PDGF A and B chains showed that the PDGFR receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA.

  16. Isolation of a complementary DNA clone encoding a precursor to human eosinophil major basic protein

    PubMed Central

    1988-01-01

    A 14-kD protein was purified from human PMNs and its NH2-terminal sequence was determined. Comparison of a portion of the NH2-terminal sequence of this protein to the recently reported NH2-terminal sequence of eosinophil major basic protein (MBP) showed them to be identical. To aid further characterization of the structural and functional properties of this molecule, we isolated from an HL-60 cDNA library a single class of cDNA clones whose sequence matched exactly the NH2- terminal amino acid sequence of the 14-kD polypeptide. Northern analysis of HL-60 cells suggests that MBP is constitutively expressed in HL-60 cells and is highly transcribed from a single copy gene. The sequence of the full-length cDNA clones predicts that MBP is synthesized as a 23-kD precursor form (pro-MBP) which is subsequently cleaved to release the mature 14-kD MBP. The putative pro-MBP has a predicted pI of 6.0, but both the charged and the hydrophobic residues are asymmetrically distributed, creating a bipolar molecule. The NH2- terminal half has a predicted pI of 3.7 and is hydrophilic, while the COOH-terminal half (corresponding to mature MBP) has a predicted pI of 11.1 and is hydrophobic. PMID:3199069

  17. Proteogenomic Analysis of Human Chromosome 9-Encoded Genes from Human Samples and Lung Cancer Tissues

    PubMed Central

    Ahn, Jung-Mo; Kim, Min-Sik; Kim, Yong-In; Jeong, Seul-Ki; Lee, Hyoung-Joo; Lee, Sun Hee; Paik, Young-Ki; Pandey, Akhilesh; Cho, Je-Yoel

    2014-01-01

    The Chromosome-centric Human Proteome Project (C-HPP) was recently initiated as an international collaborative effort. Our team adopted chromosome 9 (Chr 9) and performed a bioinformatics and proteogenomic analysis to catalog Chr 9-encoded proteins from normal tissues, lung cancer cell lines and lung cancer tissues. Approximately 74.7% of the Chr 9 genes of the human genome were identified, which included approximately 28% of missing proteins (46 of 162) on Chr 9 compared with the list of missing proteins from the neXtProt master table (2013-09). In addition, we performed a comparative proteomics analysis between normal lung and lung cancer tissues. Based on the data analysis, 15 proteins from Chr 9 were detected only in lung cancer tissues. Finally, we conducted a proteogenomic analysis to discover Chr 9-residing single nucleotide polymorphisms (SNP) and mutations described in the COSMIC cancer mutation database. We identified 21 SNPs and 4 mutations containing peptides on Chr 9 from normal human cells/tissues and lung cancer cell lines, respectively. In summary, this study provides valuable information of the human proteome for the scientific community as part of C-HPP. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD. PMID:24274035

  18. Construction of a genome-length cDNA clone for human astrovirus serotype 1 and synthesis of infectious RNA transcripts.

    PubMed

    Geigenmüller, U; Ginzton, N H; Matsui, S M

    1997-02-01

    We have constructed a genome-length cDNA clone for human astrovirus serotype 1. When a human colon cancer-derived cell line, CaCo-2, is transfected with RNA transcribed in vitro from this cDNA clone, infectious virus is produced at titers close to those observed after infection with intact astrovirus. A rodent cell line, BHK, which is largely refractory to astrovirus infection, was found to support efficient growth of the virus if transfected with viral RNA. The high transfection efficiency seen in the BHK cells allows studies of the viral replication in the transfected cells and thus should prove useful for the characterization of noninfectious astroviral mutants.

  19. Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens

    SciTech Connect

    Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J. )

    1990-09-01

    The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins.

  20. Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme.

    PubMed

    Dierks, Thomas; Schmidt, Bernhard; Borissenko, Ljudmila V; Peng, Jianhe; Preusser, Andrea; Mariappan, Malaiyalam; von Figura, Kurt

    2003-05-16

    C(alpha)-formylglycine (FGly) is the catalytic residue in the active site of eukaryotic sulfatases. It is posttranslationally generated from a cysteine in the endoplasmic reticulum. The genetic defect of FGly formation causes multiple sulfatase deficiency (MSD), a lysosomal storage disorder. We purified the FGly generating enzyme (FGE) and identified its gene and nine mutations in seven MSD patients. In patient fibroblasts, the activity of sulfatases is partially restored by transduction of FGE encoding cDNA, but not by cDNA carrying an MSD mutation. The gene encoding FGE is highly conserved among pro- and eukaryotes and has a paralog of unknown function in vertebrates. FGE is localized in the endoplasmic reticulum and is predicted to have a tripartite domain structure.

  1. Gene encoding human Ro-associated autoantigen Y5 RNA.

    PubMed Central

    Maraia, R; Sakulich, A L; Brinkmann, E; Green, E D

    1996-01-01

    Ro ribonucleoproteins are composed of Y RNAs and the Ro 60 kDa protein. While the Ro 60 kDa protein is implicated in an RNA discard pathway that recognizes 3'-extended 5S rRNAs, the function of Y RNAs remains unknown [O'Brien,C.A. and Wolin,S.L. (1995) Genes Dev. 8,2891-2903]. Y5 RNA occupies a large fraction of Ro 60 kDa protein in human Ro RNPs, contains an atypical 3'-extension not found on other Y RNAs, and constitutes an RNA antigen in certain autoimmune patients [Boulanger et al. (1995) Clin. Exp. Immunol. 99, 29-36]. An overabundance of Y RNA retroposed pseudogenes has previously complicated the isolation of mammalian Y RNA genes. The source gene for Y5 RNA was isolated from human DNA as well as from Galago senegalis DNA. Authenticity of the hY5 RNA gene was demonstrated in vivo and its activity was compared with the hY4 RNA gene that also uses a type 3 promoter for RNA polymerase III. The hY5 RNA gene was subsequently found to reside within a few hundred thousand base pairs of other Y RNA genes and the linear order of the four human Y RNA genes on chromosome 7q36 was determined. Phylogenetic comparative analyses of promoter and RNA structure indicate that the Y5 RNA gene has been subjected to positive selection during primate evolution. Consistent with the proposal of O'Brien and Harley [O'Brian,C.A. and Wolin,S.L. (1992) Gene 116, 285-289], analysis of flanking sequences suggest that the hY5 RNA gene may have originated as a retroposon. PMID:8836182

  2. An mRNA encoding a putative GABA-gated chloride channel is expressed in the human cardiac conduction system.

    PubMed

    Garret, M; Bascles, L; Boue-Grabot, E; Sartor, P; Charron, G; Bloch, B; Margolskee, R F

    1997-04-01

    GABA-gated chloride channels are the main inhibitory neurotransmitter receptors in the CNS. Conserved domains among members of previously described GABAA receptor subunits were used to design degenerate sense and antisense oligonucleotides. A PCR product from this amplification was used to isolate a full-length cDNA. The predicted protein has many of the features shared by other members of the ligand-gated ion channel family. This channel subunit has significant amino acid identity (25-40%) with members of GABAA and GABAC receptor subunits and thus may represent a new subfamily of the GABA receptor channel. Although we cannot rule out that this clone encodes a receptor for an unidentified ligand, it was termed GABA chi. This gene is mainly expressed in placenta and in heart; however, placenta appears to express only an unspliced mRNA. In situ hybridization reveals that the GABA chi subunit mRNA is present in the electrical conduction system of the human heart. Our results suggest that novel GABA receptors expressed outside of the CNS may regulate cardiac function.

  3. Two putative subunits of a peptide pump encoded in the human major histocompatibility complex class II region.

    PubMed Central

    Bahram, S; Arnold, D; Bresnahan, M; Strominger, J L; Spies, T

    1991-01-01

    The class II region of the human major histocompatibility complex (MHC) may encode several genes controlling the processing of endogenous antigen and the presentation of peptide epitopes by MHC class I molecules to cytotoxic T lymphocytes. A previously described peptide supply factor (PSF1) is a member of the multidrug-resistance family of transporters and may pump cytosolic peptides into the membrane-bound compartment where class I molecules assemble. A second transporter gene, PSF2, was identified 10 kilobases (kb) from PSF1, near the class II DOB gene. The complete sequences of PSF1 and PSF2 were determined from cDNA clones. The translation products are closely related in sequence and predicted secondary structure. Both contain a highly conserved ATP-binding fold and share 25% homology in a hydrophobic domain with a tentative number of eight membrane-spanning segments. Based on the principle dimeric organization of these two domains in other transporters, PSF1 and PSF2 may function as complementary subunits, independently as homodimers, or both. Taken together with previous genetic evidence, the coregulation of PSF1 and PSF2 by gamma interferon and the to-some-degree coordinate transcription of these genes suggest a common role in peptide-loading of class I molecules, although a distinct function of PSF2 cannot be ruled out. Images PMID:1946428

  4. Architecture of the human regulatory network derived from ENCODE data.

    PubMed

    Gerstein, Mark B; Kundaje, Anshul; Hariharan, Manoj; Landt, Stephen G; Yan, Koon-Kiu; Cheng, Chao; Mu, Xinmeng Jasmine; Khurana, Ekta; Rozowsky, Joel; Alexander, Roger; Min, Renqiang; Alves, Pedro; Abyzov, Alexej; Addleman, Nick; Bhardwaj, Nitin; Boyle, Alan P; Cayting, Philip; Charos, Alexandra; Chen, David Z; Cheng, Yong; Clarke, Declan; Eastman, Catharine; Euskirchen, Ghia; Frietze, Seth; Fu, Yao; Gertz, Jason; Grubert, Fabian; Harmanci, Arif; Jain, Preti; Kasowski, Maya; Lacroute, Phil; Leng, Jing; Lian, Jin; Monahan, Hannah; O'Geen, Henriette; Ouyang, Zhengqing; Partridge, E Christopher; Patacsil, Dorrelyn; Pauli, Florencia; Raha, Debasish; Ramirez, Lucia; Reddy, Timothy E; Reed, Brian; Shi, Minyi; Slifer, Teri; Wang, Jing; Wu, Linfeng; Yang, Xinqiong; Yip, Kevin Y; Zilberman-Schapira, Gili; Batzoglou, Serafim; Sidow, Arend; Farnham, Peggy J; Myers, Richard M; Weissman, Sherman M; Snyder, Michael

    2012-09-06

    Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.

  5. Dynamic Encoding of Face Information in the Human Fusiform Gyrus

    PubMed Central

    Ghuman, Avniel Singh; Brunet, Nicolas M.; Li, Yuanning; Konecky, Roma O.; Pyles, John A.; Walls, Shawn A.; Destefino, Vincent; Wang, Wei; Richardson, R. Mark

    2014-01-01

    Humans’ ability to rapidly and accurately detect, identify, and classify faces under variable conditions derives from a network of brain regions highly tuned to face information. The fusiform face area (FFA) is thought to be a computational hub for face processing, however temporal dynamics of face information processing in FFA remains unclear. Here we use multivariate pattern classification to decode the temporal dynamics of expression-invariant face information processing using electrodes placed directly upon FFA in humans. Early FFA activity (50-75 ms) contained information regarding whether participants were viewing a face. Activity between 200-500 ms contained expression-invariant information about which of 70 faces participants were viewing along with the individual differences in facial features and their configurations. Long-lasting (500+ ms) broadband gamma frequency activity predicted task performance. These results elucidate the dynamic computational role FFA plays in multiple face processing stages and indicate what information is used in performing these visual analyses. PMID:25482825

  6. Production of Human Albumin in Pigs Through CRISPR/Cas9-Mediated Knockin of Human cDNA into Swine Albumin Locus in the Zygotes.

    PubMed

    Peng, Jin; Wang, Yong; Jiang, Junyi; Zhou, Xiaoyang; Song, Lei; Wang, Lulu; Ding, Chen; Qin, Jun; Liu, Liping; Wang, Weihua; Liu, Jianqiao; Huang, Xingxu; Wei, Hong; Zhang, Pumin

    2015-11-12

    Precise genome modification in large domesticated animals is desirable under many circumstances. In the past it is only possible through lengthy and burdensome cloning procedures. Here we attempted to achieve that goal through the use of the newest genome-modifying tool CRISPR/Cas9. We set out to knockin human albumin cDNA into pig Alb locus for the production of recombinant human serum albumin (rHSA). HSA is a widely used human blood product and is in high demand. We show that homologous recombination can occur highly efficiently in swine zygotes. All 16 piglets born from the manipulated zygotes carry the expected knockin allele and we demonstrated the presence of human albumin in the blood of these piglets. Furthermore, the knockin allele was successfully transmitted through germline. This success in precision genomic engineering is expected to spur exploration of pigs and other large domesticated animals to be used as bioreactors for the production of biomedical products or creation of livestock strains with more desirable traits.

  7. Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening

    SciTech Connect

    Robertson, N.G.; Gutierrez-Espeleta, G.A.; Bieber, F.R. |

    1994-09-01

    We used a combination of subtractive hybridization and differential screening strategies to identify genes that may function normally in hearing and, when mutated, result in deafness. A human fetal cochlear (membranous labyrinth) cDNA library was subtracted against total human fetal brain RNAs by an avidin-biotin-based procedure to enrich for cochlear transcripts. Subtracted cochlear clones were differentially screened with {sup 32}P-labeled total cochlear and total brain cDNA probes. Sequence analysis of clones that hybridized more intensely with cochlear than with brain cDNA probes revealed some previously characterized genes, including mitochondrial sequences, collagen type I {alpha}-2 (COL1A2), collagen type II {alpha}-1 (COL2A1), collagen type III {alpha}-1 (COL3A1), spermidine/spermine N{sup 1}-acetyltransferase (SAT), osteonectin (SPARC), and peripheral myelin protein 22 (PMP22). Also identified were clones that are potential novel cochlear genes. Northern blots of cochlear and brain RNAs probed with COL1A2, COL2A1, COL3A1, SAT, SPARC, PMP22, and a novel sequence, designated Coch-5B2, confirm results of the subtractive procedure by showing preferential cochlear expression. A number of these genes serve structural or regulatory functions in extracellular matrix or neural conduction; defects in some of these genes are associated with disorders involving hearing loss. Partial sequence analysis of Coch-5B2 reveals a von Willebrand factor type A-like domain in this cDNA. To assess the cochlear specificity of Coch-5B2, a Northern blot panel of 14 human fetal tissue RNAs was probed with Coch-5B2, showing differential expression of this novel gene in the cochlea. 68 refs., 3 figs.

  8. GENCODE: the reference human genome annotation for The ENCODE Project.

    PubMed

    Harrow, Jennifer; Frankish, Adam; Gonzalez, Jose M; Tapanari, Electra; Diekhans, Mark; Kokocinski, Felix; Aken, Bronwen L; Barrell, Daniel; Zadissa, Amonida; Searle, Stephen; Barnes, If; Bignell, Alexandra; Boychenko, Veronika; Hunt, Toby; Kay, Mike; Mukherjee, Gaurab; Rajan, Jeena; Despacio-Reyes, Gloria; Saunders, Gary; Steward, Charles; Harte, Rachel; Lin, Michael; Howald, Cédric; Tanzer, Andrea; Derrien, Thomas; Chrast, Jacqueline; Walters, Nathalie; Balasubramanian, Suganthi; Pei, Baikang; Tress, Michael; Rodriguez, Jose Manuel; Ezkurdia, Iakes; van Baren, Jeltje; Brent, Michael; Haussler, David; Kellis, Manolis; Valencia, Alfonso; Reymond, Alexandre; Gerstein, Mark; Guigó, Roderic; Hubbard, Tim J

    2012-09-01

    The GENCODE Consortium aims to identify all gene features in the human genome using a combination of computational analysis, manual annotation, and experimental validation. Since the first public release of this annotation data set, few new protein-coding loci have been added, yet the number of alternative splicing transcripts annotated has steadily increased. The GENCODE 7 release contains 20,687 protein-coding and 9640 long noncoding RNA loci and has 33,977 coding transcripts not represented in UCSC genes and RefSeq. It also has the most comprehensive annotation of long noncoding RNA (lncRNA) loci publicly available with the predominant transcript form consisting of two exons. We have examined the completeness of the transcript annotation and found that 35% of transcriptional start sites are supported by CAGE clusters and 62% of protein-coding genes have annotated polyA sites. Over one-third of GENCODE protein-coding genes are supported by peptide hits derived from mass spectrometry spectra submitted to Peptide Atlas. New models derived from the Illumina Body Map 2.0 RNA-seq data identify 3689 new loci not currently in GENCODE, of which 3127 consist of two exon models indicating that they are possibly unannotated long noncoding loci. GENCODE 7 is publicly available from gencodegenes.org and via the Ensembl and UCSC Genome Browsers.

  9. Encoding of physics concepts: concreteness and presentation modality reflected by human brain dynamics.

    PubMed

    Lai, Kevin; She, Hsiao-Ching; Chen, Sheng-Chang; Chou, Wen-Chi; Huang, Li-Yu; Jung, Tzyy-Ping; Gramann, Klaus

    2012-01-01

    Previous research into working memory has focused on activations in different brain areas accompanying either different presentation modalities (verbal vs. non-verbal) or concreteness (abstract vs. concrete) of non-science concepts. Less research has been conducted investigating how scientific concepts are learned and further processed in working memory. To bridge this gap, the present study investigated human brain dynamics associated with encoding of physics concepts, taking both presentation modality and concreteness into account. Results of this study revealed greater theta and low-beta synchronization in the anterior cingulate cortex (ACC) during encoding of concrete pictures as compared to the encoding of both high and low imageable words. In visual brain areas, greater theta activity accompanying stimulus onsets was observed for words as compared to pictures while stronger alpha suppression was observed in responses to pictures as compared to words. In general, the EEG oscillation patterns for encoding words of different levels of abstractness were comparable but differed significantly from encoding of pictures. These results provide insights into the effects of modality of presentation on human encoding of scientific concepts and thus might help in developing new ways to better teach scientific concepts in class.

  10. Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-)

    SciTech Connect

    Hirono, A.; Beutler, E. )

    1988-06-01

    Glucose-6-phosphate dehydrogenase A(-) is a common variant in Blacks that causes sensitivity to drug- and infection-induced hemolytic anemia. A cDNA library was constructed from Epstein-Barr virus-transformed lymphoblastoid cells from a male who was G6PD A(-). One of four cDNA clones isolated contained a sequence not found in the other clones nor in the published cDNA sequence. Consisting of 138 bases and coding 46 amino acids, this segment of cDNA apparently is derived from the alternative splicing involving the 3{prime} end of intron 7. Comparison of the remaining sequences of these clones with the published sequence revealed three nucleotide substitutions: C{sup 33} {yields} G, G{sup 202} {yields} A, and A{sup 376} {yields} G. Each change produces a new restriction site. Genomic DNA from five G6PD A(-) individuals was amplified by the polymerase chain reaction. The findings of the same mutation in G6PD A(-) as is found in G6PD A(+) strongly suggests that the G6PD A(-) mutation arose in an individual with G6PD A(+), adding another mutation that causes the in vivo instability of this enzyme protein.

  11. Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus

    SciTech Connect

    Kwon, B.S.; Haq, A.K.; Pomerantz, S.H.; Halaban, R.

    1987-11-01

    Screening of a lambdagt11 human melanocyte cDNA library with antibodies against hamster tyrosinase resulted in the isolation of 16 clones. The cDNA inserts from 13 of the 16 clones cross-hybridized with each other, indicating that they were form related mRNA species. One of the cDNA clones, Pmel34, detected one mRNA species with an approximate length of 2.4 kilobases that was expressed preferentially in normal and malignant melanocytes but not in other cell types. The amino acid sequence deduced from the nucleotide sequence showed that the putative human tyrosinase is composed of 548 amino acids with a molecular weight of 62,610. The deduced protein contains glycosylation sites and histidine-rich sites that could be used for copper binding. Southern blot analysis of DNA derived from newborn mice carrying lethal albino deletion mutations revealed that Pmel34 maps near or at the c-albino locus, the position of the structural gene for tyrosinase.

  12. Interferon-induced 56,000 Mr protein and its mRNA in human cells: molecular cloning and partial sequence of the cDNA.

    PubMed Central

    Chebath, J; Merlin, G; Metz, R; Benech, P; Revel, M

    1983-01-01

    Treatment of responsive cells by interferons (IFNs) induces within a few hours a rise in the concentration of several proteins and mRNAs. In order to characterize these IFN-induced mRNA species, we have cloned in E. coli the cDNA made from a 17-18S poly(A)+ RNA of human fibroblastoid cells (SV80) treated with IFN-beta. We describe here a pBR322 recombinant plasmid (C56) which contains a 400 bp cDNA insert corresponding to a 18S mRNA species newly induced by IFN. The C56 mRNA codes for a 56,000 dalton protein easily detectable by hybridization-translation experiments. The sequence of 66 of the carboxy-terminal amino-acids of the protein can be deduced from the cDNA sequence. IFNs-alpha, beta or gamma are able to activate the expression of this gene in human fibroblasts as well as lymphoblastoid cells. The mRNA is not detectable without IFN; it reaches maximum levels (0.1% of the total poly(A)+ RNA) within 4-8 hrs and decreases after 16 hrs. Images PMID:6186990

  13. Characterization of human antibody-reactive epitopes encoded by human papillomavirus types 16 and 18.

    PubMed Central

    Jenison, S A; Yu, X P; Valentine, J M; Galloway, D A

    1991-01-01

    We have previously reported that the most common human serum immunoglobulin G antibody reactivities to human papillomavirus type 16 and type 18 (HPV16 and HPV18)-encoded proteins are directed against the minor capsid proteins (HPV16 L2 and HPV18 L2) and to the E7 protein of HPV16 (S. A. Jenison, X.-P. Yu, J. M. Valentine, L. A. Koutsky, A. E. Christiansen, A. M. Beckmann, and D. A. Galloway, J. Infect. Dis. 162:60-69, 1990). In this study, the antibody-reactive segments of the HPV16 E7, HPV16 L2, and HPV18 L2 polypeptides were mapped by using nested sets of deleted recombinant proteins. A single major immunoreactive region was identified in the HPV16 E7 polypeptide between amino acids (aa) 21 and 34 (DLYCYE-QLNDSSEE). In contrast, three distinct immunoreactive regions of the HPV16 L2 polypeptide were present in the segment between aa149 and aa204, and three distinct immunoreactive regions of the HPV18 L2 polypeptide were present in the segment between aa110 and aa211. With the exception of one serum sample, serum immunoglobulin G antibodies which reacted with HPV16 L2 polypeptides or with HPV18 L2 polypeptides were not cross-reactive. Images PMID:1704924

  14. Expression of sialyl-Tn antigen in breast cancer cells transfected with the human CMP-Neu5Ac: GalNAc alpha2,6-sialyltransferase (ST6GalNac I) cDNA.

    PubMed

    Julien, S; Krzewinski-Recchi, M A; Harduin-Lepers, A; Gouyer, V; Huet, G; Le Bourhis, X; Delannoy, P

    2001-01-01

    Sialyl-Tn antigen (STn) is a cancer associated carbohydrate antigen over-expressed in several cancers including breast cancer, and currently associated with more aggressive diseases and poor prognosis. However, the commonly used breast cancer cell lines (MDA-MB-231, T47-D and MCF7) do not express STn antigen. The key step in the biosynthesis of STn is the transfer of a sialic acid residue in alpha2,6-linkage to GalNAc alpha-O-Ser/Thr. This reaction is mainly catalyzed by a CMP-Neu5Ac GalNAc alpha2,6-sialyltransferase: ST6GalNAc I. In order to generate STn-positive breast cancer cells, we have cloned a cDNA encoding the full-length human ST6GalNAc I from HT-29-MTX cells. The stable transfection of MDA-MB-231 with an expression vector encoding ST6GalNAc I induces the expression of STn antigen at the cell surface. The expression of STn short cuts the initial O-glycosylation pattern of these cell lines, by competing with the Core-1 beta1,3-galactosyltransferase, the first enzyme involved in the elongation of O-glycan chains. Moreover, we show that STn expression is associated with morphological changes, decreased growth and increased migration of MDA-MB-231 cells.

  15. Nucleic acids encoding mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

    DOEpatents

    Korber, Bette T; Fischer, William; Liao, Hua-Xin; Haynes, Barton F; Letvin, Norman; Hahn, Beatrice H

    2015-04-21

    The present invention relates to nucleic acids encoding mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

  16. Mapping of the first preferentially expressed cDNA in human fetal cochlea to human 14q11.2-12 and to a region of homologous synteny on mouse chromosome 12

    SciTech Connect

    Robertson, N.G.; Weremowicz, S.; Kovatch, K.A.

    1994-09-01

    We have isolated a cDNA, Coch-5B2 (D14S564E) from a human fetal cochlear cDNA library by subtractive hybridization and differential screening methods. This is the first cDNA to date shown to be expressed preferentially in human fetal cochlea (membranous labyrinth). On Northern blot of a panel of 14 human fetal tissue RNAs including cochlea, brain, liver, spleen, skeletal muscle, kidney, lung, skin, thymus, adrenal, small intestine, eye, sternal cartilage, and cultured fibroblasts, very high level expression of D14S564E is seen only in cochlea; very faint bands are discernible in brain and eye. Sequence comparison of this clone to sequences in GenBank/EMBL data bases shows no match to any known genes, indicating that it represents a novel cochlear sequence. Chromosome localization of this cochlear cDNA may provide insight into a region of the human genome to which human deafness disorders may map. We have assigned D14S564E to human chromosome 14 using the NIGMS human/rodent somatic cell hybrid mapping panel 1, and regionally to q11.2-q12 by fluorescence in situ hybridization (FISH). Besides detection of the human genomic band on the hybrid panel, genomic bands were seen for mouse and hamster, demonstrating evolutionary conservation of D14S564E. By FISH, signal was detected on human 14q11.2-q12 in 20 metaphases. In 3 metaphases, signal was present on both chromosome 14s. The mouse homolog of this cochlear cDNA was also used to probe human metaphases by FISH: signal was detected in the same region, 14q11.2-12, as the human clone in 5 metaphases, confirming human mapping data and homology to the human cDNA. The human cochlear D14S564E was genetically mapped in the mouse to chromosome 12, in a region of homology with human 14q11.2-q12. This region on mouse 12 contains the asp-1 (audiogenic seizure prone) locus and future studies will be directed at determining whether D14S564E is a candidate gene for this disorder.

  17. Characterization of a female-specific cDNA derived from a developmentally regulated mRNA in the human blood fluke Schistosoma mansoni.

    PubMed

    Bobek, L; Rekosh, D M; van Keulen, H; LoVerde, P T

    1986-08-01

    We have isolated and characterized a cDNA clone that is derived from a developmentally regulated mRNA found only in mature female schistosomes. The mRNA is approximately 950 nucleotides in length and is not detectable in immature female schistosomes isolated from single-sex infections, in male worms, or in eggs. During normal bisexual infections, the mRNA species is first detected 28 days after infection (the time of worm pairing) and increases to a high level 35 days after infection, coinciding with the start of egg production. The nucleotide sequence of the cDNA shows two large open reading frames in the coding strand. Several features of the clone, including the deduced sequence of the polypeptide encoded by one of the reading frames, suggest a relationship to the silk moth chorion (egg shell) gene family. The isolation of this clone provides us with a probe for further studies of female schistosome development and is a first step toward a detailed understanding of this process at the molecular level.

  18. Human collagen genes encoding basement membrane. cap alpha. 1(IV) and. cap alpha. 2(IV) chains map to the distal long arm of chromosome 13

    SciTech Connect

    Griffin, C.A.; Emanuel, B.S.; Hansen, J.R.; Cavenee, W.K.; Myers, J.C.

    1987-01-01

    At least 20 genes encode the structurally related collagen chains that comprise > 10 homo- or heterotrimeric types. Six members of this multigene family have been assigned to five chromosomes in the human genome. The two type I genes, ..cap alpha..1 and ..cap alpha..2, are located on chromosomes 17 and 7, respectively, and the ..cap alpha..1(II) gene is located on chromosome 12. Their recent mapping of the ..cap alpha..1(III) and ..cap alpha..2(V) genes to the q24.3 ..-->.. q31 region of chromosome 2 provided the only evidence that the collagen genes are not entirely dispersed. To further determine their organization, the authors and others localized the ..cap alpha..1(IV) gene to chromosome 13 and in their experiments sublocalized the gene to band q34 by in situ hybridization. Here they show the presence of the ..cap alpha..2 type IV locus also on the distal long arm of chromosome 13 by hybridizing a human ..cap alpha..2(IV) cDNA clone to rodent-human hybrids and to metaphase chromosomes. These studies represent the only demonstration of linkage between genes encoding both polypeptide chains of the same collagen type.

  19. Timing predictability enhances regularity encoding in the human subcortical auditory pathway.

    PubMed

    Gorina-Careta, Natàlia; Zarnowiec, Katarzyna; Costa-Faidella, Jordi; Escera, Carles

    2016-11-17

    The encoding of temporal regularities is a critical property of the auditory system, as short-term neural representations of environmental statistics serve to auditory object formation and detection of potentially relevant novel stimuli. A putative neural mechanism underlying regularity encoding is repetition suppression, the reduction of neural activity to repeated stimulation. Although repetitive stimulation per se has shown to reduce auditory neural activity in animal cortical and subcortical levels and in the human cerebral cortex, other factors such as timing may influence the encoding of statistical regularities. This study was set out to investigate whether temporal predictability in the ongoing auditory input modulates repetition suppression in subcortical stages of the auditory processing hierarchy. Human auditory frequency-following responses (FFR) were recorded to a repeating consonant-vowel stimuli (/wa/) delivered in temporally predictable and unpredictable conditions. FFR amplitude was attenuated by repetition independently of temporal predictability, yet we observed an accentuated suppression when the incoming stimulation was temporally predictable. These findings support the view that regularity encoding spans across the auditory hierarchy and point to temporal predictability as a modulatory factor of regularity encoding in early stages of the auditory pathway.

  20. Timing predictability enhances regularity encoding in the human subcortical auditory pathway

    PubMed Central

    Gorina-Careta, Natàlia; Zarnowiec, Katarzyna; Costa-Faidella, Jordi; Escera, Carles

    2016-01-01

    The encoding of temporal regularities is a critical property of the auditory system, as short-term neural representations of environmental statistics serve to auditory object formation and detection of potentially relevant novel stimuli. A putative neural mechanism underlying regularity encoding is repetition suppression, the reduction of neural activity to repeated stimulation. Although repetitive stimulation per se has shown to reduce auditory neural activity in animal cortical and subcortical levels and in the human cerebral cortex, other factors such as timing may influence the encoding of statistical regularities. This study was set out to investigate whether temporal predictability in the ongoing auditory input modulates repetition suppression in subcortical stages of the auditory processing hierarchy. Human auditory frequency–following responses (FFR) were recorded to a repeating consonant–vowel stimuli (/wa/) delivered in temporally predictable and unpredictable conditions. FFR amplitude was attenuated by repetition independently of temporal predictability, yet we observed an accentuated suppression when the incoming stimulation was temporally predictable. These findings support the view that regularity encoding spans across the auditory hierarchy and point to temporal predictability as a modulatory factor of regularity encoding in early stages of the auditory pathway. PMID:27853313

  1. Human Genetic Disorders Caused by Mutations in Genes Encoding Biosynthetic Enzymes for Sulfated Glycosaminoglycans*

    PubMed Central

    Mizumoto, Shuji; Ikegawa, Shiro; Sugahara, Kazuyuki

    2013-01-01

    A number of genetic disorders are caused by mutations in the genes encoding glycosyltransferases and sulfotransferases, enzymes responsible for the synthesis of sulfated glycosaminoglycan (GAG) side chains of proteoglycans, including chondroitin sulfate, dermatan sulfate, and heparan sulfate. The phenotypes of these genetic disorders reflect disturbances in crucial biological functions of GAGs in human. Recent studies have revealed that mutations in genes encoding chondroitin sulfate and dermatan sulfate biosynthetic enzymes cause various disorders of connective tissues. This minireview focuses on growing glycobiological studies of recently described genetic diseases caused by disturbances in biosynthetic enzymes for sulfated GAGs. PMID:23457301

  2. A model organism for new gene discovery by cDNA sequencing

    SciTech Connect

    El-Saved, N.M.; Donelson, J.E.; Alarcon, C.M.

    1994-09-01

    One method of new gene discovery is single pass sequencing of cDNAs to identify expressed sequence tags (ESTs). Model organisms can have biological properties which makes their use advantageous over studies with humans. One such model organism with advantages for cDNA sequencing is the African trypanosome T. brucei rhodesiense. This organism has the same 40 nucleotide sequence (splice leader sequence) on the 5{prime} end of all mRNAs. We have constructed a 5{prime} cDNA library by priming off the splice leader sequence and have begun sequencing this cDNA library. To date, over nearly 500 such cDNA expressed sequence tags (ESTs) have been examined. Forty-three percent of the sequences sampled from the trypanosome cDNA library have significant similarities to sequences already in the protein and translated nucleic acid databases. Among these are cDNA sequences which encode previously reported T. brucej proteins such as the VSG, tubulin, calflagin, etc., and proteins previously identified in other trypanosomatids. Other cDNAs display significant similarities to genes in unrelated organisms encoding several ribosomal proteins, metabolic enzymes, GTP binding proteins, transcription factors, cyclophillin, nucleosomal histones, histone H1, and a macrophage stress protein, among others. The 57% of the cDNAs that are not similar to sequences currently in the databases likely encode both trypanosome-specific proteins and housekeeping proteins shared with other eukaryotes. These cDNA ESTs provide new avenues of research for exploring both the biochemistry and the genome organization of this parasite, as well as a resource for identifying the 5{prime} sequence of novel genes likely to have homology to genes expressed in other organisms.

  3. Encoding of relative enclosure size in a dynamic three-dimensional virtual environment by humans.

    PubMed

    Sturz, Bradley R; Kelly, Debbie M

    2009-10-01

    Human participants searched in a dynamic three-dimensional virtual-environment rectangular enclosure for a distinctly colored bin located in one of the four corners. During test trials, all bins were rendered identical in color, and the shape of the rectangular search space either remained the same or was modified to a relatively sized contracted rectangle, an expanded rectangle, or a square. Participants made one choice response during test trials. In the rectangular enclosures, more of participants' choice responses were allocated to the geometrically correct corners than to the geometrically incorrect corners. In the square enclosure, participants' choice responses were allocated equivalently to each of the four corners. Results replicate previous enclosure size studies demonstrating encoding of enclosure geometry with human and non-human animal subjects conducted in real environments and extend these results to include encoding of relative enclosure geometry. Results are discussed with respect to theoretical accounts of geometry learning.

  4. Effects of Acute Methamphetamine on Emotional Memory Formation in Humans: Encoding vs Consolidation

    PubMed Central

    Ballard, Michael E.; Weafer, Jessica; Gallo, David A.; de Wit, Harriet

    2015-01-01

    Understanding how stimulant drugs affect memory is important for understanding their addictive potential. Here we examined the effects of acute d-methamphetamine (METH), administered either before (encoding phase) or immediately after (consolidation phase) study on memory for emotional and neutral images in healthy humans. Young adult volunteers (N = 60) were randomly assigned to either an encoding group (N = 29) or a consolidation group (N = 31). Across three experimental sessions, they received placebo and two doses of METH (10, 20 mg) either 45 min before (encoding) or immediately after (consolidation) viewing pictures of emotionally positive, neutral, and negative scenes. Memory for the pictures was tested two days later, under drug-free conditions. Half of the sample reported sleep disturbances following the high dose of METH, which affected their memory performance. Therefore, participants were classified as poor sleepers (less than 6 hours; n = 29) or adequate sleepers (6 or more hours; n = 31) prior to analyses. For adequate sleepers, METH (20 mg) administered before encoding significantly improved memory accuracy relative to placebo, especially for emotional (positive and negative), compared to neutral, stimuli. For poor sleepers in the encoding group, METH impaired memory. METH did not affect memory in the consolidation group regardless of sleep quality. These results extend previous findings showing that METH can enhance memory for salient emotional stimuli but only if it is present at the time of study, where it can affect both encoding and consolidation. METH does not appear to facilitate consolidation if administered after encoding. The study also demonstrates the important role of sleep in memory studies. PMID:25679982

  5. Effects of acute methamphetamine on emotional memory formation in humans: encoding vs consolidation.

    PubMed

    Ballard, Michael E; Weafer, Jessica; Gallo, David A; de Wit, Harriet

    2015-01-01

    Understanding how stimulant drugs affect memory is important for understanding their addictive potential. Here we examined the effects of acute d-methamphetamine (METH), administered either before (encoding phase) or immediately after (consolidation phase) study on memory for emotional and neutral images in healthy humans. Young adult volunteers (N = 60) were randomly assigned to either an encoding group (N = 29) or a consolidation group (N = 31). Across three experimental sessions, they received placebo and two doses of METH (10, 20 mg) either 45 min before (encoding) or immediately after (consolidation) viewing pictures of emotionally positive, neutral, and negative scenes. Memory for the pictures was tested two days later, under drug-free conditions. Half of the sample reported sleep disturbances following the high dose of METH, which affected their memory performance. Therefore, participants were classified as poor sleepers (less than 6 hours; n = 29) or adequate sleepers (6 or more hours; n = 31) prior to analyses. For adequate sleepers, METH (20 mg) administered before encoding significantly improved memory accuracy relative to placebo, especially for emotional (positive and negative), compared to neutral, stimuli. For poor sleepers in the encoding group, METH impaired memory. METH did not affect memory in the consolidation group regardless of sleep quality. These results extend previous findings showing that METH can enhance memory for salient emotional stimuli but only if it is present at the time of study, where it can affect both encoding and consolidation. METH does not appear to facilitate consolidation if administered after encoding. The study also demonstrates the important role of sleep in memory studies.

  6. cDNA expression map of the human genome: Methods development and applications using brain cDNAs. Progress report, October 15, 1991--March 14, 1992

    SciTech Connect

    Sikela, J.M.

    1991-12-31

    The following describes progress on human brain cDNA sequencing and mapping that our laboratory has made over the past few months. It should be noted that our first funding installment for the first phase of this grant was obtained approximately two weeks ago. Therefore, the progress that is described represents efforts that were carried out without DOE Genome funds and thus largely are a continuation of pilot studies we began last year. We anticipate, now that DOE funds have arrived, that we will be able to significantly scale up our efforts and productivity.

  7. Encoding human sexual chemosensory cues in the orbitofrontal and fusiform cortices

    PubMed Central

    Zhou, Wen; Chen, Denise

    2009-01-01

    Chemosensory communication of affect and motivation is ubiquitous among animals. In humans, emotional expressions are naturally associated with faces and voices. Whether chemical signals play a role as well has hardly been addressed. Here we use functional magnetic resonance imaging (fMRI) to show that the right orbitofrontal cortex, right fusiform cortex, and right hypothalamus respond to airborne natural human sexual sweat, indicating that this particular chemosensory compound is encoded holistically in the brain. Our findings provide neural evidence that socioemotional meanings, including the sexual ones, are conveyed in the human sweat. PMID:19118174

  8. Chromosomal localization of the gene encoding the human DNA helicase RECQL and its mouse homologue

    SciTech Connect

    Puranam, K.L.; Kennington, E.; Blackshear, P.J.

    1995-04-10

    We have determined the chromosomal location of the human and mouse genes encoding the RECQL protein, a putative DNA helicase homologous to the bacterial DNA helicase, RecQ. RECQL was localized to human chromosome 12 by analysis of human-rodent somatic cell hybrid DNA, fine mapping of RECQL by fluorescence in situ hybridization revealed its chromosomal location to be 12p11-p12. The corresponding mouse gene, Recql, was mapped to the telomeric end of mouse chromosome 6 by analysis of DNA from an interspecific cross. 19 refs., 2 figs.

  9. Non-spin-echo 3D transverse hadamard encoded proton spectroscopic imaging in the human brain.

    PubMed

    Cohen, Ouri; Tal, Assaf; Goelman, Gadi; Gonen, Oded

    2013-07-01

    A non-spin-echo multivoxel proton MR localization method based on three-dimensional transverse Hadamard spectroscopic imaging is introduced and demonstrated in a phantom and the human brain. Spatial encoding is achieved with three selective 90° radiofrequency pulses along perpendicular axes: The first two create a longitudinal ±M(Z) Hadamard order in the volume of interest. The third pulse spatially Hadamard-encodes the ±M(Z)s in the volume of interest in the third direction while bringing them to the transverse plane to be acquired immediately. The approaching-ideal point spread function of Hadamard encoding and very short acquisition delay yield signal-to-noise-ratios of 20 ± 8, 23 ± 9, and 31 ± 10 for choline, creatine, and N-acetylaspartate in the human brain at 1.5 T from 1 cm(3) voxels in 21 min. The advantages of transverse Hadamard spectroscopic imaging are that unlike gradient (Fourier) phase-encoding: (i) the volume of interest does not need to be smaller than the field of view to prevent aliasing; (ii) the number of partitions in each direction can be small, 8, 4, or even 2 at no cost in point spread function; (iii) the volume of interest does not have to be contiguous; and (iv) the voxel profile depends on the available B1 and pulse synthesis paradigm and can, therefore, at least theoretically, approach "ideal" "1" inside and "0" elsewhere.

  10. Theta oscillations at encoding mediate the context-dependent nature of human episodic memory.

    PubMed

    Staudigl, Tobias; Hanslmayr, Simon

    2013-06-17

    Human episodic memory is highly context dependent. Therefore, retrieval benefits when a memory is recalled in the same context compared to a different context. This implies that items and contexts are bound together during encoding, such that the reinstatement of the initial context at test improves retrieval. Animal studies suggest that theta oscillations and theta-to-gamma cross-frequency coupling modulate such item-context binding, but direct evidence from humans is scarce. We investigated this issue by manipulating the overlap of contextual features between encoding and retrieval. Participants studied words superimposed on movie clips and were later tested by presenting the word with either the same or a different movie. The results show that memory performance and the oscillatory correlates of memory formation crucially depend on the overlap of the context between encoding and test. When the context matched, high theta power during encoding was related to successful recognition, whereas the opposite pattern emerged in the context-mismatch condition. In addition, cross-frequency coupling analysis revealed a context-dependent theta-to-gamma memory effect specifically in the left hippocampus. These results reveal for the first time that context-dependent episodic memory effects are mediated by theta oscillatory activity.

  11. Synthesis in Escherichia coli of human adenovirus type 12 transforming proteins encoded by early region 1A 13S mRNA and 12S mRNA.

    PubMed Central

    Kimelman, D; Lucher, L A; Brackmann, K H; Symington, J S; Ptashne, M; Green, M

    1984-01-01

    Human adenovirus (Ad)-encoded early region 1A (E1A) tumor (T) antigens have been implicated in the positive regulation of viral early genes, the positive and negative regulation of some cellular genes, and cell immortalization and transformation. To further study the Ad E1A T antigens and to facilitate their purification, we have cloned cDNA copies of the Ad12 E1A 13S mRNA and 12S mRNA downstream of a hybrid Escherichia coli trp-lac (tac) promoter. Up to 8% of the protein synthesized in E. coli cells transformed by each of the two different Ad12 E1A cDNA constructs were immunoprecipitated as a Mr 47,000 protein by antibody to a synthetic peptide encoded in the Ad12 E1A DNA sequence. Both proteins produced in E. coli appear to be authentic and complete Ad12 E1A T antigens because they possess (i) the Ad12 E1A NH2-terminal amino acid sequence predicted from the DNA sequence; (ii) the Ad12 E1A COOH-terminal sequence, as shown by immunoprecipitation with anti-peptide antibody; and (iii) a molecular weight and an acidic isoelectric point similar to that of the E1A T antigens synthesized in Ad12-infected and transformed mammalian cells. The T antigens were purified to near homogeneity in yields of 100-200 micrograms per g wet weight of transformed E. coli cells. Images PMID:6387701

  12. Structural characterization of the human platelet-derived growth factor A-chain cDNA and gene: Alternative exon usage predicts two different precursor proteins

    SciTech Connect

    Rorsman, F.; Bywater, M.; Knott, T.J.; Scott, J.; Betsholtz, C.

    1988-02-01

    The human platelet-derived growth factor (PDGF) A-chain locus was characterized by restriction endonuclease analysis, and the nucleotide sequence of its exons was determined. Seven exons were identified, spanning approximately 22 kilobase pairs of genomic DNA. Alternative exon usage, identified by cDNA cloning, occurs in a human glioblastoma cell line and may give rise to two types of A-chain precursors with different C termini. The exon-intron arrangement was similar to that of the PDGF B-chain/sis locus and seemed to divide the precursor proteins into functional domains. Southern blot analysis of genomic DNA showed that a single PDGF A-chain gene was present in the human genome.

  13. Subsecond dopamine fluctuations in human striatum encode superposed error signals about actual and counterfactual reward

    PubMed Central

    Kishida, Kenneth T.; Saez, Ignacio; Lohrenz, Terry; Witcher, Mark R.; Laxton, Adrian W.; Tatter, Stephen B.; White, Jason P.; Ellis, Thomas L.; Phillips, Paul E. M.; Montague, P. Read

    2016-01-01

    In the mammalian brain, dopamine is a critical neuromodulator whose actions underlie learning, decision-making, and behavioral control. Degeneration of dopamine neurons causes Parkinson’s disease, whereas dysregulation of dopamine signaling is believed to contribute to psychiatric conditions such as schizophrenia, addiction, and depression. Experiments in animal models suggest the hypothesis that dopamine release in human striatum encodes reward prediction errors (RPEs) (the difference between actual and expected outcomes) during ongoing decision-making. Blood oxygen level-dependent (BOLD) imaging experiments in humans support the idea that RPEs are tracked in the striatum; however, BOLD measurements cannot be used to infer the action of any one specific neurotransmitter. We monitored dopamine levels with subsecond temporal resolution in humans (n = 17) with Parkinson’s disease while they executed a sequential decision-making task. Participants placed bets and experienced monetary gains or losses. Dopamine fluctuations in the striatum fail to encode RPEs, as anticipated by a large body of work in model organisms. Instead, subsecond dopamine fluctuations encode an integration of RPEs with counterfactual prediction errors, the latter defined by how much better or worse the experienced outcome could have been. How dopamine fluctuations combine the actual and counterfactual is unknown. One possibility is that this process is the normal behavior of reward processing dopamine neurons, which previously had not been tested by experiments in animal models. Alternatively, this superposition of error terms may result from an additional yet-to-be-identified subclass of dopamine neurons. PMID:26598677

  14. Subsecond dopamine fluctuations in human striatum encode superposed error signals about actual and counterfactual reward.

    PubMed

    Kishida, Kenneth T; Saez, Ignacio; Lohrenz, Terry; Witcher, Mark R; Laxton, Adrian W; Tatter, Stephen B; White, Jason P; Ellis, Thomas L; Phillips, Paul E M; Montague, P Read

    2016-01-05

    In the mammalian brain, dopamine is a critical neuromodulator whose actions underlie learning, decision-making, and behavioral control. Degeneration of dopamine neurons causes Parkinson's disease, whereas dysregulation of dopamine signaling is believed to contribute to psychiatric conditions such as schizophrenia, addiction, and depression. Experiments in animal models suggest the hypothesis that dopamine release in human striatum encodes reward prediction errors (RPEs) (the difference between actual and expected outcomes) during ongoing decision-making. Blood oxygen level-dependent (BOLD) imaging experiments in humans support the idea that RPEs are tracked in the striatum; however, BOLD measurements cannot be used to infer the action of any one specific neurotransmitter. We monitored dopamine levels with subsecond temporal resolution in humans (n = 17) with Parkinson's disease while they executed a sequential decision-making task. Participants placed bets and experienced monetary gains or losses. Dopamine fluctuations in the striatum fail to encode RPEs, as anticipated by a large body of work in model organisms. Instead, subsecond dopamine fluctuations encode an integration of RPEs with counterfactual prediction errors, the latter defined by how much better or worse the experienced outcome could have been. How dopamine fluctuations combine the actual and counterfactual is unknown. One possibility is that this process is the normal behavior of reward processing dopamine neurons, which previously had not been tested by experiments in animal models. Alternatively, this superposition of error terms may result from an additional yet-to-be-identified subclass of dopamine neurons.

  15. Systematic Identification and Characterization of Novel Human Skin-Associated Genes Encoding Membrane and Secreted Proteins

    PubMed Central

    Buhren, Bettina Alexandra; Martinez, Cynthia; Schrumpf, Holger; Gasis, Marcia; Grether-Beck, Susanne; Krutmann, Jean

    2013-01-01

    Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics. PMID:23840300

  16. Cloning of the rat ErbB3 cDNA and characterization of the recombinant protein.

    PubMed

    Hellyer, N J; Kim, H H; Greaves, C H; Sierke, S L; Koland, J G

    1995-11-20

    Three cDNA fragments that encoded all but the extreme N terminus of the rat ErbB3 protein were cloned by low-stringency screening of a rat liver cDNA library with a human ERBB3 probe. The remaining 5'-end of the cDNA was generated by a reverse transcription-polymerase chain reaction method, and a single full-length rat ErbB3 cDNA was assembled. A comparison of the deduced amino acid (aa) sequences of human and rat ErbB3 was made, and the effects of certain aa substitutions in the putative protein tyrosine kinase domain were considered. The rat ErbB3 cDNA was subsequently expressed in cultured NIH-3T3 mouse fibroblasts, in which a high level of approx. 180-kDa recombinant ErbB3 (re-ErbB3) was generated. The rat re-ErbB3 produced in transfected fibroblasts was responsive to the polypeptide, heregulin, a known ligand for ErbB3. Challenge of transfected fibroblasts with heregulin stimulated the phosphorylation of rat re-ErbB3 on Tyr residues and promoted its association with the p85 subunit of phosphatidylinositol 3-kinase. Together, these results indicate that a fully functional rat ErbB3 cDNA has been isolated, and that fibroblast cells expressing this cDNA will be suitable for investigations of the signal transduction mechanism of ErbB3.

  17. Localization of the human genes encoding the two subunits of general transcription factor TFIIE.

    PubMed

    Purrello, M; Di Pietro, C; Rapisarda, A; Motta, S; Pavone, L; Grzeschik, K H; Sichel, G

    1994-09-01

    TFIIE is a general transcription factor for class II genes composed of two types of subunits, a large one of 56 kDa and a small of 34 kDa. By Southern analysis at high and at low stringency of a panel of mouse/human hybrid cell lines and by in situ chromosomal hybridization, we have demonstrated that both polypeptides are encoded by genes that are single copy in the human genome and are localized at 3q13-q21 and at 8p12, respectively. A TaqI RFLP (heterozygosity index of 0.07) was detected at the locus for the 56-kDa subunit.

  18. The human HNRPD locus maps to 4q21 and encodes a highly conserved protein.

    PubMed

    Dempsey, L A; Li, M J; DePace, A; Bray-Ward, P; Maizels, N

    1998-05-01

    The hnRNP D protein interacts with nucleic acids both in vivo and in vitro. Like many other proteins that interact with RNA, it contains RBD (or "RRM") domains and arg-gly-gly (RGG) motifs. We have examined the organization and localization of the human and murine genes that encode the hnRNP D protein. Comparison of the predicted sequences of the hnRNP D proteins in human and mouse shows that they are 96.9% identical (98.9% similar). This very high level of conservation suggests a critical function for hnRNP D. Sequence analysis of the human HNRPD gene shows that the protein is encoded by eight exons and that two additional exons specify sequences in the 3' UTR. Use of two of the coding exons is determined by alternative splicing of the HNRPD mRNA. The human HNRPD gene maps to 4q21. The mouse Hnrpd gene maps to the F region of chromosome 3, which is syntenic with the human 4q21 region.

  19. cDNA sequencing and expression analysis of Dicentrarchus labrax heme oxygenase-1.

    PubMed

    Prevot-D'Alvise, N; Pierre, S; Gaillard, S; Gouze, E; Gouze, J-N; Aubert, J; Richard, S; Grillasca, J-P

    2008-11-17

    The liver cDNA encoding heme oxygenase--1 (HO-1) was sequenced from European sea bass (Dicentrarchus labrax) (accession number no. EF139130). The HO-1 cDNA was 1250 bp in nucleotide length and the open reading frame encoded 277 amino acid residues. The deduced amino acid sequence of the European sea bass had 75% and 50% identity with the amino acid sequences of tetraodontiformes (Tetraodon nigroviridis and Takifugu rubripes) and human HO-1 proteins, respectively. A short hydrophobic transmembrane domain at the C--terminal region was found, and four histidine residues were highly conserved, including human his25 that is essential for HO catalytic activity. RT-PCR of mRNA from eight different European sea bass tissues revealed that, in a homeostatis state, the heme oxygenase--1 was abundant in the spleen and liver but not in the brain.

  20. The human subthalamic nucleus and globus pallidus internus differentially encode reward during action control.

    PubMed

    Justin Rossi, Peter; Peden, Corinna; Castellanos, Oscar; Foote, Kelly D; Gunduz, Aysegul; Okun, Michael S

    2017-04-01

    The subthalamic nucleus (STN) and globus pallidus internus (GPi) have recently been shown to encode reward, but few studies have been performed in humans. We investigated STN and GPi encoding of reward and loss (i.e., valence) in humans with Parkinson's disease. To test the hypothesis that STN and GPi neurons would change their firing rate in response to reward- and loss-related stimuli, we recorded the activity of individual neurons while participants performed a behavioral task. In the task, action choices were associated with potential rewarding, punitive, or neutral outcomes. We found that STN and GPi neurons encode valence-related information during action control, but the proportion of valence-responsive neurons was greater in the STN compared to the GPi. In the STN, reward-related stimuli mobilized a greater proportion of neurons than loss-related stimuli. We also found surprising limbic overlap with the sensorimotor regions in both the STN and GPi, and this overlap was greater than has been previously reported. These findings may help to explain alterations in limbic function that have been observed following deep brain stimulation therapy of the STN and GPi. Hum Brain Mapp 38:1952-1964, 2017. © 2017 Wiley Periodicals, Inc.

  1. A synergy-based hand control is encoded in human motor cortical areas.

    PubMed

    Leo, Andrea; Handjaras, Giacomo; Bianchi, Matteo; Marino, Hamal; Gabiccini, Marco; Guidi, Andrea; Scilingo, Enzo Pasquale; Pietrini, Pietro; Bicchi, Antonio; Santello, Marco; Ricciardi, Emiliano

    2016-02-15

    How the human brain controls hand movements to carry out different tasks is still debated. The concept of synergy has been proposed to indicate functional modules that may simplify the control of hand postures by simultaneously recruiting sets of muscles and joints. However, whether and to what extent synergic hand postures are encoded as such at a cortical level remains unknown. Here, we combined kinematic, electromyography, and brain activity measures obtained by functional magnetic resonance imaging while subjects performed a variety of movements towards virtual objects. Hand postural information, encoded through kinematic synergies, were represented in cortical areas devoted to hand motor control and successfully discriminated individual grasping movements, significantly outperforming alternative somatotopic or muscle-based models. Importantly, hand postural synergies were predicted by neural activation patterns within primary motor cortex. These findings support a novel cortical organization for hand movement control and open potential applications for brain-computer interfaces and neuroprostheses.

  2. Roughness encoding in human and biomimetic artificial touch: spatiotemporal frequency modulation and structural anisotropy of fingerprints.

    PubMed

    Oddo, Calogero Maria; Beccai, Lucia; Wessberg, Johan; Wasling, Helena Backlund; Mattioli, Fabio; Carrozza, Maria Chiara

    2011-01-01

    The influence of fingerprints and their curvature in tactile sensing performance is investigated by comparative analysis of different design parameters in a biomimetic artificial fingertip, having straight or curved fingerprints. The strength in the encoding of the principal spatial period of ridged tactile stimuli (gratings) is evaluated by indenting and sliding the surfaces at controlled normal contact force and tangential sliding velocity, as a function of fingertip rotation along the indentation axis. Curved fingerprints guaranteed higher directional isotropy than straight fingerprints in the encoding of the principal frequency resulting from the ratio between the sliding velocity and the spatial periodicity of the grating. In parallel, human microneurography experiments were performed and a selection of results is included in this work in order to support the significance of the biorobotic study with the artificial tactile system.

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

    PubMed

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

    2013-04-01

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

  4. Sequence and neuronal expression of mouse endothelin-1 cDNA.

    PubMed

    Kurama, M; Ishida, N; Matsui, M; Saida, K; Mitsui, Y

    1996-07-17

    We have isolated and sequenced a cDNA that encodes mouse endothelin-1 (ET-1). The putative protein contains 202 amino acids corresponds to the prepro-form of ET-1. Twenty-one amino acids sequence of the putative mature ET-1 was identical with that of rat, porcine, bovine, and human. In situ hybridization histochemistry indicate that ET-1 mRNA was expressed in several hypothalamic nuclei including the suprachiasmatic nucleus (SCN) in rodent brain.

  5. Receptor expression and responsiveness of human peripheral blood mononuclear cells to a human cytomegalovirus encoded CC chemokine.

    PubMed

    Zheng, Qi; Xu, Jun; Gao, Huihui; Tao, Ran; Li, Wei; Shang, Shiqiang; Gu, Weizhong

    2015-01-01

    Human cytomegalovirus is a ubiquitous pathogen that infects the majority of the world's population. After long period of time co-evolving with human being, this pathogen has developed several strategies to evade host immune surveillance. One of the major trick is encoding homologous to those of the host organism or stealing host cellular genes that have significant functions in immune system. To date, we have found several viral immune analogous which include G protein coupled receptor, class I major histocompatibility complex and chemokine. Chemokine is a small group of molecules which is defined by the presence of four cysteines in highly conserved region. The four kinds of chemokines (C, CC, CXC, and CX3C) are classified based on the arrangement of 1 or 2 N-terminal cysteine residues. UL128 protein is one of the analogous that encoded by human cytomegalovirus that has similar amino acid sequences to the human CC chemokine. It has been proved to be one of the essential particles that involved in human cytomegalovirus entry into epithelial/endothelial cells as well as macrophages. It is also the target of potent neutralizing antibodies in human cytomegalovirus-seropositive individuals. We had demonstrated the chemotactic trait of UL128 protein in our previous study. Recombinant UL128 in vitro has the ability to attract monocytes to the infection region and enhances peripheral blood mononuclear cell proliferation by activating the MAPK/ERK signaling pathway. However, the way that this viral encoded chemokine interacting with peripheral blood mononuclear cells and the detailed mechanism that involving the virus entry into host cells keeps unknown. Here we performed in vitro investigation into the effects of UL128 protein on peripheral blood mononuclear cell's activation and receptor binding, which may help us further understand the immunomodulatory function of UL128 protein as well as human cytomegalovirus diffusion mechanism.

  6. Complementary DNA encoding the human T-cell FK506-binding protein, a peptidylprolyl cis-trans isomerase distinct from cyclophilin

    SciTech Connect

    Maki, Noboru; Sekiguchi, Fumiko; Nishimaki, Junichi; Miwa, Keiko; Hayano, Toshiya; Takahashi, Nobuhiro; Suzuki, Masanori )

    1990-07-01

    The recently discovered macrolide FK506 has been demonstrated to have potent immunosuppressive activity at concentrations 100-fold lower than cyclosporin A, a cyclic undecapeptide that is used to prevent rejection after transplantation of bone marrow and organs, such as kidney, heart, and liver. After the recent discovery that the cylcosporin A-binding protein cyclophilin is identical to peptidylprolyl cis-trans isomerase, a cellular binding protein for FK506 was found to be distinct from cyclophilin but to have the same enzymatic activity. In this study, the authors isolated a cDNA coding for FK506-binding protein (FKBP) from human peripheral blood T cells by using mixed 20-mer oligonucleotide probes synthesized on the basis of the sequence, Glu-Asp-Gly-Lys-Lys-Phe-Asp, reported for bovine FKBP. The DNA isolated contained an open reading frame encoding 108 amino acid residues. The first 40 residues of the deduced amino acid sequence were identical to those of the reported amino-terminal sequence of bovine FKBP, indicating that the DNA sequence isolated represents the gene coding for FKBP. This result suggests that two catalytically similar proteins, cyclophilin and FKBP, evolved independently. In Northern blot analysis, mRNA species of {approx}1.8 kilobases that hybridized with human FKBP cDNA were detected in poly(A){sup +} RNAs from brain, lung, liver, and placental cells and leukocytes. Induction of Jurkat leukemic T cells with phorbol 12-myristate 13-acetate and ionomycin did not affect the level of FKBP mRNA.

  7. Characterization of the in vitro expressed autoimmune rippling muscle disease immunogenic domain of human titin encoded by TTN exons 248-249

    SciTech Connect

    Zelinka, L.; McCann, S.; Budde, J.; Sethi, S.; Guidos, M.; Giles, R.; Walker, G.R.

    2011-08-05

    Highlights: {yields} Affinity purification of the autoimmune rippling muscle disease immunogenic domain of titin. {yields} Partial sequence analysis confirms that the peptides is in the I band region of titin. {yields} This region of the human titin shows high degree of homology to mouse titin N2-A. -- Abstract: Autoimmune rippling muscle disease (ARMD) is an autoimmune neuromuscular disease associated with myasthenia gravis (MG). Past studies in our laboratory recognized a very high molecular weight skeletal muscle protein antigen identified by ARMD patient antisera as the titin isoform. These past studies used antisera from ARMD and MG patients as probes to screen a human skeletal muscle cDNA library and several pBluescript clones revealed supporting expression of immunoreactive peptides. This study characterizes the products of subcloning the titin immunoreactive domain into pGEX-3X and the subsequent fusion protein. Sequence analysis of the fusion gene indicates the cloned titin domain (GenBank ID: (EU428784)) is in frame and is derived from a sequence of N2-A spanning the exons 248-250 an area that encodes the fibronectin III domain. PCR and EcoR1 restriction mapping studies have demonstrated that the inserted cDNA is of a size that is predicted by bioinformatics analysis of the subclone. Expression of the fusion protein result in the isolation of a polypeptide of 52 kDa consistent with the predicted inferred amino acid sequence. Immunoblot experiments of the fusion protein, using rippling muscle/myasthenia gravis antisera, demonstrate that only the titin domain is immunoreactive.

  8. Small gene family encoding an eggshell (chorion) protein of the human parasite Schistosoma mansoni

    SciTech Connect

    Bobek, L.A.; Rekosh, D.M.; Lo Verde, P.T.

    1988-08-01

    The authors isolated six independent genomic clones encoding schistosome chorion or eggshell proteins from a Schistosoma mansoni genomic library. A linkage map of five of the clones spanning 35 kilobase pairs (kbp) of the S. mansoni genome was constructed. The region contained two eggshell protein genes closely linked, separated by 7.5 kbp of intergenic DNA. The two genes of the cluster were arranged in the same orientation, that is, they were transcribed from the same strand. The sixth clone probably represents a third copy of the eggshell gene that is not contained within the 35-kbp region. The 5- end of the mRNA transcribed from these genes was defined by primer extension directly off the RNA. The ATCAT cap site sequence was homologous to a silkmoth chorion PuTCATT cap site sequence, where Pu indicates any purine. DNA sequence analysis showed that there were no introns in these genes. The DNA sequences of the three genes were very homologous to each other and to a cDNA clone, pSMf61-46, differing only in three or four nucleotices. A multiple TATA box was located at positions -23 to -31, and a CAAAT sequence was located at -52 upstream of the eggshell transcription unit. Comparison of sequences in regions further upstream with silkmoth and Drosophila sequences revealed very short elements that were shared. One such element, TCACGT, recently shown to be an essential cis-regulatory element for silkmoth chorion gene promoter function, was found at a similar position in all three organisms.

  9. Dual temporal encoding mechanisms in human auditory cortex: Evidence from MEG and EEG.

    PubMed

    Tang, Huizhen; Crain, Stephen; Johnson, Blake W

    2016-03-01

    Current hypotheses about language processing advocate an integral relationship between encoding of temporal information and linguistic processing in the brain. All such explanations must accommodate the evident ability of the perceptual system to process both slow and fast time scales in speech. However most cortical neurons are limited in their capability to precisely synchronise to temporal modulations at rates faster than about 50Hz. Hence, a central question in auditory neurophysiology concerns how the full range of perceptually relevant modulation rates might be encoded in the cerebral cortex. Here we show with concurrent noninvasive magnetoencephalography (MEG) and electroencephalography (EEG) measurements that the human auditory cortex transitions between a phase-locked (PL) mode of responding to modulation rates below about 50Hz, and a non-phase-locked (NPL) mode at higher rates. Precisely such dual response modes are predictable from the behaviours of single neurons in auditory cortices of non-human primates. Our data point to a common mechanistic explanation for the single neuron and MEG/EEG results and support the hypothesis that two distinct types of neuronal encoding mechanisms are employed by the auditory cortex to represent a wide range of temporal modulation rates. This dual encoding model allows slow and fast modulations in speech to be processed in parallel and is therefore consistent with theoretical frameworks in which slow temporal modulations (such as rhythm or syllabic structure) are akin to the contours or edges of visual objects, whereas faster modulations (such as periodicity pitch or phonemic structure) are more like visual texture.

  10. Human posterior parietal cortex encodes the movement goal in a pro-/anti-reach task

    PubMed Central

    Gertz, Hanna

    2015-01-01

    Previous research on reach planning in humans has implicated a frontoparietal network, including the precuneus (PCu), a putative human homolog of the monkey parietal reach region (PRR), and the dorsal premotor cortex (PMd). Using a pro-/anti-reach task, electrophysiological studies in monkeys have demonstrated that the movement goal rather than the location of the visual cue is encoded in PRR and PMd. However, if only the effector but not the movement goal is specified (underspecified condition), the PRR and PMd have been shown to represent all potential movement goals. In this functional magnetic resonance imaging study, we investigated whether the human PCu and PMd likewise encode the movement goal, and whether these reach-related areas also engage in situations with underspecified compared with specified movement goals. By using a pro-/anti-reach task, we spatially dissociated the location of the visual cue from the location of the movement goal. In the specified conditions, pro- and anti-reaches activated similar parietal and premotor areas. In the PCu contralateral to the moving arm, we found directionally selective activation fixed to the movement goal. In the underspecified conditions, we observed activation in reach-related areas of the posterior parietal cortex, including PCu. However, the activation was substantially weaker in parietal areas and lacking in PMd. Our results suggest that human PCu encodes the movement goal rather than the location of the visual cue if the movement goal is specified and even engages in situations when only the visual cue but not the movement goal is defined. PMID:25904714

  11. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human

    SciTech Connect

    Blatt, C.; Eversole-Cire, P.; Cohn, V.H.; Zollman, S.; Fournier, R.E.K.; Mohandas, L.T.; Nesbitt, M.; Lugo, T.; Jones, D.T.; Reed, R.R.; Weiner, L.P.; Sparkes, R.S.; Simon, M.I. )

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding {alpha}-subunit proteins, two different {beta} subunits, and one {gamma} subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The {beta} subunits were also assigned-GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extend of the G{alpha} gene family and may help in attempts to correlate specific genetic diseases and with genes corresponding to G proteins.

  12. Sulforaphane-induced apoptosis in human leukemia HL-60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray.

    PubMed

    Shang, Hung-Sheng; Shih, Yung-Luen; Lee, Ching-Hsiao; Hsueh, Shu-Ching; Liu, Jia-You; Liao, Nien-Chieh; Chen, Yung-Liang; Huang, Yi-Ping; Lu, Hsu-Feng; Chung, Jing-Gung

    2017-01-01

    Sulforaphane (SFN), one of the isothiocyanates, is a biologically active compound extracted from cruciferous vegetables, and has been shown to induce cytotoxic effects on many human cancer cells including human leukemia cells. However, the exact molecular mechanism and altered gene expression associated with apoptosis is unclear. In this study, we investigated SFN-induced cytotoxic effects and whether or not they went through cell-cycle arrest and induction of apoptosis and further examined molecular mechanism and altered gene expression in human leukemia HL-60 cells. Cell viability, cell-cycle distribution, sub-G1 (apoptosis), reactive oxygen species (ROS) and Ca(2+) production, levels of mitochondrial membrane potential (ΔΨm ), and caspase-3, -8, and -9 activities were assayed by flow cytometry. Apoptosis-associated proteins levels and gene expressions were examined by Western blotting and cDNA microarray assays, respectively. Results indicated that SFN decreased viable cells, induced G2/M phase arrest and apoptosis based on sub-G1 phase development. Furthermore, SFN increased ROS and Ca(2+) production and decreased the levels of ΔΨm and activated caspase-3, -8, and -9 activities in HL-60 cells. SFN significantly upregulated the expression of BAX, Bid, Fas, Fas-L, caspase-8, Endo G, AIF, and cytochrome c, and inhibited the antiapoptotic proteins such as Bcl-x and XIAP, that is associated with apoptosis. We also used cDNA microarray to confirm several gene expressions such as caspase -8, -3, -4, -6, and -7 that are affected by SFN. Those results indicated that SFN induced apoptosis in HL-60 cells via Fas- and mitochondria-dependent pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 311-328, 2017.

  13. A brief review on the Human Encyclopedia of DNA Elements (ENCODE) project.

    PubMed

    Qu, Hongzhu; Fang, Xiangdong

    2013-06-01

    The ENCyclopedia Of DNA Elements (ENCODE) project is an international research consortium that aims to identify all functional elements in the human genome sequence. The second phase of the project comprised 1640 datasets from 147 different cell types, yielding a set of 30 publications across several journals. These data revealed that 80.4% of the human genome displays some functionality in at least one cell type. Many of these regulatory elements are physically associated with one another and further form a network or three-dimensional conformation to affect gene expression. These elements are also related to sequence variants associated with diseases or traits. All these findings provide us new insights into the organization and regulation of genes and genome, and serve as an expansive resource for understanding human health and disease.

  14. Expression of the gene encoding growth hormone in the human mammary gland

    SciTech Connect

    Mol, J.A.; Misdorp, W.; Rijnberk, A.

    1995-10-01

    Progestins cause a syndrome of growth hormone (GH) excess and enhanced mammary tumorigenesis in the dog. This has been regarded as being specific for the dog. Recently we reported that progestin-induced GH excess originates from foci of hyperplastic ductular epithelium of the mammary gland in the dog. In the present report we demonstrate by reverse-transcriptase PCR and immunohistochemistry that a main factor involved in tissue growth, i.e. GH, is also expressed in normal and neoplastic human mammary glands. The gene expressed in the human mammary gland proved to be identical to the gene encoding GH in the pituitary gland. The role of progesterone in the GH expression of the human mammary gland needs, however, to be proven. It is hypothesized that this locally produced hGH may play a pathogenetic role in breast cancer. 21 refs., 2 figs., 1 tab.

  15. The Roles of Human Lateral Temporal Cortical Neuronal Activity in Recent Verbal Memory Encoding

    PubMed Central

    Schoenfield-McNeill, Julie; Corina, David

    2009-01-01

    Activity of 98 single neurons in human lateral temporal cortex was measured during memory encoding for auditory words, text, or pictures and compared with identification of material of the same modality in extracellular recordings during awake neurosurgery for epilepsy. Frequency of activity was divided into early or late epochs or activity sustained throughout both; 44 neurons had significant changes in one or more categories. Polymodal and sustained changes lateralized to dominant hemisphere and late changes to nondominant. The majority of polymodal neurons shifted categories for different modalities. In dominant hemisphere, the timing and nature of changes in activity provide the basis for a model of the roles of temporal cortex in encoding. Superior temporal gyrus excitatory activity was related to the early epoch, when perception and processing occur, and middle gyrus to the late epoch, when semantic labeling occurs. The superior two-thirds of middle gyrus also demonstrated sustained inhibition. In a subset of lateral temporal neurons, memory-encoding activity reflected simultaneous convergence of sustained attentional and early perceptual inputs. PMID:18469317

  16. Direct evidence for encoding of motion streaks in human visual cortex

    PubMed Central

    Apthorp, Deborah; Schwarzkopf, D. Samuel; Kaul, Christian; Bahrami, Bahador; Alais, David; Rees, Geraint

    2013-01-01

    Temporal integration in the visual system causes fast-moving objects to generate static, oriented traces (‘motion streaks’), which could be used to help judge direction of motion. While human psychophysics and single-unit studies in non-human primates are consistent with this hypothesis, direct neural evidence from the human cortex is still lacking. First, we provide psychophysical evidence that faster and slower motions are processed by distinct neural mechanisms: faster motion raised human perceptual thresholds for static orientations parallel to the direction of motion, whereas slower motion raised thresholds for orthogonal orientations. We then used functional magnetic resonance imaging to measure brain activity while human observers viewed either fast (‘streaky’) or slow random dot stimuli moving in different directions, or corresponding static-oriented stimuli. We found that local spatial patterns of brain activity in early retinotopic visual cortex reliably distinguished between static orientations. Critically, a multivariate pattern classifier trained on brain activity evoked by these static stimuli could then successfully distinguish the direction of fast (‘streaky’) but not slow motion. Thus, signals encoding static-oriented streak information are present in human early visual cortex when viewing fast motion. These experiments show that motion streaks are present in the human visual system for faster motion. PMID:23222445

  17. Color signal encoding for high dynamic range and wide color gamut based on human perception

    NASA Astrophysics Data System (ADS)

    Nezamabadi, Mahdi; Miller, Scott; Daly, Scott; Atkins, Robin

    2014-01-01

    A new EOTF based on human perception, called PQ (Perceptual Quantizer), was proposed in a previous work (SMPTE Mot. Imag. J 2013, 122:52-59) and its performance was evaluated for a wide range of luminance levels and encoding bitdepth values. This paper is an extension of that previous work to include the color aspects of the PQ signal encoding. The efficiency of the PQ encoding and bit-depth requirements were evaluated and compared for standard color gamuts of Rec 709 (SRGB), and the wide color gamuts of Rec 2020, P3, and ACES for a variety of signal representations as RGB, YCbCr, and XYZ. In a selected color space for any potential local gray level 26 color samples were simulated by deviating one quantization step from the original color in each signal dimension. The quantization step sizes were simulated based on the PQ and gamma curves for different bit-depth values and luminance ranges for each of the color gamut spaces and signal representations. Color differences between the gray field and the simulated color samples were computed using CIE DE2000 color difference equation. The maximum color difference values (quantization error) were used as a metric to evaluate the performance of the corresponding EOTF curve. Extended color gamuts were found to require more bits to maintain low quantization error. Extended dynamic range required fewer additional bits in to maintain quantization error. Regarding the visual detection thresholds, the minimum bit-depth required by the PQ and gamma encodings are evaluated and compared through visual experiments.

  18. Human inhibitor of the first component of complement, C1: characterization of cDNA clones and localization of the gene to chromosome 11.

    PubMed Central

    Davis, A E; Whitehead, A S; Harrison, R A; Dauphinais, A; Bruns, G A; Cicardi, M; Rosen, F S

    1986-01-01

    C1 inhibitor is a heavily glycosylated plasma protein that regulates the activity of the first component of complement (C1) by inactivation of the serine protease subcomponents, C1r and C1s. C1 inhibitor cDNA clones have been isolated, and one of these (pC1INH1, 950 base pairs) has been partially sequenced. Sequence analysis demonstrates that the C1 inhibitor is a member of the serpin "superfamily" of protease inhibitors. In the region sequenced, C1 inhibitor has 22% identity with antithrombin III, 26% with alpha 1-antitrypsin and alpha 1-antichymotrypsin, and 18% with human angiotensinogen. C1 inhibitor has a larger amino-terminal extension than do the other plasma protease inhibitors. In addition, inspection of residues that are invariant among the other protease inhibitors shows that C1 inhibitor differs at 14 of 41 of these positions. Thus, it appears that C1 inhibitor diverged from the group relatively early in evolution, although probably after the divergence of angiotensinogen. Southern blot analysis of BamHI-digested DNA from normal individuals and from rodent-human somatic cell hybrid cell lines (that contain a limited but varied human chromosome complement) was used to localize the human C1 inhibitor gene to chromosome 11. Images PMID:3458172

  19. Interacting noradrenergic and corticosteroid systems shift human brain activation patterns during encoding.

    PubMed

    van Stegeren, Anda H; Roozendaal, Benno; Kindt, Merel; Wolf, Oliver T; Joëls, Marian

    2010-01-01

    Emotionally arousing experiences are usually well retained, an effect that depends on the release of adrenal stress hormones. Animal studies have shown that corticosterone and noradrenaline - representing the two main stress hormone systems - act in concert to enhance memory formation by actions involving the amygdala, hippocampus and prefrontal cortex (PFC). Here we test whether interactions between these two stress hormone systems also affect human memory formation as well as the associated pattern of brain activation. To this end, forty-eight male human subjects received hydrocortisone, yohimbine or both before presentation of emotional and neutral pictures. Activity in the amygdala, hippocampus and PFC was monitored with functional Magnetic Resonance Imaging (fMRI) during encoding of these stimuli, when hormonal levels were elevated. Memory performance was tested 1 week later. We investigated whether an increased level of one of the two hormone systems would lead to differential effects compared to the combined application of the drugs on brain activation and memory performance. We report that the application of cortisol led to an overall enhancing effect on recognition memory, with no significant additional effect of yohimbine. However, during encoding the brain switched from amygdala/hippocampus activation with either hormone alone, to a strong deactivation of prefrontal areas under the influence of the combination of both exogenous hormones. Although we did not find evidence that exogenous stimulation of the noradrenergic and corticosteroid systems led to significant interaction effects on memory performance in this experiment, we conclude that stress hormone levels during encoding did differentially determine the activation pattern of the brain circuits here involved.

  20. A Gene Encoding Antigenic Peptides of Human Squamous Cell Carcinoma Recognized by Cytotoxic T Lymphocytes

    PubMed Central

    Shichijo, Shigeki; Nakao, Masanobu; Imai, Yasuhisa; Takasu, Hideo; Kawamoto, Mayumi; Niiya, Fumihiko; Yang, Damu; Toh, Yuji; Yamana, Hideaki; Itoh, Kyogo

    1998-01-01

    Except for melanomas, tumor antigens recognized by cytotoxic T lymphocytes (CTLs) are yet unidentified. We have identified a gene encoding antigenic peptides of human squamous cell carcinomas (SCCs) recognized by human histocompatibility leukocyte antigens (HLA)- A2601–restricted CTLs. This gene showed no similarity to known sequences, and encoded two (125- and 43-kilodalton [kD]) proteins. The 125-kD protein with the leucine zipper motif was expressed in the nucleus of the majority of proliferating cells tested, including normal and malignant cells. The 43-kD protein was expressed in the cytosol of most SCCs from various organs and half of lung adenocarcinomas, but was not expressed in other cancers nor in a panel of normal tissues. The three nonapeptides shared by the two proteins were recognized by the KE4 CTLs, and one of the peptides induced in vitro from peripheral blood mononuclear cells (PBMCs) the CTLs restricted to the autologous tumor cells. The 43-kD protein and this nonapeptide (KGSGKMKTE) may be useful for the specific immunotherapy of HLA-A2601+ epithelial cancer patients. PMID:9449708

  1. Localization of a bacterial group II intron-encoded protein in human cells.

    PubMed

    Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; García Pérez, José Luis; Toro, Nicolás

    2015-08-05

    Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

  2. Localization of a bacterial group II intron-encoded protein in human cells

    PubMed Central

    Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; Pérez, José Luis García; Toro, Nicolás

    2015-01-01

    Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells. PMID:26244523

  3. The habenula encodes negative motivational value associated with primary punishment in humans

    PubMed Central

    Lawson, Rebecca P.; Seymour, Ben; Loh, Eleanor; Lutti, Antoine; Dolan, Raymond J.; Dayan, Peter; Weiskopf, Nikolaus; Roiser, Jonathan P.

    2014-01-01

    Learning what to approach, and what to avoid, involves assigning value to environmental cues that predict positive and negative events. Studies in animals indicate that the lateral habenula encodes the previously learned negative motivational value of stimuli. However, involvement of the habenula in dynamic trial-by-trial aversive learning has not been assessed, and the functional role of this structure in humans remains poorly characterized, in part, due to its small size. Using high-resolution functional neuroimaging and computational modeling of reinforcement learning, we demonstrate positive habenula responses to the dynamically changing values of cues signaling painful electric shocks, which predict behavioral suppression of responses to those cues across individuals. By contrast, negative habenula responses to monetary reward cue values predict behavioral invigoration. Our findings show that the habenula plays a key role in an online aversive learning system and in generating associated motivated behavior in humans. PMID:25071182

  4. The habenula encodes negative motivational value associated with primary punishment in humans.

    PubMed

    Lawson, Rebecca P; Seymour, Ben; Loh, Eleanor; Lutti, Antoine; Dolan, Raymond J; Dayan, Peter; Weiskopf, Nikolaus; Roiser, Jonathan P

    2014-08-12

    Learning what to approach, and what to avoid, involves assigning value to environmental cues that predict positive and negative events. Studies in animals indicate that the lateral habenula encodes the previously learned negative motivational value of stimuli. However, involvement of the habenula in dynamic trial-by-trial aversive learning has not been assessed, and the functional role of this structure in humans remains poorly characterized, in part, due to its small size. Using high-resolution functional neuroimaging and computational modeling of reinforcement learning, we demonstrate positive habenula responses to the dynamically changing values of cues signaling painful electric shocks, which predict behavioral suppression of responses to those cues across individuals. By contrast, negative habenula responses to monetary reward cue values predict behavioral invigoration. Our findings show that the habenula plays a key role in an online aversive learning system and in generating associated motivated behavior in humans.

  5. Human cDNA mapping using fluorescence in situ hybridization. Final progress report, April 1, 1994--July 31, 1997

    SciTech Connect

    Korenberg, J.R.

    1997-12-31

    The ultimate goal of this research is to generate and apply novel technologies to speed completion and integration of the human genome map and sequence with biomedical problems. To do this, techniques were developed and genome-wide resources generated. This includes a genome-wide Mapped and Integrated BAC/PAC Resource that has been used for gene finding, map completion and anchoring, breakpoint definition and sequencing. In the last period of the grant, the Human Mapped BAC/PAC Resource was also applied to determine regions of human variation and to develop a novel paradigm of primate evolution through to humans. Further, in order to more rapidly evaluate animal models of human disease, a BAC Map of the mouse was generated in collaboration with the MTI Genome Center, Dr. Bruce Birren.

  6. Identification of a novel candidate gene in the iron-sulfur pathway implicated in ataxia-susceptibility: human gene encoding HscB, a J-type co-chaperone.

    PubMed

    Sun, Guifeng; Gargus, J Jay; Ta, Dennis T; Vickery, Larry E

    2003-01-01

    Iron-sulfur proteins participate in a wide range of biochemical processes, including many that are central to mitochondrial electron transfer and energy metabolism. Mutations in two such proteins, frataxin and ABCB7, cause Friedreich ataxia and X-linked sideroblastic anemia with ataxia, respectively, rendering other participants in this pathway functional candidates for hereditary ataxia syndromes. Recently frataxin was shown to have an identical phylogenetic distribution with two genes and was most likely specifically involved in the same sub-process in iron-sulfur cluster assembly as one gene, designated hscB, in bacteria. To set the stage for an analysis of the potential role of this candidate gene in human disease, we defined the human HscB cDNA, its genomic locus, and its pattern of expression in normal human tissues. The isolated human HscB cDNA spans 785 bp and encodes a conserved 235-amino-acid protein, including a putative mitochondrial import leader. The HscB gene is found at chromosome 22q11-12 and is composed of six exons and five introns. Northern blot analyses of RNA from adult and fetal tissues defined a pattern of expression in mitochondria-rich tissues similar to that of frataxin, an expression pattern compatible with its implied role in mitochondrial energetics and related disease phenotypes.

  7. Molecular mechanisms deployed by virally encoded G protein-coupled receptors in human diseases.

    PubMed

    Montaner, Silvia; Kufareva, Irina; Abagyan, Ruben; Gutkind, J Silvio

    2013-01-01

    G protein-coupled receptors (GPCRs) represent the largest family of cell surface molecules involved in signal transduction. Surprisingly, open reading frames for multiple GPCRs were hijacked in the process of coevolution between Herpesviridae family viruses and their human and mammalian hosts. Virally encoded GPCRs (vGPCRs) evolved as parts of viral genomes, and this evolution allowed the power of host GPCR signaling circuitries to be harnessed in order to ensure viral replicative success. Phylogenetically, vGPCRs are distantly related to human chemokine receptors, although they feature several unique characteristics. Here, we describe the molecular mechanisms underlying vGPCR-mediated viral pathogenesis. These mechanisms include constitutive activity, aberrant coupling to human G proteins and β-arrestins, binding and activation by human chemokines, and dimerization with other GPCRs expressed in infected cells. The likely structural basis for these molecular events is described for the two closest viral homologs of human GPCRs. This information may aid in the development of novel targeted therapeutic strategies against viral diseases.

  8. Succinyl CoA: 3-oxoacid CoA transferase (SCOT): human cDNA cloning, human chromosomal mapping to 5p13, and mutation detection in a SCOT-deficient patient.

    PubMed Central

    Kassovska-Bratinova, S.; Fukao, T.; Song, X. Q.; Duncan, A. M.; Chen, H. S.; Robert, M. F.; Pérez-Cerdá, C.; Ugarte, M.; Chartrand, C.; Vobecky, S.; Kondo, N.; Mitchell, G. A.

    1996-01-01

    Succinyl CoA: 3-oxoacid CoA transferase (SCOT; E.C.2.8.3.5) mediates the rate-determining step of ketolysis in extrahepatic tissues, the esterification of acetoacetate to CoA for use in energy production. Hereditary SCOT deficiency in humans causes episodes of severe ketoacidosis. We obtained human-heart SCOT cDNA clones spanning the entire 1,560-nt coding sequence. Sequence alignment of the human SCOT peptides with other known CoA transferases revealed several conserved regions of potential functional importance. A single approximately 3.2-kb SCOT mRNA is present in human tissues (heart > leukocytes >> fibroblasts), but no signal is detectable in the human hepatoma cell line HepG2. We mapped the human SCOT locus (OXCT) to the cytogenetic band 5p13 by in situ hybridization. From fibroblasts of a patient with hereditary SCOT deficiency, we amplified and cloned cDNA fragments containing the entire SCOT coding sequence. We found a homozygous C-to-G transversion at nt 848, which changes the Ser 283 codon to a stop codon. This mutation (S283X) is incompatible with normal enzyme function and represents the first documentation of a pathogenic mutation in SCOT deficiency. Images Figure 2 Figure 6 PMID:8751852

  9. Genomic organization and complete cDNA sequence of the human phosphoinositide-specific phospholipase C {beta}3 gene (PLCB3)

    SciTech Connect

    Lagercrantz, J.; Carson, E.; Phelan, C.

    1995-04-10

    We have characterized the complete cDNA sequence, genomic structure, and expression of the human phosphoinositide-specific phospholipase C {beta}3 (PLC {beta}3) gene (gene symbol PLCB3). PLC {beta}3 plays an important role in initiating receptor-mediated signal transduction. Activation of PLC takes place in many cells as a response to stimulation by hormones, growth factors, neurotransmitters, and other ligands. The partial cDNA sequence of PLC {beta}3, previously published, was extended with 876 bp in the 5{prime} direction, giving a transcript of 4400 bp and a total open reading frame of 1234 amino acids. This was in accordance with expression analysis by Northern blotting that revealed a single 4.4-kb transcript in all tissues tested. Genomic data were obtained by sequencing plasmid subclones of a cosmid that contained the whole gene. The size of the complete transcription unit was estimated to be on the order of 15 kb. The gene contains 31 exons, with all splice donor and acceptor sites conforming to the GT/AG rule. No exon exceeds 571 bp in length, and the shortest exon spans only 36 bp. More than half of the introns are smaller than 200 bp, with the smallest being only 79 bp long. The transcription initiation site was determined to be within an 8-bp cluster 328-321 bp upstream of the translation initiation site. The 5{prime} flanking region is highly GC rich, with multiple CpG doublets, and contains multiple binding sites for Sp1. Lacking typical transcriptional regulatory sequences such as TATA and CAAT boxes, the putative promoter region conforms to the group of housekeeping promoters. 28 refs., 4 figs., 1 tab.

  10. The impact of RNA standardization and heterogeneous gene expression on the results of cDNA array of human breast carcinoma.

    PubMed

    Khoshnoud, Reza; He, Qimin; Sylván, Maria; Khoshnoud, Aida; Ivarsson, Madleen; Fornander, Tommy; Bergh, Jonas; Frisell, Jan; Rutqvist, Lars-Erik; Skog, Sven

    2010-05-01

    cDNA microarray is an established technique. However, difficulties such as handling tissue samples under RNase-free conditions, the heterogeneous tumor composition, i.e. non-malignant versus malignant cells and different pathologic types of malignant cells, and lack of appropriate reference may limit the potentially benefit of this method in clinical use. In this study, we examined how standardization of gene expression to total mg RNA or mg tissue and tumor heterogeneity affect the final results. We found that the gene expression of human breast tumors was approximately 9 times higher in malignant tissue as compared to the non-malignant tissue when expressed per total mg RNA, but approximately 40 times higher when expressed per mg tissue. Genes that were expected to act as housekeeping genes (PUC18, RPL and beta-actin) varied between different parts of the tumor and also between non-malignant and malignant tissues, excluding them as reference genes. We also found that the gene expression differed in various parts of the breast tumor, probably due to a mixture of different types of cells, i.e. non-malignant and malignant cells. To find out if the variations in the gene expression were due to cell heterogeneity we used microdissection to collect malignant cells separately. We found that the gene expression was markedly different in the isolated malignant cells as compared to the gene expression of the bulk tumor tissue. Thus, to be able to evaluate results from cDNA array gene expression experiments it is, to our opinion, necessary to work with pure tumor cell populations, until solid information is available on the impact of stromal component. Housekeeping genes should be handling with care and mg tissue may be preferred instead of microg RNA for standardization.

  11. Rapid amplification of cDNA ends (RACE) improves the PCR-based isolation of immunoglobulin variable region genes from murine and human lymphoma cells and cell lines.

    PubMed

    Doenecke, A; Winnacker, E L; Hallek, M

    1997-10-01

    The isolation of rearranged immunoglobulin (Ig) variable region (V) genes is usually performed by PCR with consensus primers binding to conserved regions within the V sequences. However, the isolation of Ig genes by this method is hampered in 15-35% by technical difficulties, mostly mismatches of oligonucleotide primers to V sequences. In order to obtain DNA sequences from V heavy chain (VH) genes which could not be amplified with consensus primers, we used a modified PCR technique, the rapid amplification of cDNA ends (RACE) PCR in combination with new heavy chain constant region primers for the isolation of human and murine VH genes. In comparison, consensus primer PCR with different sets of previously published oligonucleotide primers was used. Both methods were applied to isolate VH genes from murine B cell lymphoma (A20 and BCL1), myeloma (NS1) and hybridoma (SP6) cell lines and from freshly isolated human chronic lymphocytic leukemia and lymphoma cells. RACE PCR allowed the amplification and subsequent cloning of the complete VH gene in all cases. In contrast, consensus primer PCR failed to isolate the VH sequence of the murine A20 cell line; this was explained by a mismatch of consensus primers with VH sequences. When both PCR methods amplified VH sequences, the DNA sequences obtained were identical. Taken together, RACE PCR represents a reliable and versatile method for the isolation of VH genes from human and murine lymphoma cells, in particular if consensus primer PCR fails.

  12. The gene encoding human glutathione synthetase (GSS) maps to the long arm of chromosome 20 at band 11.2

    SciTech Connect

    Webb, G.C.; Vaska, V.L.; Ford, J.H.

    1995-12-10

    Two forms of glutathione synthetase deficiency have been described. While one form is mild, causing hemolytic anemia, the other more severe form causes 5-oxoprolinuria with secondary neurological involvement. Despite the existence of two deficiency phenotypes, Southern blots hybridized with a glutathione synthetase cDNA suggest that there is a single glutathione synthetase gene in the human genome. Analysis of somatic cell hybrids showed the human glutathione synthetase gene (GSS) to be located on chromosome 20, and this assignment has been refined to subband 20q11.2 using in situ hybridization. 16 refs., 2 figs.

  13. Expression cloning of the murine interferon gamma receptor cDNA.

    PubMed

    Munro, S; Maniatis, T

    1989-12-01

    A cDNA encoding a receptor for murine interferon gamma (IFN-gamma) was isolated from an expression library made from murine thymocytes. The clone was identified by transfecting the library into monkey COS cells and probing the transfected monolayer with radiolabeled murine IFN-gamma. Cells expressing the receptor were identified by autoradiography and plasmids encoding the receptor were directly rescued from those cells producing a positive signal. A partial cDNA so obtained was used to isolate a full-length cDNA from mouse L929 cells by conventional means. When this cDNA was expressed in COS cells it produced a specific binding site for murine IFN-gamma with an affinity constant similar to that of the receptor found on L929 cells. The predicted amino acid sequence of the murine IFN-gamma receptor shows homology to that previously reported for the human IFN-gamma receptor. However, although the two proteins are clearly related, they show less than 60% identity in both the putative extracellular domain and the intracellular domain.

  14. Encoding of nested levels of acoustic regularity in hierarchically organized areas of the human auditory cortex.

    PubMed

    Recasens, Marc; Grimm, Sabine; Wollbrink, Andreas; Pantev, Christo; Escera, Carles

    2014-11-01

    Our auditory system is able to encode acoustic regularity of growing levels of complexity to model and predict incoming events. Recent evidence suggests that early indices of deviance detection in the time range of the middle-latency responses (MLR) precede the mismatch negativity (MMN), a well-established error response associated with deviance detection. While studies suggest that only the MMN, but not early deviance-related MLR, underlie complex regularity levels, it is not clear whether these two mechanisms interplay during scene analysis by encoding nested levels of acoustic regularity, and whether neuronal sources underlying local and global deviations are hierarchically organized. We registered magnetoencephalographic evoked fields to rapidly presented four-tone local sequences containing a frequency change. Temporally integrated local events, in turn, defined global regularities, which were infrequently violated by a tone repetition. A global magnetic mismatch negativity (MMNm) was obtained at 140-220 ms when breaking the global regularity, but no deviance-related effects were shown in early latencies. Conversely, Nbm (45-55 ms) and Pbm (60-75 ms) deflections of the MLR, and an earlier MMNm response at 120-160 ms, responded to local violations. Distinct neuronal generators in the auditory cortex underlay the processing of local and global regularity violations, suggesting that nested levels of complexity of auditory object representations are represented in separated cortical areas. Our results suggest that the different processing stages and anatomical areas involved in the encoding of auditory representations, and the subsequent detection of its violations, are hierarchically organized in the human auditory cortex.

  15. Encoding of Touch Intensity But Not Pleasantness in Human Primary Somatosensory Cortex

    PubMed Central

    Laubacher, Claire M.; Olausson, Håkan; Wang, Binquan; Spagnolo, Primavera A.; Bushnell, M. Catherine

    2016-01-01

    Growing interest in affective touch has delineated a neural network that bypasses primary somatosensory cortex (S1). Several recent studies, however, have cast doubt on the segregation of touch discrimination and affect, suggesting that S1 also encodes affective qualities. We used functional magnetic resonance imaging (fMRI) and repetitive transcranial magnetic stimulation (rTMS) to examine the role of S1 in processing touch intensity and pleasantness. Twenty-six healthy human adults rated brushing on the hand during fMRI. Intensity ratings significantly predicted activation in S1, whereas pleasantness ratings predicted activation only in the anterior cingulate cortex. Nineteen subjects also received inhibitory rTMS over right hemisphere S1 and the vertex (control). After S1 rTMS, but not after vertex rTMS, sensory discrimination was reduced and subjects with reduced sensory discrimination rated touch as more intense. In contrast, rTMS did not alter ratings of touch pleasantness. Our findings support divergent neural processing of touch intensity and pleasantness, with affective touch encoded outside of S1. SIGNIFICANCE STATEMENT Growing interest in affective touch has identified a neural network that bypasses primary somatosensory cortex (S1). Several recent studies, however, cast doubt on the separation of touch discrimination and affect. We used functional magnetic resonance imaging and repetitive transcranial magnetic stimulation to demonstrate the representation of touch discrimination and intensity in S1, but the representation of pleasantness in the anterior cingulate cortex, not S1. Our findings support divergent neural processing of touch intensity and pleasantness, with affective touch encoded outside of S1. Our study contributes to growing delineation of the affective touch system, a crucial step in understanding its dysregulation in numerous clinical conditions such as autism, eating disorders, depression, and chronic pain. PMID:27225773

  16. A synergy-based hand control is encoded in human motor cortical areas

    PubMed Central

    Leo, Andrea; Handjaras, Giacomo; Bianchi, Matteo; Marino, Hamal; Gabiccini, Marco; Guidi, Andrea; Scilingo, Enzo Pasquale; Pietrini, Pietro; Bicchi, Antonio; Santello, Marco; Ricciardi, Emiliano

    2016-01-01

    How the human brain controls hand movements to carry out different tasks is still debated. The concept of synergy has been proposed to indicate functional modules that may simplify the control of hand postures by simultaneously recruiting sets of muscles and joints. However, whether and to what extent synergic hand postures are encoded as such at a cortical level remains unknown. Here, we combined kinematic, electromyography, and brain activity measures obtained by functional magnetic resonance imaging while subjects performed a variety of movements towards virtual objects. Hand postural information, encoded through kinematic synergies, were represented in cortical areas devoted to hand motor control and successfully discriminated individual grasping movements, significantly outperforming alternative somatotopic or muscle-based models. Importantly, hand postural synergies were predicted by neural activation patterns within primary motor cortex. These findings support a novel cortical organization for hand movement control and open potential applications for brain-computer interfaces and neuroprostheses. DOI: http://dx.doi.org/10.7554/eLife.13420.001 PMID:26880543

  17. Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire

    PubMed Central

    Yeung, Yik Andy; Foletti, Davide; Deng, Xiaodi; Abdiche, Yasmina; Strop, Pavel; Glanville, Jacob; Pitts, Steven; Lindquist, Kevin; Sundar, Purnima D.; Sirota, Marina; Hasa-Moreno, Adela; Pham, Amber; Melton Witt, Jody; Ni, Irene; Pons, Jaume; Shelton, David; Rajpal, Arvind; Chaparro-Riggers, Javier

    2016-01-01

    Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition. PMID:27857134

  18. Human anterior prefrontal cortex encodes the 'what' and 'when' of future intentions.

    PubMed

    Momennejad, Ida; Haynes, John-Dylan

    2012-05-15

    On a daily basis we form numerous intentions to perform specific actions. However, we often have to delay the execution of intended actions while engaging in other demanding activities. Previous research has shown that patterns of activity in human prefrontal cortex (PFC) can reveal our current intentions. However, two fundamental questions have remained unresolved: (a) how does the PFC encode information about future tasks while we are busy engaging in other activities, and (b) how does the PFC enable us to commence a stored task at the intended time? Here we investigate how the brain stores and retrieves future intentions during occupied delays, i.e. while a person is busy performing a different task. For this purpose, we conducted a neuroimaging study with a time-based prospective memory paradigm. Using multivariate pattern classification and fMRI we show that during an occupied delay, activity patterns in the anterior PFC encode the content of 'what' subjects intend to do next, and 'when' they intend to do it. Importantly, distinct anterior PFC regions store the 'what' and 'when' components of future intentions during occupied maintenance and self-initiated retrieval. These results show a role for anterior PFC activity patterns in storing future action plans and ensuring their timely retrieval.

  19. The active gene that encodes human High Mobility Group 1 protein (HMG1) contains introns and maps to chromosome 13

    SciTech Connect

    Ferrari, S.; Finelli, P.; Rocchi, M.

    1996-07-15

    The human genome contains a large number of sequences related to the cDNA for High Mobility Group 1 protein (HMG1), which so far has hampered the cloning and mapping of the active HMG1 gene. We show that the human HMG1 gene contains introns, while the HMG1-related sequences do not and most likely are retrotransposed pseudogenes. We identified eight YACs from the ICI and CEPH libraries that contain the human HMG1 gene. The HMG1 gene is similar in structure to the previously characterized murine homologue and maps to human chromosome 13 and q12, as determined by in situ hybridization. The mouse Hmg1 gene maps to the telomeric region of murine Chromosome 5, which is syntenic to the human 13q12 band. 18 refs., 3 figs.

  20. Expression of human basic fibroblast growth factor cDNA in baby hamster kidney-derived cells results in autonomous cell growth

    PubMed Central

    1988-01-01

    Growth factor over-production by responsive cells might contribute to their autonomous proliferation as well as their acquisition of a transformed phenotype in culture. Basic fibroblast growth factor (bFGF) has been shown to induce transient changes in cell behavior that resemble those encountered in transformed cells. In addition, several types of human tumor cells have been shown to produce bFGF. To determine directly the role that bFGF might play in the induction of the transformed phenotype, we have introduced a human bFGF cDNA expression vector into baby hamster kidney-derived (BHK-21) fibroblasts. One of the BHK transfectants, termed clone 19, expresses the bFGF mRNA and produces biologically active bFGF that accumulates to a high concentration inside the cells. These properties correlate with the ability of the cells to grow in serum-free medium without the addition of exogenous bFGF. Clone 19 cells also proliferated in soft agar, indicating that constitutive expression of the bFGF gene results in a loss of anchorage-dependent growth. PMID:3360856

  1. Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA clone library

    SciTech Connect

    Teitz, T.; Naiman, T.; Avissar, S.S.; Bar, S.; Okayama, H.; Canaani, D.

    1987-12-01

    In previous work, a xeroderma pigmentosum cell line belonging to complementation group C was established by transformation with origin-defective simian virus 40. We now report the complementation of the UV sensitivity of this cell line by gene transfer. A human cDNA clone library constructed in a mammalian expression vector, and itself incorporated in a lambda phage vector, was introduced into the cells as a calcium phosphate precipitate. Following selection to G418 resistance, provided by the neo gene of the vector, transformants were selected for UV resistance. Twenty-one cell clones were obtained with UV-resistance levels typical of normal human fibroblasts. All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-resistant. Secondary transformants were generated by transfection with a partial digest of total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-resistant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV resistance by secondary transformants derived by transfection of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants acquired UV resistance through DNA-mediated gene transfer and not by reversion.

  2. Mapping of an autoreactive epitope within glutamate decarboxylase using a diabetes-associated human monoclonal autoantibody and an epitope cDNA library.

    PubMed

    Richter, W; Northemann, W; Müller, M; Böhm, B O

    1996-04-01

    Glutamate decarboxylase (GAD65) is a major autoantigen in insulin-dependent diabetes (IDDM) and the neurological disorder Stiff-Man-Syndrome (SMS). We derived a human monoclonal autoantibody (MICA 2) from peripheral blood of a patient newly diagnosed with IDDM, which reacted with GAD65 in Western blots. This indicated that a linear epitope is recognized by MICA 2. Using an epitope cDNA library we mapped the MICA 2 epitope to a contiguous stretch of 26 amino acids (506-531) in the C-terminus of GAD65. Neither blocking experiments with synthetic peptides nor analysis of overlapping decapeptides expressed as fusion proteins allowed us to further narrow down the epitope to the typical size of linear epitopes of 6-8 amino acids. We suggest that a miniconformational epitope provided by amino acids 506-531 is recognized by MICA 2, which withstands SDS gel electrophoresis without destruction or partially refolds during the Western blot procedure. A sequence homology with human heat shock protein 60 (HSP60) maps to this region of GAD65 but no cross-reactivity of MICA 2 with HSP60 occurred. Our data demonstrate that reactivity of an antibody in Western blots does not necessarily define a classic linear epitope of 6-8 amino acids and describe a new autoreactive epitope in GAD65 different from those reported for sera from patients with SMS.

  3. A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice

    SciTech Connect

    Pittius, C.W.; Hennighausen, L.; Lee, E.; Westphal, H.; Nicols, E.; Vitale, J.; Gordon, K. )

    1988-08-01

    Whey acidic protein (WAP) is a major whey protein in mouse milk. Its gene is expressed in the lactating mammary gland and is inducible by steroid and peptide hormones. A series of transgenic mice containing a hybrid gene in which human tissue plasminogen activator (tPA) cDNA is under the control of the murine WAP gene promoter had previously been generated. In this study, 21 tissues from lactating and virgin transgenic female mice containing the WAP-tPA hybrid gene were screened for the distribution of murine WAP and human tPA transcripts. Like the endogenous WAP RNA, WAP-tPA RNA was expressed predominantly in mammary gland tissue and appeared to be inducible by lactation. Whereas WAP transcripts were not detected in 22 tissues of virgin mice, low levels of WAP-tPA RNA, which were not modulated during lactation, were found in tongue, kidney, and sublingual gland. These studies demonstrate that the WAP gene promoter can target the expression of a transgene to the mammary gland and that this expression is inducible during lactation.

  4. Evidence that the SRY protein is encoded by a single exon on the human Y chromosome

    SciTech Connect

    Behlke, M.A. Women's Hospital and Harvard Medical School, Boston, MA ); Bogan, J.S.; Beer-Romero, P.; Page, D.C. )

    1993-09-01

    To facilitate studies of the SRY gene, a 4741-bp portion of the sex-determining region of the human Y chromosome was sequenced and characterized. Two RNAs were found to hybridize to this genomic segment, one transcript deriving from SRY and the second cross-hybridizing to a pseudogene located 2.5 kb 5[prime] of the SRY open reading frame (ORF). Analysis of the SRY transcript using 3[prime] and 5[prime] rapid amplification and cloning of ends suggested that the entire SRY protein is encoded by a single exon. A 700-bp CpG island is located immediately 5[prime] of the pseudogene (and 2 kb 5[prime] of the SRY ORF). Within this CpG island lies the sequence CGCCCCCGC, a potential binding site for the EGR-1/WT1 family of transcription factors, some of which appear to function in gonadal development. 19 refs., 1 fig.

  5. Evidence for joint encoding of motion and disparity in human visual perception.

    PubMed

    Neri, Peter; Levi, Dennis M

    2008-12-01

    Electrophysiological recordings have established that motion and disparity signals are jointly encoded by subpopulations of neurons in visual cortex. However, the question of whether these neurons play a perceptual role has proven challenging and remains open. To answer this question we combined two powerful psychophysical techniques: perceptual adaptation and reverse correlation. Our results provide a detailed picture of how visual information about motion and disparity is processed by human observers, and how this processing is modified by prolonged sensory stimulation. We were able to isolate two perceptual components: a separable component, supported by separate motion and disparity signals, and an inseparable joint component, supported by motion and disparity signals that are concurrently represented at the level of the same neural mechanism. Both components are involved in the perception of stimuli containing motion and disparity information in line with the known existence of corresponding neuronal subpopulations in visual cortex.

  6. Decoding the disease-associated proteins encoded in the human chromosome 4.

    PubMed

    Chen, Lien-Chin; Liu, Mei-Ying; Hsiao, Yung-Chin; Choong, Wai-Kok; Wu, Hsin-Yi; Hsu, Wen-Lian; Liao, Pao-Chi; Sung, Ting-Yi; Tsai, Shih-Feng; Yu, Jau-Song; Chen, Yu-Ju

    2013-01-04

    Chromosome 4 is the fourth largest chromosome, containing approximately 191 megabases (~6.4% of the human genome) with 757 protein-coding genes. A number of marker genes for many diseases have been found in this chromosome, including genetic diseases (e.g., hepatocellular carcinoma) and biomedical research (cardiac system, aging, metabolic disorders, immune system, cancer and stem cell) related genes (e.g., oncogenes, growth factors). As a pilot study for the chromosome 4-centric human proteome project (Chr 4-HPP), we present here a systematic analysis of the disease association, protein isoforms, coding single nucleotide polymorphisms of these 757 protein-coding genes and their experimental evidence at the protein level. We also describe how the findings from the chromosome 4 project might be used to drive the biomarker discovery and validation study in disease-oriented projects, using the examples of secretomic and membrane proteomic approaches in cancer research. By integrating with cancer cell secretomes and several other existing databases in the public domain, we identified 141 chromosome 4-encoded proteins as cancer cell-secretable/shedable proteins. Additionally, we also identified 54 chromosome 4-encoded proteins that have been classified as cancer-associated proteins with successful selected or multiple reaction monitoring (SRM/MRM) assays developed. From literature annotation and topology analysis, 271 proteins were recognized as membrane proteins while 27.9% of the 757 proteins do not have any experimental evidence at the protein-level. In summary, the analysis revealed that the chromosome 4 is a rich resource for cancer-associated proteins for biomarker verification projects and for drug target discovery projects.

  7. Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells

    PubMed Central

    Ramunas, John; Yakubov, Eduard; Brady, Jennifer J.; Corbel, Stéphane Y.; Holbrook, Colin; Brandt, Moritz; Stein, Jonathan; Santiago, Juan G.; Cooke, John P.; Blau, Helen M.

    2015-01-01

    Telomere extension has been proposed as a means to improve cell culture and tissue engineering and to treat disease. However, telomere extension by nonviral, nonintegrating methods remains inefficient. Here we report that delivery of modified mRNA encoding TERT to human fibroblasts and myoblasts increases telomerase activity transiently (24–48 h) and rapidly extends telomeres, after which telomeres resume shortening. Three successive transfections over a 4 d period extended telomeres up to 0.9 kb in a cell type-specific manner in fibroblasts and myoblasts and conferred an additional 28 ± 1.5 and 3.4 ± 0.4 population doublings (PDs), respectively. Proliferative capacity increased in a dose-dependent manner. The second and third transfections had less effect on proliferative capacity than the first, revealing a refractory period. However, the refractory period was transient as a later fourth transfection increased fibroblast proliferative capacity by an additional 15.2 ± 1.1 PDs, similar to the first transfection. Overall, these treatments led to an increase in absolute cell number of more than 1012-fold. Notably, unlike immortalized cells, all treated cell populations eventually stopped increasing in number and expressed senescence markers to the same extent as untreated cells. This rapid method of extending telomeres and increasing cell proliferative capacity without risk of insertional mutagenesis should have broad utility in disease modeling, drug screening, and regenerative medicine.—Ramunas, J., Yakubov, E., Brady, J. J., Corbel, S. Y., Holbrook, C., Brandt, M., Stein, J., Santiago, J. G., Cooke, J. P., Blau, H. M. Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells. PMID:25614443

  8. Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations

    SciTech Connect

    Yan Qingfeng; Bykhovskaya, Yelena; Li Ronghua; Mengesha, Emebet; Shohat, Mordechai; Estivill, Xavier; Fischel-Ghodsian, Nathan; Guan Minxin . E-mail: min-xin.guan@chmcc.org

    2006-04-21

    Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations.

  9. Localization of genes encoding three distinct flavin-containing monooxygenases to human chromosome 1q

    SciTech Connect

    Shephard, E.A.; Fox, M.F.; Povey, S. ); Dolphin, C.T.; Phillips, I.R.; Smith, R. )

    1993-04-01

    The authors have used the polymerase chain reaction to map the gene encoding human flavin-containing monooxygenase (FMO) form II (N. Lomri, Q. Gu, and J. R. Cashman, 1992, Proc. Natl. Acad. Sci. USA 89: 1685--1689) to chromosome 1. They propose the designation FMO3 for this gene as it is the third FMO gene to be mapped. The two other human FMO genes identified to date, FMO1 and FMO2, are also located on chromosome 1 (C. Dolphin, E. A. Shephard, S. Povey, C. N. A. Palmer, D. M. Ziegler, R. Ayesh, R. L. Smith, and 1. R. Phillips, 1991, J. Biol. Chem. 266: 12379--12385; C. Dolphin, E. A. Shephard, S. F. Povey, R. L. Smith, and I. R. Phillips, 1992, Biochem. J. 286: 261--267). The localization of FMO1, FMO2, and FMO3 has been refined to the long arm of chromosome 1. Analysis of human metaphase chromosomes by in situ hybridization confirmed the mapping of FMO1 and localized this gene more precisely to 1 q23-q25. 28 refs., 3 figs., 2 tabs.

  10. Molecular and functional characterization of a cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.).

    PubMed

    Lasseur, Bertrand; Lothier, Jérémy; Djoumad, Abdelmadjid; De Coninck, Barbara; Smeekens, Sjef; Van Laere, André; Morvan-Bertrand, Annette; Van den Ende, Wim; Prud'homme, Marie-Pascale

    2006-01-01

    Fructans are the main storage compound in Lolium perenne. To account for the prevailing neokestose-based fructan synthesis in this species, a cDNA library of L. perenne was screened by using the onion (Allium cepa) fructan:fructan 6G-fructosyltransferase (6G-FFT) as a probe. A full length Lp6G-FFT clone was isolated with significant homologies to vacuolar type fructosyltransferases and invertases. The functionality of the cDNA was tested by heterologous expression in Pichia pastoris. The recombinant protein demonstrated both 6G-FFT and fructan:fructan 1-fructosyltransferase activities (1-FFT) with a maximum 6G-FFT/1-FFT ratio of two. The activity of 6G-FFT was investigated with respect to developmental stage, tissue distribution, and alterations in carbohydrate status expression and compared to sucrose:sucrose 1-fructosyltransferase (1-SST). Lp6G-FFT and Lp1-SST were predominantly expressed in the basal part of elongating leaves and leaf sheaths. Expression of both genes declined along the leaf axis, in parallel with the spatial occurrence of fructan and fructosyltransferase activities. Surprisingly, Lp6G-FFT was highly expressed in photosynthetically active tissues where very low extractable fructosyltransferase activity and fructan amounts were detected, suggesting a post-transcriptional regulation of expression. Lp6G-FFT gene expression increased only in elongating leaves following similar increases of sucrose content in blades, sheaths, and elongating leaf bases. Regulation of Lp6G-FFT gene expression depends on the tissue according to its sink-source status.

  11. Assignment of the gene encoding the [beta]-subunit of the electron-transfer flavoprotein (ETFB) to human chromosome 19q13. 3

    SciTech Connect

    Antonacci, R. ); Colombo, I.; Volta, M.; DiDonato, S.; Finocchiaro, G. ); Archidiacono, N.; Rocchi, M. )

    1994-01-01

    The electron-transfer flavoprotein (ETF), located in the mitochondrial matrix, is a nuclear-encoded enzyme delivering to the respiratory chain electrons by straight-chain acyl-CoA dehydrogenases and other dehydrogenases. ETF is composed of a 35-kDa [alpha]-subunit that is cleaved to a 32-kDa protein during mitochondrial import (ETFA) and a [beta]-subunit that reaches the mitochondrion unmodified (ETFB). The cDNA encoding both these subunits has been cloned and sequenced. 14 refs., 1 fig.

  12. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1, 1992--December 31, 1992

    SciTech Connect

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  13. Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

    PubMed

    Qing, K; Bachelot, T; Mukherjee, P; Wang, X S; Peng, L; Yoder, M C; Leboulch, P; Srivastava, A

    1997-07-01

    The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These

  14. Non-human lnc-DC orthologs encode Wdnm1-like protein

    PubMed Central

    Dijkstra, Johannes M.; Ballingall, Keith T.

    2014-01-01

    In a recent publication in Science, Wang et al. found a long noncoding RNA (lncRNA) expressed in human dendritic cells (DC), which they designated lnc-DC. Based on lentivirus-mediated RNA interference (RNAi) experiments in human and murine systems, they concluded that lnc-DC is important in differentiation of monocytes into DC. However, Wang et al. did not mention that their so-called “mouse lnc-DC ortholog” gene was already designated “ Wdnm1-like” and is known to encode a small secreted protein.  We found that incapacitation of the Wdnm1-like open reading frame (ORF) is very rare among mammals, with all investigated primates except for hominids having an intact ORF. The null-hypothesis by Wang et al. therefore should have been that the human lnc-DC transcript might only represent a non-functional relatively young evolutionary remnant of a protein coding locus.  Whether this null-hypothesis can be rejected by the experimental data presented by Wang et al. depends in part on the possible off-target (immunogenic or otherwise) effects of their RNAi procedures, which were not exhaustive in regard to the number of analyzed RNAi sequences and control sequences.  If, however, the conclusions by Wang et al. on their human model are correct, and they may be, current knowledge regarding the Wdnm1-like locus suggests an intriguing combination of different functions mediated by transcript and protein in the maturation of several cell types at some point in evolution. We feel that the article by Wang et al. tends to be misleading without the discussion presented here. PMID:25309733

  15. Molecular mechanism underlying differential apoptosis between human melanoma cell lines UACC903 and UACC903(+6) revealed by mitochondria-focused cDNA microarrays.

    PubMed

    Zhang, Qiuyang; Wu, Jun; Nguyen, Anhthu; Wang, Bi-Dar; He, Ping; Laurent, Georges St; Rennert, Owen M; Su, Yan A

    2008-08-01

    Human malignant melanoma cell line UACC903 is resistant to apoptosis while chromosome 6-mediated suppressed cell line UACC903(+6) is sensitive. Here, we describe identification of differential molecular pathways underlying this difference. Using our recently developed mitochondria-focused cDNA microarrays, we identified 154 differentially expressed genes including proapoptotic (BAK1 [6p21.3], BCAP31, BNIP1, CASP3, CASP6, FAS, FDX1, FDXR, TNFSF10 and VDAC1) and antiapoptotic (BCL2L1, CLN3 and MCL1) genes. Expression of these pro- and anti-apoptotic genes was higher in UACC903(+6) than in UACC903 before UV treatment and was altered after UV treatment. qRT-PCR and Western blots validated microarray results. Our bioinformatic analysis mapped these genes to differential molecular pathways that predict resistance and sensitivity of UACC903 and UACC903(+6) to apoptosis respectively. The pathways were functionally confirmed by the FAS ligand-induced cell death and by siRNA knockdown of BAK1 protein. These results demonstrated the differential molecular pathways underlying survival and apoptosis of UACC903 and UACC903(+6) cell lines.

  16. An efficient and rapid method for cDNA cloning from difficult templates using codon optimization and SOE-PCR: with human RANK and TIMP2 gene as examples.

    PubMed

    Huang, Gang; Wen, Qianjun; Gao, Qiangguo; Zhang, Fang; Bai, Yun

    2011-10-01

    As gene cloning from difficult templates with regionalized high GC content is a long recognized problem, we have developed a novel and reliable method to clone such genes. Firstly, the high GC content region of the target cDNA was synthesized directly after codon optimization and the remaining cDNA fragment without high GC content was generated by routine RT-PCR. Then the entire redesigned coding sequence of the target gene was obtained by fusing the above available two cDNA fragments with SOE-PCR (splicing by overlapping extension-PCR). We have cloned the human RANK gene (ten exons; CDS 1851 bp) using this strategy. The redesigned cDNA was transfected into an eukaryotic expression system (A459 cells) to verify its expression. RT-PCR and western blotting confirmed this. To validate our method, we also successfully cloned human TIMP2 gene (five exons; CDS 660 bp) also having a regionalized high GC content. Our strategy for combining codon optimization and SOE-PCR to clone difficult genes is thus feasible and potentially universally applicable.

  17. Preparation and optimisation of anionic liposomes for delivery of small peptides and cDNA to human corneal epithelial cells.

    PubMed

    Neves, Luís F; Duan, Jinghua; Voelker, Adrienne; Khanal, Anil; McNally, Lacey; Steinbach-Rankins, Jill; Ceresa, Brian P

    2016-06-01

    Drug delivery to corneal epithelial cells is challenging due to the intrinsic mechanisms that protect the eye. Here, we report a novel liposomal formulation to encapsulate and deliver a short sequence peptide into human corneal epithelial cells (hTCEpi). Using a mixture of Phosphatidylcholine/Caproylamine/Dioleoylphosphatidylethanolamine (PC/CAP/DOPE), we encapsulated a fluorescent peptide, resulting in anionic liposomes with an average size of 138.8 ± 34 nm and a charge of -18.2 ± 1.3 mV. After 2 h incubation with the peptide-encapsulated liposomes, 66% of corneal epithelial (hTCEpi) cells internalised the FITC-labelled peptide, demonstrating the ability of this formulation to effectively deliver peptide to hTCEpi cells. Additionally, lipoplexes (liposomes complexed with plasmid DNA) were also able to transfect hTCEpi cells, albeit at a modest level (8% of the cells). Here, we describe this novel anionic liposomal formulation intended to enhance the delivery of small cargo molecules in situ.

  18. Gene structure and chromosomal localization of the human HSD11K gene encoding the kidney (type 2) isozyme of 11{beta}-hydroxysteroid dehydrogenase

    SciTech Connect

    Agarwal, A.K.; Rogerson, F.M.; Mune, T.; White, P.C.

    1995-09-01

    11{beta}-hydroxysteroid dehydrogenase (11{beta}HSD) converts glucocorticoids to inactive products and is thus thought to confer specificity for aldosterone on the type I mineralocorticoid receptor in the kidney. Recent studies indicate the presence of at least two isozymes of 11{beta}HSD. In vitro, the NAD{sup +}-dependent kidney (type 2) isozyme catalyzes 11{beta}-dehydrogenase but not reductase reactions, whereas the NADP{sup +}-dependent liver (type 1) isozyme catalyzes both reactions. We have now characterized the human gene encoding kidney 11{beta}HSD (HSD11K). A bacteriophage P1 clone was isolated after screening a human genomic library by hybridization with sheep HSD11K cDNA. The gene consists of 5 exons spread over 6 kb. The nucleotide binding domain lies in the first exon are GC-rich (80%), suggesting that the gene may be transcriptionally regulated by factors that recognize GC-rich sequences. Fluorescence in situ hybridization of metaphase chromosomes with a positive P1 clone localized the gene to chromosome 16q22. In contrast, the HSD11L (liver isozyme) gene is located on chromosome 1 and contains 6 exons; the coding sequences of these genes are only 21% identical. HSD11K is expressed at high levels in the placenta and kidney of midgestation human fetuses and at lower levels in lung and testes. Different transcriptional start sites are utilized in kidney and placenta. These data should be applicable to genetic analysis of the syndrome of apparent mineralocorticoid excess, which may represent a deficiency of 11{beta}HSD. 25 refs., 5 figs.

  19. Novel human growth hormone like protein HGH-V encoded in the human genome

    SciTech Connect

    Seeburg, P.H.

    1987-05-12

    This patent describes the human growth hormone protein, HGH-V, having the amino acid sequence: phe pro thr ile pro leu ser arg leu phe asp asn ala met leu arg ala arg arg leu tyr gln leu ala tyr asp thr tyr gln glu phe glu glu ala tyr ile leu lys glu gln lys tyr ser phe leu gln asn pro gln thr ser leu cys phe ser glu ser ile pro thr pro ser asn arg val lys thr gln gln lys ser asn leu glu leu leu arg ile ser leu leu leu ile gln ser trp leu glu pro val gln leu leu arg ser val phe ala asn ser leu val tyr gly ala ser asp ser asn val tyr arg his leu lys asp leu glu glu gly ile gln thr leu met trp arg leu glu asp gly ser pro arg thr gly gln ile phe asn-glycosylation site gln ser tyr ser lys phe asp thr lys ser his asn asp asp ala leu leu lys asn tyr gly leu leu tyr cys Phe arg lys asp met asp lys val glu thr phe leu arg ile val gln cys arg ser val glu gly ser cys gly phe.

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

    PubMed

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

    1999-01-01

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

  1. The mouse and human genes encoding the recognition component of the N-end rule pathway

    PubMed Central

    Kwon, Yong Tae; Reiss, Yuval; Fried, Victor A.; Hershko, Avram; Yoon, Jeong Kyo; Gonda, David K.; Sangan, Pitchai; Copeland, Neal G.; Jenkins, Nancy A.; Varshavsky, Alexander

    1998-01-01

    The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3α. Mouse UBR1p (E3α) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans ≈120 kilobases of genomic DNA and contains ≈50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway. PMID:9653112

  2. Muscle spindles in human tibialis anterior encode muscle fascicle length changes.

    PubMed

    Day, James; Bent, Leah R; Birznieks, Ingvars; Macefield, Vaughan G; Cresswell, Andrew G

    2017-04-01

    Muscle spindles provide exquisitely sensitive proprioceptive information regarding joint position and movement. Through passively driven length changes in the muscle-tendon unit (MTU), muscle spindles detect joint rotations because of their in-parallel mechanical linkage to muscle fascicles. In human microneurography studies, muscle fascicles are assumed to follow the MTU and, as such, fascicle length is not measured in such studies. However, under certain mechanical conditions, compliant structures can act to decouple the fascicles, and, therefore, the spindles, from the MTU. Such decoupling may reduce the fidelity by which muscle spindles encode joint position and movement. The aim of the present study was to measure, for the first time, both the changes in firing of single muscle spindle afferents and changes in muscle fascicle length in vivo from the tibialis anterior muscle (TA) during passive rotations about the ankle. Unitary recordings were made from 15 muscle spindle afferents supplying TA via a microelectrode inserted into the common peroneal nerve. Ultrasonography was used to measure the length of an individual fascicle of TA. We saw a strong correlation between fascicle length and firing rate during passive ankle rotations of varying rates (0.1-0.5 Hz) and amplitudes (1-9°). In particular, we saw responses observed at relatively small changes in muscle length that highlight the sensitivity of the TA muscle to small length changes. This study is the first to measure spindle firing and fascicle dynamics in vivo and provides an experimental basis for further understanding the link between fascicle length, MTU length, and spindle firing patterns.NEW & NOTEWORTHY Muscle spindles are exquisitely sensitive to changes in muscle length, but recordings from human muscle spindle afferents are usually correlated with joint angle rather than muscle fascicle length. In this study, we monitored both muscle fascicle length and spindle firing from the human tibialis

  3. Characterization of gene expression changes associated with MNNG, arsenic, or metal mixture treatment in human keratinocytes: application of cDNA microarray technology.

    PubMed Central

    Bae, Dong-Soon; Hanneman, William H; Yang, Raymond S H; Campain, Julie A

    2002-01-01

    The identification of molecular markers related to critical biological processes during carcinogenesis may aid in the evaluation of carcinogenic potentials of chemicals and chemical mixtures. Work from our laboratory demonstrated that a single treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) enhanced spontaneous malignant transformation of the human keratinocyte cell line RHEK-1. In contrast, chronic low-level exposure of cells to arsenic alone or in a mixture containing arsenic, cadmium, chromium, and lead inhibited malignant conversion. To identify changes in gene expression that influence these different outcomes, cDNA microarray technology was used. Analysis of multiple human arrays in MNNG-transformed RHEK-1 cells, designated OM3, and those treated with arsenic or the arsenic-containing metal mixture showed unique patterns of gene expression. Genes that were overexpressed in OM3 included oncogenes, cell cycle regulators, and those involved in signal transduction, whereas genes for DNA repair enzymes and inhibitors of transformation and metastasis were suppressed. In arsenic-treated cells, multiple DNA repair proteins were overexpressed. Mixture-treated cells showed increased expression of a variety of genes including metallothioneins and integrin 4. These cells showed decreased expression of oncogenes, DNA repair proteins, and genes involved in the mitogen-activated protein kinase pathway. For comparison we are currently analyzing gene expression changes in RHEK-1 cells transformed by other means. The goal of these studies is to identify common batteries of genes affected by chemical modulators of the carcinogenic process. Mechanistic studies may allow us to correlate alterations in their expression with sequential stages in the carcinogenic process and may aid in the risk assessment of other xenobiotics. PMID:12634122

  4. Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

    PubMed

    Zay, Agnes; Choy, Francis Y M; Patrick, Chelsea; Sinclair, Graham

    2011-06-01

    The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to

  5. Identification, characterization, and precise mapping of a human gene encoding a novel membrane-spanning protein from the 22q11 region deleted in velo-cardio-facial syndrome.

    PubMed

    Sirotkin, H; Morrow, B; Saint-Jore, B; Puech, A; Das Gupta, R; Patanjali, S R; Skoultchi, A; Weissman, S M; Kucherlapati, R

    1997-06-01

    Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are characterized by a wide spectrum of phenotypes including cleft palate, conotruncal heart defects, and facial dysmorphology. Hemizygosity for a portion of chromosome 22q11 has been detected in 80-85% of VCFS/DGS patients. Using a cDNA selection protocol, we have identified a new gene, TMVCF (transmembrane protein deleted in VCFS), which maps to the deleted interval. The genomic locus is positioned between polymorphic markers D22S944 and D22S941. TMVCF encodes a small protein of 219 amino acids that is predicted to contain two membrane-spanning domains. TMVCF is expressed abundantly in human adult lung, heart, and skeletal muscle, and transcripts can be detected at least as early as Day 9 of mouse development.

  6. Flavin reductase: sequence of cDNA from bovine liver and tissue distribution.

    PubMed Central

    Quandt, K S; Hultquist, D E

    1994-01-01

    Flavin reductase catalyzes electron transfer from reduced pyridine nucleotides to methylene blue or riboflavin, and this catalysis is the basis of the therapeutic use of methylene blue or riboflavin in the treatment of methemoglobinemia. A cDNA for a mammalian flavin reductase has been isolated and sequenced. Degenerate oligonucleotides, with sequences based on amino acid sequences of peptides derived from bovine erythrocyte flavin reductase, were used as primers in PCR to selectively amplify a partial cDNA that encodes the bovine reductase. The template used in the PCR was first strand cDNA synthesized from bovine liver total RNA using oligo(dT) primers. A PCR product was used as a specific probe to screen a bovine liver cDNA library. The sequence determined from two overlapping clones contains an open reading frame of 621 nucleotides and encodes 206 amino acids. The amino acid sequence deduced from the bovine liver flavin reductase cDNA matches the amino acid sequences determined for erythrocyte reductase-derived peptides, and the predicted molecular mass of 22,001 Da for the liver reductase agrees well with the molecular mass of 21,994 Da determined for the erythrocyte reductase by electrospray mass spectrometry. The amino acid sequence at the N terminus of the reductase has homology to sequences of pyridine nucleotide-dependent enzymes, and the predicted secondary structure, beta alpha beta, resembles the common nucleotide-binding structural motif. RNA blot analysis indicates a single 1-kilobase reductase transcript in human heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle. Images PMID:7937764

  7. Molecular cloning of MER-2, a human chromosome-11-encoded red blood cell antigen, using linkage of cotransfected markers.

    PubMed

    Bill, J; Palmer, E; Jones, C

    1987-09-01

    We report the molecular cloning of a human gene MER-2 located on chromosome 11 that encodes a cell surface antigen which is polymorphic on red blood cells. An essential element of the cloning strategy was cotransfection-induced linkage of pSV2-neo, which encodes resistance to the antibiotic G418, to the human MER-2 gene. An important feature of the pSV2-neo construct is that the same gene (the transposon, Tn5) that encodes G418 resistance in eukaryotic cells confers neomycin resistance in bacteria. Chinese hamster ovary (CHO) cells were cotransfected with pSV2-neo and genomic DNA from a CHO X human cell hybrid containing a single human chromosome (chromosome 11). Transfectants expressing both the human MER-2 gene and G418 resistance were isolated by selection in the antibiotic G418, followed by indirect immunofluorescence using the monoclonal antibody 1D12, which recognizes the MER-2 antigen, manual enrichment, and single-cell cloning. Genomic DNA from a primary transfectant positive for MER-2 expression and G418 resistance was used to construct a cosmid library and cosmid clones able to grow in neomycin were isolated. Of 150,000 cosmid clones screened, 90 were resistant to neomycin and of these, 11 contained human repetitive sequences. Five neomycin-resistant cosmid clones containing human repetitive DNA were able to transfect CHO cells for G418 resistance and MER-2 expression.

  8. Lectin cDNA and transgenic plants derived therefrom

    DOEpatents

    Raikhel, Natasha V.

    1994-01-04

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties. GOVERNMENT RIGHTS This application was funded under Department of Energy Contract DE-AC02-76ER01338. The U.S. Government has certain rights under this application and any patent issuing thereon.

  9. Lectin cDNA and transgenic plants derived therefrom

    DOEpatents

    Raikhel, N.V.

    1994-01-04

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties. GOVERNMENT RIGHTS This application was funded under Department of Energy Contract DE-AC02-76ER01338. The U.S. Government has certain rights under this application and any patent issuing thereon. .

  10. Genetic analysis of the variable region genes encoding a monospecific human natural anti-DNA antibody.

    PubMed Central

    Daley, M D; Misener, V; Olee, T; Chen, P P; Siminovitch, K A

    1993-01-01

    Recent evidence suggests that natural autoantibodies may play an integral role in the development of the normal immune repertoire. To explore the genetic origins of these antibodies, we have isolated and sequenced the variable (V) region genes encoding both the heavy (H) and light (L) chains of a natural anti-DNA antibody, Kim11.4. The genes appear to be derived from the VH4.18 (subgroup VHIV), JH5, Hum1L1 (subgroup V lambda I) and J lambda 3 germline genes. The origin of the H chain diversity gene is more obscure, being potentially derived from one or more of several germline genes, arranged in either the forward or reverse orientations. Both the Kim11.4 VH and VL genes share significant degrees of similarity with those utilized in other autoantibodies, indicating that at least some degree of V restriction may exist in human autoreactive B cells. The pattern of nucleotide differences between the Kim11.4 VH and VL genes and their putative germline counterparts suggests that the Kim11.4 genes may have undergone somatic mutation and arisen as a result of antigen selection. PMID:8324896

  11. US28, a Virally-Encoded GPCR as an Antiviral Target for Human Cytomegalovirus Infection

    PubMed Central

    Lee, Sungjin; Chung, Yoon Hee; Lee, Choongho

    2017-01-01

    Viruses continue to evolve a new strategy to take advantage of every aspect of host cells in order to maximize their survival. Due to their central roles in transducing a variety of transmembrane signals, GPCRs seem to be a prime target for viruses to pirate for their own use. Incorporation of GPCR functionality into the genome of herpesviruses has been demonstrated to be essential for pathogenesis of many herpesviruses-induced diseases. Here, we introduce US28 of human cytomegalovirus (HCMV) as the best-studied example of virally-encoded GPCRs to manipulate host GPCR signaling. In this review, we wish to summarize a number of US28-related topics including its regulation of host signaling pathways, its constitutive internalization, its structural and functional analysis, its roles in HCMV biology and pathogenesis, its proliferative activities and role in oncogenesis, and pharmacological modulation of its biological activities. This review will aid in our understanding of how pathogenic viruses usurp the host GPCR signaling for successful viral infection. This kind of knowledge will enable us to build a better strategy to control viral infection by normalizing the virally-dysregulated host GPCR signaling. PMID:28035083

  12. Direction of Movement Is Encoded in the Human Primary Motor Cortex

    PubMed Central

    Toxopeus, Carolien M.; de Jong, Bauke M.; Valsan, Gopal; Conway, Bernard A.; Leenders, Klaus L.; Maurits, Natasha M.

    2011-01-01

    The present study investigated how direction of hand movement, which is a well-described parameter in cerebral organization of motor control, is incorporated in the somatotopic representation of the manual effector system in the human primary motor cortex (M1). Using functional magnetic resonance imaging (fMRI) and a manual step-tracking task we found that activation patterns related to movement in different directions were spatially disjoint within the representation area of the hand on M1. Foci of activation related to specific movement directions were segregated within the M1 hand area; activation related to direction 0° (right) was located most laterally/superficially, whereas directions 180° (left) and 270° (down) elicited activation more medially within the hand area. Activation related to direction 90° was located between the other directions. Moreover, by investigating differences between activations related to movement along the horizontal (0°+180°) and vertical (90°+270°) axis, we found that activation related to the horizontal axis was located more anterolaterally/dorsally in M1 than for the vertical axis, supporting that activations related to individual movement directions are direction- and not muscle related. Our results of spatially segregated direction-related activations in M1 are in accordance with findings of recent fMRI studies on neural encoding of direction in human M1. Our results thus provide further evidence for a direct link between direction as an organizational principle in sensorimotor transformation and movement execution coded by effector representations in M1. PMID:22110768

  13. Cloning and expression of cDNA for a human Gal(beta1-3)GalNAc alpha2,3-sialyltransferase from the CEM T-cell line.

    PubMed

    Giordanengo, V; Bannwarth, S; Laffont, C; Van Miegem, V; Harduin-Lepers, A; Delannoy, P; Lefebvre, J C

    1997-07-15

    Complementary DNA encoding a human Gal(beta1-3)GalNAc alpha2,3-sialyltransferase type II (hST3Gal II) was cloned from a CEM T-cell cDNA library using a 23-base oligonucleotide probe. The sequence of this probe was established on the basis of a slightly divergent sialylmotif L that was obtained by polymerase chain reaction with degenerate oligonucleotide primers based on the conserved sialylmotif L of mammalian Gal(beta1-3)GalNAc alpha2,3-sialyltransferases. It was thus confirmed that a short oligonucleotide probe may be sensitive and highly specific. The nucleotide and amino acid sequences of hST3Gal II show, respectively, 56.3% and 49.3% similarity to hST3Gal I [Kitagawa, H. & Paulson, J. C. (1994) J. Biol. Chem. 269, 17872-17878] and 88.1% and 93.7% similarity to murine ST3Gal II [Lee, Y. C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T., Hamamoto, T. & Tsuji, S. (1994) J. Biol. Chem. 269, 10028-10033]. hST3Gal II mRNA was highly expressed in heart, liver, skeletal muscle and various lymphoid tissues but not in brain and kidney. A soluble form of hST3Gal II expressed in COS-7 cells was tested in vitro for substrate specificity and kinetic properties. Asialofetuin and asialo-bovine submaxillary mucin appeared better substrates for hST3Gal II than for its murine counterpart as previously reported [Kojima, N., Lee, Y.-C., Hamamoto, T., Kurosawa, N. & Tsuji, S. (1994) Biochemistry 33, 5772-5776]. In previous studies, we have shown hyposialylation of O-glycans attached to two major lymphocyte CD43 and CD45 cell surface molecules in human-immunodeficiency-virus-1(HIV-1)-infected T-cell lines. Since comparable levels of hST3Gal I and hST3Gal II mRNA and enzymatic activity were observed in parental and HIV-1-infected CEM T-cell lysates, the sialylation defect associated with HIV infection of this cell line is probably due to a mechanism different from a simple altered catalytic activity of these sialyltransferases.

  14. Molecular cloning of the cDNA encoding the Epstein-Barr virus/C3d receptor (complement receptor type 2) of human B lymphocytes

    SciTech Connect

    Moore, M.D.; Cooper, N.R.; Tack, B.F.; Nemerow, G.R.

    1987-12-01

    Complementary DNA clones for complement receptor type 2 (CR2), the B-lymphocyte membrane protein that serves as the receptor for Epstein-Barr virus and the C3d complement fragment, were obtained by screening a lambda gt11 library generated from Raji B lymphoblastoid cell mRNA. A 4.2-kilobase (kb) clone, representing the entire coding sequence of the protein plus untranslated 5' and 3' nucleotide sequences was obtained and sequenced. The 4.2-kb clone, which contains all but about 500 base pairs (bp) of the 5' untranslated region of the full-length CR2 mRNA, consists of 63 bp of 5' untranslated nucleotide sequence followed successively by a start codon, a 20-amino acid hydrophobic signal peptide, 1005 amino acids having a repeating motif, a 28-amino acid probable transmembrane domain, and a 34-amino acid cytoplasmic tail. The deduced amino acid sequence of the protein indicates that the extracellular domain consists entirely of 16 tandemly arranged repeating elements, each 60-75 amino acids in length, which are identified by multiple conserved residues. This repeating motif also occurs in the C3b/C4b receptor, several complement proteins, and a number of noncomplement proteins. In CR2, the 16 repeats occur in four clusters of four repeats each. Approximately 10% of the deduced amino acid sequence, including the amino and carboxyl termini, was confirmed by amino acid sequencing of tryptic peptides derived from purified CR2. The nucleotide and derived amino acid sequence of CR2 and related studies are presented here.

  15. Modulation of endogenous β-tubulin isotype expression as a result of human βIII cDNA transfection into prostate carcinoma cells

    PubMed Central

    Ranganathan, S; McCauley, R A; Dexter, D W; Hudes, G R

    2001-01-01

    Increases of individual β tubulin isotypes in antimicrotubule drug resistant cell lines have been reported by several laboratories. We have previously described elevations in βIII and βIVa isotypes in estramustine and paclitaxel resistant human prostate carcinoma cells. To investigate further the function of β tubulin isotypes in antimicrotubule drug response, human prostate carcinoma cells that normally have very low to undetectable levels of βIII were stably transfected with βIII cDNA in pZeoSV system. An 18 bp haemagglutinin (HA) epitope tag was added at the 3′ end prior to cloning into the vector. Cells were transfected with pZeoSV or pZeoSV-βIII plasmids and selected in the presence of Zeocin. Immunofluorescent staining of the transfectant cells have shown significant expression and incorporation of HA-tagged βIII tubulin into cellular microtubules. Quantitation of Western blots revealed the HA-tagged βIII levels to be approximately 7-fold higher than the vector control cells. RT-PCR analysis confirmed the increase at the transcript level and also revealed a collateral increase of βII and βIVb transcripts. Cell viability assays indicated that sensitivity of βIII transfected cells to various antimicrotubule agents was similar to vector transfected cells: IC50 values for estramustine, paclitaxel, colchicine and vinblastine were 4 μM, 4 nM, 22 nM and 2 nM, respectively for both cell lines. Thus, overexpression of βIII isotype in human prostate carcinoma cells by stable transfection failed to confer antimicrotubule drug resistance to these cells. Counterregulatory increases of endogenous βII and βIVb tubulin isotypes in these βIII transfected cells may be a compensatory mechanism used by the cells to overcome the effects of elevated βIII levels on the cellular microtubules. These results highlight the difficulty in isolating the contribution of single tubulin isotypes in drug response studies. © 2001 Cancer Research Campaign http

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

    SciTech Connect

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

    2005-04-22

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

  17. Encoding/retrieval dissociation in working memory for human body forms.

    PubMed

    Bauser, Denise A Soria; Mayer, Kerstin; Daum, Irene; Suchan, Boris

    2011-06-20

    The present study was conducted to investigate the effect of working memory (WM) load on body processing mechanisms by using event-related potentials (ERPs). It is well known that WM load modulates the P3b (amplitude decreases as WM load increases). Additionally, WM load for faces modulates earlier ERPs like the N170. The present study aimed to investigate the effect of WM load for bodies on the P3b which is associated with WM. Additionally, we explored the effect of WM load on the N170, which is thought to be associated with configural processing, and P1, which has been observed in body as well as in face processing. Effects were analyzed during the encoding and retrieval phases. WM load was modulated by presenting one to four unfamiliar bodies simultaneously for memory encoding. The present study showed that early encoding processes (reflected by the P1 and N170) might not be modulated by WM load, whereas during the retrieval phase, early processes associated with structural encoding (N170) were affected by WM load. A possible explanation of the encoding/retrieval differences might be that subjects used distinct processing strategies in both phases. Parallel encoding of the simultaneously presented bodies might play an important role during the encoding phase where one to four bodies have to be stored, whereas serial matching might be used to compare the probe with the stored pictures during the retrieval phase. Additionally, WM load modulations were observed in later processing steps, which might be associated with stimulus identification and matching processes (reflected by the early P3b) during the encoding but not during the retrieval phase. The current findings further showed for both the encoding and the retrieval phase that the late P3b amplitude decreased as WM load for body images increased indicating that the late P3b is involved in WM processes which do not appear to be category-specific.

  18. The human clotting factor VIII cDNA contains an autonomously replicating sequence consensus- and matrix attachment region-like sequence that binds a nuclear factor, represses heterologous gene expression, and mediates the transcriptional effects of sodium butyrate.

    PubMed Central

    Fallaux, F J; Hoeben, R C; Cramer, S J; van den Wollenberg, D J; Briët, E; van Ormondt, H; van Der Eb, A J

    1996-01-01

    Expression of the human blood-clotting factor VIII (FVIII) cDNA is hampered by the presence of sequences located in the coding region that repress transcription. We have previously identified a 305-bp fragment within the FVIII cDNA that is involved in the repression (R.C. Hoeben, F.J. Fallaux, S.J. Cramer, D.J.M. van den Wollenberg, H. van Ormondt, E. Briet, and A.J. van der Eb, Blood 85:2447-2454, 1995). Here, we show that this 305-bp region of FVIII cDNA contains sequences that resemble the yeast (Saccharomyces cerevisiae) autonomously replicating sequence consensus. Two of these DNA elements coincide with AT-rich sequences that are often found in matrix attachment regions or scaffold-attached regions. One of these elements, consisting of nucleotides 1569 to 1600 of the FVIII cDNA (nucleotide numbering is according to the system of Wood et al. (W.I. Wood, D.J. Capon, C.C. Simonsen, D.L. Eaton, J. Gitschier, D. Keyt, P.H. Seeburg, D.H. Smith, P. Hollingshead, K.L. Wion, et al., Nature [London] 312:330-337,1984), binds a nuclear factor in vitro but loses this capacity after four of its base pairs have been changed. A synthetic heptamer of this segment can repress the expression of a chloramphenicol acetyltransferase (CAT) reporter gene and also loses this capacity upon mutation. Furthermore, we demonstrate that repression by FVIII sequences can be relieved by sodium butyrate. We demonstrate that the synthetic heptamer (FVIII nucleotides 1569 to 1600), when placed upstream of the Moloney murine leukemia virus long terminal repeat promoter that drives the CAT reporter, can render the CAT reporter inducible by butyrate. This effect was absent when the same element was mutated. The stimulatory effect of butyrate could not be attributed to butyrate-responsive elements in the studied long terminal repeat promoters. Our data provide a functional characterization of the sequences that repress expression of the FVIII cDNA. These data also suggest a link between

  19. Heterogeneity of rat type I 5 alpha-reductase cDNA: cloning, expression and regulation by pituitary implants and dihydrotestosterone.

    PubMed

    Lopez-Solache, I; Luu-The, V; Séralini, G E; Labrie, F

    1996-03-01

    Primer extension analysis reveals the presence of different forms of mRNA species for rat type I 5 alpha-reductase. Using a 5 alpha-reductase cDNA probe to screen the rat liver lambda gt11 cDNA library, we isolated cDNA clones that have 4 additional amino acids in the NH2-terminal region as compared with the previously reported sequence for rat type I 5 alpha-reductase. These four additional amino acids elongate the rat type I 5 alpha-reductase amino acid sequence to 259 amino acids, the same number as in human type I 5 alpha-reductase, with which it shares 60% identity. Expression of the long and short rat type I 5 alpha-reductase by transfection in human adrenal adenocarcinoma cells, SW-13 cells, indicated that the long cDNA encoded a protein with a higher affinity for the substrate than the short cDNA. To determine the effect of pituitary hormones and dihydrotestosterone (DHT), the mRNA levels in the livers of rats treated with pituitary implants, hypophysectomized, castrated, and castrated coupled with DHT treatment were quantified by dot-blot hybridization assay using rat type I 5 alpha-reductase cDNA as probes. The results demonstrated that rat type I 5 alpha-reductase mRNA is stimulated by pituitary hormones and castration but is decreased by DHT and hypophysectomy.

  20. The gene for human glutaredoxin (GLRX) is localized to human chromosome 5q14

    SciTech Connect

    Padilla, C.A.; Holmgren, A.; Bajalica, S.; Lagercrantz, J.

    1996-03-05

    Glutaredoxin is a small protein (12 kDa) catalyzing glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH, and glutathione reductase. A cDNA encoding the human glutaredoxin gene (HGMW-approved symbol GLRX) has recently been isolated and cloned from a human fetal spleen cDNA library. The screening of a human fetal spleen cDNA library. The screening of a human genomic library in Charon 4A led to the identification of three genomic clones. Using fluorescence in situ hybridization to metaphase chromosomes with one genomic clone as a probe, the human glutaredoxin gene was localized to chromosomal region 5q14. This localization at chromosome 5 was in agreement with the somatic cell hybrid analysis, using DNA from a human-hamster and a human-mouse hybrid panel and using a human glutaredoxin cDNA as a probe. 13 refs., 2 figs.

  1. Induced and Evoked Human Electrophysiological Correlates of Visual Working Memory Set-Size Effects at Encoding

    PubMed Central

    Berryhill, Marian E.; Caplovitz, Gideon P.

    2016-01-01

    The ability to encode, store, and retrieve visually presented objects is referred to as visual working memory (VWM). Although crucial for many cognitive processes, previous research reveals that VWM strictly capacity limited. This capacity limitation is behaviorally observable in the set size effect: the ability to successfully report items in VWM asymptotes at a small number of items. Research into the neural correlates of set size effects and VWM capacity limits in general largely focus on the maintenance period of VWM. However, we previously reported that neural resources allocated to individual items during VWM encoding correspond to successful VWM performance. Here we expand on those findings by investigating neural correlates of set size during VWM encoding. We hypothesized that neural signatures of encoding-related VWM capacity limitations should be differentiable as a function of set size. We tested our hypothesis using High Density Electroencephalography (HD-EEG) to analyze frequency components evoked by flickering target items in VWM displays of set size 2 or 4. We found that set size modulated the amplitude of the 1st and 2nd harmonic frequencies evoked during successful VWM encoding across frontal and occipital-parietal electrodes. Frontal sites exhibited the most robust effects for the 2nd harmonic (set size 2 > set size 4). Additionally, we found a set-size effect on the induced power of delta-band (1–4 Hz) activity (set size 2 > set size 4). These results are consistent with a capacity limited VWM resource at encoding that is distributed across to-be-remembered items in a VWM display. This resource may work in conjunction with a task-specific selection process that determines which items are to be encoded and which are to be ignored. These neural set size effects support the view that VWM capacity limitations begin with encoding related processes. PMID:27902738

  2. Induced and Evoked Human Electrophysiological Correlates of Visual Working Memory Set-Size Effects at Encoding.

    PubMed

    Gurariy, Gennadiy; Killebrew, Kyle W; Berryhill, Marian E; Caplovitz, Gideon P

    2016-01-01

    The ability to encode, store, and retrieve visually presented objects is referred to as visual working memory (VWM). Although crucial for many cognitive processes, previous research reveals that VWM strictly capacity limited. This capacity limitation is behaviorally observable in the set size effect: the ability to successfully report items in VWM asymptotes at a small number of items. Research into the neural correlates of set size effects and VWM capacity limits in general largely focus on the maintenance period of VWM. However, we previously reported that neural resources allocated to individual items during VWM encoding correspond to successful VWM performance. Here we expand on those findings by investigating neural correlates of set size during VWM encoding. We hypothesized that neural signatures of encoding-related VWM capacity limitations should be differentiable as a function of set size. We tested our hypothesis using High Density Electroencephalography (HD-EEG) to analyze frequency components evoked by flickering target items in VWM displays of set size 2 or 4. We found that set size modulated the amplitude of the 1st and 2nd harmonic frequencies evoked during successful VWM encoding across frontal and occipital-parietal electrodes. Frontal sites exhibited the most robust effects for the 2nd harmonic (set size 2 > set size 4). Additionally, we found a set-size effect on the induced power of delta-band (1-4 Hz) activity (set size 2 > set size 4). These results are consistent with a capacity limited VWM resource at encoding that is distributed across to-be-remembered items in a VWM display. This resource may work in conjunction with a task-specific selection process that determines which items are to be encoded and which are to be ignored. These neural set size effects support the view that VWM capacity limitations begin with encoding related processes.

  3. Brain-specific expression of MAP2 detected using a cloned cDNA probe

    PubMed Central

    1986-01-01

    We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low

  4. Cloning and partial nucleotide sequence of human immunoglobulin mu chain cDNA from B cells and mouse-human hybridomas.

    PubMed Central

    Dolby, T W; Devuono, J; Croce, C M

    1980-01-01

    Purified mRNAs coding for mu and kappa human immunoglobulin polypeptides were translated in vitro and their products were characterized. The mu-specific mRNAs, derived from both human lymphoblastoid cells (GM607) and from a mouse-human somatic cell hybrid secreting human mu chains (alpha D5-H11-BC11), were copied into cDNAs and inserted into the plasmid pBR322. Several recombinant cDNAs that were obtained were identified by a combination of colony hybridization with labeled probes, in vitro translation of plasmid-selected mu mRNAs, and DNA nucleotide sequence determination. One recombinant DNA, for which the sequence has been partially determined, contains the codons for part of the C3 constant region domain through the carboxy-terminal piece (155 amino acids total) as well as the entire 3' noncoding sequence up to the poly(A) site of the human mu mRNA. The sequence A-A-U-A-A occurs 12 nucleotides prior to the poly(A) addition site in the human mu mRNA. Considerable sequence homology is observed in the mouse and human mu mRNA 3' coding and noncoding sequences. Images PMID:6777778

  5. Interaction of human TNF and beta2-microglobulin with Tanapox virus-encoded TNF inhibitor, TPV-2L.

    PubMed

    Rahman, Masmudur M; Jeng, David; Singh, Rajkumari; Coughlin, Jake; Essani, Karim; McFadden, Grant

    2009-04-10

    Tanapox virus (TPV) encodes and expresses a secreted TNF-binding protein, TPV-2L or gp38, that displays inhibitory properties against TNF from diverse mammalian species, including human, monkey, canine and rabbit. TPV-2L also has sequence similarity with the MHC-class I heavy chain and interacts differently with human TNF as compared to the known cellular TNF receptors or any of the known virus-encoded TNF receptor homologs derived from many poxviruses. In order to determine the TNF binding region in TPV-2L, various TPV-2L C-terminal truncations and internal deletions were created and the muteins were expressed using recombinant baculovirus vectors. C-terminal deletions from TPV-2L resulted in reduced binding affinity for human TNF and specific mutants of TNF that discriminate between TNF-R1 and TNF-R2. However, deletion of C-terminal 42 amino acid residues totally abolished the binding of human TNF and its mutants. Removal of any of the predicted internal domains resulted in a mutant TPV-2L protein incapable of binding to human TNF. Deletion of C-terminal residues also affected the ability of TPV-2L to block TNF-induced cellular cytotoxicity. In addition to TNF, TPV-2L can also form complexes with human beta2-microglobulin to form a novel macromolecular complex. In summary, the TPV-2L protein is a bona fide MHC-1 heavy chain family member that binds and inhibits human TNF in a fashion very distinct from other known poxvirus-encoded TNF inhibitors, and also can form a novel complex with the human MHC-1 light chain, beta2-microglobulin.

  6. T cell-based functional cDNA library screening identified SEC14-like 1a carboxy-terminal domain as a negative regulator of human immunodeficiency virus replication.

    PubMed

    Urano, Emiko; Ichikawa, Reiko; Morikawa, Yuko; Yoshida, Takeshi; Koyanagi, Yoshio; Komano, Jun

    2010-05-26

    Genome-wide screening of host factors that regulate HIV-1 replication has been attempted using numerous experimental approaches. However, there has been limited success using T cell-based cDNA library screening to identify genes that regulate HIV-1 replication. We have established a genetic screening strategy using the human T cell line MT-4 and a replication-competent HIV-1. With this system, we identified the C-terminal domain (CTD) of SEC14-like 1a (SEC14L1a) as a novel inhibitor of HIV-1 replication. Our T cell-based cDNA screening system provides an alternative tool for identifying novel regulators of HIV-1 replication.

  7. Human herpesviruses-encoded dUTPases: a family of proteins that modulate dendritic cell function and innate immunity

    PubMed Central

    Ariza, Maria Eugenia; Glaser, Ronald; Williams, Marshall V.

    2014-01-01

    We have previously shown that Epstein-Barr virus (EBV)-encoded dUTPase can modulate innate immune responses through the activation of TLR2 and NF-κB signaling. However, whether this novel immune function of the dUTPase is specific for EBV or a common property of the Herpesviridae family is not known. In this study, we demonstrate that the purified viral dUTPases encoded by herpes simplex virus type 2 (HSV-2), human herpesvirus-6A (HHV-6A), human herpesvirus-8 (HHV-8) and varicella-zoster virus (VZV) differentially activate NF-κB through ligation of TLR2/TLR1 heterodimers. Furthermore, activation of NF-κB by the viral dUTPases was inhibited by anti-TLR2 blocking antibodies (Abs) and the over-expression of dominant-negative constructs of TLR2, lacking the TIR domain, and MyD88 in human embryonic kidney 293 cells expressing TLR2/TLR1. In addition, treatment of human dendritic cells and PBMCs with the herpesviruses-encoded dUTPases from HSV-2, HHV-6A, HHV-8, and VZV resulted in the secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, IL-12, TNF-α, IL-10, and IFN-γ. Interestingly, blocking experiments revealed that the anti-TLR2 Ab significantly reduced the secretion of cytokines by the various herpesviruses-encoded dUTPases (p < 0.05). To our knowledge, this is the first report demonstrating that a non-structural protein encoded by herpesviruses HHV-6A, HHV-8, VZV and to a lesser extent HSV-2 is a pathogen-associated molecular pattern. Our results reveal a novel function of the virus-encoded dUTPases, which may be important to the pathophysiology of diseases caused by these viruses. More importantly, this study demonstrates that the immunomodulatory functions of dUTPases are a common property of the Herpesviridae family and thus, the dUTPase could be a potential target for the development of novel therapeutic agents against infections caused by these herpesviruses. PMID:25309527

  8. Human herpesviruses-encoded dUTPases: a family of proteins that modulate dendritic cell function and innate immunity.

    PubMed

    Ariza, Maria Eugenia; Glaser, Ronald; Williams, Marshall V

    2014-01-01

    We have previously shown that Epstein-Barr virus (EBV)-encoded dUTPase can modulate innate immune responses through the activation of TLR2 and NF-κB signaling. However, whether this novel immune function of the dUTPase is specific for EBV or a common property of the Herpesviridae family is not known. In this study, we demonstrate that the purified viral dUTPases encoded by herpes simplex virus type 2 (HSV-2), human herpesvirus-6A (HHV-6A), human herpesvirus-8 (HHV-8) and varicella-zoster virus (VZV) differentially activate NF-κB through ligation of TLR2/TLR1 heterodimers. Furthermore, activation of NF-κB by the viral dUTPases was inhibited by anti-TLR2 blocking antibodies (Abs) and the over-expression of dominant-negative constructs of TLR2, lacking the TIR domain, and MyD88 in human embryonic kidney 293 cells expressing TLR2/TLR1. In addition, treatment of human dendritic cells and PBMCs with the herpesviruses-encoded dUTPases from HSV-2, HHV-6A, HHV-8, and VZV resulted in the secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, IL-12, TNF-α, IL-10, and IFN-γ. Interestingly, blocking experiments revealed that the anti-TLR2 Ab significantly reduced the secretion of cytokines by the various herpesviruses-encoded dUTPases (p < 0.05). To our knowledge, this is the first report demonstrating that a non-structural protein encoded by herpesviruses HHV-6A, HHV-8, VZV and to a lesser extent HSV-2 is a pathogen-associated molecular pattern. Our results reveal a novel function of the virus-encoded dUTPases, which may be important to the pathophysiology of diseases caused by these viruses. More importantly, this study demonstrates that the immunomodulatory functions of dUTPases are a common property of the Herpesviridae family and thus, the dUTPase could be a potential target for the development of novel therapeutic agents against infections caused by these herpesviruses.

  9. Evaluation of JPEG 2000 encoder options: human and model observer detection of variable signals in X-ray coronary angiograms.

    PubMed

    Zhang, Yani; Pham, Binh; Eckstein, Miguel P

    2004-05-01

    Previous studies have evaluated the effect of the new still image compression standard JPEG 2000 using nontask based image quality metrics, i.e., peak-signal-to-noise-ratio (PSNR) for nonmedical images. In this paper, the effect of JPEG 2000 encoder options was investigated using the performance of human and model observers (nonprewhitening matched filter with an eye filter, square-window Hotelling, Laguerre-Gauss Hotelling and channelized Hotelling model observer) for clinically relevant visual tasks. Two tasks were investigated: the signal known exactly but variable task (SKEV) and the signal known statistically task (SKS). Test images consisted of real X-ray coronary angiograms with simulated filling defects (signals) inserted in one of the four simulated arteries. The signals varied in size and shape. Experimental results indicated that the dependence of task performance on the JPEG 2000 encoder options was similar for all model and human observers. Model observer performance in the more tractable and computationally economic SKEV task can be used to reliably estimate performance in the complex but clinically more realistic SKS task. JPEG 2000 encoder settings different from the default ones resulted in greatly improved model and human observer performance in the studied clinically relevant visual tasks using real angiography backgrounds.

  10. Genomic organization and chromosomal localization of the human and mouse genes encoding the alpha receptor component for ciliary neurotrophic factor.

    PubMed

    Valenzuela, D M; Rojas, E; Le Beau, M M; Espinosa, R; Brannan, C I; McClain, J; Masiakowski, P; Ip, N Y; Copeland, N G; Jenkins, N A

    1995-01-01

    Ciliary neurotrophic factor (CNTF) has recently been found to share receptor components with, and to be structurally related to, a family of broadly acting cytokines, including interleukin-6, leukemia inhibitory factor, and oncostatin M. However, the CNTF receptor complex also includes a CNTF-specific component known as CNTF receptor alpha (CNTFR alpha). Here we describe the molecular cloning of the human and mouse genes encoding CNTFR. We report that the human and mouse genes have an identical intron-exon structure that correlates well with the domain structure of CNTFR alpha. That is, the signal peptide and the immunoglobulin-like domain are each encoded by single exons, the cytokine receptor-like domain is distributed among 4 exons, and the C-terminal glycosyl phosphatidylinositol recognition domain is encoded by the final coding exon. The position of the introns within the cytokine receptor-like domain corresponds to those found in other members of the cytokine receptor superfamily. Confirming a recent study using radiation hybrids, we have also mapped the human CNTFR gene to chromosome band 9p13 and the mouse gene to a syntenic region of chromosome 4.

  11. A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase ζ

    PubMed Central

    Gibbs, Peter E. M.; McGregor, W. Glenn; Maher, Veronica M.; Nisson, Paul; Lawrence, Christopher W.

    1998-01-01

    To get a better understanding of mutagenic mechanisms in humans, we have cloned and sequenced the human homolog of the Saccharomyces cerevisiae REV3 gene. The yeast gene encodes the catalytic subunit of DNA polymerase ζ, a nonessential enzyme that is thought to carry out translesion replication and is responsible for virtually all DNA damage-induced mutagenesis and the majority of spontaneous mutagenesis. The human gene encodes an expected protein of 3,130 residues, about twice the size of the yeast protein (1,504 aa). The two proteins are 29% identical in an amino-terminal region of ≈340 residues, 39% identical in a carboxyl-terminal region of ≈850 residues, and 29% identical in a 55-residue region in the middle of the two genes. The sequence of the expected protein strongly predicts that it is the catalytic subunit of a DNA polymerase of the pol ζ type; the carboxyl-terminal domain possesses, in the right order, the six motifs characteristic of eukaryotic DNA polymerases, most closely resembles yeast pol ζ among all polymerases in the GenBank database, and is different from the human α, δ, and ɛ enzymes. Human cells expressing high levels of an hsREV3 antisense RNA fragment grow normally, but show little or no UV-induced mutagenesis and are slightly more sensitive to killing by UV. The human gene therefore appears to carry out a function similar to that of its yeast counterpart. PMID:9618506

  12. Generation of stable human cell lines with Tetracycline-inducible (Tet-on) shRNA or cDNA expression.

    PubMed

    Gomez-Martinez, Marta; Schmitz, Debora; Hergovich, Alexander

    2013-03-05

    A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon(1), to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells(2,3). In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here. Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells(4), vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs(5-11). However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.(12) modified the shRNA expression vector pSUPER(5) by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of

  13. Spatial auditory regularity encoding and prediction: Human middle-latency and long-latency auditory evoked potentials.

    PubMed

    Cornella, M; Bendixen, A; Grimm, S; Leung, S; Schröger, E; Escera, C

    2015-11-11

    By encoding acoustic regularities present in the environment, the human brain can generate predictions of what is likely to occur next. Recent studies suggest that deviations from encoded regularities are detected within 10-50ms after stimulus onset, as indicated by electrophysiological effects in the middle latency response (MLR) range. This is upstream of previously known long-latency (LLR) signatures of deviance detection such as the mismatch negativity (MMN) component. In the present study, we created predictable and unpredictable contexts to investigate MLR and LLR signatures of the encoding of spatial auditory regularities and the generation of predictions from these regularities. Chirps were monaurally delivered in an either regular (predictable: left-right-left-right) or a random (unpredictable left/right alternation or repetition) manner. Occasional stimulus omissions occurred in both types of sequences. Results showed that the Na component (peaking at 34ms after stimulus onset) was attenuated for regular relative to random chirps, albeit no differences were observed for stimulus omission responses in the same latency range. In the LLR range, larger chirp-and omission-evoked responses were elicited for the regular than for the random condition, and predictability effects were more prominent over the right hemisphere. We discuss our findings in the framework of a hierarchical organization of spatial regularity encoding. This article is part of a Special Issue entitled SI: Prediction and Attention.

  14. The Drosophila pigmentation gene pink (p) encodes a homologue of human Hermansky-Pudlak syndrome 5 (HPS5).

    PubMed

    Falcón-Pérez, Juan M; Romero-Calderón, Rafael; Brooks, Elizabeth S; Krantz, David E; Dell'Angelica, Esteban C

    2007-02-01

    Lysosome-related organelles comprise a group of specialized intracellular compartments that include melanosomes and platelet dense granules (in mammals) and eye pigment granules (in insects). In humans, the biogenesis of these organelles is defective in genetic disorders collectively known as Hermansky-Pudlak syndrome (HPS). Patients with HPS-2, and two murine HPS models, carry mutations in genes encoding subunits of adaptor protein (AP)-3. Other genes mutated in rodent models include those encoding VPS33A and Rab38. Orthologs of all of these genes in Drosophila melanogaster belong to the 'granule group' of eye pigmentation genes. Other genes associated with HPS encode subunits of three complexes of unknown function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2 and -3, for which the Drosophila counterparts had not been characterized. Here, we report that the gene encoding the Drosophila ortholog of the HPS5 subunit of BLOC-2 is identical to the granule group gene pink (p), which was first studied in 1910 but had not been identified at the molecular level. The phenotype of pink mutants was exacerbated by mutations in AP-3 subunits or in the orthologs of VPS33A and Rab38. These results validate D. melanogaster as a genetic model to study the function of the BLOCs.

  15. cDNA cloning and expression of the human A-type platelet-derived growth factor (PDGF) receptor establishes structural similarity to the B-type PDGF receptor

    SciTech Connect

    Claesson-Welsh, L.; Eriksson, A.; Westermark, B.; Heldin, C.H. )

    1989-07-01

    The primary structure of the human A-type receptor for platelet-derived growth factor (PDGF) has been determined. A 6.5-kilobase (kb) transcript was identified through low-stringency hybridization with a probe derived from the B-type PDGF receptor cDNA. The sequence of a cDNA clone corresponding to the 6.5-kb transcript contains an open reading frame that predicts a 1,089-amino acid growth factor receptor-like molecule, which displays 44% overall amino acid similarity with the PDGF B-type receptor. The two receptors have a similar domain organization, with five immunoglobulin-like domains extracellularly and an intracellular split protein tyrosine kinase domain. Transfection of the new cDNA into COS cells led to the expression of a protein specifically recognized by an antiserum previously shown to react with the PDGF A-type receptor. The expressed protein was shown to display high-affinity binding of all three {sup 125}I-labeled dimeric forms of PdGF A and B chains in a manner that is characteristic for the PDGF A-type receptor.

  16. Efficacy of Herpes Simplex Virus Vector Encoding the Human Preproenkephalin Gene for Treatment of Facial Pain in Mice

    PubMed Central

    Ma, Fei; Wang, Chunmei; Yoder, William E.; Westlund, Karin N.; Carlson, Charles R.; Miller, Craig S.; Danaher, Robert J.

    2016-01-01

    Aims To determine whether herpes simplex virus–based vectors can efficiently transduce mouse trigeminal ganglion (TG) neurons and attenuate preexisting nerve injury–induced whisker pad mechanical hypersensitivity in a trigeminal inflammatory compression (TIC) neuropathic pain model. Methods Tissue transduction efficiencies of replication-conditional and replication-defective vectors to mouse whisker pads after topical administration and subcutaneous injection were assessed using quantitative real-time PCR (qPCR). Tissue tropism and transgene expression were assessed using qPCR and reverse-transcriptase qPCR following topical application of the vectors. Whisker pad mechanical sensitivities of TIC-injured mice were determined using graduated von Frey fibers before and after application of human preproenkephalin expressing replication-conditional vector (KHPE). Data were analyzed using one-way analysis of variance (ANOVA) and post hoc tests. Results Transduction of target TGs was 8- to 50-fold greater after topical application than subcutaneous injection and ≥ 100-fold greater for replication-conditional than replication-defective vectors. Mean KHPE loads remained constant in TGs (4.5–9.8 × 104 copies/TG) over 3 weeks but were below quantifiable levels (10 copies/tissue) within 2 weeks of application in other nontarget cephalic tissues examined. Transgene expression in TGs was maximal during 2 weeks after topical application (100–200 cDNA copies/mL) and was below quantifiable levels (1 cDNA copy/mL) in all nontarget tissues. Topical KHPE administration reduced TIC-related mechanical hypersensitivity on whisker pads 4-fold (P < .05) for at least 1 week. Conclusion Topically administered KHPE produced a significant antinociceptive effect in the TIC mouse model of chronic facial neuropathic pain. This is the first report in which a gene therapeutic approach reduced trigeminal pain–related behaviors in an established pain state in mice. PMID:26817032

  17. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    SciTech Connect

    Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

    1987-04-01

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone lambdaHB''-1 from a phage lambdagt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone lambdaHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone lambdaHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the lambdaHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambdaHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.

  18. Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli.

    PubMed

    Zhang, Zhi-He; Zheng, Xu; Hu, Xi-lian; Zhu, Mu-Yuan; Hou, Rong; Shen, Fu-Jun; Zhang, Liang; Liao, Ming-Juan; Lv, Xiao-Ping

    2005-01-01

    cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed. through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.

  19. Involvement of the transcription factor PU.1/Spi-1 in myeloid cell-restricted expression of an interferon-inducible gene encoding the human high-affinity Fc gamma receptor.

    PubMed Central

    Perez, C; Coeffier, E; Moreau-Gachelin, F; Wietzerbin, J; Benech, P D

    1994-01-01

    Induction by gamma interferon (IFN-gamma) of the gene encoding the human high-affinity Fc gamma receptor (Fc gamma R1) in myeloid cells requires an IFN-gamma response region (GRR) and a myeloid cell-activating transcription element (MATE). GRR and MATE interact with factors to form, respectively, an IFN-gamma-activating complex (GIRE-BP), depending on the phosphorylation of the 91-kDa protein (subunit of ISGF3), and a cell-type-specific complex (MATE-BP). Although GIRE-BP is detected in cells of different origins after IFN-gamma treatment, the presence of MATE-BP was found to be restricted to B- and myeloid cell lines. Sequence analysis of a cDNA encoding a polypeptide recognizing specifically the MATE motif led to the identification of this product as the proto-oncogene PU.1/Spi-1, a transcriptional activator expressed in myeloid and B cells. Expression of this factor in nonhematopoietic cells allowed IFN-gamma-induced expression of a reporter gene under control of the GRR and MATE sequences. The presence of these motifs in other gene promoters indicates that the binding of PU.1/Spi-1 and IFN regulatory proteins to their respective motifs could be part of a general mechanism leading to cell-type-restricted and IFN-induced gene expression. Images PMID:8035786

  20. The functional role of human right hippocampal/parahippocampal theta rhythm in environmental encoding during virtual spatial navigation.

    PubMed

    Pu, Yi; Cornwell, Brian R; Cheyne, Douglas; Johnson, Blake W

    2017-03-01

    Low frequency theta band oscillations (4-8 Hz) are thought to provide a timing mechanism for hippocampal place cell firing and to mediate the formation of spatial memory. In rodents, hippocampal theta has been shown to play an important role in encoding a new environment during spatial navigation, but a similar functional role of hippocampal theta in humans has not been firmly established. To investigate this question, we recorded healthy participants' brain responses with a 160-channel whole-head MEG system as they performed two training sets of a virtual Morris water maze task. Environment layouts (except for platform locations) of the two sets were kept constant to measure theta activity during spatial learning in new and familiar environments. In line with previous findings, left hippocampal/parahippocampal theta showed more activation navigating to a hidden platform relative to random swimming. Consistent with our hypothesis, right hippocampal/parahippocampal theta was stronger during the first training set compared to the second one. Notably, theta in this region during the first training set correlated with spatial navigation performance across individuals in both training sets. These results strongly argue for the functional importance of right hippocampal theta in initial encoding of configural properties of an environment during spatial navigation. Our findings provide important evidence that right hippocampal/parahippocampal theta activity is associated with environmental encoding in the human brain. Hum Brain Mapp 38:1347-1361, 2017. © 2016 Wiley Periodicals, Inc.

  1. On the immortality of television sets: "function" in the human genome according to the evolution-free gospel of ENCODE.

    PubMed

    Graur, Dan; Zheng, Yichen; Price, Nicholas; Azevedo, Ricardo B R; Zufall, Rebecca A; Elhaik, Eran

    2013-01-01

    A recent slew of ENCyclopedia Of DNA Elements (ENCODE) Consortium publications, specifically the article signed by all Consortium members, put forward the idea that more than 80% of the human genome is functional. This claim flies in the face of current estimates according to which the fraction of the genome that is evolutionarily conserved through purifying selection is less than 10%. Thus, according to the ENCODE Consortium, a biological function can be maintained indefinitely without selection, which implies that at least 80 - 10 = 70% of the genome is perfectly invulnerable to deleterious mutations, either because no mutation can ever occur in these "functional" regions or because no mutation in these regions can ever be deleterious. This absurd conclusion was reached through various means, chiefly by employing the seldom used "causal role" definition of biological function and then applying it inconsistently to different biochemical properties, by committing a logical fallacy known as "affirming the consequent," by failing to appreciate the crucial difference between "junk DNA" and "garbage DNA," by using analytical methods that yield biased errors and inflate estimates of functionality, by favoring statistical sensitivity over specificity, and by emphasizing statistical significance rather than the magnitude of the effect. Here, we detail the many logical and methodological transgressions involved in assigning functionality to almost every nucleotide in the human genome. The ENCODE results were predicted by one of its authors to necessitate the rewriting of textbooks. We agree, many textbooks dealing with marketing, mass-media hype, and public relations may well have to be rewritten.

  2. Modulation of Oscillatory Power and Connectivity in the Human Posterior Cingulate Cortex Supports the Encoding and Retrieval of Episodic Memories.

    PubMed

    Lega, Bradley; Germi, James; Rugg, Michael

    2017-04-07

    Existing data from noninvasive studies have led researchers to posit that the posterior cingulate cortex (PCC) supports mnemonic processes: It exhibits degeneration in memory disorders, and fMRI investigations have demonstrated memory-related activation principally during the retrieval of memory items. Despite these data, the role of the PCC in episodic memory has received only limited treatment using the spatial and temporal precision of intracranial EEG, with previous analyses focused on item retrieval. Using data gathered from 21 human participants who underwent stereo-EEG for seizure localization, we characterized oscillatory patterns in the PCC during the encoding and retrieval of episodic memories. We identified a subsequent memory effect during item encoding characterized by increased gamma band oscillatory power and a low-frequency power desynchronization. Fourteen participants had stereotactic electrodes located simultaneously in the hippocampus and PCC, and with these unique data, we describe connectivity changes between these structures that predict successful item encoding and that precede item retrieval. Oscillatory power during retrieval matched the pattern we observed during encoding, with low-frequency (below 15 Hz) desynchronization and a gamma band (especially high gamma, 70-180 Hz) power increase. Encoding is characterized by synchrony between the hippocampus and PCC, centered at 3 Hz, consistent with other observations of properties of this oscillation akin to those for rodent theta activity. We discuss our findings in light of existing theories of episodic memory processing, including the information via desynchronization hypothesis and retrieved context theory, and examine how our data fit with existing theories for the functional role of the PCC. These include a postulated role for the PCC in modulating internally directed attention and for representing or integrating contextual information for memory items.

  3. Enhanced human memory consolidation with post-learning stress: interaction with the degree of arousal at encoding.

    PubMed

    Cahill, Larry; Gorski, Lukasz; Le, Kathryn

    2003-01-01

    Abundant evidence indicates that endogenous stress hormones such as epinephrine and corticosterone modulate memory consolidation in animals. We recently provided the first demonstration that an endogenous stress hormone (epinephrine) can enhance human memory consolidation. However, these findings also suggested that post-learning stress hormone activation does not uniformly enhance memory for all recently acquired information; rather, that it interacts with the degree of arousal at initial encoding of material in modulating memory for the material. Here we tested this hypothesis by administering cold pressor stress (CPS) or a control procedure to subjects after they viewed slides of varying emotional content, and assessing memory for the slides 1 wk later. CPS, which significantly elevated salivary cortisol levels, enhanced memory for emotionally arousing slides compared with the controls, but did not affect memory for relatively neutral slides. These findings further support the view that post-learning stress hormone-related activity interacts with arousal at initial encoding to modulate memory consolidation.

  4. The GOR47-1 sequence in human DNA encoding for a potential autoantigen in connection with hepatitis C--a sequence not only reserved for humans.

    PubMed

    Dennin, R H; Chen, Z; Wo, J

    1998-10-01

    The sequence 'GOR47-1' is a consistent part of human DNA; the expressed polypeptide of it 'GOR' is accepted to be an autoantigen, and the anti-GOR an autoantibody. However, GOR47-1 was originally isolated through a cDNA clone from blood of a chimpanzee. This animal belonged to a series of chimpanzees, in which human plasma of a patient with non-A, non-B hepatitis had been passaged. To date, nothing is known how it is that this 'sequence GOR47-1' without recognizable self-replicating properties and allocated to the human genome could be isolated from a chimpanzee plasma. The aim of this study was to detect by polymerase chain reaction GOR47-1 sequences in healthy, anti-HCV-negative humans, HCV-positive patients, chimpanzee, snake, and in maize and tobacco plants. The GOR47-1 sequence is present not only in human DNA but also with a high degree of homology in chimpanzee DNA. Essential parts of this sequence are also present in DNA of a snake and the two plants listed above. Our findings reveal that the GOR47-1 sequence isolated from a chimpanzee was probably of the chimpanzee origin. This fact has not yet been considered up until now, when discussing the role of GOR/anti-GOR in humans particularly suffering from chronic hepatitis C.

  5. Nucleic acids encoding modified human immunodeficiency virus type 1 (HIV-1) group M consensus envelope glycoproteins

    DOEpatents

    Haynes, Barton F [Durham, NC; Gao, Feng [Durham, NC; Korber, Bette T [Los Alamos, NM; Hahn, Beatrice H [Birmingham, AL; Shaw, George M [Birmingham, AL; Kothe, Denise [Birmingham, AL; Li, Ying Ying [Hoover, AL; Decker, Julie [Alabaster, AL; Liao, Hua-Xin [Chapel Hill, NC

    2011-12-06

    The present invention relates, in general, to an immunogen and, in particular, to an immunogen for inducing antibodies that neutralizes a wide spectrum of HIV primary isolates and/or to an immunogen that induces a T cell immune response. The invention also relates to a method of inducing anti-HIV antibodies, and/or to a method of inducing a T cell immune response, using such an immunogen. The invention further relates to nucleic acid sequences encoding the present immunogens.

  6. Dynamic changes in parietal activation during encoding: implications for human learning and memory.

    PubMed

    Elman, Jeremy A; Rosner, Zachary A; Cohn-Sheehy, Brendan I; Cerreta, Adelle G; Shimamura, Arthur P

    2013-11-15

    The ventral posterior parietal cortex (vPPC) monitors successful memory retrieval, yet its role during learning remains unclear. Indeed, increased vPPC activation during stimulus encoding is often negatively correlated with subsequent memory performance, suggesting that this region is suppressed during learning. Alternatively, the vPPC may engage in learning-related processes immediately after stimulus encoding thus facilitating retrieval at a later time. To investigate this possibility, we assessed vPPC activity during item presentation and immediately following its offset when a cue to remember was presented. We observed a dynamic change in vPPC response such that activity was negatively correlated with subsequent memory during stimulus presentation but positively correlated immediately following the stimulus during the cue phase. Furthermore, regional differences in this effect suggest a degree of functional heterogeneity within the vPPC. These findings demonstrate that the vPPC is engaged during learning and acts to facilitate post-encoding memory processes that establish long-term cortical representations.

  7. Sensitivity-encoded (SENSE) proton echo-planar spectroscopic imaging (PEPSI) in the human brain.

    PubMed

    Lin, Fa-Hsuan; Tsai, Shang-Yueh; Otazo, Ricardo; Caprihan, Arvind; Wald, Lawrence L; Belliveau, John W; Posse, Stefan

    2007-02-01

    Magnetic resonance spectroscopic imaging (MRSI) provides spatially resolved metabolite information that is invaluable for both neuroscience studies and clinical applications. However, lengthy data acquisition times, which are a result of time-consuming phase encoding, represent a major challenge for MRSI. Fast MRSI pulse sequences that use echo-planar readout gradients, such as proton echo-planar spectroscopic imaging (PEPSI), are capable of fast spectral-spatial encoding and thus enable acceleration of image acquisition times. Combining PEPSI with recent advances in parallel MRI utilizing RF coil arrays can further accelerate MRSI data acquisition. Here we investigate the feasibility of ultrafast spectroscopic imaging at high field (3T and 4T) by combining PEPSI with sensitivity-encoded (SENSE) MRI using eight-channel head coil arrays. We show that the acquisition of single-average SENSE-PEPSI data at a short TE (15 ms) can be accelerated to 32 s or less, depending on the field strength, to obtain metabolic images of choline (Cho), creatine (Cre), N-acetyl-aspartate (NAA), and J-coupled metabolites (e.g., glutamate (Glu) and inositol (Ino)) with acceptable spectral quality and localization. The experimentally measured reductions in signal-to-noise ratio (SNR) and Cramer-Rao lower bounds (CRLBs) of metabolite resonances were well explained by both the g-factor and reduced measurement times. Thus, this technology is a promising means of reducing the scan times of 3D acquisitions and time-resolved 2D measurements.

  8. Nuclear-encoded factors involved in post-transcriptional processing and modification of mitochondrial tRNAs in human disease

    PubMed Central

    Powell, Christopher A.; Nicholls, Thomas J.; Minczuk, Michal

    2015-01-01

    The human mitochondrial genome (mtDNA) encodes 22 tRNAs (mt-tRNAs) that are necessary for the intraorganellar translation of the 13 mtDNA-encoded subunits of the mitochondrial respiratory chain complexes. Maturation of mt-tRNAs involves 5′ and 3′ nucleolytic excision from precursor RNAs, as well as extensive post-transcriptional modifications. Recent data suggest that over 7% of all mt-tRNA residues in mammals undergo post-transcriptional modification, with over 30 different modified mt-tRNA positions so far described. These processing and modification steps are necessary for proper mt-tRNA function, and are performed by dedicated, nuclear-encoded enzymes. Recent growing evidence suggests that mutations in these nuclear genes (nDNA), leading to incorrect maturation of mt-tRNAs, are a cause of human mitochondrial disease. Furthermore, mtDNA mutations in mt-tRNA genes, which may also affect mt-tRNA function, processing, and modification, are also frequently associated with human disease. In theory, all pathogenic mt-tRNA variants should be expected to affect only a single process, which is mitochondrial translation, albeit to various extents. However, the clinical manifestations of mitochondrial disorders linked to mutations in mt-tRNAs are extremely heterogeneous, ranging from defects of a single tissue to complex multisystem disorders. This review focuses on the current knowledge of nDNA coding for proteins involved in mt-tRNA maturation that have been linked to human mitochondrial pathologies. We further discuss the possibility that tissue specific regulation of mt-tRNA modifying enzymes could play an important role in the clinical heterogeneity observed for mitochondrial diseases caused by mutations in mt-tRNA genes. PMID:25806043

  9. Mg-chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two-hybrid system, and reconstitution of the enzyme activity by co-expression of recombinant CHL D, CHL H and CHL I.

    PubMed

    Papenbrock, J; Gräfe, S; Kruse, E; Hänel, F; Grimm, B

    1997-11-01

    Mg-protoporphyrin IX chelatase catalyzes insertion of the magnesium ion into protoporphyrin IX, the last common intermediate precursor in chlorophyll and heme biosynthesis, to form Mg-protoporphyrin IX. In Rhodobacter sphaeroides, and Synechocystis, the three open reading frames bchD/chID, bchH/chIH and bchI/chII encode proteins which are required for in vitro Mg-chelatase activity. In higher plants also, three proteins are necessary for the Mg chelation, and genes homologous to bchH and bchI have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characterized. Together with the tobacco clones encoding the other two subunits, full-length cDNAs are now available for the first time for all three subunits of one plant species. The CHL D polypeptide deduced from the open reading frame encodes a protein of 758 aa (82.9 kDa) with an amino terminal extension that resembles a plastid transit peptide. Sequence comparison of tobacco CHL D revealed similarities to the D subunit of Rhodobacter and Synechocystis of 44% and 75%. The amino terminal half of CHL D shows significant similarity (46%) to the entire CHL I peptide sequence, indicating a gene duplication from an ancestral gene. The carboxy terminal half seemed to be unique. Both parts of CHL D are linked with a glutamine/asparagine/proline-rich region flanked by a highly acid-rich segment. Protein-protein interaction among the three subunits CHL D, H and I was studied using the yeast two-hybrid system. Physical interaction was demonstrated between CHL D and CHL I indicating that CHL D is part of the Mg-chelatase. Heterodimer formation of CHL H with CHL I or CHL D could not be demonstrated by transactivation of the lacZ reporter gene. Homodimerization of the CHL D subunit was indicated in the more sensitive assay on X-Gal-containing agar plates. In vitro Mg2+ insertion into protoporphyrin IX was demonstrated in protein extracts of yeast strains expressing

  10. Cloning, structural characterization, and chromosomal localization of the gene encoding the human prostaglandin E(2) receptor EP2 subtype.

    PubMed

    Smock, S L; Pan, L C; Castleberry, T A; Lu, B; Mather, R J; Owen, T A

    1999-09-17

    Northern blot analysis of human placental RNA using a probe to the 5' end of the human prostaglandin E(2) (PGE(2)) EP2 receptor subtype coding region revealed the existence of a high abundance, low molecular weight transcript. To investigate the origin of this transcript, and its possible relationship to the human EP2 mRNA, we have cloned and characterized the gene encoding the human PGE(2) EP2 receptor subtype, identified transcriptional initiation and termination sites in two tissues (spleen and thymus), and determined its chromosomal localization. The human EP2 gene consists of two exons separated by a large intron, utilizes a common initiation site in both spleen and thymus at 1113 bp upstream of the translation initiation site, and has 3' transcript termini at 1140 bp and 1149 bp downstream of the translation stop site in spleen and thymus respectively. Southern and fluorescence in situ hybridization analysis demonstrated the human EP2 gene to be a single copy gene located in band 22 of the long arm of chromosome 14 (14q22). Though our initial interest in this gene was to investigate potential differential splicing of the human EP2 gene in placenta, this work demonstrates that the atypical transcript observed in placenta probably arises from a distinct, yet related, gene. Knowledge of the sequence, structure, and transcription events associated with the human EP2 gene will enable a broader understanding of its regulation and potential role in normal physiology and disease.

  11. Identification of TSIX, Encoding an RNA Antisense to Human XIST, Reveals Differences from its Murine Counterpart: Implications for X Inactivation

    PubMed Central

    Migeon, Barbara R.; Chowdhury, Ashis K.; Dunston, Jennifer A.; McIntosh, Iain

    2001-01-01

    X inactivation is the mammalian method for X-chromosome dosage compensation, but some features of this developmental process vary among mammals. Such species variations provide insights into the essential components of the pathway. Tsix encodes a transcript antisense to the murine Xist transcript and is expressed in the mouse embryo only during the initial stages of X inactivation; it has been shown to play a role in imprinted X inactivation in the mouse placenta. We have identified its counterpart within the human X inactivation center (XIC). Human TSIX produces a >30-kb transcript that is expressed only in cells of fetal origin; it is expressed from human XIC transgenes in mouse embryonic stem cells and from human embryoid-body–derived cells, but not from human adult somatic cells. Differences in the structure of human and murine genes indicate that human TSIX was truncated during evolution. These differences could explain the fact that X inactivation is not imprinted in human placenta, and they raise questions about the role of TSIX in random X inactivation. PMID:11555794

  12. Molecular cloning of cDNA for double-stranded RNA adenosine deaminase, a candidate enzyme for nuclear RNA editing.

    PubMed Central

    Kim, U; Wang, Y; Sanford, T; Zeng, Y; Nishikura, K

    1994-01-01

    We have cloned human cDNA encoding double-stranded RNA adenosine deaminase (DRADA). DRADA is a ubiquitous nuclear enzyme that converts multiple adenosines to inosines in double-helical RNA substrates without apparent sequence specificity. The A --> I conversion activity of the protein encoded by the cloned cDNA was confirmed by recombinant expression in insect cells. Use of the cloned DNA as a molecular probe documented sequence conservation across mammals and detected a single transcript of 7 kb in RNA of all human tissues analyzed. The deduced primary structure of human DRADA revealed a bipartite nuclear localization signal, three repeats of a double-stranded RNA binding motif, and the presence of sequences conserved in the catalytic center of other deaminases, including a cytidine deaminase involved in the RNA editing of apolipoprotein B. These structural properties are consistent with the enzymatic signature of DRADA, and strengthen the hypothesis that DRADA carries out the RNA editing of transcripts encoding glutamate-gated ion channels in brain. Images PMID:7972084

  13. The attentional blink reveals serial working memory encoding: evidence from virtual and human event-related potentials.

    PubMed

    Craston, Patrick; Wyble, Brad; Chennu, Srivas; Bowman, Howard

    2009-03-01

    Observers often miss a second target (T2) if it follows an identified first target item (T1) within half a second in rapid serial visual presentation (RSVP), a finding termed the attentional blink. If two targets are presented in immediate succession, however, accuracy is excellent (Lag 1 sparing). The resource sharing hypothesis proposes a dynamic distribution of resources over a time span of up to 600 msec during the attentional blink. In contrast, the ST(2) model argues that working memory encoding is serial during the attentional blink and that, due to joint consolidation, Lag 1 is the only case where resources are shared. Experiment 1 investigates the P3 ERP component evoked by targets in RSVP. The results suggest that, in this context, P3 amplitude is an indication of bottom-up strength rather than a measure of cognitive resource allocation. Experiment 2, employing a two-target paradigm, suggests that T1 consolidation is not affected by the presentation of T2 during the attentional blink. However, if targets are presented in immediate succession (Lag 1 sparing), they are jointly encoded into working memory. We use the ST(2) model's neural network implementation, which replicates a range of behavioral results related to the attentional blink, to generate "virtual ERPs" by summing across activation traces. We compare virtual to human ERPs and show how the results suggest a serial nature of working memory encoding as implied by the ST(2) model.

  14. Genome-Wide Analysis Reveals Loci Encoding Anti-Macrophage Factors in the Human Pathogen Burkholderia pseudomallei K96243

    PubMed Central

    Dowling, Andrea J.; Wilkinson, Paul A.; Holden, Matthew T. G.; Quail, Michael A.; Bentley, Stephen D.; Reger, Julia; Waterfield, Nicholas R.; Titball, Richard W.; ffrench-Constant, Richard H.

    2010-01-01

    Burkholderia pseudomallei is an important human pathogen whose infection biology is still poorly understood. The bacterium is endemic to tropical regions, including South East Asia and Northern Australia, where it causes melioidosis, a serious disease associated with both high mortality and antibiotic resistance. B. pseudomallei is a Gram-negative facultative intracellular pathogen that is able to replicate in macrophages. However despite the critical nature of its interaction with macrophages, few anti-macrophage factors have been characterized to date. Here we perform a genome-wide gain of function screen of B. pseudomallei strain K96243 to identify loci encoding factors with anti-macrophage activity. We identify a total of 113 such loci scattered across both chromosomes, with positive gene clusters encoding transporters and secretion systems, enzymes/toxins, secondary metabolite, biofilm, adhesion and signal response related factors. Further phenotypic analysis of four of these regions shows that the encoded factors cause striking cellular phenotypes relevant to infection biology, including apoptosis, formation of actin ‘tails’ and multi-nucleation within treated macrophages. The detailed analysis of the remaining host of loci will facilitate genetic dissection of the interaction of this important pathogen with host macrophages and thus further elucidate this critical part of its infection cycle. PMID:21203527

  15. Molecular cloning, expression, and chromosomal localization of the gene encoding a human myeloid membrane antigen (gp150).

    PubMed Central

    Look, A T; Peiper, S C; Rebentisch, M B; Ashmun, R A; Roussel, M F; Lemons, R S; Le Beau, M M; Rubin, C M; Sherr, C J

    1986-01-01

    DNA from a tertiary mouse cell transformant containing amplified human sequences encoding a human myeloid membrane glycoprotein, gp150, was used to construct a bacteriophage lambda library. A single recombinant phage containing 12 kilobases (kb) of human DNA was isolated, and molecular subclones were then used to isolate the complete gp150 gene from a human placental genomic DNA library. The intact gp150 gene, assembled from three recombinant phages, proved to be biologically active when transfected into NIH 3T3 cells. Molecular probes from the gp150 locus annealed with a 4.0-kb polyadenylated RNA transcript derived from human myeloid cell lines and from tertiary mouse cell transformants. The gp150 gene was assigned to human chromosome 15, and was subchromosomally localized to bands q25-26 by in situ hybridization. The chromosomal location of the gp150 gene coincides cytogenetically with the region assigned to the c-fes proto-oncogene, another human gene specifically expressed by myeloid cells. Images PMID:2428842

  16. Molecular cloning of cDNAs encoding human GLEPP1, a membrane protein tyrosine phosphatase: characterization of the GLEPP1 protein distribution in human kidney and assignment of the GLEPP1 gene to human chromosome 12p12-p13.

    PubMed

    Wiggins, R C; Wiggins, J E; Goyal, M; Wharram, B L; Thomas, P E

    1995-05-01

    Human glomerular epithelial protein 1 (GLEPP1), a receptor-like membrane protein tyrosine phosphatase (PTPase), was cloned and sequenced from a human renal cortical cDNA library. The human nucleotide and derived amino acid sequences were, respectively, 90 and 97% identical to those of rabbit. Human GLEPP1 is predicted to contain 1188 amino acids. The predicted mature protein is 1159 amino acids long and contains a large extracellular domain, a single transmembrane domain, and a single intracellular PTPase domain. Monoclonal and polyclonal antibodies raised against a human GLEPP1 fusion protein recognized a protein with distribution restricted to the glomerulus in human kidney and with an apparent molecular weight of approximately 200 kDa. The GLEPP1 gene was assigned to human chromosome 12p12-p13 by fluorescence in situ hybridization.

  17. Human Cytomegalovirus-Encoded Human Interleukin-10 (IL-10) Homolog Amplifies Its Immunomodulatory Potential by Upregulating Human IL-10 in Monocytes

    PubMed Central

    Avdic, Selmir; McSharry, Brian P.; Steain, Megan; Poole, Emma; Sinclair, John; Abendroth, Allison

    2016-01-01

    ABSTRACT The human cytomegalovirus (HCMV) gene UL111A encodes cytomegalovirus-encoded human interleukin-10 (cmvIL-10), a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). This viral homolog exhibits a range of immunomodulatory functions, including suppression of proinflammatory cytokine production and dendritic cell (DC) maturation, as well as inhibition of major histocompatibility complex (MHC) class I and class II. Here, we present data showing that cmvIL-10 upregulates hIL-10, and we identify CD14+ monocytes and monocyte-derived macrophages and DCs as major sources of hIL-10 secretion in response to cmvIL-10. Monocyte activation was not a prerequisite for cmvIL-10-mediated upregulation of hIL-10, which was dose dependent and controlled at the transcriptional level. Furthermore, cmvIL-10 upregulated expression of tumor progression locus 2 (TPL2), which is a regulator of the positive hIL-10 feedback loop, whereas expression of a negative regulator of the hIL-10 feedback loop, dual-specificity phosphatase 1 (DUSP1), remained unchanged. Engagement of the hIL-10 receptor (hIL-10R) by cmvIL-10 led to upregulation of heme oxygenase 1 (HO-1), an enzyme linked with suppression of inflammatory responses, and this upregulation was required for cmvIL-10-mediated upregulation of hIL-10. We also demonstrate an important role for both phosphatidylinositol 3-kinase (PI3K) and STAT3 in the upregulation of HO-1 and hIL-10 by cmvIL-10. In addition to upregulating hIL-10, cmvIL-10 could exert a direct immunomodulatory function, as demonstrated by its capacity to upregulate expression of cell surface CD163 when hIL-10 was neutralized. This study identifies a mechanistic basis for cmvIL-10 function, including the capacity of this viral cytokine to potentially amplify its immunosuppressive impact by upregulating hIL-10 expression. IMPORTANCE Human cytomegalovirus (HCMV) is a large, double-stranded DNA virus that causes significant human disease

  18. Fatal autoimmunity in mice reconstituted with human hematopoietic stem cells encoding defective FOXP3.

    PubMed

    Goettel, Jeremy A; Biswas, Subhabrata; Lexmond, Willem S; Yeste, Ada; Passerini, Laura; Patel, Bonny; Yang, Siyoung; Sun, Jiusong; Ouahed, Jodie; Shouval, Dror S; McCann, Katelyn J; Horwitz, Bruce H; Mathis, Diane; Milford, Edgar L; Notarangelo, Luigi D; Roncarolo, Maria-Grazia; Fiebiger, Edda; Marasco, Wayne A; Bacchetta, Rosa; Quintana, Francisco J; Pai, Sung-Yun; Klein, Christoph; Muise, Aleixo M; Snapper, Scott B

    2015-06-18

    Mice reconstituted with a human immune system provide a tractable in vivo model to assess human immune cell function. To date, reconstitution of murine strains with human hematopoietic stem cells (HSCs) from patients with monogenic immune disorders have not been reported. One obstacle precluding the development of immune-disease specific "humanized" mice is that optimal adaptive immune responses in current strains have required implantation of autologous human thymic tissue. To address this issue, we developed a mouse strain that lacks murine major histocompatibility complex class II (MHC II) and instead expresses human leukocyte antigen DR1 (HLA-DR1). These mice displayed improved adaptive immune responses when reconstituted with human HSCs including enhanced T-cell reconstitution, delayed-type hypersensitivity responses, and class-switch recombination. Following immune reconstitution of this novel strain with HSCs from a patient with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, associated with aberrant FOXP3 function, mice developed a lethal inflammatory disorder with multiorgan involvement and autoantibody production mimicking the pathology seen in affected humans. This humanized mouse model permits in vivo evaluation of immune responses associated with genetically altered HSCs, including primary immunodeficiencies, and should facilitate the study of human immune pathobiology and the development of targeted therapeutics.

  19. Identification of a yeast artificial chromosome clone encoding an accessory factor for the human interferon gamma receptor: evidence for multiple accessory factors.

    PubMed

    Soh, J; Donnelly, R J; Mariano, T M; Cook, J R; Schwartz, B; Pestka, S

    1993-09-15

    Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by the induction of human HLA class I antigen. Human chromosome 6 encodes the receptor for Hu-IFN-gamma, and human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. A small region of human chromosome 21 that is responsible for encoding such factors was localized with hamster-human somatic cell hybrids carrying an irradiation-reduced fragment of human chromosome 21. The cell line with the minimum chromosome 21-specific DNA is Chinese hamster ovary 3x1S. To localize the genes further, 10 different yeast artificial chromosome clones from six different loci in the vicinity of the 3x1S region were fused to a human-hamster hybrid cell line (designated 16-9) that contains human chromosome 6q (supplying the Hu-IFN-gamma receptor) and the human HLA-B7 gene. These transformed 16-9 cells were assayed for induction of class I HLA antigens upon treatment with Hu-IFN-gamma. Here we report that a 540-kb yeast artificial chromosome encodes the necessary species-specific factor(s) and can substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma-receptor-mediated induction of class I HLA antigens. However, the factor encoded on the yeast artificial chromosome does not confer antiviral protection against encephalomyocarditis virus, demonstrating that an additional factor encoded on human chromosome 21 is required for the antiviral activity.

  20. Three sequential brain activations encode mental transformations of upright and inverted human bodies: a high resolution evoked potential study.

    PubMed

    Tadi, T; Overney, L S; Blanke, O

    2009-04-10

    Human bodies provide a particularly rich source of visual information. Whereas most previous studies have focused on the neural mechanisms during the perception and recognition of human bodies, the aim of the present study was to investigate the time course and location of brain activation during mental imagery of human bodies. When participants were asked to imagine themselves in the position of a visually presented human body as seen from many different angles and at two orientations (upright or inverted), their reaction times were faster for upright as compared to inverted bodies and correlated differently with the tested angles. These behavioral effects were also reflected in brain activation patterns, but only during the time period from 220 to 490 ms after stimulus onset. Evoked potential mapping and electrical neuroimaging revealed three distinct and sequential steps of processing related to mental body transformation: (1) an early activation in temporo-occipital and temporo-parietal cortex (220-360 ms) that does not distinguish between upright and inverted bodies, but closely reflects the effort of mental transformation, followed (2) by an activation in temporo-occipital and medial parieto-occipital cortex (350-460 ms) that encodes mental transformation for upright bodies, and (3) a later activation in temporo-occipital and prefrontal cortex (390-490 ms) that encodes mental transformation for inverted bodies. These data suggest that the mental transformation of human bodies is not a single process but a sequence of temporally distinct processing steps, where each step reflects a distinct aspect of the transformation process that consists of activations in a network of posterior brain areas including extrastriate cortex, temporo-parietal cortex, and medial parieto-occipital cortex, as well as an anterior brain region in prefrontal cortex.

  1. Isolation and characterization of a full length cDNA for dentatorubral-pallidoluysian atrophy (DRPLA) gene

    SciTech Connect

    Oyake, M.; Onodera, O.; Ikeuchi, T.

    1994-09-01

    Hereditary dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant spinocerebellar degeneration characterized by anticipation and variable combination of symptoms including myoclonus, epilepsy, cerebellar ataxia, choleoathetosis, and dementia. Recently, we discovered that DRPLA is caused by unstable expansion of a CAG repeat of a B37 gene on chromosome 12. To characterize functions of the DRPLA gene product, we isolated several cDNA clones for the DRPLA gene from human adult and fetus brain cDNA libraries, using an oligonucleotide flanking the CAG repeat. The cDNA spans 4247 bp in length and there is only an open reading frame coding for 986 amino acids. The CAG repeat, which is expanded in DRPLA, is located 291 bp downstream from the initiation methionine and encodes a polyglutamine tract. The deduced amino acid sequence from amino acids residues 582 to 707 has a high homology to published human hippocampus derived expressed sequence (M78755) located at chromosome 1p (63.8% identity), and 3{prime}-untranslated region of the DRPLA cDNA revealed homology to the mouse small nuclear RNA U7 gene (X54165). Northern blot analysis revealed a 4.7 knt transcript which is widely expressed in various tissues including heart, lung, kidney, placenta, skeletal muscle, and brain. In human adult brain, the transcript was broadly expressed including amygdala, caudate nucleus, corpus callosum, hippocampus, hypothalamus, substantia nigra, subthalamic nucleus and thalamus, and was not specific to the dentatorubral-pallidoluysian system. The availability of a full length cDNA will be highly useful for analyzing the pathogenesis of this unique neurodegenerative disease as well as for analyzing other CAG repeat related neurodegenerative diseases.

  2. Met-ase: Cloning and distinct chromosomal location of a serine protease preferentially expressed in human natural killer cells

    SciTech Connect

    Smyth, M.J.; Trapani, J.A. ); Sayers, T.J.; Wiltrout, T. ); Powers, J.C. )

    1993-12-01

    A cDNA clone encoding a human NK serine protease was obtained by screening a [lambda]-gt10 library from the Lopez NK leukemia with the rat natural killer Met-ase (RNK-Met-1) cDNA clone. In Northern blot analysis human Met-ase (Hu-Met-1) cDNA hybridized with a 0.9-kb mRNA in two human NK leukemia cell lines, unstimulated human PBMC, and untreated purified CD3[sup [minus

  3. Adhesion domain of human T11 (CD2) is encoded by a single exon.

    PubMed Central

    Richardson, N E; Chang, H C; Brown, N R; Hussey, R E; Sayre, P H; Reinherz, E L

    1988-01-01

    The 50-kDa T11 (CD2) T-lymphocyte surface glycoprotein facilitates physical adhesion between T-lineage cells and their cognate cellular counterparts (cytotoxic T-lymphocytes-target cells, helper T lymphocytes-antigen-presenting cells, or thymocytes-thymic epithelium) as well as signaling through the antigen-specific T3-Ti receptor complex. To examine the relationship between the structure and function of the T11 molecule, we have utilized a baculoviral expression system to produce milligram quantities of the hydrophilic extracellular T11 segment. Enzyme cleavage, microsequencing, and HPLC analyses of the expressed protein in conjunction with genomic cloning information show that the domain involved in cellular adhesion is encoded by a single 321-base-pair exon. Images PMID:2455894

  4. Rapid Encoding of New Memories by Individual Neurons in the Human Brain.

    PubMed

    Ison, Matias J; Quian Quiroga, Rodrigo; Fried, Itzhak

    2015-07-01

    The creation of memories about real-life episodes requires rapid neuronal changes that may appear after a single occurrence of an event. How is such demand met by neurons in the medial temporal lobe (MTL), which plays a fundamental role in episodic memory formation? We recorded the activity of MTL neurons in neurosurgical patients while they learned new associations. Pairs of unrelated pictures, one of a person and another of a place, were used to construct a meaningful association modeling the episodic memory of meeting a person in a particular place. We found that a large proportion of responsive MTL neurons expanded their selectivity to encode these specific associations within a few trials: cells initially responsive to one picture started firing to the associated one but not to others. Our results provide a plausible neural substrate for the inception of associations, which are crucial for the formation of episodic memories.

  5. Rapid Encoding of New Memories by Individual Neurons in the Human Brain

    PubMed Central

    Ison, Matias J.; Quian Quiroga, Rodrigo; Fried, Itzhak

    2015-01-01

    Summary The creation of memories about real-life episodes requires rapid neuronal changes that may appear after a single occurrence of an event. How is such demand met by neurons in the medial temporal lobe (MTL), which plays a fundamental role in episodic memory formation? We recorded the activity of MTL neurons in neurosurgical patients while they learned new associations. Pairs of unrelated pictures, one of a person and another of a place, were used to construct a meaningful association modeling the episodic memory of meeting a person in a particular place. We found that a large proportion of responsive MTL neurons expanded their selectivity to encode these specific associations within a few trials: cells initially responsive to one picture started firing to the associated one but not to others. Our results provide a plausible neural substrate for the inception of associations, which are crucial for the formation of episodic memories. PMID:26139375

  6. Normalized cDNA libraries

    DOEpatents

    Soares, M.B.; Efstratiadis, A.

    1997-06-10

    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{prime} 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. 4 figs.

  7. Normalized cDNA libraries

    DOEpatents

    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.

  8. Food and human gut as reservoirs of transferable antibiotic resistance encoding genes

    PubMed Central

    Rolain, Jean-Marc

    2013-01-01

    The increase and spread of antibiotic resistance (AR) over the past decade in human pathogens has become a worldwide health concern. Recent genomic and metagenomic studies in humans, animals, in food and in the environment have led to the discovery of a huge reservoir of AR genes called the resistome that could be mobilized and transferred from these sources to human pathogens. AR is a natural phenomenon developed by bacteria to protect antibiotic-producing bacteria from their own products and also to increase their survival in highly competitive microbial environments. Although antibiotics are used extensively in humans and animals, there is also considerable usage of antibiotics in agriculture, especially in animal feeds and aquaculture. The aim of this review is to give an overview of the sources of AR and the use of antibiotics in these reservoirs as selectors for emergence of AR bacteria in humans via the food chain. PMID:23805136

  9. C9ORF135 encodes a membrane protein whose expression is related to pluripotency in human embryonic stem cells

    PubMed Central

    Zhou, Shixin; Liu, Yinan; Ma, Yumin; Zhang, Xiaoyan; Li, Yang; Wen, Jinhua

    2017-01-01

    Human embryonic stem cells (hESCs) are a unique population of cells defined by their capacity for self-renewal and pluripotency. Here, we identified a previously uncharacterized gene in hESCs, C9ORF135, which is sharply downregulated during gastrulation and gametogenesis, along with the pluripotency factors OCT4, SOX2, and NANOG. Human ESCs express two C9ORF135 isoforms, the longer of which encodes a membrane-associated protein, as determined by immunostaining and western blotting of fractionated cell lysates. Moreover, the results of chromatin immunoprecipitation (ChIP), mass spectrometry (MS), and co-immunoprecipitation (co-IP) analyses demonstrated that C9ORF135 expression is regulated by OCT4 and SOX2 and that C9ORF135 interacts with non-muscle myosin IIA and myosin IIB. Collectively, these data indicated that C9ORF135 encodes a membrane-associated protein that may serve as a surface marker for undifferentiated hESCs. PMID:28345668

  10. C9ORF135 encodes a membrane protein whose expression is related to pluripotency in human embryonic stem cells.

    PubMed

    Zhou, Shixin; Liu, Yinan; Ma, Yumin; Zhang, Xiaoyan; Li, Yang; Wen, Jinhua

    2017-03-27

    Human embryonic stem cells (hESCs) are a unique population of cells defined by their capacity for self-renewal and pluripotency. Here, we identified a previously uncharacterized gene in hESCs, C9ORF135, which is sharply downregulated during gastrulation and gametogenesis, along with the pluripotency factors OCT4, SOX2, and NANOG. Human ESCs express two C9ORF135 isoforms, the longer of which encodes a membrane-associated protein, as determined by immunostaining and western blotting of fractionated cell lysates. Moreover, the results of chromatin immunoprecipitation (ChIP), mass spectrometry (MS), and co-immunoprecipitation (co-IP) analyses demonstrated that C9ORF135 expression is regulated by OCT4 and SOX2 and that C9ORF135 interacts with non-muscle myosin IIA and myosin IIB. Collectively, these data indicated that C9ORF135 encodes a membrane-associated protein that may serve as a surface marker for undifferentiated hESCs.

  11. Structural organization and splice variants of the POLE1 gene encoding the catalytic subunit of human DNA polymerase epsilon.

    PubMed Central

    Huang, D; Pospiech, H; Kesti, T; Syväoja, J E

    1999-01-01

    The catalytic subunit of human DNA polymerase epsilon, an enzyme involved in nuclear DNA replication and repair, is encoded by the POLE1 gene. This gene is composed of 51 exons spanning at least 97 kb of genomic DNA. It was found to encode three alternative mRNA splice variants that differ in their 5'-terminal sequences and in the N-termini of the predicted proteins. A CpG island covers the promoter region for the major transcript in HeLa cells. This promoter is TATA-less and contains several putative binding sites for transcription factors typical of S-phase-up-regulated and serum-responsive promoters. Potential promoter regions were also identified for the two other alternative transcripts. Interestingly, no nuclear polyadenylation signal sequence was detected in the 3'-untranslated region, although a poly(A) tail was present. These results suggest a complicated regulatory machinery for the expression of the human POLE1 gene, including three alternative transcripts expressed from three promoters. PMID:10215605

  12. The genomic structure of the gene encoding the human transforming growth factor {beta} type II receptor (TGF-{beta} RII)

    SciTech Connect

    Takenoshita, Seiichi; Hagiwara, Koichi; Nagashima, Makoto; Gemma, Akihiko

    1996-09-01

    The genomic structure of the human transforming growth factor-{beta} type II receptor gene (TGF-{beta} RII) was determined by two PCR-based methods, the {open_quotes}long distance sequencer{close_quotes} method and the {open_quotes}promoter finder{close_quotes} method. Genomic fragments containing exons and adjacent introns were amplified by PCR, and the nucleotide sequences were determined by direct sequencing and subcloning sequencing. The TGF-{beta} RII protein is encoded by 567 codons in 7 exons. This is the first report about the genomic structure of a gene that belongs to the serine/threonine kinase type II receptor subfamily. Knowledge of the genomic structure of the TGF-{beta} RII gene will facilitate investigation of the TGF-{beta} RII gene will facilitate investigation of the TGF-{beta} signaling pathway in normal human cells and of the aberrations occurring during carcinogenesis. 18 refs., 2 figs., 1 tab.

  13. Assignment of the gene encoding human galanin receptor (GALNR) to 18q23 by in situ hybridization

    SciTech Connect

    Nicholl, J.; Sutherland, G.R.; Shine, J.

    1995-12-10

    The neuropeptide galanin is widely distributed throughout the central and peripheral nervous systems of mammalian, avian, reptilian, and fish species and has a broad range of physiological and behavioral effects. Human galanin is a 30-amino-acid non-C-terminally amidated peptide that potently stimulates growth hormone secretion, inhibits cardiac vagal slowing of heart rate, abolishes sinus arrhythmia, and inhibits postprandial gastrointestinal motility. The actions of galanin are mediated through interaction with specific membrane receptors that are members of the seven transmembrane family of G-protein-coupled receptors. A functional human galanin receptor has recently been cloned, and we report here the localization of the gene encoding this receptor (GALNR) to chromosome 18q23. 5 refs., 1 fig.

  14. Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

    DOEpatents

    Freimuth, Paul I.

    2010-04-06

    The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

  15. Characterization of verotoxin-encoding phages from Escherichia coli O103:H2 strains of bovine and human origins.

    PubMed

    Karama, Musafiri; Gyles, Carlton L

    2008-08-01

    The objectives of this study were to induce and characterize verotoxin-encoding phages from a collection of 91 verotoxin-producing Escherichia coli (VTEC) O103:H2 strains of human and bovine origins. All the strains carried the vt1 gene, and two carried the vt2 gene as well. The phages were induced by UV irradiation and characterized by DNA restriction fragment length polymorphism (RFLP), genome size, morphology, and Q and P genes, characteristic of lambdoid phages. A total of 32 vt-positive phages were induced and isolated from 31 VTEC O103:H2 strains. Thirty phages were vt1 positive, and two were vt2 positive. Ten of the 30 vt1-positive phages (33.3%) were from cattle strains, and 20 (66.6%) were from human strains. The two vt2-positive phages were from human strains. Phages belonged to 21 RFLP profiles, of which 17 were single-phage profiles and 4 were multiple-phage profiles. The estimated genome size of the phages ranged from 34 to 84 kb. Two phages that were examined by electron microscopy possessed hexagonal heads with long tails, and one had an elongated head with a long tail. The Q and P genes were amplified in all 32 phages, and the Q-stxA(1) gene region yielded an amplicon in 19 phages (59.3%). It is concluded that the VTEC O103:H2 strains of human origin were more readily inducible than those of bovine origin and that the genotypic profiles of verotoxin-encoding phages were highly diverse, as revealed by their RFLP profiles.

  16. Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1.

    PubMed Central

    Kominami, K; Okura, N; Kawamura, M; DeMartino, G N; Slaughter, C A; Shimbara, N; Chung, C H; Fujimuro, M; Yokosawa, H; Shimizu, Y; Tanahashi, N; Tanaka, K; Toh-e, A

    1997-01-01

    Nin1p, a component of the 26S proteasome of Saccharomyces cerevisiae, is required for activation of Cdc28p kinase at the G1-S-phase and G2-M boundaries. By exploiting the temperature-sensitive phenotype of the nin1-1 mutant, we have screened for genes encoding proteins with related functions to Nin1p and have cloned and characterized two new multicopy suppressors, SUN1 and SUN2, of the nin1-1 mutation. SUN1 can suppress a null nin1 mutation, whereas SUN2, an essential gene, does not. Sun1p is a 268-amino acid protein which shows strong similarity to MBP1 of Arabidopsis thaliana, a homologue of the S5a subunit of the human 26S proteasome. Sun1p binds ubiquitin-lysozyme conjugates as do S5a and MBP1. Sun2p (523 amino acids) was found to be homologous to the p58 subunit of the human 26S proteasome. cDNA encoding the p58 component was cloned. Furthermore, expression of a derivative of p58 from which the N-terminal 150 amino acids had been removed restored the function of a null allele of SUN2. During glycerol density gradient centrifugation, both Sun1p and Sun2p comigrated with the known proteasome components. These results, as well as other structural and functional studies, indicate that both Sun1p and Sun2p are components of the regulatory module of the yeast 26S proteasome. Images PMID:9017604

  17. Locations of human and mouse genes encoding the RFX1 and RFX2 transcription factor proteins.

    PubMed

    Doyle, J; Hoffman, S; Ucla, C; Reith, W; Mach, B; Stubbs, L

    1996-07-01

    RFX transcription factors constitute a highly conserved family of site-specific DNA binding proteins involved in the expression of a variety of cellular and viral genes, including major histocompatibility complex class II genes and genes in human hepatitis B virus. Five members of the RFX gene family have been isolated from human and mouse, and all share a highly characteristic DNA binding domain that is distinct from other known DNA binding motifs. The human RFX1 and RFX2 genes have been assigned by in situ hybridization to chromosome 19p13.1 and 19p13.3, respectively. In this paper, we present data that localize RFX1 and RFX2 precisely within the detailed physical map of human chromosome 19 and genetic data that assign Rfx1 and Rfx2 to homologous regions of mouse chromosomes 8 and 17, respectively. These data define the established relationships between these homologous mouse and human regions in further detail and provide new tools for linking cloned genes to phenotypes in both species.

  18. Induction of human dendritic cell maturation using transfection with RNA encoding a dominant positive toll-like receptor 4.

    PubMed

    Cisco, Robin M; Abdel-Wahab, Zeinab; Dannull, Jens; Nair, Smita; Tyler, Douglas S; Gilboa, Eli; Vieweg, Johannes; Daaka, Yehia; Pruitt, Scott K

    2004-06-01

    Maturation of dendritic cells (DC) is critical for the induction of Ag-specific immunity. Ag-loaded DC matured with LPS, which mediates its effects by binding to Toll-like receptor 4 (TLR4), induce Ag-specific CTL in vitro and in vivo in animal models. However, clinical use of LPS is limited due to potential toxicity. Therefore, we sought to mimic the maturation-inducing effects of LPS on DC by stimulating TLR4-mediated signaling in the absence of exogenous LPS. We developed a constitutively active TLR4 (caTLR4) and demonstrated that transfection of human DC with RNA encoding caTLR4 led to IL-12 and TNF-alpha secretion. Transfection with caTLR4 RNA also induced a mature DC phenotype. Functionally, transfection of DC with caTLR4 RNA enhanced allostimulation of CD4(+) T cells. DC transfected with RNA encoding the MART (Melan-A/MART-1) melanoma Ag were then used to stimulate T cells in vitro. Cotransfection of these DC with caTLR4 RNA enhanced the generation of MART-specific CTL. This CTL activity was superior to that seen when DC maturation was induced using either LPS or a standard mixture of cytokines (TNF-alpha, IL-6, IL-1beta, and PGE(2)). We conclude that transfection of DC with RNA encoding a functional signaling protein, such as caTLR4, may provide a new tool for studying TLR signaling in DC and may be a promising approach for the induction of DC maturation for tumor immunotherapy.

  19. A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines

    PubMed Central

    Sciamanna, Ilaria; Gualtieri, Alberto; Cossetti, Cristina; Osimo, Emanuele Felice; Ferracin, Manuela; Macchia, Gianfranco; Aricò, Eleonora; Prosseda, Gianni; Vitullo, Patrizia; Misteli, Tom; Spadafora, Corrado

    2013-01-01

    LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the ‘normal’ small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the

  20. A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines.

    PubMed

    Sciamanna, Ilaria; Gualtieri, Alberto; Cossetti, Cristina; Osimo, Emanuele Felice; Ferracin, Manuela; Macchia, Gianfranco; Aricò, Eleonora; Prosseda, Gianni; Vitullo, Patrizia; Misteli, Tom; Spadafora, Corrado

    2013-12-01

    LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the 'normal' small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the

  1. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

    PubMed

    Birney, Ewan; Stamatoyannopoulos, John A; Dutta, Anindya; Guigó, Roderic; Gingeras, Thomas R; Margulies, Elliott H; Weng, Zhiping; Snyder, Michael; Dermitzakis, Emmanouil T; Thurman, Robert E; Kuehn, Michael S; Taylor, Christopher M; Neph, Shane; Koch, Christoph M; Asthana, Saurabh; Malhotra, Ankit; Adzhubei, Ivan; Greenbaum, Jason A; Andrews, Robert M; Flicek, Paul; Boyle, Patrick J; Cao, Hua; Carter, Nigel P; Clelland, Gayle K; Davis, Sean; Day, Nathan; Dhami, Pawandeep; Dillon, Shane C; Dorschner, Michael O; Fiegler, Heike; Giresi, Paul G; Goldy, Jeff; Hawrylycz, Michael; Haydock, Andrew; Humbert, Richard; James, Keith D; Johnson, Brett E; Johnson, Ericka M; Frum, Tristan T; Rosenzweig, Elizabeth R; Karnani, Neerja; Lee, Kirsten; Lefebvre, Gregory C; Navas, Patrick A; Neri, Fidencio; Parker, Stephen C J; Sabo, Peter J; Sandstrom, Richard; Shafer, Anthony; Vetrie, David; Weaver, Molly; Wilcox, Sarah; Yu, Man; Collins, Francis S; Dekker, Job; Lieb, Jason D; Tullius, Thomas D; Crawford, Gregory E; Sunyaev, Shamil; Noble, William S; Dunham, Ian; Denoeud, France; Reymond, Alexandre; Kapranov, Philipp; Rozowsky, Joel; Zheng, Deyou; Castelo, Robert; Frankish, Adam; Harrow, Jennifer; Ghosh, Srinka; Sandelin, Albin; Hofacker, Ivo L; Baertsch, Robert; Keefe, Damian; Dike, Sujit; Cheng, Jill; Hirsch, Heather A; Sekinger, Edward A; Lagarde, Julien; Abril, Josep F; Shahab, Atif; Flamm, Christoph; Fried, Claudia; Hackermüller, Jörg; Hertel, Jana; Lindemeyer, Manja; Missal, Kristin; Tanzer, Andrea; Washietl, Stefan; Korbel, Jan; Emanuelsson, Olof; Pedersen, Jakob S; Holroyd, Nancy; Taylor, Ruth; Swarbreck, David; Matthews, Nicholas; Dickson, Mark C; Thomas, Daryl J; Weirauch, Matthew T; Gilbert, James; Drenkow, Jorg; Bell, Ian; Zhao, XiaoDong; Srinivasan, K G; Sung, Wing-Kin; Ooi, Hong Sain; Chiu, Kuo Ping; Foissac, Sylvain; Alioto, Tyler; Brent, Michael; Pachter, Lior; Tress, Michael L; Valencia, Alfonso; Choo, Siew Woh; Choo, Chiou Yu; Ucla, Catherine; Manzano, Caroline; Wyss, Carine; Cheung, Evelyn; Clark, Taane G; Brown, James B; Ganesh, Madhavan; Patel, Sandeep; Tammana, Hari; Chrast, Jacqueline; Henrichsen, Charlotte N; Kai, Chikatoshi; Kawai, Jun; Nagalakshmi, Ugrappa; Wu, Jiaqian; Lian, Zheng; Lian, Jin; Newburger, Peter; Zhang, Xueqing; Bickel, Peter; Mattick, John S; Carninci, Piero; Hayashizaki, Yoshihide; Weissman, Sherman; Hubbard, Tim; Myers, Richard M; Rogers, Jane; Stadler, Peter F; Lowe, Todd M; Wei, Chia-Lin; Ruan, Yijun; Struhl, Kevin; Gerstein, Mark; Antonarakis, Stylianos E; Fu, Yutao; Green, Eric D; Karaöz, Ulaş; Siepel, Adam; Taylor, James; Liefer, Laura A; Wetterstrand, Kris A; Good, Peter J; Feingold, Elise A; Guyer, Mark S; Cooper, Gregory M; Asimenos, George; Dewey, Colin N; Hou, Minmei; Nikolaev, Sergey; Montoya-Burgos, Juan I; Löytynoja, Ari; Whelan, Simon; Pardi, Fabio; Massingham, Tim; Huang, Haiyan; Zhang, Nancy R; Holmes, Ian; Mullikin, James C; Ureta-Vidal, Abel; Paten, Benedict; Seringhaus, Michael; Church, Deanna; Rosenbloom, Kate; Kent, W James; Stone, Eric A; Batzoglou, Serafim; Goldman, Nick; Hardison, Ross C; Haussler, David; Miller, Webb; Sidow, Arend; Trinklein, Nathan D; Zhang, Zhengdong D; Barrera, Leah; Stuart, Rhona; King, David C; Ameur, Adam; Enroth, Stefan; Bieda, Mark C; Kim, Jonghwan; Bhinge, Akshay A; Jiang, Nan; Liu, Jun; Yao, Fei; Vega, Vinsensius B; Lee, Charlie W H; Ng, Patrick; Shahab, Atif; Yang, Annie; Moqtaderi, Zarmik; Zhu, Zhou; Xu, Xiaoqin; Squazzo, Sharon; Oberley, Matthew J; Inman, David; Singer, Michael A; Richmond, Todd A; Munn, Kyle J; Rada-Iglesias, Alvaro; Wallerman, Ola; Komorowski, Jan; Fowler, Joanna C; Couttet, Phillippe; Bruce, Alexander W; Dovey, Oliver M; Ellis, Peter D; Langford, Cordelia F; Nix, David A; Euskirchen, Ghia; Hartman, Stephen; Urban, Alexander E; Kraus, Peter; Van Calcar, Sara; Heintzman, Nate; Kim, Tae Hoon; Wang, Kun; Qu, Chunxu; Hon, Gary; Luna, Rosa; Glass, Christopher K; Rosenfeld, M Geoff; Aldred, Shelley Force; Cooper, Sara J; Halees, Anason; Lin, Jane M; Shulha, Hennady P; Zhang, Xiaoling; Xu, Mousheng; Haidar, Jaafar N S; Yu, Yong; Ruan, Yijun; Iyer, Vishwanath R; Green, Roland D; Wadelius, Claes; Farnham, Peggy J; Ren, Bing; Harte, Rachel A; Hinrichs, Angie S; Trumbower, Heather; Clawson, Hiram; Hillman-Jackson, Jennifer; Zweig, Ann S; Smith, Kayla; Thakkapallayil, Archana; Barber, Galt; Kuhn, Robert M; Karolchik, Donna; Armengol, Lluis; Bird, Christine P; de Bakker, Paul I W; Kern, Andrew D; Lopez-Bigas, Nuria; Martin, Joel D; Stranger, Barbara E; Woodroffe, Abigail; Davydov, Eugene; Dimas, Antigone; Eyras, Eduardo; Hallgrímsdóttir, Ingileif B; Huppert, Julian; Zody, Michael C; Abecasis, Gonçalo R; Estivill, Xavier; Bouffard, Gerard G; Guan, Xiaobin; Hansen, Nancy F; Idol, Jacquelyn R; Maduro, Valerie V B; Maskeri, Baishali; McDowell, Jennifer C; Park, Morgan; Thomas, Pamela J; Young, Alice C; Blakesley, Robert W; Muzny, Donna M; Sodergren, Erica; Wheeler, David A; Worley, Kim C; Jiang, Huaiyang; Weinstock, George M; Gibbs, Richard A; Graves, Tina; Fulton, Robert; Mardis, Elaine R; Wilson, Richard K; Clamp, Michele; Cuff, James; Gnerre, Sante; Jaffe, David B; Chang, Jean L; Lindblad-Toh, Kerstin; Lander, Eric S; Koriabine, Maxim; Nefedov, Mikhail; Osoegawa, Kazutoyo; Yoshinaga, Yuko; Zhu, Baoli; de Jong, Pieter J

    2007-06-14

    We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

  2. Human Dorsal Striatum Encodes Prediction Errors during Observational Learning of Instrumental Actions

    ERIC Educational Resources Information Center

    Cooper, Jeffrey C.; Dunne, Simon; Furey, Teresa; O'Doherty, John P.

    2012-01-01

    The dorsal striatum plays a key role in the learning and expression of instrumental reward associations that are acquired through direct experience. However, not all learning about instrumental actions require direct experience. Instead, humans and other animals are also capable of acquiring instrumental actions by observing the experiences of…

  3. Human cortical θ during free exploration encodes space and predicts subsequent memory.

    PubMed

    Snider, Joseph; Plank, Markus; Lynch, Gary; Halgren, Eric; Poizner, Howard

    2013-09-18

    Spatial representations and walking speed in rodents are consistently related to the phase, frequency, and/or amplitude of θ rhythms in hippocampal local field potentials. However, neuropsychological studies in humans have emphasized the importance of parietal cortex for spatial navigation, and efforts to identify the electrophysiological signs of spatial navigation in humans have been stymied by the difficulty of recording during free exploration of complex environments. We resolved the recording problem and experimentally probed brain activity of human participants who were fully ambulant. On each of 2 d, electroencephalography was synchronized with head and body movement in 13 subjects freely navigating an extended virtual environment containing numerous unique objects. θ phase and amplitude recorded over parietal cortex were consistent when subjects walked through a particular spatial separation at widely separated times. This spatial displacement θ autocorrelation (STAcc) was quantified and found to be significant from 2 to 8 Hz within the environment. Similar autocorrelation analyses performed on an electrooculographic channel, used to measure eye movements, showed no significant spatial autocorrelations, ruling out eye movements as the source of STAcc. Strikingly, the strength of an individual's STAcc maps from day 1 significantly predicted object location recall success on day 2. θ was also significantly correlated with walking speed; however, this correlation appeared unrelated to STAcc and did not predict memory performance. This is the first demonstration of memory-related, spatial maps in humans generated during active spatial exploration.

  4. Response-Modality-Specific Encoding of Human Choices in Upper Beta Band Oscillations during Vibrotactile Comparisons

    PubMed Central

    Herding, Jan; Ludwig, Simon; Blankenburg, Felix

    2017-01-01

    Perceptual decisions based on the comparison of two vibrotactile frequencies have been extensively studied in non-human primates. Recently, we obtained corresponding findings from human oscillatory electroencephalography (EEG) activity in the form of choice-selective modulations of upper beta band amplitude in medial premotor areas. However, the research in non-human primates as well as its human counterpart was so far limited to decisions reported by button presses. Thus, here we investigated whether the observed human beta band modulation is specific to the response modality. We recorded EEG activity from participants who compared two sequentially presented vibrotactile frequencies (f1 and f2), and decided whether f2 > f1 or f2 < f1, by performing a horizontal saccade to either side of a computer screen. Contrasting time-frequency transformed EEG data between both choices revealed that upper beta band amplitude (∼24–32 Hz) was modulated by participants’ choices before actual responses were given. In particular, “f2 > f1” choices were always associated with higher beta band amplitude than “f2 < f1” choices, irrespective of whether the choice was correct or not, and independent of the specific association between saccade direction and choice. The observed pattern of beta band modulation was virtually identical to our previous results when participants responded with button presses. In line with an intentional framework of decision making, the most likely sources of the beta band modulation were now, however, located in lateral as compared to medial premotor areas including the frontal eye fields. Hence, we could show that the choice-selective modulation of upper beta band amplitude is on the one hand consistent across different response modalities (i.e., same modulation pattern in similar frequency band), and on the other hand effector specific (i.e., modulation originating from areas involved in planning and executing saccades). PMID:28360848

  5. A mutation of MET, encoding hepatocyte growth factor receptor, is associated with human DFNB97 hearing loss

    PubMed Central

    Mujtaba, Ghulam; Schultz, Julie M; Imtiaz, Ayesha; Morell, Robert J; Friedman, Thomas B; Naz, Sadaf

    2015-01-01

    Background Hearing loss is a heterogeneous neurosensory disorder. Mutations of 56 genes are reported to cause recessively inherited nonsyndromic deafness. Objective We sought to identify the genetic lesion causing hearing loss segregating in a large consanguineous Pakistani family. Methods and Results Mutations of GJB2 and all other genes reported to underlie recessive deafness were ruled out as the cause of the phenotype in the affected members of the participating family. Homozygosity mapping with a dense array of one million SNP markers allowed us to map the gene for recessively inherited severe hearing loss to chromosome 7q31.2, defining a new deafness locus designated DFNB97 (maximum LOD score of 4.8). Whole-exome sequencing revealed a novel missense mutation c.2521T>G (p.F841V) in MET, which encodes the receptor for hepatocyte growth factor. The mutation co-segregated with the hearing loss phenotype in the family and was absent from 800 chromosomes of ethnically matched control individuals as well as from 136,602 chromosomes in public databases of nucleotide variants. Analyses by multiple prediction programs indicated that p.F841V is likely damaging to MET function. Conclusion We identified a missense mutation of MET, encoding the hepatocyte growth factor receptor, as a likely cause of hearing loss in humans. PMID:25941349

  6. Endogenous microRNAs in human microvascular endothelial cells regulate mRNAs encoded by hypertension-related genes.

    PubMed

    Kriegel, Alison J; Baker, Maria Angeles; Liu, Yong; Liu, Pengyuan; Cowley, Allen W; Liang, Mingyu

    2015-10-01

    The goal of this study was to systematically identify endogenous microRNAs (miRNAs) in endothelial cells that regulate mRNAs encoded by genes relevant to hypertension. Small RNA deep sequencing was performed in cultured human microvascular endothelial cells. Of the 50 most abundant miRNAs identified, 30 had predicted target mRNAs encoded by genes with known involvement in hypertension or blood pressure regulation. The cells were transfected with anti-miR oligonucleotides to inhibit each of the 30 miRNAs and the mRNA abundance of predicted targets was examined. Of 95 miRNA-target pairs examined, the target mRNAs were significantly upregulated in 35 pairs and paradoxically downregulated in 8 pairs. The result indicated significant suppression of the abundance of mRNA encoded by ADM by endogenous miR-181a-5p, ATP2B1 by the miR-27 family, FURIN by miR-125a-5p, FGF5 by the let-7 family, GOSR2 by miR-27a-3p, JAG1 by miR-21-5p, SH2B3 by miR-30a-5p, miR-98, miR-181a-5p, and the miR-125 family, TBX3 by the miR-92 family, ADRA1B by miR-22-3p, ADRA2A by miR-30a-5p and miR-30e-5p, ADRA2B by miR-30e-5p, ADRB1 by the let-7 family and miR-98, EDNRB by the miR-92 family, and NOX4 by the miR-92 family, miR-100-5p, and miR-99b-5p (n=3-9; P<0.05 versus scrambled anti-miR). Treatment with anti-miR-21 decreased blood pressure in mice fed a 4% NaCl diet. Inhibition of the miRNAs targeting NOX4 mRNA increased H2O2 release from endothelial cells. The findings indicate widespread, tonic control of mRNAs encoded by genes relevant to blood pressure regulation by endothelial miRNAs and provide a novel and uniquely informative basis for studying the role of miRNAs in hypertension.

  7. Encoding of frequency-modulation (FM) rates in human auditory cortex.

    PubMed

    Okamoto, Hidehiko; Kakigi, Ryusuke

    2015-12-14

    Frequency-modulated sounds play an important role in our daily social life. However, it currently remains unclear whether frequency modulation rates affect neural activity in the human auditory cortex. In the present study, using magnetoencephalography, we investigated the auditory evoked N1m and sustained field responses elicited by temporally repeated and superimposed frequency-modulated sweeps that were matched in the spectral domain, but differed in frequency modulation rates (1, 4, 16, and 64 octaves per sec). The results obtained demonstrated that the higher rate frequency-modulated sweeps elicited the smaller N1m and the larger sustained field responses. Frequency modulation rate had a significant impact on the human brain responses, thereby providing a key for disentangling a series of natural frequency-modulated sounds such as speech and music.

  8. Encoding of frequency-modulation (FM) rates in human auditory cortex

    PubMed Central

    Okamoto, Hidehiko; Kakigi, Ryusuke

    2015-01-01

    Frequency-modulated sounds play an important role in our daily social life. However, it currently remains unclear whether frequency modulation rates affect neural activity in the human auditory cortex. In the present study, using magnetoencephalography, we investigated the auditory evoked N1m and sustained field responses elicited by temporally repeated and superimposed frequency-modulated sweeps that were matched in the spectral domain, but differed in frequency modulation rates (1, 4, 16, and 64 octaves per sec). The results obtained demonstrated that the higher rate frequency-modulated sweeps elicited the smaller N1m and the larger sustained field responses. Frequency modulation rate had a significant impact on the human brain responses, thereby providing a key for disentangling a series of natural frequency-modulated sounds such as speech and music. PMID:26656920

  9. Human gene encoding prostacyclin synthase (PTGIS): Genomic organization, chromosomal localization, and promoter activity

    SciTech Connect

    Yokoyama, Chieko; Yabuki, Tomoko; Inoue, Hiroyasu

    1996-09-01

    The prostacyclin synthase gene isolated from human genomic libraries (PTGIS) consists of 10 exons spanning approximately 60 kb. All the splice donor and acceptor sites conform to the GT/AG rule. Genomic Southern blot and fluorescence in situ hybridization analyses revealed that the human prostacyclin synthase gene is present as a single copy per haploid genome and is localized on chromosome 20q13.11-q13.13. The 1.5-kb sequence of the 5{prime} of the translational initiation site contained both GC-rich and pyrimidine-rich regions and consensus sequences of the transcription factor recognition sites such as Sp1, AP-2, the interferon-{gamma} response element, GATA, NF-{kappa}B, the CACCC box, and the glucocorticoid response element. The core binding sequence (GAGACC) of the shear stress responsive element was also found in the 5{prime}-flanking region of the gene. The major product of the primer extension analysis suggested that the transcription of the gene started from the positions around 49 bp upstream of the translational initiation codon. Transient transfection experiments using human aortic and bovine arterial endothelial cells demonstrated that the GC-rich region (positions -145 to -10) possessed a significant promoter activity. The 6-kb downstream sequence of the translational termination codon contained multiple polyadenylation signals, Alu repeat sequences, and the consensus sequence of the primate-repetitive DNA element, MER1. Two sizes of the prostacyclin synthase mRNAs (approximately 6 and 3.3 kb) were detected with the human aorta and lung. RNA blot hybridization analysis using the 3{prime}-untranslated region as probe indicated that the sizes of the 3{prime}-flanking regions were different in the major 6-kb and minor 3.3-kb mRNAs. 54 refs., 7 figs.

  10. A physiologically based model for temporal envelope encoding in human primary auditory cortex.

    PubMed

    Dugué, Pierre; Le Bouquin-Jeannès, Régine; Edeline, Jean-Marc; Faucon, Gérard

    2010-09-01

    Communication sounds exhibit temporal envelope fluctuations in the low frequency range (<70 Hz) and human speech has prominent 2-16 Hz modulations with a maximum at 3-4 Hz. Here, we propose a new phenomenological model of the human auditory pathway (from cochlea to primary auditory cortex) to simulate responses to amplitude-modulated white noise. To validate the model, performance was estimated by quantifying temporal modulation transfer functions (TMTFs). Previous models considered either the lower stages of the auditory system (up to the inferior colliculus) or only the thalamocortical loop. The present model, divided in two stages, is based on anatomical and physiological findings and includes the entire auditory pathway. The first stage, from the outer ear to the colliculus, incorporates inhibitory interneurons in the cochlear nucleus to increase performance at high stimuli levels. The second stage takes into account the anatomical connections of the thalamocortical system and includes the fast and slow excitatory and inhibitory currents. After optimizing the parameters of the model to reproduce the diversity of TMTFs obtained from human subjects, a patient-specific model was derived and the parameters were optimized to effectively reproduce both spontaneous activity and the oscillatory part of the evoked response.

  11. Tumor-specific gene therapy for pancreatic cancer using human neural stem cells encoding carboxylesterase

    PubMed Central

    Choi, Seon-A; Yoon, Seung-Bin; Kim, Seung U.; Lee, Hong J.

    2016-01-01

    Advanced pancreatic cancer is one of the most lethal malignant human diseases lacking effective treatment. Its extremely low survival rate necessitates development of novel therapeutic approach. Human neural stem cells (NSCs) are known to have tumor-tropic effect. We genetically engineered them to express rabbit carboxyl esterase (F3.CE), which activates prodrug CPT-11(irinotecan) into potent metabolite SN-38. We found significant inhibition of the growth of BxPC3 human pancreatic cancer cell line in vitro by F3.CE in presence of CPT-11. Apoptosis was also markedly increased in BxPC3 cells treated with F3.CE and CPT-11. The ligand VEGF and receptor VEGF-1(Flt1) were identified to be the relevant tumor-tropic chemoattractant. We confirmed in vivo that in mice injected with BxPC3 on their skin, there was significant reduction of tumor size in those treated with both F3.CE and BxPC3 adjacent to the cancer mass. Administration of F3.CE in conjunction with CPT-11 could be a new possibility as an effective treatment regimen for patients suffering from advanced pancreatic cancer. PMID:27659534

  12. Human Dorsal Striatum Encodes Prediction Errors during Observational Learning of Instrumental Actions

    PubMed Central

    Cooper, Jeffrey C.; Dunne, Simon; Furey, Teresa; O’Doherty, John P.

    2013-01-01

    The dorsal striatum plays a key role in the learning and expression of instrumental reward associations that are acquired through direct experience. However, not all learning about instrumental actions require direct experience. Instead, humans and other animals are also capable of acquiring instrumental actions by observing the experiences of others. In this study, we investigated the extent to which human dorsal striatum is involved in observational as well as experiential instrumental reward learning. Human participants were scanned with fMRI while they observed a confederate over a live video performing an instrumental conditioning task to obtain liquid juice rewards. Participants also performed a similar instrumental task for their own rewards. Using a computational model-based analysis, we found reward prediction errors in the dorsal striatum not only dur