Science.gov

Sample records for human cdna encoding

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

    SciTech Connect

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III ); Billheimer, J.T. )

    1991-01-15

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

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

    SciTech Connect

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

    1987-01-01

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

  3. 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.

  4. Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.

    PubMed Central

    Calhoun, D H; Bishop, D F; Bernstein, H S; Quinn, M; Hantzopoulos, P; Desnick, R J

    1985-01-01

    Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A (alpha-Gal A; alpha-D-galactoside galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of alpha-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human alpha-Gal A was isolated from a lambda gt11 human liver cDNA expression library. To facilitate screening, an improved affinity purification procedure was used to obtain sufficient homogeneous enzyme for production of monospecific antibodies and for amino-terminal and peptide microsequencing. On the basis of an amino-terminal sequence of 24 residues, two sets of oligonucleotide mixtures were synthesized corresponding to adjacent, but not overlapping, amino acid sequences. In addition, an oligonucleotide mixture was synthesized based on a sequence derived from an alpha-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 X 10(7) plaques. Of these, only one clone (designated lambda AG18) demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to synthetic oligonucleotide mixtures corresponding to amino-terminal and internal amino acid sequences. Nucleotide sequencing of the 5' end of the 1250-base-pair EcoRI insert of clone lambda AG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone lambda AG18 appears to contain the full-length coding region of the processed, enzymatically active alpha-Gal A, as well as sequences coding for five amino acids of the amino-terminal propeptide, which is posttranslationally cleaved during enzyme maturation. Images PMID:2997789

  5. 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.

  6. 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.

  7. Nucleotide and predicted amino acid sequence of a cDNA clone encoding part of human transketolase.

    PubMed

    Abedinia, M; Layfield, R; Jones, S M; Nixon, P F; Mattick, J S

    1992-03-31

    Transketolase is a key enzyme in the pentose-phosphate pathway which has been implicated in the latent human genetic disease, Wernicke-Korsakoff syndrome. Here we report the cloning and partial characterisation of the coding sequences encoding human transketolase from a human brain cDNA library. The library was screened with oligonucleotide probes based on the amino acid sequence of proteolytic fragments of the purified protein. Northern blots showed that the transketolase mRNA is approximately 2.2 kb, close to the minimum expected, of which approximately 60% was represented in the largest cDNA clone. Sequence analysis of the transketolase coding sequences reveals a number of homologies with related enzymes from other species. PMID:1567394

  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 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

  10. Isolation and characterization of human cDNA clones encoding the. alpha. and the. alpha. prime subunits of casein kinase II

    SciTech Connect

    Lozeman, F.J.; Litchfield, D.W.; Piening, C.; Takio, Koji; Walsh, K.A.; Krebs E.G. )

    1990-09-11

    Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two {alpha} or {alpha}{prime} subunits (or one of each) and two {beta} subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell {lambda}gt 10 library using cDNA clones isolated from Drosophila melanogasten. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5{prime} untranslated region) and followed by 871 bp (3{prime} untranslated region). The hT4.1 close was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 by (5{prime} untranslated region) and followed by 550 bp (3{prime} untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of {alpha} and {alpha}{prime} subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the {alpha} and {alpha}{prime} subunits of casein kinase II. These studies show that there are two distinct catalytic subunits for casein II ({alpha} and {alpha}{prime}) and that the sequence of these subunits is largely conserved between the bovine and the human.

  11. Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C.

    PubMed Central

    Dewaste, V; Pouillon, V; Moreau, C; Shears, S; Takazawa, K; Erneux, C

    2000-01-01

    Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] 3-kinase catalyses the phosphorylation of Ins(1,4,5)P(3) to Ins(1,3,4,5)P(4). cDNAs encoding two isoenzymes of Ins(1,4,5)P(3) 3-kinase (3-kinases A and B) have been described previously. In the present study, we report the cloning of a full-length 2052 bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P(3) 3-kinase family, referred to as isoform C. This novel enzyme has a calculated molecular mass of 75. 207 kDa and a K(m) for Ins(1,4,5)P(3) of 6 microM. Northern-blot analysis showed the presence of a transcript of approx. 3.9 kb in various human tissues. Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5alphaF' bacteria or COS-7 cells. Calcium alone decreases the Ins(1,4,5)P(3) 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells. This inhibitory effect is reversed in the presence of calmodulin. The recombinant bacterial 3-kinase C can be adsorbed on calmodulin-Sepharose in the presence of calcium. The present data show that Ins(1,4,5)P(3) 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin-Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin. PMID:11085927

  12. Cloning of cDNA encoding human rapsyn and mapping of the RAPSN gene locus to chromosome 11p11.2-p11.1

    SciTech Connect

    Buckel, A.; Beeson, D.; Vincent, A.

    1996-08-01

    We have isolated and sequenced cDNA clones for the human 43-kDa acetylcholine receptor-associated protein rapsyn. The cDNA encodes a 412-amino-acid protein that has a predicted molecular mass of 46,330 Da and shows 96% sequence identity with mouse rapsyn. Analysis of PCR amplifications, first from somatic cell hybrids and subsequently from radiation hybrids, localizes the human RAPSN gene locus to chromosome 11p11.2-p11.1 in close proximity to ACP2. 12 refs., 2 figs.

  13. 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

  14. 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.

  15. In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I.

    PubMed Central

    Kodama, K; Barnes, D E; Lindahl, T

    1991-01-01

    Human cDNAs encoding fragments of DNA ligase I, the major replicative DNA ligase in mammalian cells, have been expressed as lacZ fusion proteins in Escherichia coli. A cDNA encoding the carboxyl-terminal catalytic domain of human DNA ligase I was able to complement a conditional-lethal DNA ligase mutation in E. coli as measured by growth of the mutant strain at the non-permissive temperature. Targeted deletions of the amino and carboxyl termini of the catalytic domain identified a minimum size necessary for catalytic function and a maximum size for optimal complementing activity in E. coli. The human cDNA was subjected to systematic site-directed mutagenesis in vitro and mutant polypeptides assayed for functional expression in the E. coli DNA ligase mutant. Such functional analysis of the active site of DNA ligase I identified specific residues required for the formation of an enzyme-adenylate reaction intermediate. Images PMID:1956768

  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. Molecular characterization of hNRP, a cDNA encoding a human nucleosome-assembly-protein-I-related gene product involved in the induction of cell proliferation.

    PubMed Central

    Simon, H U; Mills, G B; Kozlowski, M; Hogg, D; Branch, D; Ishimi, Y; Siminovitch, K A

    1994-01-01

    We have isolated from a human thymus cDNA library a cDNA clone encoding a potential protein with 54% amino acid similarity to that encoded by a previously identified cDNA for yeast nucleosome assembly protein I (NAP-I). The deduced amino acid sequence for this newly identified cDNA, designated hNRP (human NAP-related protein), contains a potential seven-residue nuclear localization motif, three clusters of highly acidic residues and other structural features found in various proteins implicated in chromatin formation. When expressed as a fusion protein in Escherichia coli, hNRP reacted specifically with a monoclonal antibody raised against human NAP-I. The hNRP transcript was detected in all tissues and cell lines studied, but levels were somewhat increased in rapidly proliferating cells. Moreover, levels of both hNRP mRNA and protein increased rapidly in cultured T-lymphocytes induced to proliferate by incubation with phorbol ester and ionomycin. Phorbol 12-myristate 13-acetate/ionomycin-induced increases in both hNRP mRNA and mitogenesis, as measured by thymidine incorporation, were markedly inhibited, however, in cells treated with an hNRP antisense oligonucleotide. These results demonstrate a correlation between induction of hNRP expression and mitogenesis and taken together with the structural similarities between hNRP and yeast NAP-I suggest that the hNRP gene product participates in DNA replication and thereby plays an important role in the process of cell proliferation. Images Figure 4 Figure 5 Figure 6 PMID:8297347

  18. Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O sup 6 -alkylguanine

    SciTech Connect

    Tano, K.; Shiota, S.; Collier, J.; Foote, R.S.; Mitra, S. )

    1990-01-01

    O{sup 6}-Methylguanine-DNA methyltransferase a unique DNA repair protein present in most organisms, removes the carcinogenic and mutagenic adduct O{sup 6}-alkylguanine from DNA by stoichiometrically accepting the alkyl group on a cysteine residue in a suicide reaction. The mammalian protein is highly regulated in both somatic and germ-like cells. In addition, the toxicity of certain alkylating drugs in tumor and normal cells is inversely related to the levels of this protein. The cDNA of the human gene, henceforth named MGMT, has been cloned in an expression vector on the basis of its rescue of a methyltransferase-deficient (ada{sup {minus}}) Escherichia coli host. A 22-kDa active methyltransferase encoded entirely by the cDNA contains an amino acid sequence of 61 residues that bears 60-65% similarity with segments of E. coli methyltransferase which encompass the alkyl-acceptor residues. The human cDNA has no sequence similarity with the ada and ogt genes, due in part to differences in codon usage, and shows no detectable homology with E. coli genomic DNA. However, it hybridizes with distinct restriction fragments of human, mouse, and rat DNAs. The lack of methyltransferase observed in many human cell lines is due to the absence of the MGNT gene or to lack of synthesis and/or stability of its 0.95-kilobase poly(A){sup +} RNA transcript.

  19. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins

    SciTech Connect

    Matviw, Yu, G.; Young, D. )

    1992-11-01

    The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: The N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expressions of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals. 42 refs., 5 figs.

  20. 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.

  1. Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription.

    PubMed Central

    DeJong, J; Bernstein, R; Roeder, R G

    1995-01-01

    The human general transcription factor TFIIA is one of several factors involved in specific transcription by RNA polymerase II, possibly by regulating the activity of the TATA-binding subunit (TBP) of TFIID. TFIIA purified from HeLa extracts consists of 35-, 19-, and 12-kDa subunits. Here we describe the isolation of a cDNA clone (hTFIIA gamma) encoding the 12-kDa subunit. Using expression constructs derived from hTFIIA gamma and TFIIA alpha/beta (which encodes a 55-kDa precursor to the alpha and beta subunits of natural TFIIA), we have constructed a synthetic TFIIA with a polypeptide composition similar to that of natural TFIIA. The recombinant complex supports the formation of a DNA-TBP-TFIIA complex and mediates both basal and Gal4-VP16-activated transcription by RNA polymerase II in TFIIA-depleted nuclear extracts. In contrast, TFIIA has no effect on tRNA and 5S RNA transcription by RNA polymerase III in this system. We also present evidence that both the p55 and p12 recombinant subunits interact with TBP and that the basic region of TBP is critical for the TFIIA-dependent function of TBP in nuclear extracts. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7724559

  2. Characterization of a cDNA encoding a novel human Golgi alpha 1, 2-mannosidase (IC) involved in N-glycan biosynthesis.

    PubMed

    Tremblay, L O; Herscovics, A

    2000-10-13

    A human cDNA encoding a 70.9-kDa type II membrane protein with sequence similarity to class I alpha1,2-mannosidases was isolated. The enzymatic properties of the novel alpha1,2-mannosidase IC were studied by expressing its catalytic domain in Pichia pastoris as a secreted glycoprotein. alpha1,2-Mannosidase IC sequentially hydrolyzes the alpha1,2-linked mannose residues of [(3)H]mannose-labeled Man(9)GlcNAc to form [(3)H]Man(6)GlcNAc and a small amount of [(3)H]Man(5)GlcNAc. The enzyme requires calcium for activity and is inhibited by both 1-deoxymannojirimycin and kifunensine. The order of mannose removal was determined by separating oligosaccharide isomers formed from pyridylaminated Man(9)GlcNAc(2) by high performance liquid chromatography. The terminal alpha1,2-linked mannose residue from the middle branch is the last mannose removed by the enzyme. This residue is the mannose cleaved from Man(9)GlcNAc(2) by the endoplasmic reticulum alpha1, 2-mannosidase I to form Man(8)GlcNAc(2) isomer B. The order of mannose hydrolysis from either pyridylaminated Man(9)GlcNAc(2) or Man(8)GlcNAc(2) isomer B differs from that previously reported for mammalian Golgi alpha1,2-mannosidases IA and IB. The full-length alpha1,2-mannosidase IC was localized to the Golgi of MDBK and MDCK cells by indirect immunofluorescence. Northern blot analysis showed tissue-specific expression of a major transcript of 3.8 kilobase pairs. The expression pattern is different from that of human Golgi alpha1,2-mannosidases IA and IB. Therefore, the human genome contains at least three differentially regulated Golgi alpha1, 2-mannosidase genes encoding enzymes with similar, but not identical specificities. PMID:10915796

  3. Molecular cloning and characterization of the human KIN17 cDNA encoding a component of the UVC response that is conserved among metazoans.

    PubMed

    Kannouche, P; Mauffrey, P; Pinon-Lataillade, G; Mattei, M G; Sarasin, A; Daya-Grosjean, L; Angulo, J F

    2000-09-01

    We describe the cloning and characterization of the human KIN17 cDNA encoding a 45 kDa zinc finger nuclear protein. Previous reports indicated that mouse kin17 protein may play a role in illegitimate recombination and in gene regulation. Furthermore, overproduction of mouse kin17 protein inhibits the growth of mammalian cells, particularly the proliferation of human tumour-derived cells. We show here that the KIN17 gene is remarkably conserved during evolution. Indeed, the human and mouse kin17 proteins are 92.4% identical. Furthermore, DNA sequences from fruit fly and filaria code for proteins that are 60% identical to the mammalian kin17 proteins, indicating conservation of the KIN17 gene among metazoans. The human KIN17 gene, named (HSA)KIN17, is located on human chromosome 10 at p15-p14. The (HSA)KIN17 RNA is ubiquitously expressed in all the tissues and organs examined, although muscle, heart and testis display the highest levels. UVC irradiation of quiescent human primary fibroblasts increases (HSA)KIN17 RNA with kinetics similar to those observed in mouse cells, suggesting that up-regulation of the (HSA)KIN17 gene after UVC irradiation is a conserved response in mammalian cells. (HSA)kin17 protein is concentrated in intranuclear focal structures in proliferating cells as judged by indirect immunofluorescence. UVC irradiation disassembles (HSA)kin17 foci in cycling cells, indicating a link between the intranuclear distribution of (HSA)kin17 protein and the DNA damage response. PMID:10964102

  4. 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.

  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. 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.

  7. Cloning of cDNA encoding steroid 11. beta. -hydroxylase (P450c11)

    SciTech Connect

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

    1987-10-01

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

  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

    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.

  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

    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.

  13. Isolation of the cDNA and chromosomal localization of the gene (TAX1) encoding the human axonal glycoprotein TAG-1

    SciTech Connect

    Tsiotra, P.C.; Karagogeos, D.; Theodorakis, K.; Michaelidis, T.M.; Papamatheakis, J. ); Modi, W.S. ); Furley, A.J.; Jessell, T.M. )

    1993-12-01

    The transient axonal glycoprotein (TAG-1) is a cell adhesion molecule that promotes neurite outgrowth and belongs to the immunoglobulin superfamily. The authors have isolated cDNAs encoding TAX1, the human homologue of TAG-1. Human TAX1 shows a high degree of homology to rat TAX1 and less to its chick counterpart, axonin-1, with 91 and 75% identity at the amino acid level, respectively. The numbers of immunoglobulin (IgC2) domains and fibronectin repeats present in TAG-1 are conserved among the three species. The highest degree of conservation occurs in the second IgC2 domain (98% with the rat and 82% with the chick). The human homologue also contains a putative N-terminal signal sequence and a C-terminal hydrophobic sequence, suggestive of linkage to the cell membrane via phosphatidylinositol. In addition, the two mammalian TAG-1 proteins share the RDG tripeptide, a motif known to mediate recognition of fibronection by integrins. In situ hybridization to human metaphase chromosomes maps the TAX1 gene encoding human TAG-1 to a single location on chromosome 1q32. 35 refs., 3 figs.

  14. Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation

    SciTech Connect

    Ninomiya-Tsuji, Jun ); Nomoto, Satoshi; Matsumoto, Kunihiro ); Yasuda, Hideyo ); Reed, S.I. )

    1991-10-15

    The authors have cloned two different human cDNAs that can complement cdc28 mutations of budding yeast Saccharomyces cerevisiae. One corresponds to a gene encoding human p34{sup CDC2} kinase, and the other to a gene (CDK2; cell division kinase) that has not been characterized previously. The CDK2 protein is highly homologous to p34{sup CDC2} kinase and more significantly is homologous to Xenopus Eg1 kinase, suggesting that CDK2 is the human homolog of Eg1. The human CDC2 and CDK2 genes were both able to complement the inviability of a null allele of S. cerevisiae CDC28. This result indicates that the CDK2 protein has a biological activity closely related to the CDC28 and p34{sup CDC2} kinases. However, CDK2 was unable to complement cdc2 mutants in fission yeast Schizosaccharomyces pombe under the condition where the human CDC2 gene could complement them. CDK2 mRNA appeared late in G{sub 1} or in early S phase, slightly before CDC2 mRNA, after growth stimulation in normal human fibroblast cells. These results suggest that in human cells, two different CDC2-like kinases may regulate the cell cycle at distinct stages.

  15. Comparative mapping on the mouse and human X chromosomes of a human cDNA clone encoding the vasopressin renal-type receptor (AVP2R)

    SciTech Connect

    Faust, C.J.; Gonzales, J.C.; Seibold, A.; Birnbaumer, M.; Herman, G.E. )

    1993-02-01

    Mutation in the gene for the human renal-type vasopressin receptor (V2R) have recently been identified in patients with nephrogenic diabetes insipidus (NDI). Both V2R and NDI have been independently mapped to Xq28. Using a combination of genetic and physical mapping, we have localized the murine V2r locus to within 100 kb of L1Cam on the mouse X chromosome in a region syntenic with human Xq28. Based on conserved gene order of mouse and human loci in this region, physical mapping using DNA derived form human lymphoblasts has established that the corresponding human loci V2R and L1CAM are linked within 210 kb. The efficiency and precision of genetic mapping of V2r and other loci in the mouse suggest that it might be easier to map additional human genes in the mouse first and infer the corresponding human location. More precise physical mapping in man could then be performed using pulsed-field gel electrophoresis and/or yeast artificial chromosomes. 16 refs., 1 fig. 1 tab.

  16. 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.

  17. A cDNA clone encoding a peptide highly specific for hepatitis C infection.

    PubMed

    Arima, T; Mori, C; Takamizawa, A; Shimomura, H; Tsuji, T

    1990-04-01

    A random primed lambda gt11-cDNA library was constructed from donors plasma presumably infected by blood-borne non-A, non-B hepatitis (hepatitis C:HC) agent and immunoscreened with serum pooled from patients with acute or chronic HC. Twelve lambda gt11-cDNA clones encoding antigens associated with HC infection in Japan as well as in the USA were isolated. Of these one clone consisting of 114 nucleotides and showing a discrete band on an immunoblot analysis, was extensively studied. The clone is not derived from the host DNA encoding one polypeptide specific and highly sensitive for serum from patients with HC and has no homology to the nucleotide sequences of known human viruses including hepatitis A,B and D viruses, Ebstein-Barr virus, coxsackievirus, immunodeficiency virus type 1 or Japanese encephalitis virus. These results suggest that this clone is derived from the genome of HC agent. PMID:1693349

  18. Characterization of Cxorf5 (71-7A), a novel human cDNA mapping to Xp22 and encoding a protein containing coiled-coil alpha-helical domains.

    PubMed

    de Conciliis, L; Marchitiello, A; Wapenaar, M C; Borsani, G; Giglio, S; Mariani, M; Consalez, G G; Zuffardi, O; Franco, B; Ballabio, A; Banfi, S

    1998-07-15

    The human X chromosome is known to contain several disease genes yet to be cloned. In the course of a project aimed at the construction of a transcription map of the Xp22 region, we fully characterized a novel cDNA, Cxorf5 (HGMW-approved symbol, alias 71-7A), previously mapped to this region but for which no sequence information was available. We isolated and sequenced the full-length transcript, which encodes a predicted protein of unknown function containing a large number of coiled-coild domains, typically presented in a variety of different molecules, from fibrous proteins to transcription factors. We showed that the Cxorf5 cDNA is ubiquitously expressed, undergoes alternative splicing, and escapes X inactivation. Furthermore, we precisely mapped two additional Cxorf5-related loci on the Y chromosome and on chromosome 5. By virtue of its mapping assignment to the Xp22 region, Cxorf5 represents a candidate gene for at least four human diseases, namely spondyloepiphiseal dysplasia late, oral-facial-digital syndrome type 1, craniofrontonasal syndrome, and a nonsyndromic sensorineural deafness. PMID:9722947

  19. 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

  20. 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.

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

    PubMed

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

    2002-07-01

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

  2. 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. PMID:7665074

  3. Isolation of distinct cDNA clones encoding HLA-DR beta chains by use of an expression assay.

    PubMed Central

    Long, E O; Wake, C T; Strubin, M; Gross, N; Accolla, R S; Carrel, S; Mach, B

    1982-01-01

    cDNA clones encoding different human Ia antigen beta chains were isolated by use of a complementation-expression assay in Xenopus oocytes. The assay was based on two previous findings. First, oocytes injected with mRNA from a human B-cell line express HLA-DR antigen. The three intracellular DR chains are assembled in oocytes and can be immunoprecipitated with anti-DR monoclonal antibodies. Second, we have isolated cDNA clones encoding DR alpha and intermediate chains. In order to identify beta-chain cDNA clones, mRNA was hybrid-selected with pools of cDNA clones, mixed with mRNA for the alpha and intermediate chains, and injected into oocytes. We isolated two distinct clones that could select DR beta-chain mRNA as demonstrated by assembly of the translation product with DR alpha chains and immunoprecipitation with DR-specific monoclonal antibodies. One clone is specific for a beta chain of the DR locus. The other clone, much weaker in its ability to select DR mRNA, encodes another Ia-like beta chain. Full-length cDNA clones corresponding to the DR and Ia-like beta chains were isolated and compared. Cross-hybridization was detectable in the coding regions but not in the 3' untranslated regions. Distinct RNAs homologous to the DR and the Ia-like beta-chain clones were present in B cells but were undetectable in three T-cell lines. Images PMID:6818545

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

    PubMed

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

    2011-12-01

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

  5. 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.

  6. 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

  7. 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.

  8. Human DNA ligase I cDNA: Cloning and functional expression in Saccharomyces cerevisiae

    SciTech Connect

    Barnes, D.E.; Kodama, Kenichi; Tomkinson, A.E.; Lindahl, T.; Lasko, D.D. ); Johnston, L.H. )

    1990-09-01

    Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomuces cerevisiae. The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I. The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S. cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.

  9. Analysis of a cDNA encoding the major vault protein from the electric ray Discopyge ommata.

    PubMed

    Herrmann, C; Zimmermann, H; Volknandt, W

    1997-03-25

    The major vault protein is the predominant constituent of vaults ubiquitous large cytosolic ribonucleoprotein particles. A cDNA clone encoding the 100-kDa major vault protein (MVP100) was isolated from an electric lobe library of Discopyge ommata. The complete nucleotide sequence was determined. Northern blot analysis revealed a 2.8-kb transcript with a high expression in neural tissue. Southern blot analysis indicates that the electric ray MVP100 is a single copy-gene with at least two introns. The primary structure of major vault proteins characterized in slime mold, ray, rat and human is evolutionary highly conserved. PMID:9099863

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

    PubMed Central

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

    1991-01-01

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

  11. 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.

  12. Cloning and characterization of a cDNA encoding hexokinase from tomato.

    PubMed

    Menu, T; Rothan, C; Dai, N; Petreikov, M; Etienne, C; Destrac-Irvine, A; Schaffer, A; Granot, D; Ricard, B

    2001-01-01

    Two different partial sequences encoding putative hexokinase (HXK, ATP: hexose-6-phosphotransferase, EC 2.7.1.1) were isolated from tomato (Lycopersicon esculentum) by RT-PCR using degenerate primers. Southern blot analysis suggested the existence of two divergent HXK genes. A complete cDNA of one HXK was isolated by screening a cDNA library prepared from young cherry tomato fruit. The 1770 bp cDNA of LeHXK2 contained an open reading frame encoding a 496 amino acid protein that has 69% identity with the two Arabidopsis HXKs, 83 and 85% identity with potato StHXK1 and tobacco NtHXK, respectively. However, this clone had 97% amino acid identity with potato StHXK2 and, therefore, was named LeHXK2. LeHXK2 cDNA was expressed in a triple mutant yeast (Saccharomyces cerevisiae) strain which lacked the ability to phosphorylate glucose and fructose and, therefore, was unable to grow on these sugars as carbon sources. Mutant cells expressing LeHXK2 grew on both glucose and fructose with shorter doubling time on glucose. The kinetic properties of LeHXK2 expressed in yeast were determined after the purification of LeHXK2 by HPLC-ion exchange chromatography, confirming the identity of LeHXK2 as hexokinase with higher affinity to glucose. LeHXK2 mRNA was detected by RT-PCR expression analysis in all organs and tissues and at all stages of fruit development. However, semi-quantitative RT-PCR analysis showed that LeHXK2 was most highly expressed in flowers. PMID:11164592

  13. Characterization of cDNA clones selected by the GeneMark analysis from size-fractionated cDNA libraries from human brain.

    PubMed

    Hirosawa, M; Nagase, T; Ishikawa, K; Kikuno, R; Nomura, N; Ohara, O

    1999-10-29

    We have conducted a sequencing project of human cDNAs which encode large proteins in brain. For selection of cDNA clones to be sequenced in this project, cDNA clones have been experimentally examined by in vitro transcription/translation prior to sequencing. In this study, we tested an alternative approach for picking up cDNA clones having a high probability of carrying protein coding region. This approach exploited 5'-end single-pass sequence data and the GeneMark program for assessing protein-coding potential, and allowed us to select 74 clones out of 14,804 redundant cDNA clones. The complete sequence data of these 74 clones revealed that 45% of them encoded proteins consisting of more than 500 amino acid residues while all the clones thus selected carried possible protein coding sequences as expected. The results indicated that the GeneMark analysis of 5'-end sequences of cDNAs offered us a simple and effective means to select cDNA clones with protein-coding potential although the sizes of the encoded proteins could not be predicted. PMID:10574461

  14. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease.

    PubMed Central

    Uéda, K; Fukushima, H; Masliah, E; Xia, Y; Iwai, A; Yoshimoto, M; Otero, D A; Kondo, J; Ihara, Y; Saitoh, T

    1993-01-01

    A neuropathological hallmark of Alzheimer disease (AD) is a widespread amyloid deposition. We analyzed the entire amino acid sequences in an amyloid preparation and found, in addition to the major beta/A4-protein (A beta) fragment, two unknown peptides. We raised antibodies against synthetic peptides using subsequences of these peptides. These antibodies immunostained amyloid in neuritic and diffuse plaques as well as vascular amyloid. Electron microscopic analysis demonstrated that the immunostaining was localized on amyloid fibrils. We have isolated an apparently full-length cDNA encoding a 140-amino-acid protein within which two previously unreported amyloid sequences are encoded in tandem in the most hydrophobic domain. We tentatively named this 35-amino acid peptide NAC (non-A beta component of AD amyloid) and its precursor NACP. NAC is the second component, after A beta, identified chemically in the purified AD amyloid preparation. Secondary structure predictions indicate that the NAC peptide sequence has a strong tendency to form beta-structures consistent with its association with amyloid. NACP is detected as a M(r) 19,000 protein in the cytosolic fraction of brain homogenates and comigrates on immunoblots with NACP synthesized in Escherichia coli from NACP cDNA. NACP mRNA is expressed principally in brain but is also expressed in low concentrations in all tissues examined except in liver, suggesting its ubiquitous and brain-specific functions. The availability of the cDNA encoding full-length NACP should help to elucidate the mechanisms of amyloidosis in AD. Images Fig. 1 Fig. 3 Fig. 5 PMID:8248242

  15. 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.

  16. Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na+/nucleoside cotransporter (hCNT2) selective for purine nucleosides and uridine.

    PubMed

    Ritzel, M W; Yao, S Y; Ng, A M; Mackey, J R; Cass, C E; Young, J D

    1998-01-01

    Two Na(+)-dependent nucleoside transporters implicated in adenosine and uridine transport in mammalian cells are distinguished functionally on the basis of substrate specificity: CNT1 is selective for pyrimidine nucleosides but also transports adenosine; CNT2 (also termed SPNT) is selective for purine nucleosides but also transports uridine. Both proteins belong to a gene family that includes the NupC proton/nucleoside symporter of E. coli. cDNAs encoding members of the CNT family have been isolated from rat tissues (jejunum, brain, liver; rCNT1 and rCNT2/SPNT) and, most recently, human kidney (hCNT1 and hSPNT1). Here, the molecular cloning and functional characterization of a CNT2/SPNT-type transporter from human small intestine are described. The encoded 658-residue protein (hCNT2 in the nomenclature) had the same predicted amino acid sequence as human kidney hSPNT1, except for a polymorphism at residue 75 (Arg substituted by Ser), and was 83 and 72% identical to rCNT2 and hCNT1, respectively. Sequence differences between hCNT2 and rCNT2 were greatest at the N-terminus. In Xenopus oocytes, recombinant hCNT2 exhibited the functional characteristics of a Na(+)-dependent nucleoside transporter with selectivity for adenosine, other purine nucleosides and uridine (adenosine and uridine K(m) app values 8 and 40 microM, respectively). hCNT2 transcripts were found in kidney and small intestine but, unlike rCNT2, were not detected in liver. Deoxyadenosine, which undergoes net renal secretion in humans, was less readily transported than adenosine. hCNT2 also mediated small, but significant, fluxes of the antiviral purine nucleoside analogue 2',3'-dideoxyinosine. hCNT2 is, therefore potentially involved in both the intestinal absorption and renal handling of purine nucleosides (including adenosine), uridine and purine nucleoside drugs. The gene encoding hCNT2 was mapped to chromosome 15q15. PMID:10087507

  17. 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

  18. 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.

  19. Nucleotide sequence of the cDNA encoding the precursor of the beta subunit of rat lutropin.

    PubMed Central

    Chin, W W; Godine, J E; Klein, D R; Chang, A S; Tan, L K; Habener, J F

    1983-01-01

    We have determined the nucleotide sequences of cDNAs encoding the precursor of the beta subunit of rat lutropin, a polypeptide hormone that regulates gonadal function, including the development of gametes and the production of steroid sex hormones. The cDNAs were prepared from poly(A)+ RNA derived from the pituitary glands of rats 4 weeks after ovariectomy and were cloned in bacterial plasmids. Bacterial colonies containing transfected plasmids were screened by hybridization with a 32P-labeled cDNA encoding the beta subunit of human chorionic gonadotropin, a protein that is related in structure to lutropin. Several recombinant plasmids were detected that by nucleotide sequence analyses contained coding sequences for the precursor of the beta subunit of lutropin. Complete determination of the nucleotide sequences of these cDNAs, as well as of cDNA reverse-transcribed from pituitary poly(A)+ RNA by using a synthetic pentadecanucleotide as a primer of RNA, provided the entire 141-codon sequence of the precursor of the beta subunit of rat lutropin. The precursor consists of a 20 amino acid leader (signal) peptide and an apoprotein of 121 amino acids. The amino acid sequence of the rat lutropin beta subunit shows similarity to the beta subunits of the ovine/bovine, porcine, and human lutropins (81, 86, and 74% of amino acids identical, respectively). Blot hybridization of pituitary RNAs separated by electrophoresis on agarose gels showed that the mRNA encoding the lutropin beta subunit consists of approximately 700 bases. The availability of cDNAs for both the alpha and beta subunits of lutropin will facilitate studies of the regulation of lutropin expression. Images PMID:6192440

  20. 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.

  1. Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

    PubMed

    Jikuya, Hiroyuki; Takano, Jun; Kikuno, Reiko; Hirosawa, Makoto; Nagase, Takahiro; Nomura, Nobuo; Ohara, Osamu

    2003-02-28

    To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility. PMID:12693554

  2. cDNA cloning of human plasminogen activator-inhibitor from endothelial cells.

    PubMed Central

    Ginsburg, D; Zeheb, R; Yang, A Y; Rafferty, U M; Andreasen, P A; Nielsen, L; Dano, K; Lebo, R V; Gelehrter, T D

    1986-01-01

    Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HUVEC) lambda gt11 cDNA library. Three overlapping clones were identified by immunologic screening of 10(6) recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-1 mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-1 gene is located on human chromosome 7. Images PMID:3097076

  3. Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize.

    PubMed Central

    Wu, S; Kriz, A L; Widholm, J M

    1994-01-01

    The cloning and analysis of two different cDNA clones encoding putative maize (Zea mays L.) chitinases obtained by polymerase chain reaction (PCR) and cDNA library screening is described. The cDNA library was made from poly(A)+ RNA from leaves challenged with mercuric chloride for 2 d. The two clones, pCh2 and pCh11, appear to encode class I chitinase isoforms with cysteine-rich domains (not found in pCh11 due to the incomplete sequence) and proline-/glycine-rich or proline-rich hinge domains, respectively. The pCh11 clone resembles a previously reported maize seed chitinase; however, the deduced proteins were found to have acidic isoelectric points. Analysis of all monocot chitinase sequences available to date shows that not all class I chitinases possess the basic isoelectric points usually found in dicotyledonous plants and that monocot class II chitinases do not necessarily exhibit acidic isoelectric points. Based on sequence analysis, the pCh2 protein is apparently synthesized as a precursor polypeptide with a signal peptide. Although these two clones belong to class I chitinases, they share only about 70% amino acid homology in the catalytic domain region. Southern blot analysis showed that pCh2 may be encoded by a small gene family, whereas pCh11 was single copy. Northern blot analysis demonstrated that these genes are differentially regulated by mercuric chloride treatment. Mercuric chloride treatment caused rapid induction of pCh2 from 6 to 48 h, whereas pCh11 responded only slightly to the same treatment. During seed germination, embryos constitutively expressed both chitinase genes and the phytohormone abscisic acid had no effect on the expression. The fungus Aspergillus flavus was able to induce both genes to comparable levels in aleurone layers and embryos but not in endosperm tissue. Maize callus growth on the same plate with A. flavus for 1 week showed induction of the transcripts corresponding to pCh2 but not to pCh11. These studies indicate that

  4. 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.

  5. Molecular cloning of cDNA encoding a novel platelet-endothelial cell tetra-span antigen, PETA-3.

    PubMed

    Fitter, S; Tetaz, T J; Berndt, M C; Ashman, L K

    1995-08-15

    Platelet-endothelial cell tetra-span antigen (PETA-3) was originally identified as a novel human platelet surface glycoprotein, gp27, which was detected by a monoclonal antibody (MoAb), 14A2.H1. Although this glycoprotein is present in low abundance on the platelet surface, MoAb 14A2.H1 stimulates platelet aggregation and mediator release. We now report isolation of a cDNA clone encoding PETA-3 from a library derived from the megakaryoblastic leukemia cell line MO7e. The clone encodes an open reading frame of 253 amino acids that displays 25% to 30% amino acid sequence identity with several members of the newly defined Tetraspan, or Transmembrane 4 superfamily. These proteins consist of four conserved putative transmembrane domains with a large divergent extracellular loop between the third and fourth membrane-spanning regions. PETA-3 has a single consensus sequence for N-linked glycosylation located in this extracellular loop. A single PETA-3 RNA transcript (1.6 kb) was detected in RNA isolated from MO7e cells, bone marrow stromal cells, the C11 endothelial cell line, and several myeloid leukemia cell lines. No transcript was detected in the lymphoblastoid cell lines MOLT-4 and BALM-1. This pattern correlates well with previous protein expression data. Northern blot analysis of RNA from a range of human tissues indicated that the transcript was present in most tissues, the notable exception being brain. PMID:7632941

  6. Suppression of the chemically transformed phenotype of BHK cells by a human cDNA

    SciTech Connect

    Eiden, M.V.; MacArthur, L.; Okayama, Hiroto )

    1991-10-01

    Transformation of baby hamster kidney cell line BHK SN-10 by chemical carcinogens such as nitrosylmethylurea (NMU) is mediated by the loss of a gene product critical for the suppression of malignant transformation. Somatic cell hybrids between chemically transformed BHK SN-10 cells and either normal hamster kidney or human fibroblast cells are nontransformed; therefore, a recessive mechanism underlies the malignant transformation of BHK SN-10 cells after chemical carcinogenesis. A human fibroblast cDNA library was constructed and introduced into NMU-transformed BHK SN-10 cells (NMU 34m) in order to identify a human cDNA capable of suppressing cellular transformation. NMU-transformed BHK cells were analyzed for reversion to an anchorage-dependent stable reversion of NMU 34m cells encodes the intermediate filament protein vimentin, which is apparently required for maintenance of the normal phenotype in BHK SN-10 cells.

  7. Serine protease variants encoded by Echis ocellatus venom gland cDNA: cloning and sequencing analysis.

    PubMed

    Hasson, S S; Mothana, R A; Sallam, T A; Al-balushi, M S; Rahman, M T; Al-Jabri, A A

    2010-01-01

    Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs) from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization) the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent. PMID:20936075

  8. Brain cDNA clone for human cholinesterase.

    PubMed Central

    McTiernan, C; Adkins, S; Chatonnet, A; Vaughan, T A; Bartels, C F; Kott, M; Rosenberry, T L; La Du, B N; Lockridge, O

    1987-01-01

    A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase (EC 3.1.1.8). Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum. The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase (EC 3.1.1.8) rather than acetylcholinesterase (EC 3.1.1.7). It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes, coded for cholinesterase. Images PMID:3477799

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

    SciTech Connect

    Barbry, P.; Chassande, O.; Champigny, G.; Lingueglia, E.; Frelin, C.; Lazdunski, M. ); Champe, M.; Munemitsu, S.; Ullrich, A. ); Maes, P.; Tartar, A. Institut Pasteur de Lille )

    1990-10-01

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

  10. Cloning of the cDNA and gene for a human D sub 2 dopamine receptor

    SciTech Connect

    Grady, D.K.; Makam, H.; Stofko, R.E.; Bunzow, J.R.; Civelli, O. ); Marchionni, M.A.; Alfano, M.; Frothingham, L.; Fischer, J.B.; Burke-Howie, K.J.; Server, A.C. )

    1989-12-01

    A clone encoding a human D{sub 2} dopamine receptor was isolated from a pituitary cDNA library and sequenced. The deduced protein sequence is 96% identical with that of the cloned rat receptor with one major difference: the human receptor contains an additional 29 amino acids in its putative third cytoplasmic loop. Southern blotting demonstrated the presence of only one human D{sub 2} receptor gene. Two overlapping phage containing the gene were isolated and characterized. DNA sequence analysis of these clones showed that the coding sequence is interrupted by six introns and that the additional amino acids present in the human pituitary receptor are encoded by a single exon of 87 base pairs. The involvement of this sequence in alternative splicing and its biological significance are discussed.

  11. Isolation of cDNA from Jacaratia mexicana encoding a mexicain-like cysteine protease gene.

    PubMed

    Ramos-Martínez, Erick M; Herrera-Ramírez, Alejandra C; Badillo-Corona, Jesús Agustín; Garibay-Orijel, Claudio; González-Rábade, Nuria; Oliver-Salvador, María Del Carmen

    2012-07-01

    Cysteine proteases (CPs) from the C1 family, which are similar to papain, can be found in animals and plants, as well as some viruses and prokaryotes. These enzymes have diverse physiological functions and are thus very attractive for science and industry. Jacaratia mexicana, a member of the Caricaceae plant family, contains several CPs, the principal being mexicain, found to favorably compete against papain for many industrial applications due to its high stability and specific activity. In this study, leaves of J. mexicana were used to isolate a CP-coding gene, similar to those that code for mexicain and chymomexicain. By using rapid amplification of cDNA ends (RACE) as well as oligonucleotide design from papain-like conserved amino acids (aa), a sequence of 1404 bp consisting of a 5' terminal untranslated region (UTR) of 153 bp, a 3' terminal UTR of 131 bp, with a polyadenylation (poly(A)) signal sequence and a poly(A) tail, and an open reading frame (ORF) of 1046 bp, was obtained by overlapping three partial sequences. Two full-length cDNA sequences that encode for mexicain-like proteases were cloned from mRNA (JmCP4 and JmCP5). JmCP4 is predicted to have an ORF of 1044 bp, which codifies for polypeptides that have a 26 aa signal peptide region, a 108 aa propeptide region and a mature enzyme of 214 aa. A 969 bp fragment (JmCP5) encodes for a partial sequence of a CP gene, without the signal peptide region but with a full-length propeptide region. The sequence analysis showed that this protease presented a high similarity to other plant CPs from J. mexicana, Vasconcellea cundinamarcensis, Vasconcellea stipulata, and Carica papaya, among others, mainly at the conserved catalytic site. Obtaining the sequence of this CP gene from J. mexicana provides an alternative for production in a standard system and could be an initial step towards the commercialization of this enzyme. PMID:22543019

  12. Isolation and characterization of cDNA clones for human apolipoprotein A-I.

    PubMed Central

    Breslow, J L; Ross, D; McPherson, J; Williams, H; Kurnit, D; Nussbaum, A L; Karathanasis, S K; Zannis, V I

    1982-01-01

    We have isolated cDNA clones encoding human apolipoprotein (apo) A-I. Twenty putative apo A-I cDNA clones were selected by screening 10,000 clones of an adult human liver cDNA library with an oligonucleotide probe. The probe was a mixture of synthetic 14-base-long DNA oligomers constructed to correspond to the codons for apo A-I amino acids 105-109. Four of these clones were examined further and showed 600- to 800-base-pair (bp) inserts. Preliminary restriction mapping and partial DNA sequence analysis indicated that the shorter inserts were a subset of the longer DNA inserts. DNA sequence analysis of the clone with an insert of approximately equal to 600 bp, designated pAI-113, revealed that it contained a DNA sequence corresponding to apo A-I amino acids 94-243. The DNA base sequence of this clone also contained a standard termination codon, polyadenylylation signal, and poly(A) tail. Partial DNA sequence of a second clone that contained an 800-bp insert, designated pAI-107, showed that it corresponded to apo A-I amino acids 18-243 and also included the 3' untranslated region. Isolation of these cDNA clones will facilitate molecular analyses of apolipoproteins in normal and disease states. PMID:6294659

  13. 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.

  14. Molecular Cloning and Characterization of cDNA Encoding a Putative Stress-Induced Heat-Shock Protein from Camelus dromedarius

    PubMed Central

    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. PMID:21845074

  15. 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.

  16. Isolation and characterization of cDNA clones for human erythrocyte. beta. -spectrin

    SciTech Connect

    Prchal, J.T.; Morley, B.J.; Yoon, S.H.; Coetzer, T.L.; Palek, J.; Conboy, J.G.; Kan, Y.W.

    1987-11-01

    Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical ..cap alpha.. (M/sub r/ 240,000) and ..beta.. (M/sub r/ 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. The authors report here the isolation and characterization of a human erythroid-specific ..beta..-spectrin cDNA clone that encodes parts of the ..beta..-9 through ..beta..-12 repeat segments. This cDNA was used as a hybridization probe to assign the ..beta..-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte ..beta..-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the ..beta..-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities.

  17. Characterization of cDNA clones for the human c-yes gene.

    PubMed Central

    Sukegawa, J; Semba, K; Yamanashi, Y; Nishizawa, M; Miyajima, N; Yamamoto, T; Toyoshima, K

    1987-01-01

    Three c-yes cDNA clones were obtained from poly(A)+ RNA of human embryo fibroblasts. Sequence analysis of the clones showed that they contained inserts corresponding to nearly full-length human c-yes mRNA, which could encode a polypeptide of 543 amino acids with a relative molecular weight (Mr) of 60,801. The predicted amino acid sequence of the protein has no apparent membrane-spanning region or suspected ligand binding domain and closely resembles pp60c-src. Comparison of the sequences of c-yes and v-yes revealed that the v-yes gene contains most of the c-yes coding sequence except the region encoding its extreme carboxyl terminus. The region missing from the v-yes protein is the part that is highly conserved in cellular gene products of the protein-tyrosine kinase family. PMID:2436037

  18. 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. PMID:21334357

  19. Characterisation and expression of a cDNA encoding the 80-kDa large subunit of Schistosoma japonicum calpain.

    PubMed

    Scott, J C; McManus, D P

    2000-01-01

    We describe the cloning of a full length calpain-encoding cDNA constructed from two truncated cDNAs isolated from a cDNA library prepared with mRNA isolated from adult worms of the Philippine strain of Schistosoma japonicum. The cDNA sequence is 2.456 kb in length and predicts a protein of 758 residues with a molecular mass of 86.61 kDa and an isoelectric point of 5.34. Probes spanning the entire calpain cDNA hybridised to multiple bands in genomic DNAs of Philippine (SjP) and Chinese (SjC) S. japonicum, with some restriction fragment length polymorphisms evident between the two strains. Northern hybridisation analysis indicated that the cDNA codes for a single RNA transcript between 2.6 and 3.6 kb in size in the SjP and SjC genomes. After subcloning in the QIA express vectors pQE-31 and pQE-40 and subsequent expression, the recombinant protein was purified and shown to bind calcium. The availability of recombinant S. japonicum calpain will allow its future evaluation as a vaccine candidate, especially in light of recent work with the S. mansoni homologue which has provided evidence that this protein may be a target of protective immunity. PMID:11227760

  20. Identification of a proglucagon cDNA from Rana tigrina rugulosa that encodes two GLP-1s and that is alternatively spliced in a tissue-specific manner.

    PubMed

    Yeung, C M; Chow, B K

    2001-11-01

    Glucagon plays a pivotal role in the regulation of metabolism. A glucagon receptor has been previously characterized in the frog, Rana tigrina rugulosa, and the frog and human glucagon receptors have been shown to possess similar binding affinities toward human glucagon. To study the structural evolution of glucagon peptide and its receptor in vertebrates, in the current study, a proglucagon cDNA from the same frog species was cloned. Interestingly, in contrast to the mammalian proglucagons that contain only one GLP-1 peptide, the frog proglucagon cDNA encodes two GLP-1 peptides (GLP-1A and GLP-1B) in addition to a glucagon peptide and a glucagon-like peptide 2 (GLP-2). By reverse transcriptase-PCR (RT-PCR) analysis, the proglucagon gene expression was widely detected in the brain, colon, small intestine, liver, lung, and pancreas, suggesting that the proglucagon-derived peptides have diverse functions in frogs. Moreover, tissue-specific alternative mRNA splicing was observed in the brain, colon, and pancreas. In these tissues, proglucagon transcripts with a 135 bp in frame deletion encoding GLP-1A were found. This splicing event in R. tigrina rugulosa is novel because it deletes a GLP-1 encoding sequence instead of the GLP-2 observed in other vertebrates. These findings should enhance understanding of the proglucagon evolution, structure, and expression in vertebrates. PMID:11703080

  1. Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer's disease: Identification as the microtubule-associated protein tau

    SciTech Connect

    Goedert, M.; Wischik, C.M.; Crowther, R.A.; Walker, J.E.; Klug, A. )

    1988-06-01

    Screening of cDNA libraries prepared from the frontal cortex of an Alzheimer's disease patient and from fetal human brain has led to isolation of the cDNA for a core protein of the paired helical filament of Alzheimer's disease. The partial amino acid sequence of this core protein was used to design synthetic oligonucleotide probes. The cDNA encodes a protein of 352 amino acids that contains a characteristic amino acid repeat in its carboxyl-terminal half. This protein is highly homologous to the sequence of the mouse microtubule-associated protein tau and thus constitutes the human equivalent of mouse tau. RNA blot analysis indicates the presence of two major transcripts, 6 and 2 kilobases long, with a wide distribution in normal human brain. Tau protein mRNAs were found in normal amounts in the frontal cortex from patients with Alzheimer's disease. The proof that at least part of tau protein forms a component of the paired helical filament core opens the way to understanding the mode of formation of paired helical filaments and thus, ultimately, the pathogenesis of Alzheimer's disease.

  2. Isolation of a cDNA for HSF 2: Evidence for two heat shock factor genes in humans

    SciTech Connect

    Schuetz, T.J.; Gallo, G.J.; Sheldon, L.; Kingston, R.E. Harvard Medical School, Boston, MA ); Tempst, P. )

    1991-08-15

    The heat shock response is transcriptionally regulated by an evolutionarily conserved protein termed heat shock factor (HSF). The authors report the purification to homogeneity and the partial peptide sequence of HSF from HeLa cells. The peptide sequence was used to isolate a human cDNA with a predicted open reading frame that has homology to the DNA binding domains of both Saccharomyces cerevisiae and Drosophila HSFs. The cDNA directs the synthesis of a protein that binds to the heat shock element with specificity identical to HeLa HSF and stimulates transcription from a heat shock promoter. The expressed protein cross-reacts with anti-HSF antibodies. Surprisingly, however, this cDNA does not encode all of the peptides obtained from purified HeLa HSF. These peptides are encoded by a distinct human cDNA. HSF1. It therefore appears that there is a human heat shock factor gene family and that at least two separate but related HSF proteins regulate the stress response in humans.

  3. A mesocarp-and species-specific cDNA clone from oil palm encodes for sesquiterpene synthase.

    PubMed

    Shah; Cha

    2000-05-29

    The differential display method was used to isolate cDNAs corresponding to transcripts that accumulate during the period of lipid synthesis, 12-20 weeks after anthesis (WAA) in the mesocarp of two oil palms, Elaeis oleifera and Elaeis guineensis, Tenera. DNA-free total RNA from mesocarp and kernel of E. guineensis, Tenera and E. oleifera (15 WAA) were used to obtain differential gene expression patterns between these tissues from the two species. In this report, we describe the isolation and characterization of a specific cDNA clone, MO1 (434 bp) which was shown to be mesocarp-specific as well as species-specific for E. oleifera Sequencing of this fragment showed homology to the enzyme sesquiterpene synthase. Its longer cDNA clone, pMO1 (1072 bp), isolated from a 15-week E. oleifera mesocarp cDNA library confirmed that it encodes for sesquiterpene synthase. The complete sequence of 1976 bp was obtained using 5'RACE method. Northern hybridization showed that MO1 and pMO1 mRNA transcripts are highly expressed only in the mesocarp of E. oleifera from 5 to 20 WAA. No expression was detected in the kernel (12-17 WAA) and vegetative tissues of both species nor in the mesocarp of E. guineensis. This is the first communication to document on the isolation and characterisation of a mesocarp-and species-specific cDNA clone from oil palm. PMID:10729614

  4. 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

  5. Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family

    SciTech Connect

    Hanks, S.K.

    1987-01-01

    Mixed /sup 32/P-labeled oligonucleotide probes were used to screen a HeLa cDNA library for clones encoding amino acid contiguities whose conservation is characteristic of the protein-serine kinase family. Eighty thousand clones were screened, from which 19 were identified as showing strong hybridization to two distinct probes. Four clones were chosen for characterization by partial DNA sequence analysis and 3 of these were found to encode amino acid sequences typical of protein-serine kinases. One deduced amino acid sequence shares 72% identify with rabbit skeletal muscle phosphorylase kinase ..gamma..-subunit, while another is closely related to the yeast protein-serine kinases CDC2 in Schizosaccharomyces pombe and CDC28 in Saccharomyces cerevisiae. This screening approach should have applications in the identification of clones encoding previously unknown or poorly characterized members of other protein families.

  6. Characterization of the shrimp eyestalk cDNA encoding a novel fushi tarazu-factor 1 (FTZ-F1).

    PubMed

    Chan, S M; Chan, K M

    1999-07-01

    To study the role of ecdysone and the ecdysone inducible gene in the regulation of molting and development in crustaceans, we have cloned a cDNA encoding an orphan nuclear receptor family member from the eyestalk of the shrimp Metapenaeus ensis. The size of the cDNA is 4.3 kb with the longest open reading frame (ORF) encoding a protein of 545 amino acid residues. The deduced amino acid sequence of the shrimp cDNA consists of regions that are characteristic of those of the nuclear hormone receptors. It shows a high degree of amino acid sequence identity in the DNA binding domain, ligand binding domain and the FTZ box as compared to those of invertebrates and vertebrates. Unlike the insects Drosophila melanogaster and Bombyx mori, an AF2 transactivation domain was present in the shrimp FTZ-F1. Northern blot analysis using total RNA indicated that the FTZ-F1 mRNA could also be detected in the mature ovary. Northern blot analysis and RT-PCR analysis showed that the shrimp FTZ-F1 transcripts could be detected in the ovary, newly hatched nauplius, testis, eyestalk and epidermis of the adult shrimp. Although the cDNA clone was isolated from the eyestalk library, the shrimp FTZ-F1 appeared to express most abundantly in the mature oocytes. The presence of abundant FTZ-F1 specific maternal message in the late vitellogenic ovary and early nauplius indicates that it may be important for the early embryonic and larval development of the shrimp. Interestingly, shrimp FTZ-F1 can also be found in testis of the male shrimp. The presence of FTZ-F1 in other tissues such as epidermis suggests that it may also be involved in other physiological processes such as molting. PMID:10413106

  7. Molecular and Biochemical Analysis of Two cDNA Clones Encoding Dihydroflavonol-4-Reductase from Medicago truncatula1

    PubMed Central

    Xie, De-Yu; Jackson, Lisa A.; Cooper, John D.; Ferreira, Daneel; Paiva, Nancy L.

    2004-01-01

    Dihydroflavonol-4-reductase (DFR; EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Two DFR cDNA clones (MtDFR1 and MtDFR2) were isolated from the model legume Medicago truncatula cv Jemalong. Both clones were functionally expressed in Escherichia coli, confirming that both encode active DFR proteins that readily reduce taxifolin (dihydroquercetin) to leucocyanidin. M. truncatula leaf anthocyanins were shown to be cyanidin-glucoside derivatives, and the seed coat proanthocyanidins are known catechin and epicatechin derivatives, all biosynthesized from leucocyanidin. Despite high amino acid similarity (79% identical), the recombinant DFR proteins exhibited differing pH and temperature profiles and differing relative substrate preferences. Although no pelargonidin derivatives were identified in M. truncatula, MtDFR1 readily reduced dihydrokaempferol, consistent with the presence of an asparagine residue at a location known to determine substrate specificity in other DFRs, whereas MtDFR2 contained an aspartate residue at the same site and was only marginally active on dihydrokaempferol. Both recombinant DFR proteins very efficiently reduced 5-deoxydihydroflavonol substrates fustin and dihydrorobinetin, substances not previously reported as constituents of M. truncatula. Transcript accumulation for both genes was highest in young seeds and flowers, consistent with accumulation of condensed tannins and leucoanthocyanidins in these tissues. MtDFR1 transcript levels in developing leaves closely paralleled leaf anthocyanin accumulation. Overexpression of MtDFR1 in transgenic tobacco (Nicotiana tabacum) resulted in visible increases in anthocyanin accumulation in flowers, whereas MtDFR2 did not. The data reveal unexpected properties and differences in two DFR proteins from a single species. PMID:14976232

  8. 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...

  9. 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.

  10. Isolation and partial characterization of cDNA clone of human ceruloplasmin receptor.

    PubMed

    Sasina, L K; Tsymbalenko, N V; Platonova, N A; Puchkova, L V; Voronina, O V; Gyulikhandanova, N E; Gaitskhoki, V S

    2000-05-01

    An individual clone, presumably carrying a 3 bp fragment of ceruloplasmin receptor cDNA was isolated from the expression library of human placenta cDNA using polyclonal specific antibodies to ceruloplasmin receptors. EcoR1-hydrolysate of isolated DNA was cloned in a pTZ19 bacterial vector and sequenced in the forward and reverse direction. The comparison of the revealed sequence with known sequences of human genome revealed its high similarity to ceruloplasmin cDNA. PMID:10977961

  11. Molecular cloning and expression of the porcine trigeminal ganglion cDNA encoding a 5-ht(1F) receptor.

    PubMed

    Bhalla, Pankaj; Sharma, Hari S; Wurch, Thierry; Pauwels, Petrus J; Saxena, Pramod R

    2002-02-01

    Using a combination of reverse transcription polymerase chain reaction (RT-PCR) and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-hydroxytryptamine 1F (5-ht(1F)) receptor complementary DNA (cDNA) derived from porcine trigeminal ganglion. Sequence analysis revealed 1101 base pairs (bp) encoding an open reading frame of 366 amino acids showing a high similarity (>90%) with the 5-ht(1F) receptor sequences from other species, including human. The recombinant porcine 5-ht(1F) receptor was expressed in African green monkey kidney cell lines (COS-7 cells) and its ligand binding profile was determined using [3H]5-HT. The affinities of several agonists (LY334370 (5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate)>CP122638 (N-methyl-3 [pyrrolidin 2(R)-yl methyl]-1H-indol-5-ylmethyl sulphonamide)=naratriptan =5HT>eletriptan>sumatriptan>frovatriptan =avitriptan>dihydroergotamine>zolmitriptan>5-carboxamidotryptamine>rizatriptan>alniditan=donitriptan>L694247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine) and putative antagonists (methiothepin>GR127935 (N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2'-methyl 4'-(5-methyl-1,2,4-oxadiazol-3-yl) [1,1-biphenyl]-4-carboxamide hydrochloride)>ritanserin>SB224289 (2,3,6,7-tetrahydro-1'-methyl-5-[2'-methyl-4'(5-methyl-1,2,4-oxadiazol-3-yl) biphenyl-4-carbonyl] furo [2,3-f] indole-3-spiro-4'-piperidine hydrochloride)>BRL155572 ([1-(3-chlorophenyl)-4-[3,3-diphenyl (2-(S,R) hydroxypropanyl)piperazine] hydrochloride)>ketanserin=pindolol) correlated highly with those described for the recombinant human 5-ht(1F) receptor (Spearman correlation coefficient; r(s)=0.942). Nevertheless, as compared to the human homologue, some triptans (i.e. sumatriptan, zolmitriptan and rizatriptan) displayed a 10- to 15-fold lower affinity for the porcine 5-ht(1F) receptor. Using RT-PCR technique, the expression of porcine 5-ht(1F) receptor mRNA was observed in

  12. Cloning of a Locusta cDNA encoding a precursor peptide for two structurally related proteinase inhibitors.

    PubMed

    Kromer, E; Nakakura, N; Lagueux, M

    1994-03-01

    Two peptides of respectively 35 and 36 residues were recently isolated from Locusta migratoria and their full structural characteristics were established by Edman degradation and mass spectrometry. These peptides were subsequently shown to have a proteinase inhibiting activity. We report here the cloning and characterization of a cDNA encoding a 92-residue precursor with three distinct domains: (I) a typical signal peptide of 19 residues; (II) the peptide sequence of the 35-residue inhibitor separated by a Lys-Arg dipeptide cleavage site from (III) the peptide sequence of the 36-residue inhibitor. We show by Northern blot analysis that the gene encoding this precursor is mainly transcribed in the cells of the fat body. PMID:8019577

  13. Isolation and characterization of cDNA encoding Argonaute, a component of RNA silencing in shrimp (Penaeus monodon).

    PubMed

    Unajak, Sasimanas; Boonsaeng, Vichai; Jitrapakdee, Sarawut

    2006-10-01

    We have identified a cDNA clone that encodes a protein with high sequence homology to Argonaute proteins of mammals and Drosophila melanogaster. The cDNA of Penaeus monodon (Pm Ago) consisted of 3178 nucleotides encoding 939-amino acid residues with a calculated molecular weight of 104 kDa. The primary structure of Pm Ago showed the presence of two signature domains, PAZ and PIWI domains that exhibit highest homology to their counterparts in D. melanogaster. The inferred protein sequence of Pm Ago was 80.8% identical with D. melanogaster and 82.1% identical with Anopheles gambiae Ago proteins. Phylogenetic analysis of Pm Ago with other invertebrate and vertebrate Argonaute proteins suggested that Pm Ago belongs to the Ago1 subfamily that plays crucial roles in stem cell differentiation or RNA interference (RNAi). Semi-quantitative RT-PCR analysis showed that the gene is highly expressed in the lymphoid organ and moderately expressed in intestine, muscle, pleopods and hemocytes. The expression of Pm Ago1 mRNA was 2-3-fold increased during the early period of viral infection but declined rapidly at 30 hour post infection. By contrast, infection of shrimp by a bacterial pathogen, Vibrio harveyi did not induce a reduction of Pm Ago1 mRNA suggesting that its expression is associated with virus infection. PMID:16938476

  14. Molecular cloning and sequence analysis of the cDNA encoding rat liver cysteine sulfinate decarboxylase (CSD).

    PubMed

    Reymond, I; Sergeant, A; Tappaz, M

    1996-06-01

    The taurine biosynthesis enzyme, cysteine sulfinate decarboxylase (CSD), was purified to homogeneity from rat liver. Three CSD peptides generated by tryptic cleavage were isolated and partially sequenced. Two of them showed a marked homology with glutamate decarboxylase and their respective position on the CSD amino acid sequence was postulated accordingly. Using appropriate degenerated primers derived from these two peptides, a PCR amplified DNA fragment was generated from liver poly(A)+ mRNA, cloned and used as a probe to screen a rat liver cDNA library. Three cDNAs, length around 1800 bp, were isolated which all contained an open reading frame (ORF) encoding a 493 amino acid protein with a calculated molecular mass of 55.2 kDa close to the experimental values for CSD. The encoded protein contained the sequence of the three peptides isolated from homogenous liver CSD. Our data confirm and significantly extend those recently published (Kaisaki et al. (1995) Biochim. Biophys. Acta 1262, 79-82). Indeed, an additional base pair found 1371 bp downstream from the initiation codon led to a shift in the open reading frame which extended the carboxy-terminal end by 15 amino acid residues and altogether modified 36 amino acids. The validity of this correction is supported by the finding that the corrected reading frame encoded a peptide issued from CSD tryptic cleavage that was not encoded anywhere in the CSD sequence previously reported. PMID:8679699

  15. Expression cloning of genes encoding human peroxisomal proteins

    SciTech Connect

    Spathaky, J.M.; Tate, A.W.; Cox, T.M.

    1994-09-01

    Numerous metabolic disorders associated with diverse peroxisomal defects have been identified but their molecular characterization has been hampered by difficulties associated with the purification of proteins from this fragile organelle. We have utilized antibodies directed against the C-terminal tripeptide peroxisomal targeting signal to detect hitherto unknown peroxisomal proteins in tissue fractions and to isolate genes encoding peroxisonal proteins from human expression libraries. We immunized rabbits with a peptide conjugate encompassing the C-terminal nine amino acids of rat peroxisomal acyl CoA oxidase. Immunoprecipitation assays using radio-labelled peptide showed that the antibody specifically recognizes the terminal SKL motif as well as C-terminal SHL and SRL but not SHL at an internal position. Affinity-purified antibody was used to probe Western blots of crude and peroxisome-enriched monkey liver preparations and detected 8-10 proteins specifically in the peroxisome fractions. 100 positive clones were identified on screening a human liver cDNA expression library in {lambda}-gt11. Sequence analysis has confirmed the identity of cDNA clones for human acyl CoA oxidase and epoxide hydrolase. Four clones show no sequence identity and their putative role in the human peroxisome is being explored.

  16. 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.

  17. Identification of cDNA clones encoding HMG 2, a major protein of the mexican axolotl hydrocortisone-sensitive thymocytes.

    PubMed

    de Guerra, A; Guillet, F; Charlemagne, J; Fellah, J S

    1995-01-01

    We have identified and analyzed cDNA clones encoding a major 26 kDa protein of the HMG1-2 family which is abundant in the cytoplasm and nucleus of axolotl hydrocortisone-sensitive thymocytes. The axolotl HMG2 protein is very similar to proteins belonging to the HMG1-2 family, from teleost fish to mammals. All the molecular features of the HMG1-2 proteins are conserved, including the high proportion of basic and aromatic residues, and the characteristic acidic C-terminus tail. The 3'-untranslated region of the HMG2 axolotl cDNA is also similar to the avian and mammalian HMG2 3'-UT sequences, suggesting that some selective events have acted at the DNA level to conserve this region, which could be important in the differential expression of the HMG1 and HMG2 genes. The axolotl HMG2 protein contains the two well conserved HMG boxes which are thought to be the DNA-binding domains of the molecule. Axolotl thymocytes and spleen cells contain almost identical amounts of HMG2 mRNAs but HMG2 polypeptide is undetectable in spleen cells using anti-26 kDa antibodies. The reason for the accumulation of HMG1-2 molecules in vertebrate hydrocortisone-sensitive thymocytes is discussed, as well as their possible role in apoptosis. PMID:8654668

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

    PubMed

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

    2002-03-01

    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

  19. Sequence analysis and mapping of a novel human mitochondrial ATP synthase subunit 9 cDNA (ATP5G3)

    SciTech Connect

    Yan, W.L.; Gusella, J.F. |; Haines, J.L. |

    1994-11-15

    The authors describe the cloning, sequence analysis, and chromosomal mapping of a novel mitochondrial ATP synthase subunit 9 cDNA, P3. Subunit 9 transports protons across the inner mitochondrial membrane to the F{sub 1}-ATPase protruding on the matrix side, resulting in the generation of ATP. Sequence analysis of the P3 cDNA reveals only 80% identity with the human subunit 9 genes P1 and P2 in the DNA sequence encoding the mature protein identical to P1 and P2. The predicted sequence of the P3 leader peptide differs from the P1 and P2 leaders, but retains the {open_quotes}RFS{close_quotes} motif critical for mitochondrial import and maturation. The P3 gene (ATP5G3) maps to chromosome 2. 8 refs., 1 fig., 1 tab.

  20. Human cytoplasmic actin proteins are encoded by a multigene family

    SciTech Connect

    Engel, J.; Gunning, P.; Kedes, L.

    1982-06-01

    The authors characterized nine human actin genes that they isolated from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and ..cap alpha..-, ..beta..-, and ..gamma..-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria they show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken ..beta..-actin cDNA. They conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.

  1. Sequence and expression of an Eisenia-fetida-derived cDNA clone that encodes the 40-kDa fetidin antibacterial protein.

    PubMed

    Lassegues, M; Milochau, A; Doignon, F; Du Pasquier, L; Valembois, P

    1997-06-15

    Fetidins are 40-kDa and 45-kDa hemolytic and antibacterial glycoproteins present in the coelomic fluid of the earthworm Eisenia fetida andrei. By screening a cDNA library with a polyclonal antifetidin serum, we have cloned a cDNA that encoded the 40-kDa fetidin. The clone contains an insert of 1.44 kb encoding a protein of 34 kDa, which corresponds to the size of deglycosylated fetidins. The recombinant protein inhibits Bacillus megaterium growth. Restriction fragment polymorphisms were observed on Southern blots and correspond to a known protein polymorphism. The sequence of the cDNA contains a peroxidase signature and fetidins from earthworm coelomic fluid have peroxidase activity. The 40-kDa and 45-kDa fetidins therefore represent two related polymorphic defence factors in invertebrates. PMID:9219536

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

    PubMed

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

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A2, 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 its gene regulation. PMID:1907252

  3. 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.

  4. 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. PMID:18786582

  5. Cloning and analysis of human gastric mucin cDNA reveals two types of conserved cysteine-rich domains.

    PubMed Central

    Klomp, L W; Van Rens, L; Strous, G J

    1995-01-01

    Human gastric mucin was isolated by successive CsCl-gradient ultracentrifugation in the presence of guanidinium hydrochloride to prevent degradation of the polypeptide moieties of the molecules. The amino acid sequence of a tryptic fragment of this molecule was identical to that of a tryptic fragment of tracheobronchial mucin. An oligonucleotide based on this sequence hybridized specifically to human stomach mRNA and was subsequently used to screen a human stomach lambda ZAPII cDNA library. The largest of 10 positive clones encoded 850 amino acid residues, including the tryptic fragment, with high amounts of threonine, serine and proline residues. Interestingly, cysteine accounted for almost 8% of the amino acid residues. The 3' part of the sequence was very similar but not identical to the 3' region of human tracheobronchial cDNA. No tandem repeated sequences were present and the deduced polypeptide sequence contained two potential N-linked glycosylation sites. Four cysteine-rich clusters were detected, one of which was apparently homologous to the D-domains present in other mucins and in von Willebrand factor. The arrangement of the cysteines in three other cysteine-rich clusters was conserved in the human gastric mucin cDNA in a similar fashion as in two domains in the MUC2 gene product. The cysteine-rich domains were separated by short stretches of non-repetitive amino acid residues with a very high content of threonine and serine residues. These data suggest that the encoded polypeptide of this clone may be involved in disulphide-bond-mediated oligomerization of the mucin, and provide new insights into the molecular organization of mammalian apomucins. Images Figure 1 PMID:8948439

  6. The complete cDNA sequence of laminin alpha 4 and its relationship to the other human laminin alpha chains.

    PubMed

    Richards, A; Al-Imara, L; Pope, F M

    1996-06-15

    We previously localised the gene (LAMA4) encoding a novel laminin alpha 4 chain to chromosome 6q21. In this study, we describe the complete coding sequence and compare the protein with the other three known human laminin alpha chains. Although closely linked to LAMA2, the LAMA4 product most closely resembles laminin alpha 3, a constituent of laminin 5. Like laminin alpha 3A, the alpha 4 chain is a truncated version of the alpha 1 and alpha 2 chains, with a much reduced short arm. While the alpha 4 molecule is most similar to alpha 3, it shares some features of the C-terminal domains G4 and G5 in common with alpha 2. Unlike the LAMA3 gene, LAMA4 appears to encode only a single transcript, as determined by 5' rapid amplification of cDNA ends. The cDNA sequence encodes 1816 amino acids, which include a 24-residue signal peptide. The gene is expressed in skin, placenta, heart, lung, skeletal muscle, and pancreas. We have also shown that the mRNA can be readily reverse transcribed and amplified from cultured dermal fibroblasts. PMID:8706685

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

    SciTech Connect

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. )

    1991-01-01

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

  8. Isolation and Characterization of cDNA Encoding Three Dehydrins Expressed During Coffea canephora (Robusta) Grain Development

    PubMed Central

    HINNIGER, CÉCILE; CAILLET, VICTORIA; MICHOUX, FRANCK; BEN AMOR, MOHAMED; TANKSLEY, STEVE; LIN, CHENWEI; MCCARTHY, JAMES

    2006-01-01

    • Background and Aims Dehydrins, or group 2 late embryogenic abundant proteins (LEA), are hydrophilic Gly-rich proteins that are induced in vegetative tissues in response to dehydration, elevated salt, and low temperature, in addition to being expressed during the late stages of seed maturation. With the aim of characterizing and studying genes involved in osmotic stress tolerance in coffee, several full-length cDNA-encoding dehydrins (CcDH1, CcDH2 and CcDH3) and an LEA protein (CcLEA1) from Coffea canephora (robusta) were isolated and characterized. • Methods The protein sequences deduced from the full-length cDNA were analysed to classify each dehydrin/LEA gene product and RT–PCR was used to determine the expression pattern of all four genes during pericarp and grain development, and in several other tissues of C. arabica and C. canephora. Primer-assisted genome walking was used to isolate the promoter region of the grain specific dehydrin gene (CcDH2). • Key Results The CcDH1 and CcDH2 genes encode Y3SK2 dehydrins and the CcDH3 gene encodes an SK3 dehydrin. CcDH1 and CcDH2 are expressed during the final stages of arabica and robusta grain development, but only the CcDH1 transcripts are clearly detected in other tissues such as pericarp, leaves and flowers. CcDH3 transcripts are also found in developing arabica and robusta grain, in addition to being detected in pericarp, stem, leaves and flowers. CcLEA1 transcripts were only detected during a brief period of grain development. Finally, over 1 kb of genomic sequence potentially encoding the entire grain-specific promoter region of the CcDH2 gene was isolated and characterized. • Conclusions cDNA sequences for three dehydrins and one LEA protein have been obtained and the expression of the associated genes has been determined in various tissues of arabica and robusta coffees. Because induction of dehydrin gene expression is associated with osmotic stress in other plants, the dehydrin sequences

  9. 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).

  10. Isolation and characterisation of cDNA encoding a wheat heavy metal-associated isoprenylated protein involved in stress responses.

    PubMed

    Zhang, X; Feng, H; Feng, C; Xu, H; Huang, X; Wang, Q; Duan, X; Wang, X; Wei, G; Huang, L; Kang, Z

    2015-11-01

    In cells, metallochaperones are important proteins that safely transport metal ions. Heavy metal-associated isoprenylated plant proteins (HIPPs) are metallochaperones that contain a metal binding domain and a CaaX isoprenylation motif at the carboxy-terminal end. To investigate the roles of wheat heavy metal-associated isoprenylated plant protein (TaHIPP) genes in plant development and in stress responses, we isolated cDNA encoding the wheat TaHIPP1 gene, which contains a heavy metal-associated domain, nuclear localisation signals and an isoprenylation motif (CaaX motif). Quantitative real-time PCR analysis indicated that the TaHIPP1 gene was differentially expressed under biotic and abiotic stresses. Specifically, TaHIPP1 expression was up-regulated by ABA exposure or wounding. Additionally, TaHIPP1 over-expression in yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under Cu(2+) and high salinity stresses. The nuclear localisation of the protein was confirmed with confocal laser scanning microscopy of epidermal onion cells after particle bombardment with chimeric TaHIPP1-GFP constructs. In addition, TaHIPP1 was shown to enhance the susceptibility of wheat to Pst as determined by virus-induced gene silencing. These data indicate that TaHIPP1 is an important component in defence signalling pathways and may play a crucial role in the defence response of wheat to biotic and certain abiotic stresses. PMID:25951496

  11. Characterization of a cDNA clone encoding the calmodulin-binding domain of mouse brain calcineurin.

    PubMed Central

    Kincaid, R L; Nightingale, M S; Martin, B M

    1988-01-01

    A cDNA clone corresponding to a portion of the catalytic subunit of calmodulin (CaM)-dependent phosphoprotein phosphatase (calcineurin) was isolated from a murine brain library by expression vector immunoscreening. A beta-galactosidase fusion protein that reacted on Western blots with anti-calcineurin antibodies and biotinylated CaM was purified in preparative amounts using CaM-Sepharose affinity chromatography. Partial digestion of the hybrid protein with Staphylococcus aureus V-8 protease produced several immunoreactive peptides that appeared identical to fragments generated from authentic brain calcineurin. The 1111-base-pair (bp) EcoRI insert contained an open reading frame encoding a protein of 35 kDa followed by a 190-bp 3' noncoding region; seven peptides obtained by partial amino acid sequencing of the bovine brain enzyme were found in the deduced sequence. A domain approximately 12 kDa from the carboxyl terminus was deduced to be the CaM-binding site based on consensus structural features and a sequence of seven amino acids highly related to smooth muscle myosin light-chain kinase. Two regions with identity to protein phosphatases 1 and 2A were found in the amino half of the cloned sequence; however, the intervening sequence contained apparent insertions, suggesting splicing of subdomains. Thus, the structure of calcineurin is chimeric, consisting of conserved catalytic elements and a regulatory CaM-binding domain. Images PMID:2848250

  12. Isolation of a cDNA encoding 5T4 oncofetal trophoblast glycoprotein. An antigen associated with metastasis contains leucine-rich repeats.

    PubMed

    Myers, K A; Rahi-Saund, V; Davison, M D; Young, J A; Cheater, A J; Stern, P L

    1994-03-25

    The monoclonal antibody 5T4 defines a human oncotrophoblast antigen expressed by a variety of carcinomas but with a restricted pattern of expression in normal adult tissues. The 5T4 antigen has been isolated from term placenta as a 72-kDa glycoprotein consisting of a 42-kDa core protein with extensive N-linked glycosylation. A cDNA has been isolated from a human placental library using pools of oligonucleotides based on amino acid sequence obtained from purified 5T4 molecules. The predicted open reading frame encodes a protein of 420 amino acids with a molecular mass of 46 kDa and 8 potential N-glycosylation sites. There are N- and C-terminal hydrophobic segments corresponding to putative signal and membrane anchorage sequences, respectively. Northern analysis has demonstrated a major 2.5-kilobase mRNA present in cell lines serologically reactive with the monoclonal antibody 5T4. Comparison of the 5T4 protein sequence with current sequence data bases has identified the presence of leucine-rich repeats, which are found in a variety of proteins from yeast, insects, and mammals. The 5T4 antigen expression is strongly associated with metastasis in colorectal and gastric cancer, and, hence, the possible functions of the gene product and its relationship to tumor growth and progression are discussed. PMID:8132670

  13. Cloning, sequencing, and expression of cDNA for human. beta. -glucuronidase

    SciTech Connect

    Oshima, A.; Kyle, J.W.; Miller, R.D.; Hoffmann, J.W.; Powell, P.P.; Grubb, J.H.; Sly, W.S.; Tropak, M.; Guise, K.S.; Gravel, R.A.

    1987-02-01

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

  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. Cataloging of the genes expressed in human keratinocytes: analysis of 607 randomly isolated cDNA sequences.

    PubMed

    Konishi, K; Morishima, Y; Ueda, E; Kibe, Y; Nonomura, K; Yamanishi, K; Yasuno, H

    1994-07-29

    The partial nucleotide sequences of 607 cDNAs randomly isolated from a cDNA library of cultured human epidermal keratinocytes were determined by single pass sequencing. Homology search of the sequences to the non-redundant nucleotide databases revealed that 27% of the cDNAs matched registered human-or non-human genes encoding not only keratinocyte specific genes, but also a variety of functional proteins, the expression of which had not been identified in keratinocytes. Non-matching cDNAs covering 49% of the cDNAs were not homologous even to ESTs from other organs, suggesting that these cDNAs include novel genes expressed in the cells. The large scale sequencing of keratinocyte cDNAs provides a useful molecular source for research into biology and diseases of the skin. PMID:8048971

  16. Identification and expression analysis of a full-length cDNA encoding a Kandelia candel tonoplast intrinsic protein.

    PubMed

    Huang, Wei; Fang, Xiao-Dong; Lin, Qi-Fen; Li, Guan-Yi; Zhao, Wen-Ming

    2003-03-01

    Soil salinity is an important issue, as most crop plants are low in salt tolerance. Salt tolerance, a complex, multifactorial, and multigenic process, has been known to be a quantitative trait. The identification of the salt stress responsive genes or salt tolerance genes is essential for the breeding programs. Most recent efforts have been focused on the products of structural genes (transport proteins, ion channels, enzymes of solute synthesis) while little attention were paid to the regulatory aspects of these proteins. Since the first aquaporin gene from plants was cloned and functionally expressed in 1993, there has been a growing interest in the molecular biology of MIPs (membrane intrinsic proteins) and their bearing on the biophysics of water flow across plant membranes. In the last decades, studies on Mangroves, a special kind of wood plants, grow in high-salt and flooding conditions have been concentrated almost exclusively on their physiological and ecological characteristics. Kandelia candel, one of the dominant species of mangroves along the Chinese coast, lacks salt glands or salt hairs used for removal of excess salt in other mangroves. This makes K. candel a perfect model to study the molecular mechanism of salt tolerance in mangrove plants. Using cDNA RDA, a cDNA-specific modification of genomic representational difference analysis, a series of salt responsive genes of Kandelia candel were cloned. Among these gene fragments, a 183 bp fragment (termed as SRGKC1) encoding a tonoplast intrinsic protein (TIP) in Kandelia candel (KCTIP1) was identified. Based on the sequence of SRGKC1, two gene specific primers were designed, and the 3' and 5' end of the KCTIP1 gene were obtained using the SMART RACE cDNA Amplification Kit. RACE products were purified from low-melting agarose, and sequenced directly with GSPs as the sequencing primers. A 500-bp fragment corresponding to the 3'end of this gene was obtained using the GSP1 primer, and a 690 bp fragment

  17. 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.

  18. Cloning and sequence analysis of cDNA for human cathepsin D.

    PubMed Central

    Faust, P L; Kornfeld, S; Chirgwin, J M

    1985-01-01

    An 1110-base-pair cDNA clone for human cathepsin D was obtained by screening a lambda gt10 human hepatoma G2 cDNA library with a human renin exon 3 genomic fragment. Poly(A)+ RNA blot analysis with this cathepsin D clone demonstrated a message length of about 2.2 kilobases. The partial clone was used to screen a size-selected human kidney cDNA library, from which two cathepsin D recombinant plasmids with inserts of about 2200 and 2150 base pairs were obtained. The nucleotide sequences of these clones and of the lambda gt10 clone were determined. The amino acid sequence predicted from the cDNA sequence shows that human cathepsin D consists of 412 amino acids with 20 and 44 amino acids in a pre- and a prosegment, respectively. The mature protein region shows 87% amino acid identity with porcine cathepsin D but differs in having nine additional amino acids. Two of these are at the COOH terminus; the other seven are positioned between the previously determined junction for the light and heavy chains of porcine cathepsin D. A high degree of sequence homology was observed between human cathepsin D and other aspartyl proteases, suggesting a conservation of three-dimensional structure in this family of proteins. Images PMID:3927292

  19. Recovery of avian metapneumovirus subgroup C from cDNA: cross-recognition of avian and human metapneumovirus support proteins.

    PubMed

    Govindarajan, Dhanasekaran; Buchholz, Ursula J; Samal, Siba K

    2006-06-01

    Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with "swollen head syndrome" in chickens, contributing to significant economic losses for the U.S. poultry industry. With a long-term goal of developing a better vaccine for controlling AMPV in the United States, we established a reverse genetics system to produce infectious AMPV of subgroup C entirely from cDNA. A cDNA clone encoding the entire 14,150-nucleotide genome of AMPV subgroup C strain Colorado (AMPV/CO) was generated by assembling five cDNA fragments between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a transcription plasmid, pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1, and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. Characterization of the recombinant AMPV/CO showed that its growth properties in tissue culture were similar to those of the parental virus. The potential of AMPV/CO to serve as a viral vector was also assessed by generating another recombinant virus, rAMPV/CO-GFP, that expressed the enhanced green fluorescent protein (GFP) as a foreign protein. Interestingly, GFP-expressing AMPV and GFP-expressing human metapneumovirus (HMPV) could be recovered using the support plasmids of either virus, denoting that the genome promoters are conserved between the two metapneumoviruses and can be cross-recognized by the polymerase complex proteins of either virus. These results indicate a close functional relationship between AMPV/CO and HMPV. PMID:16731918

  20. Sequence of human hexokinase III cDNA and assignment of the human hexokinase III gene (HK3) to chromosome band 5q35.2 by fluorescence in situ hybridization

    SciTech Connect

    Furuta, Hiroto; Le Beau, M.M.; Fernald, A.A.

    1996-08-15

    Complementary DNA clones encoding human hexokinase III were isolated from a liver cDNA library. There was 84.7% identity between the amino acid sequences of human and rat hexokinase III. RNA blotting showed the presence of hexokinase III mRNA in liver and lung. Fluorescence in situ hybridization localized the human hexokinase III gene (HK3) to chromosome 5, band q35.2. 11 refs., 3 figs.

  1. cDNA cloning, characterization and expression analysis of DTX2, a human WWE and RING-finger gene, in human embryos.

    PubMed

    Yi, Zhengfang; Yi, Tingfang; Wu, Zirong

    2006-06-01

    The WWE domain is a conserved globular domain in several proteins and predicted to mediate specificprotein-protein interactions in ubiquitin and ADP ribose conjugation systems. The RING domain is a conserved and specialized zinc-finger motif with 40-60 residues binding to two zinc atoms, which is also probably involved in mediating protein-protein interactions. Here, from human fetal heart cDNA library, we identified DTX2, a human WWE & RING-finger gene, with high similarity with its homologues. Evaluation of full-length cDNA obtained by RACE indicated it encodes a protein composed of two WWE domains and a RING-finger region. The DTX2 gene located in human chromosome 7q11.23 spanning approximately 44.3 kb on the genome and the deduced protein is 622 amino acids. Northern analysis revealed DTX2 was expressed in the 18-week, 22.5-week human embryo hearts and adult hearts, especially with high levels in the 18-week and adult hearts. Taken together, these results indicate that DTX2 is a gene encoding a WWE-RING-finger protein and involved in regulating heart development and heart functions. PMID:17286044

  2. Fibulin-2 (FBLN2): Human cDNA sequence, mRNA expression, and mapping of the gene on human and mouse chromosomes

    SciTech Connect

    Zhang, R.Z.; Pan, T.C.; Zhang, Z.Y.

    1994-07-15

    Fibulin-2 is a new extracellular matrix protein recently identified by characterizing mouse cDNA clones. Fibulin-2 mRNA is prominently expressed in mouse heart tissue and is present in low amounts in other tissues. In this study, the authors isolated and sequenced a 4.1-kb human fibulin-2 cDNA, which encoded a mature protein of 1157 amino acids preceded by a 27-residue signal sequence. The predicted polypeptide contains three consecutive anaphylatoxin-related segments (domain I) in its central region followed by 10 EGF-like repeats (domain II), 9 of which have a consensus sequence for calcium binding. The 408-residue N-terminal region consists of two separate subdomains, a cysteine-rich segment of 150 residues (Na subdomain) and a cysteine-free segment with a stretch of acidic amino acids (Nb subdomain). The 115-residue C-terminal segment (domain III) is similar to the C variant of fibulin-1. The amino acid sequences of the human and mouse fibulin-2 share {approximately}90% identity in domains Na, I, II, and III but only 62% identity in domain Nb. The human cDNA lacks an EGF-like repeat, which is alternatively spliced in the mouse cDNA clones, and a potential cell-binding Arg-Gly-Asp sequence found in the Nb domain of the mouse counterpart. Northern blot analysis of mRNA from various human tissues reveals an abundant 4.5-kb transcript in heart, placenta, and ovary tissue. The expression pattern differs from that of fibulin-1. The fibulin-2 gene was localized by in situ hybridization to the p24-p25 region of human chromosome 3 and to the band D-E of mouse chromosome 6. 27 refs., 5 figs.

  3. Selection and sequence analysis of a cDNA clone encoding a known chorion protein of the A family.

    PubMed Central

    Tsitilou, S G; Regier, J C; Kafatos, F C

    1980-01-01

    Using as criteria the size, abundance and developmental specificity of hybridizing mRNA sequences, we have selected from our chorion cDNA library a clone corresponding to a specific chorion protein, A4--cl. Comparison between the clone sequence and the largely known sequence of A4--cl validates the use of the cDNA library for sequence analysis of the chorion multigene families. The two major chorion protein families, A and B, share certain structural similarities. Images PMID:7433133

  4. Cloning and expression of small cDNA fragment encoding strong antiviral peptide from Celosia cristata in Escherichia coli.

    PubMed

    Gholizadeh, A; Kohnehrouz, B Baghban; Santha, I M; Lodha, M L; Kapoor, H C

    2005-09-01

    A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714. PMID:16266271

  5. Sequence Characterization of cDNA Sequence of Encoding of an Antimicrobial Peptide With No Disulfide Bridge from the Iranian Mesobuthus Eupeus Venomous Glands

    PubMed Central

    Farajzadeh-Sheikh, Ahmad; Jolodar, Abbas; Ghaemmaghami, Shamsedin

    2013-01-01

    Background Scorpion venom glands produce some antimicrobial peptides (AMP) that can rapidly kill a broad range of microbes and have additional activities that impact on the quality and effectiveness of innate responses and inflammation. Objectives In this study, we reported the identification of a cDNA sequence encoding cysteine-free antimicrobial peptides isolated from venomous glands of this species. Materials and Methods Total RNA was extracted from the Iranian mesobuthus eupeus venom glands, and cDNA was synthesized by using the modified oligo (dT). The cDNA was used as the template for applying Semi-nested RT- PCR technique. PCR Products were used for direct nucleotide sequencing and the results were compared with Gen Bank database. Results A 213 BP cDNA fragment encoding the entire coding region of an antimicrobial toxin from the Iranian scorpion M. Eupeus venom glands were isolated. The full-length sequence of the coding region was 210 BP contained an open reading frame of 70 amino with a predicted molecular mass of 7970.48 Da and theoretical Pi of 9.10. The open reading frame consists of 210 BP encoding a precursor of 70 amino acid residues, including a signal peptide of 23 residues a propertied of 7 residues, and a mature peptide of 34 residues with no disulfide bridge. The peptide has detectable sequence identity to the Lesser Asian mesobuthus eupeus MeVAMP-2 (98%), MeVAMP-9 (60%) and several previously described AMPs from other scorpion venoms including mesobuthus martensii (94%) and buthus occitanus Israelis (82%). Conclusions The secondary structure of the peptide mainly consisted of α-helical structure which was generally conserved by previously reported scorpion counterparts. The phylogenetic analysis showed that the Iranian MeAMP-like toxin was similar but not identical with that of venom antimicrobial peptides from lesser Asian scorpion mesobuthus eupeus. PMID:23486842

  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. 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...

  8. 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.

  9. 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.

  10. Molecular cloning of a cDNA for human {triangle}{sup 1}-pyrroline-5-carboxylate (P5C) dehydrogenase, the gene defective in type 2 hyperprolinemia

    SciTech Connect

    Hu, C.A.; Lin, W.; Valle, D.

    1994-09-01

    P5C dehydrogenase (EC 1.5.1.12) is a mitochondrial matrix NAD(P) dependent enzyme catalyzing the conversion of P5C, derived from either proline or ornithine, to glutamate. This reaction is an important component in the pathway interconnecting the urea cycle with the tricarboxylic acid cycle. Deficiency of P5C dehydrogenase causes type 2 hyperprolinemia (HPII), an autosomal recessive disorder characterized by seizures, hyperprolinemia and accumulation of P5C. To investigate the molecular basis of HPII and the pathophysiology of gyrate atrophy, a disorder of ornithine metabolism, we have cloned a cDNA for P5C dehydrogenase. Utilizing published sequences of peptides from purified human P5C dehydrogenase and the nucleotide sequence of yeast P5C dehydrogenase, we designed degenerate PCR primers to amplify cDNAs from a HepG2 cDNA library. We identified an amplified fragment of the correct size that encoded one of the many peptides and used it to clone near full length clones of the corresponding cDNA. The longest is 1.8 kb with a 1,485 bp ORF encoding a protein corresponding to the C terminal 495 residues of yeast P5C dehydrogenase. The predicted amino acid sequence of this clone has 100% identity to published sequence of human P5C dehydrogenase peptides and 42% identity with the corresponding sequence of the yeast enzyme. This cDNA detects a 2.3 kb transcript in Northern blots of fibroblast RNA. We conclude we have cloned a near full length cDNA for human P5C dehydrogenase. Studies investigating the molecular basis of HPII are in progress.

  11. Human antisera detect a Plasmodium falciparum genomic clone encoding a nonapeptide repeat.

    PubMed

    Koenen, M; Scherf, A; Mercereau, O; Langsley, G; Sibilli, L; Dubois, P; Pereira da Silva, L; Müller-Hill, B

    Plasmodium falciparum causes malaria infections in its human host. Its wide distribution in tropical countries is a major world health problem. Before a vaccine can be produced, the identification and characterization of parasite antigens is necessary. This can be achieved by the cloning and subsequent analysis of genes coding for parasite antigens. Recently established cDNA banks allow the expression of cDNA derived from the simian parasite Plasmodium knowlesi and P. falciparum in Escherichia coli. Recombinants encoding parasite antigens have been identified by immunodetection in both banks. Two of them contain repetitive units of 11 (ref. 7) or 12 (ref. 5) amino acids. We describe here the construction of an expression bank made directly from randomly generated fragments of P. falciparum genomic DNA. We detect several clones which react strongly with human African immune sera. One clone expresses an antigenic determinant composed of occasionally degenerated repeats of a peptide nonamer. PMID:6090935

  12. Expression cloning in yeast of a cDNA encoding a broad specificity amino acid permease from Arabidopsis thaliana.

    PubMed Central

    Frommer, W B; Hummel, S; Riesmeier, J W

    1993-01-01

    To study amino acid transport in plants at the molecular level, we have isolated an amino acid permease cDNA from Arabidopsis thaliana by complementation of a yeast mutant defective in proline uptake with a cDNA. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows no significant homologies to other known transporters. Expression of the cDNA enables the yeast mutant to take up L-[14C]proline. Competition studies argue for a broad but stereospecific substrate recognition by the permease, which resembles neutral or general amino acid transport systems from Chlorella and higher plants. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism. Images Fig. 1 PMID:8327465

  13. 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.

  14. HUGE: a database for human large proteins identified in the Kazusa cDNA sequencing project.

    PubMed

    Kikuno, R; Nagase, T; Suyama, M; Waki, M; Hirosawa, M; Ohara, O

    2000-01-01

    HUGE is a database for human large proteins newly identified in the Kazusa cDNA project, the aim of which is to predict the primary structure of proteins from the sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are the current targets of the project. HUGE contains >1100 cDNA sequences and detailed information obtained through analysis of the sequences of cDNAs and the predicted proteins. Besides an increase in the number of cDNA entries, the amount of experimental data for expression profiling has been largely increased and data on chromosomal locations have been newly added. All of the protein-coding regions were examined by GeneMark analysis, and the results of a motif/domain search of each predicted protein sequence against the Pfam database have been newly added. HUGE is available through the WWW at http://www.kazusa.or.jp/huge PMID:10592264

  15. Isolation and characterization of a cDNA encoding granule-bound starch synthase in cassava (Manihot esculenta Crantz) and its antisense expression in potato.

    PubMed

    Salehuzzaman, S N; Jacobsen, E; Visser, R G

    1993-12-01

    A tuber-specific cDNA library of cassava (Manihot esculenta Crantz) was constructed and a full-length cDNA for granule-bound starch synthase (GBSS, also known as waxy protein), the enzyme responsible for the synthesis of amylose in reserve starch, was cloned. Sequencing of the cloned cDNA showed that it has 74% identity with potato GBSS and 60-72% identity with GBSS from other plant species. The cDNA encodes a 608 amino acid protein of which 78 amino acids form a chloroplast/amyloplast transit peptide of 8.37 kDa. The mature protein has a predicted molecular mass of 58.61 kDa (530 amino acids). Comparison of the GBSS proteins of various plant species and glycogen synthase of bacteria showed extensive identity among the mature form of plant GBSS proteins, in which the monocots and dicots form two separate branches in the evolutionary tree. From analysis of the genomic DNA of allotetraploid cassava, it is shown that GBSS is a low-copy-number gene. GBSS transcript is synthesized in a number of different organs, but most abundantly in tubers. Potato plants were transformed with the cassava GBSS cDNA in antisense orientation fused between the potato GBSS promoter and the nopaline synthase terminator. The expression of the endogenous GBSS gene in these transgenic potato plants was partially or completely inhibited. Complete inhibition of GBSS activity by the cassava antisense gene resulted in absence of GBSS protein and amylose giving rise to almost complete amylose-free potato starch. This shows that also heterologous genes can be used to achieve antisense effects in other plant species. PMID:8260633

  16. Expression of a full-length cDNA for the human MDR1 gene confers resistance to colchicine, doxorubicin, and vinblastine

    SciTech Connect

    Ueda, K.; Cardarelli, C.; Gottesman, M.M.; Pastan, I.

    1987-05-01

    Intrinsic and acquired multidrug resistance (MDR) is an important problem in cancer therapy. MDR in human KB carcinoma cells selected for resistance to colchicine, vinblastine, or doxorubicin (former generic name adriamycin) is associated with overexpression of the MDR1 gene, which encodes P-glycoprotein. The authors previously have isolated an overlapping set of cDNA clones for the human MDR1 gene from multidrug-resistant KB cells. Here they report the construction of a full-length cDNA for the human MDR1 gene and show that this reconstructed cDNA, when inserted into a retroviral expression vector containing the long terminal repeats of Moloney leukemia virus or Harvey sarcoma virus, functions in mouse NIH 3T3 and human KB cells to confer the complete multidrug-resistance phenotype. These results suggest that the human MDR1 gene may be used as a positive selectable marker to introduce genes into human cells and to transform human cells to multidrug resistance without introducing nonhuman antigens.

  17. Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression.

    PubMed

    Phiriyangkul, Pharima; Utarabhand, Prapaporn

    2006-04-01

    In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp. PMID:16432892

  18. Genomic organization of the human NSP gene, prototype of a novel gene family encoding reticulons

    SciTech Connect

    Roebroek, A.J.M.; Ayoubi, T.A.Y.; Velde, H.J.K. van de; Schoenmakers, E.F.P.M.; Pauli, I.G.L.; Van De Ven, W.J.M.

    1996-03-01

    Recently, cDNA cloning and expression of three mRNA variants of the human NSP gene were described. This neuroendocrine-specific gene encodes three NSP protein isoforms with unique amino-terminal parts, but common carboxy-terminal parts. The proteins, with yet unknown function, are associated with the endoplasmic reticulum and therefore are named NSP reticulons. Potentially, these proteins are neuroendocrine markers of a novel category in human lung cancer diagnosis. Here, the genomic organization of this gene was studied by analysis of genomic clones isolated from lambda phage and YAC libraries. The NSP exons were found to be dispersed over a genomic region of about 275 kb. The present elucidation of the genomic organization of the NSP gene explains the generation of NSP mRNA variants encoding NSP protein isoforms. Multiple promoters rather than alternative splicing of internal exons seem to be involved in this diversity. Furthermore, comparison of NSP genomic and cDNA sequences with databank nucleotide sequences resulted in the discovery of other human members of this novel family of reticulons encoding genes. 25 refs., 4 figs.

  19. 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.

  20. 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.

  1. Identification and molecular characterization of the cDNA encoding Cucumis melo allergen, Cuc m 3, a plant pathogenesis-related protein

    PubMed Central

    Sankian, Mojtaba; Hajavi, Jafar; Moghadam, Malihe; Varasteh, Abdol-Reza

    2014-01-01

    Background: Melon (Cucumis melo) allergy is one of the most common food allergies, characterized by oral allergy syndrome. To date, two allergen molecules, Cuc m 1 and Cuc m 2, have been fully characterized in melon pulp, but there are few reports about the molecular characteristics of Cuc m 3. Methods: The Cuc m 3 cDNA has been characterized by rapid amplification of cDNA ends (RACE), which revealed a 456 base-pair (bp) fragment encoding a 151-amino acid polypeptide with a predicted molecular mass of 16.97 kDa, and identified 79 and 178 bp untranslated sequences at the 5′ and 3´ ends, respectively. Results: In silico analysis showed strong similarities between Cuc m 3 and other plant pathogen-related protein 1s from cucumber, grape, bell pepper, and tomato. Conclusion: Here we report the identification and characterization of the Cuc m 3 cDNA, which will be utilized for further analyses of structural and allergenic features of this allergen. PMID:26989726

  2. Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

    PubMed Central

    Knutzon, D S; Lardizabal, K D; Nelsen, J S; Bleibaum, J L; Davies, H M; Metz, J G

    1995-01-01

    Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme. PMID:8552723

  3. 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.

  4. In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.

    PubMed

    Wolf, D; Laver-Rudich, Z; Rotter, V

    1985-08-01

    The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons. PMID:3018534

  5. 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.

  6. Identification of a mouse brain cDNA that encodes a protein related to the Alzheimer disease-associated amyloid beta protein precursor.

    PubMed Central

    Wasco, W; Bupp, K; Magendantz, M; Gusella, J F; Tanzi, R E; Solomon, F

    1992-01-01

    We have isolated a cDNA from a mouse brain library that encodes a protein whose predicted amino acid sequence is 42% identical and 64% similar to that of the amyloid beta protein precursor (APP). This 653-amino acid protein, which we have termed the amyloid precursor-like protein (APLP), appears to be similar to APP in overall structure as well as amino acid sequence. The amino acid homologies are concentrated within three distinct regions of the two proteins where the identities are 47%, 54%, and 56%. The APLP cDNA hybridizes to two messages of approximately 2.4 and 1.6 kilobases that are present in mouse brain and neuroblastoma cells. Polyclonal antibodies raised against a peptide derived from the C terminus of APLP stain the cytoplasm in a pattern reminiscent of Golgi staining. In addition to APP, APLP also displays significant homology to the Drosophila APP-like protein APPL and a rat testes APP-like protein. These data indicate that the APP gene is a member of a strongly conserved gene family. Studies aimed at determining the functions of the proteins encoded by this gene family should provide valuable clues to their potential role in Alzheimer disease neuropathology. Images PMID:1279693

  7. 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.

  8. The human serotonin 5-HT{sub 2C} receptor: Complete cDNA, genomic structure, and alternatively spliced variant

    SciTech Connect

    Xie, Enzhong; Zhu, Lingyu; Zhao, Lingyun

    1996-08-01

    The complete 4775-nt cDNA encoding the human serotonin 5-HT{sub 2C} receptor (5-HT{sub 2C}R), a G-protein-coupled receptor, has been isolated. It contains a 1377-nt coding region flanked by a 728-nt 5{prime}-untranslated region and a 2670-nt 3{prime}-untranslated region. By using the cloned 5-HT{sub 2C}R cDNA probe, the complete human gene for this receptor has been isolated and shown to contain six exons and five introns spanning at least 230 kb of DNA. The coding region of the human 5-HT{sub 2C}R gene is interrupted by three introns, and the positions of the intron/exon junctions are conserved between the human and the rodent genes. In addition, an alternatively spliced 5-HT{sub 2C}R RNA that contains a 95-nt deletion in the region coding for the second intracellular loop and the fourth transmembrane domain of the receptor has been identified. This deletion leads to a frameshift and premature termination so that the short isoform RNA encodes a putative protein of 248 amino acids. The ratio for the short isoform over the 5-HT{sub 2C}R RNA was found to be higher in choroid plexus tumor than in normal brain tissue, suggesting the possibility of differential regulation of the 5-HT{sub 2C}R gene in different neural tissues or during tumorigenesis. Transcription of the human 5-HT{sub 2C}R gene was found to be initiated at multiple sites. No classical TATA-box sequence was found at the appropriate location, and the 5{prime}-flanking sequence contains many potential transcription factor-binding sites. A 7.3-kb 5{prime}-flanking 5-HT{sub 2C}R DNA directed the efficient expression of a luciferase reported gene in SK-N-SH and IMR32 neuroblastoma cells, indicating that is contains a functional promoter. 69 refs., 8 figs., 1 tab.

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

    SciTech Connect

    Imanishi, Tadashi; Itoh, Takeshi; Suzuki, Yutaka; O'Donovan, Claire; Fukuchi, Satoshi; Koyanagi, Kanako O.; Barrero, Roberto A.; Tamura, Takuro; Yamaguchi-Kabata, Yumi; Tanino, Motohiko; Yura, Kei; Miyazaki, Satoru; Ikeo, Kazuho; Homma, Keiichi; Kasprzyk, Arek; Nishikawa, Tetsuo; Hirakawa, Mika; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Ashurst, Jennifer; Jia, Libin; Nakao, Mitsuteru; Thomas, Michael A.; Mulder, Nicola; Karavidopoulou, Youla; Jin, Lihua; Kim, Sangsoo; Yasuda, Tomohiro; Lenhard, Boris; Eveno, Eric; Suzuki, Yoshiyuki; Yamasaki, Chisato; Takeda, Jun-ichi; Gough, Craig; Hilton, Phillip; Fujii, Yasuyuki; Sakai, Hiroaki; Tanaka, Susumu; Amid, Clara; Bellgard, Matthew; de Fatima Bonaldo, Maria; Bono Hidemasa; Bromberg, Susan K.; Brookes, Anthony J.; Bruford, Elspeth; Carninci Piero; Chelala, Claude; Couillault, Christine; de Souza, Sandro J.; Debily, Marie-Anne; Devignes, Marie-Dominique; Dubchak, Inna; Endo, Toshinori; Estreicher, Anne; Eyras, Eduardo; Fukami-Kobayashi, Kaoru; Gopinath, Gopal R.; Graudens, Esther; Hahn, Yoonsoo; Han, Michael; Han, Ze-Guang; Hanada, Kousuke; Hanaoka, Hideki; Harada, Erimi; Hashimoto, Katsuyuki; Hinz, Ursula; Hirai, Momoki; Hishiki, Teruyoshi; Hopkinson, Ian; Imbeaud, Sandrine; Inoko, Hidetoshi; Kanapin, Alexander; Kaneko, Yayoi; Kasukawa, Takeya; Kelso, Janet; Kersey, Paul; Kikuno Reiko; Kimura, Kouichi; Korn, Bernhard; Kuryshev, Vladimir; Makalowska, Izabela; Makino Takashi; Mano, Shuhei; Mariage-Samson, Regine; Mashima, Jun; Matsuda, Hideo; Mewes, Hans-Werner; Minoshima, Shinsei; Nagai, Keiichi; Nagasaki, Hideki; Nagata, Naoki; Nigam, Rajni; Ogasawara, Osamu; Ohara, Osamu; Ohtsubo, Masafumi; Okada, Norihiro; Okido, Toshihisa; Oota, Satoshi; Ota, Motonori; Ota, Toshio; Otsuki, Tetsuji; Piatier-Tonneau, Dominique; Poustka, Annemarie; Ren, Shuang-Xi; Saitou, Naruya; Sakai, Katsunaga; Sakamoto, Shigetaka; Sakate, Ryuichi; Schupp, Ingo; Servant, Florence; Sherry, Stephen; Shiba Rie; et al.

    2004-01-15

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

  10. Characterization of a Pinus pinaster cDNA encoding an auxin up-regulated putative peroxidase in roots.

    PubMed

    Charvet-Candela, V; Hitchin, S; Reddy, M S; Cournoyer, B; Marmeisse, R; Gay, G

    2002-03-01

    As part of a study to identify host plant genes regulated by fungal auxin during ectomycorrhiza formation, we differentially screened a cDNA library constructed from roots of auxin-treated Pinus pinaster (Ait.) Sol. seedlings. We identified three cDNAs up-regulated by auxin. Sequence analysis of one of these cDNAs, PpPrx75, revealed the presence of an open reading frame of 216 amino acids with the characteristic consensus sequences of plant peroxidases. The deduced amino acid sequence showed homology with Arabidopsis thaliana (L.) Heynh., Arachis hypogaea L. and Stylosanthes humilis HBK cationic peroxidases. Amino acid sequence identities in the conserved domains of plant peroxidases ranged from 60 to 100%. In PpPrx75, there are five cysteine residues and one histidine residue that are found at conserved positions among other peroxidases. A potential glycosylation site (NTS) is present in the deduced sequence. Phylogenetic analysis showed that PpPrx75 is closely related to two A. thaliana peroxidases. The PpPrx75 cDNA was induced by active auxins, ethylene, abscisic acid and quercetin, a flavonoid possibly involved in plant-microorganism interactions. Transcript accumulation was detected within 3 h following root induction by auxin, and the amount of mRNA increased over the following 24 h. The protein synthesis inhibitor cycloheximide did not inhibit indole-3-acetic acid-induced transcript accumulation, suggesting that PpPrx75 induction is a primary (direct) response to auxin. This cDNA can be used to study expression of an auxin-regulated peroxidase during ectomycorrhiza formation. PMID:11874719

  11. cDNA sequence and chromosomal localization of a novel human protein, RBQ-1 (RBBP6), that binds to the retinoblastoma gene product

    SciTech Connect

    Sakai, Yoshihisa; Saijo, Masafumi; Taya, Yoichi

    1995-11-01

    We have previously isolated cDNA of a novel protein (RBQ-1, HGMW-approved symbol RBBP6) that binds to the retinoblastoma gene product (pRB). Total nucleotide sequence of the cDNA has now been determined. It encoded a protein of 140 kDa that consists of 948 amino acids and contains multiple repeated sequences like SRS, YRE, and VPPP. The region used for pRB binding was identified on a small region near the C-terminus. We have mapped this gene to 16p11.2-p12 using polymerase chain reaction analysis on a human-hamster hybrid cell panel and chromosomal fluorescence in situ hybridization. 24 refs., 3 figs.

  12. Human proviral mRNAs down regulated in choriocarcinoma encode a zinc finger protein related to Krüppel.

    PubMed Central

    Kato, N; Shimotohno, K; VanLeeuwen, D; Cohen, M

    1990-01-01

    RNA transcripts of the HERV-R (ERV3) human provirus that are abundant in placenta but absent in choriocarcinoma contain nonproviral genomic sequences at their 3' ends. We report here the isolation of cDNA clones of these genomic sequences. The transcripts encode a Krüppel-related zinc finger protein consisting of a unique leader region and more than 12 28-amino-acid finger motifs. Images PMID:2115127

  13. Isolation and identification of the cDNA encoding the pheromone biosynthesis activating neuropeptide and additional neuropeptides in the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae).

    PubMed

    Choi, M Y; Tanaka, M; Kataoka, H; Boo, K S; Tatsuki, S

    1998-10-01

    The present study is concerned with cloning and characterizing Has-PBAN cDNA which is 756 nucleotides long, isolated from the brain and suboesophageal ganglion complex (Br-Sg) of Helicoverpa assulta adults. The 194-amino acid sequence deduced from this cDNA possessed the proteolytic endocleavage sites to generate multiple peptides. From the processing of the prepro-hormone, it can be predicted that the cDNA has a PBAN domain with 33 amino acids and four additional peptide domains: 24 amino acid-, 7 amino acid-, 18 amino acid- and 8 amino acid-long sequences, with FXPR (or K) L (X = G, T or S) amidated at their C-termini. The amino acid sequence of all five predicted peptides, including the PBAN, are identical to that of Helicoverpa zea (Raina, A.K., Jaffe, H., Kempe, T.G., Keim, P., Blacher, R.W., Fales, H.M., Riley, C.T., Klun, J.A., Ridgway, R.L., Hayes, D.K., 1989. Identification of a neuropeptide hormone that regulates sex pheromone production in female moths. Science 244, 796-798 and Ma, P.W.K., Knipple, D.C., Roelofs, W.L., 1994. Structural organization of the Helicoverpa zea gene encoding the precursor protein for pheromone biosynthesis-activating neuropeptide and other neuropeptides. Proc. Natl. Acad. Sci., U.S.A. 91, 506-510). A single mRNA species corresponding to the size of Has-PBAN cDNA was detected from the Br-Sg of 1-3-day old female and male adults, and their expression was also at a similar level. Pheromone production was induced upon injection of female or male Br-Sg extracts or synthetic PBAN into the haemocoel of decapitated 1-3-day old female adults during the photophase when they are not supposed to produce pheromone. From these results, H. assulta adult females seem to use their own PBAN for regulating sex pheromone biosynthesis. Functions of the four other peptides ending with FXPR (or K) L in the Has-PBAN cDNA and of the male PBAN remain to be elucidated. PMID:9807222

  14. 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

    2011-01-01

    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

  15. 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.

  16. 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.

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

    PubMed

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

    2007-08-01

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

  18. 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. PMID:25038276

  19. Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy.

    PubMed Central

    Shi, J; Dixon, R A; Gonzales, R A; Kjellbom, P; Bhattacharyya, M K

    1995-01-01

    An approach was developed for the isolation and characterization of soybean plasma membrane-associated proteins by immunoscreening of a cDNA expression library. An antiserum was raised against purified plasma membrane vesicles. In a differential screening of approximately 500,000 plaque-forming units with the anti-(plasma membrane) serum and DNA probes derived from highly abundant clones isolated in a preliminary screening, 261 clones were selected from approximately 1,200 antiserum-positive plaques. These clones were classified into 40 groups by hybridization analysis and 5'- and 3'-terminal sequencing. By searching nucleic acid and protein sequence data bases, 11 groups of cDNAs were identified, among which valosin-containing protein (VCP), clathrin heavy chain, phospholipase C, and S-adenosylmethionine:delta 24-sterol-C-methyltransferase have not to date been cloned from plants. The remaining 29 groups did not match any current data base entries and may, therefore, represent additional or yet uncharacterized genes. A full-length cDNA encoding the soybean VCP was sequenced. The high level of amino acid identity with vertebrate VCP and yeast CDC48 protein indicates that the soybean protein is a plant homolog of vertebrate VCP and yeast CDC48 protein. Images Fig. 1 Fig. 2 PMID:7753826

  20. From Plant Extract to a cDNA Encoding a Glucosyltransferase Candidate: Proteomics and Transcriptomics as Tools to Help Elucidate Saponin Biosynthesis in Centella asiatica.

    PubMed

    de Costa, Fernanda; Barber, Carla J S; Reed, Darwin W; Covello, Patrick S

    2016-01-01

    Centella asiatica (L.) Urban (Apiaceae), a small annual plant that grows in India, Sri Lanka, Malaysia, and other parts of Asia, is well-known as a medicinal herb with a long history of therapeutic uses. The bioactive compounds present in C. asiatica leaves include ursane-type triterpene sapogenins and saponins-asiatic acid, madecassic acid, asiaticoside, and madecassoside. Various bioactivities have been shown for these compounds, although most of the steps in the biosynthesis of triterpene saponins, including glycosylation, remain uncharacterized at the molecular level. This chapter describes an approach that integrates partial enzyme purification, proteomics methods, and transcriptomics, with the aim of reducing the number of cDNA candidates encoding for a glucosyltransferase involved in saponin biosynthesis and facilitating the elucidation of the pathway in this medicinal plant. PMID:26843164

  1. 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.

  2. 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.

  3. Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence

    SciTech Connect

    Millan, J.L.; Driscoll, C.E.; LeVan, K.M.; Goldberg, E.

    1987-08-01

    The sequence and structure of human testis-specific L-lactate dehydrogenase (LDHC/sub 4/, LDHX; (L)-lactate:NAD/sup +/ oxidoreductase, EC 1.1.1.27) has been derived from analysis of a complementary DNA (cDNA) clone comprising the complete protein coding region of the enzyme. From the deduced amino acid sequence, human LDHC/sub 4/ is as different from rodent LDHC/sub 4/ (73% homology) as it is from human LDHA/sub 4/ (76% homology) and porcine LDHB/sub 4/ (68% homology). Subunit homologies are consistent with the conclusion that the LDHC gene arose by at least two independent duplication events. Furthermore, the lower degree of homology between mouse and human LDHC/sub 4/ and the appearance of this isozyme late in evolution suggests a higher rate of mutation in the mammalian LDHC genes than in the LDHA and -B genes. Comparison of exposed amino acid residues of discrete anti-genic determinants of mouse and human LDHC/sub 4/ reveals significant differences. Knowledge of the human LDHC/sub 4/ sequence will help design human-specific peptides useful in the development of a contraceptive vaccine.

  4. 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.

  5. Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs

    SciTech Connect

    Kimura, S.; Kotani, T.; McBride, O.W.; Umeki, K.; Hirai, K.; Nakayama, T.; Ohtaki, S.

    1987-08-01

    Two forms of human thyroid peroxidase cDNAs were isolated from a lambdagt11 cDNA library, prepared from Graves disease thyroid tissue mRNA, by use of oligonucleotides. The longest complete cDNA, designated phTPO-1, has 3048 nucleotides and an open reading frame consisting of 933 amino acids, which would encode a protein with a molecular weight of 103,026. Five potential asparagine-linked glycosylation sites are found in the deduced amino acid sequence. The second peroxidase cDNA, designated phTPO-2, is almost identical to phTPO-1 beginning 605 base pairs downstream except that it contains 1-base-pair difference and lacks 171 base pairs in the middle of the sequence. This results in a loss of 57 amino acids corresponding to a molecular weight of 6282. Interestingly, this 171-nucleotide sequence has GT and AG at its 5' and 3' boundaries, respectively, that are in good agreement with donor and acceptor splice site consensus sequences. Using specific oligonucleotide probes for the mRNAs derived from the cDNA sequences hTOP-1 and hTOP-2, the authors show that both are expressed in all thyroid tissues examined and the relative level of two mRNAs is different in each sample. The results suggest that two thyroid peroxidase proteins might be generated through alternate splicing of the same gene. By using somatic cell hybrid lines, the thyroid peroxidase gene was mapped to the short arm of human chromosome 2.

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

    SciTech Connect

    Parente, M.G.; Chung, L.C.; Ryynaenen, J.; Monli Chu; Uitto, J. ); Woodley, D.T.; Wynn, K.C.; Bauer, E.A. ); Mattei, M.G. )

    1991-08-15

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

  7. A human alcohol dehydrogenase gene (ADH6) encoding an additional class of isozyme.

    PubMed Central

    Yasunami, M; Chen, C S; Yoshida, A

    1991-01-01

    The human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) gene family consists of five known loci (ADH1-ADH5), which have been mapped close together on chromosome 4 (4q21-25). ADH isozymes encoded by these genes are grouped in three distinct classes in terms of their enzymological properties. A moderate structural similarity is observed between the members of different classes. We isolated an additional member of the ADH gene family by means of cross-hybridization with the ADH2 (class I) cDNA probe. cDNA clones corresponding to this gene were derived from PCR-amplified libraries as well. The coding sequence of a 368-amino-acid-long open reading frame was interrupted by introns into eight exons and spanned approximately 17 kilobases on the genome. The gene contains a glucocorticoid response element at the 5' region. The transcript was detected in the stomach and liver. The deduced amino acid sequence of the open reading frame showed about 60% positional identity with known human ADHs. This extent of homology is comparable to interclass similarity in the human ADH family. Thus, the newly identified gene, which is designated ADH6, governs the synthesis of an enzyme that belongs to another class of ADHs presumably with a distinct physiological role. Images PMID:1881901

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

    PubMed Central

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

    1991-01-01

    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. Images PMID:2027750

  9. 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.

  10. Cloning of murine interferon gamma receptor cDNA: expression in human cells mediates high-affinity binding but is not sufficient to confer sensitivity to murine interferon gamma.

    PubMed Central

    Hemmi, S; Peghini, P; Metzler, M; Merlin, G; Dembic, Z; Aguet, M

    1989-01-01

    A full-length cDNA encoding the murine interferon gamma (IFN-gamma) receptor was isolated from a lambda gt11 library using a human IFN-gamma receptor cDNA probe. The deduced amino acid sequence of the murine IFN-gamma receptor shows approximately 53% homology to its human counterpart but no homology to other known proteins. Murine IFN-gamma receptor cDNA was expressed in human HEp-2 cells, which do not bind murine IFN-gamma and are insensitive to its action. Transfectants displayed the same binding properties as mouse cells. The biological responsiveness of such transfectants to various biological effects of both human and murine IFN-gamma was investigated, including modulation of major histocompatibility complex class I and class II antigen expression, inhibition of cell growth, and antiviral activity. Like parental HEp-2 cells, these transfectants responded only to human, but not to murine, IFN-gamma. Inversely, mouse L929 cells transfected with human IFN-gamma receptor cDNA were insensitive to human IFN-gamma. These results confirm and extend previous findings, suggesting that species-specific cofactors are needed for IFN-gamma-mediated signal transduction. Images PMID:2532365

  11. Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

    NASA Astrophysics Data System (ADS)

    Jiang, Keyong; Sun, Shujuan; Liu, Mei; Wang, Baojie; Meng, Xiaolin; Wang, Lei

    2013-01-01

    AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) ( P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) ( P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

  12. Cloning, expression and protective immunity evaluation of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum.

    PubMed

    Yu, JunLong; Wang, ShiPing; Li, WenKai; Dai, Gan; Xu, ShaoRui; He, Zhuo; Peng, XianChu; Zhou, SongHua; Liu, XueQin

    2007-04-01

    1071-bp fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3' and 5' ends of the incomplete expression sequence tag (EST) of succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) were amplified by the anchored PCR with 2 pairs of primers designed according to the EST of SjSDISP and the sequence of multiclone sites of the library vector. Sequence analysis indicated that the fragment was a full-length cDNA with a complete open reading frame (ORF), encoding 278 amino acid residues. The fragment was cloned into prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE and Western-blot analyses showed that the recombinant protein was about 32 kD and could be recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Compared with the FCA controls, mice vaccinated with rSjSDISP (test) or rSjGST (positive control) all revealed high levels of specific antibody and significant reduction in worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs. These results suggest that SjSDISP may be a novel and partially protective vaccine candidate against schistosomiasis. In contrast to the worm burden reduction rate, the higher degree of egg reduction rate in the test group also suggested that SjSDISP vaccine may primarily play a role in anti-embryonation or anti-fecundity immunity. PMID:17447029

  13. 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.

  14. Cloning of human genes encoding novel G protein-coupled receptors

    SciTech Connect

    Marchese, A.; Docherty, J.M.; Heiber, M.

    1994-10-01

    We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

  15. 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.

  16. Human acidic ribosomal phosphoproteins P0, P1, and P2: Analysis of cDNA clones, in vitro synthesis, and assembly

    SciTech Connect

    Rich, B.E.; Steitz, J.A.

    1987-11-01

    cDNA clones encoding three antigenically related human ribosomal phosophoproteins (P-proteins) P0, P1, and P2 were isolated and sequenced. P1 and P2 are analogous to Escherichia coli ribosomal protein L7/L12, and P0 is likely to be an analog of L10. The three proteins have a nearly identical carboxy-terminal 17-amino-acid sequence (KEESEESD(D/E)DMGFGLFD-COOH) that is the basis of their immunological cross-reactivity. The identifies of the P1 and P2 cDNAs were confirmed by the strong similarities of their encoded amino acid sequences to published primary structures of the homologous rat, brine shrimp, and Saccharomyces cerevisiae proteins. The P0 cDNA was initially identified by translation of hybrid-selected mRNA and immunoprecipitation of the products. To demonstrate that the coding sequences are full length, the P0, P1, and P2 cDNAs were transcribed in vitro by bacteriophage T7 RNA polymerase and the resulting mRNAs were translated in vitro. The synthetic P0, P1, and P2 proteins were serologically and electrophoretically identical to P-proteins extracted from HeLa cells. These synthetic P-proteins were incorporated into 60S but not 40S ribosomes and also assembled into a complex similar to that described for E. coli L7/L12 and L10.

  17. A human cDNA library for high-throughput protein expression screening.

    PubMed

    Büssow, K; Nordhoff, E; Lübbert, C; Lehrach, H; Walter, G

    2000-04-01

    We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses. PMID:10777659

  18. Cloning and expression of a cDNA encoding epitopes shared by 15- and 60-kilodalton proteins of Cryptosporidium parvum sporozoites.

    PubMed Central

    Jenkins, M C; Fayer, R; Tilley, M; Upton, S J

    1993-01-01

    A cDNA (CP15/60) encoding epitopes of Cryptosporidium parvum 15- and 60-kDa sporozoite proteins was isolated and expressed in Escherichia coli toward the goal of developing an immunogen for producing high-titer anticryptosporidial colostrum. Antisera prepared in rats to native C. parvum 15-kDa protein and used to identify the CP15/60 bacteriophage clone recognized both 15- and 60-kDa in vitro translation products derived from sporozoite RNA. Antisera specific for recombinant CP15/60 antigen recognized native 15- and 60-kDa C. parvum sporozoite proteins by immunoblotting and identified both surface and internal antigens on C. parvum sporozoites by immunofluorescence staining. Northern (RNA) and Southern blot hybridization experiments using sporozoite RNA and DNA indicated that CP15/60 DNA is transcribed as a single 1.4-kb RNA species from a single-copy gene. Recombinant CP15/60 antigen was recognized by hyperimmune colostrum from cows immunized with C. parvum oocyst-sporozoite protein and by convalescent-phase sera from C. parvum-infected calves. Images PMID:7684726

  19. Primary structure and functional expression of a cDNA encoding the bile canalicular, purine-specific Na(+)-nucleoside cotransporter.

    PubMed

    Che, M; Ortiz, D F; Arias, I M

    1995-06-01

    We previously characterized a purine-specific Na(+)-nucleoside cotransport system in bile canalicular membrane. The function of this transport system may be related to conserving nucleosides and preventing cholestasis. We report here the isolation of a cDNA encoding a Na(+)-dependent nucleoside transporter from rat liver using an expression cloning strategy. The substrate specificities and kinetic characteristics of the cloned cotransporter are consistent with the properties of the Na(+)-dependent, purine-selective nucleoside transporter in bile canalicular membranes. The nucleotide sequence predicts a protein of 659 amino acids (72 kDa) with 14 putative membrane-spanning domains. Northern blot analysis showed that the transcripts are present in liver and several other tissues. Data base searches indicate significant sequence similarity to the pyrimidine-selective nucleoside transporter (cNT1) of rat jejunum. Although these two subtypes of Na(+)-nucleoside cotransporter have different substrate specificities and tissue localizations, they are members of a single gene family. PMID:7775409

  20. Characterization of a novel growth hormone receptor-encoding cDNA in rainbow trout and regulation of its expression by nutritional state.

    PubMed

    Walock, Chad N; Kittilson, Jeffrey D; Sheridan, Mark A

    2014-01-01

    To clarify the divergence of the growth hormone receptor (GHR) family, we characterized a novel GHR from a teleost fish (rainbow trout). A 2357-nt cDNA was isolated and found to contain a single initiation site 71 nt from the most 5' end, an open reading frame of 1971 nt encoding a 657-amino acid protein, and a single polyadenylation site 229 nt from the poly-A tail. Based on structural analysis, the protein was identified as a type 1 GHR (GHR1). The new GHR1 shares 42% and 43% amino acid identity, respectively, with GHR2a and GHR2b, the two type 2 GHRs isolated from trout previously. GHR1 mRNA was found in a wide array of tissues with the highest expression in the liver, red muscle, and white muscle. Fasting animals for 4 weeks reduced steady state levels of GHR1 in the liver, adipose, and red muscle. These findings help clarify the divergence and nomenclature of GHRs and provide insight into the function of duplicated GHR types. PMID:24080484

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

    SciTech Connect

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. |

    1994-09-01

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

  2. Phonetic Feature Encoding in Human Superior Temporal Gyrus

    PubMed Central

    Mesgarani, Nima; Cheung, Connie; Johnson, Keith; Chang, Edward F.

    2015-01-01

    During speech perception, linguistic elements such as consonants and vowels are extracted from a complex acoustic speech signal. The superior temporal gyrus (STG) participates in high-order auditory processing of speech, but how it encodes phonetic information is poorly understood. We used high-density direct cortical surface recordings in humans while they listened to natural, continuous speech to reveal the STG representation of the entire English phonetic inventory. At single electrodes, we found response selectivity to distinct phonetic features. Encoding of acoustic properties was mediated by a distributed population response. Phonetic features could be directly related to tuning for spectrotemporal acoustic cues, some of which were encoded in a nonlinear fashion or by integration of multiple cues. These findings demonstrate the acoustic-phonetic representation of speech in human STG. PMID:24482117

  3. [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. PMID:16240713

  4. Cloning of Human IgG Fc cDNA and Expression of Whole Human Anti-HBsAg Antibody in CHO Cells.

    PubMed

    Tong, Yi-Gang; Xu, Jing; Liu, Guo-Qi; Zhang, Yong-Guo; Cheng, Wan-Rong; Liu, Shu-Ling; Wang, Hai-Tao

    2000-01-01

    Messenger RNA was extracted from human peripheral lymphocytes and first strand cDNA was prepared by reverse-transciption. The cDNA of Fc fragment of human IgG1 was then obtained by PCR and was cloned into the pGEM T-vector. The DNA sequences encoding signal peptides of both light and heavy chains were synthesized and cloned respectively. For construction of the light chain expression plasmid, the light chain signal sequence was linked with the light chain variable and constant regions (VL-CL) which had been cloned previously by screening of phage display libraries with HBsAg. The resulting full-lenth light chain sequence was then inserted into pcDNA3.1, a mammalian expression vector. For construction of the heavy chain expression plasmid, the heavy chain signal sequence, the variable region, the first constant region (VH-CH1, cloned previously by screening of phage display libraries with HBsAg) and Fc fragment sequence were ligated to form a full-length heavy chain ORF, which was then cloned into another mammalian expression vector, pCI-DHFR1. CHO(dhfr(-)) cells were cotransfected with the above light and heavy chain expression plasmids, and cell clones expressing human anti-HBsAg antibodies were selected by G418 and methotrexate (MTX). The recombinant human antibodies were purified with protein L affinity chromatography from the cell culture medium. As human serum IgG, the recombinant IgG exhibited only one band with a molecular weight of more than 100 kD in non-reducing SDS-PAGE in reducing SDS-PAGE, however, it turned out to be two bands of approximately 50 kD and 25 kD respectively. Western-blot analysis demonstrated that the whole IgG in the non-reducing SDS-PAGE, and the heavy chain in the reducing SDS-PAGE both reacted with goat anti-human Fc antiserum. PMID:12075430

  5. The Human Brain Encodes Event Frequencies While Forming Subjective Beliefs

    PubMed Central

    d’Acremont, Mathieu; Schultz, Wolfram; Bossaerts, Peter

    2015-01-01

    To make adaptive choices, humans need to estimate the probability of future events. Based on a Bayesian approach, it is assumed that probabilities are inferred by combining a priori, potentially subjective, knowledge with factual observations, but the precise neurobiological mechanism remains unknown. Here, we study whether neural encoding centers on subjective posterior probabilities, and data merely lead to updates of posteriors, or whether objective data are encoded separately alongside subjective knowledge. During fMRI, young adults acquired prior knowledge regarding uncertain events, repeatedly observed evidence in the form of stimuli, and estimated event probabilities. Participants combined prior knowledge with factual evidence using Bayesian principles. Expected reward inferred from prior knowledge was encoded in striatum. BOLD response in specific nodes of the default mode network (angular gyri, posterior cingulate, and medial prefrontal cortex) encoded the actual frequency of stimuli, unaffected by prior knowledge. In this network, activity increased with frequencies and thus reflected the accumulation of evidence. In contrast, Bayesian posterior probabilities, computed from prior knowledge and stimulus frequencies, were encoded in bilateral inferior frontal gyrus. Here activity increased for improbable events and thus signaled the violation of Bayesian predictions. Thus, subjective beliefs and stimulus frequencies were encoded in separate cortical regions. The advantage of such a separation is that objective evidence can be recombined with newly acquired knowledge when a reinterpretation of the evidence is called for. Overall this study reveals the coexistence in the brain of an experience-based system of inference and a knowledge-based system of inference. PMID:23804108

  6. Genetically encoded optical activation of DNA recombination in human cells.

    PubMed

    Luo, J; Arbely, E; Zhang, J; Chou, C; Uprety, R; Chin, J W; Deiters, A

    2016-06-30

    We developed two tightly regulated, light-activated Cre recombinase enzymes through site-specific incorporation of two genetically-encoded photocaged amino acids in human cells. Excellent optical off to on switching of DNA recombination was achieved. Furthermore, we demonstrated precise spatial control of Cre recombinase through patterned illumination. PMID:27277957

  7. 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.

  8. Identification of a cDNA for a human high-molecular-weight B-cell growth factor.

    PubMed Central

    Ambrus, J L; Pippin, J; Joseph, A; Xu, C; Blumenthal, D; Tamayo, A; Claypool, K; McCourt, D; Srikiatchatochorn, A; Ford, R J

    1993-01-01

    Proliferation is necessary for many of the phenotypic changes that occur during B-cell maturation. Further differentiation of mature B cells into plasma cells or memory B cells requires additional rounds of proliferation. In this manuscript, we describe a cDNA for a human B-cell growth factor we call high-molecular-weight B-cell growth factor (HMW-BCGF). Purified HMW-BCGF has been shown to induce B-cell proliferation, inhibit immunoglobulin secretion, and selectively expand certain B-cell subpopulations. Studies using antibodies to HMW-BCGF and its receptor have suggested that HMW-BCGF, while produced by T cells and some malignant B cells, acts predominantly on normal and malignant B cells. The HMW-BCGF cDNA was identified by expression cloning using a monoclonal antibody and polyclonal antisera to HMW-BCGF. Protein produced from the cDNA induced B-cell proliferation, inhibited immunoglobulin secretion, and was recognized in immunoblots by anti-HMW-BCGF antibodies. The amino acid sequence of HMW-BCGF deduced from the cDNA predicts a secreted protein of 53 kDa with three potential N-linked glycosylation sites. The identification of this cDNA will allow further studies examining physiologic roles of this cytokine. We propose to call it interleukin 14. Images Fig. 2 Fig. 4 Fig. 6 PMID:8327514

  9. 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

  10. Characterization of cDNA clones for human myeloperoxidase: predicted amino acid sequence and evidence for multiple mRNA species.

    PubMed Central

    Johnson, K R; Nauseef, W M; Care, A; Wheelock, M J; Shane, S; Hudson, S; Koeffler, H P; Selsted, M; Miller, C; Rovera, G

    1987-01-01

    Myeloperoxidase is a component of the microbicidal network of polymorphonuclear leukocytes. The enzyme is a tetramer consisting of two heavy and two light subunits. A large proportion of humans demonstrate genetic deficiencies in the production of myeloperoxidase. As a first step in analyzing these deficiencies in more detail, we have isolated cDNA clones for myeloperoxidase from an expression library of the HL-60 human promyelocytic leukemia cell line. Two overlapping plasmids (pMP02 and pMP062) were identified as myeloperoxidase cDNA clones based on the detection with myeloperoxidase antiserum of 70 kDa protein expressed in pMP02-containing bacteria and a 75 kDa polypeptide produced by hybridization selection and translation using pMP062 and HL-60 RNA. Formal identification of the clones was made by matching the predicted amino acid sequences with the amino terminal sequences of the heavy and light subunits. Both subunits are encoded by one mRNA in the following order: pre-pro-sequences--light subunit--heavy subunit. The molecular weight of the predicted primary translation product is 83.7 kDa. Northern blots reveal two size classes of hybridizing RNAs (approximately 3.0-3.3 and 3.5-4.0 kilobases) whose expression is restricted to cells of the granulocytic lineage and parallels the changes in enzymatic activity observed during differentiation. Images PMID:3031585

  11. CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p.

    PubMed Central

    Yokota, K; Kagawa, S; Shimizu, Y; Akioka, H; Tsurumi, C; Noda, C; Fujimuro, M; Yokosawa, H; Fujiwara, T; Takahashi, E; Ohba, M; Yamasaki, M; DeMartino, G N; Slaughter, C A; Toh-e, A; Tanaka, K

    1996-01-01

    The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease. The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1. NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase. Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth. The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p. Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation. The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome. Images PMID:8816993

  12. A human papilloma virus type 11 transcript encoding an E1--E4 protein.

    PubMed

    Nasseri, M; Hirochika, R; Broker, T R; Chow, L T

    1987-08-01

    The human papilloma virus (HPV) associated with a genital wart (condyloma acuminatum) was determined to be type 11. The majority of the viral DNA molecules were monomeric circles present in the cells at high copy number, as demonstrated by one- and two-dimensional agarose gell electrophoretic separation followed by Southern blot analysis. A cDNA library in phage lambda gt11 was constructed from poly(A)-selected mRNA recovered from the tissue. Recombinant clones corresponding to the most abundant 1.2-kb viral mRNA species detected by Northern blot hybridization and by electron microscopic analysis of R loops were isolated and their nucleotide sequence was determined. Comparison to the prototype HPV-11 DNA sequence revealed that this message consisted of two exons. The promotor-proximal exon spanned nucleotides 716 through 847 and the distal exon included nucleotides 3325 through 4390 or 4392. The mRNAs were alternatively polyadenylated after either of these latter two sites, in both cases following a G and preceding a U residue. Fourteen or sixteen bases upstream from the poly(A) was the hexanucleotide AGUAAA, which apparently serves as the signal for cleavage and polyadenylation of the nascent message. The splice donor and acceptor sites conformed to the usual /GU. . .AG/pattern. The exons joined open reading frame (ORF) E1, which contributed the initiation codon and four additional triplets, to ORF E4, which specified 85 amino acids to encode a protein of 10,022 Da. The cDNA also contained the ORFs E5a and E5b toward the 3' end. The complete sequence of the cDNA revealed three single-base changes from the prototype HPV-11, two resulting in altered amino acids in E4. Neither affects the coding potential of the overlapping E2 ORF. The function of the E1--E4 protein is unknown. PMID:2887066

  13. Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase

    SciTech Connect

    Hjalmarsson, K.; Marklund, S.L.; Engstroem, A.; Edlund, T.

    1987-09-01

    A complementary DNA (cDNA) clone from a human placenta cDNA library encoding extracellular superoxide dismutase has been isolated and the nucleotide sequence determined. The cDNA has a very high G + C content. EC-SOD is synthesized with a putative 18-amino acid signal peptide, preceding the 222 amino acids in the mature enzyme, indicating that the enzyme is a secretory protein. The first 95 amino acids of the mature enzyme show no sequence homology with other sequenced proteins and there is one possible N-glycosylation site (Asn-89). The amino acid sequence from residues 96-193 shows strong homology (approx. 50%) with the final two-thirds of the sequences of all know eukaryotic CuZn SODs, whereas the homology with the P. leiognathi CuZn SOD is clearly lower. The ligands to Cu and Zn, the cysteines forming the intrasubunit disulfide bridge in the CuZn SODs, and the arginine found in all CuZn SODs in the entrance to the active site can all be identified in EC-SOD. A comparison with bovine CuZn SOD, the three-dimensional structure of which is known, reveals that the homologies occur in the active site and the divergencies are in the part constituting the subunit contact area in CuZn SOD. Amino acid sequence 194-222 in the carboxyl-terminal end of EC-SOD is strongly hydrophilic and contains nine amino acids with a positive charge. This sequence probably confers the affinity of EC-SOD for heparin and heparan sulfate. An analysis of the amino acid sequence homologies with CuZn SODs from various species indicates that the EC-SODs may have evolved form the CuZn SODs before the evolution of fungi and plants.

  14. Identification of an NADH-Cytochrome b5 Reductase Gene from an Arachidonic Acid-Producing Fungus, Mortierella alpina 1S-4, by Sequencing of the Encoding cDNA and Heterologous Expression in a Fungus, Aspergillus oryzae

    PubMed Central

    Sakuradani, Eiji; Kobayashi, Michihiko; Shimizu, Sakayu

    1999-01-01

    Based on the sequence information for bovine and yeast NADH-cytochrome b5 reductases (CbRs), a DNA fragment was cloned from Mortierella alpina 1S-4 after PCR amplification. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 298 amino acid residues which show marked sequence similarity to CbRs from other sources, such as yeast (Saccharomyces cerevisiae), bovine, human, and rat CbRs. These results suggested that this cDNA is a CbR gene. The results of a structural comparison of the flavin-binding β-barrel domains of CbRs from various species and that of the M. alpina enzyme suggested that the overall barrel-folding patterns are similar to each other and that a specific arrangement of three highly conserved amino acid residues (i.e., arginine, tyrosine, and serine) plays a role in binding with the flavin (another prosthetic group) through hydrogen bonds. The corresponding genomic gene, which was also cloned from M. alpina 1S-4 by means of a hybridization method with the above probe, had four introns of different sizes. These introns had GT at the 5′ end and AG at the 3′ end, according to a general GT-AG rule. The expression of the full-length cDNA in a filamentous fungus, Aspergillus oryzae, resulted in an increase (4.7 times) in ferricyanide reduction activity involving the use of NADH as an electron donor in the microsomes. The M. alpina CbR was purified by solubilization of microsomes with cholic acid sodium salt, followed by DEAE-Sephacel, Mono-Q HR 5/5, and AMP-Sepharose 4B affinity column chromatographies; there was a 645-fold increase in the NADH-ferricyanide reductase specific activity. The purified CbR preferred NADH over NADPH as an electron donor. This is the first report of an analysis of this enzyme in filamentous fungi. PMID:10473389

  15. Molecular characterization of the body site-specific human epidermal cytokeratin 9: cDNA cloning, amino acid sequence, and tissue specificity of gene expression.

    PubMed

    Langbein, L; Heid, H W; Moll, I; Franke, W W

    1993-12-01

    Differentiation of human plantar and palmar epidermis is characterized by the suprabasal synthesis of a major special intermediate-sized filament (IF) protein, the type I (acidic) cytokeratin 9 (CK 9). Using partial amino acid (aa) sequence information obtained by direct Edman sequencing of peptides resulting from proteolytic digestion of purified CK 9, we synthesized several redundant primers by 'back-translation'. Amplification by polymerase chain reaction (PCR) of cDNAs obtained by reverse transcription of mRNAs from human foot sole epidermis, including 5'-primer extension, resulted in multiple overlapping cDNA clones, from which the complete cDNA (2353 bp) could be constructed. This cDNA encoded the CK 9 polypeptide with a calculated molecular weight of 61,987 and an isoelectric point at about pH 5.0. The aa sequence deduced from cDNA was verified in several parts by comparison with the peptide sequences and showed the typical structure of type I CKs, with a head (153 aa), and alpha-helical coiled-coil-forming rod (306 aa), and a tail (163 aa) domain. The protein displayed the highest homology to human CK 10, not only in the highly conserved rod domain but also in large parts of the head and the tail domains. On the other hand, the aa sequence revealed some remarkable differences from CK 10 and other CKs, even in the most conserved segments of the rod domain. The nuclease digestion pattern seen on Southern blot analysis of human genomic DNA indicated the existence of a unique CK 9 gene. Using CK 9-specific riboprobes for hybridization on Northern blots of RNAs from various epithelia, a mRNA of about 2.4 kb in length could be identified only in foot sole epidermis, and a weaker cross-hybridization signal was seen in RNA from bovine heel pad epidermis at about 2.0 kb. A large number of tissues and cell cultures were examined by PCR of mRNA-derived cDNAs, using CK 9-specific primers. But even with this very sensitive signal amplification, only palmar

  16. 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

  17. [Cloning and analysis of cDNA encoding key enzyme gene (dxr) of the non-MVA pathway in Taxus chinensis cells].

    PubMed

    Zheng, Qing-Ping; Yu, Long-Jiang; Liu, Zhi; Li, Mo-Yi; Xiang, Fu; Yang, Qin

    2004-07-01

    Two distinct routes (classical mevalonate pathway and a novel mevalonate-independent pathway) are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids (Fig. 1). Present researches indicated that taxol was synthesized mainly via non-mevalonate pathway, but not genetic evidence was showed. The second step in non-mevalonate pathway involves an intramolecular rearrangement and subsequent reduction of deoxyxylulose phosphate to yield 2-C-methyl-D-erythritol-4-phosphate, and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) with responsibility for this reaction was considered as a key enzyme. As a tool for the isolation of genes in terpenoid biosynthesis in plants, total RNA was prepared from Taxus chinensis suspension cells, a cell type highly specialized for diterpene (taxol). A reverse transcription-PCR strategy based on the design of degenerated oligonucleotides was developed for isolating the gene encoding a gymnosperm homolog of this enzyme from Taxus chinensis. Through sequence analysis by Blast P online, the resulting cDNA showed highly homologous to 1-deoxy-D-xylulose 5-phosphate reductoisomerases, with 95% identification compared with Arabidopsis thaliana (Q9XFS9), 94% with Mentha x piperita (Q9XESO), 80% with Synechococcus elongatus (Q8DK30), 78% with Synechocystis sp. PCC 6803 (Q55663) and Nostoc sp. PCC 7120 (Q8YP49), and 73% with Synechococcus leopoliensis (Q9RKT1). Deduced amino acid sequences were also analyzed by PROSITE, ClustalX (1.81) and Phylio (3.6 alpha), and data present evidence for the existence of this deoxyxyluose phosphate reductoisomerase in Taxus chinensis. This is the first report of the dxr gene cloned from gymnosperm. PMID:15968987

  18. Isolation and sequence analysis of a cDNA encoding the c subunit of a vacuolar-type H(+)-ATPase from the CAM plant Kalanchoë daigremontiana.

    PubMed

    Bartholomew, D M; Rees, D J; Rambaut, A; Smith, J A

    1996-05-01

    We report the sequence of a cDNA clone encoding the c ("16 kDa') subunit of a vacuolar-type H(+)-ATPase (V-ATPase) from Kalanchoë daigremontiana, a plant in which the cell vacuole plays a pivotal role in crassulacean acid metabolism. The clone, pKVA211, was isolated from a K. daigremontiana leaf cDNA library constructed in lambda ZAP II using a homologous PCR-generated cDNA probe for the V-ATPase c subunit. The KVA211 cDNA was 839 nucleotides long and included a 20 bp poly(A)+ tail together with a complete 495 bp coding region for a polypeptide with a predicted molecular mass of 16659 Da. The deduced amino acid sequence was highly conserved across the wide range of eukaryotes (vertebrates, invertebrates, fungi, plants and protozoa) in which this gene has now been identified. Sequence comparison of several PCR products and genomic Southern analysis indicated that the V-ATPase c subunit in K. daigremontiana is encoded by a small multi-gene family. Steady-state levels of the KVA211 mRNA were much higher in leaves than in roots or flowers, and expression of this transcript in leaves was shown to be strongly light-dependent. PMID:8756609

  19. Analysis of the C4 genes in baleen whales using a human cDNA probe.

    PubMed

    Spilliaert, R; Palsdottir, A; Arnason, A

    1990-01-01

    We have used a human C4 cDNA probe to investigate the complement component C4 gene in four members of the family Balaenopteridae: fin whale (Balaenoptera physalus), sei whale (B. borealis), minke whale (B. acutorostrata), and bryde's whale (B. edeni). Restriction mapping of genomic DNA from the first three species suggests the presence of only one locus in these species, and also shows that the C4 genes in the three species are very similar. We have used 14 restriction endonucleases to investigate the restriction fragment length polymorphism (RFLP) of fin whales, 13 enzymes for sei whales, and 8 enzymes for the minke whale. No polymorphism was seen in DNA from the five minke whale samples, but Rsa I and Taq I restriction enzymes gave polymorphism in fin and sei whales whereas Hind III and Msp I restriction enzymes showed polymorphism in sei whales only. Only one bryde's whale sample was available for investigation. The study of DNA available from mother-fetus pairs from the two polymorphic species demonstrated a simple, two-allele transmission of RFLP alleles. PMID:1975799

  20. Cloning and expression of a human kidney cDNA for an /alpha//sub 2/-adrenergic receptor subtype

    SciTech Connect

    Regan, J.W.; Kobilka, T.S.; Yang-Feng, T.L.; Caron, M.G.; Lefkowitz, R.J.; Kobilka, B.K.

    1988-09-01

    An /alpha//sub 2/-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet /alpha//sub 2/-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet /alpha//sub 2/-adrenergic receptor and is consistent with the structure of other members of he family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the /alpha//sub 2/-adrenergic ligand (/sup 3/H)rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the /alpha//sub 2/B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet /alpha//sub 2/-adrenergic receptor (/alpha//sub 2/A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective /alpha/-adrenergic ligands.

  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. cap alpha. /sub 2/-HS-glycoprotein: the A and B chains with a connecting sequence are encoded by a single mRNA transcript

    SciTech Connect

    Lee, C.C.; Bowman, B.H.; Yang, F.

    1987-07-01

    The ..cap alpha../sub 2/-HS-glycoprotein (AHSG) is a plasma protein reported to play roles in bone mineralization and in the immune response. It is composed of two subunits, the A and B chains. Recombinant plasmids containing human cDNA AHSG have been isolated by screening an adult human liver library with a mixed oligonucleotide probe. The cDNA clones containing AHSG inserts span approximately 1.5 kilobase pairs and include the entire AHSG coding sequence, demonstrating that the A and B chains are encoded by a single mRNA transcript. The cDNA sequence predicts an 18-amino-acid signal peptide, followed by the A-chain sequence of AHSG. A heretofore unseen connecting sequence of 40 amino acids was deduced between the A- and B-chain sequences. The connecting sequence demonstrates the unique amino acid doublets and collagen triplets found in the A and B chains; it is not homologous with other reported amino acid sequences. The connecting sequence may be cleaved in a posttranslational step by limited proteolysis before mature AHSG is released into the circulation or may vary in its presence because of alternative processing. The AHSG cDNA was utilized for mapping the AHSG gene to the 3q21..-->..qter region of human chromosome 3. The availability of the AHSG cDNA clone will facilitate the analysis of its genetic control and gene expression during development and bone formation.

  3. Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein

    SciTech Connect

    Hickey, E.; Brandon, S.E.; Weber, L.A.; Lloyd, D.

    1989-06-01

    Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89/alpha/ and hspio/beta/) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89/alpha/, is induced by the adenovirus E1A gene product. The authors have isolated a human hsp89/alpha/ gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression n a /beta/-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89/alpha/ protein sequence differed from the human hsp89/beta/ sequence reported elsewhere in at least 99 out of the 732 amino acids. Transcription of the hsp89/alpha/ gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycles. hsp89/alpha/ mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.

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

    PubMed

    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

    1996-11-01

    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

  5. [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. PMID:26152174

  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. 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. PMID:25594487

  8. 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

    1995-04-01

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

  9. 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.

  10. cDNA cloning and gene structure of a novel water channel expressed exclusively in human kidney: Evidence for a gene cluster of aquaporins at chromosome locus 12q13

    SciTech Connect

    Ma, Tonghui; Yang, Baoxue; Verkman, A.S.

    1996-08-01

    A 1.8-kb cDNA clone (designed hKID, gene symbol AQP2L) with homology to the aquaporins was isolated from a human kidney cDNA library. The longest open reading frame of 846 bp encoded a 282-amino-acid hydrophobic protein that contained the conserved NPA motifs of MIP family members. Cell-free translation produced a nonglycosylated protein migrating at 29 kDa. Northern blot analysis revealed a 2.2-kb transcript expressed only in human kidney. PCR/Southern blot analysis of human kidney cDNA using primers flanking the hKID coding sequence revealed expression of a full-length mRNA and short transcripts with partial exon 1 and partial exon 4 deletions. Genomic Southern blot indicted a single-copy hKID gene. PCR analysis of a human/rodent somatic hybrid panel localized the hKID gene to chromosome 12. Chromosomal fluorescence in situ hybridization mapped the hKID (AQP2L) gene to chromosome locus 12q13, the same location a as the AQP-2 and MIP genes. The high sequence homology, similar genomic structure, and identical chromosomal loci of hKID, MIP, and AQP-2 suggest a MIP family gene cluster at chromosome locus 12q13. Further work is needed to establish the physiological significance of hKID. 43 refs., 6 figs.

  11. 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

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

    PubMed Central

    Adjaye, James; Herwig, Ralf; Herrmann, Doris; Wruck, Wasco; BenKahla, Alia; Brink, Thore C; Nowak, Monika; Carnwath, Joseph W; Hultschig, Claus; Niemann, Heiner; Lehrach, Hans

    2004-01-01

    Background Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. The objective of this study was to investigate the feasibility and reproducibility of cross-species analysis employing a human cDNA microarray as probe. Results As a proof of principle, total RNA derived from human and bovine fetal brains was used as a source of labelled targets for hybridisation onto a human cDNA microarray composed of 349 characterised genes. Each gene was spotted 20 times representing 6,980 data points thus enabling highly reproducible spot quantification. Employing high stringency hybridisation and washing conditions, followed by data analysis, revealed slight differences in the expression levels and reproducibility of the signals between the two species. We also assigned each of the genes into three expression level categories- i.e. high, medium and low. The correlation co-efficient of cross hybridisation between the orthologous genes was 0.94. Verification of the array data by semi-quantitative RT-PCR using common primer sequences enabled co-amplification of both human and bovine transcripts. Finally, we were able to assign gene names to previously uncharacterised bovine ESTs. Conclusions Results of our study demonstrate the harnessing and utilisation power of comparative genomics and prove the feasibility of using human microarrays to facilitate the identification of co-expressed orthologous genes in common tissues derived from different

  13. Isolation and characterization of human cDNAs encoding a cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase.

    PubMed

    Rosman, G J; Martins, T J; Sonnenburg, W K; Beavo, J A; Ferguson, K; Loughney, K

    1997-05-20

    Human cyclic GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE2A3) cDNAs were cloned from hippocampus and fetal brain cDNA libraries. A 4.2-kb composite DNA sequence constructed from overlapping cDNA clones encodes a 941 amino acid protein with a predicted molecular mass of 105,715 Da. Extracts prepared from yeast expressing the human PDE2A3 hydrolyzed both cyclic AMP (cAMP) and cyclic GMP (cGMP). This activity was inhibited by EHNA, a selective PDE2 inhibitor, and was stimulated three-fold by cGMP. Human PDE2A is expressed in brain and to a lesser extent in heart, placenta, lung, skeletal muscle, kidney and pancreas. The human PDE2A3 differs from the bovine PDE2A1 and rat PDE2A2 proteins at the amino terminus but its amino-terminal sequence is identical to the bovine PDE2A3 sequence. The different amino termini probably arise from alternative exon splicing of the PDE2A mRNA. PMID:9210593

  14. 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

  15. 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. PMID:26529408

  16. A neural circuit encoding sexual preference in humans.

    PubMed

    Poeppl, Timm B; Langguth, Berthold; Rupprecht, Rainer; Laird, Angela R; Eickhoff, Simon B

    2016-09-01

    Sexual preference determines mate choice for reproduction and hence guarantees conservation of species in mammals. Despite this fundamental role in human behavior, current knowledge on its target-specific neurofunctional substrate is based on lesion studies and therefore limited. We used meta-analytic remodeling of neuroimaging data from 364 human subjects with diverse sexual interests during sexual stimulation to quantify neural regions associated with sexual preference manipulations. We found that sexual preference is encoded by four phylogenetically old, subcortical brain structures. More specifically, sexual preference is controlled by the anterior and preoptic area of the hypothalamus, the anterior and mediodorsal thalamus, the septal area, and the perirhinal parahippocampus including the dentate gyrus. In contrast, sexual non-preference is regulated by the substantia innominata. We anticipate the identification of a core neural circuit for sexual preferences to be a starting point for further sophisticated investigations into the neural principles of sexual behavior and particularly of its aberrations. PMID:27339689

  17. YAC contig mapping of six expressed sequences encoded by human chromosome 21

    SciTech Connect

    Yu, J.; Cox, M.; Patterson, D. |

    1995-03-01

    Six cDNA clones from human chromosome 21 have been mapped in a set of complete YAC contig spanning the entire chromosome 21q. The mapping positions between two STSs on the YAC contig and the NotI coordinates starting from the telomere of 21q were determined for the cDNA clones. The YAC contig mapping positions agree well with those using a comprehensive somatic cell hybrid mapping panel. 6 refs., 1 fig., 2 tabs.

  18. Immunohistochemical detection of the human cytochrome P4507B1: production of a monoclonal antibody after cDNA immunization.

    PubMed

    Trap, Catherine; Nato, Farida; Chalbot, Sonia; Kim, Sae-Bom; Lafaye, Pierre; Morfin, Robert

    2005-02-01

    The cytochrome P4507B1 (P4507B1) is responsible for the 7alpha-hydroxylation of dehydroepiandrosterone (DHEA) and other 3beta-hydroxysteroids in the brain and other organs. The cDNA of human P4507B1 was used for DNA immunization of mice. The best responding mouse led to the production of monoclonal antibodies (mAbs). The clone D16-37 produced an IgM specific for P4507B1 with no cross-reaction with other human P450s. This antibody permitted the immunohistochemical detection of P4507B1 in slices of human hippocampus. P4507B1 was expressed in neurons only. This new tool will be used for the extensive examination of the P4507B1 presence and determination of its levels in slices of human normal and diseased brain and in other human tissues. PMID:15652401

  19. EGASP: the human ENCODE Genome Annotation Assessment Project

    PubMed Central

    Guigó, Roderic; Flicek, Paul; Abril, Josep F; Reymond, Alexandre; Lagarde, Julien; Denoeud, France; Antonarakis, Stylianos; Ashburner, Michael; Bajic, Vladimir B; Birney, Ewan; Castelo, Robert; Eyras, Eduardo; Ucla, Catherine; Gingeras, Thomas R; Harrow, Jennifer; Hubbard, Tim; Lewis, Suzanna E; Reese, Martin G

    2006-01-01

    Background We present the results of EGASP, a community experiment to assess the state-of-the-art in genome annotation within the ENCODE regions, which span 1% of the human genome sequence. The experiment had two major goals: the assessment of the accuracy of computational methods to predict protein coding genes; and the overall assessment of the completeness of the current human genome annotations as represented in the ENCODE regions. For the computational prediction assessment, eighteen groups contributed gene predictions. We evaluated these submissions against each other based on a 'reference set' of annotations generated as part of the GENCODE project. These annotations were not available to the prediction groups prior to the submission deadline, so that their predictions were blind and an external advisory committee could perform a fair assessment. Results The best methods had at least one gene transcript correctly predicted for close to 70% of the annotated genes. Nevertheless, the multiple transcript accuracy, taking into account alternative splicing, reached only approximately 40% to 50% accuracy. At the coding nucleotide level, the best programs reached an accuracy of 90% in both sensitivity and specificity. Programs relying on mRNA and protein sequences were the most accurate in reproducing the manually curated annotations. Experimental validation shows that only a very small percentage (3.2%) of the selected 221 computationally predicted exons outside of the existing annotation could be verified. Conclusion This is the first such experiment in human DNA, and we have followed the standards established in a similar experiment, GASP1, in Drosophila melanogaster. We believe the results presented here contribute to the value of ongoing large-scale annotation projects and should guide further experimental methods when being scaled up to the entire human genome sequence. PMID:16925836

  20. Identification of endothelial antigens relevant to transplant coronary artery disease from a human endothelial cell cDNA expression library.

    PubMed

    Ationu, A

    1998-06-01

    Accelerated transplant coronary artery disease (TxCAD) results in increased expression of antiendothelial antibodies whose target antigens remain largely unidentified. One of these endothelial antigens has been identified as vimentin, a cytoskeletal protein present in cells of the blood vessel walls. In the present study, SDS-PAGE and Western blot analysis of human endothelial cell (EAHy 926) lysates probed with sera from a TxCAD patient were used to confirm immunoreactivity of antiendothelial antibodies towards several endothelial proteins. To further elucidate the identity of these putative antigens, a human endothelial cell (EAHy 926) cDNA expression library was immunoscreened with serum obtained from a TxCAD patient. Two positive cDNA clones were identified by partial nucleotide sequence analysis and GenBank/EMBL database searches for homology as the 85 kDa human CD36 antigen (a cell surface glycoprotein expressed in various cells including epithelial and endothelial cells) and a 50 kDa keratin-like protein (a member of the intermediate filament protein expressed in epithelial cells). These results are the first to demonstrate that human CD36 antigen and a keratin-like protein may be additional target proteins for the anti-endothelial antibodies associated with TxCAD. PMID:9852639

  1. Human glutamate pyruvate transaminase (GPT): Localization to 8q24.3, cDNA and genomic sequences, and polymorphic sites

    SciTech Connect

    Sohocki, M.M.; Sullivan, L.S.; Daiger, S.P.

    1997-03-01

    Two frequent protein variants of glutamate pyruvate transaminase (GPT) (E.C.2.6.1.2) have been used as genetic markers in humans for more than two decades, although chromosomal mapping of the GPT locus in the 1980s produced conflicting results. To resolve this conflict and develop useful DNA markers for this gene, we isolated and characterized cDNA and genomic clones of GPT. We have definitively mapped human GPT to the terminus of 8q using several methods. First, two cosmids shown to contain the GPT sequence were derived from a chromosome 8-specific library. Second, by fluorescence in situ hybridization, we mapped the cosmid containing the human GPT gene to chromosome band 8q24.3. Third, we mapped the rat gpt cDNA to the syntenic region of rat chromosome 7. Finally, PCR primers specific to human GPT amplify sequences contained within a {open_quotes}half-YAC{close_quotes} from the long arm of chromosome 8, that is, a YAC containing the 8q telomere. The human GPT genomic sequence spans 2.7 kb and consists of 11 exons, ranging in size from 79 to 243 bp. The exonic sequence encodes a protein of 495 amino acids that is nearly identical to the previously reported protein sequence of human GPT-1. The two polymorphic GPT isozymes are the result of a nucleotide substitution in codon 14. In addition, a cosmid containing the GPT sequence also contains a previously unmapped, polymorphic microsatellite sequence, D8S421. The cloned GPT gene and associated polymorphisms will be useful for linkage and physical mapping of disease loci that map to the terminus of 8q, including atypical vitelliform macular dystrophy (VMD1) and epidermolysis bullosa simplex, type Ogna (EBS1). In addition, this will be a useful system for characterizing the telomeric region of 8q. Finally, determination of the molecular basis of the GPT isozyme variants will permit PCR-based detection of this world-wide polymorphism. 22 refs., 3 figs.

  2. Encoding of Sensory Prediction Errors in the Human Cerebellum

    PubMed Central

    Schlerf, John; Ivry, Richard B.; Diedrichsen, Jörn

    2015-01-01

    A central tenet of motor neuroscience is that the cerebellum learns from sensory prediction errors. Surprisingly, neuroimaging studies have not revealed definitive signatures of error processing in the cerebellum. Furthermore, neurophysiologic studies suggest an asymmetry, such that the cerebellum may encode errors arising from unexpected sensory events, but not errors reflecting the omission of expected stimuli. We conducted an imaging study to compare the cerebellar response to these two types of errors. Participants made fast out-and-back reaching movements, aiming either for an object that delivered a force pulse if intersected or for a gap between two objects, either of which delivered a force pulse if intersected. Errors (missing the target) could therefore be signaled either through the presence or absence of a force pulse. In an initial analysis, the cerebellar BOLD response was smaller on trials with errors compared with trials without errors. However, we also observed an error-related decrease in heart rate. After correcting for variation in heart rate, increased activation during error trials was observed in the hand area of lobules V and VI. This effect was similar for the two error types. The results provide evidence for the encoding of errors resulting from either the unexpected presence or unexpected absence of sensory stimulation in the human cerebellum. PMID:22492047

  3. Prefrontal Gamma Oscillations Encode Tonic Pain in Humans

    PubMed Central

    Schulz, Enrico; May, Elisabeth S.; Postorino, Martina; Tiemann, Laura; Nickel, Moritz M.; Witkovsky, Viktor; Schmidt, Paul; Gross, Joachim; Ploner, Markus

    2015-01-01

    Under physiological conditions, momentary pain serves vital protective functions. Ongoing pain in chronic pain states, on the other hand, is a pathological condition that causes widespread suffering and whose treatment remains unsatisfactory. The brain mechanisms of ongoing pain are largely unknown. In this study, we applied tonic painful heat stimuli of varying degree to healthy human subjects, obtained continuous pain ratings, and recorded electroencephalograms to relate ongoing pain to brain activity. Our results reveal that the subjective perception of tonic pain is selectively encoded by gamma oscillations in the medial prefrontal cortex. We further observed that the encoding of subjective pain intensity experienced by the participants differs fundamentally from that of objective stimulus intensity and from that of brief pain stimuli. These observations point to a role for gamma oscillations in the medial prefrontal cortex in ongoing, tonic pain and thereby extend current concepts of the brain mechanisms of pain to the clinically relevant state of ongoing pain. Furthermore, our approach might help to identify a brain marker of ongoing pain, which may prove useful for the diagnosis and therapy of chronic pain. PMID:25754338

  4. Characterization of cDNAs encoding human pyruvate dehydrogenase alpha subunit.

    PubMed Central

    Ho, L; Wexler, I D; Liu, T C; Thekkumkara, T J; Patel, M S

    1989-01-01

    A cDNA clone (1423 base pairs) comprising the entire coding region of the precursor form of the alpha subunit of pyruvate dehydrogenase (E1 alpha) has been isolated from a human liver cDNA library in phage lambda gt11. The first 29 amino acids deduced from the open reading frame correspond to a typical mitochondrial targeting leader sequence. The remaining 361 amino acids, starting at the N terminus with phenylalanine, represent the mature mitochondrial E1 alpha peptide. The cDNA has 43 base pairs in the 5' untranslated region and 210 base pairs in the 3' untranslated region, including a polyadenylylation signal and a short poly(A) tract. The nucleotide sequence of human liver E1 alpha cDNA was confirmed by the nucleotide sequences of three overlapping fragments generated from human liver and fibroblast RNA by reverse transcription and DNA amplification by the polymerase chain reaction. This consensus nucleotide sequence of human liver E1 alpha cDNA resolves existing discrepancies among three previously reported human E1 alpha cDNAs and provides the unambiguous reference sequence needed for the characterization of genetic mutations in pyruvate dehydrogenase-deficient patients. Images PMID:2748588

  5. 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

  6. 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.

  7. 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. PMID:8771778

  8. The human CCGl gene, essential for progression of the G sub 1 phase, encodes a 210-kilodalton nuclear DNA-binding protein

    SciTech Connect

    Sekiguchi, Takeshi; Nohiro, Yukiko; Hisamoto, Naoki; Nishimoto, Takeharu ); Nakamura, Yasuhara )

    1991-06-01

    The human CCGl gene complements tsBN462, a temperature-sensitive G{sup 1} mutant of the BHK21 cell line. The previously cloned cDNA turned out to be a truncated form of the actual CCGl cDNA. The newly cloned CCGl cDNA was 6.0 kb and encoded a protein with a molecular mass of 210 kDa. Using an antibody to a predicted peptide from the CCGl protein, a protein with a molecular mass of over 200 kDa was identified in human, monkey, and hamster cell lines. In the newly defined C-terminal region, an acidic domain was found. It contained four consensus target sequences for casein kinase 2 and was phosphorylated by this enzyme in vitro. However, this C-terminal region was not required to complement tsBN462 mutation since the region encoding the C-terminal part was frequently missing in complemented clones derived by DNA-mediated gene transfer, CCGl contains a sequence similar to the putative DNA-binding domain of HMGl in addition to the previously detected amino acid sequences common in nuclear proteins, such as a proline cluster and a nuclear translocation signal. Consistent with these predictions, CCGl was present in nuclei, possessed DNA-binding activity, and was eluted with similar concentrations of salt, 0.3 to 0.4 M NaCl either from isolated nuclei or from a DNA-cellulose column.

  9. The vitellogenin cDNA of Cherax quadricarinatus encodes a lipoprotein with calcium binding ability, and its expression is induced following the removal of the androgenic gland in a sexually plastic system.

    PubMed

    Abdu, Uri; Davis, Claytus; Khalaila, Isam; Sagi, Amir

    2002-07-01

    Oocyte maturation in decapod crustaceans is a two step process. Primary vitellogenesis is followed by a variable hiatus that lasts up to the onset of secondary vitellogenesis, which is marked by the rapid accumulation of yolk proteins in the oocytes. We have cloned a complete Cherax quadricarinatus vitellogenin cDNA. The sequenced cDNA contains a 2584 aa open reading frame which shows sequence similarity to vitellogenins from other crustaceans. The mRNA encodes at least two of the previously identified vitellin components, indicating that the primary translation product is subject to post-translational modification, including proteolytic cleavage. The region close to the 3(') end of the mRNA encodes a previously characterized negatively charged protein (provisionally designated P(106)). We show here that the negative charge of P(106) could be due to its ability to bind calcium. Northern blot data show that this gene is expressed as a single 8000 nt transcript and is present in the hepatopancreas of secondary-vitellogenic females. Primary vitellogenic and other tissues examined in male and female animals were negative. In sexually plastic intersex animals, removal of the androgenic gland results in vitellogenin transcription, indicating that the gene is negatively regulated by the androgenic gland. PMID:12225768

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

    PubMed Central

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

    1988-01-01

    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 hygromycin 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 lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-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 hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(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. Images PMID:2841588

  11. Auditory modulation of visual stimulus encoding in human retinotopic cortex

    PubMed Central

    de Haas, Benjamin; Schwarzkopf, D. Samuel; Urner, Maren; Rees, Geraint

    2013-01-01

    Sounds can modulate visual perception as well as neural activity in retinotopic cortex. Most studies in this context investigated how sounds change neural amplitude and oscillatory phase reset in visual cortex. However, recent studies in macaque monkeys show that congruence of audio-visual stimuli also modulates the amount of stimulus information carried by spiking activity of primary auditory and visual neurons. Here, we used naturalistic video stimuli and recorded the spatial patterns of functional MRI signals in human retinotopic cortex to test whether the discriminability of such patterns varied with the presence and congruence of co-occurring sounds. We found that incongruent sounds significantly impaired stimulus decoding from area V2 and there was a similar trend for V3. This effect was associated with reduced inter-trial reliability of patterns (i.e. higher levels of noise), but was not accompanied by any detectable modulation of overall signal amplitude. We conclude that sounds modulate naturalistic stimulus encoding in early human retinotopic cortex without affecting overall signal amplitude. Subthreshold modulation, oscillatory phase reset and dynamic attentional modulation are candidate neural and cognitive mechanisms mediating these effects. PMID:23296187

  12. 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

  13. Localization of the gene encoding peptidylglycine [alpha]-amidating monooxygenase (PAM) to human chromosome 5q14-5q21

    SciTech Connect

    Ouafik, L.H.; Giraud, P.; Oliver, C. ); Mattei, M.G. ); Eipper, B.A.; Mains, R.E. )

    1993-11-01

    Peptidylglycine [alpha]-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the [alpha]-amidation of neuroendocrine peptides. Southern blot analysis of human placental DNA demonstrated that PAM is encoded by a single gene. The chromosomal localization of the PAM gene was established using in situ hybridization. A 2.2-kb human PAM cDNA hybridized to human metaphase chromosomes revealed a significant clustering of silver grains over chromosome 5 bands q14-q21. The gene encoding another enzyme important in the post-translational processing of neuroendocrine precursors, prohormone convertase 1 (PC1), is localized in the same region (5q15-q21). 14 refs., 2 figs.

  14. Complete cDNA sequence of human complement C1s and close physical linkage of the homologous genes C1s and C1r

    SciTech Connect

    Tosi, M.; Duponchel, C.; Meo, T.; Julier, C.

    1987-12-29

    Overlapping molecular clones encoding the complement subcomponent C1s were isolated from a human liver cDNA library. The nucleotide sequence reconstructed from these clones spans about 85% of the length of the liver C1s messenger RNAs, which occur in three distinct size classes around 3 kilobases in length. Comparisons with the sequence of C1r, the other enzymatic subcomponent of C1, reveal 40% amino acid identity and conservation of all the cysteine residues. Beside the serine protease domain, the following sequence motifs, previously described in C1r, were also found in C1s: (a) two repeats of the type found in the Ba fragment of complement factor B and in several other complement but also noncomplement proteins, (b) a cysteine-rich segment homologous to the repeats of epidermal growth factor precursor, and (c) a duplicated segment found only in C1r and C1s. Differences in each of these structural motifs provide significant clues for the interpretation of the functional divergence of these interacting serine protease zymogens. Hybridizations of C1r and C1s probes to restriction endonuclease fragments of genomic DNA demonstrate close physical linkage of the corresponding genes. The implications of this finding are discussed with respect to the evolution of C1r and C1s after their origin by tandem gene duplication and to the previously observed combined hereditary deficiencies of Clr and Cls.

  15. Cloning and expresion of cDNA for rat O6-methylguanine-DNA methyltransferase.

    PubMed Central

    Sakumi, K; Shiraishi, A; Hayakawa, H; Sekiguchi, M

    1991-01-01

    cDNA for O6-methylguanine-DNA methyltransferase was isolated by screening rat liver cDNA libraries, using as a probe the human cDNA sequence for methyltransferase. The rat cDNA encodes a protein with 209 amino acid residues. The predicted amino acid sequence of the rat methyltransferase exhibits considerable homology with those of the human, yeast and bacterial enzymes, especially around putative methyl acceptor sites. When the cDNA was placed under control of the lac promoter and expressed in methyltransferase-deficient Escherichia coli (ada-, ogt-) cells, a characteristic methyltransferase protein was produced. The rat DNA methyltransferase thus expressed could complement the biological defects of the E. coli cell caused by lack of its own DNA methyltransferases; e.g. increased sensitivity to alkylating agents in terms of both cell death and mutation induction. Images PMID:1945835

  16. Bioinformatics Annotation of Human Y Chromosome-Encoded Protein Pathways and Interactions.

    PubMed

    Rengaraj, Deivendran; Kwon, Woo-Sung; Pang, Myung-Geol

    2015-09-01

    We performed a comprehensive analysis of human Y chromosome-encoded proteins, their pathways, and their interactions using bioinformatics tools. From the NCBI annotation release 107 of human genome, we retrieved a total of 66 proteins encoded on Y chromosome. Most of the retrieved proteins were also matched with the proteins listed in the core databases of the Human Proteome Project including neXtProt, PeptideAtlas, and the Human Protein Atlas. When we examined the pathways of human Y-encoded proteins through KEGG database and Pathway Studio software, many of proteins fall into the categories related to cell signaling pathways. Using the STRING program, we found a total of 49 human Y-encoded proteins showing strong/medium interaction with each other. While using the Pathway studio software, we found that a total of 16 proteins interact with other chromosome-encoded proteins. In particular, the SRY protein interacted with 17 proteins encoded on other chromosomes. Additionally, we aligned the sequences of human Y-encoded proteins with the sequences of chimpanzee and mouse Y-encoded proteins using the NCBI BLAST program. This analysis resulted in a significant number of orthologous proteins between human, chimpanzee, and mouse. Collectively, our findings provide the scientific community with additional information on the human Y chromosome-encoded proteins. PMID:26279084

  17. Molecular characterization of cDNA encoding oxygen evolving enhancer protein 1 increased by salt treatment in the mangrove Bruguiera gymnorrhiza.

    PubMed

    Sugihara, K; Hanagata, N; Dubinsky, Z; Baba, S; Karube, I

    2000-11-01

    Young plants of the common Okinawa mangrove species Bruguiera gymnorrhiza were transferred from freshwater to a medium with seawater salt level (500 mM NaCl). Two-dimensional gel electrophoresis revealed in the leaf extract of the plant a 33 kDa protein with pI 5.2, whose quantity increased as a result of NaCl treatment. The N-terminal amino acids sequence of this protein had a significant homology with mature region of oxygen evolving enhancer protein 1 (OEE1) precursor. The cloning of OEE1 precursor cDNA fragment was carried out by means of reverse transcription-PCR (RT-PCR) using degenerated primers. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA ends (RACE) method. The deduced amino acid sequence consisted of 322 amino acids and was 87% identical to that of Nicotiana tabacum. In B. gymnorrhiza, the predicted amino acid sequence of the mature protein starts at the residue number 85 of the open reading frame. The first 84-amino acid residues correspond to a typical transit sequence for the signal directing OEE1 to its appropriate compartment of chloroplast. The expression of OEE1 was analyzed together with other OEE subunits and D1 protein of photosystem II. The transcript levels of all the three OEEs were enhanced by NaCl treatment, but the significant increase of D1 protein was not observed. PMID:11092914

  18. Characterization of a cDNA encoding RP43, a CUB-domain-containing protein from the tube of Riftia pachyptila (Vestimentifera), and distribution of its transcript.

    PubMed Central

    Chamoy, L; Nicolaï, M; Quennedey, B; Gaill, F; Delachambre, J

    2000-01-01

    A major 43 kDa protein from the protective tube of Riftia pachyptila (Vestimentifera), named RP43, was partly microsequenced after isolation by SDS/PAGE from the protein fraction of tubes collected around the hydrothermal vents at the East Pacific Rise. On the basis of the partial peptide sequences obtained, experiments using reverse-transcriptase-mediated PCR and rapid amplification of cDNA ends led to the complete cDNA sequence. Analysis of deduced amino acid sequence of RP43 showed the presence of CUB domains (100-110-residue-spanning domains first reported in the complement subcomponents C1r/C1s, epidermal-growth-factor-related sea urchin protein and bone morphogenetic protein 1) that seem to be involved in protein-protein and glycosaminoglycan-protein interactions. This peculiarity strongly suggests that RP43 might have a crucial role in tightening the different elements of the worm tube. However, the absence of chitin-binding motifs inclines us to favour a role in protein-protein interactions during assembly of the tube components. The RP43 mRNA was found to be present in specific epidermal cells from the worm body wall but never in the chitin-synthesizing gland cells. This unexpected result clearly indicates that the major tube protein is synthesized in specialized areas of the outer epithelium and that at least two different tissues are involved in the synthesis of the exoskeleton. PMID:10947956

  19. 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

  20. Isolation and properties of Drosophila melanogaster ferritin--molecular cloning of a cDNA that encodes one subunit, and localization of the gene on the third chromosome.

    PubMed

    Charlesworth, A; Georgieva, T; Gospodov, I; Law, J H; Dunkov, B C; Ralcheva, N; Barillas-Mury, C; Ralchev, K; Kafatos, F C

    1997-07-15

    Ferritin was purified from iron-fed Drosophila melanogaster extracts by centrifugation in a gradient of potassium bromide. On polyacrylamide gel electrophoresis, the product showed two protein bands corresponding to the ferritin monomer and dimer. Electrophoresis following dissociation with SDS and 2-mercaptoethanol revealed three strong bands of approximately 25, 26, and 28 kDa. N-terminal amino acid sequences were identical for the 25-kDa and 26-kDa subunits, but different for the 28-kDa subunit. Conserved ferritin PCR primers were used to amplify a 360-bp cDNA product, which was used to isolate a clone from a D. melanogaster cDNA library that contained the complete coding sequence for a ferritin subunit. Additional 5' sequence obtained by the RACE method revealed the presence of a putative iron regulatory element. The PCR product was also used to locate the position of the ferritin subunit gene at region 99F on the right arm of the third chromosome. The deduced amino acid sequence of the D. melanogaster ferritin subunit contained a signal sequence and resembled most closely ferritin of the mosquito Aedes aegypti. The evolution of ferritin sequences is discussed. PMID:9266686

  1. Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells

    SciTech Connect

    Davidson, D.J.; Fraser, M.J.; Castellino, F.J. )

    1990-06-12

    A comparison has been made between the Asn{sup 289}-linked oligosaccharide structures of human plasma plasminogen and a recombinant human plasminogen, expressed in lepidopteran insect (Spodoptera frugiperda) cells, after infection of these cells with a recombinant baculovirus containing the entire human plasminogen cDNA. Using anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the proteins by glycopeptidase F, compared with elution positions of standard oligosaccharide structures, coupled with monosaccharide compositional analysis, the authors find that the human plasma protein contained only bisialo-biantennary complex-type carbohydrate and asialo-biantennary complex carbohydrate, confirming earlier work published by this laboratory. The glycosylation pattern of the insect cell expressed recombinant human plasminogen showed considerable microheterogeneity, with identifiable high-mannose carbohydrate and truncated high-mannose oligosaccharide. Of major importance, approximately 40% of the oligosaccharide population consisted of complex carbohydrate (bisialo-biantennary), identical in structure with that of the human plasma protein. This the first direct identification of complex carbohydrate in proteins produced in insect cells and demonstrates that trimming and processing of high-mannose carbohydrate into complex-type oligosaccharide can occur. The data indicate that both normal and alternate pathways exist in these cells for incorporation and trimming of high-mannose oligosaccharides and that mannosidases, as well as galactosyl-, hexosaminidasyl-, and sialyltransferases are present, and/or can be induced, in these cells. From these observations, the authors conclude that amino acid sequences and/or protein conformational properties can control oligosaccharide processing events.

  2. Human ESP1/CRP2, a member of the LIM domain protein family: Characterization of the cDNA and assignment of the gene locus to chromosome 14q32.3

    SciTech Connect

    Karim, Mohammad Azharul; Ohta, Kohji; Matsuda, Ichiro

    1996-01-15

    The LIM domain is present in a wide variety of proteins with diverse functions and exhibits characteristic arrangements of Cys and His residues with a novel zinc-binding motif. LIM domain proteins have been implicated in development, cell regulation, and cell structure. A LIM domain protein was identified by screening a human cDNA library with rat cysteine-rich intestinal protein (CRIP) as a probe, under conditions of low stringency. Comparison of the predicted amino acid sequence with several LIM domain proteins revealed 93% of the residues to be identical to rat LIM domain protein, termed ESP1 or CRP2. Thus, the protein is hereafter referred to as human ESP1/CRP2. The cDNA encompasses a 1171-base region, including 26, 624, and 521 bases in the 5{prime}-noncoding region, coding region, and 3{prime}-noncoding regions, respectively, and encodes the entire ESP1/CRP2 protein has two LIM domains, and each shares 35.1% and 77 or 79% identical residues with human cysteine-rich protein (CRP) and rat CRIP, respectively. Northern blot analysis of ESP1/CRP2 in various human tissues showed distinct tissue distributions compared with CRP and CRIP, suggesting that each might serve related but specific roles in tissue organization or function. Using a panel of human-rodent somatic cell hybrids, the ESP1/CRP2 locus was assigned to chromosome 14. Fluorescence in situ hybridization, using cDNA and a genome DNA fragment of the ESP1/CRP2 as probes, confirms this assignment and relegates regional localization to band 14q32.3 47 refs., 7 figs.

  3. 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.

  4. Cloning of a cDNA encoding a plasma membrane-associated, uronide binding phosphoprotein with physical properties similar to viral movement proteins.

    PubMed Central

    Reymond, P; Kunz, B; Paul-Pletzer, K; Grimm, R; Eckerskorn, C; Farmer, E E

    1996-01-01

    Oligogalacturonides are structural and regulatory homopolymers from the extracellular pectic matrix of plants. In vitro micromolar concentrations of oligogalacturonates and polygalacturonates were shown previously to stimulate the phosphorylation of a small plasma membrane-associated protein in potato. Immunologically cross-reactive proteins were detected in plasma membrane-enriched fractions from all angiosperm subclasses in the Cronquist system. Polygalacturonate-enhanced phosphorylation of the protein was observed in four of the six dicotyledon subclasses but not in any of the five monocotyledon subclasses. A cDNA for the protein was cloned from potato. The deduced protein is extremely hydrophilic and has a proline-rich N terminus. The C-terminal half of the protein was predicted to be a coiled coil, suggesting that the protein interacts with other macromolecules. The recombinant protein was found to bind both simple and complex galacturonides. The behavior of the protein suggests several parallels with viral proteins involved in intercellular communication. PMID:8989883

  5. 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.

  6. Use of PCR amplification of cDNA to study mechanisms of human cell mutagenesis and malignant transformation

    SciTech Connect

    Maher, V.M.; Yang, Jialing; Chen, Rueyhwa; McGregor, W.G.; Lukash, L.; Scheid, J.M.; Reinhold, D.S.; McCormick, J.J. )

    1991-01-01

    PCR is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. But some investigators need to sequence the entire coding region of mammalian genes to determine what specific changes have occurred. In 1989, the authors described a method to copy mRNA of the hypoxanthine (guanine) phosphoribosyl transferase (HPRT) gene directly from the lysate of a clone of 6-thioguanine-resistant mutant diploid human fibroblasts without the need for RNA extraction or DNA template purification. The consensus sequence of the cDNA is determined by direct nucleotide sequencing. Using this method, they have investigated the kinds of mutations induced by carcinogens in the coding region of the HPRT gene and their location in the gene and examined the role of DNA repair were exposed to mutagens in exponential growth or synchronized and exposed at the beginning of S phase or in G{sub 1} phase several hr prior to DNA replication.

  7. 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.

  8. 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.

  9. 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

  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. PMID:23313486

  11. 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.

  12. Human liver mitochondrial carnitine palmitoyltransferase I: characterization of its cDNA and chromosomal localization and partial analysis of the gene.

    PubMed Central

    Britton, C H; Schultz, R A; Zhang, B; Esser, V; Foster, D W; McGarry, J D

    1995-01-01

    Using the cDNA for rat liver mitochondrial carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) as a probe, we isolated its counterpart as three overlapping clones from a human liver cDNA library. Both the nucleotide sequence of the human cDNA and the predicted primary structure of the protein (773 aa) proved to be very similar to those of the rat enzyme (82% and 88% identity, respectively). The CPT I mRNA size was also found to be the same (approximately 4.7 kb) in both species. Screening of a human genomic library with the newly obtained cDNA yielded a positive clone of approximately 6.5 kb which, upon partial analysis, was found to contain at least two complete exons linked by a 2.3-kb intron. Oligonucleotide primers specific to upstream and downstream regions of one of the exon/intron junctions were tested in PCRs with DNA from a panel of somatic cell hybrids, each containing a single human chromosome. The results allowed unambiguous assignment of the human liver CPT I gene to the q (long) arm of chromosome 11. Additional experiments established that liver and fibroblasts express the same isoform of mitochondrial CPT I, legitimizing the use of fibroblast assays in the differential diagnosis of the "muscle" and "hepatic" forms of CPT deficiency. The data provide insights into the structure of a human CPT I isoform and its corresponding gene and establish unequivocally that CPT I and CPT II are distinct gene products. Availability of the human CPT I cDNA should open the way to an understanding of the genetic basis of inherited CPT I deficiency syndromes, how the liver CPT I gene is regulated, and which tissues other than liver express this particular variant of the enzyme. Images Fig. 4 Fig. 5 PMID:7892212

  13. Induction of human beta-interferon synthesis with poly(rI . rC) in mouse cells transfected with cloned cDNA plasmids.

    PubMed Central

    Pitha, P M; Ciufo, D M; Kellum, M; Raj, N B; Reyes, G R; Hayward, G S

    1982-01-01

    Human genomic DNA and plasmids carrying portions of the cDNA gene for human beta-interferon have been introduced into mouse Ltk- cells by cotransfection with a herpes simplex virus thymidine kinase (TK) gene. One plasmid contains 840 base pairs of human DNA complementary to pre-beta-interferon mRNA inserted into pBR322, whereas the other plasmids have hybrid genes containing only the 560-base pair coding region inserted under the transcriptional control of the TK promoter. Constitutive interferon production could not be detected in any of the mouse TK+ cell lines tested. Nevertheless, synthesis of interferon could be induced by poly(rI . rC) treatment in at least 16 of these cell lines, including clones transfected with genomic DNA, the beta-interferon cDNA, and the TK-beta-interferon cDNA hybrid gene. The interferon produced was specific for human cells and could be neutralized by antiserum against human beta-interferon. In contrast to human fibroblast cells, in which the synthesis of induced beta-interferon is transient, the poly(rI . rC)-induced TK+ lines continued to produce beta-interferon for prolonged periods of time and did not respond to superinduction conditions. Therefore, in transfected mouse cells, the coding DNA sequence from the human beta-interferon gene, without any of the adjacent 3' or 5' flanking human DNA sequences, was sufficient both to direct synthesis of biologically active product and to respond to the specific induction system that operates in human cells. However, the mechanism that switches off the synthesis of induced interferon in human cells appears not to operate in mouse cells transfected with beta-interferon cDNA. PMID:6956863

  14. 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.

  15. Rescue of a genotype 4 human hepatitis E virus from cloned cDNA and characterization of intergenotypic chimeric viruses in cultured human liver cells and in pigs

    PubMed Central

    Córdoba, Laura; Feagins, Alicia R.; Opriessnig, Tanja; Cossaboom, Caitlin M.; Dryman, Barbara A.; Huang, Yao-Wei

    2012-01-01

    Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 are zoonotic, infecting both humans and pigs. This report describes, for the first time, the successful rescue of infectious HEV in vitro and in vivo from cloned cDNA of a genotype 4 human HEV (strain TW6196E). The complete genomic sequence of the TW6196E virus was determined and a full-length cDNA clone (pHEV-4TW) was assembled. Capped RNA transcripts from the pHEV-4TW clone were replication competent in Huh7 cells and infectious in HepG2/C3A cells. Pigs inoculated intrahepatically with capped RNA transcripts from pHEV-4TW developed an active infection, as evidenced by faecal virus shedding and seroconversion, indicating the successful rescue of infectious genotype 4 HEV and cross-species infection of pigs by a genotype 4 human HEV. To demonstrate the utility of the genotype 4 HEV infectious clone and to evaluate the potential viral determinant(s) for species tropism, four intergenotypic chimeric clones were constructed by swapping various genomic regions between genotypes 1 and 4, and genotypes 1 and 3. All four chimeric clones were replication competent in Huh7 cells, but only the two chimeras with sequences swapped between genotypes 1 and 4 human HEVs produced viruses capable of infecting HepG2/C3A cells. None of the four chimeras was able to establish a robust infection in pigs. The availability of a genotype 4 HEV infectious clone affords an opportunity to delineate the molecular mechanisms of HEV cross-species infection in the future. PMID:22837416

  16. 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.

  17. Molecular cloning and heterologous expression of a cDNA encoding a mouse glutathione S-transferase Yc subunit possessing high catalytic activity for aflatoxin B1-8,9-epoxide.

    PubMed Central

    Hayes, J D; Judah, D J; Neal, G E; Nguyen, T

    1992-01-01

    Resistance to the carcinogenic effects of aflatoxin B1 (AFB1) in the mouse is due to the constitutive expression of an Alpha-class glutathione S-transferase (GST), YcYc, with high detoxification activity towards AFB1-8,9-epoxide. A cDNA clone (pmusGST Yc) for a murine GST Yc polypeptide has been isolated. Sequencing has shown the cDNA insert of pmusGST Yc to be 922 bp in length, with an open reading frame of 663 bp that encodes a polypeptide of M(r) 25358. The primary structure of the murine GST Yc subunit predicted by pmusGST Yc is in complete agreement with the partial amino acid sequence of the aflatoxin-metabolizing mouse liver GST described previously [McLellan, Kerr, Cronshaw & Hayes (1991) Biochem. J. 276, 461-469]. A plasmid, termed pKK-musGST Yc, which permits the expression of the murine Yc subunit in Escherichia coli, has been constructed. The murine GST expressed in E. coli was purified and found to be catalytically active towards several GST substrates, including AFB1-8,9-epoxide. This enzyme was also found to possess electrophoretic and immunochemical properties closely similar to those of the GST Yc subunit from mouse liver. However, the GST synthesized in E. coli and the constitutive mouse liver Alpha-class GST exhibited small differences in their chromatographic behaviour during reverse-phase h.p.l.c. Automated Edman degradation revealed alanine to be the N-terminal amino acid in the GST Yc subunit expressed in E. coli, whereas the enzyme in mouse liver possesses a blocked N-terminus. Although sequencing showed that the purified Yc subunit from E. coli lacked the initiator methionine, the amino acid sequence obtained over the first eleven N-terminal residues agreed with that predicted from the cDNA clone, pmusGST Yc. Comparison of the deduced amino acid sequence of the mouse Yc polypeptide with the primary structures of the rat Alpha-class GST enzymes revealed that it is more closely related to the ethoxyquin-induced rat liver Yc2 subunit than to

  18. Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor.

    PubMed Central

    Ny, T; Sawdey, M; Lawrence, D; Millan, J L; Loskutoff, D J

    1986-01-01

    A lambda gt11 expression library containing cDNA inserts prepared from human placental mRNA was screened immunologically using an antibody probe developed against the beta-migrating plasminogen activator inhibitor (beta-PAI) purified from cultured bovine aortic endothelial cells. Thirty-four positive clones were isolated after screening 7 X 10(5) phages. Three clones (lambda 1.2, lambda 3, and lambda 9.2) were randomly picked and further characterized. These contained inserts 1.9, 3.0, and 1.9 kilobases (kb) long, respectively. Escherichia coli lysogenic for lambda 9.2, but not for lambda gt11, produced a fusion protein of 180 kDa that was recognized by affinity-purified antibodies against the bovine aortic endothelial cell beta-PAI and had beta-PAI activity when analyzed by reverse fibrin autography. The largest cDNA insert was sequenced and shown to be 2944 base pairs (bp) long. It has a large 3' untranslated region [1788 bp, excluding the poly(A) tail] and contains the entire coding region of the mature protein but lacks the initiation codon and part of the signal peptide coding region at the 5' terminus. The two clones carrying the 1.9-kb cDNA inserts were partially sequenced and shown to be identical to the 3.0-kb cDNA except that they were truncated, lacking much of the 3' untranslated region. Blot hybridization analysis of electrophoretically fractionated RNA from the human fibrosarcoma cell line HT-1080 was performed using the 3.0-kb cDNA as hybridization probe. Two distinct transcripts, 2.2 and 3.0 kb, were detected, suggesting that the 1.9-kb cDNA may have been copied from the shorter RNA transcript. The amino acid sequence deduced from the cDNA was aligned with the NH2-terminal sequence of the human beta-PAI. Based on this alignment, the mature human beta-PAI is 379 amino acids long and contains an NH2-terminal valine. The deduced amino acid sequence has extensive (30%) homology with alpha 1-antitrypsin and antithrombin III, indicating that the beta

  19. Reconstitution of a MEC1-independent checkpoint in yeast by expression of a novel human fork head cDNA.

    PubMed Central

    Pati, D; Keller, C; Groudine, M; Plon, S E

    1997-01-01

    A novel human cDNA, CHES1 (checkpoint suppressor 1), has been isolated by suppression of the mec1-1 checkpoint mutation in Saccharomyces cerevisiae. CHES1 suppresses a number of DNA damage-activated checkpoint mutations in S. cerevisiae, including mec1, rad9, rad24, dun1, and rad53. CHES1 suppression of sensitivity to DNA damage is specific for checkpoint-defective strains, in contrast to DNA repair-defective strains. Presence of CHES1 but not a control vector resulted in G2 delay after UV irradiation in checkpoint-defective strains, with kinetics, nuclear morphology, and cycloheximide resistance similar to those of a wild-type strain. CHES1 can also suppress the lethality, UV sensitivity, and G2 checkpoint defect of a mec1 null mutation. In contrast to this activity, CHES1 had no measurable effect on the replication checkpoint as assayed by hydroxyurea sensitivity of a mec1 strain. Sequence analysis demonstrates that CHES1 is a novel member of the fork head/Winged Helix family of transcription factors. Suppression of the checkpoint-defective phenotype requires a 200-amino-acid domain in the carboxy terminus of the protein which is distinct from the DNA binding site. Analysis of CHES1 activity is most consistent with activation of an alternative MEC1-independent checkpoint pathway in budding yeast. PMID:9154802

  20. 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

  1. YAC contig and cell hybrid mapping of six expressed sequences encoded by human chromosome 21

    SciTech Connect

    Yu, J.; Cox, M.; Patterson, D.

    1994-09-01

    The candidate gene approach for positional cloning requires a sufficient number of expressed gene sequences from the chromosomal region of interest. Trisomy for human chromosome 21 results in Down syndrome (DS). However, only a limited number of genes on chromosome 21 have been identified and cloned. We used 1,000 single-copy microclones from a microdissection library of chromosome 21 to screen various cDNA libraries and isolated 9 cDNA clones, of which 6 contain unique sequences: 21E-C1, C3, C4, C5, C7, C10. Using a refined regional mapping panel of chromosome 21 which comprised 24 cell hybrids and divided the chromosome into 33 subregions, we assigned 21E-C1 and C7 to subregion No. 22 (distal q22.1), 21E-C3 to No. 25 (proximal q22.2), 21E-C4 to No. 23 (very distal q22.1), 21E-C5 to No. 31 (proximal q22.3), and 21E-C10 to No. 28 (middle q22.2). In addition, we identified YAC clones corresponding to these cDNA clones using the complete YAC contig spanning the entire chromosome 21q. On the average, 10 positive YAC clones were identified for each cDNA. The mapping positions for the 6 cDNAs determined by the STSs in the YAC contig agree well with the cytogenetic map constructed by the hybrid panel. These cDNA clones with refined mapping positions on chromosome 21 should be useful as candidate genes for the specific component phenotypes of DS assigned to the region.

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

    PubMed Central

    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. PMID:26606454

  3. [Construction and identification of mammary expressional vector for cDNA of human lactoferrin].

    PubMed

    Meng, Li; Zhang, Yanli; Xu, Xin; Wang, Ziyu; Yan, Yibo; Pang, Xunsheng; Zhong, Bushuai; Huang, Rong; Song, Yang; Wang, Jinyu; Wang, Feng

    2011-02-01

    The aim of this study was to construct a mammary gland-specific expressional vector pBC1-hLF-Neo for Human Lactoferrin (hLF) gene and then investigate its expression in the mammary gland epithelium cells. The constructed vector contained the 6.2 kb long 5' flank regulation region including promoter, other elements and the 7.1 kb long 3' flank regulation region including transcriptional ending signal of a goat's beta-casein gene. A cassette of Neo gene was also inserted into the vector which gave a total length of 26.736 kb identified by restriction fragment analysis and partial DNA sequencing. The results revealed that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, we transfected the lined vector DNA into the dairy goat's mammary gland epithelium cells and C127 cells of a mouse's mammary epithelium by Lipofectamine. After selection with G418 for 8-10 days, G418-risistant clones were obtained. PCR analysis demonstrated that hLF gene cassette had been integrated into the genomic DNA of G418-risistant clones. After proliferation culture, the two kinds of transgenic cells were cultured in serum-free DMEM-F12 medium with prolactin, insulin and hydrocortisone- a medium capable of inducing recombinant hLF expression. RT-PCR, Western blotting and anti-bacteria bioactivity experiments demonstrated that the constructed mammary gland specific vector pBC1-hLF-Neo possessed the desirable bioactivity to efficiently express and could secrete hLF in both mammary gland cells and have the effect of E. coli proliferation inhibition. Paramount to everything, this study laid a firm foundation for preparing the hLF gene transgenic goat fetal-derived fibroblast cells. PMID:21650051

  4. The human and mouse homologs of the yeat RAD52 gene: cDNA cloning, sequence analysis, assignment to human chromosome 12p12.2-p13, and mRNA expression in mouse tissues

    SciTech Connect

    Shen, Z.; Chen, D.J.; Denison, K.

    1995-01-01

    The yeast Saccharomyces cerevisiae RAD52 gene is involved in DNA double-strand break repair and mitotic/meiotic recombination. The N-terminal amino acid sequence of yeast S. cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis and chicken is highly conserved. Using the technology of mixed oligonucleotide primed amplification of cDNA (MOPAC), two mouse RAD52 homologous cDNA fragments were amplified and sequenced. Subsequently, we have cloned the cDNA of the human and mouse homologs of yeast RAD52 gene by screening cDNA libraries using the identified mouse cDNA fragments. Sequence analysis of cDNA derived amino acid revealed a highly conserved N-terminus among human, mouse, chicken, and yeast RAD52 genes. The human RAD52 gene was assigned to chromosome 12p12.2-p13 by fluorescence in situ hybridization, R-banding, and DNA analysis of somatic cell hybrids. Unlike chicken RAD52 and mouse RAD51, no significant difference in mouse RAD52 mRNA level was found among mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. In addition to an {approximately}1.9-kb RAD52 mRNA band that is present in all of the tested tissues, an extra mRNA species of {approximately}0.85 kb was detectable in mouse testis. 40 refs., 7 figs., 1 tab.

  5. Human xeroderma pigmentosum group G gene encodes a DNA endonuclease.

    PubMed Central

    Habraken, Y; Sung, P; Prakash, L; Prakash, S

    1994-01-01

    Because of defective nucleotide excision repair of ultraviolet damaged DNA, xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers. Cell fusion studies have identified seven XP complementation groups, A to G. Previous studies have implicated the products of these seven XP genes in the recognition of ultraviolet-induced DNA damage and in incision of the damage-containing DNA strand. Here, we express the XPG-encoded protein in Sf9 insect cells and purify it to homogeneity. We demonstrate that XPG is a single-strand specific DNA endonuclease, thus identifying the catalytic role of the protein in nucleotide excision repair. We suggest that XPG nuclease acts on the single-stranded region created as a result of the combined action of the XPB helicase and XPD helicase at the DNA damage site. Images PMID:8078765

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

    SciTech Connect

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A.

    1994-10-01

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

  7. 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.

  8. 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.

  9. Human flavin-containing monooxygenase form 3: cDNA expression of the enzymes containing amino acid substitutions observed in individuals with trimethylaminuria.

    PubMed

    Cashman, J R; Bi, Y A; Lin, J; Youil, R; Knight, M; Forrest, S; Treacy, E

    1997-08-01

    Trimethylaminuria is an autosomal recessive human disorder affecting a small part of the population as an inherited polymorphism. Individuals diagnosed with trimethylaminuria excrete relatively large amounts of trimethylamine in their urine, sweat, and breath, and this results in a fishy odor characteristic of trimethylamine. Activity of the human flavin-containing monooxygenase (FMO) has been proposed to be deficient in trimethylaminuria patients causing a decrease in the metabolism of trimethylamine that results in a fishy body odor. Cohorts of Australian, American, and British individuals suffering from trimethylaminuria have been identified. The human FMO3 cDNA was amplified from lymphocytes of affected patients. We report preliminary evidence of substitutions detected by screening of the cDNA and genomic DNA. The variant human FMO3 cDNA was constructed from wild type human FMO3 cDNA by site-directed mutagenesis as maltose-binding protein fusions. Five distinct human FMO3 mutants were expressed as fusion proteins in Escherichia coli and compared with wild type human FMO3 maltose-binding proteins (FMO3-MBP) for the N-oxygenation of 10-[(N,N-dimethylamino)pentyl]-2-(trifluoromethyl)phenothiazine, tyramine, and trimethylamine. Human Lys158 FMO3-MBP and, to a greater extent, human Glu158 FMO3-MBP efficiently N-oxygenated the three amine substrates. Human Lys158 Ile66 FMO3-MBP, Glu158 Ile66 FMO3-MBP, Lys158 Leu153 FMO3-MBP, and Glu158 Leu153 FMO3-MBP were all constructed as mutants identified as possible FMO3 variants responsible for trimethylaminuria and were found to be inactive as N-oxygenases. The results suggest that mutations at codons 66 and 153 of FMO3 can cause trimethylaminuria in humans. We observed a common polymorphism of Lys to Glu at codon 158 of FMO3 that segregated with almost equal allele frequencies in a number of control Australian and North American samples studied. The Lys158 to Glu158 human FMO3 polymorphism does not decrease trimethylamine N

  10. 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

    2015-01-01

    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. PMID:26560187

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26560187

  12. 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. PMID:12757705

  13. 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.

  14. The human hGSTA5 gene encodes an enzymatically active protein

    PubMed Central

    Singh, Sharda P.; Zimniak, Ludwika; Zimniak, Piotr

    2009-01-01

    Background Of the five human Alpha-class glutathione transferases, expression of hGSTA5 has not been experimentally documented, even though in silico the hGSTA5 sequence can be assembled into a mRNA and translated. The present work was undertaken to determine whether hGSTA5 is functional. Methods Human K562 cells were transfected with the hGSTA5 gene driven by the CMV promoter, and hGSTA5 cDNA was recovered from mature mRNA by reverse transcription. The cDNA was used in bacterial and eukaryotic protein expression systems. The resulting protein, after purification by glutathione affinity chromatography where appropriate, was tested for glutathione transferase activity. Results Human K562 cells transfected with the hGSTA5 gene under control of a CMV promoter produced a fully spliced mRNA which, after reverse transcription and expression in E. coli, yielded a protein that catalyzed the conjugation of the lipid peroxidation product 4-hydroxynonenal to glutathione. Similarly, transfection of human HEK-293 cells with the hGSTA5 gene driven by the CMV promoter led to an elevated 4-hydroxynonenal-conjugating activity in the cell lysate. In addition, translation of hGSTA5 cDNA in a cell-free eukaryotic system gave rise to a protein with 4-hydroxynonenal-conjugating activity. Conclusions hGSTA5 can be processed to a mature mRNA which is translation-competent, producing a catalytically active enzyme. General Significance Because a functional gene would not be maintained in the absence of selective pressure, we conclude that the native hGSTA5 promoter is active but has a spatially or temporally restricted expression pattern, and/or is expressed only under specific (patho)physiological conditions. PMID:19664689

  15. Identification, cloning and regulation of cDNA encoding aldo-keto reductase 1B7 in the adrenal gland of two Saharan rodents Meriones libycus (Libyan jird) and Gerbillus gerbillus (gerbil).

    PubMed

    Mataoui-Mazari, Houria; Amirat, Zaïna; Khammar, Farida; Martinez, Antoine

    2011-12-01

    Aldo-Keto Reductase 1B7 (AKR1B7) is a mouse aldose reductase-like protein with two major sites of expression, the vas deferens and the adrenal cortex. In the adrenal cortex, Akr1b7 is an adrenocorticotropin (ACTH)-responsive-gene whose product scavenges harmful byproducts of steroidogenesis and limits stress response through the biosynthesis of prostaglandin F2α. The purpose of the present study was to explore the possible expression of AKR1B7 in the adrenal glands of two saharan rodents, Libyan jird and Lesser Egyptian gerbil. Western blot analyses demonstrated that a protein related to murine/rat AKR1B7 was highly expressed in adrenals and absent from vas deferens of both saharan species. Based on conserved sequences between mouse and rat, full length cDNA were cloned and sequenced in both species while hormonal regulation and tissue localization were explored in Libyan jird. Both cDNA encoded the expected 316 amino acids protein typical of AKR1B subfamily and contained the highly conserved catalytic tetrad consisting in Asp-44, Tyr-49, Lys-78 and His-111 residues. The deduced proteins shared higher identities with aldose reductase-like, i.e. AKR1B7 (86-94%), AKR1B8 and AKR1B10 (83-86%) than with aldose reductase group, i.e. AKR1B1 and AKR1B3 (70%). Phylogenetic analysis showed that the Libyan jird and gerbil enzymes were more closely related to murine and rat AKR1B7 than to the other AKR1B members. Northern blot analyses of total RNA from Libyan jird adrenals showed a single mRNA transcript of 1.4 kb whose expression was dependent on circulating ACTH levels. In conclusion, we demonstrate here that adrenal glands of Libyan jird and gerbil express both an ortholog of the murine/rat Akr1b7 gene and that ACTH-responsiveness is at least conserved in Libyan jird. PMID:21963864

  16. The human Ig-[beta] cDNA sequence, a homologue of murine B29, is identical in B cell and plasma cell lines producing all the human Ig isotypes

    SciTech Connect

    Hashimoto, Shiori; Gregersen, P.K.; Chiorazzi, N. Cornell Univ., New York, NY )

    1993-01-15

    The B cell Ag receptor complex consists of at least two disulfide-linked, heterodimeric structures: the clonally restricted membrane Ig (mIg) molecule and the nonpolymorphic Ig-[alpha]:Ig-[beta] protein dimer. The latter molecule is encoded by two separate genes, mb-1 and B29. The DNA sequences of murine and human mb-1 and murine B29 have been determined previously. This study describes the sequence of the full-length human cDNA homologue of the murine Ig-[beta]/B29 message. The human sequence codes for a protein that displays the typical subunit features of a transmembrane member of the Ig superfamily. The transmembrane and intracytoplasmic domains exhibit striking nucleotide and amino acid sequence similarity between the two species. These regions show almost complete conservation of areas presumed to be involved in noncovalent interactions with other members of the receptor complex and with intracellular kinases and cytoskeletal components. The only sequence dissimilarity seen in these presumed critical areas involves the Y-E-G-L-N motif, a potential target for tyrosine phosphorylation. In contrast, the extracellular portion is much more divergent. Inasmuch as similar patterns of species diversity have been reported for Ig-[alpha], the Ig-[alpha] and Ig-[beta] molecules may have coevolved to maintain species-specific extracellular interactions between one another and with mIg. Similar to the Ig-[alpha] molecule, the Ig-[beta] sequence is identical in B lineage cells expressing all five Ig isotypes. However, in contrast to the Ig-[alpha] molecule, the Ig-[beta] sequence is expressed at apparently similar levels in terminally differentiated, mIg[sup [minus

  17. Two putative subunits of a peptide pump encoded in the human major histocompatability complex class 2 region

    SciTech Connect

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

    1991-11-15

    The class 2 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 1 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 1 molecules assemble. A second transporter gene, PSF2, was identified 10 kilobases (kb) from PSF1, near the class 2 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 1 molecules, although a distinct function of PSF2 cannot be ruled out.

  18. Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex.

    PubMed

    Fritzler, M J; Hamel, J C; Ochs, R L; Chan, E K

    1993-07-01

    Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library. Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins. PMID:8315394

  19. 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

  20. 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.

  1. 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.

  2. Cloning of the cDNA for the human ATP synthase OSCP subunit (ATP5O) by exon trapping and mapping to chromosome 21q22.1-q22.2

    SciTech Connect

    Chen, Haiming; Morris, M.A.; Rossier, C.

    1995-08-10

    Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP5O gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP5O subunit is a key structural component of the stalk of the mitochondrial respiratory chain F{sub 1}F{sub 0}-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21). 39 refs., 5 figs.

  3. Expression of human. alpha. sub 2 -macroglobulin cDNA in baby hamster kidney fibroblasts: Secretion of high levels of active. alpha. sub 2 -macroglobulin

    SciTech Connect

    Boel, E.; Mortensen, S.B. ); Kristensen, T.; Sottrup-Jensen, L. ); Petersen, C.M. )

    1990-05-01

    Human {alpha}{sub 2}-macroglobulin ({alpha}{sub 2}M) is a unique 720-kDa proteinase inhibitor with a broad specificity. Unlike most other proteinase inhibitors, it does not inhibit proteolytic activity by blocking the active site of the proteinase. During complex formation with a proteinase {alpha}{sub 2}M entraps the proteinase molecule in a reaction that involves large conformational changes in {alpha}{sub 2}M. The authors describe the molecular cloning of {alpha}{sub 2}M cDNA from the human hepatoblastoma cell line HepG2. The cDNA was subcloned under control of the adenovirus major late promoter in a mammalian expression vector and introduced into the baby hamster kidney (BHK) cell line. Transformed clones were isolated and tested for production of human {alpha}{sub 2}M with a specific enzyme-linked immunosorbent assay. Human recombinant {alpha}{sub 2}M (r{alpha}{sub 2}M), secreted and purified form isolated transfected BHK cell lines, was structurally and functionally compared to {alpha}{sub 2}M purified from human serum. The results show that r{alpha}{sub 2}M was secreted from the BHK cells as an active proteinase-binding tetramer with functional thiol esters. Cleavage reactions of r{alpha}{sub 2}M with methylamine and trypsin showed that the recombinant product, which was correctly processed at the N-terminus, exhibited molecular characteristics similar to those of the human serum derived reference.

  4. Encoding of Physics Concepts: Concreteness and Presentation Modality Reflected by Human Brain Dynamics

    PubMed Central

    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. PMID:22848602

  5. 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.

  6. 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. PMID:3461449

  7. Structural/functional analysis of the human OXR1 protein: identification of exon 8 as the anti-oxidant encoding function

    PubMed Central

    2012-01-01

    Background The human OXR1 gene belongs to a class of genes with conserved functions that protect cells from reactive oxygen species (ROS). The gene was found using a screen of a human cDNA library by its ability to suppress the spontaneous mutator phenotype of an E. coli mutH nth strain. The function of OXR1 is unknown. The human and yeast genes are induced by oxidative stress and targeted to the mitochondria; the yeast gene is required for resistance to hydrogen peroxide. Multiple spliced isoforms are expressed in a variety of human tissues, including brain. Results In this report, we use a papillation assay that measures spontaneous mutagenesis of an E. coli mutM mutY strain, a host defective for oxidative DNA repair. Papillation frequencies with this strain are dependent upon a G→T transversion in the lacZ gene (a mutation known to occur as a result of oxidative damage) and are suppressed by in vivo expression of human OXR1. N-terminal, C-terminal and internal deletions of the OXR1 gene were constructed and tested for suppression of the mutagenic phenotype of the mutM mutY strain. We find that the TLDc domain, encoded by the final four exons of the OXR1 gene, is not required for papillation suppression in E. coli. Instead, we show that the protein segment encoded by exon 8 of OXR1 is responsible for the suppression of oxidative damage in E. coli. Conclusion The protein segment encoded by OXR1 exon 8 plays an important role in the anti-oxidative function of the human OXR1 protein. This result suggests that the TLDc domain, found in OXR1 exons 12–16 and common in many proteins with nuclear function, has an alternate (undefined) role other than oxidative repair. PMID:22873401

  8. Cloning of the cDNA for a human homologue of the Drosophila white gene and mapping to chromosome 21q22.3

    SciTech Connect

    Haiming Chen; Lalioti, M.D.; Perrin, G.; Antonarakis, S.E.

    1996-07-01

    In an effort to contribute to the transcript map of human chromosome 21 and the understanding of the pathophysiology of trisomy 21, we have used exon trapping to identify fragments of chromosome 21 genes. Two trapped exons, from pools of chromosome 21-specific cosmids, showed homology to the Drosophila white (w) gene. We subsequently cloned the corresponding cDNA for a human homologue of the Drosophila w gene (hW) from human retina and fetal brain cDNA libraries. The gene belongs to the ATP-binding cassette transporter gene family and is homologous to Drosophila w (and to 2 genes from other species) and to a lesser extent to Drosophila brown (bw) and scarlet (st) genes that are all involved in the transport of eye pigment precursor molecules. A DNA polymorphism with 62% heterozygosity due to variation of a poly (T) region in the 3{prime} UTR of the hW has been identified and used for the incorporation of this gene to the genetic map of chromosome 21. The hW is located at 21q22.3 between DNA markers D21S212 and D21S49 in a P1 clone that also contains marker BCEI. The gene is expressed at various levels in many human tissues. The contributions of this gene to the Down syndrome phenotypes, to human eye color, and to the resulting phenotypes of null or missense mutations are presently unknown. 56 refs., 8 figs., 1 tab.

  9. GENCODE: The reference human genome annotation for The ENCODE Project

    PubMed Central

    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.

    2012-01-01

    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. PMID:22955987

  10. 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. PMID:22955987

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

    PubMed Central

    Hirono, A; Beutler, E

    1988-01-01

    Glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) A(-) is a common variant in Blacks that causes sensitivity to drug-and infection-induced hemolytic anemia. A cDNA library was constructed from Epstein-Barr virus-transformed lymphoblastoid cells from a male who was G6PD A(-). One of four cDNA clones isolated contained a sequence not found in the other clones nor in the published cDNA sequence. Consisting of 138 bases and coding 46 amino acids, this segment of cDNA apparently is derived from the alternative splicing involving the 3' end of intron 7. Comparison of the remaining sequences of these clones with the published sequence revealed three nucleotide substitutions: C33----G, G202----A, and A376----G. Each change produces a new restriction site. Genomic DNA from five G6PD A(-) individuals was amplified by the polymerase chain reaction. The base substitution at position 376, identical to the substitution that has been reported in G6PD A(+), was present in all G6PD A(-) samples and none of the control G6PD B(+) samples examined. The substitution at position 202 was found in four of the five G6PD A(-) samples and no normal control sample. At position 33 guanine was found in all G6PD A(-) samples and seven G6PD B(+) control samples and is, presumably, the usual nucleotide found at this position. The finding 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. Images PMID:2836867

  12. Genome-wide expression profiling of 8-chloroadenosine- and 8-chloro-cAMP-treated human neuroblastoma cells using radioactive human cDNA microarray.

    PubMed

    Park, Gil Hong; Choe, Jaegol; Choo, Hyo-Jung; Park, Yun Gyu; Sohn, Jeongwon; Kim, Meyoung-kon

    2002-07-31

    Previous reports raised question as to whether 8-chloro-cyclic adenosine 3,5-monophosphate (8-Cl-cAMP) is a prodrug for its metabolite, 8-Cl-adenosine which exerts growth inhibition in a broad spectrum of cancer cells. The present study was carried out to clarify overall cellular affects of 8-Cl-cAMP and 8-Cl-adenosine on SK-N-DZ human neuroblastoma cells by systematically characterizing gene expression using radioactive human cDNA microarray. Microarray was prepared with PCR-amplified cDNA of 2,304 known genes spotted on nylon membranes, employing (33)P-labeled cDNAs of SK-N-DZ cells as a probe. The expression levels of approximately 100 cDNAs, representing about 8% of the total DNA elements on the array, were altered in 8-Cl-adenosine- or 8-Cl-cAMP-treated cells, respectively. The genome-wide expression of the two samples exhibited partial overlaps; different sets of up-regulated genes but the same set of down-regulated genes. 8-Cl-adenosine treatment up-regulated genes involved in differentiation and development (LIM protein, connexin 26, neogenin, neurofilament triplet L protein and p21(WAF1/CIP1)) and immune response such as natural killer cells protein 4, and down-regulated ones involved in proliferation and transformation (transforming growth factor-beta, DYRK2, urokinase-type plasminogen activator and proteins involved in transcription and translation) which were in close parallel with those by 8-Cl-cAMP. Our results indicated that the two drugs shared common genomic pathways for the down-regulation of certain genes, but used distinct pathways for the up-regulation of different gene clusters. Based on the findings, we suggest that the anti-cancer activity of 8-Cl-cAMP results at least in part through 8-Cl-adenosine. Thus, the systematic use of DNA arrays can provide insight into the dynamic cellular pathways involved in anticancer activities of chemotherapeutics. PMID:12216110

  13. Can visual information encoded in cortical columns be decoded from magnetoencephalography data in humans?

    PubMed

    Cichy, Radoslaw Martin; Ramirez, Fernando Mario; Pantazis, Dimitrios

    2015-11-01

    It is a principal open question whether noninvasive imaging methods in humans can decode information encoded at a spatial scale as fine as the basic functional unit of cortex: cortical columns. We addressed this question in five magnetoencephalography (MEG) experiments by investigating a columnar-level encoded visual feature: contrast edge orientation. We found that MEG signals contained orientation-specific information as early as approximately 50 ms after stimulus onset even when controlling for confounds, such as overrepresentation of particular orientations, stimulus edge interactions, and global form-related signals. Theoretical modeling confirmed the plausibility of this empirical result. An essential consequence of our results is that information encoded in the human brain at the level of cortical columns should in general be accessible by multivariate analysis of electrophysiological signals. PMID:26162550

  14. 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.

  15. Rescue of recombinant Newcastle disease virus from cDNA.

    PubMed

    Ayllon, Juan; García-Sastre, Adolfo; Martínez-Sobrido, Luis

    2013-01-01

    Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae(1), is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA(2-5). Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs. PMID:24145366

  16. Rescue of Recombinant Newcastle Disease Virus from cDNA

    PubMed Central

    Ayllon, Juan; García-Sastre, Adolfo; Martínez-Sobrido, Luis

    2013-01-01

    Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs. PMID:24145366

  17. Perceptual biases are inconsistent with Bayesian encoding of speed in the human visual system.

    PubMed

    Hassan, Omar; Hammett, Stephen T

    2015-01-01

    The notion that Bayesian processes are fundamental to brain function and sensory processing has recently received much support, and a number of Bayesian accounts of how the brain encodes the speed of moving objects have been proposed that challenge earlier mechanistic models. We measured the perceived speed of low contrast patterns at both low (2.5 cd m(-2)) and high (25 cd m(-2)) luminance in order to assess these competing models of how the human visual system encodes speed. At both luminance levels low contrast stimuli are perceptually biased such that they appear slower at slow (< 8 Hz) speeds but faster at higher (16 Hz) speeds. However, we find that the reversal of the perceptual bias from under- to overestimation occurred at slower speeds at low luminance. We also found that the bias was greater at slow speeds at high luminance but greater at fast speeds at low luminance. Moreover, discrimination thresholds were found to be similar at high and low luminance. These findings can be predicted by models in which speed is encoded by the relative activity within two broadly tuned temporal channels but are inconsistent with Bayesian models of speed encoding. We conclude that Bayesian processes cannot adequately account for speed encoding in the human visual system. PMID:25761348

  18. 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

  19. Structural of the class II enzyme of human liver alcohol dehydrogenase: combined cDNA and protein sequence determination of the. pi. subunit

    SciTech Connect

    Hoeoeg, J.O.; von Bahr-Lindstroem, H.; Heden, L.O.; Holmquist, B.; Larsson, K.; Hempel, J.; Vallee, B.L.; Joernvall, H.

    1987-04-07

    The class II enzyme of human liver alcohol dehydrogenase was isolated, carboxymethylated, and cleaved with CNBr and proteolytic enzymes. Sequence analysis of peptides established structures corresponding to the ..pi.. subunit. Two segments from the C-terminal region unique to ..pi.. were selected for synthesis of oligodeoxyribonucleotide probes to screen a human liver cDNA library constructed in plasmid pT4. Sequence analysis of two identical hybridization-positive clones with cDNA inserts of about 2000 nucleotides gave the entire coding region of the ..pi.. subunit, a 61-nucleotide 5' noncoding region and a 741-nucleotide 3' noncoding region containing four possible polyadenylation sites. Translation of the coding region yields a 391-residue polypeptide, which in all regions except the C-terminal segment corresponds to the protein structure as determined directly by peptide analysis. With the class I numbering system, the exception concerns a residue exchange at position 368, the actual C-terminus which is Phe-374 by peptide data but a 12 residue extension by cDNA data, and possibly two further residue exchanges at positions 303 and 312. The size difference might indicate the existence of posttranslational modifications of the mature protein or, in combination with the residue exchanges, the existence of polymorphism at the locus for class II subunits. The ..pi.. subunit analyzed directly results in a 379-residue polypeptide and is the only class II size thus far known to occur in the mature protein. Comparison of the ..pi.. structure with those of the class I subunits (..cap alpha.., ..beta.., and ..gamma..) reveals a homology with extensive differences. Large variations in segments affecting relationships at the active site and the area of subunit interactions account for the significant alterations of enzymatic specificities and other properties that differentiate class II from class I enzymes.

  20. Generation of a panel of antibodies against proteins encoded on human chromosome 21

    PubMed Central

    2010-01-01

    Background Down syndrome (DS) is caused by trisomy of all or part of chromosome 21. To further understanding of DS we are working with a mouse model, the Tc1 mouse, which carries most of human chromosome 21 in addition to the normal mouse chromosome complement. This mouse is a model for human DS and recapitulates many of the features of the human syndrome such as specific heart defects, and cerebellar neuronal loss. The Tc1 mouse is mosaic for the human chromosome such that not all cells in the model carry it. Thus to help our investigations we aimed to develop a method to identify cells that carry human chromosome 21 in the Tc1 mouse. To this end, we have generated a panel of antibodies raised against proteins encoded by genes on human chromosome 21 that are known to be expressed in the adult brain of Tc1 mice Results We attempted to generate human specific antibodies against proteins encoded by human chromosome 21. We selected proteins that are expressed in the adult brain of Tc1 mice and contain regions of moderate/low homology with the mouse ortholog. We produced antibodies to seven human chromosome 21 encoded proteins. Of these, we successfully generated three antibodies that preferentially recognise human compared with mouse SOD1 and RRP1 proteins on western blots. However, these antibodies did not specifically label cells which carry a freely segregating copy of Hsa21 in the brains of our Tc1 mouse model of DS. Conclusions Although we have successfully isolated new antibodies to SOD1 and RRP1 for use on western blots, in our hands these antibodies have not been successfully used for immunohistochemistry studies. These antibodies are freely available to other researchers. Our data high-light the technical difficulty of producing species-specific antibodies for both western blotting and immunohistochemistry. PMID:20727138

  1. 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. PMID:25679982

  2. 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

  3. 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

  4. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase.

    PubMed

    Bokar, J A; Shambaugh, M E; Polayes, D; Matera, A G; Rottman, F M

    1997-11-01

    The methylation of internal adenosine residues in eukaryotic mRNA, forming N6-methyladenosine (m6A), is catalyzed by a complex multicomponent enzyme. Previous studies suggested that m6A affects the efficiency of mRNA processing or transport, although the mechanism by which this occurs is not known. As a step toward better understanding the mechanism and function of this ubiquitous posttranscriptional modification, we have shown that HeLa mRNA (N6-adenosine)-methyltransferase requires at least two separate protein factors, MT-A and MT-B, and MT-A contains the AdoMet binding site on a 70-kDa subunit (MT-A70). MT-A70 was purified by conventional chromatography and electrophoresis, and was microsequenced. The peptide sequence was used to design a degenerate oligodeoxynucleotide that in turn was used to isolate the cDNA clone coding for MT-A70 from a HeLa cDNA library. Recombinant MT-A70 was expressed as a fusion protein in bacteria and was used to generate anti-MT-A70 antisera in rabbits. These antisera recognize MT-A70 in HeLa nuclear extracts by western blot and are capable of depleting (N6-adenosine)-methyltransferase activity from HeLa nuclear extract, confirming that MT-A70 is a critical subunit of (N6-adenosine)-methyltransferase. Northern blot analysis reveals that MT-A70 mRNA is present in a wide variety of human tissues and may undergo alternative splicing. MT-A70 cDNA probe hybridizes to a 2.0-kilobase (kb) polyadenylated RNA isolated from HeLa cells, whereas it hybridizes to two predominant RNA species (approximately 2.0 kb and 3.0 kb) using mRNA isolated from six different human tissues. Analysis of the cDNA sequence indicates that it codes for a 580-amino acid protein with a predicted MW = 65 kDa. The predicted protein contains sequences similar to consensus methylation motifs I and II identified in prokaryotic DNA (N6-adenosine)-methyltransferases, suggesting the functional conservation of peptide motifs. MT-A70 also contains a long region of homology to

  5. Characterization of novel Leishmania infantum recombinant proteins encoded by genes from five families with distinct capacities for serodiagnosis of canine and human visceral leishmaniasis.

    PubMed

    Oliveira, Geraldo G S; Magalhães, Franklin B; Teixeira, Márcia C A; Pereira, Andrea M; Pinheiro, Cristiane G M; Santos, Lenita R; Nascimento, Marília B; Bedor, Cheila N G; Albuquerque, Alessandra L; dos-Santos, Washington L C; Gomes, Yara M; Moreira, Edson D; Brito, Maria E F; Pontes de Carvalho, Lain C; de Melo Neto, Osvaldo P

    2011-12-01

    To expand the available panel of recombinant proteins that can be useful for identifying Leishmania-infected dogs and for diagnosing human visceral leishmaniasis (VL), we selected recombinant antigens from L. infantum, cDNA, and genomic libraries by using pools of serum samples from infected dogs and humans. The selected DNA fragments encoded homologs of a cytoplasmic heat-shock protein 70, a kinesin, a polyubiquitin, and two novel hypothetical proteins. Histidine-tagged recombinant proteins were produced after subcloning these DNA fragments and evaluated by using an enzyme-linked immunosorbent assays with panels of canine and human serum samples. The enzyme-linked immunosorbent assays with different recombinant proteins had different sensitivities (67.4-93.0% and 36.4-97.2%) and specificities (76.1-100% and 90.4-97.3%) when tested with serum samples from Leishmania-infected dogs and human patients with VL. Overall, no single recombinant antigen was sufficient to serodiagnosis all canine or human VL cases. PMID:22144438

  6. Characterisation of immune responses and protective efficacy in mice after immunisation with Rift Valley Fever virus cDNA constructs

    PubMed Central

    Lagerqvist, Nina; Näslund, Jonas; Lundkvist, Åke; Bouloy, Michèle; Ahlm, Clas; Bucht, Göran

    2009-01-01

    Background Affecting both livestock and humans, Rift Valley Fever is considered as one of the most important viral zoonoses in Africa. However, no licensed vaccines or effective treatments are yet available for human use. Naked DNA vaccines are an interesting approach since the virus is highly infectious and existing attenuated Rift Valley Fever virus vaccine strains display adverse effects in animal trials. In this study, gene-gun immunisations with cDNA encoding structural proteins of the Rift Valley Fever virus were evaluated in mice. The induced immune responses were analysed for the ability to protect mice against virus challenge. Results Immunisation with cDNA encoding the nucleocapsid protein induced strong humoral and lymphocyte proliferative immune responses, and virus neutralising antibodies were acquired after vaccination with cDNA encoding the glycoproteins. Even though complete protection was not achieved by genetic immunisation, four out of eight, and five out of eight mice vaccinated with cDNA encoding the nucleocapsid protein or the glycoproteins, respectively, displayed no clinical signs of infection after challenge. In contrast, all fourteen control animals displayed clinical manifestations of Rift Valley Fever after challenge. Conclusion The appearance of Rift Valley Fever associated clinical signs were significantly decreased among the DNA vaccinated mice and further adjustment of this strategy may result in full protection against Rift Valley Fever. PMID:19149901

  7. 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

  8. 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

    2016-01-01

    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

  9. 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

  10. cDNA cloning, expression and activity of a second human aflatoxin B1-metabolizing member of the aldo-keto reductase superfamily, AKR7A3.

    PubMed

    Knight, L P; Primiano, T; Groopman, J D; Kensler, T W; Sutter, T R

    1999-07-01

    The aflatoxin B1 (AFB1) aldehyde metabolite of AFB1 may contribute to the cytotoxicity of this hepatocarcinogen via protein adduction. Aflatoxin B1 aldehyde reductases, specifically the NADPH-dependent aldo-keto reductases of rat (AKR7A1) and human (AKR7A2), are known to metabolize the AFB1 dihydrodiol by forming AFB1 dialcohol. Using a rat AKR7A1 cDNA, we isolated and characterized a distinct aldo-keto reductase (AKR7A3) from an adult human liver cDNA library. The deduced amino acid sequence of AKR7A3 shares 80 and 88% identity with rat AKR7A1 and human AKR7A2, respectively. Recombinant rat AKR7A1 and human AKR7A3 were expressed and purified from Escherichia coli as hexa-histidine tagged fusion proteins. These proteins catalyzed the reduction of several model carbonyl-containing substrates. The NADPH-dependent formation of AFB1 dialcohol by recombinant human AKR7A3 was confirmed by liquid chromatography coupled to electrospray ionization mass spectrometry. Rabbit polyclonal antibodies produced using recombinant rat AKR7A1 protein were shown to detect nanogram amounts of rat and human AKR7A protein. The amount of AKR7A-related protein in hepatic cytosols of 1, 2-dithiole-3-thione-treated rats was 18-fold greater than in cytosols from untreated animals. These antibodies detected AKR7A-related protein in normal human liver samples ranging from 0.3 to 0.8 microg/mg cytosolic protein. Northern blot analysis showed varying levels of expression of AKR7A RNA in human liver and in several extrahepatic tissues, with relatively high levels in the stomach, pancreas, kidney and liver. Based on the kinetic parameters determined using recombinant human AKR7A3 and AFB1 dihydrodiol at pH 7.4, the catalytic efficiency of this reaction (k2/K, per M/s) equals or exceeds those reported for other enzymes, for example cytochrome P450s and glutathione S-transferases, known to metabolize AFB1 in vivo. These findings indicate that, depending on the extent of AFB1 dihydrodiol formation, AKR

  11. Population-level expression variability of mitochondrial DNA-encoded genes in humans

    PubMed Central

    Wang, Gang; Yang, Ence; Mandhan, Ishita; Brinkmeyer-Langford, Candice L; Cai, James J

    2014-01-01

    Human mitochondria contain multiple copies of a circular genome made up of double-stranded DNA (mtDNA) that encodes proteins involved in cellular respiration. Transcript abundance of mtDNA-encoded genes varies between human individuals, yet the level of variation in the general population has not been systematically assessed. In the present study, we revisited large-scale RNA sequencing data generated from lymphoblastoid cell lines of HapMap samples of European and African ancestry to estimate transcript abundance and quantify expression variation for mtDNA-encoded genes. In both populations, we detected up to over 100-fold difference in mtDNA gene expression between individuals. The marked variation was not due to differences in mtDNA copy number between individuals, but was shaped by the transcription of hundreds of nuclear genes. Many of these nuclear genes were co-expressed with one another, resulting in a module-enriched co-expression network. Significant correlations in expression between genes of the mtDNA and nuclear genomes were used to identify factors involved with the regulation of mitochondrial functions. In conclusion, we determined the baseline amount of variability in mtDNA gene expression in general human populations and cataloged a complete set of nuclear genes whose expression levels are correlated with those of mtDNA-encoded genes. Our findings will enable the integration of information from both mtDNA and nuclear genetic systems, and facilitate the discovery of novel regulatory pathways involving mitochondrial functions. PMID:24398800

  12. 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.

  13. Structural organization of the human gene (LMNB1) encoding nuclear lamin B1

    SciTech Connect

    Lin, F.; Worman, H.J.

    1995-05-20

    The authors have determined the structural organization of the human gene (LMNB1) that encodes nuclear lamin B1, an intermediate filament protein of the nuclear envelope. The transcription unit spans more than 45 kb and the transcription start site is 348 nucleotides upstream from the translation initiation codon. Lamin B1 is encoded by 11 exons. Exon 1 codes for the amino-terminal head domain and the first portion of the central rod domain, exons 2 through 6 the central rod domain, and exons 7 through 11 the carboxyl-terminal tail domain of this intermediate filament protein. Intron positions are conserved in other lamin genes from frogs, mice, and humans but different in lamin genes from Drosophila melanogaster and Caenorhabditis elegans. In the region encoding the central rod domain, intron positions are also similar to those in the gene for an invertebrate nonneuronal cytoplasmic intermediate filament protein and the genes for most vertebrate cytoplasmic intermediate filament proteins except neurofilaments and nestin. 51 refs., 3 figs.

  14. The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains

    PubMed Central

    Srivastava, Anand K.; Pispa, Johanna; Hartung, Andrew J.; Du, Yangzhu; Ezer, Sini; Jenks, Ted; Shimada, Tokihiko; Pekkanen, Maija; Mikkola, Marja L.; Ko, Minoru S. H.; Thesleff, Irma; Kere, Juha; Schlessinger, David

    1997-01-01

    Mouse Tabby (Ta) and X chromosome-linked human EDA share the features of hypoplastic hair, teeth, and eccrine sweat glands. We have cloned the Ta gene and find it to be homologous to the EDA gene. The gene is altered in two Ta alleles with a point mutation or a deletion. The gene is expressed in developing teeth and epidermis; no expression is seen in corresponding tissues from Ta mice. Ta and EDA genes both encode alternatively spliced forms; novel exons now extend the 3′ end of the EDA gene. All transcripts recovered have the same 5′ exon. The longest Ta cDNA encodes a 391-residue transmembrane protein, ectodysplasin-A, containing 19 Gly-Xaa-Yaa repeats. The isoforms of ectodysplasin-A may correlate with differential roles during embryonic development. PMID:9371801

  15. The Novelty of Human Cancer/Testis Antigen Encoding Genes in Evolution

    PubMed Central

    Dobrynin, Pavel; Matyunina, Ekaterina; Malov, S. V.; Kozlov, A. P.

    2013-01-01

    In order to be inherited in progeny generations, novel genes should originate in germ cells. Here, we suggest that the testes may play a special “catalyst” role in the birth and evolution of new genes. Cancer/testis antigen encoding genes (CT genes) are predominantly expressed both in testes and in a variety of tumors. By the criteria of evolutionary novelty, the CT genes are, indeed, novel genes. We performed homology searches for sequences similar to human CT in various animals and established that most of the CT genes are either found in humans only or are relatively recent in their origin. A majority of all human CT genes originated during or after the origin of Eutheria. These results suggest relatively recent origin of human CT genes and align with the hypothesis of the special role of the testes in the evolution of the gene families. PMID:23691492

  16. Genetically encoded impairment of neuronal KCC2 cotransporter function in human idiopathic generalized epilepsy

    PubMed Central

    Kahle, Kristopher T; Merner, Nancy D; Friedel, Perrine; Silayeva, Liliya; Liang, Bo; Khanna, Arjun; Shang, Yuze; Lachance-Touchette, Pamela; Bourassa, Cynthia; Levert, Annie; Dion, Patrick A; Walcott, Brian; Spiegelman, Dan; Dionne-Laporte, Alexandre; Hodgkinson, Alan; Awadalla, Philip; Nikbakht, Hamid; Majewski, Jacek; Cossette, Patrick; Deeb, Tarek Z; Moss, Stephen J; Medina, Igor; Rouleau, Guy A

    2014-01-01

    The KCC2 cotransporter establishes the low neuronal Cl− levels required for GABAA and glycine (Gly) receptor-mediated inhibition, and KCC2 deficiency in model organisms results in network hyperexcitability. However, no mutations in KCC2 have been documented in human disease. Here, we report two non-synonymous functional variants in human KCC2, R952H and R1049C, exhibiting clear statistical association with idiopathic generalized epilepsy (IGE). These variants reside in conserved residues in the KCC2 cytoplasmic C-terminus, exhibit significantly impaired Cl−-extrusion capacities resulting in less hyperpolarized Gly equilibrium potentials (EGly), and impair KCC2 stimulatory phosphorylation at serine 940, a key regulatory site. These data describe a novel KCC2 variant significantly associated with a human disease and suggest genetically encoded impairment of KCC2 functional regulation may be a risk factor for the development of human IGE. PMID:24928908

  17. Discovery of Human sORF-Encoded Polypeptides (SEPs) in Cell Lines and Tissue

    PubMed Central

    2015-01-01

    The existence of nonannotated protein-coding human short open reading frames (sORFs) has been revealed through the direct detection of their sORF-encoded polypeptide (SEP) products. The discovery of novel SEPs increases the size of the genome and the proteome and provides insights into the molecular biology of mammalian cells, such as the prevalent usage of non-AUG start codons. Through modifications of the existing SEP-discovery workflow, we discover an additional 195 SEPs in K562 cells and extend this methodology to identify novel human SEPs in additional cell lines and human tissue for a final tally of 237 new SEPs. These results continue to expand the human genome and proteome and demonstrate that SEPs are a ubiquitous class of nonannotated polypeptides that require further investigation. PMID:24490786

  18. Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast.

    PubMed

    Shiosaki, K; Takata, K; Omichi, K; Tomita, N; Horii, A; Ogawa, M; Matsubara, K

    1990-05-14

    A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes. PMID:2197187

  19. 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

    2016-01-01

    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. PMID:26880543

  20. 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.

  1. Expression of active iron regulatory factor from a full-length human cDNA by in vitro transcription/translation.

    PubMed Central

    Hirling, H; Emery-Goodman, A; Thompson, N; Neupert, B; Seiser, C; Kühn, L C

    1992-01-01

    Iron regulatory factor (IRF), also called iron responsive element-binding protein (IRE-BP), is a cytoplasmic RNA-binding protein which regulates post-transcriptionally transferrin receptor mRNA stability and ferritin mRNA translation. By using the polymerase chain reaction (PCR) and the sequence published by Rouault et al. (1990) a probe was derived which permitted the isolation of three human IRF cDNA clones. Hybridization to genomic DNA and mRNA, as well as sequencing data indicated a single copy gene of about 40 kb specifying a 4.0 kb mRNA that translates into a protein of 98,400 dalton. By in vitro transcription of a assembled IRF cDNA coupled to in vitro translation in a wheat germ extract, we obtained full sized IRF that bound specifically to a human ferritin IRE. In vitro translated IRF retained sensitivity to sulfhydryl oxidation by diamide and could be reactivated by beta-mercaptoethanol in the same way as native placental IRF. An IRF deletion mutant shortened by 132 amino acids at the COOH-terminus was no longer able to bind to an IRE, indicating that this region of the protein plays a role in RNA recognition. Placental IRF has previously been shown to migrate as a doublet on SDS-polyacrylamide gels. After V8 protease digestion the heterogeneity was located in a 65/70 kDa NH2-terminal doublet. The liberated 31 kDa COOH-terminal polypeptide was found to be homogeneous by amino acid sequencing supporting the conclusion of a single IRF gene. Images PMID:1738601

  2. Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer

    SciTech Connect

    Moscow, J.A.; Townsend, A.J.; Goldsmith, M.E.; Whang-Peng, J.; Vickers, P.J.; Poisson, R.; Legault-Poisson, S.; Myers, C.E.; Cowan, K.H.

    1988-09-01

    The development of multidrug resistance in MCF7 human breast cancer cells is associated with overexpression of P-glycoprotein, changes in activities of several detoxication enzymes, and loss of hormone sensitivity and estrogen receptors (ERs). The authors have cloned the cDNA for one of the drug-detoxifying enzymes overexpressed in multidrug-resistant MCF7 cells (Adr/sup R/ MCF7), the anionic isozyme of glutathione S-transferase (GST/pi/). Hybridization with this GST/pi/ cDNA, GST/pi/-1, demonstrated that increased GST/pi/ activity in Adr/sup R/ MCF7 cells is associated with overexpression but not with amplification of the gene. They mapped the GST/pi/ gene to human chromosome 11q13 by in situ hybridization. Since multidrug resistance and GST/pi/ overexpression are associated with the loss of ERs in Adr/sup R/ MCF7 cells, they examined several other breast cancer cell lines that were not selected for drug resistance. In each of these cell lines they found an inverse association between GST/pi/ expression and ER content. They also examined RNA from 21 primary breast cancers and found a similar association between GST/pi/ expression and ER content in vivo. The finding of similar patterns of expression of a drug-detoxifying enzyme and of ERs in vitro as well as in vivo suggests that ER-negative breast cancer cells may have greater protection against antineoplastic agents conferred by GST/pi/ than ER-positive tumors.

  3. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

    PubMed

    Blatt, C; Eversole-Cire, P; Cohn, V H; Zollman, S; Fournier, R E; Mohandas, L T; Nesbitt, M; Lugo, T; Jones, D T; Reed, R R

    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 extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins. PMID:2902634

  4. Validation of 4-nitrophenol as an in vitro substrate probe for human liver CYP2E1 using cDNA expression and microsomal kinetic techniques.

    PubMed

    Tassaneeyakul, W; Veronese, M E; Birkett, D J; Gonzalez, F J; Miners, J O

    1993-12-01

    The involvement of human cytochrome P450 (CYP) 2E1 in the hydroxylation of 4-nitrophenol (4NP) to 4-nitrocatechol (4NC) has been investigated using cDNA expression and liver microsomal kinetic and inhibitor techniques. 4NP hydroxylation by human liver microsomes and cDNA-expressed human CYP2E1 exhibited Michaelis-Menten kinetics; the respective apparent Km values were 30 +/- 7 and 21 microM. Mutual competitive inhibition was observed for 4NP and chlorzoxazone (CZ) (an alternative human CYP2E1 substrate) in liver microsomes, with close similarities between the calculated apparent Km and Ki values for each individual compound. 4NP and CZ hydroxylase activities in microsomes from 18 liver donors varied to a similar extent (3.3- and 3.0-fold, respectively) and 4NP hydroxylase activity correlated significantly (rs > or = 0.75, P < 0.005) with both CZ hydroxylation and immunoreactive CYP2E1 content. The prototypic CYP2E1 inhibitor, diethyldithiocarbamate, was a potent inhibitor of 4NC formation and decreased 4NP hydroxylation by cDNA-expressed CYP2E1 and human liver microsomes in parallel. Probes for other human CYP isoforms namely (alpha-naphthoflavone, coumarin, sulphaphenazole, quinidine, troleandomycin and mephenytoin) caused < 15% inhibition of liver microsomal 4NP hydroxylation. These data confirm that, as in animal species, 4NP hydroxylation is catalysed largely by CYP2E1 in human liver and 4NP may therefore be used as an in vitro substrate probe for the human enzyme. PMID:8267647

  5. 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.

  6. Isolation, characterization, and mapping of gene encoding dihydrolipoyl succinyltransferase (E2k) of human [alpha]-ketoglutarate dehydrogenase complex

    SciTech Connect

    Ali, G.; Cai, Xingang; Sheu, Kwan-Fu R.; Blass, J.P. ); Wasco, W.; Gaston, S.M.; Tanzi, R.E.; Cooper, A.J.L.; Gusella, J.F. ); Szabo, P. )

    1994-03-01

    The authors have isolated and sequenced cDNAs representing the full-length (2987-bp) gene for dihydrolipoyl succinyltransferase (E2k component) of the human [alpha]-ketoglutarate dehydrogenase complex (KHDHC) from a human fetal brain cDNA library. The E2k cDNA was mapped to human chromosome 14 using a somatic cell hybrid panel, and more precisely to band 14q24.3 by in situ hybridization. This cDNA also cross-hybridized to an apparent E2k pseudogene on chromosome 1p31. Northern analysis revealed the E2k gene to be ubiquitously expressed in peripheral tissues and brain. Interestingly, chromosome 14q24.3 has recently been reported to contain gene defects for an early-onset form of familial Alzheimer's disease and for Machado-Joseph disease. Future studies will be necessary to determine whether the E2K gene plays a role in either of these two disorders.

  7. 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.

  8. Promoter for the human ferritin heavy chain-encoding gene (FERH): structural and functional characterization.

    PubMed

    Bevilacqua, M A; Giordano, M; D'Agostino, P; Santoro, C; Cimino, F; Costanzo, F

    1992-02-15

    We conducted a functional analysis of the promoter for the human ferritin heavy chain-encoding gene (pFERH) in HepG2 and HeLa cells. The activity of pFERH is equivalent in both cell types, despite their different ferritin (Fer) isotypes. Transfections of a series of 5'-deletion mutants indicate that pFERH activity is essentially dependent on two motifs. One of them, accounting for about 50% of the total transcriptional activity, is recognized by the RNA polymerase II transcription factor, Sp1, and the other by a low-affinity factor present in both the cell types analyzed. PMID:1541403

  9. Epistatic interaction of genetic depression risk variants in the human subgenual cingulate cortex during memory encoding

    PubMed Central

    Schott, B H; Assmann, A; Schmierer, P; Soch, J; Erk, S; Garbusow, M; Mohnke, S; Pöhland, L; Romanczuk-Seiferth, N; Barman, A; Wüstenberg, T; Haddad, L; Grimm, O; Witt, S; Richter, S; Klein, M; Schütze, H; Mühleisen, T W; Cichon, S; Rietschel, M; Noethen, M M; Tost, H; Gundelfinger, E D; Düzel, E; Heinz, A; Meyer-Lindenberg, A; Seidenbecher, C I; Walter, H

    2014-01-01

    Recent genome-wide association studies have pointed to single-nucleotide polymorphisms (SNPs) in genes encoding the neuronal calcium channel CaV1.2 (CACNA1C; rs1006737) and the presynaptic active zone protein Piccolo (PCLO; rs2522833) as risk factors for affective disorders, particularly major depression. Previous neuroimaging studies of depression-related endophenotypes have highlighted the role of the subgenual cingulate cortex (CG25) in negative mood and depressive psychopathology. Here, we aimed to assess how recently associated PCLO and CACNA1C depression risk alleles jointly affect memory-related CG25 activity as an intermediate phenotype in clinically healthy humans. To investigate the combined effects of rs1006737 and rs2522833 on the CG25 response, we conducted three functional magnetic resonance imaging studies of episodic memory formation in three independent cohorts (N=79, 300, 113). An epistatic interaction of PCLO and CACNA1C risk alleles in CG25 during memory encoding was observed in all groups, with carriers of no risk allele and of both risk alleles showing higher CG25 activation during encoding when compared with carriers of only one risk allele. Moreover, PCLO risk allele carriers showed lower memory performance and reduced encoding-related hippocampal activation. In summary, our results point to region-specific epistatic effects of PCLO and CACNA1C risk variants in CG25, potentially related to episodic memory. Our data further suggest that genetic risk factors on the SNP level do not necessarily have additive effects but may show complex interactions. Such epistatic interactions might contribute to the ‘missing heritability' of complex phenotypes. PMID:24643163

  10. Expression of modified gene encoding functional human alpha-1-antitrypsin protein in transgenic tomato plants.

    PubMed

    Agarwal, Saurabh; Singh, Rahul; Sanyal, Indraneel; Amla, D V

    2008-10-01

    Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human alpha-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5' and 3' flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5' while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3' end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around approximately 50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli ( approximately 45 kDa). Our results demonstrate

  11. The porcine gene TBP10 encodes a protein homologous to the human tat-binding protein/26S protease subunit family.

    PubMed

    Leeb, T; Rettenberger, G; Breech, J; Hameister, H; Brenig, B

    1996-03-01

    We have cloned a porcine gene, designated TBP1O, that belongs to the Tat-binding protein/26S protease subunit family. The genomic structure of the porcine TBP1O gene was analyzed after isolation of three overlapping genomic phage lambda clones. The TBP10 gene harbors 12 exons spanning 4.5 kb of chromosomal DNA. The TBP1O gene was assigned to Chromosome (Chr) 12 by fluorescence in situ hybridization (FISH) on metaphase chromosomes. The chromosomal location was confirmed by PCR analysis of a porcine-rodent hybrid cell panel. The TBP1O protein is encoded by a 1221 nucleotide cDNA and has a molecular mass of 45.6 kDa. The predicted amino acid sequence has highest similarity to the human and bovine p45 subunit of the 26S protease and the human transcription factor TRIP1. Further similarities were detected to the slime mold protein DdTBP1O and the Schizosaccharomyces pombe and Saccharomyces cerevisiae protein SUG1. Like DdTBP1O and other members of the protein family, the porcine TBP1O harbors a leucine zipper motif in the N-terminal region and a domain characteristics of ATP-dependent proteases in the C-terminal region. PMID:8833236

  12. Insulin receptor-like ectodomain genes and splice variants are found in both arthropods and human brain cDNA

    PubMed Central

    VÄSTERMARK, Åke; RASK-ANDERSEN, Mathias; SAWANT, Rahul S.; REITER, Jill L.; SCHIÖTH, Helgi B.; WILLIAMS, Michael J.

    2016-01-01

    Truncated receptor ectodomains have been described for several classes of cell surface receptors, including those that bind to growth factors, cytokines, immunoglobulins, and adhesion molecules. Soluble receptor isoforms are typically generated by proteolytic cleavage of the cell surface receptor or by alternative splicing of RNA transcripts arising from the same gene encoding the full-length receptor. Both the epidermal growth factor receptor (EGFR) and the insulin receptor (INSR) families produce soluble receptor splice variants in vertebrates and truncated forms of insulin receptor-like sequences have previously been described in Drosophila. The EGFR and INSR ectodomains share significant sequence homology with each other suggestive of a common evolutionary origin. We discovered novel truncated insulin receptor-like variants in several arthropod species. We performed a phylogenetic analysis of the conserved extracellular receptor L1 and L2 subdomains in invertebrate species. While the segregation of insulin receptor-like L1 and L2 domains indicated that an internal domain duplication had occurred only once, the generation of truncated insulin receptor-like sequences has occurred multiple times. The significance of this work is the previously unknown and widespread occurrence of truncated isoforms in arthropods, signifying that these isoforms play an important functional role, potentially related to such isoforms in mammals. PMID:27375681

  13. Interaction between the human cytomegalovirus‑encoded UL142 and cellular Snapin proteins.

    PubMed

    Liu, Chang; Qi, Ying; Ma, Yanping; He, Rong; Sun, Zhengrong; Huang, Yujing; Ji, Yaohua; Ruan, Qiang

    2015-02-01

    Human cytomegalovirus (HCMV) infection can cause severe illness in immunocompromised and immunodeficient individuals. As a novel HCMV‑encoded major histocompatibility complex class I‑related molecule, the UL142‑encoded protein (pUL142) is capable of suppressing natural killer (NK) cell recognition in the course of infection. However, no host factors that directly interact with HCMV pUL142 have been reported so far. In order to understand the interactions between HCMV pUL142 and host proteins, the current study used yeast two‑hybrid screening, a GST pull‑down assay and an immunofluorescence assay. A host protein, the SNARE‑associated protein Snapin, was identified to directly interact and colocalize with HCMV pUL142 in transfected human embryonic kidney‑293 cells. Snapin is abundantly expressed in the majority of cells and mediates the release of neurotransmitters through vesicular transport in the nervous system and vesicle fusion in non‑neuronal cells. It is hypothesized that HCMV pUL142 may have an impact on the neurotransmitter release process and viral dissemination via interaction with Snapin. PMID:25369979

  14. Yeast RAD14 and human xeroderma pigmentosum group A DNA-repair genes encode homologous proteins.

    PubMed

    Bankmann, M; Prakash, L; Prakash, S

    1992-02-01

    Xeroderma pigmentosum (XP), a human autosomal recessive disorder, is characterized by extreme sensitivity to sunlight and high incidence of skin cancers. XP cells are defective in the incision step of excision repair of DNA damaged by ultraviolet light. Cell fusion studies have defined seven XP complementation groups, XP-A to XP-G. Similar genetic complexity of excision repair is observed in the yeast Saccharomyces cerevisiae. Mutations in any one of five yeast genes, RAD1, RAD2, RAD3, RAD4, and RAD10, cause a total defect in incision and an extreme sensitivity to ultraviolet light. Here we report the characterization of the yeast RAD14 gene. The available rad14 point mutant is only moderately ultraviolet-sensitive, and it performs a substantial amount of incision of damaged DNA. Our studies with the rad14 deletion (delta) mutation indicate an absolute requirement of RAD14 in incision. RAD14 encodes a highly hydrophilic protein of 247 amino acids containing zinc-finger motifs, and it is similar to the protein encoded by the human XPAC gene that complements XP group A cell lines. PMID:1741034

  15. 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

  16. 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

    2015-01-01

    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

  17. Role of Broca's area in encoding sequential human actions: a virtual lesion study.

    PubMed

    Clerget, Emeline; Winderickx, Aline; Fadiga, Luciano; Olivier, Etienne

    2009-10-28

    The exact contribution of Broca's area to motor cognition is still controversial. Here we used repetitive transcranial magnetic stimulation (5 Hz, five pulses) to interfere transiently with the function of left BA44 in 13 healthy individuals; the task consisted of reordering human actions or nonbiological events based on three pictures presented on a computer screen and extracted from a video showing the entire sequence beforehand. We found that a virtual lesion of left BA44 impairs individual performance only for biological actions, and more specifically for object-oriented syntactic actions. Our finding provides evidence that Broca's area plays a crucial role in encoding complex human movements, a process which may be crucial for understanding and/or programming actions. PMID:19809371

  18. 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.

  19. 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.

  20. Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner.

    PubMed Central

    Ney, P A; Andrews, N C; Jane, S M; Safer, B; Purucker, M E; Weremowicz, S; Morton, C C; Goff, S C; Orkin, S H; Nienhuis, A W

    1993-01-01

    The human globin locus control region-binding protein, NF-E2, was purified by DNA affinity chromatography. Its tissue-specific component, p45 NF-E2, was cloned by use of a low-stringency library screen with murine p45 NF-E2 cDNA (N. C. Andrews, H. Erdjument-Bromage, M. B. Davidson, P. Tempst, and S. H. Orkin, Nature [London] 362:722-728, 1993). The human p45 NF-E2 gene was localized to chromosome 12q13 by fluorescent in situ hybridization. Human p45 NF-E2 and murine p45 NF-E2 are highly homologous basic region-leucine zipper (bZIP) proteins with identical DNA-binding domains. Immunoprecipitation experiments demonstrated that p45 NF-E2 is associated in vivo with an 18-kDa protein (p18). Because bZIP proteins bind DNA as dimers, we infer that native NF-E2 must be a heterodimer of 45- and 18-kDa subunits. Although AP-1 and CREB copurified with NF-E2, no evidence was found for heterodimer formation between p45 NF-E2 and proteins other than p18. Thus, p18 appears to be the sole specific partner of p45 NF-E2 in erythroid cells. Cloning of human p45 NF-E2 should permit studies of the role of NF-E2 in globin gene regulation and erythroid differentiation. Images PMID:8355703

  1. Detection of Regulatory SNPs in Human Genome Using ChIP-seq ENCODE Data

    PubMed Central

    Matveeva, Marina Yu.; Shilov, Alexander G.; Kashina, Elena V.; Mordvinov, Viatcheslav A.; Merkulova, Tatyana I.

    2013-01-01

    A vast amount of SNPs derived from genome-wide association studies are represented by non-coding ones, therefore exacerbating the need for effective identification of regulatory SNPs (rSNPs) among them. However, this task remains challenging since the regulatory part of the human genome is annotated much poorly as opposed to coding regions. Here we describe an approach aggregating the whole set of ENCODE ChIP-seq data in order to search for rSNPs, and provide the experimental evidence of its efficiency. Its algorithm is based on the assumption that the enrichment of a genomic region with transcription factor binding loci (ChIP-seq peaks) indicates its regulatory function, and thereby SNPs located in this region are more likely to influence transcription regulation. To ensure that the approach preferably selects functionally meaningful SNPs, we performed enrichment analysis of several human SNP datasets associated with phenotypic manifestations. It was shown that all samples are significantly enriched with SNPs falling into the regions of multiple ChIP-seq peaks as compared with the randomly selected SNPs. For experimental verification, 40 SNPs falling into overlapping regions of at least 7 TF binding loci were selected from OMIM. The effect of SNPs on the binding of the DNA fragments containing them to the nuclear proteins from four human cell lines (HepG2, HeLaS3, HCT-116, and K562) has been tested by EMSA. A radical change in the binding pattern has been observed for 29 SNPs, besides, 6 more SNPs also demonstrated less pronounced changes. Taken together, the results demonstrate the effective way to search for potential rSNPs with the aid of ChIP-seq data provided by ENCODE project. PMID:24205329

  2. 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.

  3. 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.

  4. Segregated encoding of reward-identity and stimulus-reward associations in human orbitofrontal cortex.

    PubMed

    Klein-Flügge, Miriam Cornelia; Barron, Helen Catharine; Brodersen, Kay Henning; Dolan, Raymond J; Behrens, Timothy Edward John

    2013-02-13

    A dominant focus in studies of learning and decision-making is the neural coding of scalar reward value. This emphasis ignores the fact that choices are strongly shaped by a rich representation of potential rewards. Here, using fMRI adaptation, we demonstrate that responses in the human orbitofrontal cortex (OFC) encode a representation of the specific type of food reward predicted by a visual cue. By controlling for value across rewards and by linking each reward with two distinct stimuli, we could test for representations of reward-identity that were independent of associative information. Our results show reward-identity representations in a medial-caudal region of OFC, independent of the associated predictive stimulus. This contrasts with a more rostro-lateral OFC region encoding reward-identity representations tied to the predicate stimulus. This demonstration of adaptation in OFC to reward specific representations opens an avenue for investigation of more complex decision mechanisms that are not immediately accessible in standard analyses, which focus on correlates of average activity. PMID:23407973

  5. 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. PMID:22418393

  6. 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

  7. Four phosphoproteins with common amino termini are encoded by human cytomegalovirus AD169

    SciTech Connect

    Wright, D.A.; Staprans, S.I.; Spector, D.H.

    1988-01-01

    In this report, the authors identify the proteins encoded by the 2.2-kilobase class of early transcripts arising from a region of the strain AD169 human cytomegalovirus genome (map units 0.682 to 0.713) which contains cell-related sequences. These transcripts, encoded by adjacent EcoRI fragments R and d, have a complex spliced structure with 5' and 3' coterminal ends. Antiserum directed against a synthetic 11-amino-acid peptide corresponding to the predicted amino terminus of the proteins was generated and found to immunoprecipitate four-infected-cell proteins of 84, 50, 43, and 34 kilodaltons. These proteins were phosphorylated and were associated predominantly with the nuclei of infected cells. The 43-kilodalton protein was the most abundant of the four proteins, and its level of expression remained relatively constant throughout the infection. Expression of the other proteins increased as the infection progressed. Pulse-chase analysis failed to show a precursor-product relationship between any of the proteins. A comparison of the (/sup 35/S)methionine-labeled tryptic peptide maps of the four proteins from infected cells and an in vitro-generated polypeptide derived from the putative first exon showed that all four infected-cell proteins were of viral origin and contained a common amino-terminal region.

  8. The relationship between transcript expression levels of nuclear encoded (TFAM, NRF1) and mitochondrial encoded (MT-CO1) genes in single human oocytes during oocyte maturation

    PubMed Central

    Novin, M Ghaffari; Allahveisi, A; Noruzinia, M; Farhadifar, F; Yousefian, E; Fard, A Dehghani; Salimi, M

    2015-01-01

    In some cases of infertility in women, human oocytes fail to mature when they reach the metaphase II (MII) stage. Mitochondria plays an important role in oocyte maturation. A large number of mitochondrial DNA (mtDNA), copied in oocytes, is essential for providing adenosine triphosphate (ATP) during oocyte maturation. The purpose of this study was to identify the relationship between transcript expression levels of the mitochondrial encoded gene (MT-CO1) and two nuclear encoded genes, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) in various stages of human oocyte maturation. Nine consenting patients, age 21–35 years old, with male factors were selected for ovarian stimulation and intracytoplasmic sperm injection (ICSI) procedures. mRNA levels of mitochondrial-related genes were performed by singlecell TaqMan® quantitative real-time polymerase chain reaction (qRT-PCR). There was no significant relationship between the relative expression levels in germinal vesicle (GV) stage oocytes (p = 0.62). On the contrary, a significant relationship was seen between the relative expression levels of TFAM and NRF1 and the MT-CO1 genes at the stages of metaphase I (MI) and MII (p = 0.03 and p = 0.002). A relationship exists between the transcript expression levels of TFAM and NRF1, and MT-CO1 genes in various stages of human oocyte maturation. PMID:26929904

  9. 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.

  10. PiggyBac transposon vectors: the tools of the human gene encoding

    PubMed Central

    Zhao, Shuang; Jiang, Enze; Chen, Shuangshuang; Gu, Yuan; Shangguan, Anna Junjie; Lv, Tangfeng

    2016-01-01

    A transposon is a DNA segment, which is able to change its relative position within the entire genome of a cell. The piggyBac (PB) transposon is a movable genetic element that efficiently transposes between vectors and chromosomes through a “cut-and-paste” mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeats (ITRs) sequences located on both ends of the transposon vector and eight efficiently moves the contents from its original positions and efficiently integrates them into TTAA chromosomal sites. PB has drawn much attention because of its transposition efficiency, safety and stability. Due to its priorities, PB can be used as a new genetic vehicle, a new tool for oncogene screening and a new method for gene therapy. PB has created a new outlook for human gene encoding. PMID:26958506

  11. Identification of genes expressed in human CD34+ hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning

    PubMed Central

    Mao, Mao; Fu, Gang; Wu, Ji-Sheng; Zhang, Qing-Hua; Zhou, Jun; Kan, Li-Xin; Huang, Qiu-Hua; He, Kai-Li; Gu, Bai-Wei; Han, Ze-Guang; Shen, Yu; Gu, Jian; Yu, Ya-Ping; Xu, Shu-Hua; Wang, Ya-Xin; Chen, Sai-Juan; Chen, Zhu

    1998-01-01

    Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34+ HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34+ cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2,603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5′ ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation. PMID:9653160

  12. Human ubiquitin-activating enzyme, E1. Indication of potential nuclear and cytoplasmic subpopulations using epitope-tagged cDNA constructs.

    PubMed

    Handley-Gearhart, P M; Stephen, A G; Trausch-Azar, J S; Ciechanover, A; Schwartz, A L

    1994-12-30

    The ubiquitin-activating enzyme E1 catalyzes the first step in the ubiquitin conjugation pathway. Previously, we have cloned and sequenced the cDNA for human E1. Expression of the E1 cDNA in the ts20 cell line, which harbors a thermolabile E1, abrogated the phenotypic defects associated with this line. However, little is known of the cell biology of the E1 protein or the nature of the E1 doublet. Thus, we constructed epitope-tagged E1 cDNAs in which the HA monoclonal antibody epitope tag sequence (from influenza hemagglutinin and recognized by the 12CA5 monoclonal antibody) was fused to the amino terminus of E1. Because the amino-terminal amino acid sequence of E1 is unknown, three constructs were made in which the HA tag was placed at each of the first three ATGs in the open reading frame (HA-1E1, HA-2E1, and HA-3E1). Western analysis of HeLa cells transfected with the constructs revealed that HA-1E1 closely comigrated with the upper band of the E1 doublet, and HA-2E1 comigrated with the lower band of the E1 doublet; HA-3E1 appeared smaller than either of the E1 bands. Metabolic labeling with 32P and immunoprecipitation with anti-HA antibody revealed that only the HA-1E1 protein product is phosphorylated; polyclonal anti-E1 antibody showed that only the upper band of the endogenous E1 doublet is phosphorylated. Each of the constructs was able to rescue the mutant phenotype of the ts20 cell line. Immunofluorescence studies showed that HA-2E1 and HA-3E1 were distributed in the cytoplasm with both negative and positive nuclei. This pattern of distribution has also been observed when immunostaining with a monoclonal antibody to E1 (1C5). However, the staining pattern associated with a polyclonal anti-E1 antibody (JJJ) is characterized by positive staining cytoplasm and nuclei in all cells. The HA-1E1 construct exhibited apparently exclusive nuclear distribution in HeLa cells. The difference between the staining patterns of the polyclonal and monoclonal anti-E1

  13. Human chromosome 16 encodes a factor involved in induction of class II major histocompatibility antigens by interferon gamma.

    PubMed Central

    Bono, M R; Alcaïde-Loridan, C; Couillin, P; Letouzé, B; Grisard, M C; Jouin, H; Fellous, M

    1991-01-01

    Interferon gamma (IFN-gamma) induces expression of class II major histocompatibility complex (MHC)-encoded antigens in immunocompetent cells. To gain further insight into the mechanism of this induction, we prepared somatic cell hybrids between different human cell lines and a murine cell line, RAG, that does not express murine class II MHC antigens before or after treatment with murine IFN-gamma. Some of the resulting cell hybrids express murine class II MHC antigens when treated with murine IFN-gamma. This inducible phenotype is correlated with the presence of human chromosome 16. It has been shown previously that the induction of class I MHC antigens by human IFN-gamma in human-rodent hybrids requires the presence of species-specific factors encoded by chromosome 6, which bears the gene for the human IFN-gamma receptor, and chromosome 21, whose product(s) is necessary for the transduction of human IFN-gamma signals. In this report, we show that the induction of murine class II MHC antigens by human IFN-gamma in the human-RAG cell hybrids requires, likewise, the presence of human chromosomes 6 and 21, in addition to chromosome 16. In some of these hybrids, when all three of these human chromosomes were present, induction of cell-surface HLA-DR antigens was also observed. Our results demonstrate that human chromosome 16 encodes a non-species-specific factor involved in the induction of class II MHC antigens by IFN-gamma. Images PMID:1906174

  14. 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

  15. 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.

  16. Characterization of the cDNA of a broadly reactive neutralizing human anti-gp120 monoclonal antibody.

    PubMed Central

    Marasco, W A; Bagley, J; Zani, C; Posner, M; Cavacini, L; Haseltine, W A; Sodroski, J

    1992-01-01

    The F105 mAb, identified in an HIV-1-infected individual, binds to a discontinuous epitope on the HIV-1 gp120 envelope glycoprotein, blocks the binding of gp120 to the CD4 viral receptor, and neutralizes a broad range of HIV-1 isolates. This study reports the primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chains of the mAb F105. This IgG1k mAb uses a VH gene member of the VH4 gene family (V71-4) and is productively rearranged with a D-D fusion product of the dlr4 and da4 germline DH genes and the JH5 gene. This rearranged heavy chain gene expresses the VH4-HV2a idiotope, which is seen in human monoclonal IgM cold agglutinins. The F105 Vk appears to be derived from the Humvk325 germline gene and is rearranged with a Jk2 gene. For both chains, the mutational pattern in the rearranged VH and VL genes is indicative of an antigen-driven process. These studies show that production of a broadly neutralizing anti-HIV-1 antibody that recognizes determinants within the CD4 recognition site of the envelope glycoprotein is achieved by rearrangement of the V71-4 and Humvk325 germline variable region genes along with selected individual point mutations in the rearranged genes. PMID:1401079

  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. PMID:27530524

  18. 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.

  19. Phosphoproteome of Human Glioblastoma Initiating Cells Reveals Novel Signaling Regulators Encoded by the Transcriptome

    PubMed Central

    Kozuka-Hata, Hiroko; Nasu-Nishimura, Yukiko; Koyama-Nasu, Ryo; Ao-Kondo, Hiroko; Tsumoto, Kouhei; Akiyama, Tetsu; Oyama, Masaaki

    2012-01-01

    Background Glioblastoma is one of the most aggressive tumors with poor prognosis. Although various studies have been performed so far, there are not effective treatments for patients with glioblastoma. Methodology/Principal Findings In order to systematically elucidate the aberrant signaling machinery activated in this malignant brain tumor, we investigated phosphoproteome dynamics of glioblastoma initiating cells using high-resolution nanoflow LC-MS/MS system in combination with SILAC technology. Through phosphopeptide enrichment by titanium dioxide beads, a total of 6,073 phosphopeptides from 2,282 phosphorylated proteins were identified based on the two peptide fragmentation methodologies of collision induced dissociation and higher-energy C-trap dissociation. The SILAC-based quantification described 516 up-regulated and 275 down-regulated phosphorylation sites upon epidermal growth factor stimulation, including the comprehensive status of the phosphorylation sites on stem cell markers such as nestin. Very intriguingly, our in-depth phosphoproteome analysis led to identification of novel phosphorylated molecules encoded by the undefined sequence regions of the human transcripts, one of which was regulated upon external stimulation in human glioblastoma initiating cells. Conclusions/Significance Our result unveils an expanded diversity of the regulatory phosphoproteome defined by the human transcriptome. PMID:22912867

  20. Explicit Encoding of Multimodal Percepts by Single Neurons in the Human Brain

    PubMed Central

    Quiroga, Rodrigo Quian; Kraskov, Alexander; Koch, Christof; Fried, Itzhak

    2010-01-01

    Summary Different pictures of Marilyn Monroe can evoke the same percept, even if greatly modified as in Andy Warhol’s famous portraits. But how does the brain recognize highly variable pictures as the same percept? Various studies have provided insights into how visual information is processed along the “ventral pathway,” via both single-cell recordings in monkeys [1, 2] and functional imaging in humans [3, 4]. Interestingly, in humans, the same “concept” of Marilyn Monroe can be evoked with other stimulus modalities, for instance by hearing or reading her name. Brain imaging studies have identified cortical areas selective to voices [5, 6] and visual word forms [7, 8]. However, how visual, text, and sound information can elicit a unique percept is still largely unknown. By using presentations of pictures and of spoken and written names, we show that (1) single neurons in the human medial temporal lobe (MTL) respond selectively to representations of the same individual across different sensory modalities; (2) the degree of multimodal invariance increases along the hierarchical structure within the MTL; and (3) such neuronal representations can be generated within less than a day or two. These results demonstrate that single neurons can encode percepts in an explicit, selective, and invariant manner, even if evoked by different sensory modalities. PMID:19631538

  1. Explicit encoding of multimodal percepts by single neurons in the human brain.

    PubMed

    Quian Quiroga, Rodrigo; Kraskov, Alexander; Koch, Christof; Fried, Itzhak

    2009-08-11

    Different pictures of Marilyn Monroe can evoke the same percept, even if greatly modified as in Andy Warhol's famous portraits. But how does the brain recognize highly variable pictures as the same percept? Various studies have provided insights into how visual information is processed along the "ventral pathway," via both single-cell recordings in monkeys and functional imaging in humans. Interestingly, in humans, the same "concept" of Marilyn Monroe can be evoked with other stimulus modalities, for instance by hearing or reading her name. Brain imaging studies have identified cortical areas selective to voices and visual word forms. However, how visual, text, and sound information can elicit a unique percept is still largely unknown. By using presentations of pictures and of spoken and written names, we show that (1) single neurons in the human medial temporal lobe (MTL) respond selectively to representations of the same individual across different sensory modalities; (2) the degree of multimodal invariance increases along the hierarchical structure within the MTL; and (3) such neuronal representations can be generated within less than a day or two. These results demonstrate that single neurons can encode percepts in an explicit, selective, and invariant manner, even if evoked by different sensory modalities. PMID:19631538

  2. Pharmacovirological impact of an integrase inhibitor on human immunodeficiency virus type 1 cDNA species in vivo.

    PubMed

    Goffinet, Christine; Allespach, Ina; Oberbremer, Lena; Golden, Pamela L; Foster, Scott A; Johns, Brian A; Weatherhead, Jason G; Novick, Steven J; Chiswell, Karen E; Garvey, Edward P; Keppler, Oliver T

    2009-08-01

    Clinical trials of the first approved integrase inhibitor (INI), raltegravir, have demonstrated a drop in the human immunodeficiency virus type 1 (HIV-1) RNA loads of infected patients that was unexpectedly more rapid than that with a potent reverse transcriptase inhibitor, and apparently dose independent. These clinical outcomes are not understood. In tissue culture, although their inhibition of integration is well documented, the effects of INIs on levels of unintegrated HIV-1 cDNAs have been variable. Furthermore, there has been no report to date on an INI's effect on these episomal species in vivo. Here, we show that prophylactic treatment of transgenic rats with the strand transfer INI GSK501015 reduced levels of viral integrants in the spleen by up to 99.7%. Episomal two-long-terminal-repeat (LTR) circles accumulated up to sevenfold in this secondary lymphoid organ, and this inversely correlated with the impact on the proviral burden. Contrasting raltegravir's dose-ranging study with HIV patients, titration of GSK501015 in HIV-infected animals demonstrated dependence of the INI's antiviral effect on its serum concentration. Furthermore, the in vivo 50% effective concentration calculated from these data best matched GSK501015's in vitro potency when serum protein binding was accounted for. Collectively, this study demonstrates a titratable, antipodal impact of an INI on integrated and episomal HIV-1 cDNAs in vivo. Based on these findings and known biological characteristics of viral episomes, we discuss how integrase inhibition may result in additional indirect antiviral effects that contribute to more rapid HIV-1 decay in HIV/AIDS patients. PMID:19458008

  3. From PREDs and open reading frames to cDNA isolation: revisiting the human chromosome 21 transcription map.

    PubMed

    Reymond, A; Friedli, M; Henrichsen, C N; Chapot, F; Deutsch, S; Ucla, C; Rossier, C; Lyle, R; Guipponi, M; Antonarakis, S E

    2001-11-01

    A supernumerary copy of human chromosome 21 (HC21) causes Down syndrome. To understand the molecular pathogenesis of Down syndrome, it is necessary to identify all HC21 genes. The first annotation of the sequence of 21q confirmed 127 genes, and predicted an additional 98 previously unknown "anonymous" genes (predictions (PREDs) and open reading frames (C21orfs)), which were foreseen by exon prediction programs and/or spliced expressed sequence tags. These putative gene models still need to be confirmed as bona fide transcripts. Here we report the characterization and expression pattern of the putative transcripts C21orf7, C21orf11, C21orf15, C21orf18, C21orf19, C21orf22, C21orf42, C21orf50, C21orf51, C21orf57, and C21orf58, the GC-rich sequence DNA-binding factor candidate GCFC (also known as C21orf66), PRED12, PRED31, PRED34, PRED44, PRED54, and PRED56. Our analysis showed that most of the C21orfs originally defined by matching spliced expressed sequence tags were correctly predicted, whereas many of the PREDs, defined solely by computer prediction, do not correspond to genuine genes. Four of the six PREDs were incorrectly predicted: PRED44 and C21orf11 are portions of the same transcript, PRED31 is a pseudogene, and PRED54 and PRED56 were wrongly predicted. In contrast, PRED12 (now called C21orf68) and PRED34 (C21orf63) are now confirmed transcripts. We identified three new genes, C21orf67, C21orf69, and C21orf70, not previously predicted by any programs. This revision of the HC21 transcriptome has consequences for the entire genome regarding the quality of previous annotations and the total number of transcripts. It also provides new candidates for genes involved in Down syndrome and other genetic disorders that map to HC21. PMID:11707072

  4. Expression of angiostatin cDNA in human gallbladder carcinoma cell line GBC-SD and its effect on endothelial proliferation and growth

    PubMed Central

    Yang, Ding-Zhong; He, Jing; Zhang, Ji-Cheng; Wang, Zuo-Ren

    2006-01-01

    AIM: To explore the influence of angiostatin up-regulation on the biologic behavior of gallbladder carcinoma cells in vitro and in vitro, and the potential value of angiostatin gene therapy for gallbladder carcinoma. METHODS: A eukaryotic expression vector of pcDNA3.1(+) containing murine angiostatin was constructed and identified by restriction endonuclease digestion and sequencing. The recombinant vector pcDNA3.1-angiostatin was transfected into human gallbladder carcinoma cell line GBC-SD with Lipofectamine 2000, and paralleled with the vector and mock control. The resistant clone was screened by G418 filtration. Angiostatin transcription and protein expression were examined by RT-PCR, immunofluorescence and Western-blot. The supernatant was collected to treat endothelial cells. Cell proliferation and growth in vitro were observed under microscope. RESULTS: Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). After 14 d of transfection and selection with G418, macroscopic resistant cell cloning was formed in the experimental group transfected with pcDNA 3.1(+)-angiostatin and vector control. But untreated cells died in the mock control. Angiostatin was detected by RT-PCR and protein expression was detected in the experimental group by immunofluorescence and Western-blot. Cell proliferation and growth in vitro in the three groups were observed respectively under microscope. No significant difference was observed in the growth speed of GBC-SD cells between groups that were transfected with and without angiostatin. After treatment with supernatant, significant differences were observed in endothelial cell (ECV-304) growth in vitro. The cell proliferation and growth were inhibited. CONCLUSION: Angiostatin does not directly inhibit human gallbladder carcinoma cell proliferation and growth in vitro, but the secretion of angiostatin inhabits endothelial cell proliferation and growth. PMID:16718765

  5. Transfection of Human Keratinocytes with Nucleoside-Modified mRNA Encoding CPD-Photolyase to Repair DNA Damage.

    PubMed

    Boros, Gábor; Karikó, Katalin; Muramatsu, Hiromi; Miko, Edit; Emri, Eszter; Hegedűs, Csaba; Emri, Gabriella; Remenyik, Éva

    2016-01-01

    In vitro-synthesized mRNA containing nucleoside modifications has great therapeutical potential to transiently express proteins with physiological importance. One such protein is photolyase which rapidly removes UV-induced DNA damages, but this enzyme is absent in humans. Here, we apply a novel mRNA-based platform to achieve functional nonhuman photolyase production in cultured human keratinocytes. Transfection of nucleoside-modified mRNA encoding photolyase leads to accelerated repair of DNA photolesions in human keratinocytes. PMID:27236802

  6. Celsr1, a neural-specific gene encoding an unusual seven-pass transmembrane receptor, maps to mouse chromosome 15 and human chromosome 22qter.

    PubMed

    Hadjantonakis, A K; Sheward, W J; Harmar, A J; de Galan, L; Hoovers, J M; Little, P F

    1997-10-01

    We have identified Celsr1, a gene that encodes a developmentally regulated vertebrate seven-pass transmembrane protein. The extracellular domain of Celsr1 contains two regions each with homology to distinct classes of well-characterized motifs found in the extra-cellular domains of many cell surface molecules. The most N-terminal region contains a block of contiguous cadherin repeats, and C-terminal to this is a region containing seven epidermal growth factor-like repeats interrupted by two laminin A G-type repeats. Celsr1 is unique in that it contains this combination of repeats coupled to a seven-pass transmembrane domain. As part of the characterization of the Celsr1 gene, we have determined its chromosomal map location in both mouse and human. The European Collaborative Interspecific Backcross (EUCIB) and BXD recombinant inbred strains were used for mapping Celsr1 cDNA clones in the mouse, and fluorescence in situ hybridization was used to map human Celsr1 cosmid clones on metaphase chromosomes. We report that Celsr1 maps to proximal mouse Chromosome 15 and human chromosome 22qter, a region of conserved synteny. Reverse transcriptase-polymerase chain reaction analysis and in situ hybridization were used to determine the spatial restriction of Celsr1 transcripts in adult and embryonic mice. The results presented here extend our previous finding of expression of the Celsr1 receptor in the embryo and show that expression continues into adult life when expression in the brain is localized principally in the ependymal cell layer, choroid plexus, and the area postrema. PMID:9339365

  7. 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.

  8. 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

  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. 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.

  11. Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

    PubMed Central

    Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

    2015-01-01

    Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

  12. 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.

  13. Structure and chromosomal localization of the gene (BDKRB2) encoding human bradykinin B{sub 2} receptor

    SciTech Connect

    Jian-Xing Ma; Dan-Zhao Wang; Limei Chen

    1994-09-15

    The bradykinin B{sub 2} receptor (BDKRB2) has high affinity for the intact kinins, which mediate a wide spectrum of biological effects, including pain, inflammation, vasodilation, and smooth muscle contraction and relaxation. In the present study, the authors have cloned and sequenced the gene encoding human bradykinin B{sub 2} receptor from a human genomic library. The B{sub 2} receptor gene contains three exons separated by two introns. The first and second exons are noncoding, while the third exon contains the full-length coding region, which encodes a protein of 364 amino acids forming 7 transmembrane domains. The human B{sub 2} gene shares high sequence identity with rat and mouse B{sub 2} receptor genes and significant similarity with the gene encoding the angiotensin II type I receptor in the nucleotide sequence and exon-intron arrangement. In the 5` flanking region, a consensus TATA box and several putative transcription factor-binding sites have been identified. Genomic Southern blot analysis showed that the B{sub 2} receptor is encoded by a single-copy gene that was localized to chromosome 14q32 by in situ hybridization. In a Southern blot analysis following reverse transcription and polymerase chain reaction, the human B{sub 2} receptor was found to be expressed in most human tissues. 30 refs., 7 figs.

  14. 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.

  15. 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

  16. A low-frequency oscillatory neural signal in humans encodes a developing decision variable.

    PubMed

    Kubanek, Jan; Snyder, Lawrence H; Brunton, Bingni W; Brody, Carlos D; Schalk, Gerwin

    2013-12-01

    We often make decisions based on sensory evidence that is accumulated over a period of time. How the evidence for such decisions is represented in the brain and how such a neural representation is used to guide a subsequent action are questions of considerable interest to decision sciences. The neural correlates of developing perceptual decisions have been thoroughly investigated in the oculomotor system of macaques who communicated their decisions using an eye movement. It has been found that the evidence informing a decision to make an eye movement is in part accumulated within the same oculomotor circuits that signal the upcoming eye movement. Recent evidence suggests that the somatomotor system may exhibit an analogous property for choices made using a hand movement. To investigate this possibility, we engaged humans in a decision task in which they integrated discrete quanta of sensory information over a period of time and signaled their decision using a hand movement or an eye movement. The discrete form of the sensory evidence allowed us to infer the decision variable on which subjects base their decision on each trial and to assess the neural processes related to each quantum of the incoming decision evidence. We found that a low-frequency electrophysiological signal recorded over centroparietal regions strongly encodes the decision variable inferred in this task, and that it does so specifically for hand movement choices. The signal ramps up with a rate that is proportional to the decision variable, remains graded by the decision variable throughout the delay period, reaches a common peak shortly before a hand movement, and falls off shortly after the hand movement. Furthermore, the signal encodes the polarity of each evidence quantum, with a short latency, and retains the response level over time. Thus, this neural signal shows properties of evidence accumulation. These findings suggest that the decision-related effects observed in the oculomotor system

  17. 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.

  18. Studies on the expression and processing of human proinsulin derivatives encoded by different DNA constructs.

    PubMed

    Aslam, Farheen; Gardner, Qurra-tul Ann Afza; Zain, Hina; Nadeem, Muhammad Shahid; Ali, Muhammad; Rashid, Naeem; Akhtar, Muhammad

    2013-10-01

    A synthetic gene encoding human proinsulin, containing Escherichia coli preferred codons, with an additional N-terminal methionine, was used for the expression, of M-proinsulin and construction of nine derivatives. No improvement in expression was noted, relative to that of M-proinsulin, when the 5'- of the gene was appended to codons for seven amino acids of a well expressed E. coli protein (threonine dehydrogenase), or the constructs contained multiple copies of the proinsulin gene. That in the latter constructs only the gene adjacent to the prometer sequence is expressed, was shown by a construct containing a proinsulin gene followed by that for interferon α-2b. With the latter construct, the proinsulin was, predominantly, expressed. The availability of data on the constructs prompted, subjecting these to analysis by two models designed to predict the expression of proteins from the sequences, of putative mRNA, around the start of translation but no significant relationship was noted. In all cases the proteins were expressed as inclusion bodies, which were refolded to give products of desired masses and successfully converted into insulin derivatives. Of all the constructs containing a trypsin sensitive site before phenylalanine (F), the N-terminal sequence, MKR↓F, was most efficiently processed, by a cocktail of trypsin and buffalo carboxypeptidase B, to give insulin with the removal of the N-terminus linker as well as the C-peptide in a single step, without cleaving the trypsin sensitive K(29)T(30) peptide bond. PMID:23872484

  19. Human cytomegalovirus-encoded US28 may act as a tumor promoter in colorectal cancer

    PubMed Central

    Cai, Zhen-Zhai; Xu, Jian-Gang; Zhou, Yu-Hui; Zheng, Ji-Hang; Lin, Ke-Zhi; Zheng, Shu-Zhi; Ye, Meng-Si; He, Yun; Liu, Chang-Bao; Xue, Zhan-Xiong

    2016-01-01

    AIM: To assess human cytomegalovirus-encoded US28 gene function in colorectal cancer (CRC) pathogenesis. METHODS: Immunohistochemical analysis was performed to determine US28 expression in 103 CRC patient samples and 98 corresponding adjacent noncancerous samples. Patient data were compared by age, sex, tumor location, histological grade, Dukes’ stage, and overall mean survival time. In addition, the US28 gene was transiently transfected into the CRC LOVO cell line, and cell proliferation was assessed using a cell counting kit-8 assay. Cell cycle analysis by flow cytometry and a cell invasion transwell assay were also carried out. RESULTS: US28 levels were clearly higher in CRC tissues (38.8%) than in adjacent noncancerous samples (7.1%) (P = 0.000). Interestingly, elevated US28 amounts in CRC tissues were significantly associated with histological grade, metastasis, Dukes’ stage, and overall survival (all P < 0.05); meanwhile, US28 expression was not significantly correlated with age, sex or tumor location. In addition, multivariate Cox regression data revealed US28 level as an independent CRC prognostic marker (P = 0.000). LOVO cells successfully transfected with the US28 gene exhibited higher viability, greater chemotherapy resistance, accelerated cell cycle progression, and increased invasion ability. CONCLUSION: US28 expression is predictive of poor prognosis and may promote CRC. PMID:26973417

  20. Varicella-zoster virus (VZV) transcription during latency in human ganglia: detection of transcripts mapping to genes 21, 29, 62, and 63 in a cDNA library enriched for VZV RNA.

    PubMed Central

    Cohrs, R J; Barbour, M; Gilden, D H

    1996-01-01

    Information on the extent of virus DNA transcription and translation in infected tissue is crucial to an understanding of herpesvirus latency. To detect low-abundance latent varicella-zoster virus (VZV) transcripts, poly(A)+ RNA extracted from latently infected human trigeminal ganglia was enriched for VZV transcripts by hybridization to biotinylated VZV DNA. After hybridization, the RNA-DNA hybrid was isolated by binding to avidin-coated beads and extensively washed, and the RNA was released by heat denaturation. A lambda-based cDNA library was then constructed from the enriched RNA. PCR and DNA sequencing of DNA extracted from the cDNA library revealed the presence of VZV genes 21, 29, 62, and 63, but not VZV genes 4, 10, 40, 51, and 61, in the enriched cDNA library. These findings confirm the detection of VZV gene 29 and 62 transcripts on Northern (RNA) blots prepared from latently infected human ganglia (J.L. Meier, R.P. Holman, K.D. Croen, J.E. Smialek, and S.E. Straus, Virology 193:193-200, 1993) and the presence of VZV gene 21 transcripts in a cDNA library from mRNA of latently infected ganglia (R.J. Cohrs, K. Srock, M.B. Barbour, G. Owens, R. Mahalingam, M.E. Devlin, M. Wellish and D.H. Gilden, J. Virol. 68:7900-7908,1994) and also reveal, for the first time, the presence of VZV gene 63 RNA in latently infected human ganglia. PMID:8627753

  1. 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

  2. Functional Analysis of the env Open Reading Frame in Human Endogenous Retrovirus IDDMK1,222 Encoding Superantigen Activity

    PubMed Central

    Lapatschek, Matthias; Dürr, Susanne; Löwer, Roswitha; Magin, Christine; Wagner, Hermann; Miethke, Thomas

    2000-01-01

    Mice harbor a family of endogenous retroviruses, the mouse mammary tumor viruses (MMTV), which encode superantigens. These superantigens are responsible for the deletion of T cells expressing certain Vβ chains of the T-cell receptor in the thymus. Human T cells are able to recognize MMTV-encoded superantigens presented by human major histocompatibility complex class II-positive cells. Owing to this and to the similarity of the human and murine immune systems, it was speculated that human endogenous retroviruses might also code for superantigens. Recently, it was reported that a proviral clone (IDDMK1,222) of the human endogenous retrovirus family HTDV/HERV-K encodes a superantigen. The putative superantigen gene was located within the env region of the virus. Stimulated by these findings, we amplified by PCR and cloned into eucaryotic expression vectors open reading frames (ORFs) which were identical or very similar to IDDMK1,222. When we transfected these vectors into A20 cells, a murine B-cell lymphoma, we were able to demonstrate mRNA expression and protein production. However, we did not find any evidence that the ORF stimulated human or murine T cells in a Vβ-specific fashion, the most prominent feature of superantigens. PMID:10864649

  3. Pathogenic Entamoeba histolytica: cDNA cloning of a histone H3 with a divergent primary structure.

    PubMed

    Födinger, M; Ortner, S; Plaimauer, B; Wiedermann, G; Scheiner, O; Duchêne, M

    1993-06-01

    Entamoeba histolytica has an unusual nuclear structure characterized by a low degree of chromatin condensation and the absence of stainable metaphase chromosomes. Although nucleosome-like particles were observed, no information about histones was available so far. In this paper we describe a cDNA clone with significant homology to H3 histones that was isolated from a library of pathogenic E. histolytica. The complete cDNA encodes a 15-kDa polypeptide, which like the histone sequence from Volvox carteri is shorter by one residue than the human homologue. The amino acid sequence has only 69% identity with human H3.3 histone and 67% identity with the human H3.1 histone. This is the highest degree of sequence divergence observed for any eukaryote H3 histone sequence. Our results indicate that this divergence may contribute to the unusual chromatin structure of E. histolytica. PMID:8341328

  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. Androgen regulation of the human FERM domain encoding gene EHM2 in a cell model of steroid-induced differentiation

    PubMed Central

    Chauhan, Sanjay; Pandey, Ritu; Way, Jeffrey F.; Sroka, Thomas C.; Demetriou, Manolis C.; Kunz, Susan; Cress, Anne E.; Mount, David W.; Miesfeld, Roger L.

    2009-01-01

    We have developed a cell model to investigate steroid control of differentiation using a subline of HT1080 cells (HT-AR1) that have been engineered to express the human androgen receptor. Dihydrotestosterone (DHT) treatment of HT-AR1 cells induced growth arrest and cytoskeletal reorganization that was associated with the expression of fibronectin and the neuroendocrine markers chromogranin A and neuron-specific enolase. Expression profiling analysis identified the human FERM domain-encoding gene EHM2 as uniquely induced in HT-AR1 cells as compared to 16 other FERM domain containing genes. Since FERM domain proteins control cytoskeletal functions in differentiating cells, and the human EHM2 gene has not been characterized, we investigated EHM2 steroid-regulation, genomic organization, and sequence conservation. We found that DHT, but not dexamethasone, induced the expression of a 3.8 kb transcript in HT-AR1 cells encoding a 504 amino acid protein, and moreover, that human brain tissue contains a 5.8 kb transcript encoding a 913 amino acid isoform. Construction of an unrooted phylogenetic tree using 98 FERM domain proteins revealed that the human EHM2 gene is a member of a distinct subfamily consisting of nine members, all of which contain a highly conserved 325 amino acid FERM domain. PMID:14521927

  6. Androgen regulation of the human FERM domain encoding gene EHM2 in a cell model of steroid-induced differentiation.

    PubMed

    Chauhan, Sanjay; Pandey, Ritu; Way, Jeffrey F; Sroka, Thomas C; Demetriou, Manolis C; Kunz, Susan; Cress, Anne E; Mount, David W; Miesfeld, Roger L

    2003-10-17

    We have developed a cell model to investigate steroid control of differentiation using a subline of HT1080 cells (HT-AR1) that have been engineered to express the human androgen receptor. Dihydrotestosterone (DHT) treatment of HT-AR1 cells induced growth arrest and cytoskeletal reorganization that was associated with the expression of fibronectin and the neuroendocrine markers chromogranin A and neuron-specific enolase. Expression profiling analysis identified the human FERM domain-encoding gene EHM2 as uniquely induced in HT-AR1 cells as compared to 16 other FERM domain containing genes. Since FERM domain proteins control cytoskeletal functions in differentiating cells, and the human EHM2 gene has not been characterized, we investigated EHM2 steroid-regulation, genomic organization, and sequence conservation. We found that DHT, but not dexamethasone, induced the expression of a 3.8 kb transcript in HT-AR1 cells encoding a 504 amino acid protein, and moreover, that human brain tissue contains a 5.8 kb transcript encoding a 913 amino acid isoform. Construction of an unrooted phylogenetic tree using 98 FERM domain proteins revealed that the human EHM2 gene is a member of a distinct subfamily consisting of nine members, all of which contain a highly conserved 325 amino acid FERM domain. PMID:14521927

  7. Structure of the gene encoding the 14.5 kDa subunit of human RNA polymerase II.

    PubMed Central

    Acker, J; Wintzerith, M; Vigneron, M; Kedinger, C

    1993-01-01

    The structure of the gene encoding the 14.5 kDa subunit of the human RNA polymerase II (or B) has been elucidated. The gene consists of six exons, ranging from 52 to over 101 bp, interspaced with five introns ranging from 84 to 246 bp. It is transcribed into three major RNA species, present at low abundance in exponentially growing HeLa cells. The corresponding messenger RNAs contain the same open reading frame encoding a 125 amino acid residue protein, with a calculated molecular weight of 14,523 Da. This protein (named hRPB14.5) shares strong homologies with the homologous polymerase subunits encoded by the Drosophila (RpII15) and yeast (RPB9) genes. Cysteines characteristic of two zinc fingers are conserved in all three corresponding sequences and, like the yeast protein, the hRPB14.5 subunit exhibits zinc-binding activity. Images PMID:8265347

  8. Differential cytokine modulation of the genes LAMA3, LAMB3, and LAMC2, encoding the constitutive polypeptides, alpha 3, beta 3, and gamma 2, of human laminin 5 in epidermal keratinocytes.

    PubMed

    Korang, K; Christiano, A M; Uitto, J; Mauviel, A

    1995-07-24

    Laminin 5, an anchoring filament protein previously known as nicein/kalinin/epiligrin, consists of three polypeptide chains, alpha 3, beta 3, and gamma 2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. The expression of laminin 5 was detected by Northern hybridization with specific cDNA probes in various epidermal keratinocyte cultures, whereas no expression of any of the three genes could be detected in foreskin fibroblast cultures. Transforming growth factor-beta (TGF-beta) enhanced LAMA3, LAMB3, and LAMC2 gene expression in human epidermal keratinocytes, as well as in HaCaT and Balb/K cells in culture, although the extent of enhancement was greater for LAMA3 and LAMC2 genes than for LAMB3. Interestingly, tumor necrosis factor-alpha, (TNF-alpha) alone did not alter the expression of LAMB3 and LAMC2 genes in human epidermal keratinocytes, whereas it inhibited the expression of LAMA3. These results suggest that the expression of the three genes encoding the laminin 5 subunits is not coordinately regulated by the cytokines tested. PMID:7635220

  9. 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. PMID:21277335

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

    PubMed

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

    2005-03-23

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

  11. Psychophysical and EEG responses to repeated experimental muscle pain in humans: pain intensity encodes EEG activity.

    PubMed

    Chang, Peng-Fei; Arendt-Nielsen, Lars; Graven-Nielsen, Thomas; Chen, Andrew C N

    2003-02-15

    Clinical pain is often characterized by repetitive and persistent occurrence in deep structures, but few studies investigated repetitive tonic pain in humans. To determine cerebral responses to repetitive tonic pain, psychophysical responses, and electroencephalographic (EEG) activation to five trials of repeated tonic muscle pain induced by hypertonic saline were examined and analyzed in 13 male subjects. The study was composed of two experimental sessions performed in separate days. Five sequential injections of hypertonic saline (5.8%) were used to induce repeated muscle pain in the left forearm, and five sequential injections of isotonic saline (0.9%) acted as control. Visual analogue scales (VAS) for pain intensity and 32-channels EEG activities were recorded simultaneously. Five trials of relatively stable muscle pain were induced by intramuscular injections of hypertonic saline, but no evident pain was induced by the injections of isotonic saline. Significant decreases in alpha-1 and -2 activities in posterior part of the head were found during repeated muscle pain in comparison with non-pain. In comparison with baseline, alpha-1 and -2 activities reduced significantly during the first two trials, and gradually resumed in the following three trials of muscle pain. However, beta-2 activity increased consistently throughout the five trials of muscle pain compared to baseline. Alpha-1 activity was negatively, but beta-2 activity was positively correlated to the pain intensity and pain area on the skin. Throughout five injections, the reduction of alpha-1 activity was contrary to the changes of pain intensity. These results indicates that pain-related EEG activities were encoded by the pain intensity. The thalamo-cortical system and descending inhibitory neuronal networks may be involved in the regulation of pain intensity. PMID:12576151

  12. Molecular Cloning of cDNA Encoding an Aquaglyceroporin, AQP-h9, in the Japanese Tree Frog, Hyla japonica: Possible Roles of AQP-h9 in Freeze Tolerance.

    PubMed

    Hirota, Atsushi; Takiya, Yu; Sakamoto, Joe; Shiojiri, Nobuyoshi; Suzuki, Masakazu; Tanaka, Shigeyasu; Okada, Reiko

    2015-06-01

    In order to study the freeze-tolerance mechanism in the Japanese tree frog, Hyla japonica, wecloned a eDNA encoding aquaporin (AQP) 9 from its liver. The predicted amino acid sequence ofH. japonica AQP9 (AQP-h9) contained six putative transmembrane domains and two conservedAsn-Pro-Aia motifs, which are characteristic of AQPs. A swelling assay using Xenopus laevisoocytes injected with AQP-h9 cRNA showed that AQP-h9 facilitated water and glycerol permeation,confirming its property as an aquaglyceroporin. Subsequently, glycerol concentrations in serumand tissue extracts were compared among tree frogs that were hibernating, frozen, or thawed afterfreezing. Serum glycerol concentration of thawed frogs was significantly higher than that of hibernatingfrogs. Glycerol content in the liver did not change in the freezing experiment, whereas thatin the skeletal muscle was elevated in thawed frogs as compared with hibernating or frozen frogs. Histological examination of the liver showed that erythrocytes aggregated in the sinusoids during hibernation and freezing, and immunoreactive AQP-h9 protein was detected over the erythrocytes. The AQP-h9 labeling was more intense in frozen frogs than in hibernating frogs, but nearly undetectable in thawed frogs. For the skeletal muscle, weak labels for AQP-h9 were observed in the cytoplasm of myocytes of hibernating frogs. AQP-h9 labeling was markedly enhanced by freezing and was decreased by thawing. These results indicate that glycerol may act as a c;:ryoprotectant in H. japonica and that during hibernation, particularly during freezing, AQP-h9 may be involved in glycerol uptake in erythrocytes in the liver and in intracellular glycerol transport in the skeletal muscle cells. PMID:26402924

  13. Complete sequence of an HLA-dR beta chain deduced from a cDNA clone and identification of multiple non-allelic DR beta chain genes.

    PubMed Central

    Long, E O; Wake, C T; Gorski, J; Mach, B

    1983-01-01

    At least three polymorphic class II antigens are encoded in the human major histocompatibility complex (HLA): DR, DC and SB. cDNA clones encoding beta chains of HLA-DR antigen, derived from mRNA of a heterozygous B-cell line, were isolated and could be divided into four subsets, clearly distinct from cDNA clones encoding DC beta chains. Therefore, at least two non-allelic DR beta chain genes exist. The complete sequence of one of the DR beta chain cDNA clones is presented. It defines a putative signal sequence, two extracellular domains, a trans-membrane region and a cytoplasmic tail. Comparison with a DC beta chain cDNA clone revealed a homology of 70% between the two beta chains and that the two genes diverged under relatively little selective pressure. A set of amino acids conserved in immunoglobulin molecules was found to be identical in both DR and DC beta chains. Comparison of the DR beta chain sequence with the amino acid sequence of another DR beta chain revealed a homology of 87% and that most differences are single amino acid substitutions. Allelic polymorphism in DR beta chains has probably not arisen by changes in long blocks of sequence. PMID:11894954

  14. Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

    PubMed Central

    2010-01-01

    Background The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS) cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry. Results Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture. Conclusions Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development. PMID:20682060

  15. 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.

  16. 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

  17. 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. PMID:25912975

  18. Prolegomena to a neurocomputational architecture for human grammatical encoding and decoding.

    PubMed

    Kempen, Gerard

    2014-01-01

    This study develops a neurocomputational architecture for grammatical processing in language production and language comprehension (grammatical encoding and decoding, respectively). It seeks to answer two questions. First, how is online syntactic structure formation of the complexity required by natural-language grammars possible in a fixed, preexisting neural network without the need for online creation of new connections or associations? Second, is it realistic to assume that the seemingly disparate instantiations of syntactic structure formation in grammatical encoding and grammatical decoding can run on the same neural infrastructure? This issue is prompted by accumulating experimental evidence for the hypothesis that the mechanisms for grammatical decoding overlap with those for grammatical encoding to a considerable extent, thus inviting the hypothesis of a single "grammatical coder." The paper answers both questions by providing the blueprint for a syntactic structure formation mechanism that is entirely based on prewired circuitry (except for referential processing, which relies on the rapid learning capacity of the hippocampal complex), and can subserve decoding as well as encoding tasks. The model builds on the "Unification Space" model of syntactic parsing developed by Vosse and Kempen (Cognition 75:105-143, 2000; Cognitive Neurodynamics 3:331-346, 2009a). The design includes a neurocomputational mechanism for the treatment of an important class of grammatical movement phenomena. PMID:23872869

  19. The Drosophila melanogaster homologue of the human histo-blood group Pk gene encodes a glycolipid-modifying α1,4-N-acetylgalactosaminyltransferase

    PubMed Central

    2004-01-01

    Insects express arthro-series glycosphingolipids, which contain an α1,4-linked GalNAc residue. To determine the genetic basis for this linkage, we cloned a cDNA (CG17223) from Drosophila melanogaster encoding a protein with homology to mammalian α1,4-glycosyltransferases and expressed it in the yeast Pichia pastoris. Culture supernatants from the transformed yeast were found to display a novel UDP-GalNAc:GalNAcβ1,4GlcNAcβ1-R α-N-acetylgalactosaminyltransferase activity when using either a glycolipid, p-nitrophenylglycoside or an N-glycan carrying one or two terminal β-N-acetylgalactosamine residues. NMR and MS in combination with glycosidase digestion and methylation analysis indicate that the cloned cDNA encodes an α1,4-N-acetylgalactosaminyltransferase. We hypothesize that this enzyme and its orthologues in other insects are required for the biosynthesis of the N5a and subsequent members of the arthro-series of glycolipids as well as of N-glycan receptors for Bacillus thuringiensis crystal toxin Cry1Ac. PMID:15130086

  20. pap-2-encoded fimbriae adhere to the P blood group-related glycosphingolipid stage-specific embryonic antigen 4 in the human kidney.

    PubMed Central

    Karr, J F; Nowicki, B J; Truong, L D; Hull, R A; Moulds, J J; Hull, S I

    1990-01-01

    A subtype of P fimbriae, encoded by the pap-2 gene cluster, has been analyzed for agglutination of erythrocytes and for binding to cryostat sections of the human kidney. We have demonstrated that pap-2-encoded fimbriae are capable of binding to erythrocytes from some animal species and to human erythrocytes which express globoside and the LKE (stage-specific embryonic antigen 4 [SSEA-4]) antigen. The pap-2 fimbriae bind to Bowman's capsule in the human kidney. Monoclonal antibodies directed against glycosphingolipids were used for the detection of specific P blood group-related antigens in the human kidney and on erythrocytes. Preincubation of kidney sections with monoclonal antibody MC813-70, which binds to the SSEA-4 antigen, inhibited adherence of purified pap-2-encoded fimbriae to Bowman's capsule. We suggest that one receptor for pap-2-encoded fimbriae is the antigen known as LKE (Luke) on human erythrocytes or SSEA-4 in the tissues. Images PMID:1979319

  1. 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

  2. Neurons in the human amygdala encode face identity, but not gaze direction.

    PubMed

    Mormann, Florian; Niediek, Johannes; Tudusciuc, Oana; Quesada, Carlos M; Coenen, Volker A; Elger, Christian E; Adolphs, Ralph

    2015-11-01

    The amygdala is important for face processing, and direction of eye gaze is one of the most socially salient facial signals. Recording from over 200 neurons in the amygdala of neurosurgical patients, we found robust encoding of the identity of neutral-expression faces, but not of their direction of gaze. Processing of gaze direction may rely on a predominantly cortical network rather than the amygdala. PMID:26479589

  3. Neurons in the human amygdala encode face identity but not gaze direction

    PubMed Central

    Mormann, Florian; Niediek, Johannes; Tudusciuc, Oana; Quesada, Carlos M.; Coenen, Volker; Elger, Christian; Adolphs, Ralph

    2015-01-01

    The amygdala is a key structure in face processing, and direction of eye gaze is one of the most socially salient facial signals. Recording from over 200 neurons in the amygdala of neurosurgical patients, we here find robust encoding of the identity of neutral-expression faces, but not to their direction of gaze. Processing of gaze direction may rely on a predominantly cortical network rather than the amygdala. PMID:26479589

  4. Nucleic acids encoding modified human immunodeficiency virus type 1 (HIV-1) group M consensus envelope glycoproteins

    DOEpatents

    Haynes, Barton F.; Gao, Feng; Korber, Bette T.; Hahn, Beatrice H.; Shaw, George M.; Kothe, Denise; Li, Ying Ying; Decker, Julie; Liao, Hua-Xin

    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.

  5. 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

  6. Distinguishing informational from value-related encoding of rewarding and punishing outcomes in the human brain.

    PubMed

    Jessup, Ryan K; O'Doherty, John P

    2014-06-01

    There is accumulating evidence implicating a set of key brain regions in encoding rewarding and punishing outcomes, including the orbitofrontal cortex, medial prefrontal cortex, ventral striatum, anterior insula, and anterior cingulate. However, it has proved challenging to reach consensus concerning the extent to which different brain areas are involved in differentially encoding rewarding and punishing outcomes. Here, we show that many of the brain areas involved in outcome processing represent multiple outcome components: encoding the value of outcomes (whether rewarding or punishing) and informational coding, i.e. signaling whether a given outcome is rewarding or punishing, ignoring magnitude or experienced utility. In particular, we report informational signals in the lateral orbitofrontal cortex and anterior insular cortex that respond to both rewarding and punishing feedback, even though value-related signals in these areas appear to be selectively driven by punishing feedback. These findings highlight the importance of taking into account features of outcomes other than value when characterising the contributions of different brain regions in outcome processing. PMID:24863104

  7. 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.

  8. 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.

  9. 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.

  10. YY1 and Sp1 activate transcription of the human NDUFS8 gene encoding the mitochondrial complex I TYKY subunit.

    PubMed

    Lescuyer, Pierre; Martinez, Pascal; Lunardi, Joël

    2002-03-19

    Complex I is the most complicated of the multimeric enzymes that constitute the mitochondrial respiratory chain. It is encoded by both mitochondrial and nuclear genomes. We have previously characterized the human NDUFS8 gene that encodes the TYKY subunit. This essential subunit is thought to participate in the electron transfer and proton pumping activities of complex I. Here, we have analyzed the transcriptional regulation of the NDUFS8 gene. Using primer extension assays, we have identified two transcription start sites. The basal promoter was mapped to a 247 bp sequence upstream from the main transcription start site by reporter gene analysis in HeLa cells and in differentiated or non-differentiated C2C12 cells. Three Sp1 sites and one YY1 site were identified in this minimal promoter. Through gel shift analysis, all sites were shown to bind to their cognate transcription factors. Site-directed mutagenesis revealed that the YY1 site and two upstream adjacent Sp1 sites drive most of the promoter activity. This work represents the first promoter analysis for a complex I gene. Together with previous studies, our results indicate that YY1 and Sp1 control the expression of genes encoding proteins that are involved in almost all steps of the oxidative phosphorylation metabolism. PMID:11955626

  11. Cystic Fibrosis Gene Encodes a cAMP-Dependent Chloride Channel in Heart

    NASA Astrophysics Data System (ADS)

    Hart, Padraig; Warth, John D.; Levesque, Paul C.; Collier, Mei Lin; Geary, Yvonne; Horowitz, Burton; Hume, Joseph R.

    1996-06-01

    cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significnatly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.

  12. Integrating Memories in the Human Brain: Hippocampal–Midbrain Encoding of Overlapping Events

    PubMed Central

    Shohamy, Daphna; Wagner, Anthony D.

    2008-01-01

    SUMMARY Decisions are often guided by generalizing from past experiences. Fundamental questions remain regarding the cognitive and neural mechanisms by which generalization takes place. Prior data suggest that generalization may stem from inference-based processes that occur at the time of generalization. By contrast, it has been hypothesized that generalization may emerge from mnemonic processes that occur while premise events are being encoded. Here, participants engaged in a two-phase learning and generalization task, wherein they initially learned a series of overlapping associations, and were subsequently probed to generalize what they learned to novel stimulus combinations. Functional magnetic resonance imaging (fMRI) revealed that subsequent generalization performance was associated with coupled changes in learning-phase activity in the hippocampus and midbrain (ventral tegmental area/substantia nigra). These findings provide novel evidence for generalization based on integrative encoding, whereby overlapping past events are integrated into a linked mnemonic representation. Hippocampal–midbrain interactions support the dynamic integration of experiences, providing a powerful mechanism for building a rich associative history that extends beyond individually experienced events. PMID:18957228

  13. Architecture and anatomy of the chromosomal locus in human chromosome 21 encoding the Cu/Zn superoxide dismutase.

    PubMed Central

    Levanon, D; Lieman-Hurwitz, J; Dafni, N; Wigderson, M; Sherman, L; Bernstein, Y; Laver-Rudich, Z; Danciger, E; Stein, O; Groner, Y

    1985-01-01

    The SOD-1 gene on chromosome 21 and approximately 100 kb of chromosomal DNA from the 21q22 region have been isolated and characterized. The gene which is present as a single copy per haploid genome spans 11 kb of chromosomal DNA. Heteroduplex analysis and DNA sequencing reveals five rather small exons and four introns that interrupt the coding region. The donor sequence at the first intron contains an unusual variant dinucleotide 5'-G-C, rather than the highly conserved 5'-GT. The unusual splice junction is functional in vivo since it was detected in both alleles of the SOD-1 gene, which were defined by differences in the length of restriction endonuclease fragments (RFLPs) that hybridize to the cDNA probe. Genomic blots of human DNA isolated from cells trisomic for chromosome 21 (Down's syndrome patients) show the normal pattern of bands. At the 5' end of gene there are the 'TATA' and 'CAT' promoter sequences as well as four copies of the -GGCGGG- hexanucleotide. Two of these -GC- elements are contained within a 13 nucleotide inverted repeat that could form a stem-loop structure with stability of -33 kcal. The 3'-non coding region of the gene contains five short open reading-frames starting with ATG and terminating with stop codons. Images Fig. 1. Fig. 3. Fig. 7. PMID:3160582

  14. GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development.

    PubMed

    Kuwahara, Kazuhiko; Yamamoto-Ibusuki, Mutsuko; Zhang, Zhenhuan; Phimsen, Suchada; Gondo, Naomi; Yamashita, Hiroko; Takeo, Toru; Nakagata, Naomi; Yamashita, Daisuke; Fukushima, Yoshimi; Yamamoto, Yutaka; Iwata, Hiroji; Saya, Hideyuki; Kondo, Eisaku; Matsuo, Keitaro; Takeya, Motohiro; Iwase, Hirotaka; Sakaguchi, Nobuo

    2016-04-01

    Human chromosome 21 is known to be associated with the high risk of hematological malignancy but with resistance to breast cancer in the study of Down syndrome. In human cancers, we previously observed the significant alterations of the protein expression encoded by the ganp/MCM3AP gene on human chromosome 21q22.3. Here, we investigated GANP protein alterations in human breast cancer samples (416 cases) at various stages by immunohistochemical analysis. This cohort study clearly showed that expression of GANP is significantly decreased in human breast cancer cases with poor prognosis as an independent risk factor (relapse-free survival, hazard ratio = 2.37, 95% confidence interval, 1.27-4.42, P = 0.007 [univariate analysis]; hazard ratio = 2.70, 95% confidence interval, 1.42-5.13, P = 0.002 [multivariate analysis]). To investigate whether the altered GANP expression is associated with mammary tumorigenesis, we created mutant mice that were conditionally deficient in the ganp/MCM3AP gene using wap-cre recombinase transgenic mice. Mammary gland tumors occurred at a very high incidence in female mammary gland-specific GANP-deficient mice after severe impairment of mammary gland development during pregnancy. Moreover, tumor development also occurred in female post parous GANP-heterodeficient mice. GANP has a significant role in the suppression of DNA damage caused by estrogen in human breast cancer cell lines. These results indicated that the GANP protein is associated with breast cancer resistance. PMID:26749495

  15. An E1M--E2C fusion protein encoded by human papillomavirus type 11 is asequence-specific transcription repressor.

    PubMed Central

    Chiang, C M; Broker, T R; Chow, L T

    1991-01-01

    We have isolated a putative, spliced E5 cDNA of human papillomavirus type 11 (HPV-11) by polymerase chain reaction amplification of cDNAs from an experimental condyloma. Using retrovirus-mediated gene transfer, we isolated two novel HPV-11 cDNAs, one of which had a splice linking nucleotides 1272 and 3377. This transcript also existed in experimental condylomata and in cervical carcinoma cells transfected with cloned genomic HPV-11 DNAs. The 5' end of the transcript in transfected cells originated upstream of the initiation codon of the E1 open reading frame (ORF). It could conceptually encode a fusion protein consisting of the amino-terminal 23% of the E1 ORF and the carboxy-terminal 40% of the E2 ORF. This E1M--E2C fusion protein contained both the DNA replication modulator domain E1M, as defined in the bovine papillomavirus system, and the DNA binding domain of the E2 protein, which regulates viral transcriptional activities. Indirect immunofluorescence with polyclonal antibodies raised against the bacterially expressed TrpE-HPV-11 E2 protein demonstrated nuclear localization of the E1M--E2C protein in cells transiently transfected with an expression plasmid. Immunoprecipitation revealed a specific protein with an apparent molecular weight of 42,000 in transfected cells. The chloramphenicol acetyltransferase assay established that the putative E1M--E2C protein was a potent transcriptional repressor of both E2-dependent and E2-independent HPV-11 enhancer/promoter activities. Northern (RNA) blot hybridization indicated the repression was on the transcriptional level. Mutational analysis suggested that the E1M--E2C protein is an E2-binding site-specific repressor. The fusion protein also repressed bovine papillomavirus type 1 (BPV-1) E2 protein-dependent BPV-1 enhancer activity. When constitutively expressed in mouse C127 cells, the E1M--E2C protein inhibited BPV-1 transformation and episomal DNA replication, consistent with a role in the modulation of replication

  16. Down-regulation of messenger ribonucleic acid encoding an importer of sulfoconjugated steroids during human chorionic gonadotropin-induced follicular luteinization in vivo.

    PubMed

    Brown, Kristy A; Bouchard, Nadine; Lussier, Jacques G; Sirois, Jean

    2007-01-01

    Members of the organic anion transporting polypeptide (SLCO/OATP) superfamily are capable of importing anionic compounds across the lipid bilayer in a sodium-independent manner. Member 2B1 has been shown to transport few substrates, two of which are dihydroepiandrosterone-3-sulfate (DHEA-S) and estrone-3-sulfate. Steroid sulfatase (STS) catalyses the hydrolysis of these steroids into their unconjugated counterparts. The objective of this study was to investigate the regulation of SLCO2B1 and STS mRNAs during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine SLCO2B1 cDNA was cloned and shown to encode a 709-amino acid protein (OATP2B1) that is highly conserved when compared to mammalian orthologs. RT-PCR/Southern blot analyses were performed to study the regulation of SLCO2B1 and STS transcripts in equine preovulatory follicles isolated between 0 and 39h after hCG treatment. Results showed high levels of SLCO2B1 mRNA expression before hCG, with a marked decrease observed in follicles obtained 24-39h post-hCG (P<0.05). Analyses of isolated granulosa and theca interna cells identified high mRNA expression in both cell types prior to hCG treatment, with granulosa cells showing a more rapid SLCO2B1 mRNA down-regulation. No significant change in STS mRNA was observed in intact follicle walls. However, when both cell types were isolated, a significant decrease in STS mRNA was observed in granulosa cells 24-39h post-hCG. Collectively, these results demonstrate that the hCG-dependent induction of follicular luteinization is accompanied by the down-regulation of SLCO2B1 and STS transcripts. Considering that OATP2B1 can import sulfoconjugated DHEA and estrogens, and that STS can remove the sulfonate moiety from these steroids, their down-regulation in luteinizing preovulatory follicles may provide an additional biochemical basis for the decrease in ovarian 17beta-estradiol biosynthesis after the LH surge. PMID:17049229

  17. A partial cDNA clone of trypomastigote decay-accelerating factor (T-DAF), a developmentally regulated complement inhibitor of Trypanosoma cruzi, has genetic and functional similarities to the human complement inhibitor DAF.

    PubMed Central

    Tambourgi, D V; Kipnis, T L; da Silva, W D; Joiner, K A; Sher, A; Heath, S; Hall, B F; Ogden, G B

    1993-01-01

    Resistance to complement-mediated lysis in Trypanosoma cruzi is due to the expression of complement-regulatory factors by the virulent developmental forms of this protozoan parasite. An 87- to 93-kDa molecule, which we have termed T-DAF (trypomastigote decay-accelerating factor), is present on the surface of the parasite and inhibits complement activation in a manner functionally similar to the mammalian complement regulatory component, decay-accelerating factor. In this report, we characterized monospecific polyclonal and monoclonal antibodies which were obtained from mice and rabbits immunized with fast protein liquid chromatography-purified T-DAF. These polyclonal antibodies were shown to inhibit T-DAF activity and were capable of inducing lysis of the parasites. Both the polyclonal and monoclonal antibodies were used to screen a cDNA expression library prepared from T. cruzi trypomastigote mRNA. From this library, we obtained a partial lambda gt11 cDNA clone which showed genetic and functional similarity to the human C3 convertase inhibitor DAF (A. Nicholson-Weller, J. Burge, D. T. Fearon, P. F. Weller, and K. F. Austen, J. Immunol. 129:184-189, 1982). Images PMID:7689538

  18. 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. PMID:23431001

  19. Fatal autoimmunity in mice reconstituted with human hematopoietic stem cells encoding defective FOXP3

    PubMed Central

    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.

    2015-01-01

    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. PMID:25833964

  20. Conservation of structure in the human gene encoding argininosuccinate synthetase and the argG genes of the archaebacteria Methanosarcina barkeri MS and Methanococcus vannielii

    SciTech Connect

    Morris, C.J.; Reeve, J.N.

    1988-07-01

    The DNA sequences of the argG genes of Methanosarcina barkeri MS and Methanococcus vannielii were determined. The polypeptide products of these methanogen genes have amino acid sequences which are 50% identical to each other and 38% identical to the amino acid sequence encoded by the exons of the human argininosuccinate synthetase gene. Introns in the human chromosomal gene separate regions which encode amino acids conserved in both the archaebacterial and human gene products. An open reading frame immediately upstream of argG in Methanosarcina barkeri MS codes for an amino acid sequence which is 45 and 31% identical to the sequences of the large subunits of carbamyl phosphate synthetase in Escherichia coli and Saccharomyces cerevisiae, respectively. If this gene encodes carbamyl phosphate synthetase in Methanosarcina barkeri, this is the first example, in an archaebacterium, of physical linkage of genes that encode enzymes which catalyze reactions in the same amino acid biosynthetic pathway.

  1. Monitoring Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes with Genetically Encoded Calcium and Voltage Fluorescent Reporters

    PubMed Central

    Shinnawi, Rami; Huber, Irit; Maizels, Leonid; Shaheen, Naim; Gepstein, Amira; Arbel, Gil; Tijsen, Anke J.; Gepstein, Lior

    2015-01-01

    Summary The advent of the human-induced pluripotent stem cell (hiPSC) technology has transformed biomedical research, providing new tools for human disease modeling, drug development, and regenerative medicine. To fulfill its unique potential in the cardiovascular field, efficient methods should be developed for high-resolution, large-scale, long-term, and serial functional cellular phenotyping of hiPSC-derived cardiomyocytes (hiPSC-CMs). To achieve this goal, we combined the hiPSC technology with genetically encoded voltage (ArcLight) and calcium (GCaMP5G) fluorescent indicators. Expression of ArcLight and GCaMP5G in hiPSC-CMs permitted to reliably follow changes in transmembrane potential and intracellular calcium levels, respectively. This allowed monitoring short- and long-term changes in action-potential and calcium-handling properties and the development of arrhythmias in response to several pharmaceutical agents and in hiPSC-CMs derived from patients with different inherited arrhythmogenic syndromes. Combining genetically encoded fluorescent reporters with hiPSC-CMs may bring a unique value to the study of inherited disorders, developmental biology, and drug development and testing. PMID:26372632

  2. Transgenic Expression of the Chemokine Receptor Encoded by Human Herpesvirus 8 Induces an Angioproliferative Disease Resembling Kaposi's Sarcoma

    PubMed Central

    Yang, Tong-Yuan; Chen, Shu-Cheng; Leach, Michael W.; Manfra, Denise; Homey, Bernhard; Wiekowski, Maria; Sullivan, Lee; Jenh, Chung-Her; Narula, Satwant K.; Chensue, Stephen W.; Lira, Sergio A.

    2000-01-01

    Human herpesvirus 8 (HHV8, also known as Kaposi's sarcoma [KS]-associated herpesvirus) has been implicated as an etiologic agent for KS, an angiogenic tumor composed of endothelial, inflammatory, and spindle cells. Here, we report that transgenic mice expressing the HHV8-encoded chemokine receptor (viral G protein–coupled receptor) within hematopoietic cells develop angioproliferative lesions in multiple organs that morphologically resemble KS lesions. These lesions are characterized by a spectrum of changes ranging from erythematous maculae to vascular tumors, by the presence of spindle and inflammatory cells, and by expression of vGPCR, CD34, and vascular endothelial growth factor. We conclude that vGPCR contributes to the development of the angioproliferative lesions observed in these mice and suggest that this chemokine receptor may play a role in the pathogenesis of KS in humans. PMID:10662790

  3. Transgenic expression of the chemokine receptor encoded by human herpesvirus 8 induces an angioproliferative disease resembling Kaposi's sarcoma.

    PubMed

    Yang, T Y; Chen, S C; Leach, M W; Manfra, D; Homey, B; Wiekowski, M; Sullivan, L; Jenh, C H; Narula, S K; Chensue, S W; Lira, S A

    2000-02-01

    Human herpesvirus 8 (HHV8, also known as Kaposi's sarcoma [KS]-associated herpesvirus) has been implicated as an etiologic agent for KS, an angiogenic tumor composed of endothelial, inflammatory, and spindle cells. Here, we report that transgenic mice expressing the HHV8-encoded chemokine receptor (viral G protein-coupled receptor) within hematopoietic cells develop angioproliferative lesions in multiple organs that morphologically resemble KS lesions. These lesions are characterized by a spectrum of changes ranging from erythematous maculae to vascular tumors, by the presence of spindle and inflammatory cells, and by expression of vGPCR, CD34, and vascular endothelial growth factor. We conclude that vGPCR contributes to the development of the angioproliferative lesions observed in these mice and suggest that this chemokine receptor may play a role in the pathogenesis of KS in humans. PMID:10662790

  4. 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.

  5. Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

    PubMed Central

    Leyton-Mange, Jordan S.; Mills, Robert W.; Macri, Vincenzo S.; Jang, Min Young; Butte, Faraz N.; Ellinor, Patrick T.; Milan, David J.

    2014-01-01

    Summary In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity. PMID:24527390

  6. Rapid cellular phenotyping of human pluripotent stem cell-derived cardiomyocytes using a genetically encoded fluorescent voltage sensor.

    PubMed

    Leyton-Mange, Jordan S; Mills, Robert W; Macri, Vincenzo S; Jang, Min Young; Butte, Faraz N; Ellinor, Patrick T; Milan, David J

    2014-02-11

    In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity. PMID:24527390

  7. A novel human gene encoding a G-protein-coupled receptor (GPR15) is located on chromosome 3

    SciTech Connect

    Heiber, M.; Marchese, A.; O`Dowd, B.F.

    1996-03-05

    We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor encoded by GPR15 with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1. GPR15 was mapped to human chromosome 3q11.2-q13.1. 12 refs., 2 figs.

  8. 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.

  9. ChromNet: Learning the human chromatin network from all ENCODE ChIP-seq data.

    PubMed

    Lundberg, Scott M; Tu, William B; Raught, Brian; Penn, Linda Z; Hoffman, Michael M; Lee, Su-In

    2016-01-01

    A cell's epigenome arises from interactions among regulatory factors-transcription factors and histone modifications-co-localized at particular genomic regions. We developed a novel statistical method, ChromNet, to infer a network of these interactions, the chromatin network, by inferring conditional-dependence relationships among a large number of ChIP-seq data sets. We applied ChromNet to all available 1451 ChIP-seq data sets from the ENCODE Project, and showed that ChromNet revealed previously known physical interactions better than alternative approaches. We experimentally validated one of the previously unreported interactions, MYC-HCFC1. An interactive visualization tool is available at http://chromnet.cs.washington.edu. PMID:27139377

  10. 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

  11. 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

  12. Human hTM. cap alpha. gene: Expression in muscle and nonmuscle tissue

    SciTech Connect

    MacLeod, A.R.; Gooding, C.

    1988-01-01

    The authors isolated a cDNA clone from a human skeletal muscle library which contains the complete protein-coding sequence of a skeletal muscle ..cap alpha..-tropomyosin. This cDNA sequence defines a fourth human tropomyosin gene, the hTM..cap alpha.. gene, which is distinct from the hTM/sub nm/ gene encoding a closely related isoform of skeletal muscle ..cap alpha..-tropomyosin. In cultured human fibroblasts, the hTM..cap alpha.. gene encodes both skeletal-muscle- and smooth-muscle-type ..cap alpha..-tropomyosins by using an alternative mRNA-splicing mechanism.

  13. The mammalian single-minded (SIM) gene: Mouse cDNA structure and diencephalic expression indicate a candidate gene for Down syndrome

    SciTech Connect

    Yamaki, Akiko |; Kudoh, Jun; Shindoh, Nobuaki

    1996-07-01

    We have recently isolated a human homolog (hSIM) of the Drosophila single-minded (sim) gene from the Down syndrome critical region of chromosome 21 using the exon trapping method. The Drosophila sim gene encodes a transcription factor that regulates the development of the central nervous system midline cell lineage. To elucidate the structure of the mammalian SIM protein, we have isolated cDNA clones from a mouse embryo cDNA library. The cDNA clones encode a polypeptide of 657 amino acids with a bHLH (basic-helix-loop-helix) domain, characteristic of a large family of transcription factors, and a PAS (Per-Arnt-Sim) domain in the amino-terminal half region. Both of these domains have striking sequence homology with human SIM and Drosophila SIM proteins. In contrast, the carboxy-terminal half of the mouse SIM protein consists of a proline-rich region with no sequence homology to the Drosophila SIM provator domain of a number of transcription factors. Whole-mount embryo in situ hybridization experiments revealed that the SIM mRNA is expressed prominently in the diencephalon during embryogenesis strongly suggest that the newly isolated mammalian SIM homolog may play a critical role in the development of the mammalian central nervous system. We propose that the human SIM gene may be one of the pathogenic genes of Down syndrome. 36 refs., 6 figs.

  14. Acetylcholinesterase of Stomoxys calcitrans (L.) (Diptera: Muscidae): cDNA sequence, baculovirus expression, and biochemical properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 2193-nucleotide cDNA encoding acetylcholinesterase (AChE) of the stable fly, Stomoxys calcitrans (L.) was expressed in the baculovirus system. The open reading frame encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 96% and 94% identity to the AChEs of Haema...

  15. A testis-specific gene, TPTE, encodes a putative transmembrane tyrosine phosphatase and maps to the pericentromeric region of human chromosomes 21 and 13, and to chromosomes 15, 22, and Y.

    PubMed

    Chen, H; Rossier, C; Morris, M A; Scott, H S; Gos, A; Bairoch, A; Antonarakis, S E

    1999-11-01

    To contribute to the creation of a transcription map of human chromosome 21 (HC21) and to the identification of genes that may be involved in the pathogenesis of Down syndrome, exon trapping was performed from HC21-specific cosmids covering the entire chromosome. More than 700 exons have been identified to date. One such exon, hmc01a06, maps to YAC 831B6 which contains marker D21Z1 (alphoid repeats) and had previously been localized to the pericentromeric region of HC21. Northern-blot analysis revealed a 2.5-kb mRNA species strongly and exclusively expressed in the testis. We cloned the corresponding full-length cDNA, which encodes a predicted polypeptide of 551 amino acids with at least two potential transmembrane domains and a tyrosine phosphatase motif. The cDNA has sequence homology to chicken tensin, bovine auxilin and rat cyclin-G associated kinase (GAK). The entire polypeptide sequence also has significant homology to tumor suppressor PTEN/MMAC1 protein. We termed this novel gene/protein TPTE (transmembrane phosphatase with tensin homology). Polymerase chain reaction amplification, fluorescent in situ hybridization, Southern-blot and sequence analysis using monochromosomal somatic cell hybrids showed that this gene has highly homologous copies on HC13, 15, 22, and Y, in addition to its HC21 copy or copies. The estimated minimum number of copies of the TPTE gene in the haploid human genome is 7 in male and 6 in female. Zoo-blot analysis showed that TPTE is conserved between humans and other species. The biological function of the TPTE gene is presently unknown; however, its expression pattern, sequence homologies, and the presence of a potential tyrosine phosphatase domain suggest that it may be involved in signal transduction pathways of the endocrine or spermatogenetic function of the testis. It is also unknown whether all copies of TPTE are functional or whether some are pseudogenes. TPTE is, to our knowledge, the gene located closest to the human

  16. Molecular cloning of cDNAs encoding lamp A, a human lysosomal membrane glycoprotein with apparent M sub r approx 120,000

    SciTech Connect

    Viitala, J.; Carlsson, S.R.; Siebert, P.D.; Fukuda, M. )

    1988-06-01

    Although several lysosomal membrane glycoproteins have been characterized by using specific antibodies, none of the studies so far elucidated the amino acid sequence of a lysosomal membrane glycoprotein. Here we describe cDNA clones encoding for one of the lysosome-associated membrane proteins with apparent M{sub r} {approx} 120,000, lamp A. The amino acid sequence based on the fully coded cDNA shows that as many as 18 potential N-glycosylation sites can be found in the total of 385 amino acid residues. The results obtained by endoglycosidase F digestion support the conclusion that this glycoprotein contains 18 N-glycans. These N-glycosylation sites are clustered in two domains; one contains 10 and the other contains 8 N-glycosylation sites. These domains are separated by a (proline-serine)-rich region that has a distinct homology to the IgA hinge structure. The first N-glycosylated domain is elongated to a potential leader peptide toward the NH{sub 2}-terminal end. The second N-glycosylated domain, on the other hand, is connected to a putative transmembrane portion consisting of hydrophobic amino acids. This segment, in turn, is elongated to a short cytoplasmic segment composed of 11 amino acid residues at the COOH-terminal end.

  17. The human GARS-AIRS-GART gene encodes two proteins which are differentially expressed during human brain development and temporally overexpressed in cerebellum of individuals with Down syndrome.

    PubMed

    Brodsky, G; Barnes, T; Bleskan, J; Becker, L; Cox, M; Patterson, D

    1997-11-01

    Purines are critical for energy metabolism, cell signalling and cell reproduction. Nevertheless, little is known about the regulation of this essential biochemical pathway during mammalian development. In humans, the second, third and fifth steps of de novo purine biosynthesis are catalyzed by a trifunctional protein with glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS) and glycinamide ribonucleotide formyltransferase (GART) enzymatic activities. The gene encoding this trifunctional protein is located on chromosome 21. The enzyme catalyzing the intervening fourth step of de novo purine biosynthesis, phosphoribosylformylglycineamide amidotransferase (FGARAT), is encoded by a separate gene on chromosome 17. To investigate the regulation of these proteins, we have generated monoclonal and/or polyclonal antibodies specific to each of these enzymatic domains. Using these antibodies on western blots of Chinese hamster ovary (CHO) cells transfected with the human GARS-AIRS-GART gene, we show that this gene encodes not only the trifunctional protein of 110 kDa, but also a monofunctional GARS protein of 50 kDa. This carboxy-truncated human GARS protein is produced by alternative splicing resulting in the use of a polyadenylation site in the intron between the terminal GARS and the first AIRS exons. The expression of both the GARS and GARS-AIRS-GART proteins are regulated during development of the human cerebellum, while the expression of FGARAT appears to be constitutive. All three proteins are expressed at high levels during normal prenatal cerebellum development while the GARS and GARS-AIRS-GART proteins become undetectable in this tissue shortly after birth. In contrast, the GARS and GARS-AIRS-GART proteins continue to be expressed during the postnatal development of the cerebellum in individuals with Down syndrome. PMID:9328467

  18. 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

  19. Plasmid-encoding vasostatin inhibited the growth and metastasis of human hepatocellular carcinoma cells.

    PubMed

    Peng, Xing-Chen; Wang, Ming; Chen, Xu-Xia; Liu, Jing; Xiao, Gui-Hua; Liao, Hong-Li

    2014-10-01

    The growth and metastasis of solid tumors depends on angiogenesis. Anti-angiogenesis therapy may represent a promising therapeutic option. Vasostatin, the N-terminal domain of calreticulin, is a very potent endogenous inhibitor of angiogenesis and tumor growth. In this study, we attempted to investigate whether plasmid-encoding vasostatin complexed with cationic liposome could suppress the growth and metastasis of hepatocellular carcinoma in vivo and discover its possible mechanism of action. Apoptosis induction of pSecTag2B-vasostatin plasmid on murine endothelial cells (MS1) was examined by flow cytometric analysis in vitro. Nude mice bearing HCCLM3 tumor received pSecTag2B-vasostatin, pSecTag2B-Null, and 0.9 % NaCl solution, respectively. Tumor net weight was measured and survival time was observed. Microvessel density within tumor tissues was determined by CD31 immunohistochemistry. H&E staining of lungs and TUNEL assay of primary tumor tissues were also conducted. The results displayed that pSecTag2B-vasostatin could inhibit the growth and metastasis of hepatocellular carcinoma xenografts and prolong survival time compared with the controls in vivo. Moreover, histologic analysis revealed that pSecTag2B-vasostatin treatment increased apoptosis and inhibited angiogenesis. The present data may be of importance to the further exploration of this new anti-angiogenesis approach in the treatment of hepatocellular cancer. PMID:24997628

  20. 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. PMID:17571346

  1. Structure and chromosomal localization of the gene encoding the human myelin protein zero (MPZ)

    SciTech Connect

    Hayasaka, Kiyoshi; Himoro, Masato; Takada, Goro ); Wang, Yimin; Takata, Mizuho; Minoshima, Shinsei; Shimizu, Nobuyoshi; Miura, Masayuki; Uyemura, Keiichi )

    1993-09-01

    The authors describe the cloning, characterization, and chromosomal mapping of the human myelin protein zero (MPZ) gene (a structural protein of myelin and an adhesive glycoprotein of the immunoglobulin superfamily). The gene is about 7 kb long and consists of six exons corresponding of the functional domains. All exon-intron junction sequences conform to the GT/AG rule. The 5[prime]-flanking region of the gene has a TA-rich element (TATA-like box), two CAAT boxes, and a single defined transcription initiation site detected by the primer extension method. The gene for human MPZ was assigned to chromosome 1q22-q23 by spot blot hybridization of flow-sorted human chromosomes and fluorescence in situ hybridization. The localization of the MPZ gene coincides with the locus for Charcot-Marie-Tooth disease type 1B, determined by linkage analysis. 20 refs., 3 figs., 1 tab.

  2. Structure and localization of the gene encoding human peripheral myelin protein 2 (PMP2)

    SciTech Connect

    Hayasaka, Kiyoshi; Himoro, Masato; Takada, Goro ); Takahashi, Ei-Ichi ); Minoshima, Shinsei; Shimizu, Nobuyoshi )

    1993-11-01

    Peripheral myelin protein 2 (PMP2) is a small, basic, and cytoplasmic lipid-binding protein of peripheral myelin. In this paper, the authors describe the cloning, characterization, and chromosomal mapping of the human PMP2 gene. The gene is about 8 kb long and consists of four exons. All exon-intron junction sequences conform to the GT/AG rule. The 5[prime]-flanking region of the gene has a TA-rich element (TATA-like box) and a single defined transcription initiation site detected by the primer extension method. The gene for human PMP2 was assigned to chromosome 8q21.3-q22.1 by spot hybridization of flow-sorted human chromosomes and fluorescence in situ hybridization. 29 refs., 4 figs., 1 tab.

  3. Development and Validation of a Microarray for the Investigation of the CAZymes Encoded by the Human Gut Microbiome

    PubMed Central

    Leroy, Quentin; Vialettes, Bernard; Million, Matthieu; Raoult, Didier; Henrissat, Bernard

    2013-01-01

    Distal gut bacteria play a pivotal role in the digestion of dietary polysaccharides by producing a large number of carbohydrate-active enzymes (CAZymes) that the host otherwise does not produce. We report here the design of a custom microarray that we used to spot non-redundant DNA probes for more than 6,500 genes encoding glycoside hydrolases and lyases selected from 174 reference genomes from distal gut bacteria. The custom microarray was tested and validated by the hybridization of bacterial DNA extracted from the stool samples of lean, obese and anorexic individuals. Our results suggest that a microarray-based study can detect genes from low-abundance bacteria better than metagenomic-based studies. A striking example was the finding that a gene encoding a GH6-family cellulase was present in all subjects examined, whereas metagenomic studies have consistently failed to detect this gene in both human and animal gut microbiomes. In addition, an examination of eight stool samples allowed the identification of a corresponding CAZome core containing 46 families of glycoside hydrolases and polysaccharide lyases, which suggests the functional stability of the gut microbiota despite large taxonomical variations between individuals. PMID:24391873

  4. Mutations in CSPP1, Encoding a Core Centrosomal Protein, Cause a Range of Ciliopathy Phenotypes in Humans

    PubMed Central

    Shaheen, Ranad; Shamseldin, Hanan E.; Loucks, Catrina M.; Seidahmed, Mohammed Zain; Ansari, Shinu; Ibrahim Khalil, Mohamed; Al-Yacoub, Nadya; Davis, Erica E.; Mola, Natalie A.; Szymanska, Katarzyna; Herridge, Warren; Chudley, Albert E.; Chodirker, Bernard N.; Schwartzentruber, Jeremy; Majewski, Jacek; Katsanis, Nicholas; Poizat, Coralie; Johnson, Colin A.; Parboosingh, Jillian; Boycott, Kym M.; Innes, A. Micheil; Alkuraya, Fowzan S.

    2014-01-01

    Ciliopathies are characterized by a pattern of multisystem involvement that is consistent with the developmental role of the primary cilium. Within this biological module, mutations in genes that encode components of the cilium and its anchoring structure, the basal body, are the major contributors to both disease causality and modification. However, despite rapid advances in this field, the majority of the genes that drive ciliopathies and the mechanisms that govern the pronounced phenotypic variability of this group of disorders remain poorly understood. Here, we show that mutations in CSPP1, which encodes a core centrosomal protein, are disease causing on the basis of the independent identification of two homozygous truncating mutations in three consanguineous families (one Arab and two Hutterite) affected by variable ciliopathy phenotypes ranging from Joubert syndrome to the more severe Meckel-Gruber syndrome with perinatal lethality and occipital encephalocele. Consistent with the recently described role of CSPP1 in ciliogenesis, we show that mutant fibroblasts from one affected individual have severely impaired ciliogenesis with concomitant defects in sonic hedgehog (SHH) signaling. Our results expand the list of centrosomal proteins implicated in human ciliopathies. PMID:24360803

  5. 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…

  6. Human Cortical θ during Free Exploration Encodes Space and Predicts Subsequent Memory

    PubMed Central

    Snider, Joseph; Plank, Markus; Lynch, Gary; Halgren, Eric

    2013-01-01

    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. PMID:24048836

  7. 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. PMID:24048836

  8. The encoding of category-specific versus nonspecific information in human inferior temporal cortex.

    PubMed

    Guo, Bingbing; Meng, Ming

    2015-08-01

    Several brain areas in the inferior temporal (IT) cortex, such as the fusiform face area (FFA) and parahippocampal place area (PPA), are hypothesized to be selectively responsive to a particular category of visual objects. However, how category-specific and nonspecific information may be encoded at this level of visual processing is still unclear. Using fMRI, we compared averaged BOLD activity as well as multi-voxel activation patterns in the FFA and PPA corresponding to high-contrast and low-contrast face and house images. The averaged BOLD activity in the FFA and PPA was modulated by the image contrast regardless of the stimulus category. Interestingly, unlike the univariate averaged BOLD activity, multi-voxel activation patterns in the FFA and PPA were barely affected by variations in stimulus contrast. In both the FFA and PPA, decoding the categorical information about whether participants saw faces or houses was independent of stimulus contrast. Moreover, the multivariate pattern analysis (MVPA) results were highly stable when either the voxels that were more sensitive to stimulus contrast or the voxels that were less sensitive were used. Taken together, these findings demonstrate that both category-specific (face versus house) information and nonspecific (image contrast) information are available to be decoded orthogonally in the same brain areas (FFA and PPA), suggesting that complementary neural mechanisms for processing visual features and categorical information may occur in the same brain areas but respectively be revealed by averaged activity and multi-voxel activation patterns. Whereas stimulus strength, such as contrast, modulates overall activity amplitudes in these brain areas, activity patterns across populations of neurons appear to underlie the representation of object category. PMID:25869859

  9. Identification of a partial cDNA clone for the C3d/Epstein-Barr virus receptor of human B lymphocytes: homology with the receptor for fragments C3b and C4b of the third and fourth components of complement.

    PubMed

    Weis, J J; Fearon, D T; Klickstein, L B; Wong, W W; Richards, S A; de Bruyn Kops, A; Smith, J A; Weis, J H

    1986-08-01

    Human complement receptor type 2 (CR2) is the B-lymphocyte receptor both for the C3d fragment of the third component of complement and for the Epstein-Barr virus. Amino acid sequence analysis of tryptic peptides of CR2 revealed a strong degree of homology with the human C3b/C4b receptor, CR1. This homology suggested that CR1 gene sequences could be used to detect the CR2 sequences at conditions of low-stringency hybridization. Upon screening a human tonsillar cDNA library with CR1 cDNA sequences, two clones were identified that hybridized at low, but not at high, stringency. Redundant oligonucleotides specific for CR2 sequences were synthesized and used to establish that the two cDNA clones weakly hybridizing with the CR1 cDNA contained CR2 sequences. One of these CR2 cDNA clones hybridized to oligonucleotides derived from two distinct CR2 tryptic peptides, whereas the other, smaller cDNA clone hybridized to oligonucleotides derived from only one of the CR2 peptides. Nucleotide sequence analysis of the CR2 cDNA confirmed that the site of oligonucleotide hybridization was identical to that predicted from the peptide sequence, including flanking sequences not included within the oligonucleotide probes. The CR2-specific cDNA sequences identified a poly(A)+ RNA species of 5 kilobases in RNA extracted from human B cells but did not hybridize to any RNA obtained from the CR2-negative T-cell line HSB-2, thus confirming the appropriate size and tissue-specific distribution for the CR2 mRNA. The striking peptide sequence homology between CR2 and CR1 and the cross-hybridization of the CR2 cDNA with the CR1-specific sequences allow the placement of CR2 in a recently defined gene family of C3- and C4-binding proteins consisting of CR1, C4-binding protein, factor H, and now, CR2. PMID:3016712

  10. 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. PMID:26283043

  11. Cloning of a parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR 106) cell line: Chromosomal assignment of the gene in the human, mouse, and rat genomes

    SciTech Connect

    Pausova, Z.; Bourdon, J.; Clayton, D.; Janicic, N.; Goltzman, D.; Hendy, G.N. ); Mattei, M.G. ); Seldin, M.F. ); Riviere, M.; Szpirer, J. )

    1994-03-01

    Complementary DNAs spanning the entire coding region of the rat parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) were isolated from a rat osteosarcoma (UMR 106) cell-line cDNA library. The longest of these clones (rPTHrec4) was used to chromosomally assign the PTHR gene in the human, rat, and mouse genomes. By somatic cell hybrid analysis, the gene was localized to human chromosome 3 and rat chromosome 8; by in situ hybridization, the gene was mapped to human chromosome 3p21.1-p22 and to mouse chromosome 9 band F; and by interspecific backcross analysis, the Pthr gene segregated with the transferrin (Trf) gene in chromosome 9 band F. Mouse chromosome 9 and rat chromosome 8 are known to be highly homologous and to also show synteny conservation with human chromosome 3. These three chromosomes share the transferrin gene (TF), the myosin light polypeptide 3 gene (MYL3), and the acelpeptide hydrolase gene (APEH). These results add a fourth gene, the PTHR gene, to the synteny group conserved in these chromosomes. 34 refs., 7 figs. 1 tab.

  12. Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

    1992-01-01

    We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

  13. Assignment of the gene encoding glycogen synthase (GYS) to human chromosome 19, band q13,3

    SciTech Connect

    Lehto, M. Helsinki Univ. ); Stoffel, M.; Espinosa, R. III; Beau, M.M. le; Bell, G.I. ); Groop, L. )

    1993-02-01

    The enzyme glycogen synthase (UDP glocose:glycogen 4-[alpha]-D-glucosyltransferase, EC 2.4.1.11) catalyzes the formation of glycogen from uridine diphosphate glucose (UPDG). Impaired activation of muscle glycogen synthase by insulin has been noted in patients with genetic risk of developing non-insulin-dependent diabets mellitus (NIDDM) and this may represent an early defect in the pathogenesis of this disorder. As such, glycogen synthase represents a candidate gene for contributing to genetic susceptibility. As a first step in studying the role of glycogen synthase in the genetics of NIDDM, we have isolated a cosmid encoding the human glycogen synthase gene (gene symbol GYS) and determined its chromosomal localization by fluorescence in situ hybridization. 4 refs., 1 fig.

  14. The complete exon-intron structure of the 156-kb human gene NFKB1, which encodes the p105 and p50 proteins of transcription factors NF-{kappa}B and I{kappa}B-{gamma}: Implications for NF-{kappa}B-mediated signal transduction

    SciTech Connect

    Heron, E.; Deloukas, P.; van Loon, A.P.G.M.

    1995-12-10

    The NFKB1 gene encodes three proteins of the NF-{kappa}/Rel and I{kappa}B families: p105, p50, and (in mouse) I{kappa}B-{gamma}. We determined the complete genomic structure of human NFKB1. NFKB1 spans 156 kb and has 24 exons with introns varying between 40,000 and 323 bp in length. Although NFKB2, which encodes p100 and p52, also has 24 exons and has a comparable exon-intron structure, it is 20 times shorter than NFKB1. We propose that the long size of NFKB1 is important for transient activation of NF-{kappa}B complexes containing p50. I{kappa}B-{gamma} corresponds to the carboxyl-terminal half of p105. DNA sequence analysis showed that the 3{prime}-end of human intron 11 and the 5{prime}-end of exon 12 of NFKB1 are colinear with the 5{prime}-untranslated region of mouse I{kappa}B-{gamma} cDNA. I{kappa}B-{gamma} is thus likely to be generated by transcription starting within intron 11 and not by alternative splicing of the mouse mRNA encoding p105 and p50. 71 refs., 5 figs., 1 tab.

  15. 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

  16. Extensive Cochleotopic Mapping of Human Auditory Cortical Fields Obtained with Phase-Encoding fMRI

    PubMed Central

    Amedi, Amir

    2011-01-01

    The primary sensory cortices are characterized by a topographical mapping of basic sensory features which is considered to deteriorate in higher-order areas in favor of complex sensory features. Recently, however, retinotopic maps were also discovered in the higher-order visual, parietal and prefrontal cortices. The discovery of these maps enabled the distinction between visual regions, clarified their function and hierarchical processing. Could such extension of topographical mapping to high-order processing regions apply to the auditory modality as well? This question has been studied previously in animal models but only sporadically in humans, whose anatomical and functional organization may differ from that of animals (e.g. unique verbal functions and Heschl's gyrus curvature). Here we applied fMRI spectral analysis to investigate the cochleotopic organization of the human cerebral cortex. We found multiple mirror-symmetric novel cochleotopic maps covering most of the core and high-order human auditory cortex, including regions considered non-cochleotopic, stretching all the way to the superior temporal sulcus. These maps suggest that topographical mapping persists well beyond the auditory core and belt, and that the mirror-symmetry of topographical preferences may be a fundamental principle across sensory modalities. PMID:21448274

  17. Chromosome locations of genes encoding human signal transduction adapter proteins, Nck (NCK), Shc (SHC1), and Grb2 (GRB2)

    SciTech Connect

    Huebner, K.; Kastury, K.; Druck, T.

    1994-07-15

    Abnormalities due to chromosomal aberration or point mutation in gene products of growth factor receptors or in ras gene products, which lie on the same signaling pathway, can cause disease in animals and humans. Thus, it can be important to determine chromosomal map positions of genes encoding {open_quotes}adapter{close_quotes} proteins, which are involved in transducing signals from receptor tyrosine kinases to downstream signal recipients such as ras, because adaptor protein genes could also, logically, serve as targets of mutation, rearrangement, or other aberration in disease. Therefore, DNAs from panels of rodent-human hybrids carrying defined complements of human chromosomes were assayed for the presence of the cognate genes for NCK, SHC, and GRB2, three SH2 or SH2/SH3 (Src homology 2 and 3) domain-containing adapter proteins. Additionally, NCK and SHC genes were more narrowly localized by chromosomal in situ hybridization. The NCK locus is at chromosome region 3q21, a region involved in neoplasia-associated changes; the SHC cognate locus, SHC1, is at 1q21, and the GRB2 locus is at 17q22-qter telomeric to the HOXB and NGFR loci. Both SHC1 and GRB2 are in chromosome regions that may be duplicated in some tumor types. 41 refs., 4 figs.

  18. Breast cancer gene therapy using an adenovirus encoding human IL-2 under control of mammaglobin promoter/enhancer sequences.

    PubMed

    Chaurasiya, S; Hew, P; Crosley, P; Sharon, D; Potts, K; Agopsowicz, K; Long, M; Shi, C; Hitt, M M

    2016-06-01

    Interleukin-2 (IL-2) has been used clinically for the treatment of some malignancies, but the toxicities associated with systemic IL-2 therapy are a major challenge. Here we have determined whether transcriptional targeting of IL-2 to breast cancer (BrCa) using an engineered human mammaglobin promoter/enhancer (MPE2) is a feasible option for reducing IL-2-associated toxicities while still achieving a meaningful antitumor effect. We have constructed nonreplicating adenovirus vectors encoding either a reporter gene (luciferase) or human IL-2 (hIL-2) complementary DNA under control of the MPE2 sequence, the murine cytomegalovirus immediate early (MCMV) promoter or the human telomerase reverse transcriptase (hTERT) promoter. Luciferase and hIL-2 complementary DNAs under the control of the MPE2 sequence in adenovirus vectors were expressed at high levels in BrCa cells and at lower levels in normal cells of human and murine origin. Cancer specificity of the hTERT promoter was found to be similar to that of the MPE2 promoter in cells of human origin, but reduced specificity in murine cells. The MPE2 regulatory sequence demonstrated excellent tissue specificity in a mouse tumor model. Whereas the MCMV promoter-controlled IL-2 vector generated high liver toxicity in mice, the MPE2-controlled IL-2 vector generated little or no liver toxicity. Both IL-2 vectors exerted significant tumor growth delay; however, attempts to further enhance antitumor activity of the IL-2 vectors by combining with the proapoptotic drug procaspase activating compound 1 (PAC1) were unsuccessful. PMID:27151235

  19. Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b/sub 5/ reductase

    SciTech Connect

    Yubisui, T.; Naitoh, Y.; Zenno, S.; Tamura, M.; Takeshita, M.; Sakaki, Y.

    1987-06-01

    A cDNA coding for human liver NADH-cytochrome b/sub 5/ reductase was cloned from a human liver cDNA library constructed in phage lambdagt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b/sub 5/ reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb/sub 5/R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb/sub 5/R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme

  20. Locus heterogeneity disease genes encode proteins with high interconnectivity in the human protein interaction network

    PubMed Central

    Keith, Benjamin P.; Robertson, David L.; Hentges, Kathryn E.

    2014-01-01

    Mutations in genes potentially lead to a number of genetic diseases with differing severity. These disease genes have been the focus of research in recent years showing that the disease gene population as a whole is not homogeneous, and can be categorized according to their interactions. Locus heterogeneity describes a single disorder caused by mutations in different genes each acting individually to cause the same disease. Using datasets of experimentally derived human disease genes and protein interactions, we created a protein interaction network to investigate the relationships between the products of genes associated with a disease displaying locus heterogeneity, and use network parameters to suggest properties that distinguish these disease genes from the overall disease gene population. Through the manual curation of known causative genes of 100 diseases displaying locus heterogeneity and 397 single-gene Mendelian disorders, we use network parameters to show that our locus heterogeneity network displays distinct properties from the global disease network and a Mendelian network. Using the global human proteome, through random simulation of the network we show that heterogeneous genes display significant interconnectivity. Further topological analysis of this network revealed clustering of locus heterogeneity genes that cause identical disorders, indicating that these disease genes are involved in similar biological processes. We then use this information to suggest additional genes that may contribute to diseases with locus heterogeneity. PMID:25538735

  1. Cloning and expression of CTP:phosphoethanolamine cytidylyltransferase cDNA from rat liver.

    PubMed Central

    Bladergroen, B A; Houweling, M; Geelen, M J; van Golde, L M

    1999-01-01

    CTP:phosphoethanolamine cytidylyltransferase (ET) is a key regulatory enzyme in the CDP-ethanolamine pathway for phosphatidylethanolamine synthesis. As a first step in the elucidation of the structure-function relationship and the regulation of ET, an ET cDNA was cloned from rat liver. The cloned cDNA encodes a protein of 404 amino acid residues with a calculated molecular mass of 45.2 kDa. The deduced amino acid sequence is very similar to that of human ET (89% identity). Furthermore, it shows less, but significant, similarity to yeast ET as well as to other cytidylyltransferases, including rat CTP:phosphocholine cytidylyltransferase and Bacillus subtilis glycerol-3-phosphate cytidylyltransferase. Like human and yeast ET, rat ET has a large repetitive internal sequence in the N- and C-terminal halves of the protein. Both parts of the repeat contain the HXGH motif, the most conserved region in the N-terminal active domain of other cytidylyltransferases, indicating the existence of two catalytic domains in ET. The hydropathy profile revealed that rat ET is largely hydrophilic and lacks a hydrophobic stretch long enough to span a bilayer membrane. There was no prediction for an amphipathic alpha-helix. Transfection of COS cells with the cDNA clone resulted in an 11-fold increase in ET activity, corresponding to an increase in the amount of ET protein as detected on a Western blot. Determination of the ET activity during liver development showed a 2. 5-fold increase between day 17 of gestation and birth (day 22) and the amount of ET protein changed accordingly. Northern blot analysis showed that this was accompanied by an increase in the amount of ET mRNA. Between day 17 of gestation and birth, the amount of mRNA in fetal rat liver increased approx. 6-fold, suggesting the regulation of ET at both pretranslational and post-translational levels during rat liver development. PMID:10493918

  2. Genomic organization of the human SCN5A gene encoding the cardiac sodium channel

    SciTech Connect

    Wang, Qing; Li, Zhizhong; Shen, Jiaxiang; Keating, M.T.

    1996-05-15

    The voltage-gated cardiac sodium channel, SCN5A, is responsible for the initial upstroke of the action potential. Mutations in the human SCN5A gene cause susceptibility to cardiac arrhythmias and sudden death in the long QT syndrome (LQT). In this report we characterize the genomic structure of SCN5A. SCN5A consists of 28 exons spanning approximately 80 kb on chromosome 3p21. We describe the sequences of all intron/exon boundaries and a dinucleotide repeat polymorphism in intron 16. Oligonucleotide primers based on exon-flanking sequences amplify all SCN5A exons by PCR. This work establishes the complete genomic organization of SCN5A and will enable high-resolution analyses of this locus for mutations associated with LQT and other phenotypes for which SCN5A may be a candidate gene. 40 refs., 4 figs., 2 tabs.

  3. Cloning and expression of two human genes encoding calcium-binding proteins that are regulated during myeloid differentiation.

    PubMed Central

    Lagasse, E; Clerc, R G

    1988-01-01

    The cellular mechanisms involved in chronic inflammatory processes are poorly understood. This is especially true for the role of macrophages, which figure prominently in the inflammatory response. Two proteins, MRP8 and MRP14, which are expressed in infiltrate macrophages during inflammatory reactions but not in normal tissue macrophages, have been characterized. Here we report that MRP8 and MRP14 mRNAs are specifically expressed in human cells of myeloid origin and that their expression is regulated during monocyte-macrophage and granulocyte differentiation. To initiate the analysis of cis-acting elements governing the tissue-specific expression of the MRP genes, we cloned the human genes encoding MRP8 and MRP14. Both genes contain three exons, are single copy, and have a strikingly similar organization. They belong to a novel subfamily of highly homologous calcium-binding proteins which includes S100 alpha, S100 beta, intestinal calcium-binding protein, P11, and calcyclin (2A9). A transient expression assay was devised to investigate the tissue-specific regulatory elements responsible for MRP gene expression after differentiation in leukemia HL60 cells. The results of this investigation demonstrated that the cis-acting elements responsible for MRP expression are present on the cloned DNA fragment containing the MRP gene loci. Images PMID:3405210

  4. Distribution of Genes Encoding the Trypsin-Dependent Lantibiotic Ruminococcin A among Bacteria Isolated from Human Fecal Microbiota

    PubMed Central

    Marcille, F.; Gomez, A.; Joubert, P.; Ladiré, M.; Veau, G.; Clara, A.; Gavini, F.; Willems, A.; Fons, M.

    2002-01-01

    Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance active against Clostridium perfringens were isolated from human fecal samples of various origins (from healthy adults and children, as well as from adults with chronic pouchitis). Identification of these strains showed that they belonged to Ruminococcus gnavus, Clostridium nexile, and Ruminococcus hansenii species or to new operational taxonomic units, all from the Clostridium coccoides phylogenetic group. In hybridization experiments with a probe specific for the structural gene encoding the trypsin-dependent lantibiotic ruminococcin A (RumA) produced by R. gnavus, seven strains gave a positive response. All of them harbored three highly conserved copies of rumA-like genes. The deduced peptide sequence was identical to or showed one amino acid difference from the hypothetical precursor of RumA. Our results indicate that the rumA-like genes have been disseminated among R. gnavus and phylogenetically related strains that can make up a significant part of the human fecal microbiota. PMID:12089024

  5. The human herpes virus 8-encoded chemokine receptor is required for angioproliferation in a murine model of Kaposi's sarcoma.

    PubMed

    Jensen, Kristian K; Manfra, Denise J; Grisotto, Marcos G; Martin, Andrea P; Vassileva, Galya; Kelley, Kevin; Schwartz, Thue W; Lira, Sergio A

    2005-03-15

    Kaposi's sarcoma (KS)-associated herpesvirus or human herpes virus 8 is considered the etiological agent of KS, a highly vascularized neoplasm that is the most common tumor affecting HIV/AIDS patients. The KS-associated herpesvirus/human herpes virus 8 open reading frame 74 encodes a constitutively active G protein-coupled receptor known as vGPCR that binds CXC chemokines with high affinity. In this study, we show that conditional transgenic expression of vGPCR by cells of endothelial origin triggers an angiogenic program in vivo, leading to development of an angioproliferative disease that resembles KS. This angiogenic program consists partly in the expression of the angiogenic factors placental growth factor, platelet-derived growth factor B, and inducible NO synthase by the vGPCR-expressing cells. Finally, we show that continued vGPCR expression is essential for progression of the KS-like phenotype and that down-regulation of vGPCR expression results in reduced expression of angiogenic factors and regression of the lesions. Together, these findings implicate vGPCR as a key element in KS pathogenesis and suggest that strategies to block its function may represent a novel approach for the treatment of KS. PMID:15749907

  6. Isolation of a gene encoding a Chlamydia sp. strain TWAR protein that is recognized during infection of humans.

    PubMed

    Campbell, L A; Kuo, C C; Thissen, R W; Grayston, J T

    1989-01-01

    Chlamydia sp. strain TWAR is a unique Chlamydia sp. that causes acute respiratory disease. A gene bank consisting of TWAR isolate AR-39 DNA in pUC19 was screened with anti-AR-39 rabbit immune sera. Two positive clones were isolated that contained 7.3-kilobase (pLC1) and 14.9-kilobase (pLC2) plasmids. Restriction mapping and hybridization studies showed that both pLC1 and pLC2 contained a common 4.2-kilobase PstI fragment. Plasmids were used as templates of in vitro transcription-translation. All three plasmids had a novel protein product of ca. 75 kilodaltons not found in the vector alone. Western blots showed that this protein reacted with anti-TWAR rabbit immune sera and with human immune serum from an individual who had proven TWAR infection. Whole-cell lysates of TWAR demonstrated a protein having the same molecular weight and immunoreactivity as the recombinant gene product. This protein was also recognized by rabbit immune serum against Chlamydia psittaci or Chlamydia trachomatis. Southern hybridizations with the cloned fragment as a probe of digests of other Chlamydia spp. showed weakly hybridizing fragments. These results suggest that we have isolated a gene encoding a protein recognized during human TWAR infection that contains some sequences shared among Chlamydia spp. PMID:2909493

  7. Cloning, expression and characterization of a lipase encoding gene from human oral metagenome.

    PubMed

    Preeti, Arivaradarajan; Hemalatha, Devaraj; Rajendhran, Jeyaprakash; Mullany, Peter; Gunasekaran, Paramasamy

    2014-09-01

    The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6-7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes. PMID:24891735

  8. Murine and human b locus pigmentation genes encode a glycoprotein (gp75) with catalase activity

    SciTech Connect

    Halaban, R.; Moellmann, G. )

    1990-06-01

    Melanogenesis is regulated in large part by tyrosinase, and defective tyrosinase leads to albinism. The mechanisms for other pigmentation determinants (e.g., those operative in tyrosinase-positive albinism and in murine coat-color mutants) are not yet known. One murine pigmentation gene, the brown (b) locus, when mutated leads to a brown (b/b) or hypopigmentated (B{sup lt}/B{sup lt}) coat versus the wild-type black (B/B). The authors show that the b locus codes for a glycoprotein with the activity of a catalase (catalase B). Only the c locus protein is a tyrosinase. Because peroxides may be by-products of melanogenic activity and hydrogen peroxide in particular is known to destroy melanin precursors and melanin, they conclude that pigmentation is controlled not only by tyrosinase but also by a hydroperoxidase. The studies indicate that catalase B is identical with gp75, a known human melanosomal glycoprotein; that the b mutation is in a heme-associated domain; and that the B{sup lt} mutation renders the protein susceptible to rapid proteolytic degradation.

  9. Adaptation to shifted interaural time differences changes encoding of sound location in human auditory cortex.

    PubMed

    Trapeau, Régis; Schönwiesner, Marc

    2015-09-01

    The auditory system infers the location of sound sources from the processing of different acoustic cues. These cues change during development and when assistive hearing devices are worn. Previous studies have found behavioral recalibration to modified localization cues in human adults, but very little is known about the neural correlates and mechanisms of this plasticity. We equipped participants with digital devices, worn in the ear canal that allowed us to delay sound input to one ear, and thus modify interaural time differences, a major cue for horizontal sound localization. Participants wore the digital earplugs continuously for nine days while engaged in day-to-day activities. Daily psychoacoustical testing showed rapid recalibration to the manipulation and confirmed that adults can adapt to shifted interaural time differences in their daily multisensory environment. High-resolution functional MRI scans performed before and after recalibration showed that recalibration was accompanied by changes in hemispheric lateralization of auditory cortex activity. These changes corresponded to a shift in spatial coding of sound direction comparable to the observed behavioral recalibration. Fitting the imaging results with a model of auditory spatial processing also revealed small shifts in voxel-wise spatial tuning within each hemisphere. PMID:26054873

  10. The expression of the human steroid sulfatase-encoding gene is driven by alternative first exons.

    PubMed

    Dalla Valle, Luisa; Toffolo, Vania; Nardi, Alessia; Fiore, Cristina; Armanini, Decio; Belvedere, Paola; Colombo, Lorenzo

    2007-10-01

    We have analyzed steroid sulfatase (STS) gene transcription in 10 human tissues: ovary, adrenal cortex, uterus, thyroid, liver, pancreas, colon, mammary gland, dermal papilla of the hair follicle, and peripheral mononuclear leukocytes. Overall, six different promoters were found to drive STS expression, giving rise to transcripts with unique first exons that were labeled 0a, 0b, 0c, 1a, 1c, and 1d, of which the last two and 0c are newly reported. All of them, except exon 1d, vary in length owing to the occurrence of multiple transcriptional start sites. While placental exon 1a is partially coding, the other five first exons are all untranslated. Three of these (0a, 0b, and 0c) are spliced to the common partially coding exon 1b, whereas the other two (1c and 1d) are spliced to the coding exon 2, which occurs in all transcripts. Whatever the ATG actually used, the differences are restricted to the signal peptide which is post-transcriptionally cleaved. Transcripts with exons 0a and 0b have the broadest tissue distribution, occurring, in 6 out of the 12 tissues so far investigated, while the other first exons are restricted to one or two tissues. The proximal promoter of each first exon was devoid of TATA box or initiator element and lacked consensus elements for transcription factors related to steroidogenesis, suggesting that regulatory sequences are probably placed at greater distance. In conclusion, the regulation of STS transcription appears to be more complex than previously thought, suggesting that this enzyme plays a substantial role in intercellular integration. PMID:17601726

  11. Purification and cDNA cloning of HeLa cell p54nrb, a nuclear protein with two RNA recognition motifs and extensive homology to human splicing factor PSF and Drosophila NONA/BJ6.

    PubMed Central

    Dong, B; Horowitz, D S; Kobayashi, R; Krainer, A R

    1993-01-01

    While searching for a human homolog of the S.cerevisiae splicing factor PRP18, we found a polypeptide that reacted strongly with antibodies against PRP18. We purified this polypeptide from HeLa cells using a Western blot assay, and named it p54nrb (for nuclear RNA-binding protein, 54 kDa). cDNAs encoding p54nrb were cloned with probes derived from partial sequence of the purified protein. These cDNAs have identical coding sequences but differ as a result of alternative splicing in the 5' untranslated region. The cDNAs encode a 471 aa polypeptide that contains two RNA recognition motifs (RRMs). Human p54nrb has no homology to yeast PRP18, except for a common epitope, but is instead 71% identical to human splicing factor PSF within a 320 aa region that includes both RRMs. In addition, both p54nrb and PSF are rich in Pro and Gln residues outside the main homology region. The Drosophila puff-specific protein BJ6, one of three products encoded by the alternatively spliced no-on-transient A gene (nonA), which is required for normal vision and courtship song, is 42% identical to p54nrb in the same 320 aa region. The striking homology between p54nrb, PSF, and NONA/BJ6 defines a novel phylogenetically conserved protein segment, termed DBHS domain (for Drosophila behavior, human splicing), which may be involved in regulating diverse pathways at the level of pre-mRNA splicing. Images PMID:8371983

  12. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2. alpha. (eIF-2. alpha. ) kinase of rabbit reticulocytes: Homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2. alpha. kinase

    SciTech Connect

    Chen, J.J.; Throop, M.S.; Kuo, I.; Pal, J.K.; Brodsky, M.; London, I.M. ); Gehrke, L. Harvard Medical School, Boston, MA )

    1991-09-01

    The authors have cloned the cDNA of the heme-regulated eIF-2{alpha} kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2{alpha} kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of {approx} 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2{alpha} kinase. This observation suggests that GCN2 protein kinase may be an eIF-2{alpha} kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle.

  13. Localization of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II {beta} subunit (CSNK2B)

    SciTech Connect

    Albertella, M.R.; Jones, H.; Thomson, W.

    1996-09-01

    A wide range of autoimmune and other diseases are known to be associated with the major histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility antigens in the class I and class II regions, but some appear to be more strongly associated with genes in the central 1100-kb class III region, making it important to characterize this region fully for the presence of novel genes. An {approximately}220-kb segment of DNA in the class III region separating the Hsp70 (HSPA1L) and BAT1 (D6S8IE) genes, which was previously known to contain 14 genes. Genomic DNA fragments spanning the gaps between the known genes were used as probes to isolate cDNAs corresponding to five new genes within this region. Evidence from Northern blot analysis and exon trapping experiments that suggested the presence of at least two more new genes was also obtained. Partial cDNA and complete exonic genomic sequencing of one of the new genes has identified it as the casein kinase II{beta} subunit (CSNK2B). Two of the other novel genes lie within a region syntenic to that implicated in susceptibility to experimental allergic orchitis in the mouse, an autoimmune disease of the testis, and represent additional candidates for the Orch-1 locus associated with this disease. In addition, characterization of the 13-kb intergenic gap separating the RD (D6545) and G11 (D6S60E) genes has revealed the presence of a gene encoding a 1246-amino-acid polypeptide that shows significant sequence similarity to the yeast anti-viral Ski2p gene product. 49 refs., 8 figs.

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

    PubMed

    Heo, Yunwi; Kwon, Young Chul; Bae, Seong Kyeong; Hwang, Duhyeon; Yang, Hye Ryeon; Choudhary, Indu; Lee, Hyunkyoung; Yum, Seungshic; Shin, Kyoungsoon; Yoon, Won Duk; Kang, Changkeun; Kim, Euikyung

    2016-01-01

    An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His(69), Asp(117), and Ser(216). The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5' donor splice (GT) and 3' acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai. PMID:27399771

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

    PubMed Central

    Heo, Yunwi; Kwon, Young Chul; Bae, Seong Kyeong; Hwang, Duhyeon; Yang, Hye Ryeon; Choudhary, Indu; Lee, Hyunkyoung; Yum, Seungshic; Shin, Kyoungsoon; Yoon, Won Duk; Kang, Changkeun; Kim, Euikyung

    2016-01-01

    An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His69, Asp117, and Ser216. The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5′ donor splice (GT) and 3′ acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai. PMID:27399771

  16. cDNA cloning, characterization and expression analysis of the antioxidant enzyme gene, catalase, of Chinese shrimp Fenneropenaeus chinensis.

    PubMed

    Zhang, Qingli; Li, Fuhua; Zhang, Xiaojun; Dong, Bo; Zhang, Jiquan; Xie, Yusu; Xiang, Jianhai

    2008-05-01

    Catalase is an important antioxidant protein that protects organisms against various oxidative stresses by eliminating hydrogen peroxide. The full-length catalase cDNA of Chinese shrimp Fenneropenaeus chinensis was cloned from the hepatopancreas using degenerate primers by the method of 3' and 5' rapid amplification of cDNA ends PCR. The cDNA sequence consists of 1892 bp with a 1560 bp open reading frame, encoding 520 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases. The sequence includes the catalytic residues His71, Asn144, and Tyr354. The molecular mass of the predicted protein is 58824.04 Da with an estimated pI of 6.63. Sequence comparison showed that the deduced amino acid sequence of F. chinensis catalase shares 96%, 73%, 71% and 70% identity with that of Pacific white shrimp Litopenaeus vannamei, Abalone Haliotis discus hannai, Zhikong scallop Chlamys farreri and Human Homo sapiens, respectively. Catalase transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill by real-time PCR. The variation of catalase mRNA transcripts in hemocytes and hepatopancreas was also quantified by real-time PCR and the result indicated that the catalase showed up-regulated expression trends in hemocytes at 14 h and in hepatopancreas at 37 h after injection with white spot syndrome virus (WSSV). PMID:18353680

  17. Identification of the polypeptides encoded in the unassigned reading frames 2, 4, 4L, and 5 of human mitochondrial DNA

    SciTech Connect

    Mariottini, P.; Chomyn, A.; Riley, M.; Cottrell, B.; Doolittle, R.F.; Attardi, G.

    1986-03-01

    In previous work, antibodies prepared against chemically synthesized peptides predicted from the DNA sequence were used to identify the polypeptides encoded in three of the eight unassigned reading frames (URFs) of human mitochondrial DNA (mtDNA). In the present study, this approach has been extended to other human mtDNA URFs. In particular, antibodies directed against the NH/sub 2/-terminal octapeptide of the putative URF2 product specifically precipitated component 11 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative URF4 product reacted specifically with components 4 and 5, and antibodies against a COOH-terminal heptapeptide of the presumptive URF4L product reacted specifically with component 26. Antibodies against the NH/sub 2/-terminal heptapeptide of the putative product of URF5 reacted with component 1, but only to a marginal extent; however, the results of a trypsin fingerprinting analysis of component 1 point strongly to this component as being the authentic product of URF5. The polypeptide assignments to the mtDNA URFs analyzed here are supported by the relative electrophoretic mobilities of proteins 11, 4-5, 26, and 1, which are those expected for the molecular weights predicted from the DNA sequence for the products of URF2, URF4, URF4L, and URF5, respectively. With the present assignment, seven of the eight human mtDNA URFs have been shown to be expressed in HeLa cells.

  18. The Human Cytomegalovirus-Specific UL1 Gene Encodes a Late-Phase Glycoprotein Incorporated in the Virion Envelope

    PubMed Central

    Shikhagaie, Medya; Mercé-Maldonado, Eva; Isern, Elena; Muntasell, Aura; Albà, M. Mar; López-Botet, Miguel; Hengel, Hartmut

    2012-01-01

    We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is ex