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Artigos de revistas sobre o assunto "Amino Acid Sequence Homology"
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Giorda, R., e H. L. Ennis. "Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes". Molecular and Cellular Biology 7, n.º 6 (junho de 1987): 2097–103. http://dx.doi.org/10.1128/mcb.7.6.2097-2103.1987.
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A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.
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Giorda, R., e H. L. Ennis. "Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes." Molecular and Cellular Biology 7, n.º 6 (junho de 1987): 2097–103. http://dx.doi.org/10.1128/mcb.7.6.2097.
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A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.
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Day, A. A., C. I. McQuillan, J. D. Termine e M. R. Young. "Molecular cloning and sequence analysis of the cDNA for small proteoglycan II of bovine bone". Biochemical Journal 248, n.º 3 (15 de dezembro de 1987): 801–5. http://dx.doi.org/10.1042/bj2480801.
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The cDNA for the full-length core protein of the small chondroitin sulphate proteoglycan II of bovine bone was cloned and sequenced. A 1.3 kb clone (lambda Pg28) was identified by plaque hybridization with a previously isolated 1.0 kb proteoglycan cDNA clone (lambda Pg20), positively identified previously by polyclonal and monoclonal antibody reactivity and by hybrid-selected translation in vitro [Day, Ramis, Fisher, Gehron Robey, Termine & Young (1986) Nucleic Acids Res. 14, 9861-9876]. The cDNA sequences of both clones were identical in areas of overlap. The 360-amino-acid-residue protein contains a 30-residue propeptide of which the first 15 residues are highly hydrophobic. The mature protein consists of 330 amino acid residues corresponding to an Mr of 36,383. The core protein contains three potential glycosaminoglycan-attachment sites (Ser-Gly), only one of which is within a ten-amino-acid-residue homologous sequence seen at the known attachment sites of related small proteoglycans. Comparisons of the published 24-residue N-terminal protein sequence of bovine skin proteoglycan II core protein with the corresponding region in the deduced sequence of the bovine core protein reveals complete homology. Comparison of the cDNA-derived sequences of bovine bone and human embryonic fibroblast proteoglycans shows a hypervariable region near the N-terminus. Nucleotide homology between bone and fibroblast core proteins was 87% and amino acid homology was 90%.
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Qaisar, U., M. Irfan, A. Meqbool, M. Zahoor, M. Y. Khan, B. Rashid, S. Riazuddin e T. Husnain. "Identification, sequencing and characterization of a stress induced homologue of fructose bisphosphate aldolase from cotton". Canadian Journal of Plant Science 90, n.º 1 (1 de janeiro de 2010): 41–48. http://dx.doi.org/10.4141/cjps08056.
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A drought-induced homologue of fructose-1, 6 bisphosphate aldolase full length cDNA (GaAldp) was isolated and sequenced by gene homology and rapid amplification of cDNA ends (RACE) from cotton variety FDH-786 (Gossypium arboretum). Quantitative polymerase chain reaction (PCR) analysis showed that drought stress, salinity and exogenous treatment with abscisic acid (ABA) enhanced the accumulation of GaAldp transcripts in the leaf and stem tissues of the plant. An alignment of the 1413 bp cDNA and 1937 bp genomic DNA sequences revealed that GaAldp has an open reading frame (ORF) encoding 394 amino acids and contains five introns. The predicted amino acid sequence is homologous to a heat-induced isoform of oat chlorplastic fructose bisphosphate aldolase (FBP) (AF216582) and NPALDP1 from Nicotiana paniculata. The GaAldp sequence includes a novel stroma targeting N-terminal transit peptide (TP) of 45 amino acids, which is 63-76% similar to other chloroplastic TPs.Key words: Fructose-1, 6 bisphosphate aldolase, Gossypium arboreum, drought, abscisic acid, transit peptide, salt stress
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Gotschlich, E. C., M. Seiff e M. S. Blake. "The DNA sequence of the structural gene of gonococcal protein III and the flanking region containing a repetitive sequence. Homology of protein III with enterobacterial OmpA proteins." Journal of Experimental Medicine 165, n.º 2 (1 de fevereiro de 1987): 471–82. http://dx.doi.org/10.1084/jem.165.2.471.
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The insert of a lambda gt11 clone expressing gonococcal protein III was sequenced. The deduced amino acid sequence showed a coding frame of 236 amino acids with a typical 22-amino-acid signal peptide, followed by the known NH2-terminal sequence of PIII. The mature protein has a molecular weight of 23,298. It was found that PIII had extensive and very striking homology to the carboxy-terminal portion of enterobacterial OmpA proteins. The homology encompasses the OmpA domain that is believed to be located in the periplasmic space. If the disposition of PIII across the OM is analogous, then the surface-exposed domain consists of less than 40 amino acids. These include a potential 15-amino-acid disulfide loop, a feature not found in OmpA proteins. Hybridization studies with the sequenced insert indicated that it contained a repetitive sequence that occurred at least 20 times in the genome. By additional hybridization studies the area containing the repetitive sequence was narrowed to a region of 43 bp. This region contained an exact copy of the consensus sequence of a 26-bp repetitive sequence recently described. An analogous sequence recurs in an inverted orientation 53 bp downstream.
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Xia-Yun, Jiang, Zou Shu-Ming e Zhou Pei-Gen. "Cloning and sequence analysis of complete cDNA of chitin deacetylase from Mucor racemosus". Chinese Journal of Agricultural Biotechnology 4, n.º 2 (agosto de 2007): 167–72. http://dx.doi.org/10.1017/s1479236207001489.
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AbstractA complete chitin deacetylase (CDA) complementary DNA (cDNA) from Mucor racemosus was cloned and sequenced by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification cDNA end (RACE) with gene special conserved primers. The cDNA sequence was submitted to GenBank (DQ538514). The complete cDNA with full-length of 1506 bp contained a 67 bp 5′-untranslated region, an open reading frame of 1344 bp and 95 bp 3′-untranslated region including tailing site AATAAA. The gene encoded a sequence of 448 amino acid residues and consisted of core nucleotides encoding a polysaccharide deacetylase domain covering 32% of the entire sequence. The CDA gene shared sequence homology with those of several fungi. The corresponding homology of the deduced amino acid sequences varied from 21 to 69%. Phylogenetic analysis according to the deduced amino acid sequences matched the classical fungi taxonomy. The three-dimensional structure of this protein was predicted. The protein had a whole CDA functional domain and a polysaccharide deacetylase domain.
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Reenan, R. A., e R. D. Kolodner. "Isolation and characterization of two Saccharomyces cerevisiae genes encoding homologs of the bacterial HexA and MutS mismatch repair proteins." Genetics 132, n.º 4 (1 de dezembro de 1992): 963–73. http://dx.doi.org/10.1093/genetics/132.4.963.
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Abstract Homologs of the Escherichia coli (mutL, S and uvrD) and Streptococcus pneumoniae (hexA, B) genes involved in mismatch repair are known in several distantly related organisms. Degenerate oligonucleotide primers based on conserved regions of E. coli MutS protein and its homologs from Salmonella typhimurium, S. pneumoniae and human were used in the polymerase chain reaction (PCR) to amplify and clone mutS/hexA homologs from Saccharomyces cerevisiae. Two DNA sequences were amplified whose deduced amino acid sequences both shared a high degree of homology with MutS. These sequences were then used to clone the full-length genes from a yeast genomic library. Sequence analysis of the two MSH genes (MSH = mutS homolog), MSH1 and MSH2, revealed open reading frames of 2877 bp and 2898 bp. The deduced amino acid sequences predict polypeptides of 109.3 kD and 109.1 kD, respectively. The overall amino acid sequence identity with the E. coli MutS protein is 28.6% for MSH1 and 25.2% for MSH2. Features previously found to be shared by MutS homologs, such as the nucleotide binding site and the helix-turn-helix DNA binding motif as well as other highly conserved regions whose function remain unknown, were also found in the two yeast homologs. Evidence presented in this and a companion study suggest that MSH1 is involved in repair of mitochondrial DNA and that MSH2 is involved in nuclear DNA repair.
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Mcintyre, CL, AL Rae, MD Curtis e JM Manners. "Sequence and Expression of a Caffeic Acid O-Methyl Transferase Cdna Homologue in the Tropical Forage Legume Stylosanthes Humilis." Functional Plant Biology 22, n.º 3 (1995): 471. http://dx.doi.org/10.1071/pp9950471.
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The isolation and characterisation of a cDNA encoding a caffeic acid 0-methyl transferase cDNA homologue (COMT) from Stylosanthes humilis are described. The clone is 1391 nucleotides in length, with an open reading frame encoding a predicted protein of 366 amino acids. Cluster analysis of the deduced amino acid sequence revealed extensive homology to other published O-methyl transferase sequences. Maximum levels of homology were seen with COMTs from alfalfa (87%) and aspen (84%). Southern analysis suggested that this enzyme is encoded by two genes in S. humilis. The mRNA is most strongly expressed in stem tissue, with intermediate levels of expression in young leaves and roots, and does not appear to be induced upon fungal infection or wounding.
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Saxena, A., R. Padmanabha e C. V. Glover. "Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II". Molecular and Cellular Biology 7, n.º 10 (outubro de 1987): 3409–17. http://dx.doi.org/10.1128/mcb.7.10.3409-3417.1987.
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Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.
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Saxena, A., R. Padmanabha e C. V. Glover. "Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II." Molecular and Cellular Biology 7, n.º 10 (outubro de 1987): 3409–17. http://dx.doi.org/10.1128/mcb.7.10.3409.
Texto completo da fonteResumo:
Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.
Teses / dissertações sobre o assunto "Amino Acid Sequence Homology"
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Menlove, Kit J. "Model Detection Based upon Amino Acid Properties". BYU ScholarsArchive, 2010. https://scholarsarchive.byu.edu/etd/2253.
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Similarity searches are an essential component to most bioinformatic applications. They form the bases of structural motif identification, gene identification, and insights into functional associations. With the rapid increase in the available genetic data through a wide variety of databases, similarity searches are an essential tool for accessing these data in an informative and productive way. In our chapter, we provide an overview of similarity searching approaches, related databases, and parameter options to achieve the best results for a variety of applications. We then provide a worked example and some notes for consideration. Homology detection is one of the most basic and fundamental problems at the heart of bioinformatics. It is central to problems currently under intense investigation in protein structure prediction, phylogenetic analyses, and computational drug development. Currently discriminative methods for homology detection, which are not readily interpretable, are substantially more powerful than their more interpretable counterparts, particularly when sequence identity is very low. Here I present a computational graph-based framework for homology inference using physiochemical amino acid properties which aims to both reduce the gap in accuracy between discriminative and generative methods and provide a framework for easily identifying the physiochemical basis for the structural similarity between proteins. The accuracy of my method slightly improves on the accuracy of PSI-BLAST, the most popular generative approach, and underscores the potential of this methodology given a more robust statistical foundation.
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Shah, Anuj R. "Improving protein remote homology detection using supervised and semi-supervised support vector machines". Online access for everyone, 2008. http://www.dissertations.wsu.edu/Dissertations/Spring2008/A_Shah_042408.pdf.
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Wistrand, Markus. "Hidden Markov models for remote protein homology detection /". Stockholm, 2005. http://diss.kib.ki.se/2006/91-7140-598-4/.
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Chelliah, Vijayalakshmi. "Functional restraints on amino acid substitution patterns : application to definition of binding sites and sequence-structure homology recognition". Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615170.
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Lindholm, Cecilia K. "Shb and its homologues : signaling in T lymphocytes and fibroblasts /". Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2002. http://publications.uu.se/theses/91-554-5260-4/.
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Santos-Ciminera, Patricia Dantas Ciminera Patricia Dantas Santos Santos Patricia. "Molecular epidemiology of epidemic severe malaria caused by Plasmodium vivax in the state of Amazonas, Brazil /". Download the dissertation in PDF, 2005. http://www.lrc.usuhs.mil/dissertations/pdf/Santos2005.pdf.
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Chu, Yi-wen 1962. "Amino acid sequence requirements for ornithine decarboxylase activity". Thesis, The University of Arizona, 1988. http://hdl.handle.net/10150/276838.
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ODC activity of the altered proteins was measured and compared to that of the full length 461 amino acid containing ODC. Mouse ODC cDNA sequences were deleted from either 5' or 3' ends using exonuclease III and Mung Bean nuclease treatments. An internal deletion was obtained by Hinc II and Bcl I restriction endonuclease digestion of the full length ODC cDNA. Capped mRNAs were synthesized in vitro using the resulting deleted DNA as templates, and the protein was translated in vitro. The results indicate that the protein in which translation initiates at internal AUG start codons does not have any activity. The protein with 39 amino acids deleted from carboxy-terminus maintains 12% of the activity, while deletion of greater than 79 amino acids have no activity. An internal deletion from amino acid 290 to 331 and which may contain the suspected ornithine binding site has no activity. These results suggest that the entire amino acid sequence of mouse ODC is required for full activity of the enzyme.
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Forsberg, Hanna. "Genetic and biochemical characterization of a novel sensor of extracellular amino acids in yeast /". Stockholm : Karolinska Univ. Press, 2001. http://diss.kib.ki.se/2001/91-89428-06-4/.
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Oppermann, Madalina. "Chemical and mass spectrometrical methods in protein analysis /". Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4542-x/.
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Gallina, Serafina. "Structural Characterization of Donkey Lactoferrin: Amino Acid Sequence and Glycan Compositions". Doctoral thesis, Università di Catania, 2016. http://hdl.handle.net/10761/3880.
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Lactoferrin, a protein showing an array of biochemical properties, including immuno-modulation, iron-binding ability, as well as antioxidant, antibacterial and antiviral activities, was isolated from donkey milk by ion exchange chromatography.The characterization of its primary structure, by means of enzymatic digestions, SPITC derivatization of tryptic digest, reversed-phase high performance liquid chromatography, electrospray and matrix-assisted laser desorption/ionization mass spectrometry, is reported. Our results allowed the almost complete characterization of donkey s lactoferrin sequence, that, at least for the covered sequence, differs from the mare s genomic deduced sequence (UniProtKB Acc. Nr. O77811) by five point substitutions located at positions 91 (Arg->His), 328 (Thr->Ile/Leu), 466 (Ala->Gly), 642 (Asn->Ser) and 668 (Ser->Ala).
Glycosylation, one of the most common post-translational modifications, can modify the structural conformation of the protein and consequently its biological activity. A comprehensive glycosylation profile by chymotrypsin digestion, TiO2 and HILIC enrichment strategy, reversed-phase high performance liquid chromatography, electrospray mass spectrometry, high collision dissociation fragmentation, is reported. 26 different glycan compositions were identified, linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476
Livros sobre o assunto "Amino Acid Sequence Homology"
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Morrison, Mary Elizabeth. Murine homolog of the poliovirus receptor (PVR): Molecular cloning, expression, and mapping of poliovirus-receptor interactions. [New York]: [Columbia University], 1993.
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D, Baxevanis Andreas, ed. Current protocols in bioinformatics. New York: Wiley, 2003.
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Hans, Jörnvall, Höög J. -O, Gustavsson A. -M e International Conference on Methods in Protein Sequence Analysis (8th : 1990 : Kiruna, Sweden), eds. Methods in protein sequence analysis. Basel: Birkhäuser Verlag, 1991.
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Ray, Gribskov Michael, e Devereux John 1947-, eds. Sequence analysis primer. New York: Oxford University Press, 1994.
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Aitken, Alastair. Identification of protein consensus sequences: Active site motifs, phosphorylation, and other post-translational modifications. New York: Ellis Horwood, 1990.
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1958-, Gribskov Michael, e Devereux John 1947-, eds. Sequence analysis primer. Oxford: Oxford University Press, 1995.
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1958-, Gribskov Michael, e Devereux John, eds. Sequence analysis primer. New York: Freeman, 1992.
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J, L'Italien James, e Symposium of American Protein Chemists on Modern Methods in Protein Chemistry (1st : 1985 : San Diego, Calif.), eds. Proteins: Structure and function. New York: Plenum Press, 1987.
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G, Allen. Sequencing of proteins and peptides. Amsterdam: Elsevier/North-Holland, 1989.
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1934-, Bhown Ajit S., ed. Protein/peptide sequence analysis: Current methodologies. Boca Raton, Fla: CRC Press, 1988.
Encontre o texto completo da fonteCapítulos de livros sobre o assunto "Amino Acid Sequence Homology"
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Toh, Hiroyuki. "Homology Search and Prediction of Biological Function of Protein from Amino Acid Sequences". In Methods in Protein Sequence Analysis, 233–40. Boston, MA: Springer US, 1993. http://dx.doi.org/10.1007/978-1-4899-1603-7_30.
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Ericsson, L. H., D. Atherton, R. Kutny, A. J. Smith e J. W. Crabb. "Realistic Expectations for Amino Acid Analysis". In Methods in Protein Sequence Analysis, 143–50. Basel: Birkhäuser Basel, 1991. http://dx.doi.org/10.1007/978-3-0348-5678-2_13.
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Moorad, Deborah R., Gregory E. Garcia e B. P. Doctor. "Amino Acid Sequence of Horse Serum Butyrylcholinesterase". In Structure and Function of Cholinesterases and Related Proteins, 145–46. Boston, MA: Springer US, 1998. http://dx.doi.org/10.1007/978-1-4899-1540-5_37.
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Bidlingmeyer, Brian A., Thomas L. Tarvin e Steven A. Cohen. "Amino Acid Analysis of Submicrogram Hydrolyzate Samples". In Methods in Protein Sequence Analysis · 1986, 229–45. Totowa, NJ: Humana Press, 1987. http://dx.doi.org/10.1007/978-1-59259-480-1_15.
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Hare, P. E. "Detection Limits in Amino Acid Analysis: An Overview". In Methods in Protein Sequence Analysis, 2–9. Berlin, Heidelberg: Springer Berlin Heidelberg, 1989. http://dx.doi.org/10.1007/978-3-642-73834-0_1.
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Li, Weiming, Bin Ma e Kaizhong Zhang. "Amino Acid Classification and Hash Seeds for Homology Search". In Bioinformatics and Computational Biology, 44–51. Berlin, Heidelberg: Springer Berlin Heidelberg, 2009. http://dx.doi.org/10.1007/978-3-642-00727-9_6.
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Martin-Ponthieu, Annie, Danièle Wouters-Tyrou, Denise Bélaiche e Pierre Sautière. "Cuttelfish Protamines: Amino-acid Sequences of Three Distinct Variants". In Methods in Protein Sequence Analysis, 289–92. Berlin, Heidelberg: Springer Berlin Heidelberg, 1989. http://dx.doi.org/10.1007/978-3-642-73834-0_38.
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Sternberg, Michael J. E. "Prediction of Protein Structure from Amino Acid Sequence". In Crystallography in Molecular Biology, 141–49. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4684-5272-3_12.
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Abiko, Takashi, e Hiroshi Sekino. "Syntheses of thymopoietins and their immunological effects: Relationship of amino acid sequence to immunological activity". In Amino Acids, 730–37. Dordrecht: Springer Netherlands, 1990. http://dx.doi.org/10.1007/978-94-011-2262-7_88.
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Wang, Yuhuan, Alexander W. Bell, Mark A. Hermodson e Peter J. Roach. "Identification of Phosphorylated Amino Acid Residues During Gas Phase Sequencing". In Methods in Protein Sequence Analysis · 1986, 479–82. Totowa, NJ: Humana Press, 1987. http://dx.doi.org/10.1007/978-1-59259-480-1_39.
Texto completo da fonteTrabalhos de conferências sobre o assunto "Amino Acid Sequence Homology"
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Jenny, Richard J., Debra D. Pittman, John J. Toole, Ronald W. Kriz, Randal J. Kaufman e Kenneth G. Mann. "THE COMPLETE AMINO ACID SEQUENCE OF HUMAN FACTOR V". In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643887.
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cDNA clones encoding human factor V have been isolated and sequenced. The cDNA sequence of factor V obtained from overlapping clones includes a 6672 bp coding region, a 90 bp 5'-untranslated region and a 163 bp 3’-untranslated region including a poly-A tail. The deduced amino acid sequence consists of 2224 amino acids including a 28 amino acid leader peptide. A direct comparison to human factor VIII reveals considerable homology between both proteins with respect to amino acid sequence and domain structure. A triplicated "A" domain and duplicated "C" domain show an approximate 40% identity to the corresponding domains in factor VIII. Factor V and Factor VIII both possess a heavily glycosylated B domain that separates the heavy and light chains of the activated cofactors, although no significant homology is observed in this region. The B domain of factor V contains 35 tandem and approximately 9 additional semi - conserved repeats of nine amino acids of the form (D-L-S-Q-T-T-L-S-P) and 2 additional semi-conserved 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. By direct comparison to amino acid sequence obtained from both human and bovine factor V, the thrombin (IIa) cleavage sites have been assigned as Arg-709/Ser-710, Arg-1018/Thr-1019, and Are-1545/Ser-1546.(Supported by NIH Grant HL-34575)
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Stenflo, J., A.-K. öhlin, Å. Lundvall e B. Dahlback. "β-HYDROXY ASPARTIC ACID AND ft-HYDROXYASPARAGINE IN THEEGF-HOMOLOGY REGIONS OF PROTEIN C AND PROTEINS". In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643995.
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The amino acid sequence has been determined for all of the vitamin K-dependent proteins and the gene structure is known for some of them. These findings have shown the proteins to consist of four clearly discernible domains, except protein S which has six domains. The protein domains seem to be coded on separate exons (Foster, D. C. et. al. 1985 Proc. Natl. Acad. Sci. USA 82,4673). The vitamin K-dependent γ-carboxyglutamic acid (Gla) containing domain isthe common structural denominator of the members of this protein family. In addition, all of these proteins except prothrombin contain domains that are homologous to the precursor of the epidermal growth factor (EGF). Such domains arealso found in proteins that are not vitamin K-dependent, such as the low density lipoprotein receptor, thrombomodulin, factor XII, plasminogen, the tissue type plasminogen activator, urokinase and the complement protein Clr. The vitamin K-dependent proteins can be dividedinto three groups. Factors VII, IX, X, protein C and protein Z form one group, which in addition to the Gla-region have two EGF-homology regions and one domain that is homologous to the serine proteases. Prothrombin has two 'kringle' structures and a serine protease domain and constitutes a group of its own. Protein S is also unique in that it has four EGF-homology regions and a COOH-terminal region that is homologous to the sexual hormone binding globulin (see poster by Edenbrand et. al.).Recently a posttranslationally modified amino acid, B-hydroxyaspatic acid (Hya), was identified in position 71 in the NH2-terminal EGF-homology region ofbovine protein C. The amino acid is formed by hydroxylation of aspartic acid. It has also been identified in the corresponding positions in factors VII, IX,X and protein Z (i. e. proteins which like protein C have two EGF-homology regions each). In protein S the N2-terminal of four EGF-homology regions has hydroxy lated aspartic acid .whereas the following three EGF-like domains have B-hydroxyasparagine. The nucleotide sequence codes for asparagine in the three latter positions. Neither vitamin K nor vitamin C seem to be involvedin the formation of the two hydroxylated amino acids. Recently, Hya was identified in acid hydrolysates of the complement protein Clr. Hya and Hyn have onlybeen found in domains that are homologous to the EGF precursor. In an attempt to identify the structural requirement of the hydroxylating enzyme, we have compared the sequences of EGF-homology regions that contain Hya or Hyn with the corresponding sequences that have been shown not to contain the modified amino acids. The domains that have Hya or Hyn have the consensus sequence Cx xxxx xCxC. This sequence has been found in three EGF-like domains in the EGF-precursor, in two in the LDL-receptor and in two in thrombomodulin. Furthermore, the neurogenic Notch locus in Drosophila melanogaster codes for 36 EGF-homolgy regions, 22 of which contain the consensussequence, whereas the Lin-12 locus in Caenorhabditis elegans codes for at least 11 EGF-like repeats, two of which comply with the consensus sequence. Whether any of these proteins contain Hya orHyn is not yet known with certainty.It has been hypothesized that Hya isinvolved in the Gla independent Ca2+binding of factors IX, X and protein C. In an attempt to resolve this issue, we have isolated the EGF-homology region from human protein C and been able to demonstrate that it binds Ca2+ (see poster by öhlin and Stenflo). However, we do not yet know whether Hya is directly involved in the Ca2+binding.
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Sumi, Y., Y. Nakamura, M. Sakai, M. Muramatsu e N. Aoki. "STRUCTURE OF HUMAN α2-PLASMIN INHIBITOR DEDUCED FROM THAT OF cDNA". In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644371.
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The complete amino acid sequence of α2-plasmin inhibitor (α2PI) was determined by cDNA cloning. A Agt 10 cDNA library was prepared from poly(A)+mRNA isolated from cultured human liver cells. The labeled oligonucleotides, corresponding to the reported partial amino acid sequences of α2PI, were used as probes to screen the library. One of the positive clones was subcloned into plasmid pUC8. A 2.2 kilobase cDNA clone thus isolated contains a region coding for a portion of a leader sequence, the mature protein, a stop codon (TGA), a 3' noncoding region (733 nucleotides), and a poly(A)tail (37 nucleotides). The amino acid sequence deduced from the cDNA is composed of 452 amino acids starting with an amino-terminal sequence of Asn-Gln-Glu-Gln and ending with a carboxyl-terminal sequence of Gly-Ser-Pro-Lys. The sequence shows approximately 30% homology with those of other plasma serine protease inhibitors. However, α2PI extends 50-52 amino acids beyond the carboxyl-terminal ends of the other inhibitors. This 50-52 carboxyl-terminal amino acid sequence is therefore specific to α2PI, and contains the sequence that is exactly the same as that of the peptide containing the plasminogen binding site. There are three lysine residues possibly involved in the binding to plasminogen in this region. From the homology with the other inhibitors, the inhibitor's reactive-site peptide bond was suggested to be Met-Ser and the same as that of ai-antitrypsin. The Met residue is located at the 362 position from the amino-terminal end.
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Edgington, T. S., J. H. Morrissey e H. Fakhrai. "MOLECULAR CLONING OF HUMAN TISSUE FACTOR cDNA". In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643740.
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Tissue factor (TF), the cell-surface receptor and allo-steric activator for factor Vll/VIIa, is important in hemostasis andinflammation. The TF apoprotein was purifiedfrom human brain using factor Vll-affinitychromatography and SDS gel electrophoresis,and was found to consist of a 47 kDa heavy chain plus a 12.5 kDa light chain. Approximately one-third of the heavy chain amino acid sequence was determined for four regions by microsequencing the intact protein and peptides derived from V8-protease digestion. A λgtll cDNA library, made from mRNA derived from the human fibroblastic cell line WI38,was screened with (a) affinity-purified rabbit antibodies to human tissue factor, and (b) a 45-mer oligonucleotide probe based on TF heavy chain amino acid sequence. Five overlapping cDNA clones were identifiedand sequenced which confirmed all four partial TF amino acid sequences. Together these clones span the entire heavy chain coding sequence as well as 5" and 3" nontranslated regions. The N-terminusof the TF heavy chain is preceded by an unusually long signal peptide which appears to be cleaved at alternative sites two amino acids apart. This results in two variants of TF heavy chains which differ slightly in length and amino-terminal sequence.The deduced protein sequence shows no major homology to known protein sequences. However,a relatively uncommon tripeptide sequence, Trp-Lys-Ser (WKS), appears three times in the TF heavy chain. This tripeptidesequence also occurs in HMW kininogen, factor VIII,von Willebrand"s factor andant ithrombin-III. Limited sequence similarity is observed in flanking sequences,andthis may indicate a possible functional domain for the recognition of members ofthe vitamin K-dependent serine protease famil.Supported by NIH grants HL-16411 andCA-41085.
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Foster, D., B. Schach, M. Rudinsky, K. Berkner, A. Kumar, C. Sprecher, F. Hagen e E. W. bavie. "The Effect of Changes in the Leader Sequence of Human Protein C on Biosynthetic Processing and Gamma-Carboxylation". In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643648.
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Protein C is the precursor to a serine protease in plasma which contains gamma-carboxy glutamic acid and functions as a potent anticoagulant. Protein C shows considerable structural homology with the other vitamin K-dependent coagulation factors including prothrombin, factor VII, factor IX and factor X. This homology includes the putative pro-peptide region of the prepro leader sequences for these proteins, as well as the leader sequences for gamma-carboxylated proteins from bone. Deletion mutants have been constructed in the cDNA for human protein C in order to test the possibility that the pro-peptide portion of the 42 amino acid leader sequence serves as a molecular signal for gamma-carboxylation. Accordingly, these mutants contain the pre-peptide (hydrophobic leader) plus portions of the pro-peptide at the amino terminus of the light chain. The mutant proteins were expressed in carboxylation-competent mammalian cells and analyzed by barium citrate precipitation and N-terminal amino acid sequencing. These studies have shown that deletions in the pro-peptide region interfere with gamma-carboxylation and removal of the pro-peptide. Deletion of residues −1 through −12 had little effect on the carboxylation or secretion. Deletion of −1 through −17 completely abolished gamma-carboxylation, but had no measurable effect on secretion. Amino terminal sequence analysis of the latter mutant showed that the light chain began with Thr-Pro-Ala-Pro... This corresponds to a sequence in the prepro leader starting at −24. This indicates that the signal peptidase cleavage site for human protein C is between residues −25 and −24 and removal of the pro-peptide had been blocked by the deletion.
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Lopez, J. A., D. W. Chung, K. Fujikawa, F. S. Hagen, T. Papavannopoulou e G. J. Roth. "MOLECULAR CLONING OF HUMAN PLATELET GLYCOPROTEIN Ib". In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642927.
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Glycoprotein Ib (GPIb) mediates von Willebrand factor-dependent platelet adhesion and participates in the resulting platelet activation process. In the present investigation, the primary structure of human platelet GPIb was studied. GPIb and its proteolytic fragment glycocalicin were purified to near homogeneity from human platelets by affinity chromatography using wheat germ agglutinin and anti-GPIb monoclonal antibody (D. Nugent, University of Washington) coupled to Sepharose. GPIba chain, β chain, and glycocalicin were isolated, reduced and carboxymethylated, and then fragmented by trypsin and S. aureus V8 protease. Peptides were isolated by HPLC and subjected to amino acid sequence analysis. Approximately 200 amino acid residues were identified. Affinity purified rabbit antibodies directed against the a chain, the ft chain, and glycocalicin were prepared and shown to be monospecific by Western blot analysis. Total RNA was prepared from human erythroleukemia cells grown in the presence of phorbol acetate. Poly(A)+ RNA was selected and used to prepare a cDNA library in λgt11. The library was screened with [125]I-labeled polyclonal antibody to glycocalicin. The clone with the largest cDNA insert was sequenced and shown to code for amino acid sequences corresponding to those determined by Edman degradation of glycocalicin. The predicted amino acid sequence contains at least six tandem repeats of 24 amino acids that are highly homologous with 13 repeats present in leucine rich α2 glycoprotein of human plasma. Another region in the protein contains a second repeat rich in threonine and serine, which shows some homology to a 9 amino acid repeat in the connecting region of human factor V. This region is probably the major site of attachment of clusters of O-linked carbohydrate in GPIbα. These results indicate that human platelet glycoprotein Ibα has a multi-domain structure composed of a number of repetitive sequences. Supported in part by grants from the American Heart Association, Robert Wood Johnson Foundation, Veterans Administration, and National Institutes of Health.
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Fuhlendorff, J., I. Clemmensen e S. Magnusson. "PRIMARY STRUCTURE OF TETRANECTIN. SEQUENCE HOMOLOGY WITH ASIALOGLYCOPROTEIN RECEPTORS AND WITH PROTEOGLYCAN CORE PROTEIN FROM CARTILAGE". In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644380.
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Tetranectin (Mr = 68,000) is a tetrameric blood plasma protein, which binds to plasminogen and also to the lysine-binding site of the isolated kringle 4 from plasminogen. Its four polypeptide chains, which are non-covalently bound, each consists of 181 amino acid residues. We have determined the complete amino acid sequence and the disulfide bonds. Each position corresponds to a single amino acid residue except 34 which contains Ala and Ser and 37 which contains Val and Met in equimolar amounts. The three disulfide bonds connect Cys-50 to Cys-60, Cys-77 to Cys-176 and Cys-152 to Cys-168. The sequence of tetranectin was found to be homologous, to an extent indicating common ancestry, with the extracellular part of the asialoglyco-protein receptors and with the C-terminal globular domain of the cartilage proteoglycan core protein. Conserved residues include the six half-cystines of tetranectin. Therefore, we can now propose disulfide bond patterns for the proteins homologous with tetranectin. Supported by NIH-grant HL-16238 (S.M.), the Danish Science and Medical Research Councils, the Danish Cancer Society and NOVO Foundation.
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Kaida, S., T. Miyata, S. Kawabata, T. Morita, Y. Yoshizawa, H. Igarashi e S. Iwanaga. "NUCLEOTIDE SEQUENCE OF THE STAPHYLOCOAGULASE GENE FROM STAPHYLOCOCCUS AUREUS STRAIN BB". In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644607.
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Staphylocoagulase (SC) is a secretary protein produced by several strains of Staphylococcus aureus (S. aureus). This protein forms a molecular complex ("staphylothrombin") with human prothrombin in a molar ratio of 1:1. The complex displays the ability to clot fibrinogen and to hydrolyze the synthetic tripeptide substrates for α-thrombin. The formation of staphylothrombin does not require proteolytic cleavage of the prothrombin molecule, and this mechanism differs markedly from the activation process by either blood-clotting factor Xa or snake venom procoagulant.In the present studies, a pAT153 library containing partial Mbo I-digested DNA prepared from aureus strain BB has been screened with a fibrin gel formation method. The identity of these clones with SC was confirmed by DNA sequence analysis and by comparison of the derived amino acid sequence with that determined for the purified SC protein. One of the positive colonies was isolated and 3.1 Kb of the insert DNA was determined by the dideoxy chain termination method. The results indicated that the insert DNA consists of 148 bp 5' flanking region, protein coding region of 715 amino acids and 746 bp 3' flanking region, and that SC from strain BB is synthesized as a precursor with a signal peptide of 26 amino acids. Thus, the mature form was composed of 689 amino acids with a molecular weight of 77,337- The NH2-terminal sequence (324 amino acids) of SC isolated from S. aureus strain 213 (S. Kawabata et al. (1986): J. Biol. Chem. 261, 527-531) was compared with that of SC derived from strain BB. The sequence homology between them was found to show 57 %. It was also found that SC derived from strain BB was composed of 8 tandem repeats (27 amino acid residues in length) in the COOH-terminal region, although their functions are not known.
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Vieira, Diogo Munaro, Elvismary Molina de Armas, Maria L. G. Jaramillo, Marcos Catanho, Antonio B. Miranda, Edward Hermann Haeusler e Sérgio Lifschitz. "A New Data Modeling Approach for Alignment-free Biological Applications". In Simpósio Brasileiro de Banco de Dados. Sociedade Brasileira de Computação - SBC, 2023. http://dx.doi.org/10.5753/sbbd.2023.232471.
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Finding homologous proteins and grouping them are tasks of utmost importance in biology, which currently rely on tools based on information from these proteins' DNA or amino acid sequences. These tasks require identifying evolutionary patterns that are challenging to obtain automatically using traditional methods. This work proposes a data modeling approach to leverage evolutionary patterns in homology searching, ranking, and clustering tasks through an alignment-free process using image similarity algorithms. This strategy is valuable even for distant homologs and contributes to data privacy and security.
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Foster, D., B. Schach, M. Rudisky, K. Berkner, A. Kumar, A. Kumar, C. Sprecher, F. Hagen e E. W. Davie. "The Effect of Changes in the Leader Sequence of Human Protein C on Biosynthetic Processing and Gamma-Carboxylation". In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643993.
Texto completo da fonteResumo:
Protein C is the precursor to a serine protease in plasma which contains gamma-carboxy glutamic acid and functions as a potent anticoagulant. Protein C shows considerable structural homology with the other vitamin K-dependent coagulation factors including prothrombin, factor VII, factor IX and factor X. Sequence analysis ofthe cDNAs for these proteins has revealedthe presence of a prepro leader sequence that contains a pre sequence or hydrophobic signal sequence and a propeptide containing a number of highly conserved amino acids. The pre region is removed from thegrowing polypeptide chain by signal peptidase, while the pro region is subsequently removed from the protein prior to secretion. Deletion mutants have been constructed in the propeptide portion of the cDNAfor human protein C in order to test the possibility that the propeptide portion of the 42 amino acid leader sequence serves as a molecular signal for gamma-carboxylation.Accordingly, these mutants containthe pre-peptide (hydrophobic leader) plusportions of the pro-peptide at the amino terminus of the light chain. These deletions include the removal of 4, 9, 12, 15, 16 or 17 amino acids from the carboxyl end of the leader sequence of 42 amino acids. The mutant proteins were expressed in carboxylation-competent mammalian cells and were then examined by Western blotting, barium citrate adsorption and precipitation, amino acid sequence analysis, and biological activity and compared with the native protein present in normal plasma. These studies have shown that deletions inthe pro-peptide region interfere with gamma-carboxylation and removal of the pro-peptide. Deletion of residues -1 through -12 had little effect on the carboxylationor secretion. Deletion of -1 through -17 completely abolished gamma-carboxylation, but had no measurable effect on secretion.Amino terminal sequence analysis of thelatter mutant showed that the light chainbegan with Thr-Pro-Ala-Pro... This corresponds to a sequence in the prepro leader starting at -24. This indicates that the signal peptidase cleavage site for human protein C is between residues -25 and -24 and removal of the pro-peptide had been blocked by the deletion.Furthermore, during biosynthesis and secretion, the amino-terminal region of the propeptide (residues from about -12 through -17) are important for carboxylation of protein C, while the carboxyl-terminal portion of the peptide (residues -1 through -4) are important for the removal of the proleader sequence by proteolytic processing.
Relatórios de organizações sobre o assunto "Amino Acid Sequence Homology"
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Loebenstein, Gad, William Dawson e Abed Gera. Association of the IVR Gene with Virus Localization and Resistance. United States Department of Agriculture, agosto de 1995. http://dx.doi.org/10.32747/1995.7604922.bard.
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We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi. During the present grant we found that TMV production is enhanced in protoplasts and plants of local lesion responding tobacco cultivars exposed to 35oC, parallel to an almost complete suppression of the production of IVR. We also found that IVR is associated with resistance mechanisms in pepper cultivars. We succeeded to clone the IVR gene. In the first attempt we isolated a clone - "101" which had a specific insert of 372 bp (the full length gene for the IVR protein of 23 kD should be around 700 bp). However, attempts to isolate the full length gene did not give clear cut results, and we decided not to continue with this clone. The amino acid sequence of the N-terminus of IVR was determined and an antiserum was prepared against a synthetic peptide representing amino acids residues 1-20 of IVR. Using this antiserum as well as our polyclonal antiserum to IVR a new clone NC-330 was isolated using lamba-ZAP library. This NC-330 clone has an insert of about 1 kB with an open reading frame of 596 bp. This clone had 86.6% homology with the first 15 amino acids of the N-terminal part of IVR and 61.6% homology with the first 23 amino acids of IVR. In the QIA expression system and western blotting of the expressed protein, a clear band of about 21 kD was obtained with IVR antiserum. This clone was used for transformation of Samsun tobacco plants and we have presently plantlets which were rooted on medium containing kanamycin. Hybridization with this clone was also obtained with RNA from induced resistant tissue of Samsun NN but not with RNA from healthy control tissue of Samsun NN, or infected or healthy tissue of Samsun. This further strengthens the previous data that the NC 330 clone codes for IVR. In the U.S. it was shown that IVR is induced in plants containing the N' gene when infected with mutants of TMV that elicit the HR. This is a defined system in which the elicitor is known to be due to permutations of the coat protein which can vary in elicitor strength. The objective was to understand how IVR synthesis is induced after recognition of elicitor coat protein in the signal transduction pathway that leads to HR. We developed systems to manipulate induction of IVR by modifying the elicitor and are using these elicitor molecules to isolate the corresponding plant receptor molecules. A "far-western" procedure was developed that found a protein from N' plants that specifically bind to elicitor coat proteins. This protein is being purified and sequenced. This objective has not been completed and is still in progress. We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi.
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Mevarech, Moshe, Jeremy Bruenn e Yigal Koltin. Virus Encoded Toxin of the Corn Smut Ustilago Maydis - Isolation of Receptors and Mapping Functional Domains. United States Department of Agriculture, setembro de 1995. http://dx.doi.org/10.32747/1995.7613022.bard.
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Ustilago maydis is a fungal pathogen of maize. Some strains of U. maydis encode secreted polypeptide toxins capable of killing other susceptible strains of U. maydis. Resistance to the toxins is conferred by recessive nuclear genes. The toxins are encoded by genomic segments of resident double-strande RNA viruses. The best characterized toxin, KP6, is composed of two polypeptides, a and b, which are not covalently linked. It is encoded by P6M2 dsRNA, which has been cloned, sequenced and expressed in a variety of systems. In this study we have shown that the toxin acts on the membranes of sensitive cells and that both polypeptides are required for toxin activity. The toxin has been shown to function by creating new pores in the cell membrane and disrupting ion fluxes. The experiments performed on artificial phospholipid bilayers indicated that KP6 forms large voltage-independent, cation-selective channels. Experiments leading to the resolution of structure-function relationship of the toxin by in vitro analysis have been initiated. During the course of this research the collaboration also yielded X-ray diffracion data of the crystallized a polypeptide. The effect of the toxin on the pathogen has been shown to be receptor-mediated. A potential receptor protein, identified in membrane fractions of sensitive cells, was subjected to tryptic hydrolysis followed by amino-acid analysis. The peptides obtained were used to isolate a cDNA fragment by reverse PCR, which showed 30% sequence homology to the human HLA protein. Analysis of other toxins secreted by U. maydis, KP1 and KP4, have demonstrated that, unlike KP6, they are composed of a single polypeptide. Finally, KP6 has been expressed in transgenic tobacco plants, indicating that accurate processing by Kex2p-like activity occurs in plants as well. Using tobacco as a model system, we determined that active antifungal toxins can be synthesized and targeted to the outside of transgenic plant cells. If this methodology can be applied to other agronomically crop species, then U. maydis toxins may provide a novel means for biological control of pathogenic fungi.
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Sadot, Einat, Christopher Staiger e Zvi Kam Weizmann. functional genomic screen for new plant cytoskeletal proteins and the determination of their role in actin mediated functions and guard cells regulation. United States Department of Agriculture, janeiro de 2003. http://dx.doi.org/10.32747/2003.7587725.bard.
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The original objectives of the approved proposal were: 1. To construct a YFP fused Arabidopsis cDNA library in a mammalian expression vector. 2. To infect the library into a host fibroblast cell line and to screen for new cytoskeletal associated proteins using an automated microscope. 3. Isolate the new genes. 4. Characterize their role in plants. The project was approved as a feasibility study to allow proof of concept that would entail building the YFP library and picking up a couple of positive clones using the fluorescent screen. We report here on the construction of the YFP library, the development of the automatic microscope, the establishment of the screen and the isolation of positive clones that are plant cDNAs encoding cytoskeleton associated proteins. The rational underling a screen of plant library in fibroblasts is based on the high conservation of the cytoskeleton building blocks, actin and tubulin, between the two kingdoms (80-90% homology at the level of amino acids sequence). In addition, several publications demonstrated the recognition of mammalian cytoskeleton by plant cytoskeletal binding proteins and vice versa. The major achievements described here are: 1. The development of an automated microscope equipped with fast laser auto-focusing for high magnification and a software controlling 6 dimensions; X, Y position, auto focus, time, color, and the distribution and density of the fields acquired. This system is essential for the high throughput screen. 2. The construction of an extremely competent YFP library efficiently cloned (tens of thousands of clones collected, no empty vectors detected) with all inserts oriented 5't03'. These parameters render it well representative of the whole transcriptome and efficient in "in-frame" fusion to YFP. 3. The strategy developed for the screen allowing the isolation of individual positive cDNA clones following three rounds of microscopic scans. The major conclusion accomplished from the work described here is that the concept of using mammalian host cells for fishing new plant cytoskeletal proteins is feasible and that screening system developed is complete for addressing one of the major bottlenecks of the plant cytoskeleton field: the need for high throughput identification of functionally active cytoskeletal proteins. The new identified plant cytoskeletal proteins isolated in the pilot screen and additional new proteins which will be isolated in a comprehensive screen will shed light on cytoskeletal mediated processes playing a major role in cellular activities such as cell division, morphogenesis, and functioning such as chloroplast positioning, pollen tube and root hair elongation and the movement of guard cells. Therefore, in the long run the screen described here has clear agricultural implications.
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Sengupta-Gopalan, Champa, Shmuel Galili e Rachel Amir. Improving Methionine Content in Transgenic Forage Legumes. United States Department of Agriculture, fevereiro de 2001. http://dx.doi.org/10.32747/2001.7580671.bard.
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Leguminous forage crops are high in proteins but deficient in S- amino acids. It has been shown that both wool quality and milk production can be limited by the post-ruminal supply of sulfur-containing amino acids. Efforts to use conventional plant breeding and cell selection techniques to increase the S-amino acid content of alfalfa have met with little success. With the objective to increase the S-amino acid content of forage legumes, the goal of this project was to co- express the methionine rich zein genes from corn along with a gene for a key enzyme in methionine biosynthesis, aspartate kinase(AK). The zeins are seed storage proteins from corn and are groupec into four distinct classes based on their amino acid sequence homologies. The b-zein (15kd) and the 6zein (10kD and 18kD) have proportionately high levels of methionine (10%, 22% and 28%, respectively). Initial studies from our lab had shown that while the 15kD zein accumulated to high levels in vegetative tissues of transgenic tobacco the l0kD zein did not. However, co-expression of the 10kD zein with the 15kD zein genes in tobacco showed stabilization of the 10kD zein and the co-localization of the 10kD and 15kD zein proteins in unique ER derived protein bodies. AK is the key enzyme for producing carbon skeletons for all amino acids of the aspartate family including methionine. It is, however, regulated by end-product feedback inhibition. The specific objectives of this proposal were: i. to co-express the 15kD zein with the 10/18kD zein genes in alfalfa in order to enhance the level of accumulation of the 10/18kD zein; ii. to increase methionine pools by expressing a feedback insensitive AK gene in transformants co-expressing the 15kD and 10/18kD zein genes. The Israeli partners were successful in expressing the AK gene in alfalfa which resulted in an increase in free and bound threonine but not in methionine (Galili et al., 2000). Since our target was to increase methionine pools, we changed our second objective to replace the AK gene with the gene for cystathionine gamma synthase (CGS) in the co-expression studies. The first methionine specific reaction is catalyzed by CGS. An additional objective was to develop a transformation system for Berseem clover, and to introduce the appropriate gene constructs into it with the goal of improving their methionine content. Genes for the 15kD zein along with the genes for either the 10kD or 18kD zein have been introduced into the same alfalfa plant both by sexual crosses and by re-transformation. Analysis of these zein co-expressors have shown that both the IOkD and 18kD zein levels go up 5 to 10 fold when co-expressed with the 15kD zein (Bagga et al., MS in preparation). Incubation of the leaves of transgenic alfalfa co-expressing the 15kD and 10kD zein genes, in the rumen of cows have shown that the zein proteins are stable in the rumen. To increase the level of zein accumulation in transgenic alfalfa different promoters have been used to drive the zein genes in alfalfa and we have concluded that the CaMV 35S promoter is superior to the other strong leaf -specific promoters. By feeding callus tissue of alfalfa plants co-expressing the 15kD and 10kD zein genes with methionine and its precursors, we have shown that the zein levels could be significantly enhanced by increasing the methionine pools. We have now introduced the CGS gene (from Arabidopsis; kindly provided to us by Dr. Leustek), into the 15kD zein transformants and experiments are in progress to check if the expression of the CGS gene indeed increases the level of zein accumulation in alfalfa. We were not successful in developing a transformation protocol for Berseem clover.
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Barefoot, Susan F., Bonita A. Glatz, Nathan Gollop e Thomas A. Hughes. Bacteriocin Markers for Propionibacteria Gene Transfer Systems. United States Department of Agriculture, junho de 2000. http://dx.doi.org/10.32747/2000.7573993.bard.
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The antibotulinal baceriocins, propionicin PLG-1 and jenseniin G., were the first to be identified, purified and characterized for the dairy propionibaceria and are produced by Propionibacterium thoenii P127 and P. thoenii/jensenii P126, respectively. Objectives of this project were to (a) produce polyclonal antibodies for detection, comparison and monitoring of propionicin PLG-1; (b) identify, clone and characterize the propionicin PLG-1 (plg-1) and jenseniin G (jnG) genes; and (3) develop gene transfer systems for dairy propionibacteria using them as models. Polyclonal antibodies for detection, comparison and monitoring of propionicin PLG-1 were produced in rabbits. Anti-PLG-1 antiserum had high titers (256,000 to 512,000), neutralized PLG-1 activity, and detected purified PLG-1 at 0.10 mg/ml (indirect ELISA) and 0.033 mg/ml (competitive indirect ELISA). Thirty-nine of 158 strains (most P. thoenii or P. jensenii) yielded cross-reacting material; four strains of P. thoenii, including two previously unidentified bacteriocin producers, showed biological activity. Eight propionicin-negative P127 mutants produced neither ELISA response nor biological activity. Western blot analyses of supernates detected a PLG-1 band at 9.1 kDa and two additional protein bands with apparent molecular weights of 16.2 and 27.5 kDa. PLG-1 polyclonal antibodies were used for detection of jenseniin G. PLG-1 antibodies neutralized jenseniin G activity and detected a jenseniin G-sized, 3.5 kDa peptide. Preliminary immunoprecipitation of crude preparations with PLG-1 antibodies yielded three proteins including an active 3-4 kDa band. Propionicin PLG-1 antibodies were used to screen a P. jensenii/thoenii P126 genomic expression library. Complete sequencing of a cloned insert identified by PLG-1 antibodies revealed a putative response regulator, transport protein, transmembrane protein and an open reading frame (ORF) potentially encoding jenseniin G. PCR cloning of the putative plg-1 gene yielded a 1,100 bp fragment with a 355 bp ORF encoding 118 amino acids; the deduced N-terminus was similar to the known PLG-1 N-terminus. The 118 amino acid sequence deduced from the putative plg-1 gene was larger than PLG-1 possibly due to post-translational processing. The product of the putative plg-1 gene had a calculated molecular weight of 12.8 kDa, a pI of 11.7, 14 negatively charged residues (Asp+Glu) and 24 positively charged residues (Arg+Lys). The putative plg-1 gene was expressed as an inducible fusion protein with a six-histidine residue tag. Metal affinity chromatography of the fused protein yielded a homogeneous product. The fused purified protein sequence matched the deduced putative plg-1 gene sequence. The data preliminarily suggest that both the plg-1 and jnG genes have been identified and cloned. Demonstrating that antibodies can be produced for propionicin PLG-1 and that those antibodies can be used to detect, monitor and compare activity throughout growth and purification was an important step towards monitoring PLG-1 concentrations in food systems. The unexpected but fortunate cross-reactivity of PLG-1 antibodies with jenseniin G led to selective recovery of jenseniin G by immunoprecipitation. Further refinement of this separation technique could lead to powerful affinity methods for rapid, specific separation of the two bacteriocins and thus facilitate their availability for industrial or pharmaceutical uses. Preliminary identification of genes encoding the two dairy propionibacteria bacteriocins must be confirmed; further analysis will provide means for understanding how they work, for increasing their production and for manipulating the peptides to increase their target species. Further development of these systems would contribute to basic knowledge about dairy propionibacteria and has potential for improving other industrially significant characteristics.
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Wicker, Louise, e Nissim Garti. Entrapment and controlled release of nutraceuticals from double emulsions stabilized by pectin-protein hybrids. United States Department of Agriculture, outubro de 2004. http://dx.doi.org/10.32747/2004.7695864.bard.
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Original Objectives Specific objectives are to: (1) modify charge and hydrophobicity of pectins to improve emulsion stabilizing properties (2) develop emulsions that can be sterically stabilized using modified pectins and/or pectin/protein hybrids (3) obtain submicronal inner emulsion droplets (10-50 nanometers) with small and monodispersed double emulsion (1-2 μm) droplets with long-term stability (possibly by emulsified microemulsions) and (4) trigger and control the release at will. Background Methodology for encapsulation and controlled release of selected addenda, e.g. drugs, vitamins, phytochemicals, flavors, is of major impact in the food industries. Stable double emulsions with desired solubilization and release properties of selected addenda are formed using charge modified pectin or pectin-protein hybrids. Major conclusions, solutions, achievements * We developed methodology to isolate PME isozymes and prepared modified pectins in sufficient quantity to characterize, form single and double emulsions and test stability. *Amino acid sequence of PME isozymes was estimated and will facilitate cloning of PME for commercial application * The contribution of total charge and distribution of charge of modified pectin was determined *Soluble complexes or modified pectins and whey isolates are formed * Stable W/O/W double emulsions were formed that did not cream, had small particle size * Inner phase of double emulsions are nano-sized and stable. These new structures were termed emulsified microemulsions (EME) * Release of bioactives were controlled between a few days to months depending on layering on droplets by hybrids * Commercial testing by Israeli company of stability and release of Vitamin C showed good chemical stability Implications Resolved the major stability limitation of W/O/W emulsions. Resolved the questions regarding citrus PMEs and tailored pilot scale modification of pectins.
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Yalovsky, Shaul, e Julian Schroeder. The function of protein farnesylation in early events of ABA signal transduction in stomatal guard cells of Arabidopsis. United States Department of Agriculture, janeiro de 2002. http://dx.doi.org/10.32747/2002.7695873.bard.
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Loss of function mutations in the farnesyltransferase β subunit gene ERA1 (enhanced response to abscisic acid), cause abscisic acid hypersensitivity in seedlings and in guard cells. This results in slowed water loss of plants in response to drought. Farnesyltransferase (PFT) catalyses the attachment of the 15-carbon isoprenoid farnesyl to conserved cysteine residues located in a conserved C-terminal domain designated CaaX box. PFT is a heterodimeric protein comprised of an a and b sununits. The a subunit is shared between PFT and geranylgeranyltransferase-I (PGGTI) which catalyses the attachemt of the 20-carbon isoprenoid geranylgeranyl to CaaX box proteins in which the last amino acid is almost always leucine and in addition have a polybasic domain proximal to the CaaL box. Preliminary data presented in the proposal showed that increased cytoplasmic Ca2+ concentration in stomal guard cells in response to non-inductive ABA treatements. The goals set in the proposal were to characterize better how PFT (ERA1) affects ABA induced Ca2+ concentrations in guard cells and to identify putative CaaX box proteins which function as negative regulators of ABA signaling and which function is compromised in era1 mutant plants. To achieve these goals we proposed to use camelion Ca2+ sensor protein, high throughput genomic to identify the guard cell transcriptome and test prenylation of candidate proteins. We also proposed to focus our efforts of RAC small GTPases which are prenylated proteins which function in signaling. Our results show that farnesyltransferaseprenylates protein/s that act between the points of ABA perception and the activation of plasma membrane calcium influx channels. A RAC protein designated AtRAC8/AtRop10 also acts in negative regulation of ABA signaling. However, we discovered that this protein is palmitoylated and not prenylated although it contains a C-terminal CXXX motif. We further discovered a unique C-terminal sequence motif required for membrane targeting of palmitoylatedRACs and showed that their function is prenylation independent. A GC/MS based method for expression in plants, purification and analysis of prenyl group was developed. This method would allow highly reliable identification of prenylated protein. Mutants in the shared α subunit of PFT and PGGT-I was identified and characterized and was shown to be ABA hypersensitive but less than era1. This suggested that PFT and PGGT-I have opposing functions in ABA signaling. Our results enhanced the understanding of the role of protein prenylation in ABA signaling and drought resistance in plants with the implications of developing drought resistant plants. The results of our studies were published 4 papers which acknowledge support from BARD.
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Rafaeli, Ada, e Russell Jurenka. Molecular Characterization of PBAN G-protein Coupled Receptors in Moth Pest Species: Design of Antagonists. United States Department of Agriculture, dezembro de 2012. http://dx.doi.org/10.32747/2012.7593390.bard.
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The proposed research was directed at determining the activation/binding domains and gene regulation of the PBAN-R’s thereby providing information for the design and screening of potential PBAN-R-blockers and to indicate possible ways of preventing the process from proceeding to its completion. Our specific aims included: (1) The identification of the PBAN-R binding domain by a combination of: (a) in silico modeling studies for identifying specific amino-acid side chains that are likely to be involved in binding PBAN with the receptor and; (b) bioassays to verify the modeling studies using mutant receptors, cell lines and pheromone glands (at tissue and organism levels) against selected, designed compounds to confirm if compounds are agonists or antagonists. (2) The elucidation ofthemolecular regulationmechanisms of PBAN-R by:(a) age-dependence of gene expression; (b) the effect of hormones and; (c) PBAN-R characterization in male hair-pencil complexes. Background to the topic Insects have several closely related G protein-coupled receptors (GPCRs) belonging to the pyrokinin/PBAN family, one with the ligand pheromone biosynthesis activating neuropeptide or pyrokinin-2 and another with diapause hormone or pyrokinin-1 as a ligand. We were unable to identify the diapause hormone receptor from Helicoverpa zea despite considerable effort. A third, related receptor is activated by a product of the capa gene, periviscerokinins. The pyrokinin/PBAN family of GPCRs and their ligands has been identified in various insects, such as Drosophila, several moth species, mosquitoes, Triboliumcastaneum, Apis mellifera, Nasoniavitripennis, and Acyrthosiphon pisum. Physiological functions of pyrokinin peptides include muscle contraction, whereas PBAN regulates pheromone production in moths plus other functions indicating the pleiotropic nature of these ligands. Based on the alignment of annotated genomic sequences, the primary and secondary structures of the pyrokinin/PBAN family of receptors have similarity with the corresponding structures of the capa or periviscerokinin receptors of insects and the neuromedin U receptors found in vertebrates. Major conclusions, solutions, achievements Evolutionary trace analysisof receptor extracellular domains exhibited several class-specific amino acid residues, which could indicate putative domains for activation of these receptors by ligand recognition and binding. Through site-directed point mutations, the 3rd extracellular domain of PBAN-R was shown to be critical for ligand selection. We identified three receptors that belong to the PBAN family of GPCRs and a partial sequence for the periviscerokinin receptor from the European corn borer, Ostrinianubilalis. Functional expression studies confirmed that only the C-variant of the PBAN-R is active. We identified a non-peptide agonist that will activate the PBAN-receptor from H. zea. We determined that there is transcriptional control of the PBAN-R in two moth species during the development of the pupa to adult, and we demonstrated that this transcriptional regulation is independent of juvenile hormone biosynthesis. This transcriptional control also occurs in male hair-pencil gland complexes of both moth species indicating a regulatory role for PBAN in males. Ultimate confirmation for PBAN's function in the male tissue was revealed through knockdown of the PBAN-R using RNAi-mediated gene-silencing. Implications, both scientific and agricultural The identification of a non-peptide agonist can be exploited in the future for the design of additional compounds that will activate the receptor and to elucidate the binding properties of this receptor. The increase in expression levels of the PBAN-R transcript was delineated to occur at a critical period of 5 hours post-eclosion and its regulation can now be studied. The mysterious role of PBAN in the males was elucidated by using a combination of physiological, biochemical and molecular genetics techniques.
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Ginzberg, Idit, Richard E. Veilleux e James G. Tokuhisa. Identification and Allelic Variation of Genes Involved in the Potato Glycoalkaloid Biosynthetic Pathway. United States Department of Agriculture, agosto de 2012. http://dx.doi.org/10.32747/2012.7593386.bard.
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Steroidal glycoalkaloids (SGAs) are secondary metabolites being part of the plant defense response. The two major SGAs in cultivated potato (Solanum tuberosum) are α-chaconine and α-solanine, which exhibit strong cellular lytic properties and inhibit acetylcholinesterase activity, and are poisonous at high concentrations for humans. As SGAs are not destroyed during cooking and frying commercial cultivars have been bred to contain low levels, and their content in tubers should not exceed 20 mg/100 g fresh weight. However, environmental factors can increase tuber SGA content above the safe level. The focus of the proposed research was to apply genomic approaches to identify candidate genes that control potato SGA content in order to develop tools for potato improvement by marker-assisted selection and/or transgenic approaches. To this end, the objectives of the proposal included identification of genes, metabolic intermediates and allelic variations in the potato SGAbiosynthetic pathway. The SGAs are biosynthesized by the sterol branch of the mevalonic acid/isoprenoid pathway. Transgenic potato plants that overexpress 3-hydroxy-3-methylglutaryl-CoA reductase 1 (HMG1) or squalene synthase 1 (SQS1), key enzymes of the mevalonic acid/isoprenoid pathway, exhibited elevated levels of solanine and chaconine as well as induced expression of genes downstream the pathway. These results suggest of coordinated regulation of isoprenoid (primary) metabolism and SGA secondary metabolism. The transgenic plants were further used to identify new SGA-related candidate genes by cDNA-AFLP approach and a novel glycosyltransferase was isolated. In addition, genes involved in phytosterol biosynthesis may have dual role and synthesize defense-related steroidal metabolites, such as SGAs, via lanosterol pathway. Potato lanosterol synthase sequence (LAS) was isolated and used to prepare transgenic plants with overexpressing and silencing constructs. Plants are currently being analyzed for SGA content. The dynamics of SGA accumulation in the various organs of a potato species with high SGA content gave insights into the general regulation of SGA abundance. Leaf SGA levels in S. chacoense were 10 to 20-fold greater than those of S. tuberosum. The leptines, SGAs with strong antifeedant properties against Colorado potato beetles, were present in all aerial tissues except for early and mid-developmental stages of above ground stolons, and accounted for the high SGA content of S. chacoense. These results indicate the presence of regulatory mechanisms in most tissues except in stolons that limit the levels of α-solanine and α-chaconine and confine leptine accumulation to the aerial tissues. The genomes of cultivated and wild potato contain a 4-member gene family coding for SQS. Three orthologs were cloned as cDNAs from S. chacoense and heterologously expressed in E. coli. Squalene accumulated in all E. coli lines transformed with each of the three gene constructs. Differential transcript abundance in various organs and amino acid sequence differences in the conserved domains of three isoenzymes indicate subfunctionalization of SQS activity and triterpene/sterol metabolism. Because S. chacoense and S. phureja differ so greatly for presence and accumulation of SGAs, we selected four candidate genes from different points along the biosynthetic pathway to determine if chcor phuspecific alleles were associated with SGA expression in a segregating interspecific diploid population. For two of the four genes (HMG2 and SGT2) F2 plants with chcalleles expressed significantly greater total SGAs compared with heterozygotes and those with phualleles. Although there are other determinants of SGA biosynthesis and composition in potato, the ability of allelic states at two genes to affect SGA levels confirms some of the above transgenic work where chcalleles at two other loci altered SGA expression in Desiree. Present results reveal new opportunities to manipulate triterpene/sterol biosynthesis in more targeted ways with the objective of altering SGA content for both human health concerns and natural pesticide content without disrupting the essential metabolism and function of the phytosterol component of the membranes and the growth regulating brassinosteroids.
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Gafni, Yedidya, e Vitaly Citovsky. Molecular interactions of TYLCV capsid protein during assembly of viral particles. United States Department of Agriculture, abril de 2007. http://dx.doi.org/10.32747/2007.7587233.bard.
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Tomato yellow leaf curl geminivirus (TYLCV) is a major pathogen of cultivated tomato, causing up to 100% crop loss in many parts of the world. The present proposal, a continuation of a BARD-funded project, expanded our understanding of the molecular mechanisms by which CP molecules, as well as its pre-coat partner V2, interact with each other (CP), with the viral genome, and with cellular proteins during assembly and movement of the infectious virions. Specifically, two major objectives were proposed: I. To study in detail the molecular interactions between CP molecules and between CP and ssDNA leading to assembly of infectious TYLCV virions. II. To study the roles of host cell factors in TYLCV assembly. Our research toward these goals has produced the following major achievements: • Characterization of the CP nuclear shuttling interactor, karyopherin alpha 1, its pattern of expression and the putative involvement of auxin in regulation of its expression. (#1 in our list of publication, Mizrachy, Dabush et al. 2004). • Identify a single amino acid in the capsid protein’s sequence that is critical for normal virus life-cycle. (#2 in our list of publications, Yaakov, Levy et al. in preparation). • Development of monoclonal antibodies with high specificity to the capsid protein of TYLCV. (#3 in our list of publications, Solmensky, Zrachya et al. in press). • Generation of Tomato plants resistant to TYLCV by expressing transgene coding for siRNA targeted at the TYLCV CP. (#4 in our list of publications, Zrachya, Kumar et al. in press). •These research findings provided significant insights into (i) the molecular interactions of TYLCV capsid protein with the host cell nuclear shuttling receptor, and (ii) the mechanism by which TYLCV V2 is involved in the silencing of PTGS and contributes to the virus pathogenicity effect. Furthermore, the obtained knowledge helped us to develop specific strategies to attenuate TYLCV infection, for example, by blocking viral entry into and/or exit out of the host cell nucleus via siRNA as we showed in our publication recently (# 4 in our list of publications). Finally, in addition to the study of TYLCV nuclear import and export, our research contributed to our understanding of general mechanisms for nucleocytoplasmic shuttling of proteins and nucleic acids in plant cells. Also integration for stable transformation of ssDNA mediated by our model pathogen Agrobacterium tumefaciens led to identification of plant specific proteins involved.