DOCSLIB.ORG

Cloning and Structure Determination of Cdna for Cutinase, an Enzyme

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Cloning and Structure Determination of Cdna for Cutinase, an Enzyme Proc. Natl. Acad. Sci. USA Vol. 81, pp. 3939-3943, July 1984 Biochemistry Cloning and structure determination of cDNA for cutinase, an enzyme involved in fungal penetration of plants (cDNA cloning/nucleotide sequencing/amino acid sequencing/fungal cutinase) C. L. SOLIDAY, W. H. FLURKEY, T. W. OKITA, AND P. E. KOLATTUKUDY* Institute of Biological Chemistry and Biochemistry/Biophysics Program, Washington State University, Pullman, WA 99164-6340 Communicated by Harold J. Evans, February 27, 1984 ABSTRACT The primary structure of cutinase, an extra- cloning of cutinase cDNA and the complete nucleotide se- cellular fungal enzyme involved in the penetration of plants by quence of the coding region. The amino acid sequence pre- pathogenic fungi, has been determined from the nucleotide se- dicted from the nucleotide sequence was verified by amino quence of cloned cDNA. Clones containing cDNA made from acid sequence determination of a significant portion of the poly(A)+ RNA isolated from fungal cultures induced to syn- enzyme. thesize cutinase were screened for their ability to hybridize with the [32P]cDNA for mRNA unique to the induced culture. The 75 cDNA clones thus identified were screened for the cu- MATERIALS AND METHODS tinase genetic code by hybrid-selected translation and exami- Construction of a Fusarium solani pisi cDNA Library. nation of products with anti-cutinase IgG. This method yielded Poly(A)+ RNA from Fusarium solani f. .sp. pisi, T8 strain, 15 clones containing cDNA for cutinase, and Southern blots was isolated as described (7). Single- (8) and double- (9) showed that the size of the cDNA inserts ranged from 279 to stranded cDNA was synthesized and the hairpin loops were 950 nucleotides. Blot analysis showed that cutinase mRNA cleaved with nuclease S1 and the termini were repaired to contained 1050 nucleotides, indicating that the clone contain- blunt ends with the large fragment of DNA polymerase I. A ing 950 nucleotides represented nearly the entire mRNA. This size fraction of duplex DNA >500 base pairs (bp) in length near-full-length cDNA and the restriction fragments subcloned was obtained by preparative electrophoresisfin a 1% agarose from it were sequenced by a combination of the Maxam-Gil- gel. Homopolymer tracts of dC were added to the 3' ends of bert and the phage M13-dideoxy techniques. cDNAs from two this cDNA with calf-thymus terminal transferase (10). The other clones, containing the bulk of the coding region for cu- oligo(dC)-tailed cDNA and oligo(dG)-tailed pBR322 plasmid tinase, were also completely sequenced, and the results con- were annealed (11), and this modified plasmid was used to firmed the sequence obtained with the first clone. A peptide transform Escherichia coli K-12 RR1. Colonies that con- isolated from a trypsin digest of cutinase was sequenced and tained pBR322 plasmid with cDNA inserts were selected by the amino acid sequence as well as the initiation and termina- their tetracycline resistance and ampicillin sensitivity. The tion codons were used to identify the coding region of the enzymes and plasmids used in this cloning procedure were cDNA. The primary structure of the enzyme so far determined purchased from New England Nuclear. by amino acid sequencing (=40% of the total) agreed com- Isolation of Plasmid DNA. Plasmid DNA, extracted from pletely with the nucleotide sequencing results. Thus, the com- selected clones by using either a Triton X-100 (12) or an alka- plete primary structure of the mature enzyme and that of the line lysis procedure (13), was purified by CsCl/ethidium bro- signal peptide region were ascertained. mide density gradient centrifugation. Translation of Hybrid-Selected mRNA. From the total Cutinase, a glycoprotein excreted by phytopathogenic fungi, poly(A)+ RNA isolated from induced fungal cultures, specif- catalyzes the hydrolysis of cutin, the structural polyester of ic mRNA was purified by hybridization to cloned DNA that the plant cuticle (1, 2). Cutinase was shown to be present at had been denatured and immobilized on nitrocellulose filters the site of fungal penetration of the host plant cuticle and (14). The translation products of the hybridized mRNA gen- specific inhibition of cutinase was shown to protect plants erated in a wheat germ translation system (15) with [35S]me- against fungal penetration and consequently infection (1, 2). thionine as the label were examined (7). The active site of cutinase is composed of a catalytic triad Colony Hybridizations. Colony hybridization was per- involving serine, histidine, and a carboxyl group (3). The en- formed with [32P]cDNA probes (16, 17). Two cDNA probes zyme contains one disulfide bridge, which is essential for the were generated from poly(A)+ RNA isolated from induced activity of the enzyme (2). Recently the amino acid sequence and noninduced fungal cultures with reverse transcriptase of the active-serine-containing tryptic peptide of cutinase and [a-32P]dATP (17). Hybridization probes were also made was determined (4). An understanding of the structure and from cDNA inserts isolated from cutinase clones that re- mechanism of action of cutinase could help in the develop- sponded positively to the hybrid-selected translation tests in- ment of effective inhibitors for use as antipenetrants to pro- dicated above. Plasmid isolated from a cutinase cDNA clone tect plants against fungal attack. was treated with Pst I restriction endonuclease and the di- Recent evidence suggested that the infecting capacity of gest was subjected to electrophoresis in 1% low-melting certain pathogenic fungi can be determined by the ability of agarose. The agarose containing the DNA insert was cut the pathogen to produce cutinase (5, 6). Thus the regulation from the gel and melted in 2 vol of 0.01 M Tris HCl, pH 7.5, of expression of cutinase gene could be highly relevant to containing 0.001 M EDTA at 650C, and the agarose was re- pathogenesis. To investigate these molecular aspects of moved from the solution by phenol extraction. After the ad- host-pathogen interaction, labeled cDNA probes for cutin- dition of NaCl to 0.2 M and 2 vol of ethanol, the DNA was ase would be highly beneficial. In this paper we report the precipitated by cooling the solution to -70°C for 15 min. A 32P-labeled probe with a specific activity in excess of 108 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: bp, base pair(s). in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 3939 Downloaded by guest on September 27, 2021 3940 Biochemistry: Soliday et al. Proc. NatL Acad Sci. USA 81 (1984) dpm/4g was prepared by subjecting 1 tug of the DNA insert to produce, cutinase by the addition of low quantities of to a nick-translation reaction with 60 tkCi (1 Ci = 37 GBq) of chemically prepared cutin hydrolysate. Under such condi- [a-32P]dATP (800 Ci/mmol, New England Nuclear) as de- tions cutinase is the major (>70%) extracellular protein (30). scribed (18). Therefore, the poly(A)+ RNA from such induced cultures, Blot Hybridization Analysis of Inserted DNA and of Fungal already enriched in cutinase-specific mRNA, could be readi- RNA. Plasmid was isolated from cutinase cDNA clones by a ly used to generate cutinase cDNA by reverse transcribing rapid miniscreen procedure described for determination of the total poly(A)+ RNA. This inducibility was also useful in the size of the inserted DNA (13). Restriction enzyme di- the initial selection of possible cutinase cDNA clones in the gests of the plasmid preparations were subjected to electro- total cDNA library. Replica nitrocellulose filters containing phoresis through 1% agarose gels, transferred to nitrocellu- lysed colonies of the fungal cDNA library were hybridized lose filters by the method of Southern (19), and hybridized with labeled cDNA probes transcribed from poly(A)+ RNA with a 32P-labeled probe (17). The hybridized filter was sub- isolated from either cultures induced to produce cutinase or jected to autoradiography and the sizes of the labeled DNA control cultures. From 600 colonies selected as containing inserts were determined by their mobilities as compared to recombinant DNA, 75 colonies showed hybridization with those of DNA standards. the probe generated only from induced cultures. Screening The size of cutinase mRNA was determined by subjecting for clones containing cutinase DNA sequences in these 75 3.5 gg of poly(A)+ RNA isolated from induced fungal cul- colonies was done by hybrid-selected translation. The colo- tfres to electrophoresis through a 1.5% agarose gel.contain- nies were pooled into nine groups, and the plasmid DNA ing 2.2 M formaldehyde (20). The formaldehyde-denatured isolated from five of these groups hybridized with fungal RNA was transferred from the gel to nitrocellulose filters mRNA that upon cell-free translation generated protein that (21) and hybridized with a 32P-labeled' probe. The location of cross-reacted with anti-cutinase IgG. Electrophoresis of the the hybridized mRNA was determined by autoradiography immunoprecipitated translation product in a NaDodSO4/ and the size of the cutinase mRNA was determined by com- polyacrylamide gel revealed a single band with a molecular parison of its mobility to that of known DNA fragments gen- weight of 24,200 compared to the 21,600 molecular weight of erated by Alu I and Hae III digestion of pBR322. the mature cutinase. Evaluation of the group of colonies that Subcloning of DNA Fragments from Restriction Endonucle- displayed the most efficient hybrid-selected translation ase Digests of Isolated Cloned Inserts. Taq I, Hpa II, and Sau- showed two individual colonies that contained cutinase 3A digests were selected for subcloning in cohesive Acc I, DNA sequences inserted in their plasmids.
Load more

Recommended publications
  • Endogenous Reverse Transcriptase and Rnase H-Mediated Antiviral Mechanism in Embryonic Stem Cells
    www.nature.com/cr www.cell-research.com ARTICLE Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells Junyu Wu1, Chunyan Wu1, Fan Xing1, Liu Cao1, Weijie Zeng1, Liping Guo1, Ping Li1, Yongheng Zhong1, Hualian Jiang1, Manhui Luo1, Guang Shi2, Lang Bu1, Yanxi Ji1, Panpan Hou1, Hong Peng1, Junjiu Huang2, Chunmei Li1 and Deyin Guo 1 Nucleic acid-based systems play important roles in antiviral defense, including CRISPR/Cas that adopts RNA-guided DNA cleavage to prevent DNA phage infection and RNA interference (RNAi) that employs RNA-guided RNA cleavage to defend against RNA virus infection. Here, we report a novel type of nucleic acid-based antiviral system that exists in mouse embryonic stem cells (mESCs), which suppresses RNA virus infection by DNA-mediated RNA cleavage. We found that the viral RNA of encephalomyocarditis virus can be reverse transcribed into complementary DNA (vcDNA) by the reverse transcriptase (RTase) encoded by endogenous retrovirus-like elements in mESCs. The vcDNA is negative-sense single-stranded and forms DNA/RNA hybrid with viral RNA. The viral RNA in the heteroduplex is subsequently destroyed by cellular RNase H1, leading to robust suppression of viral growth. Furthermore, either inhibition of the RTase activity or depletion of endogenous RNase H1 results in the promotion of virus proliferation. Altogether, our results provide intriguing insights into the antiviral mechanism of mESCs and the antiviral function of endogenized retroviruses and cellular RNase H. Such a natural nucleic acid-based antiviral mechanism in mESCs is referred to as ERASE (endogenous RTase/RNase H-mediated antiviral system), which is an addition to the previously known nucleic acid-based antiviral mechanisms including CRISPR/Cas in bacteria and RNAi in plants and invertebrates.
    [Show full text]
  • Specific Principles of Genome-Wide RNA-Chromatin Interactions
    ARTICLE There are amendments to this paper https://doi.org/10.1038/s41467-020-14337-6 OPEN RADICL-seq identifies general and cell type–specific principles of genome-wide RNA-chromatin interactions Alessandro Bonetti 1,2,18*, Federico Agostini 3,18, Ana Maria Suzuki1,4, Kosuke Hashimoto1, Giovanni Pascarella1, Juliette Gimenez 5, Leonie Roos6,7, Alex J. Nash6,7, Marco Ghilotti1, Christopher J. F. Cameron8,9, Matthew Valentine 1, Yulia A. Medvedeva10,11,12, Shuhei Noguchi1, Eneritz Agirre 2, Kaori Kashi1, Samudyata2, Joachim Luginbühl1, Riccardo Cazzoli13, Saumya Agrawal1, Nicholas M. Luscombe 3,14,15, Mathieu Blanchette8, Takeya Kasukawa 1, Michiel de Hoon1, Erik Arner1, 1234567890():,; Boris Lenhard 6,7,16, Charles Plessy 1, Gonçalo Castelo-Branco 2, Valerio Orlando5,17* & Piero Carninci 1* Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding tran- scripts exhibit nuclear localization and several have been shown to play a role in the reg- ulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA–chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of tran- scripts as well as cell type–specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure.
    [Show full text]
  • Pharmacokinetics, Pharmacodynamics and Metabolism Of
    PHARMACOKINETICS, PHARMACODYNAMICS AND METABOLISM OF GTI-2040, A PHOSPHOROTHIOATE OLIGONUCLEOTIDE TARGETING R2 SUBUNIT OF RIBONUCLEOTIDE REDUCTASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Xiaohui Wei, M.S. * * * * * * The Ohio State University 2006 Approved by Dissertation Committee: Dr. Kenneth K. Chan, Adviser Adviser Dr. Guido Marcucci, Co-adviser Graduate Program in Pharmacy Dr. Thomas D. Schmittgen Dr. Robert J. Lee Co-Adviser Graduate Program in Pharmacy ABSTRACT Over the last several decades, antisense therapy has been developed into a promising gene-targeted strategy to specifically inhibit the gene expression. Ribonucleotide reductase (RNR), composing of subunits R1 and R2, is an important enzyme involved in the synthesis of all of the precursors used in DNA replication. Over- expression of R2 has been found in almost every type of cancer studied. GTI-2040 is a 20-mer phosphorothioate oligonucleotide targeting the coding region in mRNA of the R2 component of human RNR. In this project, clinical pharamcokinetics (PK), pharmacodynamics (PD) and metabolism of this novel therapeutics were investigated in patients with acute myeloid leukemia (AML). A picomolar specific hybridization-ligation ELISA method has been developed and validated for quantification of GTI-2040. GTI-2040 and neophectin complex was found to enhance drug cellular uptake and exhibited sequence- and dose-dependent down-regulation of R2 mRNA and protein in K562 cells. Robust intracellular concentrations (ICs) of GTI-2040 were achieved in peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells from treated AML patients. GTI-2040 concentrations in the nucleus of BM cells were found to correlate with the R2 mRNA down-regulation and disease response.
    [Show full text]
  • Termin Translat Trna Utr Mutat Protein Signal
    Drugs & Chemicals 1: Tumor Suppressor Protein p53 2: Heterogeneous-Nuclear Ribonucleo- (1029) proteins (14) activ apoptosi arf cell express function inactiv induc altern assai associ bind mdm2 mutat p53 p73 pathwai protein regul complex detect exon famili genom respons suppress suppressor tumor wild-typ interact intron isoform nuclear protein sensit site specif splice suggest variant 3: RNA, Transfer (110) 4: DNA Primers (1987) codon contain differ eukaryot gene initi amplifi analysi chain clone detect dna express mrna protein region ribosom rna fragment gene genotyp mutat pcr sequenc site speci suggest synthesi polymorph popul primer reaction region restrict sequenc speci termin translat trna utr 5: Saccharomyces cerevisiae Proteins 6: Apoptosis Regulatory Proteins (291) (733) activ apoptosi apoptosis-induc albican bud candida cerevisia complex encod apoptot bcl-2 caspas caspase-8 cell eukaryot fission function growth interact involv death fasl induc induct ligand methyl necrosi pathwai program sensit surviv trail mutant pomb protein requir saccharomyc strain suggest yeast 7: Plant Proteins (414) 8: Membrane Proteins (1608) access arabidopsi cultivar flower hybrid leaf leav apoptosi cell conserv domain express function gene human identifi inhibitor line maiz plant pollen rice root seed mammalian membran mice mous mutant seedl speci thaliana tomato transgen wheat mutat protein signal suggest transport 1 9: Tumor Suppressor Proteins (815) 10: 1-Phosphatidylinositol 3-Kinase activ arrest cell cycl cyclin damag delet dna (441) 3-kinas activ
    [Show full text]
  • Supplementary Table S1 List of Proteins Identified with LC-MS/MS in the Exudates of Ustilaginoidea Virens Mol
    Supplementary Table S1 List of proteins identified with LC-MS/MS in the exudates of Ustilaginoidea virens Mol. weight NO a Protein IDs b Protein names c Score d Cov f MS/MS Peptide sequence g [kDa] e Succinate dehydrogenase [ubiquinone] 1 KDB17818.1 6.282 30.486 4.1 TGPMILDALVR iron-sulfur subunit, mitochondrial 2 KDB18023.1 3-ketoacyl-CoA thiolase, peroxisomal 6.2998 43.626 2.1 ALDLAGISR 3 KDB12646.1 ATP phosphoribosyltransferase 25.709 34.047 17.6 AIDTVVQSTAVLVQSR EIALVMDELSR SSTNTDMVDLIASR VGASDILVLDIHNTR 4 KDB11684.1 Bifunctional purine biosynthetic protein ADE1 22.54 86.534 4.5 GLAHITGGGLIENVPR SLLPVLGEIK TVGESLLTPTR 5 KDB16707.1 Proteasomal ubiquitin receptor ADRM1 12.204 42.367 4.3 GSGSGGAGPDATGGDVR 6 KDB15928.1 Cytochrome b2, mitochondrial 34.9 58.379 9.4 EFDPVHPSDTLR GVQTVEDVLR MLTGADVAQHSDAK SGIEVLAETMPVLR 7 KDB12275.1 Aspartate 1-decarboxylase 11.724 112.62 3.6 GLILTLSEIPEASK TAAIAGLGSGNIIGIPVDNAAR 8 KDB15972.1 Glucosidase 2 subunit beta 7.3902 64.984 3.2 IDPLSPQQLLPASGLAPGR AAGLALGALDDRPLDGR AIPIEVLPLAAPDVLAR AVDDHLLPSYR GGGACLLQEK 9 KDB15004.1 Ribose-5-phosphate isomerase 70.089 32.491 32.6 GPAFHAR KLIAVADSR LIAVADSR MTFFPTGSQSK YVGIGSGSTVVHVVDAIASK 10 KDB18474.1 D-arabinitol dehydrogenase 1 19.425 25.025 19.2 ENPEAQFDQLKK ILEDAIHYVR NLNWVDATLLEPASCACHGLEK 11 KDB18473.1 D-arabinitol dehydrogenase 1 11.481 10.294 36.6 FPLIPGHETVGVIAAVGK VAADNSELCNECFYCR 12 KDB15780.1 Cyanovirin-N homolog 85.42 11.188 31.7 QVINLDER TASNVQLQGSQLTAELATLSGEPR GAATAAHEAYK IELELEK KEEGDSTEKPAEETK LGGELTVDER NATDVAQTDLTPTHPIR 13 KDB14501.1 14-3-3
    [Show full text]
  • Subject Index Proc
    13088 Subject Index Proc. Natl. Acad. Sci. USA 91 (1994) Apoptosis in substantia nigra following developmental striatal excitotoxic Brine shrimp injury, 8117 See Artemia Visualizing hippocampal synaptic function by optical detection of Ca2l Broccol entry through the N-methyl-D-aspartate channel, 8170 See Brassica Amygdala modulation of hippocampal-dependent and caudate Bromophenacyl bromide nucleus-dependent memory processes, 8477 Bromophenacyl bromide binding to the actin-bundling protein I-plastin Distribution of corticotropin-releasing factor receptor mRNA expression inhibits inositol trisphosphate-independent increase in Ca2l in human in the rat brain and pituitary, 8777 neutrophils, 3534 Brownian dynamics Preproenkephalin promoter yields region-specific and long-term Adhesion of hard spheres under the influence of double-layer, van der expression in adult brain after direct in vivo gene transfer via a Waals, and gravitational potentials at a solid/liquid interface, 3004 defective herpes simplex viral vector, 8979 Browsers Intravenous administration of a transferrin receptor antibody-nerve Thorn-like prickles and heterophylly in Cyanea: Adaptations to extinct growth factor conjugate prevents the degeneration of cholinergic avian browsers on Hawaii?, 2810 striatal neurons in a model of Huntington disease, 9077 Bruton agammaglobulinemia Axotomy induces the expression of vasopressin receptors in cranial and Genomic organization and structure of Bruton agammaglobulinemia spinal motor nuclei in the adult rat, 9636 tyrosine kinase: Localization
    [Show full text]
  • A Suppressor of a Centromere DNA Mutation Encodes a Putative Protein Kinase (MCK1)
    Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press A suppressor of a centromere DNA mutation encodes a putative protein kinase (MCK1) James H. Shero 1 and Philip Hieter Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 USA A new approach to identify genes involved in Saccharomyces cerevisiae kinetochore function is discussed. A genetic screen was designed to recover extragenic dosage suppressors of a CEN DNA mutation. This method identified two suppressors, designated MCK1 and CMS2. Increased dosage of MCK1 specifically suppressed two similar CEN DNA mutations in CDEIII, but not comparably defective CEN DNA mutations in CDEI or CDEII. A strain containing a null allele of MCK1 was viable under standard growth conditions, had a cold-sensitive phenotype (conditional lethality at l l°C), and grew slowly on Benomyl {a microtubule-destabilizing drug). Furthermore, when grown at 18°C or in the presence of Benomyl, the null mutant exhibited a dramatic increase in the rate of mitotic chromosome loss. The allele-specific suppression and chromosome instability phenotypes suggest that MCK1 plays a role in mitotic chromosome segregation specific to CDEIII function. The MCK1 gene encodes a putative protein-serine/threonine kinase, which suggests a possible role for the MCK1 protein in regulating the activity of centromere-binding proteins by phosphorylation. MCK1 was identified and cloned independently for its involvement in the induction of meiosis and is identical to a gene that encodes a phosphotyrosyl protein with protein kinase activity. [Key Words: S. cerevisiae; kinetochore function; CEN DNA; allele-specific suppression] Received December 12, 1990; revised version accepted January 28, 1991.
    [Show full text]
  • Identification and Characterization of a New Oncogene Derived from The
    The EMBO Journal Vol.17 No.3 pp.743–753, 1998 Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase Concepcio´ n Jimenez, David R.Jones, et al., 1991; Skolnik et al., 1991; Hiles et al., 1992; Hu Pablo Rodrı´guez-Viciana1, et al., 1993). Sequence analysis of the p110 primary Ana Gonzalez-Garcı´a, Esther Leonardo, sequence allows the definition of several regions including Stefan Wennstro¨ m1, Cayetano von Kobbe, a p85-binding region, a ras-binding domain, a region Jose L.Toran, Luis R.-Borlado, Victor Calvo2, homologous to PI4-kinases and the catalytic core (Dhand Sergio G.Copin3, Juan P.Albar, et al., 1994; Leevers et al., 1996; Zvelebil et al., 1996). Similarly, p85 includes a SH3 domain, a bcr-homologous M.Luisa Gaspar4, Emilio Diez5, 3 1 region flanked by two proline-rich regions, and two SH2 Miguel A.R.Marcos , Julian Downward , domains, separated by an inter-SH2 region (Dhand et al., Carlos Martinez-A, Isabel Me´ rida and 1994; Holt et al., 1994; Klippel et al., 1994). p85:p110 Ana C.Carrera6 mammalian PI3K, corresponding to class I PI3K, is regulated by cell surface receptors and phosphorylates Department of Immunology and Oncology, Centro Nacional de Biotecnologı´a, Universidad Auto´noma de Madrid, Cantoblanco, PtdIns, PtdIns (4)P and PtdIns (4,5)P2 in vitro, although 1 Madrid E-28049, Spain, Imperial Cancer Research Fund, 44 Lincoln’s PtdIns (4)P and PtdIns (4,5)P2 are thought to be their Inn Fields, London, WC2A 3PX, UK, 2Instituto de Investigaciones major in vivo substrates (Stephens et al., 1993).
    [Show full text]
  • Expression of a Human T-Cell Protein-Tyrosine-Phosphatase in Baby Hamster Kidney Cells (Phosphorylation/Regulation/Localization') DEBORAH E
    Proc. Nati. Acad. Sci. USA Vol. 87, pp. 7280-7284, September 1990 Biochemistry Expression of a human T-cell protein-tyrosine-phosphatase in baby hamster kidney cells (phosphorylation/regulation/localization') DEBORAH E. COOL*t, NICHOLAS K. TONKSt, HARRY CHARBONNEAUt, EDMOND H. FISCHERt, AND EDWIN G. KREBS* *Howard Hughes Medical Institute and tDepartment of Biochemistry, University of Washington, Seattle, WA 98195 Contributed by Edwin G. Krebs, June 20, 1990 ABSTRACT A human T-cell cDNA encoding a 48-kDa their conserved predicted catalytic core, might imply that this protein-tyrosine-phosphatase (PTPase; protein-tyrosine-phos- region plays an important role in regulation. phate phosphohydrolase, EC 3.1.3.48) was cloned into a mam- The physiological functions of the low molecular mass and malian expression vector and introduced into baby hamster receptor-linked PTPases remain unclear, though obviously kidney cells, and stable colonies were isolated. The expressed they must be necessary to control the overall level ofprotein- PTPase was found to be associated with the particulate fraction tyrosine phosphorylation and thus would be required for of the cells, where it was essentially inactive in an in vitro assay regulating growth, differentiation, and transformation. In this unless first subjected to limited trypsinization; trypsin treat- present study, overexpression of the T-cell PTPase trans- ment generated an active fragment of 33 kDa by the removal fected into baby hamster kidney (BHK) cells was investi- of a carboxyl-terminal segment of the full-length enzyme. Gel gated to determine whether this would have adverse effects was associated on normal cell growth, including cytotoxicity. In addition, a filtration indicated that the expressed enzyme carboxyl-terminal truncated form of the enzyme closely with a complex of >600 kDa.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 8,561,811 B2 Bluchel Et Al
    USOO8561811 B2 (12) United States Patent (10) Patent No.: US 8,561,811 B2 Bluchel et al. (45) Date of Patent: Oct. 22, 2013 (54) SUBSTRATE FOR IMMOBILIZING (56) References Cited FUNCTIONAL SUBSTANCES AND METHOD FOR PREPARING THE SAME U.S. PATENT DOCUMENTS 3,952,053 A 4, 1976 Brown, Jr. et al. (71) Applicants: Christian Gert Bluchel, Singapore 4.415,663 A 1 1/1983 Symon et al. (SG); Yanmei Wang, Singapore (SG) 4,576,928 A 3, 1986 Tani et al. 4.915,839 A 4, 1990 Marinaccio et al. (72) Inventors: Christian Gert Bluchel, Singapore 6,946,527 B2 9, 2005 Lemke et al. (SG); Yanmei Wang, Singapore (SG) FOREIGN PATENT DOCUMENTS (73) Assignee: Temasek Polytechnic, Singapore (SG) CN 101596422 A 12/2009 JP 2253813 A 10, 1990 (*) Notice: Subject to any disclaimer, the term of this JP 2258006 A 10, 1990 patent is extended or adjusted under 35 WO O2O2585 A2 1, 2002 U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS (21) Appl. No.: 13/837,254 Inaternational Search Report for PCT/SG2011/000069 mailing date (22) Filed: Mar 15, 2013 of Apr. 12, 2011. Suen, Shing-Yi, et al. “Comparison of Ligand Density and Protein (65) Prior Publication Data Adsorption on Dye Affinity Membranes Using Difference Spacer Arms'. Separation Science and Technology, 35:1 (2000), pp. 69-87. US 2013/0210111A1 Aug. 15, 2013 Related U.S. Application Data Primary Examiner — Chester Barry (62) Division of application No. 13/580,055, filed as (74) Attorney, Agent, or Firm — Cantor Colburn LLP application No.
    [Show full text]
  • Novel Modification of Human Myeloma Proteasomes and Development of Non-Active Site Directed Inhibitors
    Novel modification of Human Myeloma proteasomes and development of non-active site directed inhibitors. By David S. Pitcher A thesis submitted for the degree of Doctor of Philosophy October 2016 Centre of Haematology Imperial College London COPYRIGHT The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Figures 1.2, 1.3, 1.4 and 1.5 are from the sources stated in the legends of each figure and permission has been obtained for reproduction and republishing in an online open access format by the licences stated below: Figure 1.2 is licenced by ‘American Society of Hematology’ number #3973070918521 Figure 1.3 is licenced by ‘Nature Publishing Group’ number #3960180094287 Figures 1.4 & 1.5 are licenced by ‘Nature Publishing Group’ #3960180835983 I DECLARATION All the experiments described in this thesis were designed and performed by myself unless otherwise stated. Contributions from other people are acknowledged in the appropriate sections. I performed the experiments, analysed the data, produced the graphs and figures, and wrote the text; all with the guidance of my supervisor, Dr Maurits Kleijnen. David Pitcher October 2016 II ABSTRACT Multiple Myeloma (MM) is a plasma cell malignancy that is characterised by bone lesions and production of excessive amounts of monoclonal protein.
    [Show full text]
  • ABSTRACT SHU, XIAOMEI. Pathogenesis and Host Response
    ABSTRACT SHU, XIAOMEI. Pathogenesis and Host Response During Infection of Maize Kernels by Aspergillus flavus and Fusarium verticillioides. (Under the direction of Dr. Gary A. Payne.) Developing maize kernels are vulnerable to colonization by microbes. When colonization allows proliferation of the microbe at the expense of the host, disease occurs. The ascomycete fungal pathogens Aspergillus flavus and Fusarium verticillioides are capable of colonizing maize kernels, causing ear rots and contamination of the kernel with mycotoxins. These diseases lead to significant losses of crop yield and quality, and constitute a threat to food safety and human health. Thus, the significance of these diseases has prompted extensive research efforts to understand these plant-parasite interactions. However, pathogenesis and resistance mechanisms remain poorly characterized, hampering the development of effective control strategies. No commercial maize lines are completely resistant to these fungi. We applied an integrated approach consisting of histology, in situ gene expression and transcriptional profiling to better understand the nature of the interactions that occur between maize kernels and these fungi. Maize inbred line B73 was hand pollinated and inoculated with either A. flavus or F. verticillioides by wounding the kernel with a needle bearing conidia. Histological staining of the kernel sections revealed fungal mycelium in kernels adjacent to the inoculation site by 48 hours post inoculation (hpi), and in all tissues at 96 hpi. Compared with F. verticillioides, A. flavus more aggressively colonized kernel tissue and formed a unique biofilm-like structure around the scutellum. Transcriptome profiling using RNA-sequencing (RNA-seq) coupled with pathway analysis showed that these fungi were recognized by the kernel tissues prior to visible colonization.
    [Show full text]