DOCSLIB.ORG

Hybridisation Assay in Which Excess Probe

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Hybridisation Assay in Which Excess Probe Europäisches Patentamt *EP000975805B1* (19) European Patent Office Office européen des brevets (11) EP 0 975 805 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.7: C12Q 1/68 of the grant of the patent: 05.09.2001 Bulletin 2001/36 (86) International application number: PCT/GB98/01057 (21) Application number: 98917331.5 (87) International publication number: (22) Date of filing: 09.04.1998 WO 98/46790 (22.10.1998 Gazette 1998/42) (54) HYBRIDISATION ASSAY IN WHICH EXCESS PROBE IS DESTROYED HYBRIDISIERUNGSVERFAHREN IN DEM ÜBERSCHÜSSIGE SONDEN ABGEBAUT WERDEN TEST D’HYBRIDATION DANS LEQUEL LA SONDE EN EXCES EST DETRUITE (84) Designated Contracting States: (56) References cited: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU EP-A- 0 144 913 EP-A- 0 780 479 MC NL PT SE WO-A-90/01559 WO-A-98/19168 US-A- 4 833 084 (30) Priority: 14.04.1997 GB 9707531 • DATABASE WPI Derwent Publications Ltd., (43) Date of publication of application: London, GB; AN 92205018 XP002072949 "gene 02.02.2000 Bulletin 2000/05 detection method-comprises gene extn., denaturing to single strand, specifically bonding (73) Proprietor: Zetatronics Limited to labelled gene probe and reacting" & JP 04 135 Hatfield, Herts AL10 9AB (GB) 498 A (TOSHIBA) , 8 May 1992 • HARBRON S. ET AL.,: "Amplified assay of (72) Inventor: Harbron, Stuart alkaline phosphate using Flavinadenine Berkhamsted, Herts HP4 2LL (GB) dinucleotide phosphate as substrate" ANALYTICAL BIOCHEMISTRY, vol. 206, - 1 (74) Representative: Coates, Ian Harold et al October 1992 pages 119-124, XP002072948 Sommerville & Rushton, 45 Grosvenor Road St. Albans, Hertfordshire AL1 3AW (GB) Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 975 805 B1 Printed by Jouve, 75001 PARIS (FR) 1 EP 0 975 805 B1 2 Description ence of the label in the resulting hybrid is determined. A disadvantage of this method is that it is neither easy Background Art nor convenient to attach the single-stranded target nu- cleic acid to a solid support, the whole process involving [0001] Nucleic acid hybridisation is a widely used 5 a 12 - 15 hour incubation of the nucleic acid with a nitro- technique for identifying, detecting and quantitating tar- cellulose sheet, followed by a 2 hour baking step. This get polynucleotide sequences in a sample. This tech- makes the assay slow and unattractive for routine use. nique relies for its success on complementary base pair- It is also cumbersome, with the hybridisation and wash- ing between the two halves of a double-stranded nucleic ing steps being carried out in a sealed pouch, containing acid molecule: when single-stranded nucleic acids are 10 the membrane and the buffer solution. In addition, when incubated in solution under suitable conditions of tem- very low concentrations must be detected, the ratio of perature, pH and ionic strength, complementary base specific to non-specifically bound probe can be very low sequences pair to form double-stranded stable hybrid and repeated washing under highly stringent conditions molecules. This ability of single-stranded nucleic acid is frequently required. Under these conditions the sen- molecules to form a hydrogen-bonded structure with 15 sitivity of the assay is often compromised because of their complementary nucleic acid sequences has long substantial loss of specifically bound probe. been employed as an analytical tool in recombinant [0005] Since then a many improvements have been DNA research. made, most of which employ a sandwich approach us- [0002] In most cases the sample will contain double- ing two probes: a reporter probe and a capture probe. stranded nucleic acid and must be denatured prior to 20 The reporter probe is a nucleic acid having a sequence the hybridisation assay to render it single-stranded. A complementary to at least part of the target sequence nucleic acid having a known sequence which is comple- and which is labelled with a detectable group. The cap- mentary to the target sequence is either synthesised ture probe is a nucleic acid having a sequence comple- chemically in an automated fashion with great facility, or mentary to at least part of the target sequence, but which is isolated from the appropriate organism and rendered 25 is different to that of the reporter probe, and which is single-stranded by denaturation. It is then used as a labelled with an immobilisable group. In many applica- probe to search a sample for a target complementary tions, pairs of specific binding members (sbm's) have sequence. Detection of specific target nucleic acids en- been used for this purpose. ables accurate diagnosis of bacterial, fungal and viral [0006] For example, in U. S. Pat. No. 5,273,382 to disease states in humans, animals and plants. Addition- 30 Snitman and Stroupe a capture probe complementary ally, the ability to probe for a specific nucleotide se- to part of the target nucleic acid is labelled with an anti- quence enables the diagnosis of human genetic disor- gen or antibody. After hybridisation between this capture ders. Hybridisation produces stable hybrids, and a probe and the target, the solution is introduced to a sup- number of different approaches are known to the art for port-bound antibody or antigen which immobilises the detecting these. 35 hybrid formed between the capture probe and the target. [0003] One approach involves the use of labelled Following a washing step, a second, reporter probe, probes. By labelling a probe nucleic acid with some complementary to a different region of the target nucleic readily detectable chemical group, it is possible tc detect acid, is introduced and the triple sandwich formed is de- the polynucleotide sequence of interest in a test medium tected. containing sample nucleic acids in single-stranded form. 40 [0007] Similar approaches are described by Holtke et Nucleic acids have been labelled with radioisotopes, en- al.: in U.S. Pat. No. 5,344,757 is disclosed a method in zymes and fluorescent molecules. The use of labelled which a reporter probe is labelled with digoxin or digox- nucleic acids as probes in macromolecular analysis is ygenin, and hybrids are captured using antibodies important for clinical, veterinary and environmental di- against this hapten. In this case, a capture probe is not agnostic applications. 45 used, and the method is limited either to the detection [0004] Early methods for detecting target nucleic ac- of an immobilised target, or when the assay is used for ids involved their immobilisation on a solid support such detecting PCR products, one of the primers is immobi- as nitro-cellulose paper, cellulose paper, diazotized pa- lised. In U.S. patent 5,354,657 the method is further de- per, or a nylon membrane. For example, in U. S. Pat. veloped and involves the solution hybridisation between No. 4,358,535 to Falkow a method is disclosed in which 50 the target nucleic acid and a reporter probe labelled with the target nucleic acid is rendered single-stranded and digoxin or digoxygenin. This hybrid is captured by a sol- then immobilised onto a membrane. A labelled probe id-supported capture probe, complementary to a differ- which is complementary to the target nucleic acid is ent region of the target. A detectably labelled antibody brought into contact with the solid support and hybridis- against the hapten is then added and the hybrids formed es to the target nucleic acid. The solid support is washed 55 detected. several times at a carefully controlled temperature to re- [0008] Specific binding members other than antigens move unbound and non-specifically bound probe with- or haptens and antibodies have been used. In U.S. Pat. out removing specifically bound probe, and the pres- No. 5,374,524 to Miller is described a method for the 2 3 EP 0 975 805 B1 4 solution sandwich hybridisation, capture and detection reagent is selective for DNA-RNA or RNA-RNA hybrids of amplified nucieic acids. Amplicons are denatured and over the single-stranded nucleic acids. U.S. Pat. No. treated with an enzyme-labelled reporter probe and a 5,200,313 to Carrico further discloses a nucleic acid hy- biotinylated capture probe. Hybrids formed are captured bridisation assay employing an immobilised or immobi- using streptavidin-coated chromium dioxide particles. 5 lisable polynucleotide probe selected to form DNA-RNA [0009] Disadvantages of these approaches include or RNA-DNA hybrids with the particular polynucleotide the increased cost and complexity of using two probes. sequence to be determined. Resulting hybrids are de- For example, for each assay two probes need to be syn- tected by binding of an antibody reagent, preferably la- thesised and labelled: one for use as the capture probe, belled with a detectable chemical group, selective for and the other for use as a reporter probe. In addition, 10 binding the hybrids in the presence of the single-strand- hybridisation conditions have to be carefully chosen to ed sample and probe nucleic acids. Advantageous fea- form the sandwich of target, capture probe and reporter ture of Carrico's inventions are that no immobilisation or probe. labelling of sample nucleic acids is required and hybrid- [0010] Simpler approaches which avoid the use or a isation can be performed entirely in solution. A further capture probe have been described. Atlas and Steffan 15 advantage is that a universal detection reagent may be (Biotechniques (1990) 8:316 - 318) disclose a solution used whatever the target is.
Load more

Recommended publications
  • Purification, Crystallization and Preliminary X-Ray Diffraction Analysis of Exodeoxyribonuclease III from Crenarchaeon Sulfolobus Tokodaii Strain 7
    Crystal Structure Theory and Applications, 2013, 2, 155-158 Published Online December 2013 (http://www.scirp.org/journal/csta) http://dx.doi.org/10.4236/csta.2013.24021 Purification, Crystallization and Preliminary X-Ray Diffraction Analysis of Exodeoxyribonuclease III from Crenarchaeon Sulfolobus tokodaii Strain 7 Shuichi Miyamoto1*, Chieko Naoe2, Masaru Tsunoda3, Kazuo T. Nakamura2 1Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan 2School of Pharmacy, Showa University, Tokyo, Japan 3Faculty of Pharmacy, Iwaki Meisei University, Iwaki, Japan Email: *[email protected] Received October 13, 2013; revised November 12, 2013; accepted December 6, 2013 Copyright © 2013 Shuichi Miyamoto et al. This is an open access article distributed under the Creative Commons Attribution Li- cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Exodeoxyribonuclease III (EXOIII) acts as a 3’→5’ exonuclease and is homologous to purinic/apyrimidinic (AP) en- donuclease (APE), which plays an important role in the base excision repair pathway. To structurally investigate the reaction and substrate recognition mechanisms of EXOIII, a crystallographic study of EXOIII from Sulfolobus tokodaii strain 7 was carried out. The purified enzyme was crystallized by using the hanging-drop vapor-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 154.2, b = 47.7, c = 92.4 Å, β = 125.8˚ and diffracted to 1.5 Å resolution. Keywords: Crenarchaeon; Crystallization; Exodeoxyribonuclease; Sulfolobus tokodaii; X-Ray Diffraction 1. Introduction formational change upon protein binding that permits complex formation and activation of attacking water, A variety of mechanisms exist to repair damaged DNA leading to incision, in the presence of Mg2+ [10,11].
    [Show full text]
  • The Rnase H-Like Superfamily: New Members, Comparative Structural Analysis and Evolutionary Classification Karolina A
    4160–4179 Nucleic Acids Research, 2014, Vol. 42, No. 7 Published online 23 January 2014 doi:10.1093/nar/gkt1414 The RNase H-like superfamily: new members, comparative structural analysis and evolutionary classification Karolina A. Majorek1,2,3,y, Stanislaw Dunin-Horkawicz1,y, Kamil Steczkiewicz4, Anna Muszewska4,5, Marcin Nowotny6, Krzysztof Ginalski4 and Janusz M. Bujnicki1,3,* 1Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, ul. Ks. Trojdena 4, PL-02-109 Warsaw, Poland, 2Department of Molecular Physiology and Biological Physics, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA USA-22908, USA, 3Bioinformatics Laboratory, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Umultowska 89, PL-61-614 Poznan, Poland, 4Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Zwirki i Wigury 93, PL-02-089 Warsaw, Poland, 5Institute of Biochemistry and Biophysics PAS, Pawinskiego 5A, PL-02-106 Warsaw, Poland and 6Laboratory of Protein Structure, International Institute of Molecular and Cell Biology, ul. Ks. Trojdena 4, PL-02-109 Warsaw, Poland Received September 23, 2013; Revised December 12, 2013; Accepted December 26, 2013 ABSTRACT revealed a correlation between the orientation of Ribonuclease H-like (RNHL) superfamily, also called the C-terminal helix with the exonuclease/endo- the retroviral integrase superfamily, groups together nuclease function and the architecture of the numerous enzymes involved in nucleic acid metab- active site. Our analysis provides a comprehensive olism and implicated in many biological processes, picture of sequence-structure-function relation- including replication, homologous recombination, ships in the RNHL superfamily that may guide func- DNA repair, transposition and RNA interference.
    [Show full text]
  • Small Regulatory Rnas Controlling Complex Phenotypes in Vibrio Cholerae
    Small regulatory RNAs controlling complex phenotypes in Vibrio cholerae Dissertation Zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Roman Herzog aus Augsburg München, Juli 2020 Diese Dissertation wurde angefertigt unter der Leitung von Prof. Dr. Kai Papenfort im Bereich von Department Biologie I an der Ludwig-Maximilians-Universität München Gutachter: 1. Prof. Dr. Kai Papenfort 2. Prof. Dr. Heinrich Jung Datum der Abgabe: 21.07.2020 Tag der mündlichen Prüfung: 15.12.2020 II Eidesstattliche Erklärung Ich versichere hiermit an Eides statt, dass die vorgelegte Dissertation von mir selbstständig und ohne unerlaubte Hilfe angefertigt wurde. Des Weiteren erkläre ich, dass ich nicht anderweitig ohne Erfolg versucht habe, eine Dissertation einzureichen oder mich der Doktorprüfung zu unterziehen. Die folgende Dissertation liegt weder ganz, noch in wesentlichen Teilen einer anderen Prüfungskommission vor. Roman Herzog, München, den 20. Juli 2020 Statutory Declaration I declare that I have authored this thesis independently, that I have not used other than the declared sources/resources. As well I declare, that I have not submitted a dissertation without success and not passed the oral exam. The present dissertation (neither the entire dissertation nor parts) has not been presented to another examination board. Roman Herzog, Munich, 20 July 2020 III Contents Eidesstattliche Erklärung ........................................................................................................III
    [Show full text]
  • Ribonuclease and Deoxyribonuclease Activities in Experimental and Human Tumors by the Histochemical Substrate Film Method*
    Ribonuclease and Deoxyribonuclease Activities in Experimental and Human Tumors by the Histochemical Substrate Film Method* R. DAOUSTJANDHARUKOAMANOÕ (Laboratoires de Recherche, Institut du Cancer de Montréal,Hôpital Notre-Dame et Universitéde Montréal,Montréal,Canada) SUMMARY The ribonuclease and deoxyribonuclease activities of 65 experimental and human tu mors (32 different types) have been examined by histochemical substrate film methods. A same general pattern was obtained for the distribution of both nucleases in the various types of experimental and human tumors. The connective tissue stroma and the necrotic regions of the tumor masses showed various levels of nuclease activity, whereas the neoplastic cells showed no demonstrable activity. It appears that deficien cies in ribonuclease and deoxyribonuclease activities represent general properties of cancer cells. The possible significance of the losses of nuclease activities in carcinogenesis is dis cussed. Studies on nucleases by histochemical methods MATERIALS AND METHODS have shown that losses of ribonuclease (RNase) The experimental tumors used in the present and deoxyribonuclease (DNase) activities take study were mostly rat, mouse, and hamster trans- place in rat liver during azo-dye carcinogenesis (1, plantable tumors (see Table 1). The tumor-bearing 6). The loss of RNase activity is progressive and animals were obtained from commercial or private occurs before parenchymal cells become cancerous, sources, and the tumors were used as supplied or whereas the loss of DNase activity is abrupt and closely associated with the neoplastic transforma TABLE1 tion of parenchymal cells. EXPERIMENTALTUMORS If a loss of RNase or DNase activity plays an important role in tumor formation, the lack of SpeciesRat"""MouseHamsterTumorPrimary demonstrable nuclease activity observed in rat hepatomaNovikoff primary hepatomas should also be observed in a hepatomaWalker variety of tumors.
    [Show full text]
  • Supplementary Materials
    Supplementary Materials COMPARATIVE ANALYSIS OF THE TRANSCRIPTOME, PROTEOME AND miRNA PROFILE OF KUPFFER CELLS AND MONOCYTES Andrey Elchaninov1,3*, Anastasiya Lokhonina1,3, Maria Nikitina2, Polina Vishnyakova1,3, Andrey Makarov1, Irina Arutyunyan1, Anastasiya Poltavets1, Evgeniya Kananykhina2, Sergey Kovalchuk4, Evgeny Karpulevich5,6, Galina Bolshakova2, Gennady Sukhikh1, Timur Fatkhudinov2,3 1 Laboratory of Regenerative Medicine, National Medical Research Center for Obstetrics, Gynecology and Perinatology Named after Academician V.I. Kulakov of Ministry of Healthcare of Russian Federation, Moscow, Russia 2 Laboratory of Growth and Development, Scientific Research Institute of Human Morphology, Moscow, Russia 3 Histology Department, Medical Institute, Peoples' Friendship University of Russia, Moscow, Russia 4 Laboratory of Bioinformatic methods for Combinatorial Chemistry and Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia 5 Information Systems Department, Ivannikov Institute for System Programming of the Russian Academy of Sciences, Moscow, Russia 6 Genome Engineering Laboratory, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia Figure S1. Flow cytometry analysis of unsorted blood sample. Representative forward, side scattering and histogram are shown. The proportions of negative cells were determined in relation to the isotype controls. The percentages of positive cells are indicated. The blue curve corresponds to the isotype control. Figure S2. Flow cytometry analysis of unsorted liver stromal cells. Representative forward, side scattering and histogram are shown. The proportions of negative cells were determined in relation to the isotype controls. The percentages of positive cells are indicated. The blue curve corresponds to the isotype control. Figure S3. MiRNAs expression analysis in monocytes and Kupffer cells. Full-length of heatmaps are presented.
    [Show full text]
  • (51) International Patent Classification: A61K 8/66 (2006.01) A61Q 11/00
    ( (51) International Patent Classification: A61K 8/66 (2006.01) A61Q 11/00 (2006.01) (21) International Application Number: PCT/EP20 19/08 1186 (22) International Filing Date: 13 November 2019 (13. 11.2019) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 18206133.3 14 November 2018 (14. 11.2018) EP (71) Applicant: NOVOZYMES A/S [DK/DK]; Krogshoejvej 36, 2880 Bagsvaerd (DK). (72) Inventors: DURHUUS, Thomas, Thomasen; Krogshoe¬ jvej 36, 2880 Bagsvaerd (DK). PALMEN, Lorena, Gonzalez,; Krogshoejvej 36, 2880 Bagsvaerd (DK). REISER, Anna, Verena,; Krogshoejvej 36, 2880 Bagsvaerd (DK). STREICHER, Werner, W,; Krogshoe¬ jvej 36, 2880 Bagsvaerd (DK). (81) Designated States (unless otherwise indicated, for every kind of national protection available) : AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available) : ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG).
    [Show full text]
  • HOXB6 Homeo Box B6 HOXB5 Homeo Box B5 WNT5A Wingless-Type
    5 6 6 5 . 4 2 1 1 1 2 4 6 4 3 2 9 9 7 0 5 7 5 8 6 4 0 8 2 3 1 8 3 7 1 0 0 4 0 2 5 0 8 7 5 4 1 1 0 3 6 0 4 8 3 7 4 7 6 9 6 7 1 5 0 8 1 4 1 1 7 1 0 0 4 2 0 8 1 1 1 2 5 3 5 0 7 2 6 9 1 2 1 8 3 5 2 9 8 0 6 0 9 5 1 9 9 2 1 1 6 0 2 3 0 3 6 9 1 6 5 5 7 1 1 2 1 1 7 5 4 6 6 4 1 1 2 8 4 7 1 6 2 7 7 5 4 3 2 4 3 6 9 4 1 7 1 3 4 1 2 1 3 1 1 4 7 3 1 1 1 1 5 3 2 6 1 5 1 3 5 4 5 2 3 1 1 6 1 7 3 2 5 4 3 1 6 1 5 3 1 7 6 5 1 1 1 4 6 1 6 2 7 2 1 2 e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S HOXB6 homeo box B6 HOXB5 homeo box B5 WNT5A wingless-type MMTV integration site family, member 5A WNT5A wingless-type MMTV integration site family, member 5A FKBP11 FK506 binding protein 11, 19 kDa EPOR erythropoietin receptor SLC5A6 solute carrier family 5 sodium-dependent vitamin transporter, member 6 SLC5A6 solute carrier family 5 sodium-dependent vitamin transporter, member 6 RAD52 RAD52 homolog S.
    [Show full text]
  • Letters to Nature
    letters to nature Received 7 July; accepted 21 September 1998. 26. Tronrud, D. E. Conjugate-direction minimization: an improved method for the re®nement of macromolecules. Acta Crystallogr. A 48, 912±916 (1992). 1. Dalbey, R. E., Lively, M. O., Bron, S. & van Dijl, J. M. The chemistry and enzymology of the type 1 27. Wolfe, P. B., Wickner, W. & Goodman, J. M. Sequence of the leader peptidase gene of Escherichia coli signal peptidases. Protein Sci. 6, 1129±1138 (1997). and the orientation of leader peptidase in the bacterial envelope. J. Biol. Chem. 258, 12073±12080 2. Kuo, D. W. et al. Escherichia coli leader peptidase: production of an active form lacking a requirement (1983). for detergent and development of peptide substrates. Arch. Biochem. Biophys. 303, 274±280 (1993). 28. Kraulis, P.G. Molscript: a program to produce both detailed and schematic plots of protein structures. 3. Tschantz, W. R. et al. Characterization of a soluble, catalytically active form of Escherichia coli leader J. Appl. Crystallogr. 24, 946±950 (1991). peptidase: requirement of detergent or phospholipid for optimal activity. Biochemistry 34, 3935±3941 29. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and (1995). the thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281±296 (1991). 4. Allsop, A. E. et al.inAnti-Infectives, Recent Advances in Chemistry and Structure-Activity Relationships 30. Meritt, E. A. & Bacon, D. J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505± (eds Bently, P. H. & O'Hanlon, P. J.) 61±72 (R. Soc. Chem., Cambridge, 1997).
    [Show full text]
  • Datasheet for T7 Exonuclease (M0263; Lot 0031212)
    Source: Purified from an E. coli strain containing a Unit Assay Conditions: 50 mM potassium Physical Purity: Purified to > 95% homogeneity TYB12 intein fusion acetate, 20 mM Tris-acetate, 10 mM magnesium as determined by SDS-PAGE analysis using T7 Exonuclease acetate, 1mM dithiothreitol (pH 7.9) and 0.15 mM Coomassie Blue detection. Supplied in: 10 mM Tris-HCl (pH 8.0), sonicated duplex [3H] DNA. 0.1 mM EDTA, 5 mM DTT and 50% glycerol. RNase Activity (Extended Digestion): A 10 µl Quality Control Assays 1-800-632-7799 reaction in NEBuffer 4 containing 40 ng of [email protected] Reagents Supplied with Enzyme: Single Stranded Deoxyribonuclease Activity flourescein labeled RNA transcript and 10 units www.neb.com 10X NEBuffer 4. (FAM Labeled Oligo): A 50 µl reaction in of T7 Exonuclease incubated at 37°C. After M0263S 003121214121 NEBuffer 4 containing a 20 nM solution of a incubation for 4 hours, > 90% of the substrate Reaction Conditions: fluorescent internal labeled oligonucleotide and a RNA remains intact as determined by gel minimum of 50 units of T7 Exonuclease incubated 1X NEBuffer 4. electrophoresis using fluorescence detection. M0263S Incubate at 25°C. for 16 hours at 37°C yields < 5% degradation as determined by capillary electrophoresis. 1,000 units 10,000 U/ml Lot: 0031212 Heat Inactivation: No 1X NEBuffer 4: RECOMBINANT Store at –20°C Exp: 12/14 50 mM potassium acetate Endonuclease Activity: Incubation of a 50 µl References: reaction containing 100 units of T7 Exonuclease Description: T7 Exonuclease acts in the 5´ to 20 mM Tris-acetate 1.
    [Show full text]
  • The Microbiota-Produced N-Formyl Peptide Fmlf Promotes Obesity-Induced Glucose
    Page 1 of 230 Diabetes Title: The microbiota-produced N-formyl peptide fMLF promotes obesity-induced glucose intolerance Joshua Wollam1, Matthew Riopel1, Yong-Jiang Xu1,2, Andrew M. F. Johnson1, Jachelle M. Ofrecio1, Wei Ying1, Dalila El Ouarrat1, Luisa S. Chan3, Andrew W. Han3, Nadir A. Mahmood3, Caitlin N. Ryan3, Yun Sok Lee1, Jeramie D. Watrous1,2, Mahendra D. Chordia4, Dongfeng Pan4, Mohit Jain1,2, Jerrold M. Olefsky1 * Affiliations: 1 Division of Endocrinology & Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California, USA. 2 Department of Pharmacology, University of California, San Diego, La Jolla, California, USA. 3 Second Genome, Inc., South San Francisco, California, USA. 4 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA, USA. * Correspondence to: 858-534-2230, [email protected] Word Count: 4749 Figures: 6 Supplemental Figures: 11 Supplemental Tables: 5 1 Diabetes Publish Ahead of Print, published online April 22, 2019 Diabetes Page 2 of 230 ABSTRACT The composition of the gastrointestinal (GI) microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine (fMLF), are elevated in high fat diet (HFD)- induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon- like peptide-1 (GLP-1). Obese Fpr1-knockout (Fpr1-KO) mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota.
    [Show full text]
  • Structural Characterization and Subunit Communication Of
    STRUCTURAL CHARACTERIZATION AND SUBUNIT COMMUNICATION OF ESCHERICHIA COLI PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX By Jaeyoung Song A dissertation submitted to the Graduate School-Newark Rutgers, The State University of New Jersey In partial fulfillment of requirements for the degree of Doctor of Philosophy Graduate Program in Chemistry Written under the direction of Professor Frank Jordan and approved by Newark, New Jersey May, 2011 ABSTRACT OF THE THESIS Structural Characterization and Subunit Communication of Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex By Jaeyoung Song Thesis Director: Professor Frank Jordan The pyruvate dehydrogenase multienzyme complex (PDHc) from Escherichia coli (E. coli) is the best characterized of the 2-oxoacid dehydrogenase complexes. The complex plays a role as catalyst for the conversion of pyruvate to acetyl Coenzyme A (acetylCoA) by three enzyme components in the complex. The complex is comprised of 24 copies of the dimeric pyruvate dehydrogenase (E1ec; 99,474 Da), a cubic core of 24 copies of dihydrolipoamide acetyltransferase (E2ec; 65,959 Da), and 12 copies of dihydrolipoamide dehydrogenase (E3ec; 50,554 Da) (1-3). The crystal structure of the E. coli pyruvate dehydrogenase complex E1 subunit (E1ec) has been deterimined, and there were three missing regions (residues 1-55, 401-413, and 541-557) remaining absent in the model due to high flexibilities of these regions (4). Most bacterial pyruvate dehydrogenase complexes from either Gram-positive or Gram-negative bacteria have E1 components with an 2 homodimeric quaternary structure. In a sequel to our previous publications (5-8), the first NMR study on the flexible regions of the E1 component from Escherichia coli and its biological relevance ii was presented.
    [Show full text]
  • Prediction of Novel Inhibitors Against Exodeoxyribonuclease І of H
    International Journal of Scientific & Engineering Research, Volume 6, Issue 2, February-2015 217 ISSN 2229-5518 Prediction of Novel Inhibitors against Exodeoxyribonuclease І of H. influenzae through In Silico Approach Shaik Parveen, Natarajan Pradeep, Kanipakam Hema and Amineni Umamaheswari Abstract — These Haemophilus influenzae is a Gram-negative bacterium which causes pneumonia in humans. Due to its multidrug resistance to peni- cillin, rifampin and polymyxin and adverse effects of existing treatments, the condition became an open challenge for many researchers to discover novel antagonists for the treatment of pneumonia caused by H. influenzae. 5 potential sRNA candidates were identified in H. influenzae using sRNA predict tool, among them3 were enzymes and 2 were non-enzymes. Among the three identified enzymes exodeoxyribonuclease І of H. influenzae was non- homologous to humans was selected as novel drug target in the present study. Exodeoxyribonuclease І is an enzyme involved in the mismatch repair mechanism of H. influenzae. The 3D structure of the exodeoxyribonuclease І was modeled based on the crystal structure of 4JRP using Modeler 9v13 and built model was validated using PROCHECK analysis, ProQ and ProSA. The existing eight inhibitors of exodeoxyribonuclease І were searched in 3D ligand database through shape screening against ASINEX database using Phasev3.2 module and structural analogs were docked with exodeoxyri- bonuclease І in Maestro v9.6 virtual screening workflow, that implements three stage Glide docking protocol. The docking results revealed that 11 leads were having better docking and ∆G scores when compared with the eight existing inhibitors among them lead 1 was having ∆G score of -68.78 kcal/mol.
    [Show full text]