DOCSLIB.ORG

And Assignment of the Chromosomal Locus (Human Serum Proteins/Proteinase Inhibitors/Human Chromosome Mapping/Thiol Ester Bond) CHEN-CHEN KAN*, ELLEN Solomont, K

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
And Assignment of the Chromosomal Locus (Human Serum Proteins/Proteinase Inhibitors/Human Chromosome Mapping/Thiol Ester Bond) CHEN-CHEN KAN*, ELLEN Solomont, K Proc. Nati. Acad. Sci. USA Vol. 82, pp. 2282-2286, April 1985 Biochemistry Nucleotide sequence of cDNA encoding human a2-macroglobulin and assignment of the chromosomal locus (human serum proteins/proteinase inhibitors/human chromosome mapping/thiol ester bond) CHEN-CHEN KAN*, ELLEN SOLOMONt, K. TERTIA BELTt, AILEEN C. CHAIN*, LYNNE R. HIORNSt, AND GEORG FEY* *Department of Immunology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037; tImperial Cancer Research Funds Laboratory, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX, England; and tDepartment of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England Communicated by Frank J. Dixon, December 6, 1984 ABSTRACT Six a2-macroglobulin (a2M) cDNA clones ester sites are conserved. These observations led to the pro- were isolated from a human liver cDNA library by using syn- posal that the C3, C4, and a2M genes are derived from a thetic oligonucleotides as hybridization probes. One of these, common ancestral gene (16). The sequences of murine and pa2M1, carries a 4.6-kilobase-pair insert, which was se- human C3 and human C4 and partial cDNA sequences of quenced. The insert contains the coding sequences for the ma- murine C4 have recently been determined (17-21). Compari- ture a2M polypeptide (1451 amino acids) and for a 23-amino son of human a2M with murine C3 revealed a 25% overall acid signal peptide at the NH2 terminus of the precursor pro- sequence homology (17, 18, 22), supporting the proposal of a2M. At the 3' end of the insert a poly(A) addition signal A-A- their common evolutionary origin. The human C3 gene is lo- T-A-A-A and part of the poly(A) tail of the messenger RNA cated on chromosome 19 (23), and the human C4 genes map were found. The protein sequence deduced from the nucleotide to the histocompatibility complex on chromosome 6 (24). sequence agrees with the published a2M amino acid sequence In addition to binding to proteinases, a2M can also adhere for all except three residues. The a2M locus was assigned to to small molecules, including lectins, cations (zinc), basic human chromosome 12 by Southern blot analysis with DNA proteins (myelin basic protein, histone H4), and platelet-de- from a panel of mouse/human somatic cell hybrids, using a2M rived growth factor (PDGF) (1, 25). The binding mechanisms cDNA as a hybridization probe. involved are probably unrelated to the trapping of protein- ases because adherence of small molecules does not prevent a2-Macroglobulin (a2M) is a serum glycoprotein and a major the proteinase binding (1). A physiological role for the com- plasma proteinase inhibitor with a wide specificity. a2M-re- plex formation between a2M and these small molecules has lated proteins are present in all vertebrate species (1-4). Hu- not been established. The striking similarity of the amino man a2M is a tetramer of four identical 185-kDa subunits, acid sequences of PDGF and p28Sis, the transforming protein arranged as a pair of dimers each consisting of two disulfide- of the simian sarcoma virus (26, 27), suggests that viral and linked monomers (5, 6). The a2M polypeptide has a so-called other cellular transformation mechanisms may be mediated bait region and an internal thiol ester bond, which account by growth factor-like proteins. Thus, a2M may serve as an for its properties as a proteinase inhibitor. The bait region, important role in limiting the effects of PDGF-like mitogens. composed of a series of target peptide bonds for plasma pro- The amino acid sequence of a2M has recently been report- teinases (7, 8), is located close to the center of the monomer- ed (28). For our future studies of the structural properties of ic subunit. The thiol ester bond is a covalent linkage between a2M that underlie its functions and of the regulation of the y-carboxyl function of glutamate residue 975 and the sul- expression of the a2M gene, it is necessary to prepare cDNA hydryl group of cysteine residue 972 (9-12). Cleavage of the clones and to derive the complete cDNA sequence. Here we bait region by proteinases leads to a conformational change report the isolation and sequence analysis of human a2M of the a2M molecule. This conformational change activates cDNA clones and the assignment of the human a2M locus to the thiol ester bond, leading to its hydrolysis and as a conse- chromosome 12. quence to covalent binding between the proteinase and a2M via the reactive glutamate 975. a2M receptors are present on MATERIALS AND METHODS the surfaces of fibroblasts and macrophages. These receptor proteins preferentially bind a2M-proteinase complexes or Materials. Restriction enzymes were from Boehringer a2M molecules with hydrolyzed thiol ester bonds (13, 14). Mannheim, DNA polymerase I (Klenow fragment) was from This suggests that the conformational change, which accom- Bethesda Research Laboratories, and the 17-nucleotide uni- panies the complex formation between a2M and proteinases versal sequencing primer was from Collaborative Research. and the hydrolysis of the thiol ester bond, exposes regions of T4 DNA polymerase and dideoxy- and deoxynucleotide tri- the a2M molecule that are recognized by these receptors. phosphates were from P-L Biochemicals. The radionucleo- Receptor-mediated endocytosis of proteinase-a2M com- tides [a-32P]dCTP (>400 Ci/mmol; 1 Ci = 37 GBq) and plexes by macrophages and liver cells leads to clearance of deoxyadenosine 5'-(a-[35S]thio)triphosphate (>400 Ci/ the complexes from the circulation. mmol) were from Amersham. T4 DNA ligase and the Esche- Internal thiol ester bonds are also found in the comple- richia coli strain JM101-TG1 were gifts from D. Bentley and ment proteins C3 and C4 (15), which are derived from pre- T. Gibson. Oligonucleotide probes were synthesized by Cre- cursor polypeptides of size similar to a2M (180-200 kDa). ative Biomolecules (South San Francisco, CA). Their thiol ester sites are found in positions comparable to Synthetic Oligonucleotide Probes and Isolation of cDNA those in a2M, and the amino acid sequences of all three thiol Clones. The construction of the human liver cDNA library enriched for mRNA species over 2000 nucleotides in length The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: C3 and C4, third and fourth components of comple- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ment; a2M, a2-macroglobulin; kb, kilobase pair(s). 2282 Biochemistry: Kan et aL Proc. Natl. Acad. Sci. USA 82 (1985) 2283 with the plasmid vector pAT 153 Pvu II-8 has been previous- Schuell) (43). Filters were hyridized with nick-translated (44) ly described (19). Two methionine residues at positions 1378 a2M cDNA in 2.5x NaCl/Cit (lx NaCl/Cit is 150 mM sodi- and 1385 of the human a2M sequence (28) and their sur- um chloride/15 mM trisodium citrate), lOx Denhardt's solu- rounding sequences were chosen as probe sites because they tion (2% Ficoll/2% bovine serum albumin/2% polyvinylpyr- were encoded by nucleotide triplets with minimal degenera- rolidone), 0.1% sodium dodecyl sulfate, 6% polyethylene 1378 glycol 6000 (Fisons, Loughborough, England) or 10% dex- cy. These amino acid sequences are Asn-Met-Ala-Ile- 1385 tran sulfate, and denatured salmon sperm DNA at 50 ,ug/ml. Val-Asp and Asp-Val-Lys-Met-Val. The corresponding Hybridization was carried out at 65°C overnight. Filters oligonucleotide mixtures were were washed at 65°C successively with 2x, 1x, 0.3x, and AA 0.lx NaCl/Cit, three times for 20 min each. ST'-T-C-N-A-C-G-A-T-N-G-C-C-A-T-G _T-T-3' RESULTS (N represents all four nucleotides) and Identification and Sequence Analysis of cDNA Clones. A S5'-A-C-C-A-T- C_T-T-N-A-C-G T-C-3'. human liver cDNA library was screened with two mixed synthetic oligodeoxynucleotide probes, a 17-mer and a 14- This cDNA library consisted of about 15,000 different mer constructed on the basis of known amino acid se- recombinant clones; 30,000 colonies were plated on nitrocel- quences. Of 30,000 colonies screened, 34 clones hybridized lulose discs. A set of replica filters were prepared from the with both the 17-mer and 14-mer probes. master filters (29). Colonies on replica filters were grown, After restriction enzyme analysis of the cDNA inserts, six then transferred to chloramphenicol plates to amplify the recombinant plamids were selected for detailed analysis. plasmid. The colonies were processed (30, 31) and hybrid- One of them, pa2M1, carries a 4.6-kilobase pair (kb) insert ized with kinase 32P-labeled (32) 14-mer and 17-mer oligonu- whose nucleotide sequence was determined. The nucleotide cleotide mixtures. Positive colonies were rescreened until sequence from the 5' nontranslated region to the poly(A) tail genetic homogeneity was reached. The length of cDNA in- of the mRNA and the derived amino acid sequence are given serts was determined by restriction analysis with the en- in Fig. 1. The complete coding sequence was determined on zymes HindIII and Sal I. both DNA strands; each character was determined 3.75 DNA Sequence Analysis. Plasmid DNAs carrying the long- times on average. A methionine encoding triplet (nucleotides est cDNA inserts were digested with HindIII and Sal I. The 44 to 46) was identified as the translational initiation site. fragments were separated by electrophoresis in low-melting This methionine, together with the following 22 amino acid agarose gels and recovered from the agarose by extraction residues, forms the signal peptide of the a2M protein, which with phenol.
Load more

Recommended publications
  • Mutation Detection & Analysis
    PRO55 G-Biosciences ♦ 1-800-628-7730 ♦ 1-314-991-6034 ♦ [email protected] A Geno Technology, Inc. (USA) brand name Mutation Detection & Analysis Teacher’s Guidebook (Cat. # BE-314) think proteins! think G-Biosciences www.GBiosciences.com MATERIALS INCLUDED WITH THE KIT ................................................................................ 3 CHECKLIST .......................................................................................................................... 3 SPECIAL HANDLING INSTRUCTIONS ................................................................................... 3 ADDITIONAL EQUIPMENT REQUIRED ................................................................................ 3 TIME REQUIRED ................................................................................................................. 3 OBJECTIVES ........................................................................................................................ 4 BACKGROUND ................................................................................................................... 4 POLYMERASE CHAIN REACTION .................................................................................... 5 RESTRICTION ENZYMES ................................................................................................. 8 TEACHER’S PRE EXPERIMENT SET UP .............................................................................. 11 PREPARE PCR AND RESTRICTION DIGESTION REAGENTS ............................................ 11
    [Show full text]
  • Covalent Flexible Peptide Docking in Rosetta
    bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.441297; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Covalent Flexible Peptide Docking in Rosetta Barr Tivon1,#, Ronen Gabizon1,#, Bente A. Somsen2, Peter J. Cossar2, Christian Ottmann2 , Nir London1,* 1 Department of Chemical and Structural Biology, The Weizmann Institute of Science, Rehovot, 7610001, Israel 2 Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600MB Eindhoven, The Netherlands # equal contribution * Corresponding author: [email protected] Keywords: Covalent peptides; peptide docking; CovPepDock; FlexPepDock; 14-3-3; Electrophilic peptides; bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.441297; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Electrophilic peptides that form an irreversible covalent bond with their target have great potential for binding targets that have been previously considered undruggable. However, the discovery of such peptides remains a challenge. Here, we present CovPepDock, a computational pipeline for peptide docking that incorporates covalent binding between the peptide and a receptor cysteine. We applied CovPepDock retrospectively to a dataset of 115 disulfide-bound peptides and a dataset of 54 electrophilic peptides, for which it produced a top-five scoring, near-native model, in 89% and 100% of the cases, respectively.
    [Show full text]
  • A Global Review on Short Peptides: Frontiers and Perspectives †
    molecules Review A Global Review on Short Peptides: Frontiers and Perspectives † Vasso Apostolopoulos 1 , Joanna Bojarska 2,* , Tsun-Thai Chai 3 , Sherif Elnagdy 4 , Krzysztof Kaczmarek 5 , John Matsoukas 1,6,7, Roger New 8,9, Keykavous Parang 10 , Octavio Paredes Lopez 11 , Hamideh Parhiz 12, Conrad O. Perera 13, Monica Pickholz 14,15, Milan Remko 16, Michele Saviano 17, Mariusz Skwarczynski 18, Yefeng Tang 19, Wojciech M. Wolf 2,*, Taku Yoshiya 20 , Janusz Zabrocki 5, Piotr Zielenkiewicz 21,22 , Maha AlKhazindar 4 , Vanessa Barriga 1, Konstantinos Kelaidonis 6, Elham Mousavinezhad Sarasia 9 and Istvan Toth 18,23,24 1 Institute for Health and Sport, Victoria University, Melbourne, VIC 3030, Australia; [email protected] (V.A.); [email protected] (J.M.); [email protected] (V.B.) 2 Institute of General and Ecological Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego˙ 116, 90-924 Lodz, Poland 3 Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Kampar 31900, Malaysia; [email protected] 4 Botany and Microbiology Department, Faculty of Science, Cairo University, Gamaa St., Giza 12613, Egypt; [email protected] (S.E.); [email protected] (M.A.) 5 Institute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego˙ 116, 90-924 Lodz, Poland; [email protected] (K.K.); [email protected] (J.Z.) 6 NewDrug, Patras Science Park, 26500 Patras, Greece; [email protected] 7 Department of Physiology and Pharmacology,
    [Show full text]
  • 1 Design and Production of Specifically and with High Affinity
    1 Design and Production of Specifically and with High Affinity Reacting Peptides (SHARP®-s) by Jan C Biro HOMULUS FOUNDATION, 612 S. Flower Str., #1220, 90017 CA, USA [email protected] www.janbiro.com 2 Abstract Background A partially random target selection method was developed to design and produce affinity reagents (target) to any protein query. It is based on the recent concept of Proteomic Code (for review see Biro, 2007 [1]) which suggests that significant number of amino acids in specifically interacting proteins are coded by partially complementary codons. It means that the 1st and 3rd residues of codons coding many co-locating amino acids are complementary but the 2nd may but not necessarily complementary: like 5’-AXG-3’/3’-CXT-5’ codon pair, where X is any nucleotide. Results A mixture of 45 residue long, reverse, partially complementary oligonucleotide sequences (target pool) were synthesized to selected epitopes of query mRNA sequences. The 2nd codon residues were randomized. The target oligonucleotide pool was inserted into vectors, expressed and the protein products were screened for affinity to the query in Bacterial Two-Hybrid System. The best clones were used for larger-scale protein syntheses and characterization. It was possible to design and produce specific and with high affinity reacting (Kd: ~100 nM) oligopeptide reagents to GAL4 query oligopeptides. Conclusions Second codon residue randomization is a promising method to design and produce affinity peptides to any protein sequences. The method has the potential to be a rapid, inexpensive, high throughput, non-immunoglobulin based alternative to recent in vivo antibody generating procedures.
    [Show full text]
  • Structures of Restriction Endonuclease Hindiii in Complex with Its Cognate DNA and Divalent Cations
    Title Structures of restriction endonuclease HindIII in complex with its cognate DNA and divalent cations Author(s) Watanabe, Nobuhisa; Takasaki, Yozo; Sato, Chika; Ando, Shoji; Tanaka, Isao Acta Crystallographica. Section D, Biological Crystallography, 65(12), 1326-1333 Citation https://doi.org/10.1107/S0907444909041134 Issue Date 2009-12 Doc URL http://hdl.handle.net/2115/39986 Type article File Information watanabe_ActaCristD65.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP electronic reprint Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Editors: E. N. Baker and Z. Dauter Structures of restriction endonuclease HindIII in complex with its cognate DNA and divalent cations Nobuhisa Watanabe, Yozo Takasaki, Chika Sato, Shoji Ando and Isao Tanaka Acta Cryst. (2009). D65, 1326–1333 Copyright c International Union of Crystallography Author(s) of this paper may load this reprint on their own web site or institutional repository provided that this cover page is retained. Republication of this article or its storage in electronic databases other than as specified above is not permitted without prior permission in writing from the IUCr. For further information see http://journals.iucr.org/services/authorrights.html Acta Crystallographica Section D: Biological Crystallography welcomes the submission of papers covering any aspect of structural biology, with a particular emphasis on the struc- tures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure–function papers that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such papers should present new insights into biology, chemistry or structure.
    [Show full text]
  • A Short History of the Restriction Enzymes Wil A
    Published online 18 October 2013 Nucleic Acids Research, 2014, Vol. 42, No. 1 3–19 doi:10.1093/nar/gkt990 NAR Breakthrough Article SURVEY AND SUMMARY Highlights of the DNA cutters: a short history of the restriction enzymes Wil A. M. Loenen1,*, David T. F. Dryden2,*, Elisabeth A. Raleigh3,*, Geoffrey G. Wilson3,* and Noreen E. Murrayy 1Leiden University Medical Center, Leiden, the Netherlands, 2EaStChemSchool of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, UK and 3New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA Received August 14, 2013; Revised September 24, 2013; Accepted October 2, 2013 ABSTRACT Type II REases represent the largest group of characterized enzymes owing to their usefulness as tools for recombinant In the early 1950’s, ‘host-controlled variation in DNA technology, and they have been studied extensively. bacterial viruses’ was reported as a non-hereditary Over 300 Type II REases, with >200 different sequence- phenomenon: one cycle of viral growth on certain specificities, are commercially available. Far fewer Type I, bacterial hosts affected the ability of progeny virus III and IV enzymes have been characterized, but putative to grow on other hosts by either restricting or examples are being identified daily through bioinformatic enlarging their host range. Unlike mutation, this analysis of sequenced genomes (Table 1). change was reversible, and one cycle of growth in Here we present a non-specialists perspective on import- the previous host returned the virus to its original ant events in the discovery and understanding of REases. form. These simple observations heralded the dis- Studies of these enzymes have generated a wealth of covery of the endonuclease and methyltransferase information regarding DNA–protein interactions and catalysis, protein family relationships, control of restric- activities of what are now termed Type I, II, III and tion activity and plasticity of protein domains, as well IV DNA restriction-modification systems.
    [Show full text]
  • Promiscuous Restriction Is a Cellular Defense Strategy That Confers Fitness
    Promiscuous restriction is a cellular defense strategy PNAS PLUS that confers fitness advantage to bacteria Kommireddy Vasua, Easa Nagamalleswaria, and Valakunja Nagarajaa,b,1 aDepartment of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India; and bJawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India Edited by Werner Arber, der Universitat Basel, Basel, Switzerland, and approved March 20, 2012 (received for review November 22, 2011) Most bacterial genomes harbor restriction–modification systems, ognition and cofactor utilization, whereas the cognate MTase is encoding a REase and its cognate MTase. On attack by a foreign very site-specific (14). In addition to its recognition sequence, DNA, the REase recognizes it as nonself and subjects it to restric- GGTACC, the enzyme binds and cleaves a number of noncanoni- tion. Should REases be highly specific for targeting the invading cal sequences (i.e., TGTACC, GTTACC, GATACC, GGAACC, foreign DNA? It is often considered to be the case. However, when GGTCCC, GGTATC, GGTACG, GGTACT), with the binding bacteria harboring a promiscuous or high-fidelity variant of the affinity ranging from 8 to 35 nM (14, 15). Accordingly, many of fi REase were challenged with bacteriophages, tness was maximal these sites are cleaved efficiently with kcat values varying from 0.06 − − under conditions of catalytic promiscuity. We also delineate pos- to 0.15 min 1 vs. 4.3 min 1 for canonical sites (15). The promis- sible mechanisms by which the REase recognizes the chromosome cuous activity of the enzyme is directed by a number of cofactors. as self at the noncanonical sites, thereby preventing lethal dsDNA In the presence of the physiological levels of Mg2+, the most breaks.
    [Show full text]
  • Deep Time-Resolved Proteomic and Phosphoproteomic Profiling Of
    Deep time-resolved proteomic and phosphoproteomic profiling of cigarette smoke-induced chronic obstructive pulmonary disease David Skerrett-Byrne BSc Biochem & Mol Bio (Hons)(UCD) MSc Biotech (UU) 25th March 2019 Supervisors: Professor Phil Hansbro, Dr. Matt Dun, Laureate Professor Rodney Scott, Professor Peter Wark, Professor Darryl Knight, Laureate Professor Paul Foster Discipline of Immunology and Microbiology and Priority Research Centre for Healthy Lungs School of Biomedical Science and Pharmacy Faculty of Health and Medicine University of Newcastle and Hunter Medical Research Institute Newcastle, NSW, Australia Submitted in fulfilment of the requirements for the award of a Doctor of Philosophy Declaration Statement of Originality I hereby certify that the work embodied in the thesis is my own work, conducted under normal supervision. The thesis contains no material which has been accepted, or is being examined, for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made. I give consent to the final version of my thesis being made available worldwide when deposited in the University’s Digital Repository, subject to the provisions of the Copyright Act 1968 and any approved embargo. ________________________ David Skerrett-Byrne 25/03/2019 Acknowledgment of Authorship I hereby certify that the work embodied in this thesis contains scholarly work of which I am a joint author. I have included as part of the thesis a written declaration endorsed in writing by my supervisor, attesting to my contribution to the joint scholarly work.
    [Show full text]
  • Centenary Award and Sir Frederick Gowland Hopkins Memorial Lecture
    Centenary Award and Sir Frederick Gowland Hopkins Memorial Lecture Centenary Award and Sir Frederick Gowland Hopkins Memorial Lecture Seeing the invisible by paramagnetic and diamagnetic NMR G. Marius Clore*1 *Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, U.S.A. scale interdomain motions involved in ligand binding, and to the interaction of monomeric amyloid β-peptide with the surface of amyloid protofibrils and the internal cavity surface of Centenary Award and Sir Frederick the chaperonin GroEL. Gowland Hopkins Memorial Lecture Delivered at the MRC Laboratory of Molecular Biology, Cambridge, on Introduction 13 December 2012 Proteins and their complexes are not static but dynamic and Marius Clore are best described as an ensemble of states. The major species is located at the minimum of the free energy landscape and represents the species that has been studied with so much Abstract success by conventional structural and biophysical tech- niques, including crystallography and NMR. But the static Sparsely populated transient states of proteins and their picture revealed by these studies does not describe the complexes play an important role in many biological processes complete picture. In addition to the major species, there exist including protein–protein and protein–DNA recognition, al- highly transient sparsely populated states that arise from rare lostery, conformational selection, induced fit and self-assembly. excursions between the minimum free energy configuration These states are difficult to study as their low population and other local minima of the free energy landscape [1]. Given and transient nature makes them effectively invisible to that the populations of such sparsely populated states are so conventional structural and biophysical techniques.
    [Show full text]
  • Identification and Characterization of Posttranslational Modification-Specific Binding Proteins in Vivo by Mammalian Tethered Catalysis
    Identification and characterization of posttranslational modification-specific binding proteins in vivo by mammalian tethered catalysis Tanya M. Spektor and Judd C. Rice1 Department of Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine, Los Angeles, CA 90033 Communicated by C. David Allis, The Rockefeller University, New York, NY, July 14, 2009 (received for review February 26, 2009) Increasing evidence indicates that an important consequence of To overcome some of the limitations of in vitro approaches, a protein posttranslational modification (PTM) is the creation of a high previously undescribed in vivo method called yeast tethered catal- affinity binding site for the selective interaction with a PTM-specific ysis was developed (1). Briefly, an expressed fusion protein con- binding protein (BP). This PTM-mediated interaction is typically re- taining a target peptide sequence was tethered to an enzyme quired for downstream signaling propagation and corresponding resulting in the constitutive PTM of the peptide and, thereby, biological responses. Because the vast majority of mammalian pro- served as the bait in yeast two-hybrid screens for putative PTMBPs. teins contain PTMs, there is an immediate need to discover and Although this technique was used successfully to identify yeast characterize previously undescribed PTMBPs. To this end, we devel- PTMBPs, the ability to detect PTMBPs in higher eukaryotes is oped and validated an innovative in vivo approach called mammalian constrained by the limitations
    [Show full text]
  • NF1) Pseudogenes on Chromosomes 2, 14 and 22
    European Journal of Human Genetics (2000) 8, 209–214 © 2000 Macmillan Publishers Ltd All rights reserved 1018–4813/00 $15.00 y www.nature.com/ejhg ARTICLE Mechanism of spreading of the highly related neurofibromatosis type 1 (NF1) pseudogenes on chromosomes 2, 14 and 22 Mirjam Luijten1, YingPing Wang2, Blaine T Smith2, Andries Westerveld1, Luc J Smink3, Ian Dunham3, Bruce A Roe2 and Theo JM Hulsebos1 1Department of Human Genetics, Academic Medical Center, University of Amsterdam, The Netherlands; 2Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA; 3Sanger Centre, Wellcome Trust Genome Campus, Hinxton Hall, Cambridge, UK Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder that involves tissues derived from the embryonic neural crest. Besides the functional gene on chromosome arm 17q, NF1-related sequences (pseudogenes) are present on a number of chromosomes including 2, 12, 14, 15, 18, 21, and 22. We elucidated the complete nucleotide sequence of the NF1 pseudogene on chromosome 22. Only the middle part of the functional gene but not exons 21–27a, encoding the functionally important GAP-related domain of the NF1 protein, is presented in this pseudogene. In addition to the two known NF1 pseudogenes on chromosome 14 we identified two novel variants. A phylogenetic tree was constructed, from which we concluded that the NF1 pseudogenes on chromosomes 2, 14, and 22 are closely related to each other. Clones containing one of these pseudogenes cross-hybridised with the other pseudogenes in this subset, but did not reveal any in situ hybridisation with the functional NF1 gene or with NF1 pseudogenes on other chromosomes.
    [Show full text]
  • Anza Restriction Enzyme Cloning System Complete, One-Buffer System— for Beautifully Simple Cloning Cloning Has Never Been Simpler
    Anza Restriction Enzyme Cloning System Complete, one-buffer system— for beautifully simple cloning Cloning has never been simpler Finally, forget the frustrations of finding compatible buffers and sorting through protocols for your restriction enzyme digests—start getting reliable results for your downstream experiments. The Invitrogen™ Anza™ Restriction Enzyme Cloning System is a complete system, comprised of: DNA modifying enzymes 128 restriction enzymes + 5 DNA modifying enzymes Anza T4 DNA Ligase Master Mix Anza Thermosensitive All Anza™ restriction enzymes work together cohesively and are fully functional Alkaline Phosphatase with the single Anza™ buffer. Anza T4 Polynucleotide Kinase The system offers: Anza DNA Blunt End Kit • One buffer for all restriction enzymes Anza DNA End Repair Kit • One digestion protocol for all DNA types • Complete digestion in 15 minutes • Overnight digestion without star activity Convenient buffer formats All Anza restriction enzymes come with an Anza 10X Buffer and an Anza 10X Red Buffer to give you the flexibility you require. The red buffer includes a density reagent containing red and yellow tracking dyes that migrate with 800 bp DNA fragments and faster than the 10 bp DNA fragments, respectively, in a 1% agarose gel. This eliminates tedious dye addition steps prior to gel loading and is compatible with downstream applications.* * For applications that require analysis by fluorescence excitation, Anza 10X Buffer is recommended, as the Anza 10X Red Buffer may interfere with some fluorescence measurements. 2 A one-buffer system Eliminate the frustration of trying to find compatible buffers for all your enzymes. All Anza restriction enzymes allow for complete digestion using a single Anza buffer.
    [Show full text]