DOCSLIB.ORG

Biochemj00614-0075.Pdf

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Biochemj00614-0075.Pdf Biochem. J. (1973) 131, 237-246 237 Printed in Great Britain Multiple Forms of Nuclear Deoxyribonucleic Acid Polymerases and their Relationship with the Soluble Enzyme By R. L. P. ADAMS, MAUREEN A. L. HENDERSON, W. WOOD and J. G. LINDSAY Department ofBiochemistry, University of Glasgow, Glasgow G12 8QQ, U.K. (Received 31 July 1972) 1. DNA polymerase from nuclear and supernatant fractions of cultured mouse L929 cells was fractionated on columns of Sephadex G-200, Sepharose 4B and of DEAE- cellulose. Several peaks of activity are found on Sephadex chromatography and the distribution ofactivity between these depends on: (a) the source ofthe enzyme, i.e. nuclear or supernatant fraction; (b) the mode of extraction of the enzyme from the nucleus; (c) the amount of enzyme applied to the column. 2. The DNA polymerase activity in the lower-molecular-weight peaks (approximate molecular weights are 35000, 70000 and 140000) is firmly bound within the cell nucleus and shows a preference for native DNA as template, whereas the high-molecular-weight peak (peak I, molecular weight 250000 or greater) is found in supernatant fractions and shows greater activity with a denatured DNA template. 3. During periods of DNA synthesis the high-molecular-weight enzyme becomes more firmly bound within the nucleus. 4. Peak I enzymic activity is relatively unstable and is inhibited by thiol-blocking reagents and deoxycholate, but it is stimulated by univalent cations. 5. Very little endonuclease is present in the polymerase preparations, but a very active exonuclease and nucleoside diphosphokinase are present. On Sephadex chromatography, however, it was shown that the immediate precursors for DNA synthesis, at least by peak I enzyme, are the deoxyribonucleoside triphosphates. 6. Attempts to decrease the molecular weight of the peak I enzyme while still retaining activity failed. Several groups (Wallace et al., 1971; Haines et al., We have been unable to degrade the high- 1971; Weissbach et al., 1971) have now reported that molecular-weight enzyme to smaller active fragments, the DNA polymerase activity found in supernatant but have shown several important differences between fractions from animal cells is of higher molecular the high-molecular-weight and the lower-molecular- weight than the nuclear enzyme. However, no weight enzymes, in particular with regard to their evidence has appeared as to the function and com- relative stability, sensitivity to thiol-blocking reagents plexity of these enzymes nor as to their relationship and affinity for the DNA template. with each other. It is, however, known that the supernatant enzyme, which shows greater activity with a denatured DNA template, is more active in Materials and Methods whereas the nuclear enzyme, which growing tissues, Preparation of enzyme fractions normally prefers a native DNA template, shows considerable activity in cells and tissues not actively Mouse fibroblast (L929) cell cultures were sub- growing (Iwamura et al., 1968; Lindsay et al., 1970; cultured in minimum essential medium (Eagle) Ove et al., 1970). supplemented with 10% (v/v) of calf serum (medium We have confirmed that the supernatant enzyme is EC10). Cultures and media were obtained from larger than the nuclear enzyme and have shown that Flow Laboratories Ltd., Irvine, Ayrshire, U.K., or the latter contains at least three species of DNA from BioCult Laboratories, Glasgow, U.K. Cells, polymerase of approximate molecular weights 35000, which as a routine were tested for mycoplasma, were 70000 and 140000. harvested (a) while growing exponentially or (b) after Nuclei also contain DNA polymerase activity they had entered the stationary phase or (c) 2h after indistinguishable from the supernatant enzyme. There reversal of a 16h aminopterin blockade, i.e. in S- is increased association of the high-molecular-weight phase (Adams, 1969). Nuclear and supernatant enzyme with the nucleus during S-phase, and this fractions were prepared as previously described would account for the increased activity of S-phase (Lindsay et al., 1970) by homogenizing the washed nuclei with a denatured DNA template (Lindsay cells in 0.25M-sucrose buffered with 20mM-Tris- et al., 1970). HCI buffer, pH7.5. Vol. 131 238 R. L. P. ADAMS, M. A. L. HENDERSON, W. WOOD AND J. G. LINDSAY Table 1. Purification ofnuclear enzymes The values in parentheses represent the yields of enzymic activity. Sp. activity of DNA polymerase (units/mg) Total protein Native Denatured Fraction Sample (mg) template template I Whole nuclei 110 0.5 (100) 0.2 (100) II 0.4m-KCI extract 36 2.5 (160) 1.0 (167) III (NH4)2SO4 precipitate 19.4 5.8 (204) 3.9 (367) The homogenate was centrifuged at 800g for DNA polymerase assay 10min and the supernatant fraction, after being centrifuged at 105OO0g for 60min (Spinco model L DNA polymerase was assayed basically as de- ultracentrifuge, rotor no. 40), was used as a source scribed by Shepherd & Keir (1966). Samples (0.1 ml) of soluble enzyme. The nuclear pellet was washed in column buffer were incubated at 37°C for 60min in twice in buffered sucrose and was then extracted a total volume of 0.25ml with 4,umol of Tris-HCl with medium containing (final concentrations) 0.4M- buffer, pH7.5, 1.5,umol of MgCl2, 15,umol of KCI, KCl, 20mM-Tris-HCl buffer, pH7.5, and 5mM-2- 0.1,imol of EDTA, 3,umol of 2-mercaptoethanol, mercaptoethanol to yield the nuclear enzyme. 100l,g of DNA and 50nmol each of dATP, dGTP, Enzymic preparations were concentrated by adding dCTP and [Me-3H]dTTP (20nCi/nmol) (The Radio- (NH4)2SO4 to 80% saturation over 30min at 0°C chemical Centre, Amersham, Bucks., U.K.). The with constant stirring. The suspension was centri- reaction was terminated by the addition of 0.05ml fuged at 10000g for 20min in the MSE High Speed 18 of 2M-NaOH and the samples were reincubated for centrifuge and the pellet was dissolved in and dia- at least 1 h at 37°C. Portions (100,ul) were spotted on lysed against column buffer [containing (final con- to Whatman 3MM paper disks (2.5cm diam.), and centrations) 0.15M-KCI, 20mM-Tris-HCl buffer, these were washed six times in 5 % (w/v) trichloro- pH7.5, and 5mM-2-mercaptoethanol]. When the acetic acid in 50mM-Na4P207 and then dried with sample was to be applied to a DEAE-cellulose column ethanol and ether. The DNA was dissolved by it was further dialysed against 20mM-Tris - HCI heating with 0.5ml of 1 M-Hyamine hydroxide buffer, pH7.5, containing 5mM-2-mercaptoethanol. [Nuclear Enterprises (G.B.) Ltd., Edinburgh, U.K.] Table 1 gives details of the purification of the nuclear for 20min at 60°C in a counting vial. Radioactivity enzyme. was determined after the addition of toluene-based scintillator [0.5 % (w/v) 2,5-diphenyloxazole]. Column chromatography When diphosphates were used in the assay they replaced the triphosphates in equimolar amounts. Sephadex G-200 and Sepharose 4B were obtained When present, ATP was at 1.5mM. from Pharmacia (G.B.) Ltd., London W5 5SS, U.K. A unit of DNA polymerase activity is defined as Column chromatography was carried out at the amount required to catalyse incorporation of 4°C with a column (60cmxO.9cm diam.) that had 1 nmol of [Me-3H]dTTP into acid-insoluble material been calibrated with molecular-weight markers of in th at 37°C. cytochrome c, bovine serum albumin, haemoglobin, human y-globulin and urease. The flow rate was 5ml/h and 1 ml fractions were collected. DEAE-cellulose chromatography was carried out Nucleoside diphosphokinase assay in a column (6.8cm x 1.5cm diam.) with Whatman To assay the interconversion of di- and tri-phos- DE52 DEAE-cellulose (H. Reeve Angel and Co. phates 100,ul portions of the polymerase incubation Ltd., London EC4V 6AY, U.K.). The sample was mixtures were pipetted into 0.4ml of 5% trichloro- applied in and washed with 20mM-Tris-HCl buffer, acetic acid, and 50jul portions of the supernatant pH7.5, containing 5mM-2-mercaptoethanol, and then were chromatographed on Whatman no. 1 paper a 50ml linear gradient of 0-0.4M-KCI in the same with isobutyric acid-aq. NH3 soln. (sp.gr. 0.88)- buffer was applied. The column was run at 6.5ml/h EDTA (0.1M)-water (100:4.2:1.6:55.8, by vol.) and 1.3ml fractions were collected. (Krebs & Hems, 1952). Spots corresponding to 1973 NUCLEAR AND SOLUBLE DNA POLYMERASES 239 dTMP, dTDP and dTTP were cut out and radio- 0.5,umol of 2-mercaptoethanol and 20,tmol of either activity was counted by using toluene-based scintil- Tris-HCl buffer, pH7.5, or glycine-NaOH buffer, lator. pH9.2, in a total volume of 0.3ml. The reaction was terminated by cooling the samples to 0°C and 0.4mg Preparation of 14C-labelled DNA from L929 cells ofunlabelled salmon testis DNA was added as carrier. Acid-insoluble radioactivity was assayed with the aid L929 cells were grown in medium EC10 containing of toluene-based scintillator. Protein was determined [2-14C]thymidine [5OnCi (0.86nmol)/ml] for 3 days by the method of Lowry et al. (1951). Phospho- before they were harvested. The washed cells were lipase C and trypsin were obtained from Sigma suspended in balanced salt solution and poured into Chemical Co., St. Louis, Mo., U.S.A. Salmon testis an equal volume of 2% (w/v) sodium dodecyl and calf thymus DNA were obtained from sulphate containing 4mM-EDTA and 6% (w/v) of Worthington Biochemical Corp., Freehold, N.J., sodium p-aminosalicylate. The sample was stirred at U.S.A. 70°C for 30min and cooled to room temperature, and an equal volume of phenol-m-cresol (22:3, v/v) containing 0.1 % (w/v) 8-hydroxyquinoline was Results added.
Load more

Recommended publications
  • To Study Mutant P53 Gain of Function, Various Tumor-Derived P53 Mutants
    Differential effects of mutant TAp63γ on transactivation of p53 and/or p63 responsive genes and their effects on global gene expression. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By Shama K Khokhar M.Sc., Bilaspur University, 2004 B.Sc., Bhopal University, 2002 2007 1 COPYRIGHT SHAMA K KHOKHAR 2007 2 WRIGHT STATE UNIVERSITY SCHOOL OF GRADUATE STUDIES Date of Defense: 12-03-07 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY SHAMA KHAN KHOKHAR ENTITLED Differential effects of mutant TAp63γ on transactivation of p53 and/or p63 responsive genes and their effects on global gene expression BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science Madhavi P. Kadakia, Ph.D. Thesis Director Daniel Organisciak , Ph.D. Department Chair Committee on Final Examination Madhavi P. Kadakia, Ph.D. Steven J. Berberich, Ph.D. Michael Leffak, Ph.D. Joseph F. Thomas, Jr., Ph.D. Dean, School of Graduate Studies 3 Abstract Khokhar, Shama K. M.S., Department of Biochemistry and Molecular Biology, Wright State University, 2007 Differential effect of TAp63γ mutants on transactivation of p53 and/or p63 responsive genes and their effects on global gene expression. p63, a member of the p53 gene family, known to play a role in development, has more recently also been implicated in cancer progression. Mice lacking p63 exhibit severe developmental defects such as limb truncations, abnormal skin, and absence of hair follicles, teeth, and mammary glands. Germline missense mutations of p63 have been shown to be responsible for several human developmental syndromes including SHFM, EEC and ADULT syndromes and are associated with anomalies in the development of organs of epithelial origin.
    [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]
  • (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown Et Al
    US 20030082511A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown et al. (43) Pub. Date: May 1, 2003 (54) IDENTIFICATION OF MODULATORY Publication Classification MOLECULES USING INDUCIBLE PROMOTERS (51) Int. Cl." ............................... C12O 1/00; C12O 1/68 (52) U.S. Cl. ..................................................... 435/4; 435/6 (76) Inventors: Steven J. Brown, San Diego, CA (US); Damien J. Dunnington, San Diego, CA (US); Imran Clark, San Diego, CA (57) ABSTRACT (US) Correspondence Address: Methods for identifying an ion channel modulator, a target David B. Waller & Associates membrane receptor modulator molecule, and other modula 5677 Oberlin Drive tory molecules are disclosed, as well as cells and vectors for Suit 214 use in those methods. A polynucleotide encoding target is San Diego, CA 92121 (US) provided in a cell under control of an inducible promoter, and candidate modulatory molecules are contacted with the (21) Appl. No.: 09/965,201 cell after induction of the promoter to ascertain whether a change in a measurable physiological parameter occurs as a (22) Filed: Sep. 25, 2001 result of the candidate modulatory molecule. Patent Application Publication May 1, 2003 Sheet 1 of 8 US 2003/0082511 A1 KCNC1 cDNA F.G. 1 Patent Application Publication May 1, 2003 Sheet 2 of 8 US 2003/0082511 A1 49 - -9 G C EH H EH N t R M h so as se W M M MP N FIG.2 Patent Application Publication May 1, 2003 Sheet 3 of 8 US 2003/0082511 A1 FG. 3 Patent Application Publication May 1, 2003 Sheet 4 of 8 US 2003/0082511 A1 KCNC1 ITREXCHO KC 150 mM KC 2000000 so 100 mM induced Uninduced Steady state O 100 200 300 400 500 600 700 Time (seconds) FIG.
    [Show full text]
  • Supplementary Table 2
    Supplementary Table 2. Differentially Expressed Genes following Sham treatment relative to Untreated Controls Fold Change Accession Name Symbol 3 h 12 h NM_013121 CD28 antigen Cd28 12.82 BG665360 FMS-like tyrosine kinase 1 Flt1 9.63 NM_012701 Adrenergic receptor, beta 1 Adrb1 8.24 0.46 U20796 Nuclear receptor subfamily 1, group D, member 2 Nr1d2 7.22 NM_017116 Calpain 2 Capn2 6.41 BE097282 Guanine nucleotide binding protein, alpha 12 Gna12 6.21 NM_053328 Basic helix-loop-helix domain containing, class B2 Bhlhb2 5.79 NM_053831 Guanylate cyclase 2f Gucy2f 5.71 AW251703 Tumor necrosis factor receptor superfamily, member 12a Tnfrsf12a 5.57 NM_021691 Twist homolog 2 (Drosophila) Twist2 5.42 NM_133550 Fc receptor, IgE, low affinity II, alpha polypeptide Fcer2a 4.93 NM_031120 Signal sequence receptor, gamma Ssr3 4.84 NM_053544 Secreted frizzled-related protein 4 Sfrp4 4.73 NM_053910 Pleckstrin homology, Sec7 and coiled/coil domains 1 Pscd1 4.69 BE113233 Suppressor of cytokine signaling 2 Socs2 4.68 NM_053949 Potassium voltage-gated channel, subfamily H (eag- Kcnh2 4.60 related), member 2 NM_017305 Glutamate cysteine ligase, modifier subunit Gclm 4.59 NM_017309 Protein phospatase 3, regulatory subunit B, alpha Ppp3r1 4.54 isoform,type 1 NM_012765 5-hydroxytryptamine (serotonin) receptor 2C Htr2c 4.46 NM_017218 V-erb-b2 erythroblastic leukemia viral oncogene homolog Erbb3 4.42 3 (avian) AW918369 Zinc finger protein 191 Zfp191 4.38 NM_031034 Guanine nucleotide binding protein, alpha 12 Gna12 4.38 NM_017020 Interleukin 6 receptor Il6r 4.37 AJ002942
    [Show full text]
  • Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation
    BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in
    [Show full text]
  • An NKX2-1/ERK/WNT Feedback Loop Modulates Gastric Identity And
    RESEARCH ARTICLE An NKX2-1/ERK/WNT feedback loop modulates gastric identity and response to targeted therapy in lung adenocarcinoma Rediet Zewdu1,2, Elnaz Mirzaei Mehrabad1,3, Kelley Ingram1,4, Pengshu Fang1,4, Katherine L Gillis1,4, Soledad A Camolotto1,2, Grace Orstad1,4, Alex Jones1,2, Michelle C Mendoza1,4, Benjamin T Spike1,4, Eric L Snyder1,2,4* 1Huntsman Cancer Institute, Salt Lake City, United States; 2Department of Pathology, University of Utah, Salt Lake City, United States; 3School of Computing, University of Utah, Salt Lake City, United States; 4Department of Oncological Sciences, University of Utah, Salt Lake City, United States Abstract Cancer cells undergo lineage switching during natural progression and in response to therapy. NKX2-1 loss in human and murine lung adenocarcinoma leads to invasive mucinous adenocarcinoma (IMA), a lung cancer subtype that exhibits gastric differentiation and harbors a distinct spectrum of driver oncogenes. In murine BRAFV600E-driven lung adenocarcinoma, NKX2-1 is required for early tumorigenesis, but dispensable for established tumor growth. NKX2-1-deficient, BRAFV600E-driven tumors resemble human IMA and exhibit a distinct response to BRAF/MEK inhibitors. Whereas BRAF/MEK inhibitors drive NKX2-1-positive tumor cells into quiescence, NKX2- 1-negative cells fail to exit the cell cycle after the same therapy. BRAF/MEK inhibitors induce cell identity switching in NKX2-1-negative lung tumors within the gastric lineage, which is driven in part by WNT signaling and FoxA1/2. These data elucidate a complex, reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of lung adenocarcinoma identity that is likely to impact lineage-specific therapeutic strategies.
    [Show full text]
  • To Nucleotide Sequence Determinations (Pancreatic Dnase I/U1 Ribonuclease/U2 Ribonuclease/Pancreatic Ribonuclease A/Ribosubstituted DNA) GARY V
    Proc. Nat. Acad. Sci. USA Vol. 71, No. 12, pp. 5017-5021, December 1974 Deoxysubstitution in RNA by RNA Polymerase In Vitro: A New Approach to Nucleotide Sequence Determinations (pancreatic DNase I/U1 ribonuclease/U2 ribonuclease/pancreatic ribonuclease A/ribosubstituted DNA) GARY V. PADDOCK, HOWARD C. HEINDELL AND WINSTON SALSER Biology Department and Molecular Biology Institute, University of California at Los Angeles, 405 Hilgard Ave., Los Angeles, Calif. 90024 Communicated by Charles Yanofsky, August 12, 1974 ABSTRACT Deoxynucleotides have been incorporated substituted RNA. We have observed that Mn++ ion will not into RNA synthesized in vitro by RNA polymerase with only cause DNA polymerase to synthesize ribosubstituted either double-stranded or single-stranded DNA as a to synthesize template. By use of this technique to block or promote DNA but will also cause RNA polymerase cleavage at a particular phosphodiester bond, a variety of deoxysubstituted RNA. Subsequently we have discovered a specific cleavages may be obtained with the available ribo- number of other papers dealing with the synthesis of deoxy- nucleases and deoxyribonuclease I. These methods should substituted RNA (12-14). These papers did not, however, greatly increase the ease and rapidity of nucleotide se- point out the applicability of the approach to nucleotide quence determinations. sequencing and did not include the controls we have found The introduction of radiographic approaches by Sanger and essential for demonstrating that complete deoxysubstitution his colleagues (1-4) greatly increased the power of nucleotide has occurred. sequencing techniques, but there remain certain obstacles In this preliminary paper we discuss the theory by which whose solution could result in impressive further increases in substitution of deoxynucleotides becomes an aid in nucleotide the rapidity with which large sequences may be determined.
    [Show full text]
  • Cell-Based Osteoprotegerin Therapy for Debris-Induced Aseptic Prosthetic Loosening on a Murine Model
    Gene Therapy (2010) 17, 1262–1269 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 www.nature.com/gt ORIGINAL ARTICLE Cell-based osteoprotegerin therapy for debris-induced aseptic prosthetic loosening on a murine model L Zhang1,2, T-H Jia2, ACM Chong1, L Bai1,HYu1, W Gong2, PH Wooley1 and S-Y Yang1 1Orthopaedic Research Institute, Via Christi Regional Medical Center, Wichita, KS, USA and 2Department of Orthopaedic Surgery, Jinan Central Hospital, Shandong University School of Medicine, Jinan, China Exogenous osteoprotegerin (OPG) gene modification membranes existed ubiquitously at bone–implant interface appears a therapeutic strategy for osteolytic aseptic in control groups, whereas only observed sporadically in OPG loosening. The feasibility and efficacy of a cell-based OPG gene-modified groups. Tartrate-resistant acid phosphatase+ gene delivery approach were investigated using a murine osteoclasts and tumor necrosis factor a,interleukin-1b, model of knee prosthesis failure. A titanium pin was CD68+ expressing cells were significantly reduced in implanted into mouse proximal tibia to mimic a weight- periprosthetic tissues of OPG gene-modified mice. No bearing knee arthroplasty, followed by titanium particles transgene dissemination or tumorigenesis was detected in challenge to induce periprosthetic osteolysis. Mouse fibro- remote organs and tissues. Data suggest that cell-based blast-like synoviocytes were transduced in vitro with either ex vivo OPG gene therapy was comparable in efficacy with AAV-OPG or AAV-LacZ before transfused into the osteolytic in vivo local gene transfer technique to deliver functional prosthetic joint 3 weeks post surgery. Successful transgene therapeutic OPG activities, effectively halted the debris- expression at the local site was confirmed 4 weeks later after induced osteolysis and regained the implant stability in this killing.
    [Show full text]
  • Figure S1. GO Analysis of Genes in Glioblastoma Cases That Showed Positive and Negative Correlations with TCIRG1 in the GSE16011 Cohort
    Figure S1. GO analysis of genes in glioblastoma cases that showed positive and negative correlations with TCIRG1 in the GSE16011 cohort. (A‑C) GO‑BP, GO‑CC and GO‑MF terms of genes that showed positive correlations with TCIRG1, respec‑ tively. (D‑F) GO‑BP, GO‑CC and GO‑MF terms of genes that showed negative correlations with TCIRG1. Red nodes represent gene counts, and black bars represent negative 1og10 P‑values. TCIRG1, T cell immune regulator 1; GO, Gene Ontology; BP, biological process; CC, cellular component; MF, molecular function. Table SI. Genes correlated with T cell immune regulator 1. Gene Name Pearson's r ARPC1B Actin‑related protein 2/3 complex subunit 1B 0.756 IL4R Interleukin 4 receptor 0.695 PLAUR Plasminogen activator, urokinase receptor 0.693 IFI30 IFI30, lysosomal thiol reductase 0.675 TNFAIP3 TNF α‑induced protein 3 0.675 RBM47 RNA binding motif protein 47 0.666 TYMP Thymidine phosphorylase 0.665 CEBPB CCAAT/enhancer binding protein β 0.663 MVP Major vault protein 0.660 BCL3 B‑cell CLL/lymphoma 3 0.657 LILRB3 Leukocyte immunoglobulin‑like receptor B3 0.656 ELF4 E74 like ETS transcription factor 4 0.652 ITGA5 Integrin subunit α 5 0.651 SLAMF8 SLAM family member 8 0.647 PTPN6 Protein tyrosine phosphatase, non‑receptor type 6 0.641 RAB27A RAB27A, member RAS oncogene family 0.64 S100A11 S100 calcium binding protein A11 0.639 CAST Calpastatin 0.638 EHBP1L1 EH domain‑binding protein 1‑like 1 0.638 LILRB2 Leukocyte immunoglobulin‑like receptor B2 0.629 ALDH3B1 Aldehyde dehydrogenase 3 family member B1 0.626 GNA15 G protein
    [Show full text]
  • Deoxyribonuclease I
    Catalog Number: 100574, 100575, 190062 Deoxyribonuclease I CAS #: 9003-98-9 Description: Deoxyribonuclease from beef pancreas, DNase I, was first crystallized by Kunitz.10,11 It is an endonuclease which splits phosphodiester linkages, preferentially adjacent to a pyrimidine nucleotide yielding 5'-phosphate terminated polynucleotides with a free hydroxyl group on position 3'. The average chain of limit digest is a tetranucleotide.19 DNase I acts upon single chain DNA29, and upon double-stranded DNA and chromatin. In the latter case, although histones restrict susceptibility to nuclease action, over a period of time nearly all chromatin DNA is acted upon. According to Mirsky and Silverman20, this could result from the looseness of histone attachment to DNA. They found that lysine-rich histones more effectively block DNase access to DNA than arginine-rich histones. Billing and Bonner2 suggest that DNase attacks the histone-free strand of chromatin DNA. Schmidt, et. al.28 indicate that hydrolysis of the histone-free region of DNA strands accounts for the initial rapid action of the enzyme on chromatin. Bollum3 reports degradation of synthetic homopolymer complexes by DNase I. The intracellular functions of the enzyme are probably controlled by a DNase inhibitor17, which according to Lazarides and Lindberg13 is actin. Molecular weight: 31,000.18 Composition: There are four deoxyribonucleases of beef pancreas: A, B, C, and D.14, 25, 27 Five have been reported by Junowicz and Spencer.7 They are glycoproteins differing from each other either in carbohydrate side-chain or polypeptide component.14, 25 DNase A is the predominant form26; its amino acid sequence has been reported.16 Optimum pH: 7.8.
    [Show full text]
  • Skeletal Stem and Progenitor Cells Maintain Cranial Suture Patency and Prevent Craniosynostosis
    ARTICLE https://doi.org/10.1038/s41467-021-24801-6 OPEN Skeletal stem and progenitor cells maintain cranial suture patency and prevent craniosynostosis Siddharth Menon1,2,3,4, Ankit Salhotra 1,2,3, Siny Shailendra1,2,3, Ruth Tevlin1,2,3, Ryan C. Ransom1,2,3, Michael Januszyk1,2,3, Charles K. F. Chan 1,2,3,4, Björn Behr5, Derrick C. Wan1,2,3, ✉ ✉ Michael T. Longaker 1,2,3,4 & Natalina Quarto 1,2,3,6 Cranial sutures are major growth centers for the calvarial vault, and their premature fusion 1234567890():,; leads to a pathologic condition called craniosynostosis. This study investigates whether skeletal stem/progenitor cells are resident in the cranial sutures. Prospective isolation by FACS identifies this population with a significant difference in spatio-temporal representation between fusing versus patent sutures. Transcriptomic analysis highlights a distinct signature in cells derived from the physiological closing PF suture, and scRNA sequencing identifies transcriptional heterogeneity among sutures. Wnt-signaling activation increases skeletal stem/progenitor cells in sutures, whereas its inhibition decreases. Crossing Axin2LacZ/+ mouse, endowing enhanced Wnt activation, to a Twist1+/− mouse model of coronal cra- niosynostosis enriches skeletal stem/progenitor cells in sutures restoring patency. Co- transplantation of these cells with Wnt3a prevents resynostosis following suturectomy in Twist1+/− mice. Our study reveals that decrease and/or imbalance of skeletal stem/pro- genitor cells representation within sutures may underlie craniosynostosis. These findings have translational implications toward therapeutic approaches for craniosynostosis. 1 Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. 2 Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, CA, USA.
    [Show full text]
  • Deoxyribonuclease I, Bovine (D2821)
    Deoxyribonuclease I, bovine recombinant, expressed in Pichia pastoris lyophilized powder Catalog Number D2821 Storage Temperature 2–8 °C CAS RN 9003-98-9 Optimal pH: EC 3.1.21.1 The optimal pH of DNase I activity is dependent on the Synonyms: DNase I, divalent ion present. In the presence of both Mg2+ and Deoxyribonucleate 5¢-Oligonucleotidohydrolase Ca2+, the optimal pH is between 7–8, while in the absence of Ca2+, the optimal pH is between 5.5–6.0.6 Product Description 1% Deoxyribonuclease I (DNase I) is found in most cells Extinction Coefficient: E280 = 11.1 and tissues. In mammals, the pancreas is one of the best sources for the enzyme. Pancreatic DNase I was This recombinant bovine DNase I is a glycoprotein, the first isolated DNase. produced without the addition of any animal-derived materials. It is supplied as a lyophilized powder DNase I is an endonuclease that acts on containing a glycine stabilizer. phosphodiester bonds adjacent to pyrimidines to produce polynucleotides with terminal 5¢-phosphates. Molecular mass: ~39 kDa A tetranucleotide is the smallest average digestion product. DNase I hydrolyzes single- and double- Specific activity: ³4,000 units/mg protein stranded DNA. In the presence of Mg2+ ions, DNase I attacks each strand of DNA independently and the Unit definition: One unit will produce a change in A260 of cleavage sites are random. If Mn2+ ions are present, 0.001 per minute per ml at pH 5.0 at 25 °C using DNA, both DNA strands are cleaved at approximately the Type I or III, as the substrate.
    [Show full text]