B12 METABOLISM IN HUMANS By NICOLE AURORA LEAL A ...

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Electron Microscopy 102 For electron microscopy, cells were grown in minimal medium supplemented with 37 mM succinate and 26 mM propanediol. Cultures (10 ml) were incubated in 125-ml Erlenmeyer flasks at 37º C, with shaking at 275 rpm in a New Brunswick C24 Incubator Shaker. For immunogold localization of the PduP protein, cells were prepared as previously described (Bobik et al. 1999). The source of the primary antibody was rabbit polyclonal anti-PduP antiserum or preimmune serum diluted in 1:100 in PBS. The secondary antibody used was goat anti-rabbit IgG conjugated to12-nm colloidal gold (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) diluted 1:30 in PBS. DNA Sequencing and Analysis DNA sequencing was carried out at the University of Florida Interdisciplinary Center for Biotechnology Research DNA Sequencing Core Facility using Applied Biosystems automated sequencing equipment (Perkin Elmer, Norwalk, CT) or at the University of Florida, Department of Microbiology and Cell Science DNA Sequencing Facility using a LI-COR model 4000L DNA sequencer, automated sequencing equipment, and Base ImagIR Analysis Software version 04.1h (LI-COR, Lincoln, NE). The template for DNA sequencing was plasmid DNA purified using Qiagen 100 tips or Qiagen mini-prep kits. BLAST software was used for sequence similarity searches (Altschul et al. 1997). Chemicals and Reagents Formaldehyde, (R, S)-1,2-propanediol, and antibiotics were from Sigma Chemical Company (St. Louis, MO). Isopropyl-β-D-thiogalactopyranoside (IPTG) was from Diagnostic Chemicals Limited (Charlotteville PEI, Canada). Restriction enzymes were
103 from New England Biolabs (Beverly, MA) or Promega (Madison, WI). T4 DNA ligase was from New England Biolabs. Glutaraldehyde was from Tousimis (Rockville, MD), and uranyl acetate was from EM Sciences (Washington, PA). LR White resin was from Ted Pella Inc. (Redding, CA). Acrylamide, agarose, ammonium persulfate, Coomassie Brilliant Blue R-250, EDTA, ethidium bromide, 2-mercaptoethanol, N, N-bis-methylene-acrylamide, powdered milk, SDS, and TEMED were from Bio-Rad (Hercules, CA). Bacterial Protein Extraction Reagent II (BPER-II) was from Pierce (Rockford, IL). Other chemicals were from Fisher Scientific (Pittsburgh, PA). Results Effect of a Precise pduP Deletion on the Growth of S. enterica on Minimal 1,2-Propanediol Medium Prior biochemical studies indicated that a CoA-acylating propionaldehyde dehydrogenase was involved in the degradation of 1,2–propanediol by S. enterica (Toraya et al. 1979, Obradors et al. 1988). Recent DNA sequence analyses of the pdu operon (a cluster of genes required for 1,2-propanediol degradation by S. enterica) showed that the PduP protein has sequence similarity to a number of CoA-acylating aldehyde dehydrogenases and is 32% identical in amino acid sequence to the E. coli aldehyde/alcohol dehydrogenase, AdhE (Kessler et al. 1991, Bobik et al. 1999). This suggested that PduP is a CoA-acylating propionaldehyde dehydrogenase needed for the degradation of 1,2-propanediol by S. enterica. To examine the role of PduP in 1,2-propanediol degradation, a precise deletion of the pduP gene was constructed using a PCR-based method, and the effects of this mutation on the growth of S. enterica on 1,2-propanediol/CN-<strong>B12</strong> minimal medium were examined. The wild-type strain and strain BE191 (∆pduP) grew with generation times of
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B12 METABOLISM IN HUMANS By NICOLE
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The work in this dissertation is de
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TABLE OF CONTENTS Page ACKNOWLEDGME
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General Molecular and Protein Metho
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LIST OF TABLES Table page 1-1. Aden
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3-7. Stoichiometry of the MSR-ATR s
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DTT dithiothreitol E. coli Escheric
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Abstract of Dissertation Presented
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CHAPTER 1 INTRODUCTION History of C
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3 (DMB). The DMB moiety is covalent
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5 and deoxyadenosine. After hydroge
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7 homocysteine recycling and methio
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methylmalonic acid. Methylmalonic a
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folate-dependent reactions includin
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tetanomorphum, P. shermanii, Euglen
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enzyme. This suggests that cob(II)a
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at the 5’-C of ATP by cob(I)alami
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iochemical studies and complementat
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1997). Both cases of the cblD disor
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23 The cblB complementation group i
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(Watkins et al. 2002). Some patient
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Chapter 2 of this dissertation repo
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Corrin Ring Dimethyl benzimidazole
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Classes Eliminases Dehydratases OH
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A) B) CH 3THF 33 MS-cob(I)alamin Me
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propanol dehydrogenase (PduQ) propa
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or from mutations that impair the a
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1989). 5-bromo-4-chloro-3-indolyl-
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41 5’-GCCGCCGGTACCGATGACGACGACAAG
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43 Growth of Adenosyltransferase Ex
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anti-rabbit IgG (γ-chain specific)
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sequence similarity to a known ATR
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ATRs were produced as fusion protei
Page 67 and 68: 51 of the lacI repressor (not shown
Page 69 and 70: in which proteins bind B12, the "ba
Page 71 and 72: libraries may be particularly usefu
Page 73 and 74: 57 Figure 2-1. Multiple sequence al
Page 75 and 76: Table 2-2. Specific activities of b
Page 77 and 78: 61 1 2 3 4 5 25 kDa Figure 2-4. Wes
Page 79 and 80: 63 (cyanocobalamin, CNCbl) and hydr
Page 81 and 82: 65 5’- triphosphate (ATP), and di
Page 83 and 84: extracts of ATR 239K (160 mg protei
Page 85 and 86: Milford, MA). Samples (200 µl) wer
Page 87 and 88: Linearity of the ATR reaction 71 Th
Page 89 and 90: 73 reactions was characteristic of
Page 91 and 92: 75 MSR Produces little Cob(I)alamin
Page 93 and 94: Johnson et al. 2001, Saridakis et a
Page 95 and 96: AdoCbl by the MSR-ATR system. Exper
Page 97 and 98: propionyl-CoA PCC (2S)-methylmalony
Page 99 and 100: kDa 97 66 45 31 21 14 7 83 1 2 3 4
Page 101 and 102: 85 Table 3-3. The MSR-ATR system se
Page 103 and 104: Absorbance 0.5 0.4 0.3 0.2 0.1 0.0
Page 105 and 106: Wavelength, (525 nm) 0.24 0.23 0.22
Page 107 and 108: Activity, (nmol min-1 Activity, (nm
Page 109 and 110: al. 1988). The aldehyde is then dis
Page 111 and 112: CoA-acylating propionaldehyde dehyd
Page 113 and 114: 97 previously published DNA sequenc
Page 115 and 116: mM CHES (2-[N-cyclohexylamino]ethan
Page 117: 101 For the conjugation step, strai
Page 121 and 122: 105 Propionaldehyde Dehydrogenase A
Page 123 and 124: 107 fraction. Following centrifugat
Page 125 and 126: 109 antiserum obtained was specific
Page 127 and 128: 111 biochemical evidence was presen
Page 129 and 130: 113 Table 4-1. Bacterial strains Sp
Page 131 and 132: 115 Table 4-3. Propionaldehyde dehy
Page 133 and 134: kDa 209 80 49 35 29 117 1 2 3 4 Fig
Page 135 and 136: CHAPTER 5 CONCLUSIONS In humans, co
Page 137 and 138: 121 substitutions that are common p
Page 139 and 140: 123 Studies also indicated that hum
Page 141 and 142: LIST OF REFERENCES Aberg, A., S. Ha
Page 143 and 144: 127 Bradford, M. 1976. A rapid and
Page 145 and 146: 129 Fenton, W. A. and L. E. Rosenbe
Page 147 and 148: 131 Johnson, C. L. V. J., E. Pechon
Page 149 and 150: 133 Mellman, I., H. F. Willard and
Page 151 and 152: 135 Rossi, M., J. P. Glusker, L. Ra
Page 153 and 154: 137 Vlasie, M., S. Chowdhury and R.
Page 155 and 156: BIOGRAPHICAL SKETCH Nicole Aurora L
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