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

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100 sample was irradiated with a nitrogen laser (337 nm) and 100 individual laser shots were collected for each sample. Construction of a Nonpolar pduP Deletion Bases 28 to 1,386 of the pduP coding sequence were deleted via a PCR-based method (Miller and Mekalanos 1988). The deletion was designed to leave the predicted translational start and stop signals of all pdu genes intact. The following primers were used for PCR amplification of the flanking regions of the pduP gene: primer 1, 5’-GCTCTAGACCAGGCCAACATCATCCGTGAAGTTAG-3’; primer 2, 5’-TCATCGCGACCTCAGCAGGGTTTCGAGTTCAGAAGTAATCATTG-3’; primer 3, 5’-CGCTAACTGAGGTCGCGATGAATACC-3’; and primer 4, 5’-GAAGAGCTCAATTCTGCGGCGGTACGCTGACCACC-3’. Primers 1 and 2 were used to amplify 496 DNA bases upstream of the pduP gene, and primers 3 and 4 were used to amplify 508 DNA bases downstream of pduP gene. The upstream and downstream amplification products were purified and then fused by a PCR reaction that included 1 ng/µl of each product and primers 1 and 4. The fused product was cut with XbaI and SacI (these sites were designed into primers 1 and 4, respectively), and ligated to suicide vector pCVD442 (Miller and Mekalanos 1988) that had been similarly cut. The ligation mixture was used to transform E. coli S17.1 by electroporation and transformants were selected on LB medium supplemented with Amp (100 µg/ml). Plasmids were extracted from four of the isolated transformants, screened by restriction analysis, and all released an insert of the expected size (1004 bp). One of these transformants was used to introduce the pduP deletion into the S. enterica chromosome using the procedure of Miller and Mekelanos (1988) with the following modification.
101 For the conjugation step, strain BE47 was used as the recipient and exconjugants were selected by plating on LB agar supplemented with ampicillin (100 µg/ml) and chloramphenicol (20 µg/ml). Deletion of the pduP coding sequence was verified by PCR using chromosomal DNA as a template. Lastly, the thr-480 dCAM insertion used for selection of exconjugants was crossed-off by P22 transduction using a phage lysate prepared with the wild-type strain and by selecting for prototrophy on NCE glucose minimal medium. Antibody Preparation His8-PduP purified using Ni 2+ -affinity chromatography was resolved on a 12% Tris-HCl SDS-PAGE gel. The protein band corresponding to His8-PduP was excised and used as a source of antigen for the preparation of polyclonal antibody in a New Zealand white rabbit by Cocalico Biologicals (Reamstown, PA). To eliminate antibodies cross-reacting with E. coli proteins, the antiserum was preadsorbed using acetone powder prepared from E. coli BL21DE3 RIL containing pTA925 (the expression strain lacking pduP) as described previously (Harlow and Lane 1988). Western Blots Cultures were grown and prepared for SDS-PAGE and electroblotting as previously described (Havemann et al. 2002). Membranes were probed using adsorbed anti-PduP antiserum (diluted 1:3,500 in blocking buffer) as the source of the primary antibody, and goat anti-rabbit conjugated to alkaline phosphatase (diluted 1:3,000 in blocking buffer) as the secondary antibody. Chromogenic developing agents were used following the manufacturer’s instructions (Bio-Rad).
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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
Page 65 and 66: 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: mM CHES (2-[N-cyclohexylamino]ethan
Page 119 and 120: 103 from New England Biolabs (Bever
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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