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

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96 electrophoresis, proteins were stained with Coomassie Brilliant Blue R-250. The protein concentration of solutions was determined using Bio-Rad protein assay reagent (Bio-Rad) (Bradford 1976). P22 Transduction Transductional crosses were performed as described using P22 HT105/1 int-210 (Davis et al. 1980), a mutant phage that has high transducing ability (Schmieger 1971). For the preparation of P22 transducing lysates from strains having galE mutations, overnight cultures were grown on LB-medium supplemented with 11 mM glucose and 11 mM galactose. Transductants were tested for phage contamination and sensitivity by streaking on green plates against P22 H5. Cloning pduP into pLAC22 PCR was used to amplify pduP from the template pMGS2 (Havemann et al. 2002). The primers used for amplification were 5’-GGAATTCGGATCCTATGAATACTTCTG AACTCGAAAC-3’ (forward) and 5’-GGAATTCAAGCTTCAGTTAGCGAATAGAAA AGCC-3’ (reverse). These primers introduced BamHI and HindIII restriction sites that were used for cloning into pLAC22 cut with BglII and HindIII (Warren et al. 2000). Cloning was carried out by ligation of the complementary cohesive ends formed by cutting with BamHI and BglII since pduP contains an internal BglII site. Following ligation, clones were introduced into S. enterica strain TR6579 by electroporation and transformants were selected by plating on LB-agar supplemented with ampicillin (Amp) at 100 µg/ml. Pure cultures were prepared from selected transformants, and plasmid DNA isolated from these strains was cut with NruI, PstI, and HindIII. The DNA sequence of one clone that released products of the expected sizes following restriction (2,635, 3,322 and 943 base pairs) was determined and found to be identical to the
97 previously published DNA sequence of pduP (Bobik et al. 1999). This clone (pNL9) as well as pLAC22 without insert were moved into strain BE191 by P22 transduction and the resulting strains (BE270 and BE269) were used for complementation studies. Construction of His8-PduP PCR was used to fuse an N-terminal His-tag to the PduP protein. Template pMGS2 was used with the following primers: 5’-GGGGATCCATG(CAT)8ATGAATACTTCTGAACTCGAAAC-3’ (forward); and 5’-GGAATTCAAGCTTCAGTTAGCGA ATAGAAAAGCC-3’ (reverse). These primers introduced BamHI and HindIII restriction sites that were used for cloning into pTA925 cut with BglII and HindIII. The ligation mixture was used to transform E. coli strain DH5α and transformants were selected by plating on LB agar supplemented with 25 µg kanamycin/ml. Plasmid DNA isolated from selected transformants was analyzed by restriction mapping and DNA sequencing. One plasmid encoding His8-PduP (pNL69) was introduced into E. coli expression strain BL21 (DE3) RIL by electroporation to form expression strain BE273. Purification of His8-PduP under Denaturing and Nondenaturing Conditions For purification of PduP under denaturing conditions, E. coli strain BE273 was grown in 500 ml LB Kan (25 µg/ml) broth incubated at 37°C with shaking at 275 rpm in a 1-liter baffled Erlenmeyer flask. Cells were grown to an optical density of 0.6-0.8 at 600 nm and PduP production was induced by addition of IPTG to 1 mM. Cells were incubated for an additional 2 hours, and harvested by centrifugation at 6,690 x g for 10 minutes using a Beckman JLA-10.500 rotor. Two grams of cells (wet weight) were resuspended in 3 ml of 50 mM sodium phosphate, 300 mM NaCl, pH 7 and broken using
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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
Page 61 and 62: anti-rabbit IgG (γ-chain specific)
Page 63 and 64: 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: CoA-acylating propionaldehyde dehyd
Page 115 and 116: mM CHES (2-[N-cyclohexylamino]ethan
Page 117 and 118: 101 For the conjugation step, strai
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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