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

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42 into the pET-41a expression vector. The BglII site was positioned such that the resulting clones would express the human ATRs as fusion proteins with N-terminal GST and His6 tags. Ligation, transformation and analysis of clones were performed as described above for the bovine expression clones. Clones with the expected DNA sequence were transformed into E. coli strain BL21 (DE3) RIL for high-level expression. Cloning the Human Adenosyltransferase Coding Sequence for Complementation Studies For complementation studies, the human ATR coding sequence was amplified by PCR and cloned into plasmid pLAC22 which allows IPTG-inducible expression in S. enterica (Warren et al. 2000). The primers used for amplification were forward, 5’-GCCGCCAGATCTTATGCCTCAGGGCGTGGAAGACGGG-3’ and reverse, 5’-GCCGCCAGCTTTCAGAGTCCCTCAGACTCGGCCG-3’. These primers eliminate the putative MTS and provide the needed ATG start triplet such that the first five amino acids of the expressed protein would be MPQGV. The PCR product was restricted with BamHI and HindIII and ligated into pLAC22 that had been digested with BglII and HindIII. Ligation mixtures were used to transform S. enterica strain BE253 (ATR-deficient) by electroporation. Transformants were selected on AIM supplemented with 1,2-propanediol and Amp. Use of this medium allowed the identification of clones expressing ATR activity. Pure cultures were prepared from selected transformants and plasmid DNA isolated from these strains was analyzed by restriction digestion and DNA sequencing. Clones having the expected DNA sequences were moved into strain BE266 (pduO cobA) via P22 transduction. Pure cultures prepared from the resultant colonies were determined to be phage-free by cross-streaking against P22 H5 and used for complementation studies.
43 Growth of Adenosyltransferase Expression Strains and Preparation of Cell Extracts The E. coli strains used for expression of the bovine and human ATRs were grown on LB supplemented with 25 µg/ml Kan at 37°C with shaking at 275 rpm in a New Brunswick Scientific shaker incubator. Cells were grown to an absorbance of 0.6-0.8 at 600 nm and protein expression was induced by the addition of 1 mM IPTG. Cells were incubated at 37°C with shaking at 275 rpm for an additional 3 hours. Cells were removed from the incubator, held on ice for 10 minutes, and then harvested by centrifugation at 6,690 x gmax for 10 minutes using a Beckman JLA-10.500 rotor. The cells were resuspended in 3 ml of 50 mM sodium phosphate, 300 mM NaCl, pH 7, and broken using French Pressure Cell (SLM Aminco, Urbana, IL) at 20,000 psi. Phenylmethylsulfonyl fluoride was added to the cell extract to a concentration of 100 µg/ml to inhibit proteases. The crude cell extract was centrifuged at 16,000 rpm (31,000 x gmax) for 30 minutes using a Beckman JA20 rotor to separate the soluble and insoluble fractions. The supernatant was decanted (soluble fraction), and the pellet was resuspended in 1 ml of 50 mM sodium phosphate, 300 mM NaCl, pH 7 (inclusion body fraction). Both fractions were used for the ATR assays. Growth Curves Cells to be used as inocula for growth curves were grown overnight at 37°C in 2 ml LB medium or LB medium supplemented with Amp at 100 µg/ml for strains carrying pLAC22. A portion of the LB culture (1.5 ml) was pelleted by centrifugation, and resuspended in 1 ml of minimal medium. Then resuspended cells (125 µl) were used as the inoculum. Cells were grown in 16 x 150-mm culture tubes containing 5 ml of minimal medium and incubated at 37°C in a New Brunswick model C-24 water bath with
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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
Page 7 and 8: General Molecular and Protein Metho
Page 9 and 10: LIST OF TABLES Table page 1-1. Aden
Page 11 and 12: 3-7. Stoichiometry of the MSR-ATR s
Page 13 and 14: DTT dithiothreitol E. coli Escheric
Page 15 and 16: Abstract of Dissertation Presented
Page 17 and 18: CHAPTER 1 INTRODUCTION History of C
Page 19 and 20: 3 (DMB). The DMB moiety is covalent
Page 21 and 22: 5 and deoxyadenosine. After hydroge
Page 23 and 24: 7 homocysteine recycling and methio
Page 25 and 26: methylmalonic acid. Methylmalonic a
Page 27 and 28: folate-dependent reactions includin
Page 29 and 30: tetanomorphum, P. shermanii, Euglen
Page 31 and 32: enzyme. This suggests that cob(II)a
Page 33 and 34: at the 5’-C of ATP by cob(I)alami
Page 35 and 36: iochemical studies and complementat
Page 37 and 38: 1997). Both cases of the cblD disor
Page 39 and 40: 23 The cblB complementation group i
Page 41 and 42: (Watkins et al. 2002). Some patient
Page 43 and 44: Chapter 2 of this dissertation repo
Page 45 and 46: Corrin Ring Dimethyl benzimidazole
Page 47 and 48: Classes Eliminases Dehydratases OH
Page 49 and 50: A) B) CH 3THF 33 MS-cob(I)alamin Me
Page 51 and 52: propanol dehydrogenase (PduQ) propa
Page 53 and 54: or from mutations that impair the a
Page 55 and 56: 1989). 5-bromo-4-chloro-3-indolyl-
Page 57: 41 5’-GCCGCCGGTACCGATGACGACGACAAG
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
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al. 1988). The aldehyde is then dis
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CoA-acylating propionaldehyde dehyd
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97 previously published DNA sequenc
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mM CHES (2-[N-cyclohexylamino]ethan
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101 For the conjugation step, strai
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103 from New England Biolabs (Bever
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105 Propionaldehyde Dehydrogenase A
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107 fraction. Following centrifugat
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109 antiserum obtained was specific
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111 biochemical evidence was presen
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113 Table 4-1. Bacterial strains Sp
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115 Table 4-3. Propionaldehyde dehy
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kDa 209 80 49 35 29 117 1 2 3 4 Fig
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CHAPTER 5 CONCLUSIONS In humans, co
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121 substitutions that are common p
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123 Studies also indicated that hum
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LIST OF REFERENCES Aberg, A., S. Ha
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127 Bradford, M. 1976. A rapid and
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129 Fenton, W. A. and L. E. Rosenbe
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131 Johnson, C. L. V. J., E. Pechon
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133 Mellman, I., H. F. Willard and
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135 Rossi, M., J. P. Glusker, L. Ra
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137 Vlasie, M., S. Chowdhury and R.
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BIOGRAPHICAL SKETCH Nicole Aurora L
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