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

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studies, it was also shown that a Salmonella pduO cobA double mutant lacked 38 AdoCbl-dependent diol dehydratase activity due to the ATR deficiency. Furthermore, it was found that a plasmid-encoded source of ATR enzyme restored diol dehydratase activity to a cobA pduO double mutant (Johnson et al. 2001). Because this enzymatic activity can be readily detected on aldehyde indicator medium (AIM), we expected that the ATR-deficient Salmonella strain could be used to screen expression libraries for cDNAs that encode ATR enzymes. Such an approach would circumvent the difficulties associated with ATR purification. Here, we report the isolation of an ATR cDNA, from a bovine liver library, by complementation of an ATR-deficient Salmonella mutant for color formation on aldehyde indicator medium. Subsequently, sequence similarity searching identified the homologous human cDNA and its corresponding gene. Both the human and bovine cDNAs are shown to express ATR activity and complement an ATR-deficient bacterial mutant. In addition, Western blots were used to show that expression of the human ATR was altered in cell lines derived from three cblB patients compared with a cell line derived from a normal individual. We propose that the human gene identified here coincides with the cblB complementation group, defects in which lead to methylmalonic aciduria. The identification of genes involved in methylmalonic aciduria is important for the development of improved methods for the diagnoses and treatment of this devastating disorder. Chemicals and Reagents Materials and Methods CNCbl and HOCbl were purchased from Sigma Chemical Company (St. Louis, MO). Titanium (III) citrate was prepared as previously described (Bobik and Wolfe
1989). 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) and isopropyl-β-D-thiogalactopyranoside (IPTG) were from Diagnostic Chemicals Ltd. 39 (Charlottetown, Prince Edward Island, Canada). Restriction enzymes and T4 DNA ligase were from New England Biolabs (Beverly, MA). Other chemicals were from Fisher Scientific (Norcross, GA). Bacterial Strains and Growth Media Bacterial strains used in this study are listed in Table 2-1. The minimal medium used was NCE (Vogel and Bonner 1956, Berkowitz et al. 1968) supplemented with 0.4% 1,2-propanediol, 200 ng/ml of CNCbl, 1 mM MgSO4, 0.3 mM each of valine, isoleucine, leucine, and threonine. LB medium was the rich medium used (Difco Laboratories, Detroit, MI) (Miller 1972). MacConkey and aldehyde indicator media were supplemented with 1,2-propanediol and CNCbl and prepared as previously described (Johnson et al. 2001). General Protein and Molecular Methods and ATR Assays Bacterial transformation, polymerase chain reaction (PCR), restriction enzyme digests, and other standard molecular and protein methods were performed as previously described (Maniatis et al. 1982, Johnson et al. 2001). ATR assays were also performed as previously reported (Johnson et al. 2001). P22 Transductions Transductional crosses were performed as described previously (Davis et al. 1980). In preparing lysates of galE mutant strains, overnight cultures were grown on LB medium supplemented with 0.2% galactose and 0.2% glucose with the addition of appropriate antibiotics. A P22 HT105/1 int-201 phage was used for transductional crosses at a concentration of 2x10 8 phage/ml (Schmieger 1971).
Page 1 and 2:
B12 METABOLISM IN HUMANS By NICOLE
Page 3 and 4: The work in this dissertation is de
Page 5 and 6: 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: or from mutations that impair the a
Page 57 and 58: 41 5’-GCCGCCGGTACCGATGACGACGACAAG
Page 59 and 60: 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 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 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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