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

More documents

Recommendations

Info

46 is that ATR activity restores the ability of strain BE253 to convert CNCbl to AdoCbl, the required cofactor for diol dehydratase. Given AdoCbl, diol dehydratase converts 1,2-propanediol to propionaldehyde which reacts with components in the aldehyde indicator medium to form a red/brown precipitate (Johnson et al. 2001). Hence, we expected that BE253 would be useful for screening expression libraries for ATR activity. Accordingly, we attempted to identify a human ATR cDNA by transforming strain BE253 with a human liver cDNA expression library and screening the resultant colonies for red/brown color formation on AIM. Approximately 250,000 human cDNAs were screened, but no red/brown colonies were found. Subsequently, a bovine liver cDNA library was similarly screened and four positive clones were isolated. The DNA sequences of the four clones were determined and found to be identical. The clone was 1058 bp in length, and included a poly(A) tail indicating that its 3' end was complete. However, analyses indicated that the 5' end of the clone was incomplete. The clone included a 228-amino acid open reading frame that would be expressed as an N-terminal β-galactosidase fusion protein (as was expected given the cloning method employed), but it lacked the expected ATG triplet. Hence, the bovine clone apparently lacked a portion of its 5' end. After the DNA sequence of the bovine cDNA clones was determined, Blastp searches of the NCBI nonredundant database were conducted, and these searches showed that the 228-amino acid protein encoded by the bovine cDNA clone was 29% identical (Expect = 8 x 10 -15 ) to the N-terminal domain (165 amino acids) of the PduO ATR of S. enterica (Johnson et al. 2001). The findings that the bovine cDNA isolated both complemented an ATR-deficient mutant of S. enterica and encoded a protein with
sequence similarity to a known ATR enzyme suggested that the bovine clone also encoded an ATR enzyme. 47 Identification of a Human Gene Related to the Bovine Adenosyltransferase cDNA Blastp searches showed that the putative bovine ATR enzyme was 88% identical to a human protein encoded by IMAGE cDNA clone 2822202 (accession number BC005054). The human cDNA clone was 1128 bp in length and included an appropriate ATG triplet and a poly(A) tail indicating that it was a full-length clone. Further sequence analyses showed that the predicted protein encoded by the human cDNA was 250 amino acids in length and was homologous over its entire length to the putative bovine ATR except that it included 19 additional N-terminal amino acids (Figure 2-1). This suggested that the bovine clone was nearly full-length, lacking only a portion of its N-terminus, probably a small region of its MTS (see below). Perhaps more importantly, however, the high identity between the human and bovine sequences indicated that both encoded proteins with similar functions. Furthermore, sequence similarity searches showed human cDNA BC005054 encoded a protein with 26% amino acid identity the PduO ATR of S. enterica. Thus, these results tentatively identified sequence BC005054 as the human ATR cDNA. Subsequent sequence analyses showed human cDNA BC005054 was 99% identical to nine regions of human chromosome XII. The IMAGE clone BC005054 encoded a protein with 2 amino acid substitutions compared with the putative protein encoded by chromosome XII (Arg-19 to Gln and Met-239 M to Lys). These changes may represent natural polymorphisms or may have resulted from mutations introduced during cDNA preparation. The nine regions on chromosome XII were the only regions of the human genome that showed significant homology to human cDNA BC005054. Hence, it seems
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 and 54: or from mutations that impair the a
Page 55 and 56: 1989). 5-bromo-4-chloro-3-indolyl-
Page 57 and 58: 41 5’-GCCGCCGGTACCGATGACGACGACAAG
Page 59 and 60: 43 Growth of Adenosyltransferase Ex
Page 61: anti-rabbit IgG (γ-chain specific)
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
show all

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

Create successful ePaper yourself

Delete template?

Save as template?