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Lactobacillus leichmannii as a probe for the quantitation of vitamin B-12
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Cardy, Susan Mary (1989) Lactobacillus leichmannii as a probe for the quantitation of vitamin B-12. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3216264~S1
Abstract
Initial attempts to isolate the gene encoding Vitamin B12 receptor protein (jbtuB) from Lactobacillus leichmannii, resulted in the isolation of the JbtuB gene from E. coli, due to either a cross-over or gene exchange event. Complementation of an E. coli JbtuB mutant was demonstrated and expression analysis, using both in vivo and in vitro systems, revealed the cloned gene to encode a polypeptide with an apparent Mr of 66,400 as determined by PAGE. Nucleotide sequence data confirmed that the cloned gene was identical to the JbtuB gene from E. coli.
The initial 2.0 Kb Hindlll genomic DNA fragment from L. leichmannii, which exhibited homology to a synthetic oligonucleotide probe (derived from the btuB gene from E. coli), was re-cloned into an amplifiable high copy number vector in E. coli. The recombinant DNA was found to be stably maintained and had not undergone any physical rearrangements. Nucleotide sequence data revealed three putative open-reading frames, one of which encoded a protein which exhibited a degree of homology to the C-terminus of the Vitamin B., receptor protein (BtuB) from E. coli. The functions of the other two open-reading frames remain to be elucidated.
The B. 2 binding protein from L. leichmannii has been isolated and purified. It has an Mr of approximately 21,500 as determined by gel filtration. Polyclonal antibodies were raised to it for use in the identification of the desired gene product from the cloned L. leichmannii genomic fragment. Cross reactivity was found between, the antisera to the B12 binding protein from L. leichmannii and the B12 receptor protein (BtuB) from E. coli. The reverse case was found to be true also.
Transformation of L. leichmannii was achieved by electroporation. Vectors pSA3, pC194, pCKl, but not pAM01, transformed the organism to chloramphenicol resistance, albeit at low frequency.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QP Physiology Q Science > QR Microbiology |
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Library of Congress Subject Headings (LCSH): | Vitamin B12, Lactobacillus leichmannii, Escherichia coli, Polypeptides | ||||
Official Date: | 1989 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Biological Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Dow, Crawford S. | ||||
Sponsors: | Science and Engineering Research Council (Great Britain) | ||||
Extent: | xxvi, 279 leaves : charts | ||||
Language: | eng |
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