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Title: Molecular biology and enzymology of the hem C locus of Escherichia coli K-12
Author: Thomas, Steven David
ISNI:       0000 0001 3526 5912
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 1986
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The hem C locus of E. coli K12 encoding PBG deaminase was mapped on chromosomal DNA maintained within plasmids from the collection of Carbon and Clarke. The locus was sub-cloned as a 2.89 kb Sau3A fragment from one of the plasmids pLC41-4 into the BamHI site of pBR322. E. coli HB101 when transformed with the hybrid plasmid, designated pST46, exhibited 18 x the PBG deaminase activity of the parental strain. Selection of the recombinant plasmid harbouring hem C was by use of a novel fluorescence assay. Restriction endonuclease analysis of pST46 revealed the 2.89 kb insert was derived from a sequence adjacent to the cyaA locus on pLC41-4, as predicted from the results of comparative restriction endonuclease analysis of pLC41-4 with other plasmids harbouring the cyaA locus. The hem C locus was further sub-cloned into pBR322 as a 1.68 kb BAmHI-Sa mid I fragment. Two types of over-producing strains were obtained from the ligation and their plasmids were characterised. Both plasmids pST47 and pST48 carried the 1.68 kb BamHI-Sa mid I insert but further comparison revealed a 1.35 kb pBR322 derived sequence had been deleted from pST48 in the ligation procedure. This deletion was mapped and was shown to coincide with a gene encoding a DNA replication control protein. Enzyme activities in strains transformed with pST48 were increased as a result. The hem C locus was sequenced on both coding and non-coding strands using the methods of Sanger and Messing and necessitated the synthesis of eight oligonucleotide primers which were assembled using protected phosphoramidite nucleoside monomers. Examination of the sequence obtained revealed a large open reading frame preceded by the control sequences necessary for initiation of transcription and translation. Purification and N-terminal sequencing of E. coli PBG deaminase from the over-producing strain ST1048 identified the open reading frame as the hem C structural gene. Evidence for co-ordinate expression of protein from the hem C promoter was presented. Finally, the intergenic sequence between cyaA and hem C structural genes was defined and was found to be 387 base pairs. This information redefined the relative positions of both loci on the E. coli linkage map.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry