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Title: Cloning and analysis of the nupC gene of Escherichia coli, encoding a nucleoside permease
Author: Craig, Jane E.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1993
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This study reports the isolation of the nupC gene of E.coli from the Kohara ordered phage lambda library. Lambda clones carrying nupC were identified by their ability to restore nucleoside transport to a nucleoside transport deficient mutant by reciprocal exclusion. Subsequent subcloning, complementation and sequencing studies identified an ORF of 1,200bp which codes for a hydrophobic polypeptide of 43,560Da. A NupC-β-lactamase fusion protein was constructed and identification of the subcellular location of this hybrid confirmed the NupC protein to be membrane associated. Analysis of the nupC promoter region revealed the presence of at least two putative CRP binding sites, centred at -40bp and -89bp, which probably flank a CytR binding site. In addition, downstream from the nupC ORF an adjacent IS186 element was identified and found to reside within an underlying (potentially bidirectional) terminator structure. This arrangement is shown, by Southern blot analysis, to reflect the existing order in the chromosome of the E.coli strain W3110. The topological organisation of NupC within the cytoplasmic membrane has been analysed experimentally by construction of fusions to mature β-lactamase. From the combination of this data, with information from a computational hydropathy analysis, a two-dimensional model of NupC is presented which predicts that twelve transmembrane segments are likely to be present in this protein.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available