Nucleosides are transported across the inner membrane of <i>E. coli</i> by at least two separate energy-driven systems known as NupC and NupG. These systems are encoded by the genes <i>nupC</i> and <i>nupG</i> respectively. CTN1 (a mammalian transporter isolated form the jejunum) is a homologue of NupC. CNT1 is able to transport the nucleoside analogue AZT, used to combat HIV infection. CNT1 and NupC share substrate specificity and they have amino acid sequence identity of 27%, particularly in their C-terminal region. With the completion of the <i>E. coli</i> genome project paralogues of NupC and NupG have been found, designated as YeiM and YeiJ, and XapB and YegT respectively. To functionally characterise the putative nucleoside transporter-encoding genes <i>xapB, yegT, yeiM </i>and <i>yeiJ,</i> mutants were constructed which the coding sequence of these genes had been deleted from their chromosome. From this work it was established that all four putative nucleoside transporters XapB, YegT, YeiM and YeiJ were able to transport adenosine in the assays at similar levels as the positive control. Furthermore in kinetic analysis of the transporters it was possible to determine Km values for the four transporters with adenosine as substrate, which were in the same order of magnitude as mammalian nucleoside transporters and that shown for xanthosine uptake by XapB. The mutant strains were used to construct a bioassay, in which their ability to transport nucleoside analogues was assessed. AZT was used as model substrate. The use of multiple mutants in experiments involving nucleoside uptake as a sole carbon source further established the fact that the collection of genes studied in this project was indeed nucleoside permeases. Preliminary experiments gave an insight in developing a high throughput bioassay which can be developed further in order to produce a useful tool for nucleoside analogue drugs assessment.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:660889 |
| Date | January 2006 |
| Creators | Ralli, Marianna |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/15665 |
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