In the course of work aimed at discovering genes encoding novel sigma subunits of <I>Escherichia coli </I>RNA polymerase, two unknown open reading frames were identified by hybridisation of an <I>E. coli </I>genome library with synthetic oligonucleotide probes directed against the conserved subregion 2.1 of bacterial sigma factors. This thesis describes the mapping, sequencing, and characterisation of these genes. The open reading frame <I>f229</I> was located near 4324kb on the physical map of the <I>E. coli</I> K12 chromosome. Its sequence and that of a downstream Bacterial Interspersed Mosaic Element (BIME) were determined. Transcription of <I>f229</I> was not detectable under normal growth conditions, nor was it found to be inducible by various environmental shocks. However, <I>f229 </I>mRNA levels increased dramatically following alteration of the BIME structure, either by complete removal or by a deletion leaving just a single Palindromic Unit. A protein encoded by <I>f229 </I>was identified and had the expected molecular size of 25kDa, but was found to be poorly expressed even in the absence of the BIME. Moreover, an <I>f229::kanR </I>mutation did not affect growth of <I>E. coli</I> under a variety of conditions. Since the predicted amino acid sequence of F229 has no identify with any known protein and no similarity to members of either of the two known sigma families, the function of <I>f229</I> remains unknown. The downstream portion of a second open reading frame was located near 381kb on the physical map. The amino acid sequence has very high identity (80% range) with a family of <I>Pseudomonas </I>enzymes, the 4-hydroxy-2-oxovalerate aldolases. These participate in a metabolic pathway that converts a variety of aromatic compounds into Krebs Cycle intermediates. Since <I>E. coli</I> is known to carry out similar catabolic reactions and the corresponding genes in <I>Pseudomonas </I>are clustered into operons, it is likely that the unsequenced region upstream of this open reading frame contains further genes involved in aromatic degradation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:663609 |
| Date | January 1996 |
| Creators | Wedgwood, Stephen |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13220 |
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