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.

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A method of integrating large data sets of <i>S. cerevisiae</i> was developed here to select novel genes that possess characteristics implying their involvement in DNA processing. Form this method 54 genes were selected and mutants of these genes were screened for phenotype abnormalities associated with both meiotic and mitotic DNA processing. Eleven mutants were found to be sensitive to hydroxyurea (<i>Δsoh1, Δrtt101, Δfyv6, Δhur1Δ/pmr1, Δbre5, Δmms22, Δbrel, Δvid21, Δsgf73, Δrmd11, Δdef1, </i>and <i>Δlgel; </i>20.4%) and two (<i>Δdef1 </i>and <i>Δmms</i>22; 3.7%) were found to be sensitive to methyl methanesulfonate during vegetative growth. Six mutants were found to have low levels of nuclear division during meiosis (<i>Δbre1, Δvid21, Δsg73, Δarmd11, Δdef1, </i>and <i>Δlgel; </i>11.1%), four mutants displayed reductions in heteroallelic recombination in meiosis (<i>Δsoh1, Δbre5, Δhda2 </i>and <i>Δygl250w</i>; 7.4%) and higher levels of nondisjunction in meiosis was observed in two mutants (<i>Δsoh1, </i>and <i>Δyp1107</i>; 3.7%). Genes that were required for normal meiotic nuclear division (MND genes) were further assessed by analysing pre-meiotic DNA replication. One mutant (<i>Δvid21)</i> failed to initiate pre-meiotic DNA replication ; the other five mutants (<i>Δbre1, Δsgf73, Δrmd11, Δdef1, </i>and <i>Δlge1) </i> were observed to have a delay in entry and lengthened time to complete pre-meiotic DNA replication. From cytological analysis of the MND mutants able to undergo pre-meiotic DNA replication it was shown that a <i>Δdef1 </i>strain homologous chromosomes associate during meiosis I but chromosome synapsis is abnormal. Finally a mutation that interrupts <i>PMR1</i> was shown to over-replicate its genome during meiosis and form asci with greater then the normal four spores that are mostly inviable.

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The basis of my research has been to identify and characterise the function of ‘<i>E. coli</i> specific’ genes.  <i>E. coli</i> specific genes were identified after comparing 33 gamma proteobacterial genomes at the Microbial Genome Database (MBGD). The database was used to investigate the change in the number of <i>E. coli</i> specific genes since the completion of the first <i>E. coli</i> genome in 1997. Forty nine selected <i>E. coli </i>specific genes were deleted in 38 separate deletion events on the genome of the model <i>E. coli </i>K-12 MG1655 using the modified pKO3 deletion procedure. Gene expression levels and patterns in various phases of growth in LB broth were measured and are reported here.  Mutant and parent strains were tested for growth on a variety of conditions in agar based media. A mutant of the <i>yigE</i> ORF was found to be sensitive to high concentrations of nickel and cobalt in the growth medium. Mutation of the <i>E. coli</i> specific <i>htrC</i> gene showed no temperature sensitivity as reported in published literature and is shown here to play no part in the heat shock response of <i>E. coli. </i>The chromosomal position of the acetate utilisation gene <i>ackB</i> is experimentally demonstrated to be close to the known acetate kinase gene <i>ackA</i> at 50 minutes on the K-12 chromosome. Mutations in ORFs <i>yceP, ydeK </i>and <i>ygjMN</i> made the mutants sensitive to high levels of the basic dye gentian violet. This study shows that the group of genes specific to <i>E. coli</i> targeted here are expressed, show different patterns of expression and some are phenotypically functional. This study draws attention to a fraction of genes whose functional contribution to <i>E. coli</i> may have been underestimated due to their poor conservation in genomes other than <i>E. coli.</i>

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The <i>pcnB </i>gene encodes poly(A) polymerase I (PAP I), the major <i>Escherichia coli</i> poly(A) polymerase involved in mRNA processing, and the decay of small plasmid copy number controls RNAs. Prior to the study presented here, the transcriptional and translational organisation of the <i>pcnB</i> region was ill-defined. In this work, the <i>pcnB</i> promoter, identified by sub-cloning and primer extension analysis, was found to resemble closely a consensus s⁷⁰ promoter in both sequence and activity. Operon fusions of <i>pcnB </i>with <i>lacZ</i> are only slightly derepressed in a D<i>pcnB</i> background, providing little evidence of autoregulation at the level of transcription. Translation of the <i>pcnB</i> message is shown, by site-directed mutagenesis, to commence from the ribonucleotides AUU, a triplet that is very rarely used as an initiation codon. Furthermore, it was demonstrated that at the level of translation, the <i>in vivo </i>expression of <i>pcnB</i> is specifically subject to negative regulation, over a four-fold range, by Initiation Factor-3 (IF-3), whose own translation is also initiated from an AUU codon. Additional tests showed that a single chromosomal copy of wild-type <i>infC </i>(which encodes IF-3) can induce maximal IF-3-mediated repression of <i>pcnB</i>. Apart from the Shine-Dalgarno region, <i>pcnB</i> appears to lack any of the known sequence determinants encoded by <i>infC</i> that are believed to facilitate the use of AUU as an initiation codon. Several <i>pcnB</i> homologues are identified from other eubacteria that potentially could utilise an AUU triplet as a translational initiation codon. A conserved run of T residues (five to seven nucleotides long) exactly nine nucleotides downstream from the -10 region of these homologues suggested that the expression of <i>pcnB</i> might be subject to regulation by pyrimidine-sensitive selection of transcriptional start sites, coupled with UTP-dependent reiterative transcription. This possibility is investigated.

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