Every cell produced by the bacterial growth and division cycle must have a chromosome to be viable. Partitioning is the process which ensures this. In this work, the polyploid nature of spherical <I>Escherichia coli</I> mutants was utilised to carry out a series of experiments designed to reveal the underlying nature of the partitioning process. The experiments show that partitioning in <I>E.coli</I> is antimitotic, that is, it actively ensures that the inevitable homozygosity of a normally unichromosomal cell is maintained, even when that cell is multichromosomal. That this was shown in spherical cells also reveals that the process is an innate property of the relationship between partitioning and division, rather than a simple consequence of separating two large chromosomes in a narrow, rod-shaped cell. This subtle, but important definition of what the partitioning process actually achieves, should be useful in further characterisation of the mechanisms involved. Grouping of genes of similar function is a common feature of the <I>E.coli</I> chromosome. At least five clusters of genes involved in cell-wall synthesis, cell division and cell shape maintenance have been identified. The <I>mrd</I>-cluster of genes contains three implicated in morphological aspects of peptidoglycan synthesis. The presence of four uncharacterised genes and a portion of unsequenced DNA in the same region, together with proposals that the region could contain more genes involved in cell-morphology, led to the work presented here. Sequencing, followed by computer analysis and molecular-biological and genetic characterisation provided information about the transcriptional organisation of the region and the function of some of the genes. Also a new mutation affecting cell division was identified in an adjacent region of the chromosome.

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The <i>LPD1</i> gene of <i>Saccharomyces cerevisiae</i> encoding lipoamide dehydrogenase (LPDH) has been shown to be subject to the general control of amino acid biosynthesis mediated via the <i>CGN4</i> gene product. It is subject to catabolite repression and was shown to require the <i>HAP2, HAP3</i> andf <i>HAP4</i> gene products for release from glucose repression. The gene also appears to contain a carbon source-regulated transcriptional enhancer that lies 3' to the translational start site. A defined set of isogenic yeast strains was constructed in which each strain contained a different <i>LPD1-lacZ</i> gene fusion integrated at the <i>ura3</i> locus. These <i>LPD1-lacZ</i> fusions differed in the amount of <i>LPD1</i> gene fused to the <i>lacZ</i> reporter. Comparison of the β-galactosidase activities of each strain during growth on glucose or ethanol revealed that part of the <i>LPD1</i> coding region activates gene expression in a carbon source dependent manner. This activation occurred at the mRNA level and was not mediated by changes in mRNA stability. The 3' sequence of the <i>LPD1</i> gene contains motifs homologous to the DNA binding elements of the ABF1 and RAP1 proteins and a sequence homologous to the CDE1 element. These motifs may represent potential candidates for the <i>LPD1</i> 3' enhancer function. The <i>LPD1</i> gene promoter contains three motifs which show strong homology to the core HAP2/3/4 binding motif. LPDH activities in wild-type and <i>hap2</i> mutant strains were expressed similarly at basal levels when grown on glucose. However, LPDH activity in the wild-type was derepressed 4-fold in raffinose medium but remained at near basal levels (as seen on glucose) in the <i>hap2</i> mutant grown in similar conditions. Transcript analysis in wild-type and <i>hap2</i> mutants confirmed that the HAP2 protein regulates <i>LPD1</i> expression at the level of transcription in the same way as the <i>CYC1</i> gene. Similar studies (performed by others) comparing LPDH activities and <i>LPD1</i> gene transcription (assessed by constructing <i>hap</i> mutant strains carrying a single copy of the <i>LPD1</i> promoter fused in frame to the <i>lacZ</i> reporter gene integrated at the <i>ura3</i> locus) indicated that transcription of <i>LPD1</i> required HASP2, HAP3 and HPA4 for derepression on non-fermentative substrates. The <i>LPD1</i> gene promoter contains three matches to the consensus for control mediated by the GCN4 protein. Gel retardation analysis (performed by others) using <i>in-vitro</i> synthesized GCN4 protein revealed DNA:GCN4 complexes at two of the consensus motifs. When cells were grown on raffinose as a carbon substrate to partially relieve catabolite repression of the gene, levels of LPDH were derepressed about 2-fold in wild-type cells limited for histidine synthesis by the presence of 3-amino-1,2,4- triazole; this derepression did not occur in a <i>gcn4</i> mutant strain. Transcript analysis indicated that amino acid starvation affected levels of the <i>LPD1</i> transcript. Kinetic analysis indicated that subjecting cells to a sudden decrease in the availability of amino acids led to a marked increase in transcript levels within 30min, and that these continued to increase at a slower rate up to 6 hours after imposition of amino acid starvation. This differed from the response of <i>HIS3</i> gene transcripts which reached peak levels betwen 30 min and 1 h, and then declined gradually.

The primary objective of this work was the isolation and characterisation of gene products which interacted with Mts2p. This was achieved by the use of the yeast '2-hybrid' screen, and enabled isolation of a previously identified <I>S. pombe</I> gene <I>let1</I><SUP>+</SUP>, which is homologous to a gene from the budding yeast <I>S. cerevisiae</I>. The product of this homologue, <I>SUG1,</I> also a member of the AAA family, was thought to be in transcription or transcriptional regulation. Characterisation of the phenotype resulting from a disruption of <I>let1</I><SUP>+<I> </I></SUP>suggested that like <I>mts2</I><SUP>+</SUP>, it encoded a subunit of the 26S proteasome. Further evidence is presented in favour of this argument. In addition to <I>let1</I><SUP>+</SUP>, a novel <I>S. pombe </I>gene <I>aps1</I><SUP>+</SUP> was located. <I>Aps1</I><SUP>+</SUP> encodes the <I>S. pombe</I> homologue of the mouse MSS1 gene, which was originally isolated as a suppressor of a mutation in a yeast genes encoding a protein kinase. In this laboratory, however, MSS1 was isolated as a multicopy suppressor of the <I>ts</I> phenotype of <I>mts2-1</I>. The interactions between Mts2p, Let1P and Aps1p, the products of <I>mts2</I><SUP>+</SUP>, <I>let1</I><SUP>+ </SUP>and <I>aps1</I><SUP>+<I> </I></SUP>respectively, were studied using the yeast 2-hybrid<I><SUP> </SUP></I>system. The region of interaction between Let1p and Mts2p were defined, and the results suggest that, as is the case for two other ATPase subunits of the 26S proteasome, the N-terminus of each protein is important in mediating this interaction. In a separate screen to look for genes which were involved in the enhancement of position effect variegation at the centromere, 4 cold sensitive (<I>cs</I>) alleles of <I>mts2</I> were isolated. Mutation analysis was performed on these and on the three <I>ts</I> alleles of <I>mts2, </I>which had been isolated in the original drug resistance screen. All of the mutations lie in a 230 amino acid region which is highly conserved between all members of the AAA protein family. The phenotype of all of these mutants was studied with respect to morphology, DNA content and MBC resistance. The results indicate that the three <I>ts </I>alleles are more drug resistant and have a greater morphological deformation than the <I>cs</I> alleles.

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