On the basis of biochemical studies pyruvate kinase has been identified as a key regulatory enzyme in a number of organisms, however, there is little genetic evidence to substantiate this. In Aspergillus nidulans pyruvate kinase activity is high following growth on glycolytic carbon sources and low following growth on gluconeogenic carbon sources suggesting the enzyme is heavily regulated. This seemed an excellent model system to study the role and regulation of a key enzyme of carbon metabolism and a study was therefore undertaken involving the isolation of mutants of A.nidulans lacking pyruvate kinase activity and of strains producing pyruvate kinase constitutively. Following mutagenesis and filtration enrichment a number of mutants were isolated which failed to grow on glycolytic carbon sources but grew normally on gluconeogenic carbon sources. Ten such mutants lacked pyruvate kinase activity and identify a single gene locus, pkiA (chromosome V), probably the structural gene for the enzyme. The pyruvate kinase deficient pkiA mutants, though able to grow on gluconeogenic carbon sources, are poisoned by the addition of a glycolytic carbon source such as glucose or fructose, probably due to accumulation of a toxic metabolic product. A number of fructose-insensitive revertants of the pkiA1 strain were selected in an attempt to isolate strains defective in sugar transport. Amongst these revertants, two were shown to have altered sugar uptake characteristics, demonstrating that the reversion technique described is capable of generating bona fide sugar uptake mutants in A.nidulans. One interesting observation made during this work was that the presence of agar in growth media can generate spurious results. This was observed first for a number of acetate non-utilising (acu) mutants whose growth on a number of carbon sources was impaired by agar. It was subsequently demonstrated that A.nidulans can use agar as sole carbon source by the pathway of C2 metabolism.

No description available.

The availability of conditional-lethal yeast (Saccharomyces cerevisiae) mutants that are defective in the process of pre-mRNA splicing (precursor /?NA processing; prp) has greatly facilitated the characterisation of components of the splicing machinery. When this project was initiated nine prp complementation groups had been defined (prp2-prp]l), all of which accumulate pre-mRNA at the expense of mRNA when incubated at the non-permissive temperature. Pre-mRNA splicing is a complex and dynamic process relying on many gene products for its completion and therefore many more completion groups remained to be identified. Determination of a prp phenotype has relied on the direct measurement of protein to RNA ratios and on the Northern blot analysis of conditional-lethal mutants incubated at the restrictive temperature. Such analyses are both time-consuming and labour-intensive. In this thesis I present the development of a novel screening procedure which positively identifies prp mutants. I have fused a yeast intron-conlaining gene to the lacZ gene of E.coli such that only the pre-mRNA generated from this fusion can encode an active ^-galactosidase fusion-protein. This gene-fusion has been introduced into a prp2 strain and the encoded pre-mRNA has been shown to accumulate on incubation at the non-permissive temperature. This pre-mRNA accumulation results in an increase in /9-galactosidasc activity. Exploiting this observation a simple plate assay has been developed and used to screen a pool of temperature-sensitive mutants for defects in pre-mRNA splicing. A number of prp mutants have been identified and I present the results of their initial characterisation.

Regulation of gene expression in the human pathogen Neisseria meningitidis remains poorly understood. The meningococcus is a good model for a global analysis of transcriptional regulation as it has fewer transcriptional regulators and proteins able to modulate gene expression compared to other species with genomes of similar size. The genes encoding proteins predicted to modulate transcription in the meningococcus were identified using a dedicated database for systematical functional analysis (NeMeSys) in this species and mutated by utilising an improved in vitro transposon mutagenesis system. The resulting mutants were subjected to phenotypic analysis for growth and functions linked to one of the major virulence factors of the meningococcus, Type-four pili (Tfp). Tfp are essential for adhesion, aggregation, twitching motility and DNA competence in pathogenic Neisseria species. However, not much is known about the expression of the 16 proteins essential for Tfp biogenesis, and the seven proteins playing important roles in Tfp biology. The mutants were assessed for the Tfp-dependent phenotypes: aggregation, adhesion to human cells and twitching motility. No mutants were found to be dramatically impaired for these properties. However, two transcriptional regulators, HexR (NMV_1005) and FarR (NMV_2033), were found to influence the aggregative abilities of the meningococcus, with mutations in these genes resulting in a slow aggregating, and consequently, slow adhering phenotype. In parallel, two transcriptional regulators were chosen for further characterisation because they are encoded on genomic islands absent in the nonpathogenic Neisseria strain, Neisseria lactamica and could thus contribute to virulence. One island encoded an AraC type regulator divergently transcribed from genes encoding a putative TonB-dependent iron uptake system, whilst the other island encoded a putative LysR type regulator divergently transcribed from a putative transporter. These regulators are likely to require specific inducer molecules that may be absent from experiments performed in vitro.

To understand the rates and mechanisms of Neisseria gonorrhoeae opacity (opa) gene variation, the 11 opa genes were amplified independently so that an opa allelic profile could be defined for any isolate from the sequences at each locus. Initial examination of 14 unrelated gonococcal isolates showed that no opa alleles were shared. Analysis of closely related isolates showed these typically shared most opa alleles and so the mechanisms by which recent changes occurred at individual opa loci could be determined. The great majority of changes were due to recombination among existing alleles that either duplicated an opa allele present at another locus or resulted in a mosaic of existing opa alleles. Single nucleotide changes or the insertion/deletion of a single codon also occurred, but few of these events were assigned to mutation, the majority being assigned to localised recombination. Introduction of novel opa genes from co-infecting strains was rare and all but one occurred in the same sexual network. Changes at the eleventh opa locus (opa11) occurred much more rapidly than at other opa loci, almost always differing even between recent sexual contacts. Examination of the neighbouring pilE gene showed that changes at opa11 and pilE often occurred together, although this linkage may not be a causal one. The Opa protein sequences encoded by the opa genes were determined and the regions spanning the two hyper-variable regions (HVR1 and HVR2) were analysed. An almost equal number of Opa protein and opa nucleotide sequences were detected but there was a limited number of HVR combinations, indicating that a large proportion of differences are located elsewhere in the protein. Very few novel HVRs were generated by recombination, even at opa11 where the rate of variation was greatest. Most changes resulted in re-assortment of existing HVRs, most likely due to protein function restriction/benefits.

No description available.

No description available.

No description available.

No description available.

No description available.