No description available.

No description available.

No description available.

In the fission yeast, <I>S. pombe</I> the centromeres have been extensively characterised, and consist of a largely unique central core, surrounded by a large inverted repeat. Previous studies have shown that marker genes inserted into these regions are transcriptionally silenced. Several genes have been isolated which are required for this transcriptional silencing. One of these, <I>swi6*</I>, has been shown to localise to the centromere <I>in vivo </I>and it thought to form part of the functional kinetochore. In this thesis I describe the first molecular characterisation of another gene, <I>clr4*</I> which is also required for centromeric silencing, and for localisation of Swi6p to the centromere. This gene and several of its alleles have been fully sequenced, and shown to contain both a chromo-domain and a SET domain. This pattern of domain organisation has previously been shown to occur in the <I>Drosophila </I>gene Suvar (3)9, indicating that the silencing processes in this organisms are similar to those seen in <I>S. pombe</I>. Four of the mutant alleles sequenced result from point mutations, and of these, three alter conserved residues in the SET domain. The <I>clr4* </I>gene was disrupted leaving only 38 amino acids at the 5' end of the native open reading frame intact. This disruption was shown to have a phenotype similar or identical to that of the originally isolated <I>clr40s5 </I>allele. I also describe the characterisation of a novel gene, <I>eval<SUP>+</SUP></I> which was isolated due to its effect on transcriptional silencing of reporter genes placed within the central core of the <I>S. pombe</I> centromeres. This gene has been fully sequenced and shown to contain a SANT domain. Possible functions for this gene in the cell are discussed.

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In the present study, the cold shock response of <i>S. typhimurium</i> was investigated using a small custom made DNA microarray which allowed the transcriptional profiling of <i>cspA</i> and its five other paralogous genes (<i>cspB, cspC, cspD, cspE</i> and <i>cspH</i>) after a temperature downshift to 10°C or 4°C from optimal growth at 37°C. Changes in low temperature expression of the six genes were monitored in exponential and stationary phase cells. Global transcriptional analysis of <i>S. typhimurium</i> was also performed using genomic microarrays which could measure gene expression for 97.5% of all the open reading frames in the <i>S. typhimurium </i>LT2 genome. This allowed expression data from other cold induced genes as well as the <i>cspA</i> paralogues to be examined under the same conditions used for the smaller array. The main findings from the array experiments showed that <i>cspA</i> and <i>cspB</i> appeared to be the most highly induced of the paralogues genes upon cold shock, with significant levels of transcription increase also being observed for <i>cspC </i>and <i>cspE</i> at low temperature and at 37°C. The <i>cspH</i> gene was confirmed as being strictly cold-induced whilst, in contrast, <i>cspD</i> only exhibited significant levels of induction at 37°C. The genomic microarray studies detected cold shock induction of many other genes involved in a variety of cellular activities linked to the maintenance of cell envelope integrity, DNA structure and pathogenicity, among others. The overall levels and timing of global transcription up/down regulation was observed to be influenced by growth phase and the degree to which <i>Salmonella </i>cells were cold-shocked, with gene induction also being detected at temperatures below the minimum for growth (4°C). these findings suggested that the cold shock response of this pathogen can still adapt the cell for survival under such conditions until a more favourable temperature arises for cell division. Extensive mutational analysis of the <i>csp</i> genes in <i>S. typhimurium </i> indicated that two strains, harbouring only a functional <i>cspA</i> gene or, more interestingly, the constitutively expressed <i>cspE</i> gene, were fully capable of adapting the cell for growth at 10°C and for colony formation at 15°C, to a level comparable to that of the wild-type strain. A mutant strain with no functional <i>csp</i> genes was incapable of growth at temperatures below 20°C and exhibited a reduction in viability with extended cold incubation.  Other phenotypic tests on <i>csp </i>mutants seemed to indicate that CspC and CspE were capable of directly or indirectly enhancing the viability of cells to freeze/thaw treatment and that CspE may also play a role in enhancing the survival of stationary phase <i>Salmonella </i>cells against oxidative stress.

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No description available.

An attempt has been made to develop the genetics of Micrococcus radiodurans by analysing transformation and conjugation processes. The nature of the transformation process in M. radiodurans was studied by investigating the effect of various factors on the efficiency of transformations. M. radiodurans can be transformed by exogenous DNA at a frequency of 10 without special treatment. The presence of glucose or amino acids during growth or transformation had no significant effect on the frequency. Treatment of recipient cells with calcium chloride at the optimum concentration of 30 mM increased the frequency of transformation up to 10,000-fold for some markers, e.g. rifampicin resistance. Magnesium and strontium ions could not replace calcium while sine ions completely inhibited transformation. The efficiency of transformation was also affected by the pH, pH 7.0 being the most effective with CaCl-treated cells. The minimum time required for phenotypic expression varied wiith different markers, e.g. for acriflavin resistance and streptomycin resistance it was 35 and 170 minutes respectively. To obtain the maximum number of transformants further incubation was necessary before the addition of antibiotics. This time also varied for different markers. The high frequency of transformation obtained using the new protocol enabled studies on genetic linkage to be started.

This study concerns the analysis of elements involved in the control over entry into mitosis in the fission yeast, <i>Schizosaccharomyces pombe</i>. The initial aim was to characterise the role of the <i>win1</i> gene in this control system. The <i>win1</i>.1 mutation shows a strong interaction with <i>wee1</i> and <i>cdc25</i>, genes which had previously been shown to play an important role in the control over entry into mitosis, probably acting through the <i>cdc2</i> protein kinase. The strategy for the cloning of <i>win1</i> was to isolate sequences capable of suppressing the temperature sensitive cdc phenotype arising from the combination of <i>win1.1</i> with <i>wee1.50</i> and <i>cdc25.22</i>. Following the extensive screening of gene libraries, it proved impossible to isolate <i>win1</i> using this approach, although five new genes were isolated as multicopy suppressors of this phenotype. None of these sequences correspond to any known mitotic control gene, and therefore identify new genes that affect the control of entry into mitosis. These were named <i>wis</i> (<i>wi</i>n <i>s</i>uppressing) 1 to 5. A molecular analysis was undertaken on the pwis plasmids, and the phenotypes of various cell cycle mutant strains containing the pwis plasmids were also examined. <i>wis1</i><SUP>+</SUP> was found to be capable of reducing the cell length at division in a dosage dependent manner, suggesting that <i>wis1</i> is involved in a rate limiting step controlling entry into mitosis. A null allele of <i>wis1</i> was constructed and found to result in large cells which have poor viability upon entry into stationary phase. DNA sequence analysis of <i>wis1</i> predicts a 605 amino acid gene product with a strong homology to serine/threonine protein kinases. Strains lacking in <i>wis1</i> function are still sensitive to levels of <i>wee1</i> and <i>cdc25</i> expression, suggesting that <i>wis1</i> acts upstream of these control elements. The interaction of <i>win1.1</i> with other cell cycle mutants was studied and the <i>win1</i> locus mapped. The cloning of the closely linked gene <i>tps19</i> could provide an alternative strategy for the isolation of <i>win1</i>. Both <i>win1.1</i> and a <i>wis1</i><SUP>-</SUP> allele were found to be capable of suppressing the hypersporulation phenotype of <i>pat1</i><SUP>ts</SUP> mutations, suggesting that the <i>win1</i> and <i>wis1</i> gene products may play a role in the regulation both of mitosis and meiosis.