No description available.

The synthesis of ribosomes is a major metabolic pathway in all cells. In eukaryotes, a polycistronic pre-ribosomal RNA transcript is processed to the mature 18S, 5.8S and 25S/28S rRNAs, whilst undergoing extensive covalent nucleotide modifications and assembling with the ~80 ribosomal proteins. More than 140 known factors are required for post-transcriptional steps in ribosome synthesis in yeast and, although the processing of ribosomal RNA precursors is fairly well understood, the relationship between ribosomal processing, ribosome assembly, subunit export and subnucleolar structures is still largely unclear. To gain insights into these processes, I analysed three previously uncharacterised ribosomal factors. To study their involvement in the ribosomal RNA processing and assembly, conditional alleles were generated of the essential genes <i>NOP7, RRP12 </i>and <i>NOP15</i>, as well as epitope-tagged versions of their protein products. The nucleolar protein Nop7p has multiple roles in the synthesis of the 60S ribosomal subunit and is required for efficient 5’ to 3’ exonuclease digestion that generates the 5’ end of the major, short form of the 5.8S rRNA, assembly and export of the 60S ribosomal subunit. Nop15p is also needed for normal 5’ to 3’ exonuclease digestion and, additionally, for processing of residual 27SB to 7S pre-rRNA. Nop15p Contains an RNA recognition domain and is therefore likely to associate directly with the pre-rRNA to facilitate its processing. Rrp12p is the only <i>trans</i>-acting factor known to be required during late maturation of both ribosomal subunits. The protein appears to fulfil very different roles in the different subunits, as assembly factor and facilitator of exosome activity in the large subunit, and during export of the small subunit. Rrp12p is predicted to be composed of HEAT-repeats and is therefore structurally related to importin β-like family of export and import receptors. Several ribosomal processing factors, including Nop7 and Nop15p, seem to be multifunctional with roles in different cellular processes suggesting that major metabolic processes within the cell may be linked.

Mutations in the fission yeast genes mts5-1 and mbc]-1 were isolated in a screen for Schizosaccharomyces pombe (S.pombe) mutants that are both resistant to the microtubule destabilising drug methybenzylcarbamylate (MBCR) and temperature sensitive (t.s.) for growth. This screen has so far been specific for mutations in genes encoding subunits of the 26S proteasome (Gordon et al., 1993). This study shows that these strains contain mutations in the pad1 and crm] genes respectively. Crm] and pad] have previously been shown to be positive and negative regulators respectively of the AP-1 transcription factor Papi (Toda et al., 1992, Shimanuki et al., 1995), the S. pombe homologue of the mammalian AP-1 transcription factors fos and fun, which is involved in the transcription of multidrug resistance genes (Shimanuki et al., 1995). The mts5-1 (pad]-]) strain has a metaphase arrest phenotype and an increased level of high molecular weight ubiquitinated proteins when incubated at the restrictive temperature. This is identical to the mts2-1 (Gordon et al., 1993) and mts3-1 (Gordon et al., 1996) mutants isolated in the same screen and which have been shown to encode subunits of the 26S proteasome. This study reclassifies pad]' as a subunit of the 26S proteasome, 'and data is provided which shows genetic interactions between Padi and three other subunits of the 26S proteasome, Mts3 (Gordon et al., 1996), Mts4 (Wilkinson et al., 1997) and Pus (C. Wilkinson pers. comm.). A putative function for the 19S cap subunit Padi as an isopeptidase is also investigated. Crml has been implicated in MDR through Papi, since Papi is responsible for the transcription of genes involved in resistance to a wide variety of drugs. 26S proteasome mutants are also shown to be resistant to the same range of drugs as the mbcl-] (cnn]-I) mutant, but to a lower level, and that papli\ cells are sensitive to MBC (MBCS). 26S proteasome mutants are shown to have elevated levels of Papi when incubated at the permissive temperature indicating that this protein is not being degraded as efficiently as in wild type cells. A c.s. cnn] mutant has been shown to over-express a non-essential 25KDa protein that has been shown to be a downstream target of Papi (Adachi and Yanagida, 1989). This protein is also shown to be over expressed in S.pombe proteasome mutants and cnn]-1. paplA 26S proteasome double mutants are t.s. and MBCS. This is consistent with the 26S proteasome being involved in the degradation of Papi and hence involved in pleiotropic multi-drug resistance.

The polo-like kinases are a family of highly conserved serine/threonine protein kinases with multiple mitotic functions. Polo-like kinases are recognisable by the presence of two conserved domains: a catalytic, kinase domain at the amino terminus of the protein and at the carboxy terminus, a non-catalytic domain. In the fission yeast, <i>Schizosaccharomyces pombe</i>, there is one member of this family, Plol. The aim of this study was to define the function of the non-catalytic domain of the protein and to identify unknown mitotic roles for Plol. Site directed mutagenesis of Plol illustrated the roles of the two conserved domains in different aspects of the Plol function. Localisation, regulation of kinase activity and <i>in vivo</i> function of mutant proteins were analysed. Two hybrid analysis was carried out to identify interacting proteins. This was successful in revealing previously uncharacterised functions for Plol such as an interaction with the anaphase promoting complex subunit, Cut23. Reverse two hybrid screening combined with random mutagenesis was carried out to determine the <i>in vivo</i> consequences of eliminating specific interactions.

Flavocytochrome <I>b<SUB>2</SUB></I> from <I>Saccharomyces cerevisiae</I> is located in the mitochondrial intermembrane space where it catalyses the oxidation of L-lactate to pyruvate with concomitant electron transfer to cytochrome <I>c.</I> The enzyme is a homotetramer, each protomer having two distrinct domains; one of which binds flavin mononucleotide (FMN) and the other protohaem IX. The domains are connected by a region of polypeptide which constitutes the interdomain hinge. The pathway of electron transfer is from L-lactate to FMN to haem to cytochrome <I>c.</I> The work described in this thesis focuses on the latter two steps on the catalytic cycle, namely intramolecular electron transfer from FMN to haem and intermolecular electron transfer from flavocytochrome <I>b<SUB>2</SUB></I> to cytochrome <I>c. </I>Intramolecular electron transfer has been investigated by the generation of interspecies hybrid enzymes. These include hinge-swap<I>b<SUB>2</SUB></I>, in which the hinge region of the <I>S.cerevisiae </I>enzyme has been replaced with that from <I>Hansenula anomala</I>; and domain-swap-<I>b<SUB>2</SUB></I>, which comprises the flavodehydrogenase domain of <I>H.anomala</I> with the hinge region and haem domain from <I>S.cerevisiae.</I> For both hinge-swap enzymes, the most significant effect was a dramatic decrease in the rate of haem reduction, the rate constant for this decreases from 445<SUB>s</SUB>-<SUP>1</SUP> to 1.61<SUB>s</SUB>-<SUP>1</SUP> for the hinge-swap enzyme and to 0.99 <SUB>s</SUB>-<SUP>1</SUP> for domain swap-<I>b<SUB>2</SUB></I>. This indicates that flavin to haem electron transfer is severely affected in both hybrids, to the extent that this step is now rate-limiting, rather than <I>C-2</I> hydrogen abstraction as in the <I>S.cerevisiae</I> wild-type enzyme. Other kinetic parameters including deuterium kinetic isotope effects also support this. We can therefore conclude that the interdomain hinge, and more generally, the structural integrity of this region are crucial in ensuring recognition and efficient electron transfer between the domains.

The methylation of DNA is a widespread phenomenon found in organisms ranging from bacteria to mammals. Methylation of cytosine at the 5-position is the most common form of this modification and is catalyzed by a conserved family of enzymes. In bacteria, methylation of DNA forms part of the restricted-modification system. The role of DNA methylation in eukaryotes is less clearly defined but it has been implicated in process such as the control of gene expression and the organization of chromatin structure. Progress in the understanding of DNA methylation could be greatly enhanced by the opportunity to study this phenomenon in genetically tractable organisms such as yeasts and <I>Drosophila. </I>However, to date, 5-methylcytosine has not been detected in the DNA of these organisms. The fission yeast gene <I>cnd1 </I>(completion of nuclear division) was cloned by complementation of a temperature sensitive mutation and was found to encode a protein with striking homology to cytosine-specific DNA methyltransferase enzymes (m5C-MTases), (R. Bartlett, PhD thesis, Oxford University, 1991). This finding suggested that it might now be possible to study methylation in yeast. Also cloned at this time was an extragenic suppressor of the <I>cnd1-1</I> mutant. In current work, a more detailed methylation analysis of the fission yeast gnome has been carried out. However, it has still not been possible to detect 5-methylcytosine in fission yeast DNA. The extragenic suppressor of the <I>cnd1-1</I> mutant has been sequenced and found to encode a small polypeptide with no homology to known proteins. The m5C-MTase homologue was histidine-tagged, overexpressed in <I>E. coli</I> and purified over a nickel agarose column.

No description available.

<i>Shewanella </i>MR-1 is a Gram-negative, facultatively anaerobic bacterium isolated from Lake Oneida, New York. It can couple its anaerobic growth to the reduction of a wide variety of compounds such as nitrate, nitrite, TMAO, DMSO, fumarate, manganese(IV) and iron(III) oxides, sulfite and thiosulfate. Analysis of the genome sequence reveals the presence of a large number of respiratory enzymes. Three of these proteins were selected for further study: a decaheme cytochrome <i>c</i>, a heptaheme cytpchrome <i>c </i>and a flavoprotein. Decaheme 129 (Cyc129) is 37% similar to MtrC, a decaheme protein from the same organisms that have been shown to be involved in iron(III) and manganese(IV) respiration. The DNA sequence indicated the presence of a lipoprotein signal sequence but the protein is loosely associated to the membrane. Compared to the wild-type strain, no phenotypic differences were noted when the <i>cyc129 </i>gene was disrupted by the insertion of an antibiotic cassette. The second protein, heptaheme 202 (Cyc202) is a soluble, periplasmic protein and is the only heptaheme cytochrome <i>c </i>in <i>Shewanella </i>MR-1. Phenotypic studies indicate that it might be involved in the electron transport to the outer-membrane located iron-manganese reductases. FccA56 is similar to the flavin domain of flavocytochrome <i>c₃</i>, the fumarate reductase from <i>Shewanella</i> MR-1. The gene encoding this protein is part of a cluster that also encodes a tetraheme <i>c</i>-type cytochrome and a histidine ammonia lyase-like protein. Substitution of the highly conserved amino acids involved in substrate binding suggests that fumarate is not the physiological substrate of FcA56, but has a similar substrate that contains only one carboxylic group. The protein was purified after overexpression in <i>E. coli. </i>A UV-visible absorption spectrum confirmed that the ~52 kDa protein has absorption maxima at 450 and 380 nm, characteristic for flavoproteins.

Flavocytochrome c, a multihaem cytochrome from <i>Shewanella putrefaciens</i> induced under anaerobic conditions, was studied to investigate the physiological function of this protein. These studies comprised two facets: the cloning and sequence analysis of the structural gene for flavocytochrome c and a biochemical study of the fumarate reductase activity associated with flavocytochrome c. A <i>S. putrefaciens</i> genomic library was constructed in the expression vector pEX3. This library was screened in <i>E. coli</i> MM294 by colony hybridization of induced recombinants with antibody raised to purified flavocytochrome c protein. One clone giving a strong signal to antibody was identified from this library. Restriction digests of this recombinant showed the insert DNA to be approximately 1.5 kb. Southern blot analysis of the clone gave hybridization of flavocytochrome c antibody to a protein of approximately 45 kDa which was encoded by the recombinant pEX3 vector. This cloned fragment was proposed to encode part of the flavocytochrome c gene and investigated in more detail. The 1.5 kb cloned fragment was partially sequenced and mapped. Sequencing yielded two non-overlapping contigs of 438 bp and 966 bp respectively. The DNA fragment joining the two contigs remained unsequenced. Database analysis showed that the second contig contained 4 conserved c-type haem binding site motifs CXYCH within the first 90 residues. A second region in this contig from bases 123-151 was found to be completely homologous with a highly conserved FAD-binding fingerprint common to many flavoproteins. This molecular analysis strongly suggested that the cloned DNA fragment encoded at least 322 residues of the N-terminal region of the flavocytochrome c protein. This was further confirmed by the finding that the first 8 residues of the second contig were completely homologous with residues 6-13 of the N-terminal sequence of flavocytochrome c. Cloning the entire flavocytochrome c gene was attempted also by functional complementation of an E. coli fumarate reductase mutation.

Aristeromycin 4 and neplanocin A 5 are biologically active carbocyclic nucleosides produced by the organism <i>Streptomyces citricolor</i>. Previous studies towards elucidating their biosynthesis have led to a proposed biosynthetic pathway in which D-glucose is converted <i>via</i> a number of carbocyclic intermediates to neplanocin A and aristeromycin. This thesis describes the studies that have been carried out in order to: i. identify the first formed carbocyclic intermediate on the biosynthetic pathway (Chapter 2). ii. determine the identity of the phosphorylated intermediates prior to neplanocin A (Chapter 3). The novel (2<i>R</i>, 3<i>S</i>, 4<i>R</i>) and (2<i>R</i>, 3<i>S</i>, 4<i>S</i>) keto-tetrols 95, <i>ent-</i>95 and 116 have been prepared and their syntheses are described. To determine whether those intermediates lie on the biosynthetic pathway, feeding studies have been carried out using a mutant of <i>Streptomyces citricolor</i>. The results of these studies, described within, suggest that both the (2<i>R</i>, 3<i>S</i>, 4<i>R</i>) keto-tetrol 95 and its diastereomer (2<i>R</i>, 3<i>S</i>, 4<i>S</i>) keto-tetrol 116 lie on the biosynthetic pathway. The syntheses of the known intermediate tetrol 83a, the proposed phosphorylated intermediate 5-phosphate 83b and the corresponding 1-phosphate 152, <i>via</i> multi-step syntheses from the cyclopentenone 61, prepared from either D-ribose 126 or cyclopentadiene 36, are described.