In this study we aimed to understand the role Prp45p executes within the spliceosome and the splicing mechanism. Part of this work was a detailed investigation into the relationships between the structure and the function of this protein by employing a screen for temperature-sensitive mutants. Prp45p is known to associate with the spliceosome throughout the splicing reactions but at what stage the protein is involved in spliceosome assembly i.e. pre-spliceosome, inactive or active spliceosome remained to be determined. Using a <i>PRP45</i> conditionally regulated strain, it was found upon depletion of Prp45p from yeast cells and analysis of the <i>in vivo</i> splicing systems by native gel fractionation that the formation of the active spliceosome does not take place. This behaviour is compatible with Prp45p being a component of the Ntc-protein complex, which is known to be involved in this step of spliceosome assembly. Moreover, co-immunoprecipitation experiments with a tagged allele of the splicing factor Prp46p, confirmed the interaction between these two proteins suggested by two-hybrid screens. Using random PCR mutagenesis, there were identified two mutants with growth defects at 37°C. The mutants, named <i>prp45-57 </i>and <i>prp45-113</i> contained mutations in two regions, designated <i>A</i> and <i>B</i> and located respectively upstream and, downstream of the absolutely conserved SNWKN motif. To understand which of the mutations were responsible for the temperature-sensitive phenotype, the substitutions in region <i>A</i> and/or region <i>B </i>were recreated by site-directed mutagenesis. It was proved that the phenotype requires mutations in both segments, which strongly suggests that regions <i>A </i>and <i>B </i>together play roles in protein function, perhaps through intra- or inter-protein interactions. However, co-immunoprecipitation experiments revealed that these substitutions in Prp45p do not alter the interaction with Prp46p <i>in vitro</i>. In order to investigate the role of these mutations within the cell, two strains were created that carry these mutations in a c-Myc tagged <i>PRP45</i> ORF. When growing these strains at non-permissive temperature and employing a β-galactosidase assay, it was found that they had a mild effect on pre-mRNA splicing and do not affect the transcription/translation of the reporter genes.

No description available.

The yeast <i>Saccharomyces cerevisiae</i> utilises many transport pathways for the efficient an accurate segregation of proteins to the compartments of the cell in which they function. Of these, the secretory pathway is responsible for the localisation of proteins to the endoplasmic reticulum (ER), Golgi apparatus, endocytic and vacuolar compartments and the cell surface. The first step along this pathway is the targeting of nascent polypeptides to the ER membrane. For many proteins, the signal recognition particle (SRP), a cytosolic ribonucleoprotein, and its cognate receptor (SR) on the ER membrane are responsible for this step. SRP and the SR direct proteins to the translocon, which forms an aqueous channel through which the nascent proteins are translocated or from which they are then integrated into the ER membrane. The aim of this study was to dissect incompletely understood interactions that occur at the ER membrane between SRP, SR and the translocation and to reconstitute the SRP-dependent translocation reaction with purified proteins. In particular, through 2-hybrid analysis and pull-down assays a novel interaction was identified between SR and the major translocon component Sec61p. To facilitate the study of this and other interactions, attempts were made to reconstitute the SRP-dependent targeting pathway with SR and translocon purified from yeast. It was demonstrated that SRP-dependent translocation could be reconstituted with solubilised yeast ER membrane proteins. SR was purified and shown to be functional, and the translocation reaction was shown to be stimulated by the presence of the ER-lumenal chaperone Kar2p. Preliminary experiments were also carried out that suggested that the purified translocon was active; indicating that the goal of reconstituting SRP-dependent translocation with purified components is attainable.

The recombinant form of the <I>R. graminis </I><SUB>L</SUB>-mdh has been overexpressed in <I>E. coli</I> to levels around 14% of the total cell protein. The enzyme was purified to homogeneity after several ion-exchange and gel filtration columns. The steady state turnover of the enzyme was measured with the artificial electron acceptor, potassium ferricyanide (550 ± 25 s<SUP>-1</SUP>) and with is physiological electron acceptor, cytochrome <I>c</I> (225 ± 15 s<SUP>-1</SUP>). In both cases the k<SUB>cat</SUB> values were similar to those previously obtained with <SUB>L</SUB>-ldh with <SUB>L</SUB>-lactate as substrate. The individual electron transfer steps in the catalytic cycle of <SUB>L</SUB>-mdh were also measured using stopped-flow spectrophotometry. The flavin reduction rate (280 ± 20 s<SUP>-1</SUP>) appeared to be half that observed for the flavin reduction value obtained with <SUB>L</SUB>-ldh (604 ± 60s<SUP>-1</SUP>). The next step is an intra-molecular electron transfer step between the reduced flavin and oxidised haem, resulting in flavin semiquinone and reduced haem. The rate observed for the haem reduction (605 ± 50 s<SUP>-1</SUP>) was similar to that observed with <SUB>L</SUB>-ldh (445 ± 50 s<SUP>-1</SUP>). This intra-molecular electron-transfer step is thought to occur so rapidly in <SUB>L</SUB>-mdh, that full reduction of the flavin groups is not observed until after a disproportionation of the flavin semiquinones, generating 2 fully reduced and 2 oxidised flavins, and further reduction by 2 molecules of mandelate. Measurement of the flavin and haem midpoint potentials of <SUB>L</SUB>-mdh (-120 mV and -10mV respectively) compared to the values previously obtained for <SUB>L</SUB>-ldh (-78 mV and -17mV) confirms that there is a larger driving force between the two prosthetic groups of <SUB>L</SUB>-mdh. The third step in the catalytic cycle of <SUB>L</SUB>-mdh is the inter-molecular transfer step from the haem group to cytochrome <I>c. </I> Stopped-flow studies on the cytochrome <I>c</I> reductase activity of <SUB>L</SUB>-mdh yielded a second order rate constant of 41.5 ± 2 μM<SUP>-1</SUP>s<SUP>-1</SUP>, which represents the rate constant for cytochrome <I>c</I> association. The corresponding value with <SUB>L</SUB>-ldh is 34.8 ± 0.9 μM<SUP>-1</SUP> s<SUP>-1</SUP>.

No description available.

To enter the spliceosome, U6 snRNP must associate with the U4 snRNP to form a U4/U6 di-snRNP. This involves RNA-RNA interactions between U4 and U6 RNAs, as the two have extensive sequence complementary and form Watson-Crick base-pair interactions over their length. During splicing, the U4 snRNP dissociates from the U6 snRNP and U6 snRNA undergoes several structural rearrangements. Therefore, U4 and U6 snRNA must be reannealed following spliceosome disassembly in order to recycle the factors back into the splicing pathway. A group of seven proteins, Lsm2-Lsm8p, has previously been identified that interact with U6 snRNA and are essential for U6 snRNA stability and pre-mRNA splicing in the yeast <i>Saccharomyces cerevisiae. </i>In this thesis the Lsm (like-Sm) proteins have been produced either as recombinant protein in <i>E. coli</i> or as the product of a coupled <i>in vitro</i> transcription/translation reaction. Using these proteins it has been demonstrated that the Lsm proteins can facilitate U4/U6 RNA duplex formation. Another U6-ascoiated protein, Prp24p, did not facilitate U4/U6 duplex formation in the absence of the Lsm proteins. This was in contrast to previous reports suggesting a role for Prp24p in U4/U6 annealing. Prp24p is, however, required when the 3’ terminal Lsm binding site is deleted from U6 snRNA. Evidence is presented suggesting that the Lsm8 complex can facilitate U4/U6 duplex formation <i>in vivo</i> as well as <i>in vitro</i>. In particular, Lsm6p appears to be essential for stable U4/U6 duplex formation, whereas Lsm7p does not. The data presented, therefore, suggest a model where the Lsm8 complex, in particular Lsm6p, is sufficient and necessary for U4/U6 duplex formation.  Other factors such as Prp24p may facilitate this process, but are not essential for it to occur.

No description available.

The sodium-dependent NADH-ubiquinone oxidoreductase (Na<SUP>+</SUP>-NQR) was discovered first in the marine bacterium, <I>Vibrio alginolyticus. </I>It acts as a primary electrogenic pump for sodium translocation during aerobic respiration, generating a sodium motive force which drives ATP synthesis, solute transport and flagellar motion. Early biochemical studies indicated that Na<SUP>+</SUP>-NQR was composed of 3 subunits: α, β and γ, with apparent M<SUB>r</SUB> values of 52, 46 and 32 kDa. A proposed model suggested that the β subunit, a NADH dehydrogenase, accepts electrons from NADH and reduces menadione or quinone by a Na<SUP>+</SUP>-independent one-electron transfer reaction to produce the semiquinone. The subsequent reduction of the semiquinone is dependent on Na<SUP>+</SUP> and is catalyzed by the α subunit. Degenerate oligonucleotides designed from the <I>N-</I>terminal sequences obtained from partially purified α and γ subunits were used to isolate clones from an <I>Eco</I>RI library of wild-type <I>V. alginolyticus </I>DNA. Six genes which comprise the <I>nqr </I>operon, <I>nqr</I>A-<I>nqr</I>F, were sequenced and identified. Sequence analysis and database comparisons led to the conclusion that this enzyme complex is both structurally and evolutionarily distinct from the H<SUP>+</SUP>-translocating NADH-ubiquinone oxidoreductase. Na<SUP>+</SUP>-NQR comprises 3 hydrophilic subunits, NqrA, NqrC and NqrF and 3 highly hydrophobic membrane-spanning subunits, NqrB, NqrD and NqrE. The 3 hydrophilic subunits, NqrA, NqrC and NqrF correspond to the previously identified α, γ and β subunits respectively. Based on sequence comparisons, a [2Fe-2S] cluster region, a FAD binding site and an NADH binding domain were identified in NqrF, the proposed NADH dehydrogenase subunit. From hydropathy plots, NqrF also appeared to possess a hydrophobic <I>N</I>-terminal region. Pulse-chase radiolabelling of various clones expressing <I>nqr</I>B-<I>nqr</I>F verified that the putative products of the<I> nqr </I>operon identified from sequencing, were indeed transcribed and translated <I>in vivo. </I>The <I>nqr</I>F gene was cloned into pET16-b and expressed in <I>Escherichia coli, </I>BL21(DE3)p<I>LysS </I>as a 46 kDa polypeptide.

<i>Pythium mycoparasiticum</i> is described as a new species. Its mycoparasitic behaviour was compared with those of <i>P.oligandrum</i> and <i>P.nunn</i> against nine species of potential host fungi- <i>P.graminicola</i>, <i>P.vexans</i>, <i>Rhizoctonia solani</i> (2 isolates), <i>Fusarium culmorum</i>, <i>F.oxysporum</i> f sp <i>lycopersici</i>, <i>Botrytis cinerea</i>, <i>Botryotrichum piluliferum</i>, <i>Trichoderma aureoviride</i> and <i>Phialophora</i> sp. In interactions on filter paper or cellulose film, the degree of aggressiveness of each mycoparasite and the degrees of resistance of host fungi were assessed by the difference in cellulose breakdown caused by the hosts alone or in dual inoculations with mycoparasites. <i>P.oligandrum</i> was the most aggressive mycoparasite, active against most hosts; <i>P.nunn</i> was least aggressive, with a narrow host range and <i>P.mycoparasiticum</i> was intermediate in these respects. Of these hosts, <i>Pythium</i> spp and <i>R.solani</i> were least susceptible to parasitism, and <i>T.aureoviride</i> and <i>Phialophora</i> sp were most susceptible. <i>B.cinerea</i>, some other hosts to a lesser degree, was susceptible to <i>P.oligandrum</i> but not to <i>P.nunn</i>. Some of these findings were confirmed by the abilities of mycoparasites to grow on potato-dextrose agar precolonised by the host fungi; this assay method was not as sensitive as others, but it could be used to compare potential host ranges of mycoparasitic <i>Pythium</i> spp and the cellulolytic mycoparasites, <i>Trichoderma harzianum</i> and <i>Gliocladium roseum</i>. Interactions between individual hyphae were studied by videomicroscopy on films of water agar. The incidence and timing of different events were assessed from replicate interactions on videotapes and statistically analysed. The results generally confirmed those of interactions on cellulosic substrates, but several new details of interactions were observed. All three mycoparasitic <i>Pythium</i> spp had similar modes of parasitism, different from those of <i>T.harzianum</i> and <i>G.roseum</i>, used in a small comparative study. They did not affect host hyphae before contact, and none showed tropism to host hyphae. Soon after contacts involving parasite or host tips, the susceptible host hyphae stopped growing and either lysed at the point of contact or exhibited vacuolation/coagulation of their cytoplasm, starting at this point; this was often followed by penetration by mycoparasite. In some cases the parasites penetrated directly, somtimes after proliferating on the host surface. The parasites branched at points of contact and penetrated from these branches. Lysis and cytoplasmic coagulation typically preceded penetration or even the origin of a penetrating branch from the mycoparasites. Based on the timing of these events and the number of hosts affected, <i>P.nunn</i> was significantly the least aggressive mycoparasite, whereas <i>P.oligandrum</i> and <i>P.mycoparasiticum</i> were equally aggressive. The hosts differed in resistance as on cellulosic substrates. Mycoparasitism by <i>P.oligandrum</i> was investigated by inactivating the parasite or host hyphae with fine beams of intense light prior to contact on water agar. The results demonstrated an essential role of mycoparasite activity in host stoppage, lysis and cytoplasmic coagulation. However, lysis also required activity by the host. The findings are discussed in relation to postulated surface recognition events and the respective roles of host- and mycoparasite-derived wall-lytic enzymes. In limited tests, <i>P.nunn</i> was found to be unique among mycoparasitic <i>Pythium</i> spp in its ability to utilise inorganic (nitrate) nitrogen. Germination of oospores of <i>P.oligandrum</i> was found to depend on length of storage in culture and on conditions of aeration and illumination. Germination was stimulated by peptone and, to a lesser degree, malt extract, and also acetaldehyde among the volatile metabolites that were tested. The results of all these studies are discussed in relation to the roles or potential roles of the mycoparasites in biocontrol of plant pathogens.

No description available.