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To further investigate the role played byRrp6p in RNA metabolism, my work aimed at identifying and characterising potential Rrp6p-associated complexes. Density gradient analyses revealed no pool of free Rrp6p, but the majority of Rrp6p was apparently associated with complexes other than exosome. In addition to the exosome, Rrp6p appeared to be associated with a 10S complex and two RNase A sensitive complexes that may correspond to pre-40S and pre-60S ribosomes. Proteomic analyses identified Gbp2p and Srp1p as potential components of the 10S complex. However, genetic analyses did not reveal defects in stable RNA synthesis in <i>gbp2 </i>or <i>srp1</i> mutant strains. Microarray analysis identified a small number of mRNAs that were upregulated in the <i>gbp2</i><i>D</i> strain. Purification of exosome complexes identified the nuclear protein Rrp47p as a substoichiometric component. Rrp47p was shown to be required for most Rrp6p functions in stable RNA synthesis but not for Rrp6p function in RNA surveillance. To better understand this similarity, I analysed the Rrp47p-exosome and the Rrp47p-Rrp6p interactions. Around 10% of the exosome was found to be associated with both Rrp6p and Rrp47p. However Rrp47p did not appear to be part of the 10S Rrp6p complex, suggesting that the surveillance functions of Rrp6p may be performed in this complex. These results have provided new insights into the function of Rrp6p in nuclear pre-mRNA turnover and into its potential association with complexes other than exosome.

ICR 170, an acridine half-mustard, has been shown to induce predominantly frameshift addition mutations in the <i>am</i> genes of <i>Neurospora crassa</i> (Burns <i>et al</i>., 1986) and other eukaryotic genes. The related compound ICR 191 produces both addition and deletion mutations in prokaryotic DNA. The introduction of the <i>am</i> gene into a prokaryote such as <i>E.coli</i>, followed by the induction of frameshift mutations using ICR 170 should provide an insight into the mechanism by which frameshift mutations are induced in eukaryotic and prokaryotic DNA and their different responses to induction of frameshift mutations by acridine half-mustards. In order to study the mechanism by which mutations are induced in prokaryotic and eukaryotic DNA, it is necessary to have a system which will allow the identification of frameshift mutations in a copy of the <i>N.crassa am</i> gene which has been transformed into <i>E.coli</i>. This study was an attempt to set up such a system. This work involved the production of complementary DNA to the <i>am</i> gene with the intention of expressing the <i>am</i> gene product, NADP-dependent glutamate dehydrogenase, in an <i>E.coli</i> strain which is auxotrophic for glutamate. Expression of the <i>am</i> gene product should complement the <i>E.coli</i> mutation and revert the <i>E.coli</i> strain to glutamate prototrophy. In this study, optimum conditions for producing cDNA from <i>Neurospora crassa</i> mRNA were determined and cDNA libraries were produced both from total <i>Neurospora crassa</i> mRNA and from <i>N.crassa</i> mRNA which had been selected by its ability to hybridise to the <i>am</i> genomic sequence. Potential cDNA clones to the <i>am</i> gene were isolated and characterised. 19 clones from the cDNA library constructed using total <i>N.crassa</i> mRNA showed homology to the <i>am</i> sequence in colony blots. Characterisation of these clones identified them as <i>am</i> cDNA sequence in colony blots. Characterisation of these clones identified them as <i>am</i> cDNA clones which contained only the 3' end of the <i>am</i> coding sequence. Since all 19 clones represented the same area of the <i>am</i> coding sequence, it is likely that some form of structural constraint in the mRNA prevented full length cDNA molecules for <i>am</i> being formed. 1328 cDNA clones were identified as homologous to the <i>am</i> genomic sequence within the cDNA library constructed from <i>am</i>-selected <i>N.crassa</i> nRNA. This represented a 90-fold increase in the representation of cDNA clones to the <i>am</i> gene using the selected mRNA.

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The interactions between modified oligonucleotides and DNA binding proteins can provide important information on the mode of binding and the structure of the enzyme-substrate complex. Studies were carried out to determine the interaction of DNA with O<SUP>6</SUP>-methyl-G-transferase, a DNA repair enzyme which removes the alkyl group from guanine residues which are alkylated on the O<SUP>6</SUP>-position. A series of modified oligonucleotides have been synthesised containing O<SUP>6</SUP>-methylguanine, O<SUP>6</SUP>-ethylguanine and N<SUP>6</SUP>-methyl-2,6-diaminopurine. The oligonucleotides were obtained in high purity. UV melting (thermal denaturing) experiments have been carried out on duplexes containing the modified bases paired with cytosine and thymine respectively. These oligonucleotides were prepared in order to study their interactions with a mutant O<SUP>6</SUP>-methyl-G-transferase enzyme lacking DNA repair activity. Gel shift experiments suggest that the above oligonucleotides are recognised by the enzyme. Attempts were made to crystallise the enzyme-DNA complex. Triplex DNA is formed by the specific binding of a DNA strand in the major groove of a preformed DNA duplex. It has been suggested that such structures might have a biological role, especially in gene regulation, and that there is potential for therapeutic applications in which gene expression is repressed by triplex formation. One difficulty encountered in the studies of DNA triplexes is their low thermodynamic stability. In order to overcome this problem oligonucleotides with appropriate triplex forming sequences have been synthesised with the three strands linked by hexaethyleneglycol or 1,8-octanediol chains. These molecules are capable of folding back on themselves to give intramolecular triplexes with significantly increased stabilities compared to intermolecular triplexes. UV melting studies have been carried out to determine the thermal stability of these triplexes, in the presence and in the absence of the DNA triplex binding drug coralyne. Circular dichroism and NMR studies were carried out in collaboration with Dr A. Lane at the National Institute of Medical Research, London.