Six IS<I>61200</I>-transposants, obtained by intermolecular transposition of IS<I>6100</I> into the 'right-hand' chromosome end of <I>S. lividans</I>, and carrying amplifications and deletions were examined to investigate transposition-induced genome alterations. For this, cosmid clones containing representative regions of the chromosome ends, from two individuals genomic cosmid libraries of <I>S. lividans</I> and <I>S. coelicolor</I> were used as Southern hybridization probes. The sizes of the chromosomal amplifications, produced as a result of transposition of IS<I>6100</I>, were found to vary from 65.5kb to 350 kb. The nature of the telomeric sequences was further investigated, indicating that to a large extent sequences from the chromosome ends were retained in the mutants. Low-level amplifications in the 'right-hand' end were mapped to a large region extending between the terminal inverted repeat and the AUD Type 1 locus, but no concomitant large deletions were found. In parallel, isolation, cloning, and sequencing the putative terminal-DNA from <I>S. avermitilis </I>was attempted. Preserving the covalently attached terminal protein at the 5' end of the linear chromosome of <I>S. avermitilis, </I>total DNA was isolated. Restriction fragments obtained from this preparation were used for cloning. Four candidate clones obtained independently more than once were examined to check if any of them carry the chromosome end. Comparison of the DNA sequences from the four clones with another seven terminal DNA sequences revealed no similarity. However one clone, D showed 95% homology to the <I>rho</I> gene of <I>S. lividans</I> ZX7, indicating that the <I>S. avermitilis</I> <I>rho</I> gene had been cloned. The analysis of an oligonucleotide probe designed from the consensus sequence present in seven other terminal sequences. The probe generated a signal with two controls, DNA from <I>S. lividans</I> 1326 and ZX1 strains, whereas no signal was detected with DNA from <I>S. avermitilis</I> suggesting that the terminal sequence of <I>S. avermitilis </I>is not similar to the consensus sequence.

Genetic change in meiotic cells of Saccharomyces cerevisiae was examined for comparison to pre-existing mitotic data. The response of meiotic cells to various chemicals indicated that sporulation may be inhibited without a total inhibition of meiotic recombination, but no evidence for the induction of increased meiotic gene conversion above spontaneous levels at ~trp 5, in the strain D7, was found. However, in experiments where meiotic cells were treated before a return to mitotic growth the frequency of coloured colonies were enhanced above spontaneous levels. The coloured colonies were attributed mainly to reciprocal recombination between ads 2 and its centromere and further work will be required to establish whether this reflects a difference between recombination for these different loci, or else a difference between gene conversion and reciprocal recombination. A "meiotic effect" associated with the time of initiation of meiotic DNA synthesis was also found after the treatment of meiotic cells with ethidium bromide, which blocked meiotic recombination and meiotic DNA synthesis, but resulted in no lethality in mitotic cells. Further, it was demonstrated that in untreated cultures the mitochondrial DNA replicated synchronously with the nuclear DNA. Modification of the response of meiotic cells in comparison to mitotic cells was also found for N-methyl-N'-nitro-N-nitrosoguanidine induced mutation and sensitivity to the lethal effects of X-rays and ultraviolet light. In the case of N-methyl-N'-nitro-N-nitrosoguanidine no enhanced mutagenic activity was found during meiotic DNA synthesis unlike during mitotic DNA synthesis which may be due to an absence of enhanced reactivity of the DNA during meiotic DNA synthesis, while cells prior to meiotic DNA synthesis appeared more resistant to the lethal effects of X-rays and ultraviolet light than cells prior to mitotic DNA synthesis. These findings indicate differences do exist between the sensitivities of meiotic cells and mitotic cells of Saccharomyces cerevisiae.

The use of transposon mutagenesis for the identification of genes involved in <I>Streptomcyes </I>development has been frustrated in part by the high target site specificity of many transposable elements. This investigation reports the low insertion site specificity of the IS<I>6100</I>-based mini-transposon Tn/<I>792</I> from a temperature-sensitive delivery plasmid to the genome of the avermectin producer <I>Streptomyces avermitilis</I>. Sequencing of Tn/<I>792</I> insertion sites in <I>S. avermitilis</I> transposants revealed no meaningful shared homology of the target sites or predilection of Tn/<I>792</I> for GC or AT rich regions of the genome. Furthermore, the cointegrate structure normally formed as the end product of the IS<I>6100</I> family of transposable elements was found to undergo resolution resulting in the precise excision of one of the duplicated copies of the transposon and the delivery vector. Several insertions revealed previously uncharacterized genes, of which one termed <I>orf219</I> was implicated in morphological differentiation. Disruption of wild-type <I>S. avermitilis</I> with the cloned insertion site carrying Tn<I>1792</I> generated colonies unable to sporulate (<I>whi</I>), suggesting <I>orf219</I> was involved in development of the aerial hyphae. Complementation of the disruptants with a functional copy of the gene confirmed this involvement in development, producing reversion of the <I>whi</I> phenotype to that of wild-type. Microscopic analysis revealed that in strains in which <I>orf219</I> was disrupted the aerial hyphae are grossly elongated and unable to develop spore compartments. This suggests that <I>orf219</I> is involved in differentiation at or prior to septation of aerial hyphae.

No description available.

Strains of both <I>Streptomyces hygroscopicus </I>and <I>S. griseochromogenes</I> produce the antiparasitic compound milbemycin. Milbemycin is a macrocyclic polyketide synthesised by a Polyketide Synthase (PKS) in a manner analogous to the synthesis of long chain fatty acids by Fatty Acid Synthases (FAS). DNA libraries were constructed of <I>S. hygroscopicus</I> and <I>S. griseochromogenes</I> genomic DNA and probed with oligonucleotide probes for key PKS activities. A number of overlapping cosmids were identified with inserts potentially encoding the milbemycin PKS. The cosmids derived from <I>S. hygroscopicus</I> seemed to form three distinct groups based on homology with each other, while all of the <I>S. griseochromogenes </I>cosmids appeared to be derived from a single cluster. The DNA sequence was determined for a single fragment from each group of cosmids and the derived amino acid sequences were use to search the SwissProt protein sequence database. This analysis revealed that all of the fragments appeared to encode modular PKSs. Recombinant bacteriophage and plasmids were engineered to disrupt the chromosomal homologues of all of the sequenced fragments. Potential recombinants were isolated and assayed for milbemycin production by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). Attempts to disrupt the <I>S. griseochromogenes</I> PKS genes were unsuccessful and the function of the cloned DNA remains unproven. In <I>S. hygroscopicus</I> disruption of two of the putative PKS clusters resulted in mutant phenotypes both exhibiting partial loss of milbemycin synthesis. Hence the milbemycin PKS gene cluster has been conclusively identified and the way paved for determination of its genetic organisation.

The thiostrepton inducible P-<I>tipA</I> promoter of <I>Streptomyces</I> is widely used as a tool for gene cloning and over expression studies. This thesis describes the experiments carried out to investigate the regulation of P-<I>tipA</I> in <I>S. lividans</I> 1326 in order to improve expression of the promoter. Regulation of the promoter was studied using three different reporter genes, <I>aph</I>II, <I>luxAB</I> and <I>egfp</I> in both plasmid borne and chromosomally integrative forms. The promoter was found to be induced by soya flour as well as thiostrepton (Tsr). Expression of the promoter was determined to be growth-phase development and sensitive to extracellular osmolarity. The expression of the promoter was increased and prolonged in high osmolarity media in the presence and absence of Tsr. The transcriptional activator protein TipA<SUB>L</SUB>, which is required for thiostrepton induction was also shown to be required for induction by soya flour and increased extracellular osmolarity. P<I>-tipA</I> has several features, which suggest that it may be responsive to DNA superhelicity. These include a sub-optimally spaced promoter, which is contained within a palindromic sequence with the potential to form a cruciform secondary structure <I>in vivo</I>. The possible role of DNA superhelicity in the regulation of P-<I>tipA</I> was analysed using the gyrase inhibitors, novobiocin and coumermycin. These had little effect on P-<I>tipA</I> expression in the presence of high extracellular osmolarity. The promoter was also exploited for the production of transposon Tn<I>1798</I>, which carries an inducible outward reading P-<I>tipA</I>. The purpose of this transposon is to produce conditional lethal mutations for the study of essential genes. The construction and application of this transposon is described.

The <I>Saccharomyces cerevisiae</I> checkpoint genes <I>RAD9, RAD17, RAD24</I>, and <I>MEC3</I> are required for the transcriptional induction of a group of genes including some for DNA repair. In this thesis two of the checkpoint genes, <I>RAD9</I> and <I>RAD24</I>, and two nucleotide excision repair (NER) genes <I>RAD16</I> and <I>RAD26</I>, have been investigated for their role in inducible NER at the <I>MFA2</I> gene at nucleotide resolution. In wild type cells, enhanced NER is detected in cells received a minor UV irradiation one hour prior to a main UV dose compared to cells only treated with the main UV dose. This inducible NER is not detectable in mutant strains <I>rad9, rad24, rad9rad24, rad16</I> and <I>rad26</I>. These data suggest that inducible NER is dependent on the checkpoint genes <I>RAD9 </I>and <I>RAD24</I>, on the <I>RAD16</I> gene that is essential for global genome repair (GGR), and on the <I>RAD26 </I>gene that is required for efficient transcription-coupled repair (TCR). A second aspect of the thesis examined the role of histone acetylation in NER. Histone acetyltransferases (HATs) play an important role in remodelling chromatin structure during transcription activation. Here, the role of one HAT, Gen5, in the NER of UV induced CPDs in the upstream region of <I>MFA2 </I>has been examined. In Δ<I>gcn5</I> strains, NER of both strands in the control region of <I>MFA2</I> is slower than in wild type cells. As Gen5 regulates <I>MFA2</I> transcription, probably through chromatin modification, its role in chromatin organization in the control region of <I>MFA2</I> would appear to influence DNA repair in that region.

The role of transcription in repair of UV-induced CPD was investigated in the <I>Saccharomyces cerevisiae</I> <I>MFA2</I> gene. <I>MFA2</I> mutants were constructed for cloning into a yeast artificial chromosome (YAC) by deletion of either the 22nt TATA box sequences (T2Δ<I>MFA2</I>) or 55nt Mcm1 binding site (MΔ<I>MFA2</I>) from its promoter. <I>In vivo</I> transcription analysis using eGFP reporter system reveals a 10-fold reduction in transcription activity for the T2Δ<I>MFA2</I> a cells compared to the WT<I>MFA2 </I>a cells. This reduced transcription level is very close to the level found in α cells where <I>MFA2</I> is inactive. A 7-fold reduction in transcription activity was found for MΔ<I>MFA2</I> in both a and α cells. This result indicates the deletion of the MBS eliminates Mcm1/α2-mediated <I>MFA2</I> repression in α cells dramatically reduced <I>MFA2 </I>expression in a cells. Among 13 CPDs found in the promoter region, three CPDs are increasingly induced following the deletion of the TATA box and MBS sequences. However, their repair rates are not affected. Analysis of NER was performed on individual CPDs in both the transcribed (TS) and non-transcribed (NTS) strands by comparison between the YAC-borne <I>MFA2 </I>and the endogenous gene for each construct. The endogenous <I>MFA2</I> in YAC strains harbouring the WT<I>MFA2</I> and MΔ<I>MFA2</I> constructs, exhibit an identical repair profile. This suggests no genetic variation occurs within the isogenic strain harbouring the three promoter element deletion constructs. The repair results on the TS indicate the fastest rate with t<SUB>50%</SUB> of 1.5 hr for some CPDs in the transcription region of the transcribed strand of the WT<I>MFA2</I>.

No description available.

No description available.