To increase fluorescent activity of GFP in the mammalian environment, mutations were created in a humanised form of GFP (containing mammalian codon-usage preferences). The fluorescence and expression efficiency of these mutants was characterised in mammalian cells and three of them showed strong fluorescence. In addition, stable green fluorescence was seen in established cell lines expressing these mutants with no obvious detrimental effect. In a study of WT1 nuclear function, three distinct nuclear patterns were seen in Cos7 cells depending on the isoform of WT1 in the GFP-WT1 fusions. These patterns were very similar to those reported for native forms of WT1 and the fluorescent signal provided a means to study these patterns at a high resolution. Functional domain analysis of WT1 was performed by expressing GFP and truncated WT1 fusions which suggested that the C-terminus (zinc finger regions) of WT1 had nuclear localisation signal(s) and additional signals which determine the specificity of the nuclear pattern. Altered nuclear patterns of GFP-WT1 fusion implied that there was an association between native and truncated WT1 which may result in a dominant negative behaviour and the mutated proteins caused dysfunctions of WT1 in cells. GFP transgenic mice were generated containing double-reporter constructs with either GFP fused to β-galactosidase or a dicistronic gene expressing GFP and β-galactosidase by a viral IRES sequence. Expressions of these were driven by the Msx-1 promoter. Five GFP transgenic lines were generated and expressed both GFP fluorescence and x-gal enzymatic activity in embryos from E9.5 to E12.5. Both reporter activities were seen in many embryonic tissues including maxillary and mandibular arches, eye, dorsal neural tube, somites, limbs and mid brain.

The effect of CpG islands on transgene expression was first tested in cultured cells. In transient transfection it was demonstrated that CpG islands do not influence the expression of a transgene when not integrated into the genome. Even when integrated into the genome of cultured cells, CpG islands are not able to confer position-independent, copy number-dependent transgene expression, as confirmed by the analysis of individual cell lines. However, the results from bulk analysis of primary clones suggest that CpG islands improve the level of expression in cultured cells, and increase the proportion of highly expressing clones. Transgenic mice were used to study the effect of CpG islands on the level and pattern of transgene expression in vivo. Unexpectedly, from the nine transgenic lines generated, transgene expression was detected in only one line. In the rest of the lines transgene expression was silenced, and in these cases the transgene was densely methylated. In half of the silenced lines transgenes were found to localise in the pericentromeric chromatin. The results suggest that full size CpG islands used as promoters do not necessarily overcome the negative effects of neighbouring chromatin to give ubiquitous transgene expression independent of the integration site. During the study of stable cell lines it was observed that cells within a clone do not have the same level of transgene expression, and this heterogeneity in expression was not reduced by the presence of the full size CpG island. In order to elucidate this phenomenon, the hypothesis that intraclonal heterogeneity is caused by individual cells switching transgene expression on and off over a period of time was tested. Clones of cells expressing Green Fluorescent Protein (GFP) were monitored at regular time intervals, and in some cells rapid extinction of fluorescence was observed. It was concluded from this result that transgene expression varies with time.

ERCCl is a gene essential for the repair process of UV-induced DNA damage known as Nucleotide Excision Repair (NER). Deficiencies in this DNA repair mechanism have been associated with the skin cancer prone human syndrome Xeroderma Pigmentosum (XP) and also with Cockayne's Syndrome (CS) and Trichothiodystrophy (TTD). The main role of ERCCl is to act as a NER endonuclease but it has also been proposed that it may have a role in recombination. The ERCCl knock out (k/o) mouse model was first described by McWhir <I>et al</I>, in 1993. McWhir and co-workers described ERCCl -deficient mice as being severely runted, dying before weaning at approximately three weeks of age from hepatic failure, having severe aneuploidy and polyploidy in their liver and presenting elevated levels of p53 in this tissue. This k/o model shared no similarities with any of the already known NER deficiency-associated syndromes which led to the theory that a deficiency in the other non-NER ERCCl function(s) may be responsible for the observed differences. The aim of this project was to gain a better understanding of the precise mechanisms behind this atypical phenotype. Indeed, studies carried out on both liver tissue and primary fibroblast material have resulted in the development of a hypothesis for the ERCCl k/o phenotype. No human syndrome has yet been identified as being caused by a defect in ERCCl, so a subsidiary aim of this project was to see if any human condition could be attributed to an ERRC1 deficiency. Taking as a starting point the prematurely aged appearance of the ERCCl k/o mice, a rare human premature ageing syndrome called Hutchinson-Gilford progeria (HGP) was selected as a possible candidate for an ERCCl-related human condition. Primary fibroblasts derived from a HGP patient showed decreased levels of ERCCl protein, but no evidence for mutations in the ERCCl gene was found. However, an impaired p53 response to UV-irradiation was observed. Overall, this work has opened the doors for the study of premature ageing in humans in relationship to NER and p53.

Immobilised lactoperoxidase has been developed as a probe of the molecular organisation of the nuclear envelope. In particular, it has been used to identify proteins of the nuclear pore complex (which is believed to be the principle site for nucleocytoplasmic transport of ribonucleoprotein) and to investigate the extent to which the nuclear membranes are differentiated from rough endoplasmic reticulum. The outer annulus of the nuclear pore complex is shown to comprise at least 14 polypeptides, only two of which (N1 and N2) are major components of the nuclear envelope as a whole. A third major component of the nuclear envelope (N3) is located in the fibrous meshwork that underlies and interconnects the pore complexes, and which represents the peripheral aspect of the nuclear matrix. The polypeptides of the nuclear envelope and rough endoplasmic reticulum are examined with respect to their distribution and organisation. It is firmly established, contrary to widely-held beliefs, that the nuclear membranes are a highly specialised membrane system quite distinct from the rough endoplasmic reticulum.

The IncP-1 plasmids are of interest because of their ability to transfer into and be stably maintained in a large variety of gram negative genera. This ability is of interest because of both the evolutionary and medical implications arising from the existence of such plasmids in nature. In this thesis I have begun a genetic analysis of the transfer function of the IncP-1 plasmid RP1 (this function has been shown to be located in 3 distinctly separate areas of the plasmid). RP1 was mutagenised to isolate transfer deficient (Tra ) mutants of the RP1 plasmid. Of the 133 Tra mutants selected 97 were resistant to the IncP-l-specific phages PRR1 Pf3 and PR4. While 6 had a PRRlr Pf3{ PR4s phenotype and 30 were sensitive to the phages. Twenty one of the mutants had amber mutations of Tra; of these, 19 were RPi-specific phage resistant and two were PRRlr Pf3r PRO. None of the RP1 amber Tra mutants were sensitive to the 3 RP1-specific phages tested. Mu phage has been shown to both integrate randomly into a genome and cause mutations. Mil phage was used to try to obtain RP1 Tra-mutations caused by insertion of Mu into a Tra region. Although none of the RP1::Mu lysogens were Tra, several were of interest. Some had Mu inserted close or adjacent to Tra region of RP1 and could be used to generate Tra- deletions upon induction of the phage. These deletion mutants would be helpful in ordering representative point mutants within the Tra regions. To begin grouping the Tra mutants into complementation groups, transient hetarozygote complementation experiments using both amber mutants in a Su+ strain as donors and P1 transducing phages of RP1 Tra- mutants were attempted. These results were unsatisfactory; probably because the RP1 transfer frequencies in liquid medium are low. Qualitative transient heterozygote complementations using a replica-plate method allowed the amber RP1 fro mutants to be grouped into 10 complementation groups. Cloning of transfer genes away from the incompatibility determinants has been used to advantage in the study of the F transfer system. Therefore, the RP1 Tral region was cloned into the plasmid vector pBR325 using an RP1::Tn7 derivative of RP1. This chimera (pED800) was used to make partial diploid strains with all of the amber Tra- mutants. All the Tra mutants which showed sensitivity to the specific phages were complemented by pED800. Two of the amber Tra- mutants which were phage resistant were also complemented by pED800. Since the majority of the Tra- mutants tested were RP1-specific phage resistant and not complemented by pED800, it was concluded that most of the tra genes are not found in the Tral region. ITra homogenote derivatives of pED800 carrying the pED516 (PP.R1t Pf3r PRO amber Tra-) and pED615 (PRRlr Pf3r PR4r amber Tra) mutations were isolated. These derivatives were used to make partial diploid strains with some of the mutants which were complemented by pED800 and the diploid strains were tested for complementation. On the basis of these complementation experiments it was established that at least 4 tra genes are found in the Tra1 region of RP1. The Tra2 and Tra3 regions were not cloned due to lack of selecting markers and time. Nalidixic acid was found to interfere with RP1 conjugation to a similar extent as it did with F conjugation. This was probably due to inhibition of DNA replication by interfering with the functions of DNA gyrase. At least 20 plasmid coded proteins were found to be synthesized by RP1 in 35S-methionine labeled minicells. Once the Tra cistrons have been identified it will be possible to determine which of these proteins are RPl Tra proteins.

The bacteriophage maturation process includes a series of complex interactions between molecules of protein and DNA. Some of the essential protein components are present at very low levels in phage-infected cells. Systems have been developed to amplify the expression of these proteins in order to facilitate investigations of protein functions. Hybrid bacteriophage derived from phage A and P2 have been constructed in vitro using the R. Boo RI restriction endonuclease. The A/P2 hybrid phage have contributed essential information for the identification of restriction enzyme recognition sites in P2 DNA and have provided a link between genetic and physical maps for phage P2. The expression of P2 structural genes in the hybrid phage has been studied and in vivo manipulation has demonstrated that transcription of P2 DNA can occur from phage A promoters. A duplication phage has been formed after a series of in vitro recombination reactions. The duplicated DNA includes the T cos site and this creates the potential for 2 distinct linear monomeric phage DNA species. The patterns of protein synthesis from the duplication phage have been examined.

Current notions of genome organisation hold that chromatin is structurally and functionally partitioned into domains delimited by specialised chromatin elements. In transgenic studies a variety of chromatin domains and functional elements isolated from them have been shown to direct physiological levels of gene expression independent of the integration position of the transgene within the genome. The principal aim of this project was to assay for position independent, copy number dependent and tissue- and developmental stage-specific gene expression from the human and murine casein gene loci in transgenic mice. Two overlapping YACs covering the murine casein gene locus were restriction mapped, generating the first reported physical map of this locus. The order of the five casein genes is α-β-γ-ε-κ; as in other mammals the κ casein gene, though evolutionarily unrelated to the other members of the locus, is closely linked, which may imply that casein gene expression is under locus control. The YACs are collinear within the region of overlap, suggesting that neither is rearranged. One of these YACs was manipulated with the intention of inserting a reporter gene under the control of an ovine β-lactoglobulin promoter downstream of the murine β casein polyadenylation site, so that gene expression from the YAC could be detected against a murine background in cell culture or transgenic mice. In parallel, a YAC bearing the human casein locus was restriction mapped and used to establish transgenic mice. Unexpectedly, none expressed detectable levels of human casein RNA when analysed by northern blot, although four produced human κ casein RNA that was detectable by reverse transcriptase polymerase chain reaction, indicating basal levels of transcription of the gene. The reasons for this are unknown, though initial characterisation of genomic DNA from the transgenic animals suggests that the YAC is not intact in any of the eight lines. The human casein YAC was also transfected into HC11 murine mammary epithelial cells with a view to assessing the level of human casein gene expression from the YAC <I>in vitro, </I>but unfortunately no stably transfected cell lines were established. These results and their implications for the use of large DNA constructs in transgenic mammals are discussed.

To investigate the role of PrP in conferring susceptibility to transmissible spongiform encephalopathies and furthermore to investigate the effect of restricted hamster PrP expression on the susceptibility to the hamster scrapie agent, we attempted to generate transgenic mice by using the hamster PrP open reading frame only, linked to appropriate regulatory 5' flanking sequences of brain specific genes. Although correct mRNA expression was achieved in some of the transgenic lines generated, problems were encountered with PrP protein production, indicating translational control of PrP. It was postulated that other PrP sequences could be important for mRNA regulation and efficient translation. The examination of the role of untranslated regions of the PrP mRNA was considered of major importance. The effect of 5' and 3' untranslated sequences of hamster PrP was tested on the translation of both PrP and lacZ. The effect of other regulatory sequences (a generic intron and an altered translation of initiation consensus sequence) was also investigated. However, none of the sequences tested appeared to have a significant effect on either PrP or lacZ translation efficiencies.

Reverse transcription polymerase chain reaction (RT-PCR) of RNA from the infective larval stages of <i>Brugia malayi</i> with the nematode-specific 5' spliced leader sequence and oligo d(T) has been used to identify genes expressed during early larval stages. Products resolved on agarose gels show a number of prominent bands and cloning of these bands has revealed a total of 14 genes. Of these, four genes have been analysed in detail. One highly stage-specific transcript <i>Bm-cpi</i>-1, a cystatin-type cysteine proteinase has been characterised in parallel to a second, constitutively expressed, cystatin, <i>Bm-cpi</i>-2, identified by EST sequencing. The two inhibitors have been functionally expressed in <i>E. coli </i>and have distinct inhibitory specificities. In addition, both CPIs have been localised to the parasite surface and found in parasite secretions. An abundant transcript, <i>Bm-alt-</i>1, is a member of a large family of genes found in nematodes but lacks clear homologues outside the nematode phylum. Analysis of Expressed Sequence Tags (ESTs) deposited in dbEST has identified a total 10 family members in <i>B. malayi. </i>Genomic structures of the two most abundant family members have revealed conservation in the position of introns. However there is considerable sequence divergence within introns, with one gene containing distinct repeat units within its introns. In addition, heterogeneity is seen within this due to variation in the number of repeat units. Two further genes have been characterised. One is a homologue of the human histamine-releasing factor, a constitutively expressed, cytokine-like molecule of interest as a potential modulator of antiparasite responses. A second is a member of a family of proteins of unknown function, rich in glycine and tyrosine residues, which may form part of the nematode cuticle. The discovery of these abundant genes provides candidates for future analysis of immune evasion by filariae and identifies potential targets for vaccination or chemotherapy.

Despite disagreements over the fundamental importance of epistasis in the process of evolution there has been a great deal of interest in how epistasis may influence certain biological adaptations such as recombination and canalisation and how epistasis contributes to speciation and even the origins of life. In this thesis epistasis is examined in the unicellular, motile chlorophyte <i>Chlamydomonas reinhardtii</i> through its effects on the recombination load arising from sexual crosses. In an experiment that combined divergent selection to opposite environments with measures of between and within environment recombination loads it was found that the build up of incompatibilities between populations was not enhanced by divergent selection, and was instead the product of allopatric divergence together with genotype-be-environment interactions. Theoretical analysis is presented that shows that recombination facilitates the evolution of mutational robustness in a two-locus model.  A review of eukaryotic chromosomal linkage patterns is presented and it is argued, together with simple theoretical considerations, that epistatic selection will not cause general, large-scale patterning of gene order within chromosomes. Additionally, two more general non-linear models are developed. The first show that maternal effects may be an important component of mate choice in sexual selection models and the second demonstrates that marine snow and algal toxin production are social adaptations that can be understood as individual selection as well as group selection.