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The five manuscripts presented here concern events that occur during the early stages of mouse development, beginning with differentiation of primitive endoderm and ending with formation of the proamniotic cavity. The main focus of the work has been the role of the basement membrane in early development, with particular reference to its role in the regulation of epiblast and extraembryonic endodermal cell differentiation, parietal endodermal cell migration and programmed cell death (manuscripts 1, 3 and 4). HOW"9ver,the critical role played by the basement membrane in these processes prompted an investigation into the mechanisms regulating the deposition of this specialised extracellular matrix (manuscript 2). Central to all four manuscripts has been the use of an extremely good in vitro model system, the embryoid body. Embryoid bodies are derived from embryonic stem cells of the mouse blastocyst, and their development closely resembles that of the periimplantation embryo. Of particular use, has been the availability of LAMC1-/- ES cells that are unable to express the laminin y1 chain (manuscript 5). This defect renders the cells incapable of assembling a functional laminin type-1 trimer, which is necessary for basement membrane deposition. Hence, embryoid bodies derived from LAMC1-/- ES cells lack basement membranes, allowing the role of the basement membrane in early development to be analysed.

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It has long been a point of contention among biologists whether speciation can occur sympatrically as well as allopatrically. These experiments were set up in order to test the reactions of populations of D. melanogaster subjected to divergent selection for oviposition site under allopatric and sympatric selection regimes. Flies were presented with a choice of either a Potato based medium or a Sugar/killed yeast medium on which they could lay their eggs. The selection intensity was 50% under both regimes. Divergence occurred as quickly and to an approximately equal magnitude in all the Sympatric and Allopatric populations. This occurred despite there being up to 12.5% gene flow between the subpopulations of the Sympatric cages. Habituation was shown to have a significant effect on a females choice of oviposition site and a males courtship success. Flies selected on the Potato medium developed into adults quicker on the Potato medium than did flies selected on the Sugar medium. A small amount of reproductive isolation was observed in some of the Allopatric and Sympatric cages after 18 months of selection.

The main aim of this work was to evaluate the use of DNA-DNA pairing techniques in bacterial systematics. The genus Listeria was chosen for the study because of the small number of biochemical differences between the seven species. Also there has been a limited amount of nucleic acid studies carried out on the group using an endonuclease technique (Rocourt et al., 1982), therefore some comparisons of the two techniques were possible. Using optical DNA-DNA reassociation on a spectrophotometer with 23 Listeria strains from the seven species, a complete matrix of DNA-DNA homology values was produced. The data were analysed for reproducibility and second order kinetics. Possible distortion of the derived taxonomic structure due to choice of reference strains was investigated by analysing the structure obtained from the complete matrix and comparing it to results obtained from incomplete 'strip' matrices. An analysis was made on a published matrix of complete DNA relationships (Nakamura and Swezey, 1983a; Hartford and Sneath, 1988) as well as on the data from Listeria species produced in this study. Great distortion in apparent taxonomic structure can result unless reference strains are widely spaced and representative of the clusters present. Problems caused by the choice of reference strains and the use of incomplete matrices was also explored by generating a random normal swarm of OTUs and illustrating the often bizarre effects obtained by using incomplete data sets in bacterial systematics. DNA-DNA pairing data from a selection of published work were examined for experimental error. The average error from replications lay between 3 and 8.6 %, but the data were very limited.

Studies of Saccharomyces cerevisiae and Aspergillus nidulans have shown that the 3-phosphoglycerate kinase gene (PGK) is highly expressed from a strong promoter (Holland and Holland 1978; Clements 1986). The aim of the project was to isolate and study the potentially strong PGK gene promoter of P. chrysogenum. The P. chrysogenum PGK gene was isolated and the promoter and terminator sequences determined. The promoter contained a 40bp pyrimidine rich region in which transcription was initiated at multiple sites. Matches to both the glycolytic box and the essential region of the Aspergillus nidulans PGK promoter were found in the promoter by sequence comparison. The expression of the PGK promoter was studied using a fusion between the PGK promoter and the Esherichia coli lacZ reporter gene. The gene fusion was transformed into P. chrysogenum and a transformed strain containing two copies of the fusion at the oliC locus was monitored for the reporter activity. The PGK promoter was up to three times more active during growth in media containing carbon sources metabolised by gluconeogenesis compared to those metabolised by glycolysis, and the rate at which the promoter activity increased in logarithmic growth was also greater. The expression of the P. chrysogenum PGK promoter in Aspergillus nidulans was investigated by transformation of the gene fusion into A. nidulans at the qutE locus. The PGK promoter retained its broad pattern of control. A second gene fusion was made between the P. chrysogenum PGK promoter and the Isopenicillin-N-Synthetase gene. A transformed P. chrysogenum strain containing one copy of the gene fusion at the oliC locus was compared to the original strain in a batch fermentation. The transformed strain had twofold higher IPNS mRNA and enzyme levels during the first three days of the fermentation. The penicillin titre was slightly increased in this period.

In this study gamma-radiation-induced DNA strand breaks have been investigated using two systems: a plasmid-based assay, and a whole nuclei-, or whole cell-based, alkaline filter elution assay. Addition of alkali metal halides to DNA irradiated under frozen aqueous conditions were observed to have an effect on the radiosensitivity of the DNA. This, effect, which was not observed with DNA irradiated under fluid aqueous conditions, would appear to be due to two components; a physical component, and a chemical component which is dependent on the anions used. Addition of alkali metal halides appears to increase the volume of the hydrating layer of water which is formed around the DNA when it is frozen. This appears to increase the target volume when it is irradiated, with an observed increase in damage caused by H2O-+ and non-hydrated electrons from the ionisation of the hydration water. The chemical component, which may be a protective or a sensitising effect, is dependent on the anion in the system. The scavenging of electrons produced from the direct action of gamma radiation on DNA has been demonstrated using the intercalator, mitozantrone. This was demonstrated using plasmid DNA irradiated under frozen aqueous solutions, and compared with the e.s.r. spectroscopy results obtained by my coworkers. Finally, the protection of DNA by free-thiols has been investigated. Under indirect, dilute aqueous conditions, the amount of protection to plasmid DNA was observed to increase with increasing positive charge on the thiols. Under direct, frozen aqueous conditions, the radiosensitivity of plasmid DNA by the compounds used was less clear cut. Possible reasons for this contrast are discussed. The effect of a novel aminothiol with a +3 positive charge on the amount of damage to tissue culture cells was also investigated.

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