No description available.

No description available.

No description available.

No description available.

Bombardment of intact cells and tissue with DNA-coated microprojectiles represents a novel approach to the genetic transformation of biological material. In this thesis, a gas propulsion particle gun is developed for such a purpose. The particle gun utilises gas dynamics to control particle velocity and spread, thereby enabling optimisation of transformation efficiencies for varying types of target material. The dynamics associated with particle acceleration are shown to relate to transient, shock tube flow. Measurements from schlieren high-speed video photographs of the shock structure of an underexpanded jet demonstrate good agreement with empirical correlations of previous researchers; pitot tube measurements of the nozzle exit Mach number are made and shown to be in good agreement with theoretical contact surface velocities. A novel optical particle velocimeter is used to measure particle time-offlight between axial locations in the system's target chamber, providing a consistent, distance-averaged measurement of particle velocities. Particle velocities are measured for a range of system pressure ratios and driver gases. Variation in particle velocities is seen to be similar to theoretical variation in contact surface velocities. Analytical theory is used to predict small gas-particle velocity lag. Particle velocities with helium as a driver gas are shown to be considerably higher than those with air. High speed video recording of chalk particles exiting the nozzle is used to visualise the spatial and temporal variation in particle spread as a function of nozzle pressure ratio. This technique demonstrates that particle spread increases with increasing nozzle pressure ratio, as gas dynamics theory indicates. Conditions for bombardment of maize suspension cells are experimentally optimised. Significant rates of transient genetic expression are achieved with both air and helium as driver gases. High levels of transient genetic expression are also found with bombardment of maize coleoptiles and the leaves of various dicot species. Transformation efficiencies for bombardment of HL60 human leukaemia cells show dramatic increases over efficiencies seen with conventional techniques not involving cell bombardment. For other cell types the gas propulsion device described here appears to give rates of transient genetic expression similar to those reported for commercial systems using microprojectiles. Other data for the performance of such systems are too limited at present to allow comparisons of controllability and reproducibility of bombardment efficiency.

No description available.

No description available.

No description available.

Plasmid ColIb-P9 is a 93.2 kb self-transmissible plasmid, belonging to the I1 incompatibility group. Whilst much data had been gained concerning the molecular biology of conjugation mediated by this plasmid, a lack of information exsisted concerning the genetic organisation of the transfer genes. A physical map of the plasmid was constructed by detailed restriction analysis of DNA fragments sub-cloned from ColIb-P9. These fragments were also used to locate the positions of the transfer gene sog and the origin of transfer. Transposons Tn5 and Tnl723 were used to construct insertion mutants at defined points in ColIb-P9 and the effect of these on the expression of various transfer-related functions was studied. Using this technique, the probable location of the genes encoding the thick and thin sex pili were identified and also the site of the plasmid-encoded nuclease gene. The exact location of the entry exclusion gene was also determined. Complementation studies using the sub-cloned fragments of ColIb-P9 and a set of cosmid-clones generated from ColIbdrd-1 indicated that a positive regulator of the expression of the transfer genes exsisted and that this was composed of two genetically distinct elements. Studies involving wild type ColIb-P9 (drd+) indicated that this positive regulatory system is subject to negative control in cells containing the drd+ plasmid. The information gained from these studies was combined into a model of the organisation of the transfer genes of ColIb-P9. This defines at least three separate Tra regions, covering some 50 kb of the plasmid, with the origin of transfer located at one end of the transfer region.

Conjugation is a fundamentally important mechanism of horizontal DNA transfer between bacteria, bacteria x archea, and bacteria x eukaryotes. This work has concentrated on conjugation between bacteria x eukaryotes, specifically Escherichia coli x Saccharomyces cerevisiae. Four hypotheses were tested, investigating the barriers to this particular form of DNA transfer. The first investigated if a mutation that altered the cell-surface of the recipient S. cerevisiae could inhibit DNA transfer. The final three utilised a recombination-dependent-conjugation assay to investigate the barrier to DNA transmission through recombination. The hypotheses tested if the frequency of recombination, in this recombination-dependent-conjugation assay, differed when using similar or diverged DNA substrates, if a mismatch repair mutation within the recipient could affect the frequencies of recombination observed, and if the position on the plasmid of the gene of interest affected the frequency of transmission. Transmission of the Ura3 DNA sequence in the recipient S. cerevisiae was used to test all four hypotheses. The cell wall mutants mnn9, knr4, fks1 and kre6 were utilised to investigate if the cell-surface of the recipient could affect the frequency of transmission. The similar and diverged substrates utilised in the investigation of the affect of sequence similarity on recombination were the DNA sequences of ura3 from S. cerevisiae and Saccharomyces carlsbergensis, respectively and the MMR mutants utilised were msh2, pms1 and pol30-52. Cell wall mutants were not found to limit the frequency of transfer once donor-recipient contact was induced through the solid surface mating procedure. Sequence similarity, MMR and the relative position of the ura3 DNA sequence on the conjugative plasmids were shown to have little effect on the frequency of transmission in S. cerevisiae. This suggests that any DNA that enters the nucleus of S. cerevisiae (eukaryotes) can recombine with the chromosome and alter it to the same extent. However, trends within the data also suggest that DNA is transferred into the recipient and then transported to the nucleus to recombine with the chromosome as a single-stranded DNA molecule.