Manipulation of gene expression using the 434 repressor.

The interactions of the 434 repressor with its operator DNA have been extensively studied in vitro. The aims of the project described in this thesis have been to characterise the activity of the 434 repressor, the product of the coliphage 434 cl gene, and its variants in vivo. Work presented characterises an optimum, symmetrical 434 operator which demonstrates high levels of repression in combination with the 434 repressor in a reporter system. The combination of one half-site of this operator with a P22 operator half-site, to create a hybrid 434/P22 operator, is used to demonstrate the efficient repression by a 434 repressor/434P22 repressor heterodimer in vivo. The sequence of the non-contacted bases in the centre of the 434 operator are important for determining the affinity of the repressor in vitro and in vivo. The effect of the sequence of these bases is further characterised in vivo, demonstrating that the presence of G and C bases reduces the degree of repression in a reporter system. A variant of the 434 repressor, the 434Ala44 repressor, is shown to be ineffective in vivo at relieving the deleterious effect of G and C bases in the centre of the operator, despite its improved characteristics in vitro. The efficiency of the 434 repressor in a reporter system in Saccharomyces cerevisiae is investigated, demonstrating that a functional 434 repressor can be produced in a eukaryotic cell and that this repressor is able to repress expression from a highly active eukaryotic promoter transcribed by RNA polymerase II. Expression of the 434P22 repressor Saccharomyces cerevisiae has a deleterious effect on the viability of transformed yeast cells. A selection system for altered binding specificity 434 repressors is used to attempt the isolation of a repressor which will recognise a 434 pseudo-operator within the Saccharomyces cerevisiae PGK promoter. No repressors active against this operator have been detected.