Studies on Dictyostelium discoideum clones containing CRE-like sequence.

The data presented in this thesis relates to an analysis of three recombinant plasmids which had previously been isolated from a genomic library of Dictyostelium DNA by virtue of their ability to hybridise at moderate stringency to a rat DNA probe containing a cyclic AMP regulatory sequence (CRE) which is known to be involved in cyclic AMP regulated gene expression in mammals. Sequence analysis of the smallest region of the DNA in pKA2 and pKA11 with the rat probes indicated a region which showed striking homology to the mammalian CRE. The sequence homology between pKA2 and the rat CRE was 79% and the homology between pKA11 and the rat CRE was 72%. The homology also extended beyond the CRE into an adjacent G/C-rich region which, in at least one mammalian gene, is required along with the CRE for cyclic AMP- regulated gene expression. The sequence obtained from both sequences was unusual in having a very high G/C content. This was so unusual that it raised doubts about the assumption that the recombinant clones contained DNA from the Dictyostelium genome. Extensive analysis of the sequence cloned in pKA11 showed that the insert was in all probability of Dictyostelium origin since an identical fragment could be found in DNA prepared from axenic cells. During this analysis, data was obtained which showed that the sequence was unstable in axenic cells and was lost on prolonged cell growth, raising the possibility that the sequence may well be part of an endogenous plasmid that is difficult to maintain during axenic growth. Analysis of the remainder of the DNA cloned in pKA11 demonstrated that it contained two sequences which were transcribed during the life cycle of Dictyostelium. The disappearance of both was accelerated by prematurely treating cells with extracellular pulses of cyclic AMP, suggesting that expression of both genes is repressed by cyclic AMP during early aggregation. The fact that the CRE-like sequence is closely associated with two genes whose expression appears to be repressed by cyclic AMP raises the possibility that it could be involved in cyclic AMP regulation of gene expression in Dictyostelium.