Recently an insertional mutagenesis procedure has been developed to permit cloning of genes affected in developmental mutants of Dictyostelium discoideum (Kuspa and Loomis, Proc. Natl. Acad. Sci. USA 89, 8803-8807, 1992). In this procedure a plasmid bearing the URA (pyr5-6) gene is linearized with a restriction enzyme and electroporated into URA- amoebae (auxotrophic for uracil) together with the corresponding restriction enzyme. Transformants that can grow without uracil are screened for developmental defects resulting from insertion of the plasmid into a gene of developmental importance. We have modified this procedure to permit characterization of the promoters and structural sequences of genes that would be missed by the standard procedure because their disruption produces no obvious phenotype. Constructs carrying a promoter-less Escherichia coli lacZ gene were designed so that expression of lacZ requires insertion into an active host transcription unit. By screening restriction enzyme-generated transformants we have isolated several strains in which lacZ is under the control of a developmentally activated promoter and have cloned the 5' flanking DNA adjacent to the insertion site. Sequencing the junction between plasmid and host genome has confirmed in-frame fusion with the lacZ gene, and reintroduction of the cloned plasmids into parental cells has shown that the cloned sequences do actually contain the relevant promoters. This procedure should give ready access to a wide range of developmental promoters without the need for prior identification of the developmental genes involved.
All Science Journal Classification (ASJC) codes
- Molecular Biology