MUTAGENESIS AND GENE IDENTIFICATION IN DICTYOSTELIUM BY SHOTGUN ANTISENSE

We have developed a mutagenesis technique that uses antisense cDNA to identify genes required for development in Dictyostelium discoideum. W e transformed Dictyostelium cells with a cDNA library made from the mR NA of vegetative and developing cells. The cDNA was cloned in an antis ense orientation immediately downstream of a vegetative promoter, so t hat in transformed cells the promoter will drive the synthesis of an a ntisense RNA transcript. We find that individual transformants typical ly contain one or occasionally two antisense cDNAs. Using this mutagen esis technique, we have generated mutants that fail to aggregate, aggr egate but fail to form fruiting bodies, or aggregate but form abnormal fruiting bodies. The individual cDNA molecules from the mutants were identified and cloned using PCR Initial sequence analysis of the PCR p roducts from 35 mutants has identified six novel Dictyostelium genes, each from a transformant with one antisense cDNA. When the PCR-isolate d antisense cDNAs were ligated into the antisense vector and the resul ting constructs transformed into cells, the phenotypes of the transfor med cells matched those of the original mutants from which each cDNA w as obtained. We made homologous recombinant gene disruption transforma nts for three of the novel genes, in each case generating mutants with phenotypes indistinguishable from those of the original antisense tra nsformants. Shotgun antisense thus is a rapid way to identify genes in Dictyostelium and possibly other organisms.