Tp. Spann et al., MUTAGENESIS AND GENE IDENTIFICATION IN DICTYOSTELIUM BY SHOTGUN ANTISENSE, Proceedings of the National Academy of Sciences of the United Statesof America, 93(10), 1996, pp. 5003-5007
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.