Nonsense suppression in Dictyostelium discoideum.
We describe the generation of Dictyostelium discoideum cell lines that carry different suppressor tRNA genes. These genes were constructed by primer-directed mutagenesis changing a tRNA(Trp)(CCA) gene from D. discoideum to a tRNA(Trp)(amber) gene and changing a tRNA(Glu)(UUC) gene from D. discoideum to a tRNA(Glu)(ochre) as well as a tRNA(Glu)(amber) gene. These genes were stably integrated into the D. discoideum genome together with a reporter gene. An actin 6::lacZ gene fusion carrying corresponding translational stop signals served as a reported. Active beta-galactosidase is expressed only in D. discoideum strains that contain, in addition to the reporter, a functional suppressor tRNA. Both amber suppressors are active in D. discoideum without interfering significantly with cell growth and development. We failed, however, to establish cell lines containing a functional tRNA(Glu)(ochre) suppressor. This may be due to the fact that nearly every message from D. discoideum known so far terminates with UAA. Therefore a tRNA capable of reading this termination codon may not be compatible with cell growth.
Dingermann
T
T
Institut für Biochemie der Medizinischen Fakultät, Universität Erlangen-Nürnberg, Federal Republic of Germany.
Reindl
N
N
Brechner
T
T
Werner
H
H
Nerke
K
K
D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D004023:Dictyostelium / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005804:Genes, Lethal; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D010957:Plasmids; D012364:RNA, Transfer, Trp / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
D012364:RNA, Transfer, Trp