Expression of a ciliate gene in Escherichia coli using a suppressor tRNA to read the UAA and UAG glutamine codons.
Most ciliates use a particular genetic code where the standard stop codons UAA and UAG encode glutamine. Ciliate genes cannot therefore be expressed in heterologous systems such as Escherichia coli. To overcome this problem, we worked out a system of inducible suppression to permit efficient readthrough of UAAs and UAGs: a strong UAA tRNA suppressor that inserts glutamic acid was cloned downstream from a tac promoter whose efficiency was reduced by a transcription terminator. This system proved to be operational (1) to suppress UAG mutations by wobble pairing in an E. coli lacI-lacZ gene fusion and (2) to read through at least eight UAA glutamine codons in a Paramecium alpha-tubulin gene, as detected by Western blotting and colony hybridization. This work opens the way for cloning Ciliate genes from expression libraries and for expressing particular sequences without extended in vitro mutagenesis. A similar approach can be envisaged for expression of genes from Mycoplasma, mitochondria or other genomes that use non-standard genetic codes.
Cohen
J
J
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Dupuis
P
P
Viguès
B
B
D000818:Animals; D001483:Base Sequence; D016798:Ciliophora / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005973:Glutamine / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012343:RNA, Transfer / Q000235:genetics*; D013489:Suppression, Genetic; D013728:Terminator Regions, Genetic
D003062:Codon; D005973:Glutamine; D012343:RNA, Transfer