Käthe Nerke

Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.

We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO1 operator fragment was inserted at three different positions in front of a tRNA(Glu) (Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a beta-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 micrograms/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in beta-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.

Expression of human antithrombin III in the cellular slime mould Dictyostelium discoideum

SummaryIn order to test the biotechnological potential of the cellular slime mould Dictyostelium discoideum the cDNA coding for human antithrombin III was expressed in this microorganism. The 1392-bp antithrombin III cDNA was fused to the N-terminal coding part of the D. discoideum actin 6 gene. In constructs carrying this artificial N-terminal coding region only low amounts of antithrombin III were detected. However, constructs from which all actin coding nucleotides were removed produced significant amounts of antithrombin III, most of which was secreted into the culture broth. Stationary cultures (1.5 × 107 cells/ml) of certain stable transformants accumulated up to 1.0 μg antithrombin III/ml culture medium within 24 h. The recombinant protein has a slightly smaller molecular weight in sodium dodecyl sulphate-polyacrylamide gels than authentic plasma antithrombin III and it is glycosylated, as determined by concanavalin A labelling.

Structural requirements for the synthesis of tRNATrp from Dictyostelium discoideum in yeast

Dictyostelium tRNA genes can generally be expressed in vivo in yeast. Among tested Dictyostelium tRNA genes a tRNATrp gene containing a 13 bp intron is transcribed with particularly poor apparent efficiency and the intron is not removed. Elimination of the intron from the gene increases the amount of transcription products significantly. Splicing can only occur if minimal base-pairing of the anticodon with intron sequences is possible. Accumulation of tRNA gene transcripts decreases with the inability of intron splicing. Products of neither amber (UAG) nor opal (UGA) suppressor variants of the tRNATrp gene from Dictyostelium are able to suppress corresponding non-sense mutations in defined structural yeast genes. This also holds true for suppressor tRNA gene variants with precisely deleted intron regions.