OBJECTIVETo construct a bait plasmid containing human telomerase RNA with multiple point mutations in a yeast three-hybrid system and evaluate the toxicity of the recombinant bait plasmid.

METHODSThe primers were designed according to the hTR sequence and the target mutation sites for inducing T-->A mutations at the 41st, the 80th and 102nd nucleotides of the hTR gene using the overlapping extension PCR (OE-PCR) method. The mutant was cloned into PMD18T vector, confirmed by sequencing, sub-cloned into the bait plasmid PRH3' and identified with PCR and restriction enzyme digestion. The recombinant bait plasmid was then transformed into yeast L40 ura3/pHyblex/ZeoMS2 for toxicity test.

RESULTSSequence analysis demonstrated successful introduction of point mutations at the target sites without causing random mutation. The recombined bait plasmid constructed showed no obvious toxicity against the host yeast cells.

CONCLUSIONSThe recombinant plasmid containing the human telomerase RNA mutant (PRH3'-hTRm) has been successfully constructed and can be used as the bait plasmid in yeast three-hybrid system.