[Construction of a bait plasmid containing human telomerase RNA mutant in yeast three-hybrid system].
OBJECTIVE
To 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.


METHODS
The 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.


RESULTS
Sequence 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.


CONCLUSIONS
The 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.
Cao
Ying
Y
Center for Drug Evaluation and Research, School of Pharmacological Science, Southern Medical University, Guangzhou 510515, China. yingcao1986@163.com
Wang
Wei-guo
WG
Li
Lin
L
Zhang
Hui-min
HM
Wang
Guang-fa
GF
Pang
Jian-xin
JX
Wu
Shu-guang
SG
D001483:Base Sequence; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics*; D019098:Telomerase / Q000235:genetics*; D020798:Two-Hybrid System Techniques
C422952:telomerase RNA; D012313:RNA; D019098:Telomerase