title: [Construction of a bait plasmid containing human telomerase RNA mutant in yeast three-hybrid system].
abstract: 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.
authors.affiliation: Center for Drug Evaluation and Research, School of Pharmacological Science, Southern Medical University, Guangzhou 510515, China. yingcao1986@163.com
authors.forename: Ying
authors.initials: Y
authors.lastname: Cao
authors.forename: Wei-guo
authors.initials: WG
authors.lastname: Wang
authors.forename: Lin
authors.initials: L
authors.lastname: Li
authors.forename: Hui-min
authors.initials: HM
authors.lastname: Zhang
authors.forename: Guang-fa
authors.initials: GF
authors.lastname: Wang
authors.forename: Jian-xin
authors.initials: JX
authors.lastname: Pang
authors.forename: Shu-guang
authors.initials: SG
authors.lastname: Wu
mesh_terms: 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
chemical_list: C422952:telomerase RNA; D012313:RNA; D019098:Telomerase