[Construction of a bait plasmid containing human telomerase RNA mutant in yeast three-hybrid system].
Nan Fang Yi Ke Da Xue Xue Bao
; 29(4): 652-5, 2009 Apr.
Article
em Zh
| MEDLINE
| ID: mdl-19403387
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Plasmídeos
/
RNA
/
Mutação Puntual
/
Telomerase
/
Técnicas do Sistema de Duplo-Híbrido
Limite:
Humans
Idioma:
Zh
Revista:
Nan Fang Yi Ke Da Xue Xue Bao
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
China