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Published on: June 6, 2018
[Construction of a bait plasmid containing human telomerase RNA mutant in yeast three-hybrid system]
Ying Cao1, Wei-guo Wang, Lin Li
1Center for Drug Evaluation and Research, School of Pharmacological Science, Southern Medical University, Guangzhou 510515, China. yingcao1986@163.com
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.
Insights
Researchers successfully created a mutated human telomerase RNA (hTR) bait plasmid for yeast three-hybrid systems. The new plasmid is non-toxic to yeast cells, enabling further study of telomerase function.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Context:
- Telomerase is a crucial enzyme for maintaining telomere length.
- Understanding telomerase function requires robust experimental systems.
- Yeast three-hybrid systems offer a powerful platform for studying RNA-protein interactions.
Purpose:
- To construct a bait plasmid encoding a mutated human telomerase RNA (hTR) for use in a yeast three-hybrid system.
- To introduce specific point mutations (T to A at nucleotides 41, 80, and 102) into the hTR sequence.
- To assess the toxicity of the resulting recombinant bait plasmid in yeast host cells.
Summary:
- Point mutations were introduced into the hTR gene using overlapping extension PCR (OE-PCR).
- The mutated hTR gene was cloned, sequenced, and subcloned into the PRH3' bait plasmid.
- Sequence analysis confirmed successful mutagenesis without off-target mutations, and the recombinant plasmid (PRH3'-hTRm) exhibited no significant toxicity in yeast.
Impact:
- Successfully constructed a functional bait plasmid (PRH3'-hTRm) for the yeast three-hybrid system.
- The non-toxic nature of the plasmid facilitates its use in studying hTR interactions.
- Provides a valuable tool for investigating telomerase biology and its role in cellular processes.
