A Yeast 3-hybrid System For High-throughput Screening Of The Human Telomerase Inhibition

Background: Eukaryotic chromosomes are telomere 3 'end which is composed of the G-rich DNA repeat sequences and telomere-binding protein (telomere binding proteins, TBPS), like the end of the metal shoelace knot. The main function is to maintain chromosome stability and to prevent the occurrence of chromosome loss, rearrangements, terminal fusion and degradation, such as enzymatic digestion. When normal cell replicate, the telomeres were shortened about 50-100 bp after each cell division, because of the DNA polymerase can not complete replication of linear DNA . When the telomeres shortened to a certain extent, DNA is no longer stable solution, the double helix opened, the cells stop dividing and beginning to death. This is the normal cell's cause of there own genetic programming decisions of aging and death. Because the majority of malignant cells can activate telomerase (telomerase) and can fill up some telomere shortening, the cells divist without the telomere shortening. The telomerase become an important identification symbol of tumor cells between normal cells. Studies have shown that over 90% of malignant cells can activate telomerase, telomerase therefore become a tumor diagnosis and the design of new anticancer drug targets. set up screening tumor inhibitor model, at the molecular level on screening for telomerase inhibitors, has become a new ways to antineoplastic agents. Human telomerase consists of three components constitute: telomerase RNA (Telomerase RNA Component, TR), telomerase reverse transcriptase (Telomerase Reverse Transciptase, TERT) andtelomerase-associated protein 1 (Telomerase associated protein, TEP1 ).Telomerase activity has the smallest unit of hTERT and hTR, telomerase reorganization activity required the correct assembly of these two parts. The combination of hTERT and hTR liked as enzyme and substrate combination, at S phase cell cycle combinated and showed telomerase activity, the other phase was separated. This combination is reversible, susceptible to inhibitors impact. Therefore, if the screening model aims to the combination of hTR-hTERT, we can greatly improve the specificity of screening. Because hTR is RNA and degrade quickly, the traditional methods of RNA - protein interaction can not be easily carried out at the laboratory, and SENGUPTA, such as the yeast RNA three-hybrid system is ingeniously taken synthesis of RNA in cells and Research RNA molecules strategy is simple, but also provides a eukaryotic cell in vivo. Wan King, which can be used to be the best vector studying the protein and RNA. In the yeast three-hybrid system, the only variable thing is that we need to study the unknown protein and RNA interactions for their interactiongs is known .As long as there is interaction between them which can be used to activate reporter gene transcription, through the reporter gene phenotypic characteristics to filter positive clones. Conversely, if the study of the combination of RNA and protein are known, such as hTR-hTERT combination, the system can be used to study the factors of the hTR-hTERT resulted in dissociation, or as the drags screening model to screening inhibit hTR-hTERT combination. When the factors of inhibition combination of both (combination of hTERT and hTR competitive inhibitor), reporter gene can not be expressed. And the system is able to accommodate automation of point-like high technology, to set up high-throughput drug screening model. This study is that accordance with the principle of yeast RNA three-hybrid, firstly finding the interaction between hTR and hTERT fragments and then using these specific fragments to building a high-throughput fragment hTR-hTERT-specific inhibition of telomerase inhibitors combined with drug screening model for the specific screening of telomerase inhibitors.Objective:1. To build the prey plasmids expressing fragments of hTR and bait plasmids expressing fragments of hTERT.2. We build a good plasmid cotransfection yeast and study hTR and hTERT fragment combination using yeast three-hybrid principle.3. We cotransfect yeast by using exist hTR and hTERT interaction plasmid and build high-throughput yeast three-hybrid system (Y3H) with the best use of building a high-throughput point-like technology.Methods:1. Yeast phenotype Detect and self-activation test: use the defective mixed amino acid [tryptophan (Trp), leucine (Leu), histidine (His)] yeast strains to Detect nutritional needs of YBZ, L40 ura3.2. Different bait vector and transformation: human telomerase RNA (hTR) gene was amplificated from the tumor cells by the method of RT-PCR. Three loci were Site-directed mutagenesis. Site-directed mutagenesis were amplified after the false combination of hTR gene Area, which are CR4-CR5 and H / ACA, CR7 District hTR gene fragment .We clone the separation and purification pRH3 bait plasmid and construct that contains different hTR gene fragment of recombinant plasmid and transformed into E. coli .By using PCR, restricting enzyme digestion, sequencing will be positive recombinant plasmid transformed into yeast which can be the activation tested for toxicity.3. Different prey expression vector and transformation: We access hTERT cDNA fragments comprising RID1, RID2, RT, C-terminal areas from pCLXSN-hTERT plasmid, separately by PCR which can be cloned into the separation and purification pYESTrp3 prey plasmid. Then we construct gene fragment containing the different hTERT of recombinant and plasmid into E. coli DH5a and screening positive clones after the initial identification. After the expansion of cultivation and extraction of plasmid by double enzyme digestion DNA sequencing,we make the positive recombinant plasmid transformed into yeast and test the activation for toxicity.4. Different bait and prey plasmid cotransfected yeast by the permutation and combination to confirm hTERT and hTR interactions in yeast with the way of defects in growth.5. Using automation technology in recombinant yeast best yeast three-hybrid L40 ura3 (transfected with hTERT and hTR in RID1 binding domain of the false L40ura3R₁, transfected with hTERT and hTR in RID2 the CR4-CR5 District L40ura3R₂, transfected with hTERT and the RID1 false RID2 and hTR-binding domain and the CR4-CR5 District L40ura3R₃) to use for high-throughput screening of telomerase-specific inhibitors, seting up Y3H system.Result:1. The results showed that yeast strains YBZ, L40 ura3 gene phenotype are stability and grow gooin in the whole medium (YPD, YC) but can not grow in single-deficient medium (-Leu,-Trp,-His). When put YBZ, L40 ura3 yeast strains crossed in a single colony containing 0, 2.5, 5, 7.5, 10, 12.5, 15mM different 3-AT concentration of SD /-His agar plate, 30℃for 3 days , YPD culture can based on, YBZ growth should not and L40 ura3 yeast strains have a small amount of colony growth. IT tips YBZ is no leakage gene expression, while yeast strains L40 ura3 screening required plus 3-AT. 2. Different recombinant plasmid pRH3-hTR by Aat II, Avr II dual-enzyme digestion with the prediction results. The sequencing results confirmed that hTR gene fragments with different bait pRH3-hTR recombinant plasmid was successfully constructed and transformed yeast after the non-toxic and self-activated.3. Recombinant plasmid pYESTrp3-hTERT by Eco R I , Hind III restriction enzyme are correspond with prediction results. Sequencing results showed that the prey pYESTrp3 -hTERT fusion protein expression vector of gene sequence homology of 99% more than the correct reading frame. After transformation of yeast non-toxic and self-activated are exited.4. Different bait and prey plasmid by the permutation and combination cotransfectedyeast which are as a result of prediction. hTERT and the RID1 District District RID2 respectively are exited hTR false combination of district and CR4-CR5 interacted with each other.5. The use of three recombinant yeast setting up high-throughput system can be used for screening specific telomerase inhibitors.Conclusion:1. The two core components hTERT and hTR of telomerase of the human are combined as the only variable to build a specific telomerase inhibitor high-throughput screening model by using RNA three-hybrid theory for the first time.2. We fond a simpler and more precise technical methods to illustrate the combination of hTERT and hTR and take advantage of this method to find the fragment of hTR and hTERT interaction in yeast.3. The three recombinant yeasts can be used as setting up high-throughput system screening specific telomerase inhibitors.