Construction Of Screening Model For Telomerase Inhibitors Contained Human Telomerase RNA Mutant

Background&Objective:Human telomerase is a protein complex consisting of a human telomerase reverse transcriptase catalytic subunit that uses the human telomerase RNA component of the complex as a template for adding the missing repeated sequence during the DNA replication.Telomere shortening occurs in normal somatic cells reaching a point in which cell senesce.On the contrary,telomerase activity is present in eighty percent of cancer cells.For this reason,telomerase as a target for screening specific telomerase inhibitors becomes to be the research hotspot recently in order to discover new antitumor drugs.Heretofore,there is no specific screening model for telomerase inhibitor.Our studying team designed the yeast three-hybrid system model to research the interaction between telomerase reverse transcriptase(hTERT) and hTR which acting as template.Both of them are the main component of the telomerase.Two function parts of the yeast transcription factor are artificial separated.There are LexA(DNA-Binding Domain) and Gal4-AD(transcription activation domain). Among them,LexA binds to hTR via MS2 coat protein(bait) and Gal4-AD binds to hTERT(prey).Due to the spicfic combination of hTR and hTERT,the two function parts of the yeast transcription factor can be closed each other to direct expression of reporter gene.But,when there exist the inhibitors(Is) that hinder the combination of hTR and hTERT,the reporter gene can not be expressed.Through selective culture, we can filter the specific inhibitors that against the combination of hTR and hTERT.In this study,we made mutiple point mutations of hTR to construct the bait plasmid of the mutant,because some sequences of hTR will affect the normal transcription.And then,the bait plasmid containing hTR mutant co-transformed with the prey plasmid containing hTERT into yeast host cell.Finally,we successfully constructed the human telomerase inhibitors screening model based on yeast threehybrid system which focus on the interaction between telomerase reverse transcriptase(hTERT) and hTR which acting as template.Methods:1.Site directed mutagenesis of hTRAccording to the three places of four or more T in succession in hTR sequence and the scecond structure of the hybrid RNA,three mutation sites were designed to ensure to disrupt a string of Ts that was observed to cause transcriptional termination. We should make sure the mutant hybrid RNA secondary structure has not been great changes and the MS2 RNA'stem-loop structure of the hybrid RNA still exist independently.In other words,that is to say mutations do not affect the binding ability of the hybrid RNA with the other two hybrid proteins in the three hybrid system,and can also co-activate the RNA polymerase to transcribe the report gene. On the basis of all above requirements of the problem,making mutations at the the 41st(M41),the 80th(M80) and the 102nd(M102) of the hTR by using overlapping extension PCR(OE-PCR).In these places,nucleotide T are substituted by A.The product(hTRm) was cloned into an AT cloning vector PMD18T,the ligation product transformed into DH5αE.Coli bacteria.And then,we screened the positive clones and extracted plasmid PMD18T-hTRm by using plasmid extraction kit,and finally had it sequenced by Invitrogen on a DNA analyzer.2.Construction of the human telomerase inhibitors screening model based on the yeast three hybrid system(1) Check the phenotype of yeast strain L40 ura3/pHyblex/Zeo-MS2 and do self-activation testTo check the phenotype of the strain by streaking cells on plates supplemented with the appropriate amino acids and also deficient in histidine,tryptophan,uracil, adenine,or leucine.To observe the growth of yeast strain L40 ura3/pHyblex/Zeo-MS2 on these plates.And what's more,using an overlay assay to detectβ-galactosidase reporter activity.(2) Construction of bait plasmid containing human telomerase RNA mutantBoth of the plasmid PMD18T-hTRm and PRH3' are double-digested by restriction enzyme AvrⅡas well as AatⅡ.Then the products of digestion were connect by T4 DNA ligase.The recombined plasmid identified with PCR and restriction enzyme digestion.Then,the successfully recombined plasmid PRH3'-hTRm were introduced in yeast L40ura3/pHyblex/Zeo-MS2,observed the growth of the yeast on plates which deficient in histidine,tryptophan,uracil,adenine,or leucine.At last,we also check its toxicity.(3) Resistance analysis of 3-Amino-1,2,4-triazole(3-AT)Plasmids PRH3' co-transformed with pYESTrp3 into yeast strain L40 ura3/ pHyblex/Zeo-MS2.Positive control plasmids pRH3'/IRE and pYESTrp3/IRP as also.Then,streaking the cells on plates YC-H that contained 3-AT with different concentrations.Observe the growth of yeast strain on these plates in order to decide the concentration which can eliminate the false positive clones at maximum extent. (4) Primary construction of the human telomerase inhibitors screening model based on the yeast three hybrid systemPrey plasmid pYESTrp3-hTERT(1-608),pYESTrp3-hTERT(17-593), pYESTrp3-hTERT(292-608) or pYESTrp3-hTERT(292-952) co-transformed with bait plasmid PRH3'-hTRm into yeast strain L40 ura3/pHyblex/Zeo-MS2.Then, streaking the yeast strain on plates supplemented with the appropriate amino acids and also deficient in histidine,tryptophan,uracil,adenine,or leucine.Observe the growth of yeast strains on these plates.(5) Analysis ofβ-galactosidase reporter activityUsing an overlay assay to detectβ-galactosidase reporter activity.In this way, we can make preliminary screening of positive clones.(6) Repeatedly subculturing to exclude false positive clones Repeatedly subculturing three times.After each subculture,we should make analysis ofβ-galactosidase reporter activity.(7) Screened positive clones by using colony PCRChoose the positive clones from the former phase,using colony PCR to make the next step of the screening.Cracked the colnes by 100℃water bath for 10 minutes. And then,fetching the centrifugal supernatant as a template for PCR to verify whether the bait plasmid PRH3'-hTRm has transformed into yeast strain L40 ura3/ pHyblex/Zeo-MS2 successfully.On this basis,we verified whether the prey plasmid contained hTERT has transformed into yeast strain L40 ura3/pHyblex/Zeo-MS2 or not by using colony PCR the second time.Results:1.Site directed mutagenesis of hTRCompared the mutant with the original sequence,we can find that nucleotide T at the sites of the 41st,80th,and 102nd have successfully mutated into A.This indicated that we have successfully conduct the expected mutations and no unexpected random mutation occurred.2.Construction of the human telomerase inhibitors screening model based on the yeast three hybrid system(1) Check the phenotype of yeast strain L40 ura3/pHyblex/Zeo-MS2 and do self-activation testYeast strain L40 ura3/pHyblex/Zeo-MS2 can not grow on plates YC-U,YC-W, YC-H,YC-UWH.L40 ura3/pHyblex/Zeo-MS2 grown on YPD medium containing no adenine give red colonies,but that grown on YPAD medium give white colonies. When detecting itsβ-galactosidase reporter activity,the colonies still white,but not turn blue.It confirmed the phenotype of yeast strain L40 ura3/pHyblex/Zeo-MS2 is MATa,ura3-52 leu2-3,112,his3-200,trpl-1,ade2,LYS2::(lexA op)-HIS3,LexA-MS2 coat(TRP1).And it has no self-activation ability.(2) Construction of bait plasmid containing human telomerase RNA mutantPRH3'-hTRm has been successfully constructed.And then,Positive transformation of the recombined plasmids PRH3'-hTRm,and the same size yeast colonies which untransformated with plasmids PRH3'-hTRm,inoculated in the same condition.Finally,we compared the OD of them at 600nm and found that the recombined plasmids PRH3'-hTRm did no harm to yeast host cell.The yeast strain L40 ura3/pHyblex/ZeoMS2 in which has been transformed PRH3'-hTRm can grow on plates YC and YC-U,but can not grow on plates YC-W, YC-H and YC-UWH.This shows that the plasmid PRH3'-hTRm has selectable marker URA3.The transformation of the recombined plasmids PRH3'-hTRm make the auxotype of yeast strain turn into His~-,Trp~-,Ade~-,Leu~-.(3) Resistance analysis of 3-Amino-1,2,4-triazole(3-AT)Yeast strain L40 ura3/pHyblex/Zeo-MS2 transformed with Plasmids PRH3'and pYESTrp3 can grow on the plates YC-H that contained 3-AT When its concentration less than 10mM.Meanwhile,the colonies of the yeast strain transformed with plasmids pRH3'/IRE and pYESTrp3/IRP decreased with the the concentration of 3-AT increased(among the range from 0 to 10mM).When the the concentration of 3-AT more than 10mM,there also have colonies grown,but the colonies has almost no change in quantity.We decide 10mM is the concentration which can eliminate the false positive clones at maximum extent.(4) Primary construction of the human telomerase inhibitors screening model based on the yeast three hybrid systemAll of the yeast strains L40 ura3/pHyblex/Zeo-MS2 transformed with Prey plasmid pYESTrp3-hTERT(1-608),pYESTrp3-hTERT(17-593),pYESTrp3-hTERT (292-608) or pYESTrp3-hTERT(292-952) and bait plasmid PRH3'-hTRm can grow on plates YC-UZ,YC-WZ,YC-HZ,YC-UWHZ.(5) Analysis ofβ-galactosidase reporter activityIn addition to the plate with negative control,all of the plates have blue colonies grown on.(6) Repeatedly subculturing to exclude false positive clonesRepeatedly subculturing three times.After each subculture,making analysis ofβ-galactosidase reporter activity.Eventally,we obtained 21 positive clonies.Among them,8 colonies of pYESTrp3-hTERT(1-608)+PRH3'-hTRm,5 colonies of pYESTrp3-hTERT(17-593)+PRH3'-hTRm,5 colonies of pYESTrp3-hTERT(292-608)+PRH3'-hTRm and 3 colonies of pYESTrp3-hTERT(292-952)+PRH3'-hTRm.(7) Screened positive clones by using colony PCRAfter two times of colony PCR,we obtained 6 positive clonies.Among them,1 colonies of pYESTrp3—hTERT(1-608)+PRH3'-hTRm,2 colonies of pYESTrp3-hTERT(17-593)+PRH3'-hTRm,2 colonies of pYESTrp3-hTERT(292-608)+ PRH3'-hTRm and 1 colonies of pYESTrp3-hTERT(292-952)+PRIt3'-hTRm.Conclusion:1.The recombined plasmid of the human telomerase RNA mutant(PRH3'-hTRm) was constructed successfully and verified that can be used as bait plasmid in yeast three-hybrid system.2.This experiment has successfully constructed the human telomerase inhibitors screening model based on yeast three-hybrid system which focus on the interaction between telomerase reverse transcriptase(hTERT) and hTR which acting as template.