Telomeric DNA Identificated And Its Regulatory Proteins In T. Vaginalis

Trichomonas vaginalis(T.vaginalis)causes urogenital system infections in humans.It can not only cause trichomoniasis and cervicitis in women,but also urethritis and prostatitis in men.It could also infect mice and squirrel monkeys.It was a danger factor to the health of people and animal,which was an important parasitic protozoa.There are no effective method of prevention and treatment,the reason is due to little knowledge of the molecular and cellular biological characteristics of T.vaginalis.Telomere is a special structure at the end of eukaryotic chromosomes,is composed of tandemly repeated DNA and Telomeric binding proteins,which is regulated by telomerase.In most protozoa,the telomeric DNA and its related protein,such as Giardia lamblia,Cryptosporidium parvum,Leishmania major and Trypanosoma brucei have been studied.However,as the earliest branching eukaryotes,T.vaginalis telomeric DNA has not been reported,and based on the bioinformatics analysis has not basic domain structure of telomerase in T.vaginalis.Therefore,we studied on telomeric DNA and telomere regulated proteins in T.vaginalis.As followed:Determination of Telomeric DNA:Using S1 exonuclease and Mbo I restriction endonuclease to cleave T.vaginalis genome DNA and subject into pUC18 vector,and obtain the repeat sequence(TTTTAGGG)n,the length is about 2000 bp.By BAL 31cleavage and FISH demonstrated that the repeat sequence can be located in the T.vaginalis chromosome ends.Interaction between telomeric DNA and telomeric binding proteins TRF and TBP:Construction of prokaryotic expression of telomere binding protein pET-TRF and pET-TBP.Western blot and IIF combined with FISH analyzed the localization of TRF and TBP in T.vaginalis.The interaction between TRF and TBP and telomeric DNA in vitro was detected by EMSA.Western blot showed that both TRF and TBP were expressed in the nucleus,as the low expression nuclear protein.IIF and FISH combination experiments demonstrated that both TRF and TBP were able to co-location with the telomeric repeats DNA in T.vaginalis.Gel retardation bands in EMSA show that both TRF and TBP were capable of binding telomere double stranded DNA in vitro.Effects of TRF and TBP on telomeres DNA:TRF and TBP of Hammerhead ribozyme of TRF-HR and TBP-HR targeting TRF and TBP was constructed to study the effects of TRF and TBP on telomeres DNA in vivo.In vitro cleavage experiments showed that TRF-HR and TBP-HR were effective in cutting TRF and TBP transcripts in vitro,with efficiencies of 89.4%and 92.1%,respectively.After electroporation,TRF-HR and TBP-HR were detected by quantitative PCR,and TRF and TBP could also be cut in vivo.The cutting efficiency was 82.6%and 85.2%,respectively.Western,blot analysis,TRF and TBP were also significantly reduced at the level of protein expression.Southern blot reavealed that the length of telomeric DNA repeats increased as TRF and TBP decreased,suggesting that TRF and TBP,play a negative regulatory role in telomeric DNA extension.Effects of SPP on telomeric DNA:Construction of prokaryotic expression of pET-SPP,EMSA determined the relationship between SPP and telomeric DNA in vitro.Construction of hammerhead ribozyme SPP-HR to study the effects of SPP on telomeric repeat DNA sequences in vivo.The retardant band in EMSA shows that SPP can bind telomeric double stranded DNA in vitro.In vitro cleavage experiments showed that the cleavage efficiencies of SPP-HR on SPP transcripts was 89.5%.Electroporation transfection into T.vaginalis showed that SPP-HR could still cleave SPP in vivo,and the cutting efficiency was 84.6%by quantitative PCR analysis.Western blot also showed that SPP were significantly reduced in protein expression levels.Southern blot showed that the telomeric DNA repeat length was shortened when SPP mRNA and protein expression levels decreased,indicating that SPP played a positive regulatory role in telomeric DNA extension.Interaction between PIKK and SPP and effects of PIKK on telomeric DNA:Construction of prokaryotic expression of pGEX-PIKK and BiFC vector,which was inserted into the T.vaginalis alpha-SCSB promoter.GST-Pull down and BiFC verified the interaction between PIKK and SPP.Construction of hammerhead ribozyme PIKK-HR to study the effects of PIKK on telomeric repeat DNA sequences in vivo.GST-pull down results showed the binding bands of recombinant PIKK and SPP proteins.The BiFC detected red fluorescence more intuitively,thus confirming the interaction between PIKK and SPP in T.vaginalis.In vitro cleavage experiments showed that the cleavage efficiencies of PIKK-HR on PIKK transcripts was 91.2%.Electroporation transfection into T.vaginalis showed that PIKK-HR could still cleave PIKK in vivo,and the cutting efficiency was 86.7%by quantitative PCR analysis.Western blot also showed that PIKK were significantly reduced in protein expression levels.Southern blot showed that the telomeric DNA repeat length was shortened when PIKK mRNA and protein expression levels decreased,indicating that PIKK played a positive regulatory role in telomeric DNA extension.The present study demonstrated that,without telomerase,the elongation of the telomeric DNA(TTTTAGGG)_n of T.vaginalis can be negative regulated by TRF and TBP and positive regulated by PIKK and SPP.It provides a new way for further study of the mechanism of the regulation of telomers,the molecular biological characteristics and the immortalization of T.vaginalis.