Study The Function Of The Structural Domain Of CENP-E Protein On The Spindle Assemble Checkpoint

It is very important that chromosomes segregation ensures only when all the chromosomes have congressed to the metaphase plate to ensure the even partition of the genetic material into the two daughter nuclei.This is accomplished by a surveillance mechanism known as the spindle assemble checkpoint.The aneuploidy emerge even to induce tumorigenesis when the spindle assemble checkpoint dysfunction,so that it is very important to investigate spindle assemble checkpoint in order to explore the mechanism of tumorigenesis.Recently,more and more researcher pay attention to the mechanism of inactivation of spindle checkpoint,because the incorrect inactivation of spindle assemble checkpoint can lead to segregation of chromosome and induce anuepoliy present.There is close relationship between the mechanism of inactivation of spindle assemble checkpoint and Mps1 and CENP-E protein.In order to further understand the function of CENP-E in the spindle assemble checkpoint,we amplified the cDNA of CENP-E gene from the Hela cell line,and constructed six recombinant vectors containing the structural domain of CENP-E protein.Analysis the effect of structural domain of CENP-E protein on the spindle checkpoint was carried out by flow cytometrics,and we also measured the MI and ratio of cell death,and the ratio of aneuploid cells and Explored the interaction between the Mps1 protein and the structural domain of CENP-E protein by fluorescence resonance energy transfer(FRET) and co-immunoprecipetation method,we want to analyze the role of the structural domain of CENP-E protein in the spindle assemble checkpoint,these research will lay a foundation for further studying the mechanism of inactivation of spindle assemble checkpoint.PARTâ… CONSTRUCTING THE RECOMBINANT VECTORS OF THE STRUCTURAL DOMAIN OF CENP-E PROTEINObjective:1.Amplifying the cDNA of CENP-E.2.Constructing the recombinant vectors of the structural domain of CENP-E protein3.Constructing the prokaryotic expression vector containing the phosphorylation sites of CENP-E and full length of Mpsl protein,and purification of the recombinant proptein.Methods: 1.Amplifying the eDNA of CENP-E gene from the total RNA of Hela cells by RT-PCR method.2.Constructing recombinant vectors containing the structural domain of CENP-E.3.Analyzing the expression of recombinant vectors in the HEK293 cells using inverted fluorescence microscope.4.Identifying the recombinant protein collecting the HEK293 cells transfected with recombinant vectors respectively using western blot method(the anti-GFP monoclonal antibody).5.Constructing prokaryotic expression vectors and purification of recombinant protein.Results:1.The sequence of RT-PCR products was different from the sequence submitted by T J.Yen(ACCESSION:NM₀01813),which amplified from Hela cells,however sharing 99%homology with the sequence of the variant CENP-E protein.2.The location of recombinant protein expressed in the HEK293 cells was consistent with expected results.3.The molecular weight of recombinant proteins detected with western blot was consistent with the anticipated.4.The molecular weight of fusion protein purified by Ni2+-NTA agarose column was correct.Purity of recombinant protein was above 92%. Conclusion:1.We successfully amplified the cDNA of CENP-E gene,which was consistent with the sequence of the variant CENP-E protein and had distinct diversity with the sequence submitted by T J.Yen,which amplified from Hela cells too.2.We successfully constructed the recombinant vectors containing the structural domain of CENP-E protein.3.Prokaryotic expression vectors were constructed correctly and we get the high purity fusion protein.PARTâ…¡PRELIMINARY STUDY THE EXPRESSION OF CENP-E GENE IN THE TUMOR TISSUES AND CANCER CELL LINESObjective:1.Analyzing the expression of the mRNA of CENP-E gene in the Hela cells2.Analyzing the expression of the mRNA of CENP-E gene in two kinds of carcinoma tissues(hepatocellular carcinoma and renal carcinoma) and peri-cancerous tissues,and several kinds of cancer cell lines (Hela,T24,A549,U251,HepG₂)。3.Determining whether there is deletion of the 38^th extron in genomic DNA or not.Methods:1.Analyzing the expression of CENP-E mRNA by RT-PCR method in the Hela cells.2.The deletion mutation of CENP-E gene were Studied by PCR method using DNA as template.3.Analyzing the deletion and wild type CENP-E mRNA in carcinoma tissues and peri-cancerous tissues,and cancer cell linesResults:1.Both deletion and wild type mRNA of CENP-E existed in the Hela cells2.There was not deletion mutation of CENP-E gene in carcinoma tissues' and pare-cancerous tissues' DNA,and cancer cell lines' DNA.3.The expression of deletion mutation type of CENP-E mRNA was higher than that of wild type in carcinoma tissues and cancerous cell lines,the expression in peri-cancerous tissues was just the reverse.Conclusion:Two even more type mRNA of CENP-E gene co-existed in the same cell,CENP-E variant was associated with tumorigenesis. PARTâ…¢EFFECT OF THE STRUCTURAL DOMEIN OF CENP-E PROTEIN ON THE SPINDLE ASSEMBLE CHECKPOINTObjective:1.Examined the relationship between the structural domain of CENP-E protein and inactivation of the spindle assemble checkpoint;2.To observe whether kinetochore dependent of CENP-E play its role on the spindle assemble checkpoint or notMethods:1.HEK93 cells were cultured in the 6 cells plate;cells were transfected with recombinant vectors and p EGFP-C1 vector respectively.2.HEK293 cells were synchronized by double thymidine,the majority of cells were arrested in G1/S phase,cells cultured 7 hours using fresh medium after removed thymidine,then HEK293 cells treated by nocodazole 2 hours.3.Flow cytometry was used to analysis the percentage of G2/M phase of the 7^th and 9^th HEK293 cells.MI assay and trypan blue exclusion was employed.4.The influence of the structural domain of CENP-E protein on chromosome segregation was evaluated by chromosome numbers and counts of the chromosomes. Results:1.The majority of HEK293 cells arrested in G1/S phase treated double thymidine,after 7 hours removed thymidine,the majority of cells enter G2/M phase.2.Results of FCM showed the percentage of G2/M phase was decreased after treated by nocodazole in the cells transfected with pEGFP-E1 and pEGFP-ET1 vector.In contrast,the percentage of G2/M phase of other group was increased in the situation.The results of MI were consistent with the results of FCM.3.Results of trypan blue exclusion assay showed the ratio of cell death was increased after being treated by nocodazole in the cells transfected with pEGFP-E1 and pEGFP-ET1 vector.The ratio of cell death of other group has no difference compared with control group.4.Counts of the chromosomes showed that ratio of the cells with abnormal chromosomal numbers were significantly increased in cells transfected with pEGFP-E1 and pEGFP-ET1 vector.Conclusion:The motor domain of CENP-E protein is related with inactivation of spindle assemble checkpoint;the role of CENP-E in the spindle assemble checkpoint is kinetochore independent. Partâ…£EXPLORING THE STRUCTRAL DOMAIN OF CENP-E PROTEIN INTERACTED WITH MPS1 PROTEINObjective:Study the structural domain of CENP-E interacted with Mps1 proteinMethods:1.The protein-protein interaction between the structural domain of CENP-E protein and Mps1 protein was studied.2.Results of FRET were confirmed by CO-IP method.Results:1.The CENP-E3 domain of CENP-E(aa:1200-2134) can interacted with Mps1 protein.2.Results were consistent with results of FRET.Conclusion:the Mps1 protein binding domain of CENP-E resided between 1,200 and 2134.Result of FRET can be faithfully confirmed in the studying protein-protein interaction.