Effect of motor domain of CENP-E on cell cycle and separation of chromosome
AIM: To construct a mammalian expression vector containing the motor domain of human CENP-E protein and to observe its influence on the separation of chromosomes. METHODS: cDNA sequences encoding the motor domain of CENP-E were amplified through RT-PCR and cloned into the enhanced GFP fluorescence protein ( EGFP) expression vector. The effect of recombinant plasmids,namely pEGFP-CENPE1 (1-336aa) and pEGFP-CENPET1(1 -336 and 2078 -2492aa) was observed respectively on the separation of chromosomes by FCM and karyotype analysis. RESULTS: Two recombinant vectors,namely pEGFP-CENPE1 and pEGFP-CENPET1 were constructed correctly and their expressions in HEK293 cells were confirmed respectively. Results from both FCM and karyotype analysis showed that the cells transfected with the plasmid expressing the motor domain of CENP-E could not maintain the midphase and exited mitosis ahead of schedule when treated with reagent destructing the tubulin. The abnormal chromosomal separation led to increased aneuploid cells. CONCLUSION: The over expression vectors containing the motor domain of CENP-E cannot maintain the midphase. Chromosome misseparation occurs and leads to aneuploid cells.
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万方数据
维普
