| The first SMC protein was reported in 1993. The SMC proteins (Structural maintenance of chromosome) are members of a newly found protein family. They are involved in multiple aspects of chromosome dynamics, such as mitotic chromosome condensation and segregation, sister chromatid cohesion, sex-chromosome dosage compensation and DNA recombination and reparation. The finding of SMC proteins is a great breakthrough in the study of higher-order chromosome structure. Until now, there are at least six SMC protein subfamilies in eukaryotes and they are named SMC1 SMC2, SMC3, SMC4, SMC5, and SMC6 subfamily. Two different SMC proteins from these subfamilies can form three kinds of heterodimers: SMC2-SMC4, SMC1-SMC3 and SMC5-SMC6. These heterodimers serve as the core components of the whole functional complexes (such as Condensin) involved in the dynamics of higher order chromosome structures.Condensin is composed of one SMC2-SMC4 heterodimer and three non-SMC subunits. It involves in the process of mitotic chromosome condensation and segregation. Human condensin consists of two SMC proteins (hCAP-E and hCAP-C) and three non-SMC subunits (hCAP-G,hCAP-D2 and hCAP-H). hCAP-E and hCAP-C form a heterodimer and function as a core component of human condensin. What roles of these individual subunits contribute to the function of condensin is not very clear and thus is the focus of the current research.This report explored the function of the human SMC subunits-HCAP-E and hCAP-C using RNAi and transfection techniques. We constructed two RNAi plasmids-RHE and RHC that specifically corresponded to hCAP-E and hCAP-C of human condensin, respectively. At the same time, the C-terminal expressing plasmids of hCAP-E andhCAP-C that attached the Kozac sequence and the nuclear localization signal were also constructed. Using the Superfect reagent, in vitro cultured Hela cells were introduced with external plasmids. To identify the transfected cells, we also constructed the GFP expressing plasmids which also attached the Kozac sequence and nuclear localization signal to the GFP gene and the plasmids served as a selective marker for the transfected cells. The effectiveness of the nuclear localization signal selected was clearly tested in initial experiments. Based on this, the co-transfection systems used in the study was established.We first observed the multinucleate cells and chromatin bridges in cultured Hela cells when the cells are marked with GFP and Hochest33342, after co-transfection with the GFP expression plasmids and RNAi plasmids RHE or RHC. The results from RT-PCR indicated the knockdown of mRNA level of hCAP-E and hCAP-C in the co-transfeted Hela cells. We also conducted the co-transfection with the RNAi plasmids RHE and RHC and the selective marker GFP expressing plasmids simultaneously. There are three kinds of combination probability that could be observed by GFP marker. They are combination of co-transfection with RHE and GFP expressing plasmids, co-transfection with RHC and GFP expressing plasmids and co-transfection with plasmids RHE and RHC and GFP expressing plasmids at the same time. Whichever the combination may be, we always observed the phenomena of chromatin bridge in co-transfected cells. These results indicate that the phenomena of chromatin bridge should be the characteristic feature of deficiency in the human SMC subunits.After marking with GFP and Hochest33342, we also observed the multinuclei and chromatin bridges in Hela cells that were transfected with the C-terminal expressing plasmids corresponding to the human SMC subunits hCAP-E and hCAP-C, respectively. The results from RT-PCR indicated that the C-terminal expressing plasmids corresponding to the human SMC subunits hCAP-E and hCAP-C, respectively, overexpressed in the co-transfected Hela cells. When the internal expressing level of hCAP-E was compared between Hela cells that were transefected withGFP expressing plasmids and Hela cells that being co-transfected with the GFP expressing plasmids and C-terminal expressing pla...
|
PDF Full Text Request