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DOC-1R protein inhibits cell cycle S phase replication and CDK2 sequences affect its nuclear translocation preliminary study

Author: LiuZuo
Tutor: ZhangXue
School: China Medical University
Course: Cell Biology
Keywords: DOC-1R CDK2 DNA replication Centrosome duplication Into the nuclear translocation
CLC: Q26
Type: PhD thesis
Year: 2004
Downloads: 78
Quote: 1
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Abstract

Foreword cell cycle regulation and its relationship with cancer cell biology is currently a hot research. Which cyclin-dependent kinase (cyclin-dependent kinase, CDK) and cyclin (cyclin) complex formation, activation and inactivation of cell cycle regulation major molecular basis. CDK2 is the CDK family, an important member, it is directly involved in cell cycle G ₁ / S restriction point, S phase DNA replication and centrosome duplication regulation of its activity and functional regulation of the cell cycle operation The key link with the cancerous cells have a very close relationship. DOC-1R (Deleted in.oral cancer-1related) gene is a Professor Zhang Xue et al found a candidate tumor suppressor gene, in vitro experiments show that DOC-1R protein capable of CDK2 protein binding, and its intracellular may inhibit CDK2 protein into the nucleus transhipment activated by physiological function, so DOC-1R may be CDK2 specific inhibition factor (CDKinhibitor, CDI). We combine transfection techniques and indirect immune fluorescence technique, observe the the intracellular DOC-1R transfected gene expression For the DNA replication as well as centrosome replication the impact of. In addition, we on CDK2 - protein sequence and its into the nuclear transshipment process relations conducted a preliminary analyzed, and was constructed in-situ tracer of exogenous purpose gene expression the tools Xing carrier of pZX, transfected cells after the This vector has the to make the destination protein with the cell membrane marker protein simultaneously and independently express advantages, and to work with flow cytometry, indirect immunofluorescence technique combined for further research DOC-1R protein intracellular localization for CDK2 in cell cycle regulation and its role in providing an experimental basis. Experiment 1. pEGFP-CDK2, pEGFP-CDK2N and pEGFP-CDK2C recombinant vector pCMV-CDK2 with vector DNA as a template, respectively, through different primers by PCR amplification of wild-type and two kinds of CDK2 gene deletion in the 5 'and 3' each containing Hind Ⅲ and BamH Ⅰ restriction sites. With restriction endonuclease Hind Ⅲ and BamH Ⅰ were digested pEGFP-C3 vector and purified by phenol / chloroform extraction of the PCR product, the fragment was recovered by T4 DNA ligase to form a recombinant vector. Transformed by colony PCR screening positive colonies. PCR positive colonies, plasmid, with restriction endonuclease Hind Ⅲ and BamH Ⅰ for restriction enzyme digestion, using Sanger dideoxy chain termination method for further sequence analysis, detection of wild-type and two kinds of deletion type CDK2 gene inserted fragment sequence and the open reading frame is correct. 2.CDKZ protein sequence with human nuclear transfer process between the initial analysis will pEeFP an en coarse, pEe namely a eDKZN and pEGFp a CDKZC recombinant vectors were transfected into human cervical cancer cell line HeU and Chinese hamster ovary cell line CHO, take serum starvation and to add nuclear celecoxib acid reductase inhibitor method using cell cycle synchronization after transfected with pEGFP-CDKZ cells as the control group transfected with pEGFP a CDKZN and pEGFP a CDKZC cell as the experimental group, respectively, of its fluorescence localization observation, photography and analysis. 3 In situ tracer of exogenous gene expression instrumental carrier PzX construction and remodeling applications of recombinant DNA technology to Srnal and BamHI digestion pIRESne. Vector obtained IRES ligated to peDNA3 the EcoR WBamHI restriction sites, to form recombinant vector peD-NA3 an I rejection s. Synthesis HRAsc20A and HRAsc20B oligonucleotide fragments hybridized to connect into pEGFP-C3 vector Hindlll and BamHI restriction sites, to form a recombinant vector pEGFprascZo. In pEGFprase20 as a template, pCR amplification EGFPrascZO fragment and conventional cloned into peDNA3 an IRES vector Notl / xbal restriction sites by restriction analysis and sequencing recombinant vector pZX construction. For pZX cloning site alterations, we BamHI restriction sites of insertion of the hybridized double-stranded oligonucleotide Xi acid fragment of pzx carrier added EcoRI and EcoRV two restriction sites, to improve the cloning of exogenous fragments flexibility for future restructuring of different target genes provides a convenient. 4.DOC an IR gene expression studies for the effects of DNA replication in a DOC an IR pFLAG vector transfected Heu cells after transfection and cultured overnight. To contain 20 HS expansion nilB remember U of culture medium after lh, indirect immunofluorescence staining. Observed DOC an IR expression (FITC coloration) of the cell's DNA replication (rhodamine coloring) conditions, and FITC Unshaded cells DNA. Replication were compared by statistical process determines the experimental data significance. 5. DOC an IR Gene expression is fundamental to centrosome replication Influence of in order to pFLAG-CMv a BAP, pFLAG an DOC an IR vectors were transfected transfected CHO cells, transfected transfection the end of the use after containing 4mM HU the culture liquid for cell cycle synchronization Approach to the Cultivation , 40h after indirect immunofluorescence staining. Transfected with pFLAG a CMV a BAP vector cell control transfected pFLAG a DOC an IR carrier cell as the experimental group, the count in the transfected gene expression (FITC coloration) in the case of the experimental group and control group Replication Center body (rhodamine coloring) the number of cells and not for centrosome duplication number of cells (centrosome) 2 that the central body is a copy), and for xZ tests to determine the experimental data significance. Experimental results 1. PEeFP a eDKZ, pEGFP a CDKZN and pEGFP a CDKZC recombinant vector with restriction endonuclease Hind classes and BalrlHI the recombinant vector is digested, and thus the recombinant vectors were identified. When a target gene inserted when, pEGFP a CDKZ carrier can be digested into two approximately 0.gkb and 4.skb fragments, pEGFP a CDKZN and pEGFP a CDKZC respectively, can be digested into two approximately 0.6kb and 4. , skb fragment. Electrophoresis results with the expected results exactly. DNA sequencing results showed that the wild-type and two kinds of deletion CDKZ gene sequence and the open reading frame entirely correct, and with EGFP fusion gene. Containing the wild-type CDKZ gene vector was named pEGFP a CDKZ, missing the C-terminal 97 amino acids and the N-terminal 97 amino acids of the recombinant vectors were named pEGFP a CDKZN and pEGFP a CDKZC. 2. CDKZ protein structure and human nuclear transfer process between the preliminary analysis of HeLa cells and CHO cells through the cell cycle synchronization after the green fluorescence was observed under fluorescent microscope intracellular distribution. The transfected with pEGFP a CDKZ cell green fluorescence was indemnity

DOC-1R protein inhibits cell cycle S phase replication and CDK2 sequences affect its nuclear translocation preliminary study

Abstract

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