ABSTRACT
<p><b>OBJECTIVES</b>To study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1).</p><p><b>METHODS</b>Both HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo.</p><p><b>RESULTS</b>Both HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1.</p><p><b>CONCLUSIONS</b>HBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.</p>
Subject(s)
Humans , Histone Deacetylase 1 , Metabolism , Hydroxamic Acids , Metabolism , Immunoprecipitation , Plasmids , Protein Interaction Mapping , Trans-Activators , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the features of HBx protein distributed in liver cells and its expression in E. coli.</p><p><b>METHODS</b>The expression vectors encoding the full length HBx and its mutants were constructed by the routine molecular cloning method. HBx protein expression was detected using Western blotting. The distribution feature of HBx protein in liver cells was examined using the fluorescence confocal microscopy. A series of purified HBx fusion proteins were obtained by glutathione-sepharose 4B affinity chromatography.</p><p><b>RESULTS</b>The expression vectors were successfully constructed for the full length HBx and its mutants. HBx was found distributed uniformly in the nuclei but granularly in the cytoplasm of the liver cells. Under optimal conditions, the mutant GST-HBx (72-120aa) was easily degraded.</p><p><b>CONCLUSION</b>This study may provide a basis for further study on the biological function of HBx at the protein level.</p>
Subject(s)
Humans , Carcinoma, Hepatocellular , Pathology , Cell Line , Cloning, Molecular , Escherichia coli , Metabolism , Genetic Vectors , Glutathione Transferase , Genetics , Hepatocytes , Cell Biology , Metabolism , Liver , Cell Biology , Liver Neoplasms , Pathology , Mutation , Recombinant Fusion Proteins , Genetics , Trans-Activators , Genetics , Tumor Cells, CulturedABSTRACT
<p><b>OBJECTIVE</b>To study the effects of exogenous ER beta on the growth of breast cancer MCF-7 cells under different treatment.</p><p><b>METHODS</b>An eukaryotic expression vector containing 1.6 kb of human entire coding sequence of ER beta (pCDNA3-ER beta) was transfected into human breast cancer MCF-7 cells using lipofectamine 2000. The biological activity of ER beta was detected with the luciferase reporter containing estrogen responsive element (ERE) and the expression of ER beta protein by Western blot. The growth properties of MCF-7, pCDNA 3-transfected MCF-7 and pCDNA 3-ER beta-transfected MCF-7 cells under different treatment, including E2 (17beta-estradiol) and 4-OHT (4-hydroxytamoxifen), were observed.</p><p><b>RESULTS</b>A stronger activation of the reporter by ER beta in the presence of E2 was observed in the pCDNA 3-ER beta-transfected MCF-7 cells than in the pCDNA 3-transfected MCF-7 and in MCF-7 cells. Western blot analysis showed that the protein level of ER beta in the pCDNA 3-ER beta-transfected MCF-7 cells was markedly increased. Exogenous ER beta expression did not change the growth properties and the morphology of MCF-7 cells under normal condition. The pCDNA 3-ER beta-transfected MCF-7 cells proliferated at the same rate as naive cells in the presence of 4-OHT, whereas a strong inhibition of the proliferation of the pCDNA 3-ER beta-transfected MCF-7 cells in the presence of E2 was observed.</p><p><b>CONCLUSION</b>Exogenous ER beta expression does not increase the resistance to 4-OHT, and a strong inhibition of the proliferation may occur in the presence of E2.</p>
Subject(s)
Female , Humans , Breast Neoplasms , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , Estradiol , Pharmacology , Estrogen Antagonists , Pharmacology , Estrogen Receptor beta , Genetics , Metabolism , Tamoxifen , Pharmacology , TransfectionABSTRACT
A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.
Subject(s)
Bacteriophage lambda , Genetics , DNA, Recombinant , Genetics , Escherichia coli , Genetics , Genetic Engineering , Plasmids , Genetics , Rec A Recombinases , Genetics , Metabolism , Recombinant Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To establish a mouse fibroblastic cell line stably transfected with PC-1 gene, and using such cell line to investigate tumor development and progression imposed by the ectopic expression of PC-1 gene.</p><p><b>METHODS</b>Eukaryotic expression vector pcDNA3.1(-)/myc-his-pc-1 was transfected into mouse fibroblast cell line NIH3T3 by lipofectamine. Stable transfectants were selected by G418. The integration and expression of ectopic PC-1 gene were analyzed by PCR and RT-PCR. Cytomorphological analysis, MTT, soft agar colony formation and nude mice tumorigenesis assay were used to evaluate the effects of PC-1 gene expression on tumor development and progression.</p><p><b>RESULTS</b>NIH 3T3 cell lines stably expressing PC-1 gene were successfully established and confirmed by PCR and RT-PCR analyses of the integration and expression of ectopic PC-1 gene. Comparing with the parental cell line and cells transfected with control vector, the PC-1 gene transfectants acquired several phenotypes of transformed cells: increasing growth rate, ability to grow and form cell colonies on soft agar, and becoming tumorigenic in nude mice.</p><p><b>CONCLUSION</b>Ectopic expression of PC-1 gene in NIH3T3 cells can induce malignant transformation of mouse fibroblastic cells both in vitro and in vivo.</p>
Subject(s)
Animals , Mice , Cell Line, Transformed , Cell Transformation, Neoplastic , Gene Expression , Genes, Neoplasm , Physiology , Mice, Inbred BALB C , Mice, Nude , NIH 3T3 Cells , Neoplasm Proteins , Genetics , Physiology , RNA, Messenger , Genetics , Random Allocation , TransfectionABSTRACT
Estrogen receptor alpha (ERalpha) has been a primary target of treatment as well as a prognostic indicator for breast cancer. The level of human X-box binding protein 1 (XBP-1) mRNA was related with that of ERalpha in breast tumors and was over-expressed in some breast tumors. These previous studies suggested that XBP-1 may interact with ERalpha. XBP-1 has two isoforms, XBP-1S and XBP-1U, as the result of unique splicing. GST pull-down assay showed that both XBP-1S and XBP-1U bound to ERalpha in vitro. The binding of XBP-1S to ERalpha was stronger than that of XBP-1U to ERalpha. Co-immunoprecipitation revealed that the binding was in a ligand-independent manner. XBP-1S and XBP-1U interacted with the region of ERalpha that contains a DNA-binding domain. The ERalpha-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, the N-terminal basic region leucine zipper domain (bzip) and the C-terminal activation domain. These findings suggest that XBP-1S and XBP-1U may participate in ERalpha signaling pathway through the mediation of ERalpha.
Subject(s)
Female , Humans , Breast Neoplasms , Genetics , Metabolism , Cell Line, Tumor , DNA-Binding Proteins , Genetics , Metabolism , Estrogen Receptor alpha , Genetics , Metabolism , Protein Interaction Domains and Motifs , Physiology , RNA, Messenger , Genetics , Regulatory Factor X Transcription Factors , Signal Transduction , Transcription Factors , Genetics , Metabolism , X-Box Binding Protein 1ABSTRACT
<p><b>OBJECTIVE</b>To construct an ERbeta expression vector and study its expression and function in different cancer cells.</p><p><b>METHODS</b>Standard PCR was used to amplify the full-length coding sequence of ERbeta. The amplified ERbeta gene was cloned into the eukaryotic expression vector pCDNA3, generating pCDNA3-ERbeta. The ERbeta expression was detected by Western blot and in vitro translation. The biological activity of ERbeta was detected by transfecting the pCDNA3-ERbeta into SV40-transformed embryonic kidney cell line 293T,breast cancer cell lines MDA-MB-435, MDA-MB-436, SKBR3, and prostate cancer cell line PC-3, with reporters containing estrogen response elements.</p><p><b>RESULTS</b>The recombinant plasmid pCDNA3-ERbeta was confirmed by restriction analysis to contain the ERbeta gene. The 63 000 ERbeta expression was shown by Western blot and further confirmed by in vitro translation. The ERbeta expression in different cancer cells was demonstrated to stimulate the expression of the reporters containing estrogen response elements, ERE and C3.</p><p><b>CONCLUSION</b>ERbeta protein is successfully expressed and has biological activity, laying solid foundation for further study on its role in cancer cells.</p>
Subject(s)
Female , Humans , Male , Breast Neoplasms , Metabolism , Pathology , Cell Line , Cell Line, Tumor , Embryo, Mammalian , Epithelial Cells , Estrogen Receptor beta , Genetics , Metabolism , Genes, Reporter , Genetics , Genetic Vectors , Kidney , Cell Biology , Plasmids , Prostatic Neoplasms , Metabolism , Pathology , Recombinant Proteins , Genetics , Metabolism , Response Elements , Genetics , TransfectionABSTRACT
The transgenic mice that express Cre recombinase in a tissue specific manner is a powerful tool in generating the conditional gene knockout mice. The rat insulin promoter was cloned target the expression of Cre in pancreatic tissue. The Cre gene was modified by adding the nuclear localization signal and the sequence for initiation by eukaryotic ribosomes at 5' terminal of the Cre gene. Cre gene was linked to the intron of human growth factor gene. This construct was introduced into the mouse eggs using microinjection. Seven mice were identified as founders carrying the Cre gene by PCR. The results of RT-PCR showed that the transgenic mouse from one founder could transcribe the foreign gene in pancreas. The Southern blot analysis indicated that the Cre recombinase expressed in pancreas of the transgenic mouse was functional.
Subject(s)
Animals , Female , Mice , Rats , Blotting, Southern , Insulin , Genetics , Integrases , Genetics , Mice, Transgenic , Pancreas , Metabolism , Promoter Regions, Genetic , RNA, Messenger , Reverse Transcriptase Polymerase Chain Reaction , Viral Proteins , GeneticsABSTRACT
We have studied the proteomic changes of the serum of the Smad3 targeted deficient mice using 2-DE and PMF approaches. 7 proteins expressed at different level in wild type mice and the Smad3 deficient mice were identified. These results would benefit the research on diagnosis and therapy of osteoarthritis and provided clues to studying the important function of Smad3 mediated TGF-beta signals during the skeletal development.
Subject(s)
Animals , Mice , DNA-Binding Proteins , Blood , Genetics , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Genotype , Mice, Knockout , Peptide Mapping , Proteome , Smad3 Protein , Trans-Activators , Blood , GeneticsABSTRACT
Breast cancer susceptibility gene 1(BRCA1) plays an important role in breast cancer development and progression. BRCA1 encodes a 1863-amino acid protein with two BRCA1 C-terminal (BRCT) domains at its C-terminus, BRCT1 and BRCT2. Many cancer-predisposing mutations are located in the BRCT domains, which have been shown to induce chromatin unfolding by use of an approach that allows visualization of large-scale chromatin structure through lac repressor/lac operator recognition. To map the important region of BRCT domain (amino acid residues 1642-1736), six deletion mutant constructs were made. The chromatin structure assay showed that amino acid residues 1691-1721 are involved in the induction of chromatin unfolding. To further localize the critical amino acid residues, ten alanine scanning mutant constructs were made. The chromatin structure assay demonstrated that the 1707IAGGK1711 region is critical for the chromatin unfolding activity. Based on the mapped important region, Blast analysis identified a novel homologous protein. Mapping of the BRCT1 domain may aid in the presymptomatic risk assessment and provide a valuable tool for further study on the BRCT1 structure and function.
Subject(s)
Female , Humans , BRCA1 Protein , Chemistry , Physiology , Base Sequence , Chromatin , Chemistry , Cloning, Molecular , Genes, BRCA1 , Molecular Sequence Data , Mutation , Protein Folding , Structure-Activity RelationshipABSTRACT
Objective:To prepare a kind of G418 resistance fibroblast feeder layers.Methods: In order to prepare a stock primary embryonic fibroblast (EMFI) cells, the pregnant Smad3ex8+/- mice containing heterozygous neo gene were killed 14 days after coitus to get the embryo. EMFI cells were tested by PCR to identify their gene types. Only one EMFI cell which contained heterozygous neo gene was chosen and treated with mitomycin C.The mitomycin C-treated EMFI feeders were stored at -70℃. Results: The EMFI feeder layers could support the growth of embryonic stem cell line TC-1 efficiently and could live in G418 resistance medium for 1-2 weeks.
ABSTRACT
Objective:To choose the best vector for the expression of CS3 fimbriae. Methods: The CS3 operon was cloned into different plasmid vectors such as pUC19 and pTrc99A. The expression of CS3 was monitored by whole-cell ELISA and SDS-PAGE analysis. The assembly of CS3 fimbriae was detected with electron microscopy. Results:The expression level of CS3 fimbriae using plasmid pUC19 as carrying vector was the highest, and the insertion orientation of CS3 gene into the plasmid has a little effect on its expression level. The expression of CS3 fimbriae was confirmed by SDS-PAGE analysis and electron microscopy.Conclusions:The promotor of CS3 itself played the key role in the expression of CS3 fimbriae and the copy number of plasmid was the main factor to affect the expression level.