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@#Objective To construct eukaryotic expression plasmids of human promyelocytic leukaemia(hPML) gene of six transcripts and analyze the subcellular location of the recombinant proteins.Methods Primers were designed according to the hPML gene sequences registered in GenBank databases.Six transcripts of hPML gene fragments(hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ,Ⅵ and Ⅶ) were amplified by RT-PCR,which were linked to the eukaryotic expression vector pCAGGS respectively.The obtained eukaryotic expression plasmids of six transcripts of hPML gene were transfected into 293T cells respectively and detected for their protein expression by Western blot,while transfected into Vero cells and detected for their subcellular location by indirect immunofluorescence assay(IFA).Results The target gene fragments of the six eukaryotic expression plasmids were consistent with the hPML gene sequences registered in GenBank.All the six recombinant proteins showed specific binding with Myc antibody,among which the recombinant protein hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ and Ⅵ were located in the nucleus and cytoplasm,while the recombinant protein hPML Ⅶ was mainly located in the cytoplasm,rarely in the nucleus.Conclusion The eukaryotic expression plasmids of six transcripts of hPML gene all can be expressed correctly in mammalian cells,and the expressed recombinant proteins were located in nucleus and cytoplasm simultaneously or mainly in cytoplasm.This study provides an experimental basis for subsequent study on the antiviral and other biological functions of recombinant protein hPML.
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Objective: The exogenous gibberellin (GA) and ethylene (ET) treatment can improve the medicinal ingredients of Salvia miltiorrhiza. Interestingly, many reports pointed out that WRKY transcription factors played an important regulatory role in these treatment responses. However, whether the SmWRKY mediate these treatment signalings in S. miltiorrhiza remains largely elusive. Methods: qRT-PCR was used for SmWRKY42-like in response to exogenous GA and ethephon (Eth) treatment. The subcellular location of SmWRKY42-like was transiently transformed into onion epidermal cells by particle bombardment. The self-activating activity of SmWRKY42-like was verified in AH109 yeast strain. Results: SmWRKY42-like was a WRKY family gene in S. miltiorrhiza. The subcellular localization and transcriptional activity results of the SmWRKY42-like protein indicated that SmWRKY42-like mainly enriched in nucleus and might be a transcription factor in S. miltiorrhiza. In the meantime, the SmWRKY42-like gene significantly responded to exogenous GA and Eth treatment. Conclusion: These results collectively indicated the SmWRKY42-like gene functions, as an important hormone-responsive gene, might play a potentially role in ET and GA signaling pathways.
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Purpose To investigate the effect of subcellular location of tumor BRCA1 on the sensitivity to ionizing radiation (IR) and PARP inhibitor.Methods siRNA of BRCA1 were first used to inhibit endougenous BRCA1 expression in MCF7 cells.Then,plasmids of pCMV-3xFlag-WT-BRCA1,pCMV-3xFlag-NES-BRCA1 and pCMV-3xFlag-NLS-BRCA1 were transfected in MCF7 cells.Immunofluorescence staining was used to detect BRCA1 subcellular location as well as the formation of Rad51 and γ-H2AX foci.Apoptotic cells were analyzed by flow cytometry,and colony formation assay was performed to evaluate the survival of cells.Results There were 47％ cells with nuclear BRCA1,23％ cells with cytoplasmic BRCA1 and 30％ cell with mixed nuclear and cytoplasmic BRCA1 expression in WT-BRCA1 transfected cell.There were 87％ cells with nuclear BRCA1 in NES-BRCA1 transfected cell,and 82％ cells with cytoplasmic BRCA1 in NLS-BRCA1 transfected cell.There were 87％,84％ and 13％ Rad51 foci positive cells at 2 hours after 4 Gy radiation treatment and 22％,25％ and 59％ γ-H2AX foci positive cells at 24 hours after 4Gy radiation treatment in WT-BRCA1,NES-BRCA1 mutant and NLS-BRCA1 mutant transfected cell respectively.ABT-888 and radiation treatment induced more apoptosis and fewer colonies in NLS-BRCA1 transfected cell than WT-BRCA1,NES-BRCA1 mutant transfected cell.Conclusion Subcellular location of BRCA1 might affect homologous recombination repair of DNA double strand breaks and can be used to predict sensitivity to IR and PARP inhibitor.
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Young leaves of Kabuli chickpea as well as soybean Xiangdou No.3, which are the current plants that studied in our laboratory were selected as materials. Effects on protoplasts yield and survival rate of different enzyme combination, concentration of D-Mannitol in enzyme combinations, pH of enzyme combinations and enzymolysis time are detected. The results showed that, the best condition for Xiangdou No.3 leaf protoplasts isolation is to rotate the cut materials for 6 hours in enyzme solution under temperature of 27 ℃ and rotate speed of 45 r/min for 6 h. Onozuka R-10 (0.5%), Hemicellulase (0.8%), Macerozyme R-10 (0.8%) in combination with Pectolyase Y-23 (0.4%) dissolving in CPW solution with MES (0.1%) and Mannitol (10%), pH 6.0 was found best for protoplasts isolation of Xiangdou No.3 leaves.The best condition for protoplasts isolation of Kabuli chickpea is to put the cut materials into enzymatic hydrolysate enzymolyse for 7 to 8 hours under temperature of 27 ℃ and rotate speed of 45 r/min on water bath shaker, the optimum combination of enzyme consists of Onozuka R-10 (0.5%), Hemicellulase (0.8%), Macerozyme R-10 (0.8%), MES (0.1%) and Mannitol (10%) dissolved in CPW solution with pH 4.8. The protoplasts prepared with the methods above are used in subcellular location and the effects show well.
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Nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD) performs essential functions in cell metabolism and energy production due to its redox properties. The nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18) enzyme catalyses the key step in the biosynthesis of NAD+. Previously, the enzyme NMNAT was identified in Trypanosoma cruzi (TcNMNAT), a pathogenic agent with epidemiological importance in Latin America. To continue with the functional characterisation of this enzyme, its subcellular location and its possible post-translational modifications were examined in this study. For this, polyclonal antibodies were generated in mice, with soluble and denatured recombinant protein being used to detect the parasite’s NMNAT. Immunodetection assays were performed on whole extracts of T. cruzi, and an approximation of its intracellular location was determined using confocal microscopy on wild and transgenic parasites, which revealed the cytosol distribution patterns. This localisation occurs according to the needs of the dinucleotides that exist in this compartment. Additionally, a bioinformatics study was performed as a first approach to establish the post-translational modifications of the enzyme. Possible phosphorylation events were experimentally analysed by western blot, highlighting TcNMNAT as a potential target for serine kinases.
Subject(s)
Animals , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Protozoan Proteins/metabolism , Trypanosoma cruzi/enzymology , Amino Acid Sequence , Cytosol/parasitology , Host-Parasite Interactions , Mice , Mice, Inbred BALB C , Nicotinamide-Nucleotide Adenylyltransferase/isolation & purification , Phosphorylation , Protein Serine-Threonine Kinases , Protozoan Proteins/isolation & purificationABSTRACT
Objective: To clone an agglutinin gene from Pinellia ternata and to analyze its bioinformatics and subcellular location. Methods: Based on the published sequence GU593718.1 from Genbank, P. ternata agglutinin (PTA) was amplified and cloned from genomic DNA of the fresh leaves of P. ternata. The cloned PTA gene was further fused to the plant expression vector pI1300-CaMV35S-GFP to construct pI1300-CaMV35S-PTA-GFP, then transfered into cells of Agrobacterium tumefaciens GV3101. Its transient expression was observed in Nicotiana tabacum. Results: The full length of PTA contained 810 bp with the deduced 269 amino acid residues; It contained one signal peptide, two conversation B-lectin domains and three mannose binding sites; PTA shared 97%, 85%, and 83% identity with the amino acid sequence from PTA, and Pinellia pedatisecta agglutinin (PPA), Pinellia cordata agglutinin (PCA), respectively; The PTA was localized to the plasma membrane; Its registration number is KF154979 in NCBI. Conclusion: It would provide a stable foundation for the study on its effect against fungi, insects, and bacterium.
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Objective To identify the subcellular location of S AR S-CoV N protein in mammalian cells. Methods The gene fraction of SARS-CoV was cloned into the PGEFP-C1 plasmid to construct expression vecto r PGEFP-C1/N. The subcellular location of N in A549 and VeroE6 cells was observ ed under fluorescence microscope with the aid of transient transfection techniqu e. The expression of the fusion protein (GFP-N) was detected by Western blot. Results The PGEFP-C1/N was constructed. N protein was localiz ed in the cytoplasm of transfected cells and detected by Western blot. C onclusion N protein was localized in the cytoplasm of mammalian cells.
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Objective To study the subcellular localization and the tissue expression of EOLA1(endothelial-overexpressed lipopolysaccharide-associated factor 1). Methods The fusion protein EOLA1-EGFP expressed vector was constructed and transfected into endothelial cells. After 24 hours posttransfection, the subcellular localization of EOLA1 was detected by laser-scanning microscopy. The tissue-specific distribution of EOLA1 was assessed with Multiple Tissue Northern Blots. Results The expression of EOLA1 was tissue-specific in various human tissues. With human multiple tissue Northern blot analysis, it was shown that EOLA1 could express in the heart, skeletal muscle, kidney, liver, placenta, colon, spleen, small intestine, but did not in the brain, lung, thymus, and peripheral blood leukocyte. EOLA1 was mainly localized in cytoplasm and could move into the nucleus. Conclusion EOLA1 is one of intercellular proteins and may play a role in intercellular signal transduction.