Eukaryotic chromatin consisting of nucleosomes connected by linker DNA is organized into higher order structures,which is facilitated by linker histone H1.Formation of chromatin compacts and protects the genome,but al...Eukaryotic chromatin consisting of nucleosomes connected by linker DNA is organized into higher order structures,which is facilitated by linker histone H1.Formation of chromatin compacts and protects the genome,but also hinders DNA transactions.Cells have evolved mechanisms to modify/remodel chromatin resulting in chromatin states suitable for genome functions.The high mobility group box(HMGB)proteins are non-histone chromatin architectural factors characterized by one or more HMGB motifs that bind DNA in a sequence nonspecific fashion.They play a major role in chromatin dynamics.The Saccharomyces cerevisiae(yeast hereafter)HMGB protein Hmo1 contains two HMGB motifs.However,unlike a canonical HMGB protein that has an acidic C-terminus,Hmo1 ends with a lysine rich,basic,C-terminus,resembling linker histone H1.Hmo1 exhibits characteristics of both HMGB proteins and linker histones in its multiple functions.For instance,Hmo1 promotes transcription by RNA polymerases I and II like canonical HMGB proteins but makes chromatin more compact/stable like linker histones.Recent studies have demonstrated that Hmo1 destabilizes/disrupts nucleosome similarly as other HMGB proteins in vitro and acts to maintain a common topological architecture of genes in yeast genome.This minireview reviews the functions of Hmo1 and the underlying mechanisms,highlighting recent discoveries.展开更多
目的构建和评价HPV16 E6、E7多表位DNA疫苗诱导的特异性CTL细胞应答及其对肿瘤生长的干预作用,从而揭示其作为候选HPV治疗性疫苗的潜能。方法首先通过IEDB网站中的MHC I Processing Predictions和MHC I Binding Predictions方法,分别预...目的构建和评价HPV16 E6、E7多表位DNA疫苗诱导的特异性CTL细胞应答及其对肿瘤生长的干预作用,从而揭示其作为候选HPV治疗性疫苗的潜能。方法首先通过IEDB网站中的MHC I Processing Predictions和MHC I Binding Predictions方法,分别预测人类HLA-A^(*)02:01、HLA-A^(*)11:01、HLA-A^(*)24:02和C57BL/6小鼠H-2b的限制性CTL表位,然后根据评分以及ELISPOT实验筛选出二者共同呈递的CTL表位,并将其构建成多表位DNA疫苗(pVAX1-10P)。从预防性和治疗性二个方面研究pVAX1-10P对小鼠移植TC-1异位癌的免疫干预作用,流式细胞术检测特异性CTL应答。结果获得10条可被人与鼠MHC分子共呈递的CTL表位,ELISPOT结果表明这10条CTL表位均能诱导小鼠淋巴细胞产生特异性免疫应答;由此构建的多表位DNA疫苗pVAX1-10P无论在预防性实验还是治疗性实验中,均能诱导特异性的细胞免疫并抑制肿瘤的生长。结论构建的HPV16 E6、E7多表位DNA疫苗pVAX1-10P能够诱导特异性CTL应答,显著抑制肿瘤生长,有望作为候选HPV治疗性DNA疫苗。展开更多
Objective Abnormal expression of T-lymphokine-activated killer cell-originated protein kinase(TOPK)was reported to be closely related to the resistance of prostate cancer to radiotherapy and to targeted drug resistanc...Objective Abnormal expression of T-lymphokine-activated killer cell-originated protein kinase(TOPK)was reported to be closely related to the resistance of prostate cancer to radiotherapy and to targeted drug resistance in lung cancer.However,the role of TOPK inhibition in enhancing radiosensitivity of colorectal cancer(CRC)cells is unclear.This study aimed to evaluate the radiosensitization of TOPK knockdown in CRC cells.Methods The expression of TOPK was detected in CRC tissues by immunohistochemistry,and the effect of TOPK knockdown was detected in CRC cells by Western blotting.CCK-8 and clonogenic assays were used to detect the growth and clonogenic ability of CRC cells after TOPK knockdown combined with radiotherapy in CRC cells.Furthermore,proteomic analysis showed that the phosphorylation of TOPK downstream proteins changed after radiotherapy.DNA damage was detected by the comet assay.Changes in the DNA damage response signaling pathway were analyzed by Western blotting,and apoptosis was detected by flow cytometry.Results The expression of TOPK was significantly greater in CRC tissues at grades 2–4 than in those at grade 1.After irradiation,CRC cells with genetically silenced TOPK had shorter comet tails and reduced expression levels of DNA damage response-associated proteins,including phospho-cyclin-dependent kinase 1(p-CDK1),phospho-ataxia telangiectasia-mutated(p-ATM),poly ADP-ribose polymerase(PARP),and meiotic recombination 11 homolog 1(MRE11).Conclusions TOPK was overexpressed in patients with moderately to poorly differentiated CRC.Moreover,TOPK knockdown significantly enhanced the radiosensitivity of CRC cells by reducing the DNA damage response.展开更多
To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) gene...To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by liposome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E). BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01 strains (10 5 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected in HepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using anti-E. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization, and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE. It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of pJE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.展开更多
In this short review we describe implications of age-related changes of protein and DNA oxidation as a public mechanism of biological aging. Oxidatively modified protein and DNA have been demonstrated to increase with...In this short review we describe implications of age-related changes of protein and DNA oxidation as a public mechanism of biological aging. Oxidatively modified protein and DNA have been demonstrated to increase with advancing age in rodents. Half-life of proteins is extended and DNA repair activity declines in old animals. Dietary restriction initiated late in life can shorten the half-life of proteins to levels of young animals, thus contributing to reduce level of altered proteins in old animals by the regimen. Regular exercise reduced oxidatively modified proteins in the brain with improved cognitive functions. It attenuated oxidative stress in the liver, i.e., ameliorating activation of nuclear factor κB, increasing reduced glutathione, and decreasing oxidized guanine base in nuclear and mitochondrial DNA. These findings suggest that regular exercise has systemic effects in reducing oxidative stress. Thus, life-styles such as diet and exercise may extend health span, by up-regulating overall anti-oxidant capacities that include proteins involved in protein turnover and DNA repair, resulting in reduction of damaged proteins and DNA that potentially promote physiological and pathological aging.展开更多
Insoluble fatty surfaces are involved in many important interactions such as in biomembranes with soluble biological macro and micromolecules. In this paper we have studied the adsorption interaction of aqueous soluti...Insoluble fatty surfaces are involved in many important interactions such as in biomembranes with soluble biological macro and micromolecules. In this paper we have studied the adsorption interaction of aqueous solution of DNA, some proteins and lactose on several sparingly soluble fatty substances namely milk fat, stearic acid, palmitic acid, phosphatidyl choline and cholesterol surfaces by measuring the depletion of the adsorbates by analytical methods. Adsorption () of DNA on the soft surfaces of stearic acid, milk fat, phosphatidyl choline, palmitic acid and cholesterol was measured as a function of DNA concentration C2. In each case was found to increase with C2 until it reached the maximum value at a critical concentration . For different surfaces stands in the order: stearic acid > milk fat > phosphatidyl choline > cholesterol > palmitic acid. DNA forms multilayers on stearic acid surface. Adsorption of hemoglobin on cholesterol surface is found to be negative or zero but that of BSA on cholesterol is positive. Adsorption of gelatin on cholesterol surface is significantly higher than that of BSA. Lysozyme on cholesterol surface forms multilayers and on casein forms bilayer. The lowering of free energies ?DGo for all systems have been calculated using integrated form of the Gibbs adsorption and their values have been compared with each other. It is concluded that despite differences in the adsorption behavior of the biomolecules on various soft surfaces, free energy change expressed as Bull’s free energy change (Δ) remain nearly constant except for BSA-fatty acid interaction which may be likely due a specific interaction.展开更多
Taking advantage of the effects on DNA secondary structure of two DNA-intercalators,ethidium bromide and chloroquine,we used each of them to treat nuclei from both mature erythrocytes and reticulocytes of chicken,as a...Taking advantage of the effects on DNA secondary structure of two DNA-intercalators,ethidium bromide and chloroquine,we used each of them to treat nuclei from both mature erythrocytes and reticulocytes of chicken,as an alternative approach to study the relationships between DNA secondary structure,nuclear proteins and chromatin structure.We presented results of differential extraction of nuclear proteins from nuclei with DNA-intercalators,as well as preliminary characterization of these proteins.A 45kd protein is the major component in fractions extracted by both intercalators from nuclei from either mature erythrocytes or reticulocytes and seems to be a DNA-binding protein.Furthermore,from current concepts of functional aspects of DNA conformation and structural heterogeneity in chromatin and nuclear proteins,we have discussed both the significance of our results as well as technical aspects of this approach.展开更多
This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among ge-nomic DNA. cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells. DNA fragment...This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among ge-nomic DNA. cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells. DNA fragments encoding BR/HLH/LZ structure of Myc and Myn proteins were cloned in frame into pGEX-2T vec-tor respectively Fusion GST-Myc and GST-Myn synthe-sized in E.coli hosts showed affinity to CACGTG E-boxDNA and subsequently interacted with genomic fragments prepared through whole-genome-PCR. A PCR-assisted procedure which combines protein-DNA interaction and affinity chromatography was designed to enrich Myc/Myn bound DNA. At least two genomic DNA fragments ob- tained exhibit specifical binding capacity to Myc/Myn complex but not to GST alone. Significance of the work and of the technique itself as well as identification of the DNAs are discussed.展开更多
目的探讨丙型肝炎病毒NS5A反式调节蛋白9(hepatitis C virus NS5Atransactivated protein 9,NS5ATP9)在乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)形成与转录中的作用机制。方法利用...目的探讨丙型肝炎病毒NS5A反式调节蛋白9(hepatitis C virus NS5Atransactivated protein 9,NS5ATP9)在乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)形成与转录中的作用机制。方法利用1.3拷贝HBV表达质粒转染Huh7和HepG2细胞、整合有4拷贝HBV基因组的HepG2.2.15细胞、在诱导型四环素启动子控制下表达HBV的HepAD38细胞构建NS5ATP9过表达或干扰的HBV细胞模型,收集样品和细胞上清液,提取RNA、HBV核心DNA(coreDNA)、cccDNA和蛋白,利用酶联免疫吸附试验、实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)、Southern blot和Western blot技术检测HBV总RNA、前基因组RNA(pregenomic RNA,pgRNA)、乙型肝炎病毒s抗原(hepatitis B virus s antigene,HBsAg)、乙型肝炎病毒e抗原(hepatitis B virus e antigene,HBeAg)、松弛环状DNA(relax circular DNA,rcDNA)以及cccDNA水平。在HepG2细胞中转染乙型肝炎病毒x蛋白(hepatitis B virus x protein,HBx),通过免疫荧光成像及免疫共沉淀方法检测NS5ATP9与HBx的结合情况。双荧光素酶报告基因实验检测NS5ATP9对HBx启动子活性的影响。利用Huh7细胞转染HBV1.3及HBV稳定表达细胞株HepG2.2.15和HepAD38转染NS5ATP9过表达/干扰质粒,通过Western blot技术检测DDB1和SMC6的蛋白水平。结果在HBV病毒活跃的细胞中,NS5ATP9 mRNA水平[HepG2.2.15细胞:1.891±0.567比1.00±0.034,t=2.87,P=0.0351;HepAD38 tet+细胞:1.978±0.399比1.00±0.034,t=4.131,P=0.0091;HepAD38 tet-细胞:2.642±0.672比1.00±0.034,t=4.127,P=0.0091]和蛋白水平均显著增加。过表达NS5ATP9后可显著增加HBeAg[(5.402±0.327)S/COV比(2.68±0.552)S/COV,t=7.35,P=0.0018]、HBsAg[(2.846±0.185)S/COV比(1.512±0.221)S/COV,t=8.02,P=0.0013]、HBV pgRNA及rcDNA的表达水平,而干扰NS5ATP9后此增加作用消失[HBeAg:(2.029±0.09)S/COV比(3.733±0.445)S/COV,t=6.501,P=0.0029;HBsAg:(1.501±0.105)S/COV比(1.878±0.174)S/COV,t=3.216,P=0.0324)]。机制研究显示,NS5ATP9和HBx蛋白主要位于细胞核核仁内,并具有共定位信号,且NS5ATP9可显著提高HBx启动子(1071.06±79.44比488.47±40.12,t=13.09,P=0.00012)的转录活性。另外,过表达NS5ATP9可显著降低DDB1和SMC6的蛋白水平,而沉默NS5ATP9则可显著提高DDB1和SMC6的蛋白水平。结论HBV上调NS5ATP9的表达,形成HBV-NS5ATP9-HBV cccDNA-HBV的正反馈环路,NS5ATP9通过与HBx相互作用上调肝细胞中HBV cccDNA的形成与转录,进而促进慢性乙型肝炎的发生发展。展开更多
[Objective] The experiment was aimed to explore character variation between different families after DNA introduction and select variant plants with good stability. [Method] The method of pollen-tube-pathway was used ...[Objective] The experiment was aimed to explore character variation between different families after DNA introduction and select variant plants with good stability. [Method] The method of pollen-tube-pathway was used to introduce total DNA of soybean into normal maize inbred line 7313 for selecting generation by generation. When field characters of maize, grain colors, grain traits and panicle axis colors were stable, the crude protein, gliadin, glutelin and oil content of grains which were selected from variant strains were detected and compared. [Result] The grain crude protein, gliadin, glutelin and oil content of line 26h-4-3 were significantly different from these of control treatment. The increments of D3 and D4 generation were 10.34% and 26.70%, 6.58% and 6.28%, 15.09% and 70.34%, 55.82% and 51.52% respectively. All indexes of line 26h-3-1 were also higher than these of control treatment and the increments of D3 and D4 generation were 5.67% and 21.63%,1.91% and 2.31%, 10.85% and 62.27%,22.49% and 9.67%. [Conclusion] The crude protein, gliadin, glutelin and oil content of variant line 26h-4-3 and 26h-3-1 were stable, so variant line 26h-4-3 and 26h-3-1 were excellent variant strains which satisfied the requirement of high protein breeding.展开更多
文摘Eukaryotic chromatin consisting of nucleosomes connected by linker DNA is organized into higher order structures,which is facilitated by linker histone H1.Formation of chromatin compacts and protects the genome,but also hinders DNA transactions.Cells have evolved mechanisms to modify/remodel chromatin resulting in chromatin states suitable for genome functions.The high mobility group box(HMGB)proteins are non-histone chromatin architectural factors characterized by one or more HMGB motifs that bind DNA in a sequence nonspecific fashion.They play a major role in chromatin dynamics.The Saccharomyces cerevisiae(yeast hereafter)HMGB protein Hmo1 contains two HMGB motifs.However,unlike a canonical HMGB protein that has an acidic C-terminus,Hmo1 ends with a lysine rich,basic,C-terminus,resembling linker histone H1.Hmo1 exhibits characteristics of both HMGB proteins and linker histones in its multiple functions.For instance,Hmo1 promotes transcription by RNA polymerases I and II like canonical HMGB proteins but makes chromatin more compact/stable like linker histones.Recent studies have demonstrated that Hmo1 destabilizes/disrupts nucleosome similarly as other HMGB proteins in vitro and acts to maintain a common topological architecture of genes in yeast genome.This minireview reviews the functions of Hmo1 and the underlying mechanisms,highlighting recent discoveries.
文摘目的构建和评价HPV16 E6、E7多表位DNA疫苗诱导的特异性CTL细胞应答及其对肿瘤生长的干预作用,从而揭示其作为候选HPV治疗性疫苗的潜能。方法首先通过IEDB网站中的MHC I Processing Predictions和MHC I Binding Predictions方法,分别预测人类HLA-A^(*)02:01、HLA-A^(*)11:01、HLA-A^(*)24:02和C57BL/6小鼠H-2b的限制性CTL表位,然后根据评分以及ELISPOT实验筛选出二者共同呈递的CTL表位,并将其构建成多表位DNA疫苗(pVAX1-10P)。从预防性和治疗性二个方面研究pVAX1-10P对小鼠移植TC-1异位癌的免疫干预作用,流式细胞术检测特异性CTL应答。结果获得10条可被人与鼠MHC分子共呈递的CTL表位,ELISPOT结果表明这10条CTL表位均能诱导小鼠淋巴细胞产生特异性免疫应答;由此构建的多表位DNA疫苗pVAX1-10P无论在预防性实验还是治疗性实验中,均能诱导特异性的细胞免疫并抑制肿瘤的生长。结论构建的HPV16 E6、E7多表位DNA疫苗pVAX1-10P能够诱导特异性CTL应答,显著抑制肿瘤生长,有望作为候选HPV治疗性DNA疫苗。
基金supported by the Guangxi Zhuang Autonomous Region Program of China(No.Z-C20220797)Guangxi Science and Technology Planning Project of China(No.Guike AD20297047)National Natural Science Foundation of China(No.81902849).
文摘Objective Abnormal expression of T-lymphokine-activated killer cell-originated protein kinase(TOPK)was reported to be closely related to the resistance of prostate cancer to radiotherapy and to targeted drug resistance in lung cancer.However,the role of TOPK inhibition in enhancing radiosensitivity of colorectal cancer(CRC)cells is unclear.This study aimed to evaluate the radiosensitization of TOPK knockdown in CRC cells.Methods The expression of TOPK was detected in CRC tissues by immunohistochemistry,and the effect of TOPK knockdown was detected in CRC cells by Western blotting.CCK-8 and clonogenic assays were used to detect the growth and clonogenic ability of CRC cells after TOPK knockdown combined with radiotherapy in CRC cells.Furthermore,proteomic analysis showed that the phosphorylation of TOPK downstream proteins changed after radiotherapy.DNA damage was detected by the comet assay.Changes in the DNA damage response signaling pathway were analyzed by Western blotting,and apoptosis was detected by flow cytometry.Results The expression of TOPK was significantly greater in CRC tissues at grades 2–4 than in those at grade 1.After irradiation,CRC cells with genetically silenced TOPK had shorter comet tails and reduced expression levels of DNA damage response-associated proteins,including phospho-cyclin-dependent kinase 1(p-CDK1),phospho-ataxia telangiectasia-mutated(p-ATM),poly ADP-ribose polymerase(PARP),and meiotic recombination 11 homolog 1(MRE11).Conclusions TOPK was overexpressed in patients with moderately to poorly differentiated CRC.Moreover,TOPK knockdown significantly enhanced the radiosensitivity of CRC cells by reducing the DNA damage response.
基金This research was supported by a grant for project research from high Technology center of Kanazawa Medical University(H2000 2)
文摘To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by liposome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E). BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01 strains (10 5 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected in HepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using anti-E. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization, and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE. It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of pJE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.
文摘In this short review we describe implications of age-related changes of protein and DNA oxidation as a public mechanism of biological aging. Oxidatively modified protein and DNA have been demonstrated to increase with advancing age in rodents. Half-life of proteins is extended and DNA repair activity declines in old animals. Dietary restriction initiated late in life can shorten the half-life of proteins to levels of young animals, thus contributing to reduce level of altered proteins in old animals by the regimen. Regular exercise reduced oxidatively modified proteins in the brain with improved cognitive functions. It attenuated oxidative stress in the liver, i.e., ameliorating activation of nuclear factor κB, increasing reduced glutathione, and decreasing oxidized guanine base in nuclear and mitochondrial DNA. These findings suggest that regular exercise has systemic effects in reducing oxidative stress. Thus, life-styles such as diet and exercise may extend health span, by up-regulating overall anti-oxidant capacities that include proteins involved in protein turnover and DNA repair, resulting in reduction of damaged proteins and DNA that potentially promote physiological and pathological aging.
文摘Insoluble fatty surfaces are involved in many important interactions such as in biomembranes with soluble biological macro and micromolecules. In this paper we have studied the adsorption interaction of aqueous solution of DNA, some proteins and lactose on several sparingly soluble fatty substances namely milk fat, stearic acid, palmitic acid, phosphatidyl choline and cholesterol surfaces by measuring the depletion of the adsorbates by analytical methods. Adsorption () of DNA on the soft surfaces of stearic acid, milk fat, phosphatidyl choline, palmitic acid and cholesterol was measured as a function of DNA concentration C2. In each case was found to increase with C2 until it reached the maximum value at a critical concentration . For different surfaces stands in the order: stearic acid > milk fat > phosphatidyl choline > cholesterol > palmitic acid. DNA forms multilayers on stearic acid surface. Adsorption of hemoglobin on cholesterol surface is found to be negative or zero but that of BSA on cholesterol is positive. Adsorption of gelatin on cholesterol surface is significantly higher than that of BSA. Lysozyme on cholesterol surface forms multilayers and on casein forms bilayer. The lowering of free energies ?DGo for all systems have been calculated using integrated form of the Gibbs adsorption and their values have been compared with each other. It is concluded that despite differences in the adsorption behavior of the biomolecules on various soft surfaces, free energy change expressed as Bull’s free energy change (Δ) remain nearly constant except for BSA-fatty acid interaction which may be likely due a specific interaction.
文摘Taking advantage of the effects on DNA secondary structure of two DNA-intercalators,ethidium bromide and chloroquine,we used each of them to treat nuclei from both mature erythrocytes and reticulocytes of chicken,as an alternative approach to study the relationships between DNA secondary structure,nuclear proteins and chromatin structure.We presented results of differential extraction of nuclear proteins from nuclei with DNA-intercalators,as well as preliminary characterization of these proteins.A 45kd protein is the major component in fractions extracted by both intercalators from nuclei from either mature erythrocytes or reticulocytes and seems to be a DNA-binding protein.Furthermore,from current concepts of functional aspects of DNA conformation and structural heterogeneity in chromatin and nuclear proteins,we have discussed both the significance of our results as well as technical aspects of this approach.
文摘This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among ge-nomic DNA. cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells. DNA fragments encoding BR/HLH/LZ structure of Myc and Myn proteins were cloned in frame into pGEX-2T vec-tor respectively Fusion GST-Myc and GST-Myn synthe-sized in E.coli hosts showed affinity to CACGTG E-boxDNA and subsequently interacted with genomic fragments prepared through whole-genome-PCR. A PCR-assisted procedure which combines protein-DNA interaction and affinity chromatography was designed to enrich Myc/Myn bound DNA. At least two genomic DNA fragments ob- tained exhibit specifical binding capacity to Myc/Myn complex but not to GST alone. Significance of the work and of the technique itself as well as identification of the DNAs are discussed.
文摘目的探讨丙型肝炎病毒NS5A反式调节蛋白9(hepatitis C virus NS5Atransactivated protein 9,NS5ATP9)在乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)形成与转录中的作用机制。方法利用1.3拷贝HBV表达质粒转染Huh7和HepG2细胞、整合有4拷贝HBV基因组的HepG2.2.15细胞、在诱导型四环素启动子控制下表达HBV的HepAD38细胞构建NS5ATP9过表达或干扰的HBV细胞模型,收集样品和细胞上清液,提取RNA、HBV核心DNA(coreDNA)、cccDNA和蛋白,利用酶联免疫吸附试验、实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)、Southern blot和Western blot技术检测HBV总RNA、前基因组RNA(pregenomic RNA,pgRNA)、乙型肝炎病毒s抗原(hepatitis B virus s antigene,HBsAg)、乙型肝炎病毒e抗原(hepatitis B virus e antigene,HBeAg)、松弛环状DNA(relax circular DNA,rcDNA)以及cccDNA水平。在HepG2细胞中转染乙型肝炎病毒x蛋白(hepatitis B virus x protein,HBx),通过免疫荧光成像及免疫共沉淀方法检测NS5ATP9与HBx的结合情况。双荧光素酶报告基因实验检测NS5ATP9对HBx启动子活性的影响。利用Huh7细胞转染HBV1.3及HBV稳定表达细胞株HepG2.2.15和HepAD38转染NS5ATP9过表达/干扰质粒,通过Western blot技术检测DDB1和SMC6的蛋白水平。结果在HBV病毒活跃的细胞中,NS5ATP9 mRNA水平[HepG2.2.15细胞:1.891±0.567比1.00±0.034,t=2.87,P=0.0351;HepAD38 tet+细胞:1.978±0.399比1.00±0.034,t=4.131,P=0.0091;HepAD38 tet-细胞:2.642±0.672比1.00±0.034,t=4.127,P=0.0091]和蛋白水平均显著增加。过表达NS5ATP9后可显著增加HBeAg[(5.402±0.327)S/COV比(2.68±0.552)S/COV,t=7.35,P=0.0018]、HBsAg[(2.846±0.185)S/COV比(1.512±0.221)S/COV,t=8.02,P=0.0013]、HBV pgRNA及rcDNA的表达水平,而干扰NS5ATP9后此增加作用消失[HBeAg:(2.029±0.09)S/COV比(3.733±0.445)S/COV,t=6.501,P=0.0029;HBsAg:(1.501±0.105)S/COV比(1.878±0.174)S/COV,t=3.216,P=0.0324)]。机制研究显示,NS5ATP9和HBx蛋白主要位于细胞核核仁内,并具有共定位信号,且NS5ATP9可显著提高HBx启动子(1071.06±79.44比488.47±40.12,t=13.09,P=0.00012)的转录活性。另外,过表达NS5ATP9可显著降低DDB1和SMC6的蛋白水平,而沉默NS5ATP9则可显著提高DDB1和SMC6的蛋白水平。结论HBV上调NS5ATP9的表达,形成HBV-NS5ATP9-HBV cccDNA-HBV的正反馈环路,NS5ATP9通过与HBx相互作用上调肝细胞中HBV cccDNA的形成与转录,进而促进慢性乙型肝炎的发生发展。
基金Supported by Natural Science Foundation of Guangxi Province(0236005)Educational Commission of Guangxi Province[(2002)316]~~
文摘[Objective] The experiment was aimed to explore character variation between different families after DNA introduction and select variant plants with good stability. [Method] The method of pollen-tube-pathway was used to introduce total DNA of soybean into normal maize inbred line 7313 for selecting generation by generation. When field characters of maize, grain colors, grain traits and panicle axis colors were stable, the crude protein, gliadin, glutelin and oil content of grains which were selected from variant strains were detected and compared. [Result] The grain crude protein, gliadin, glutelin and oil content of line 26h-4-3 were significantly different from these of control treatment. The increments of D3 and D4 generation were 10.34% and 26.70%, 6.58% and 6.28%, 15.09% and 70.34%, 55.82% and 51.52% respectively. All indexes of line 26h-3-1 were also higher than these of control treatment and the increments of D3 and D4 generation were 5.67% and 21.63%,1.91% and 2.31%, 10.85% and 62.27%,22.49% and 9.67%. [Conclusion] The crude protein, gliadin, glutelin and oil content of variant line 26h-4-3 and 26h-3-1 were stable, so variant line 26h-4-3 and 26h-3-1 were excellent variant strains which satisfied the requirement of high protein breeding.