Coconut(Cocos nucifera L.),a major oil and fruit crop of the Arecaceae family,is extensively cultivated across the Asia—Pacific region.Despite its agricultural importance,genome assembly in coconut remains challengin...Coconut(Cocos nucifera L.),a major oil and fruit crop of the Arecaceae family,is extensively cultivated across the Asia—Pacific region.Despite its agricultural importance,genome assembly in coconut remains challenging due to its large genome size and high proportion of repetitive sequences.Allele-specific expression(ASE)plays a key role in regulating plant development and evolution,yet research on ASE in coconut is limited(Shao et al.,2019;Li et al.,2021;Zhang et al.,2021;Hu et al.,2022).Among phenotypic traits,fruit color is especially important as an indicator of maturity,guiding harvest timing and post-harvest processes(Kapoor et al.,2022).While prior studies have explored various coconut traits such as salt tolerance,fiber content,and plant height(Wang et al.,2021;Yang et al.,2021),investigations into ASE and fruit color remain scarce.展开更多
Arsenic(As)and chromium(Cr)are two harmful toxicants as well as carcinogens which can coexist in polluted surface water and groundwater.This coexistence leads to mixture effects in animals including fish.Both of these...Arsenic(As)and chromium(Cr)are two harmful toxicants as well as carcinogens which can coexist in polluted surface water and groundwater.This coexistence leads to mixture effects in animals including fish.Both of these heavy metals are reported to manifest reactive oxygen species(ROS)mediated toxicity.Though individual neurotoxic effects have been reported,their mixture effects,its mechanism and cellular responses against oxidative stress and DNA damages remain unknown.The present study evaluated the individual and mixture effects of As and Cr at their environmentally relevant concentrations in zebrafish(Danio rerio)brain after 15,30 and 60 days of exposure.Nrf2,a transcription factor is involved in the expressional regulation of enzymes needed to maintain cellular redox homeostasis.This study reported the expressional pattern of Nrf2 and its associated xenobiotic metabolizing enzyme Nqo1 and other markers of oxidative stress such as ROS generation,reduced glutathione level,lipid peroxidation and catalase activity.Increased malondialdehyde(MDA)content,glutathione level,and catalase activity indicated oxidative stress in exposed groups.In addition,this study revealed expressional alterations of neurotoxicity marker(ache),DNA repair(ogg1,apex1,creb1,polb,mlh1,msh2 and msh6)and tumor suppressor(p53,brca2)genes.Results of ROS generation,MDA level,histopathological analysis,gene expression and immunofluorescence study confirmed that As and Cr did not show antagonistic effects in combination rather indicated additive effects which was dose-dependent but not always linear.展开更多
Following the publication of Xu et al.(2022),an error was identified in Figure 1D.Specifically,the top left panel was inadvertently duplicated during figure preparation.To ensure the accuracy and integrity of our publ...Following the publication of Xu et al.(2022),an error was identified in Figure 1D.Specifically,the top left panel was inadvertently duplicated during figure preparation.To ensure the accuracy and integrity of our published work,we request the publication of a corrigendum with the corrected image.We apologize for this oversight and any confusion it may have caused.The amended figure is provided in the updated Supplementary Materials.展开更多
The cDNA fragment encoding caffeic acid 3_O_methyltransferase (COMT) in Chinese white poplar ( Populus tomentosa Carr.) was isolated and cloned by RT_PCR technique. The size of the cDNA fragment is 1 080 bp, which alm...The cDNA fragment encoding caffeic acid 3_O_methyltransferase (COMT) in Chinese white poplar ( Populus tomentosa Carr.) was isolated and cloned by RT_PCR technique. The size of the cDNA fragment is 1 080 bp, which almost covers the whole cDNA_encoding region. Authors’ cDNA fragment in P. tomentosa shares 98.7% homology with the reported corresponding cDNA in the P. tremuloids at nucleotide level, 99.4% homology at amino acid level, respectively. The analysis of Northern dot hybridization showed that COMT is expressed specifically in the developing secondary xylem of stem during the season of xylem differentiation, which means the linkage between the gene expression for a monolignol biosynthetic enzyme and seasonal regulation of xylem development in woody plant.展开更多
The ACC synthase is the key enzyme in ethylene biosynthesis and fruit ripening. To study the mechanism of ACC synthase in peach Prunus persica (L.) Batsch) fruit ripening, we cloned a full_length cDNA of ACC synthase ...The ACC synthase is the key enzyme in ethylene biosynthesis and fruit ripening. To study the mechanism of ACC synthase in peach Prunus persica (L.) Batsch) fruit ripening, we cloned a full_length cDNA of ACC synthase pacs from peach using 5′/3′ RACE PCR. The nucleic acid sequence of pacs was 1 848 bp, containing 177 bp of 5′untranslated sequence, 1 449 bp of an open reading frame, and 219 bp of 3′untranslated sequence (excluding the stop codon TAA). The pacs open reading frame encoded a 483_amino acid polypeptide with a predicted size of 54 kD and a calculated PI of 6.43. The deduced protein from ACC synthase cDNA pacs had 65%, 70%, 75%, and 90% homology with the other deduced proteins from tomato (S19677), plum (AB031026), papaya (U68216) and apple (AB034993), which contained the active site of ACC synthase SLSKDMGFPGFR conserved among these plant ACC synthases. RNA_based PCR amplification combined with hybridization analysis with pacs and another ACC synthase cDNApacs12 (AF467782) cloned by us before as probes, indicated that expression patterns of both clones were very similar. mRNAs of both clones expressed in the alabastrum and petal, and were induced after ethylene treatment. Wounding and IAA treatments could induce ACC synthase expression of both clones in the leaves. However, the wounding treatment of leaves has induced more abundant pacs ACC synthase expression than that ofpacs12. Pacs mRNA expressed in both green mature and ripening fruit, whilepacs12mRNA was little or undetectable in green mature fruit, but apparent in ripening fruit. Both clone mRNAs accumulated more in leaves (following wounding and IAA treatments) and flowers than in fruits.展开更多
The vectors carrying the genes coding for the proteins of interest are of unpredictable efficiency in transgenic animals. The expression vector of mammary gland (pINGG) containing GCSF genomic DNA was injected into m...The vectors carrying the genes coding for the proteins of interest are of unpredictable efficiency in transgenic animals. The expression vector of mammary gland (pINGG) containing GCSF genomic DNA was injected into mouse mammary gland, and expression was detected in the milk of mice. The result showed that mammary gland injection method could provide a convenient transient system to confirm vector validity.展开更多
Soybean mosaic virus (SMV) causes one of the most severe viral diseases in soybean ( Glycine max L.) and is known to contain many pathogenically and serologically related isolates. In the present study, the authors...Soybean mosaic virus (SMV) causes one of the most severe viral diseases in soybean ( Glycine max L.) and is known to contain many pathogenically and serologically related isolates. In the present study, the authors have obtained cDNAs to all cistrons of a Chinese SMV isolate, SMV_ZK, by RT_PCR. By analysing the nucleotide and amino acid sequence of the HC_PRO, NIb and CP cistrons, it was found that SMV_ZK was highly homologous to the G2 strain of SMV, thus confirming the existence of G2_like isolates in soybean crop in China. The amplified cDNAs were directly cloned into a bacterial expression vector. With the exception of the P3 cistron, expression of the cDNAs of all other cistrons in bacteria gave rise to polypeptides of expected molecular weight. The expressed viral proteins were subsequently purified by gel elution. The preparation of viral_specific cDNAs and gene products will be useful in future functional study of the SMV genome.展开更多
[Objective] The aim was to study the cDNA cloning of SMAT gene from Chimonanthus praecox and its expression. [Method] A novel cDNA was cloned by PT-PCR using the total RNA as template, and amplified to the desirable s...[Objective] The aim was to study the cDNA cloning of SMAT gene from Chimonanthus praecox and its expression. [Method] A novel cDNA was cloned by PT-PCR using the total RNA as template, and amplified to the desirable size. The RT-PCR products were reclaimed and transformed into E. coli DH 5o together with the PMD18-T vector after ligating by T-A cloning. Identified by colony PCR and EcoRI and Notl digestion, the recombinant plasmid with target gene was screened out and conducted the sequence analysis. [Result] Results of the sequence analysis showed that the ORF fragment of the SAMT cDNA was successfully cloned from Chimonanthus praecox gene, with the length of 1 196 bp and encoding 380 amino acids fragment which shared 99.2% homology to that of previously reported SAMT cDNA from Chimonanthus praecox （ABU88887）. The SAMT cDNA fragment was sub-cloned into the prokaryotic expression vector PGEX1-4T-1, and the obtained re- combinant plasmid was named PGSAMT. After inducting by 0.01mol/L IPTG, the re- sult of the SDS-PAGE analysis showed that the molecular weight of the fusion ex- pression SAMT protein was about 66 kDa, which was close to the predicted fusion protein derived from the 26 kDa GST band and 42.3 kDa SAMT gene of Chimo- nanthus praecox encoded protein. [Conclusion] This study successfully cloned and expressed the SAMT gene of Chimonanthus praecox.展开更多
INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivat...INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivating factor is the evidence thatstrongly supported the notion that the X gene ofHBV DNA integrated in HCC genomic DNA couldcontribute to the carcinogenesis of liver cells byactivation of some related cellular genes展开更多
The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors,and previous studies have indicated the involvement of multiple PI-related gene families during c...The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors,and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses.Through a detailed analysis of the Arabidopsis thaliana genome,82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS),PI-phosphate kinases (PIPK),phospholipases (PL),inositol polyphosphate phosphatases (IPPase),inositol polyphosphate kinases (IPK),PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK,PLC,PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly,DNA chip technology was employed to study the expression patterns of various isoforms.In total,79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf,stem and flower tissues,and leaves from plants treated with various hormones (auxin,cytokinin,gibberellin,abscisic acid and brassinosteroid) or environmental factors (temperature,calcium,sodium,drought,salicylic acid and jasmonic acid).Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions.In particular,the different isoforms of each family were specifically expressed in many cases,suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.展开更多
AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence...AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence was determined by dideoxy mediated chain termination method using T7 polymerase. HCV NS3 protein was expressed in E. coli . RESULTS Sequence analysis indicated that the HCV isolate of this study belongs to HCV Ⅱ; SDS PAGE demonstrated an M r 23800 and an M r 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately. Western blotting and ELISA showed NS3 protein possessed greater antigenicity. CONCLUSION Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents.展开更多
AIM To determine whether incorporation of the pH dependent bacterial toxin listeriolysin O (LLO) into the DNA carrier system could increase the endosomal escape of internalized DNA and result gene expression. METHODS ...AIM To determine whether incorporation of the pH dependent bacterial toxin listeriolysin O (LLO) into the DNA carrier system could increase the endosomal escape of internalized DNA and result gene expression. METHODS A multi component delivery system was prepared consisting of asialoglycoprotein (ASG), poly L lysine (PL), and LLO. Two marker genes, luciferase and β galactosidase in plasmids were complexed and administered in vitro to Huh7[ASG receptor (+) ] and SK Hep1[ASG receptor (-) ] cells. Purity, hemolytic activity, gene expression, specificity, and toxicity were evaluated. RESULTS An LLO containing conjugate retained cell targeting specificity and membranolytic activity. In ASG receptor (+) cells, luciferase gene expression was enhanced by more than 7 fold over that of conjugates without the incorporation of listeriolysin O. No significant expression occurred in ASG receptor (-) cells. Enhancement of β galactosidase gene expression was less, but still significantly increased over controls. There was no detectable toxicity at concentrations shown to be effective in transfection studies. CONCLUSIONS ASOR PL can be coupled to LLO using disulfide bonds, and successfully target and increase the gene expression of foreign DNA.展开更多
Objective: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased im- mune responses. Methods: Four recombinant plasmids constructed ...Objective: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased im- mune responses. Methods: Four recombinant plasmids constructed in- cluded the coding regions for the core protein (pC) and for the core, E_1 and E_2 together (pCE_1E_2), IL- 12 p35 and p40. These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/ C mice for measurement of specific antibodies and cytotoxic T-lymphocyte (CTL) responses. Results: All the recombinant plasmids were shown to express specific antigens stably in mammalian cells. Codelivery of pIL-12 expression cassettes with pC and pCE_1E_2 in mice resulted in the enhancement of Ag-dependent CTL responses and the reduction of specific Ab response. The CTL activity was: pC= 18.65%±5.71%, pCE_1E_2=20.07%±11.11%, pC +pIL-12=60.11%±17.37%, pCE_1E_2+pIL-12= 67.48%±15.57%, respectively. The average A val- ues of anti-HCV were pC=0.415±0.127, pCE_1E_2= 0.358±0.096, pC+pIL-12=0.210±0.086, pCE_1E_2 +pIL-12=0.258±0.125. Conclusion: Codelivery of pIL-12 with plasmid DNA can enhance the efficacy of immune responses and shift the type of immune responses.展开更多
AIM To clone core gene cDNA of Chinesehepatitis C virus(HCV)into eukaryoticexpression vector cosmid pTM3 and to expressHCV core antigen in HepG2 cells.METHODS Core gene cDNA of HCV wasintroduced into eukaryotic expres...AIM To clone core gene cDNA of Chinesehepatitis C virus(HCV)into eukaryoticexpression vector cosmid pTM3 and to expressHCV core antigen in HepG2 cells.METHODS Core gene cDNA of HCV wasintroduced into eukaryotic expression vectorcosmid pTM3.Using vaccinia virus/bacteriophage T7 hybrid expression system,HepG2 cells were transfected with therecombinant plasmid pTM3-Q534 by lipofectin.RESULTS From the transfected bacteriaTop10F’,2 pTM3-Q534 clones containing therecombinant plasmid were identified fromrandomly selected 10 ampicillin-resistantcolonies.By reverse transcription PCR andindirect immunofluorescence technique,HCVRNA and core protein was identified in HepG2cells transfected with the recombinant plasmid.CONCLUSION The construction of arecombinant plasmid and the expression of coregene cDNA of HCV in HepG2 was successful.展开更多
Testis specific protein Y-encoded（TSPY） is a Y-located proto-oncogene predominantly expressed in normal male germ cells and various types of germ cell tumor. Significantly, TSPY is frequently expressed in somatic ca...Testis specific protein Y-encoded（TSPY） is a Y-located proto-oncogene predominantly expressed in normal male germ cells and various types of germ cell tumor. Significantly, TSPY is frequently expressed in somatic cancers including liver cancer but not in adjacent normal tissues, suggesting that ectopic TSPY expression could be associated with oncogenesis in non-germ cell cancers. Various studies demonstrated that TSPY expression promotes growth and proliferation in cancer cells; however, its relationship to other oncogenic events in TSPY-positive cancers remains unknown. The present study seeks to correlate TSPY expression with other molecular features in clinical cancer samples, by analyses of RNA-seq transcriptome and DNA methylation data in the Cancer Genome Atlas（TCGA） database. A total of 53 genes,including oncogenic lineage protein 28 homolog B（LIN28B） gene and RNA-binding motif protein Y-linked（RBMY） gene, are identified to be consistently co-expressed with TSPY, and have been collectively designated as the TSPY co-expression network（TCN）. TCN genes were simultaneously activated in subsets of liver hepatocellular carcinoma（30%） and lung adenocarcinoma（10%） regardless of pathological stage, but only minimally in other cancer types. Further analysis revealed that the DNA methylation level was globally lower in the TCN-active than TCN-silent cancers. The specific expression and methylation patterns of TCN genes suggest that they could be useful as biomarkers for the diagnosis,prognosis and clinical management of cancers, especially those for liver and lung cancers, associated with TSPY co-expression network genes.展开更多
Salinity is a crucial environmental stress that severely affects fish growth and survival.Under environmental stress,DNA methylation plays an important role in gene expression and genome function.To better understand ...Salinity is a crucial environmental stress that severely affects fish growth and survival.Under environmental stress,DNA methylation plays an important role in gene expression and genome function.To better understand the epigenetic regulation mechanism of igf2 under low salinity stress,we analyzed the DNA methylation at 5’UTR,exon1,intron1,and exon2,and analyzed the relationship of DNA methylation with mRNA abundance as well as the special single CpG sites methylation patterns of igf2 in the liver of half smooth tongue sole under low salinity(15)for 0,7,and 60 d.When exposed to low salinity,DNA methylation at 5’UTR and exon2 remained stable,while it was up-regulated firstly and then down-regulated at exon1 and intron1.Some single CpG sites of igf2 associated with low salinity,and most of these sites with significantly changed methylation levels(P<0.05)are located in intron1 area.The discrepant variation of single CpG sites methylation levels and igf2 expression further revealed that females and males showed different response to low salinity.Remarkably,the 38-CpG site of intron1 servers as a sexual marker.Additionally,our integrative analysis demonstrated that regional DNA of igf2 methylation had highly complex interplay on gene expression.The single CpG sites in intron1 were indispensable epigenetic markers under external environmental stress.Above all,to resist the low salinity stress,half smooth tongue sole liver can regulate the expression of igf2 through methylation of CpG sites in intron1.展开更多
基金supported by Central Public-interest Scientific Institution Basal Research Fund(CATAS-Nos.1630152023007,1630152023011,1630152023012,1630152023013)the National Natural Science Foundation of China(Grant No.32071805).
文摘Coconut(Cocos nucifera L.),a major oil and fruit crop of the Arecaceae family,is extensively cultivated across the Asia—Pacific region.Despite its agricultural importance,genome assembly in coconut remains challenging due to its large genome size and high proportion of repetitive sequences.Allele-specific expression(ASE)plays a key role in regulating plant development and evolution,yet research on ASE in coconut is limited(Shao et al.,2019;Li et al.,2021;Zhang et al.,2021;Hu et al.,2022).Among phenotypic traits,fruit color is especially important as an indicator of maturity,guiding harvest timing and post-harvest processes(Kapoor et al.,2022).While prior studies have explored various coconut traits such as salt tolerance,fiber content,and plant height(Wang et al.,2021;Yang et al.,2021),investigations into ASE and fruit color remain scarce.
基金the Department of Zoology,Visva-Bharati for providing infrastructural supportSreejata Kamila is grateful to the Council of Scientific and Industrial Research(CSIR),India for Senior Research Fellowship(CSIR File No.09/202(0102)/2019-EMR-I)+2 种基金Koushik Kumar Dey acknowledges the Department of Biotechnology,New Delhi for research fellowship(No.BT/PR28560/AAQ/3/919/2018)Shehnaz Islam is thankful to Maulana Azad National Fellowship(UGC ref ID-201920–345938)India for her financial support.
文摘Arsenic(As)and chromium(Cr)are two harmful toxicants as well as carcinogens which can coexist in polluted surface water and groundwater.This coexistence leads to mixture effects in animals including fish.Both of these heavy metals are reported to manifest reactive oxygen species(ROS)mediated toxicity.Though individual neurotoxic effects have been reported,their mixture effects,its mechanism and cellular responses against oxidative stress and DNA damages remain unknown.The present study evaluated the individual and mixture effects of As and Cr at their environmentally relevant concentrations in zebrafish(Danio rerio)brain after 15,30 and 60 days of exposure.Nrf2,a transcription factor is involved in the expressional regulation of enzymes needed to maintain cellular redox homeostasis.This study reported the expressional pattern of Nrf2 and its associated xenobiotic metabolizing enzyme Nqo1 and other markers of oxidative stress such as ROS generation,reduced glutathione level,lipid peroxidation and catalase activity.Increased malondialdehyde(MDA)content,glutathione level,and catalase activity indicated oxidative stress in exposed groups.In addition,this study revealed expressional alterations of neurotoxicity marker(ache),DNA repair(ogg1,apex1,creb1,polb,mlh1,msh2 and msh6)and tumor suppressor(p53,brca2)genes.Results of ROS generation,MDA level,histopathological analysis,gene expression and immunofluorescence study confirmed that As and Cr did not show antagonistic effects in combination rather indicated additive effects which was dose-dependent but not always linear.
文摘Following the publication of Xu et al.(2022),an error was identified in Figure 1D.Specifically,the top left panel was inadvertently duplicated during figure preparation.To ensure the accuracy and integrity of our published work,we request the publication of a corrigendum with the corrected image.We apologize for this oversight and any confusion it may have caused.The amended figure is provided in the updated Supplementary Materials.
文摘The cDNA fragment encoding caffeic acid 3_O_methyltransferase (COMT) in Chinese white poplar ( Populus tomentosa Carr.) was isolated and cloned by RT_PCR technique. The size of the cDNA fragment is 1 080 bp, which almost covers the whole cDNA_encoding region. Authors’ cDNA fragment in P. tomentosa shares 98.7% homology with the reported corresponding cDNA in the P. tremuloids at nucleotide level, 99.4% homology at amino acid level, respectively. The analysis of Northern dot hybridization showed that COMT is expressed specifically in the developing secondary xylem of stem during the season of xylem differentiation, which means the linkage between the gene expression for a monolignol biosynthetic enzyme and seasonal regulation of xylem development in woody plant.
文摘The ACC synthase is the key enzyme in ethylene biosynthesis and fruit ripening. To study the mechanism of ACC synthase in peach Prunus persica (L.) Batsch) fruit ripening, we cloned a full_length cDNA of ACC synthase pacs from peach using 5′/3′ RACE PCR. The nucleic acid sequence of pacs was 1 848 bp, containing 177 bp of 5′untranslated sequence, 1 449 bp of an open reading frame, and 219 bp of 3′untranslated sequence (excluding the stop codon TAA). The pacs open reading frame encoded a 483_amino acid polypeptide with a predicted size of 54 kD and a calculated PI of 6.43. The deduced protein from ACC synthase cDNA pacs had 65%, 70%, 75%, and 90% homology with the other deduced proteins from tomato (S19677), plum (AB031026), papaya (U68216) and apple (AB034993), which contained the active site of ACC synthase SLSKDMGFPGFR conserved among these plant ACC synthases. RNA_based PCR amplification combined with hybridization analysis with pacs and another ACC synthase cDNApacs12 (AF467782) cloned by us before as probes, indicated that expression patterns of both clones were very similar. mRNAs of both clones expressed in the alabastrum and petal, and were induced after ethylene treatment. Wounding and IAA treatments could induce ACC synthase expression of both clones in the leaves. However, the wounding treatment of leaves has induced more abundant pacs ACC synthase expression than that ofpacs12. Pacs mRNA expressed in both green mature and ripening fruit, whilepacs12mRNA was little or undetectable in green mature fruit, but apparent in ripening fruit. Both clone mRNAs accumulated more in leaves (following wounding and IAA treatments) and flowers than in fruits.
文摘The vectors carrying the genes coding for the proteins of interest are of unpredictable efficiency in transgenic animals. The expression vector of mammary gland (pINGG) containing GCSF genomic DNA was injected into mouse mammary gland, and expression was detected in the milk of mice. The result showed that mammary gland injection method could provide a convenient transient system to confirm vector validity.
文摘Soybean mosaic virus (SMV) causes one of the most severe viral diseases in soybean ( Glycine max L.) and is known to contain many pathogenically and serologically related isolates. In the present study, the authors have obtained cDNAs to all cistrons of a Chinese SMV isolate, SMV_ZK, by RT_PCR. By analysing the nucleotide and amino acid sequence of the HC_PRO, NIb and CP cistrons, it was found that SMV_ZK was highly homologous to the G2 strain of SMV, thus confirming the existence of G2_like isolates in soybean crop in China. The amplified cDNAs were directly cloned into a bacterial expression vector. With the exception of the P3 cistron, expression of the cDNAs of all other cistrons in bacteria gave rise to polypeptides of expected molecular weight. The expressed viral proteins were subsequently purified by gel elution. The preparation of viral_specific cDNAs and gene products will be useful in future functional study of the SMV genome.
文摘[Objective] The aim was to study the cDNA cloning of SMAT gene from Chimonanthus praecox and its expression. [Method] A novel cDNA was cloned by PT-PCR using the total RNA as template, and amplified to the desirable size. The RT-PCR products were reclaimed and transformed into E. coli DH 5o together with the PMD18-T vector after ligating by T-A cloning. Identified by colony PCR and EcoRI and Notl digestion, the recombinant plasmid with target gene was screened out and conducted the sequence analysis. [Result] Results of the sequence analysis showed that the ORF fragment of the SAMT cDNA was successfully cloned from Chimonanthus praecox gene, with the length of 1 196 bp and encoding 380 amino acids fragment which shared 99.2% homology to that of previously reported SAMT cDNA from Chimonanthus praecox （ABU88887）. The SAMT cDNA fragment was sub-cloned into the prokaryotic expression vector PGEX1-4T-1, and the obtained re- combinant plasmid was named PGSAMT. After inducting by 0.01mol/L IPTG, the re- sult of the SDS-PAGE analysis showed that the molecular weight of the fusion ex- pression SAMT protein was about 66 kDa, which was close to the predicted fusion protein derived from the 26 kDa GST band and 42.3 kDa SAMT gene of Chimo- nanthus praecox encoded protein. [Conclusion] This study successfully cloned and expressed the SAMT gene of Chimonanthus praecox.
基金Projects of the Science Development Foundation of Shanghai (994919033)Tackling Key Problems in Science and Technology from the State Science and Technology Ministry(TJ99LA01)
文摘INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivating factor is the evidence thatstrongly supported the notion that the X gene ofHBV DNA integrated in HCC genomic DNA couldcontribute to the carcinogenesis of liver cells byactivation of some related cellular genes
基金supported by grants from the National Natural Science Foundation of China(No.30100101)the State Key Project of Basic Research(No.G19990-11604)
文摘The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors,and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses.Through a detailed analysis of the Arabidopsis thaliana genome,82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS),PI-phosphate kinases (PIPK),phospholipases (PL),inositol polyphosphate phosphatases (IPPase),inositol polyphosphate kinases (IPK),PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK,PLC,PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly,DNA chip technology was employed to study the expression patterns of various isoforms.In total,79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf,stem and flower tissues,and leaves from plants treated with various hormones (auxin,cytokinin,gibberellin,abscisic acid and brassinosteroid) or environmental factors (temperature,calcium,sodium,drought,salicylic acid and jasmonic acid).Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions.In particular,the different isoforms of each family were specifically expressed in many cases,suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.
文摘AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence was determined by dideoxy mediated chain termination method using T7 polymerase. HCV NS3 protein was expressed in E. coli . RESULTS Sequence analysis indicated that the HCV isolate of this study belongs to HCV Ⅱ; SDS PAGE demonstrated an M r 23800 and an M r 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately. Western blotting and ELISA showed NS3 protein possessed greater antigenicity. CONCLUSION Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents.
基金supported in part by agrant from the National Institutes of He alth,DK-42182(GYW)the Immune Response Cor por ation(GHW)the Herman Lopata Chair for He patitis Research.
文摘AIM To determine whether incorporation of the pH dependent bacterial toxin listeriolysin O (LLO) into the DNA carrier system could increase the endosomal escape of internalized DNA and result gene expression. METHODS A multi component delivery system was prepared consisting of asialoglycoprotein (ASG), poly L lysine (PL), and LLO. Two marker genes, luciferase and β galactosidase in plasmids were complexed and administered in vitro to Huh7[ASG receptor (+) ] and SK Hep1[ASG receptor (-) ] cells. Purity, hemolytic activity, gene expression, specificity, and toxicity were evaluated. RESULTS An LLO containing conjugate retained cell targeting specificity and membranolytic activity. In ASG receptor (+) cells, luciferase gene expression was enhanced by more than 7 fold over that of conjugates without the incorporation of listeriolysin O. No significant expression occurred in ASG receptor (-) cells. Enhancement of β galactosidase gene expression was less, but still significantly increased over controls. There was no detectable toxicity at concentrations shown to be effective in transfection studies. CONCLUSIONS ASOR PL can be coupled to LLO using disulfide bonds, and successfully target and increase the gene expression of foreign DNA.
文摘Objective: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased im- mune responses. Methods: Four recombinant plasmids constructed in- cluded the coding regions for the core protein (pC) and for the core, E_1 and E_2 together (pCE_1E_2), IL- 12 p35 and p40. These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/ C mice for measurement of specific antibodies and cytotoxic T-lymphocyte (CTL) responses. Results: All the recombinant plasmids were shown to express specific antigens stably in mammalian cells. Codelivery of pIL-12 expression cassettes with pC and pCE_1E_2 in mice resulted in the enhancement of Ag-dependent CTL responses and the reduction of specific Ab response. The CTL activity was: pC= 18.65%±5.71%, pCE_1E_2=20.07%±11.11%, pC +pIL-12=60.11%±17.37%, pCE_1E_2+pIL-12= 67.48%±15.57%, respectively. The average A val- ues of anti-HCV were pC=0.415±0.127, pCE_1E_2= 0.358±0.096, pC+pIL-12=0.210±0.086, pCE_1E_2 +pIL-12=0.258±0.125. Conclusion: Codelivery of pIL-12 with plasmid DNA can enhance the efficacy of immune responses and shift the type of immune responses.
基金the National Natural Science Foundation of China,No.39500129
文摘AIM To clone core gene cDNA of Chinesehepatitis C virus(HCV)into eukaryoticexpression vector cosmid pTM3 and to expressHCV core antigen in HepG2 cells.METHODS Core gene cDNA of HCV wasintroduced into eukaryotic expression vectorcosmid pTM3.Using vaccinia virus/bacteriophage T7 hybrid expression system,HepG2 cells were transfected with therecombinant plasmid pTM3-Q534 by lipofectin.RESULTS From the transfected bacteriaTop10F’,2 pTM3-Q534 clones containing therecombinant plasmid were identified fromrandomly selected 10 ampicillin-resistantcolonies.By reverse transcription PCR andindirect immunofluorescence technique,HCVRNA and core protein was identified in HepG2cells transfected with the recombinant plasmid.CONCLUSION The construction of arecombinant plasmid and the expression of coregene cDNA of HCV in HepG2 was successful.
基金partially supported by a Merit-Reviewed grant from the Department of Veterans Affairsa Peer-Reviewed Cancer Research Program grant from the Department of Defense (W81XWH-16-1-0488) to Y-FCL
文摘Testis specific protein Y-encoded（TSPY） is a Y-located proto-oncogene predominantly expressed in normal male germ cells and various types of germ cell tumor. Significantly, TSPY is frequently expressed in somatic cancers including liver cancer but not in adjacent normal tissues, suggesting that ectopic TSPY expression could be associated with oncogenesis in non-germ cell cancers. Various studies demonstrated that TSPY expression promotes growth and proliferation in cancer cells; however, its relationship to other oncogenic events in TSPY-positive cancers remains unknown. The present study seeks to correlate TSPY expression with other molecular features in clinical cancer samples, by analyses of RNA-seq transcriptome and DNA methylation data in the Cancer Genome Atlas（TCGA） database. A total of 53 genes,including oncogenic lineage protein 28 homolog B（LIN28B） gene and RNA-binding motif protein Y-linked（RBMY） gene, are identified to be consistently co-expressed with TSPY, and have been collectively designated as the TSPY co-expression network（TCN）. TCN genes were simultaneously activated in subsets of liver hepatocellular carcinoma（30%） and lung adenocarcinoma（10%） regardless of pathological stage, but only minimally in other cancer types. Further analysis revealed that the DNA methylation level was globally lower in the TCN-active than TCN-silent cancers. The specific expression and methylation patterns of TCN genes suggest that they could be useful as biomarkers for the diagnosis,prognosis and clinical management of cancers, especially those for liver and lung cancers, associated with TSPY co-expression network genes.
基金supported by the Key Laboratory of Mariculture of Ministry of Education, Ocean University of China (No. KLM2018009)the National Natural Science Foundation of China (No. 31672642)
文摘Salinity is a crucial environmental stress that severely affects fish growth and survival.Under environmental stress,DNA methylation plays an important role in gene expression and genome function.To better understand the epigenetic regulation mechanism of igf2 under low salinity stress,we analyzed the DNA methylation at 5’UTR,exon1,intron1,and exon2,and analyzed the relationship of DNA methylation with mRNA abundance as well as the special single CpG sites methylation patterns of igf2 in the liver of half smooth tongue sole under low salinity(15)for 0,7,and 60 d.When exposed to low salinity,DNA methylation at 5’UTR and exon2 remained stable,while it was up-regulated firstly and then down-regulated at exon1 and intron1.Some single CpG sites of igf2 associated with low salinity,and most of these sites with significantly changed methylation levels(P<0.05)are located in intron1 area.The discrepant variation of single CpG sites methylation levels and igf2 expression further revealed that females and males showed different response to low salinity.Remarkably,the 38-CpG site of intron1 servers as a sexual marker.Additionally,our integrative analysis demonstrated that regional DNA of igf2 methylation had highly complex interplay on gene expression.The single CpG sites in intron1 were indispensable epigenetic markers under external environmental stress.Above all,to resist the low salinity stress,half smooth tongue sole liver can regulate the expression of igf2 through methylation of CpG sites in intron1.
