Objectives: To obtain very end full-length cDNA ofhepatitis C virus (HCV) 5’ untranslated region(5’UTR) and analyze its primary and secondarystructure.Methods: A patient infected genotype 2a HCV wasidentified by rev...Objectives: To obtain very end full-length cDNA ofhepatitis C virus (HCV) 5’ untranslated region(5’UTR) and analyze its primary and secondarystructure.Methods: A patient infected genotype 2a HCV wasidentified by reverse transcription-nested polymerasechain reaction (RT-PCR) and restriction fragmentlength polymorphism (RFLP). Total RNA isolatedfrom the serum was used as template, and the cDNAof the 5’ untranslated region was amplified using rap-id amplification of cDNA ends (RACE). The frag-ments were recombinated by A-T clone strategy, andthe recombinants were confirmed by RFLP andPCR, and sequenced subsequently. Secondary struc-tures were analysed by RNAdraw.Results: Very end full-length cDNA of genotype 2aHCV 5’ UTR was obtained by RACE. In five clonesobtained, three contained full-length 5’UTR cDNA;A21G, G170A, T222C, T247C, C339T substitutionswere found as compared to HC-J6. Homological re-sults of HCV-1, HC-J6, HC-C2, HC-J8 were 93.6%-94.4%, 92.1%-93%, 98.8%-99.7%, 96.2%-96.5%, respectively; however, the substitutions didnot alter secondary structure. Two of 5 clones weredeletions of 53bp and 135bp at the 5’terminal ofHCV 5’UTR, respectively.Conclusions: RACE can be used to obtain the full-length cDNA of 2a genotype HCV 5’UTR. Genes de-leted at the 5’ terminal of HCV circulate in hepatitisC patients.展开更多
Objective:To study evolutionary relationship of the 5'untranslated regions(5'UTRs) in low passage dengue3 viruses(DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok dur...Objective:To study evolutionary relationship of the 5'untranslated regions(5'UTRs) in low passage dengue3 viruses(DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok during 24 year-evolution(1977-2000) comparing to the DEN3prototype(H87).Methods:The 5'UTRs of these Thai DEN3 and the H87 prototype were amplified by RT-PCR and sequenced.Their multiple sequence alignments were done by Codon Code Aligner v 4.0.4 software and their RNA secondary structures were predicted by MFOLD software.Replication of five Thai DEN3 candidates comparing to the 1187 prototype were done in human(HepG2) and the mosquito(C6/36) cell lines.Results:Among these Thai DEN3,the completely identical sequences of their first 89 nucleotides,their high-order secondary structure of 5'UTRs and three SNPs including the predominant C90 T,and two minor SNPs including A109 G and A112 G were found.The C90 T of Thai DEN3.Bangkok isolates was shown predominantly before 1977.Five Thai DEN3 candidates with the predominant C90 T were shown to replicate in human(HepG2) and the mosquito(C6/36) cell lines better than the H87 prototype.However,their highly conserved sequences as well as SNPs of the 5'UTR did not appear to correlate with their disease severity in human.Conclusions:Our findings highlighted evolutionary relationship of the completely identical 89 nucleotide sequence,the high-order secondary structure and the predominant C90 T of the 5'UTR of these Thai DEN3 during 24 year-evolution further suggesting to be their genetic markers and magic targets for future research on antiviral therapy as well as vaccine approaches of Thai DEN3.展开更多
Dear Editor： Increased homocysteine levels due to vitamin B6 or B12 deficiency or genetic defects in folate pathway genes are associated with an increased incidence of non-syndromic cleft lip with or without cleft p...Dear Editor： Increased homocysteine levels due to vitamin B6 or B12 deficiency or genetic defects in folate pathway genes are associated with an increased incidence of non-syndromic cleft lip with or without cleft palate （NSCLP）tlj. Thymidylate synthase （TS） is a folate-dependent enzyme that catalyzes methylation of 2＇-deoxyuridine-5＇-monophosphate （dUMP） to 2＇-deox- ythymidine-5＇-monophosphate （dTMP）, a rate-limiting step in DNA synthesis,展开更多
Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for han...Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.展开更多
The development of genetically modified crops requires new promoters and regulatory regions to achieve high gene ex- pression and/or tissue-specific expression patterns in plants. To obtain promoter sequences of plant...The development of genetically modified crops requires new promoters and regulatory regions to achieve high gene ex- pression and/or tissue-specific expression patterns in plants. To obtain promoter sequences of plants with new properties, we analyzed the expression traits of the cotton （Gossypium hirsutum） translation elongation factor 1A gene family. The results showed that the GhEF1A8 gene is highly expressed in different organs of cotton plants, and showed much higher transcript levels in stems and leaves. Its promoter （GhEFIA1.7） and the 5＂ untranslated region （5＂ UTR）, comprising a regulatory region named PGhEFIA8, were isolated from cotton and studied in stably transformed tobacco plants. The regulatory region sequences were fused to the 13-glucuronidase （GUS） reporter gene to characterize its expression pattern in tobacco. Histochemical and fiuorometric GUS activity assays demonstrated that PGhEF1A8 could direct GUS gene expression in all tissues and organs in transgenic tobacco, including leaves, stems, flowers, and roots. The level of GUS activity in the leaves and stems was significantly higher than in cauliflower mosaic virus （CaMV） 35S promoter：：GUS plants, but as same as CaMV 35S promoter：：GUS plants in flower and root tissues. GUS expression levels decreased 2-10-fold when the 5＂ UTR was absent from PGhEF1A8. Deletion analysis of the PGhEFIA8 sequence showed that the region -647 to -323 might possess negative elements that repress transgene expression in tobacco plants. The results suggested that the GhEFIA8 regulation region may represent a practical choice to direct high-level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.展开更多
基金This work was supported by two grants from National Science Foundation of China (No: 39770684, 30170844).
文摘Objectives: To obtain very end full-length cDNA ofhepatitis C virus (HCV) 5’ untranslated region(5’UTR) and analyze its primary and secondarystructure.Methods: A patient infected genotype 2a HCV wasidentified by reverse transcription-nested polymerasechain reaction (RT-PCR) and restriction fragmentlength polymorphism (RFLP). Total RNA isolatedfrom the serum was used as template, and the cDNAof the 5’ untranslated region was amplified using rap-id amplification of cDNA ends (RACE). The frag-ments were recombinated by A-T clone strategy, andthe recombinants were confirmed by RFLP andPCR, and sequenced subsequently. Secondary struc-tures were analysed by RNAdraw.Results: Very end full-length cDNA of genotype 2aHCV 5’ UTR was obtained by RACE. In five clonesobtained, three contained full-length 5’UTR cDNA;A21G, G170A, T222C, T247C, C339T substitutionswere found as compared to HC-J6. Homological re-sults of HCV-1, HC-J6, HC-C2, HC-J8 were 93.6%-94.4%, 92.1%-93%, 98.8%-99.7%, 96.2%-96.5%, respectively; however, the substitutions didnot alter secondary structure. Two of 5 clones weredeletions of 53bp and 135bp at the 5’terminal ofHCV 5’UTR, respectively.Conclusions: RACE can be used to obtain the full-length cDNA of 2a genotype HCV 5’UTR. Genes de-leted at the 5’ terminal of HCV circulate in hepatitisC patients.
基金supported by two research grants of Associate Professor Dr.W.Attatippaholkun:Grant No.493-5600-G-00-3461,Program in Science and Technology Cooperation,Human Capacity Development,Bureau for Global Programs,Field Support and Research,US Agency for International Development,Washington,DCThe Royal Golden Jubilee-Ph.D Program,Thailand Research Fund,Thailand
文摘Objective:To study evolutionary relationship of the 5'untranslated regions(5'UTRs) in low passage dengue3 viruses(DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok during 24 year-evolution(1977-2000) comparing to the DEN3prototype(H87).Methods:The 5'UTRs of these Thai DEN3 and the H87 prototype were amplified by RT-PCR and sequenced.Their multiple sequence alignments were done by Codon Code Aligner v 4.0.4 software and their RNA secondary structures were predicted by MFOLD software.Replication of five Thai DEN3 candidates comparing to the 1187 prototype were done in human(HepG2) and the mosquito(C6/36) cell lines.Results:Among these Thai DEN3,the completely identical sequences of their first 89 nucleotides,their high-order secondary structure of 5'UTRs and three SNPs including the predominant C90 T,and two minor SNPs including A109 G and A112 G were found.The C90 T of Thai DEN3.Bangkok isolates was shown predominantly before 1977.Five Thai DEN3 candidates with the predominant C90 T were shown to replicate in human(HepG2) and the mosquito(C6/36) cell lines better than the H87 prototype.However,their highly conserved sequences as well as SNPs of the 5'UTR did not appear to correlate with their disease severity in human.Conclusions:Our findings highlighted evolutionary relationship of the completely identical 89 nucleotide sequence,the high-order secondary structure and the predominant C90 T of the 5'UTR of these Thai DEN3 during 24 year-evolution further suggesting to be their genetic markers and magic targets for future research on antiviral therapy as well as vaccine approaches of Thai DEN3.
基金funding from the Indian Council of Medical Research(ICMR),Government of India(Project Ref.No.56/15/2007-BMS)
文摘Dear Editor： Increased homocysteine levels due to vitamin B6 or B12 deficiency or genetic defects in folate pathway genes are associated with an increased incidence of non-syndromic cleft lip with or without cleft palate （NSCLP）tlj. Thymidylate synthase （TS） is a folate-dependent enzyme that catalyzes methylation of 2＇-deoxyuridine-5＇-monophosphate （dUMP） to 2＇-deox- ythymidine-5＇-monophosphate （dTMP）, a rate-limiting step in DNA synthesis,
文摘Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.
基金supported by the New Genetically Modified Organisms Varieties Cultivation Project, China (2014ZX08005-004)
文摘The development of genetically modified crops requires new promoters and regulatory regions to achieve high gene ex- pression and/or tissue-specific expression patterns in plants. To obtain promoter sequences of plants with new properties, we analyzed the expression traits of the cotton （Gossypium hirsutum） translation elongation factor 1A gene family. The results showed that the GhEF1A8 gene is highly expressed in different organs of cotton plants, and showed much higher transcript levels in stems and leaves. Its promoter （GhEFIA1.7） and the 5＂ untranslated region （5＂ UTR）, comprising a regulatory region named PGhEFIA8, were isolated from cotton and studied in stably transformed tobacco plants. The regulatory region sequences were fused to the 13-glucuronidase （GUS） reporter gene to characterize its expression pattern in tobacco. Histochemical and fiuorometric GUS activity assays demonstrated that PGhEF1A8 could direct GUS gene expression in all tissues and organs in transgenic tobacco, including leaves, stems, flowers, and roots. The level of GUS activity in the leaves and stems was significantly higher than in cauliflower mosaic virus （CaMV） 35S promoter：：GUS plants, but as same as CaMV 35S promoter：：GUS plants in flower and root tissues. GUS expression levels decreased 2-10-fold when the 5＂ UTR was absent from PGhEF1A8. Deletion analysis of the PGhEFIA8 sequence showed that the region -647 to -323 might possess negative elements that repress transgene expression in tobacco plants. The results suggested that the GhEFIA8 regulation region may represent a practical choice to direct high-level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.
