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
The messenger RNA 3'-untranslated region(3'UTR)plays an important role in regulation of gene expres-sion on the posttranscriptional level. The 3'UTR con-trols gene expression via orchestrated interactionbe...The messenger RNA 3'-untranslated region(3'UTR)plays an important role in regulation of gene expres-sion on the posttranscriptional level. The 3'UTR con-trols gene expression via orchestrated interactionbetween the structural components of mRNAs(cis-ele-ment) and the specific trans-acting factors(RNA bind-ing proteins and non-coding RNAs). The crosstalk ofthese factors is based on the binding sequences and/or direct protein-protein interaction, or just functionalinteraction. Much new evidence that has accumulatedsupports the idea that several RNA binding factors canbind to common mRNA targets: to the non-overlappingbinding sites or to common sites in a competitive fash-ion. Various factors capable of binding to the sameRNA can cooperate or be antagonistic in their actions.The outcome of the collective function of all factorsbound to the same mRNA 3'UTR depends on manycircumstances, such as their expression levels, affinity to the binding sites, and localization in the cell, which can be controlled by various physiological conditions. Moreover, the functional and/or physical interactions of the factors binding to 3'UTR can change the character of their actions. These interactions vary during the cell cycle and in response to changing physiological condi-tions. Abnormal functioning of the factors can lead to disease. In this review we will discuss how alterations of these factors or their interaction can affect cancer development and promote or enhance the malignant phenotype of cancer cells. Understanding these altera-tions and their impact on 3'UTR-directed posttran-scriptional gene regulation will uncover promising new targets for therapeutic intervention and diagnostics. We will also discuss emerging new tools in cancer di-agnostics and therapy based on 3'UTR binding factors and approaches to improve them.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
Objective To screen the proteins associated with four-and-a-half LIM domains 3(FHL3) 3' untranslated region(3'UTR) in glioma cells. Methods Western blot was adopted to detect the regulatory effect of poly(C)-b...Objective To screen the proteins associated with four-and-a-half LIM domains 3(FHL3) 3' untranslated region(3'UTR) in glioma cells. Methods Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2(PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry(LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immunoprecipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1(PTBP1), a binding protein identified by LC-MS/MS. Results PCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98 G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein. Conclusion PCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.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.
文摘The messenger RNA 3'-untranslated region(3'UTR)plays an important role in regulation of gene expres-sion on the posttranscriptional level. The 3'UTR con-trols gene expression via orchestrated interactionbetween the structural components of mRNAs(cis-ele-ment) and the specific trans-acting factors(RNA bind-ing proteins and non-coding RNAs). The crosstalk ofthese factors is based on the binding sequences and/or direct protein-protein interaction, or just functionalinteraction. Much new evidence that has accumulatedsupports the idea that several RNA binding factors canbind to common mRNA targets: to the non-overlappingbinding sites or to common sites in a competitive fash-ion. Various factors capable of binding to the sameRNA can cooperate or be antagonistic in their actions.The outcome of the collective function of all factorsbound to the same mRNA 3'UTR depends on manycircumstances, such as their expression levels, affinity to the binding sites, and localization in the cell, which can be controlled by various physiological conditions. Moreover, the functional and/or physical interactions of the factors binding to 3'UTR can change the character of their actions. These interactions vary during the cell cycle and in response to changing physiological condi-tions. Abnormal functioning of the factors can lead to disease. In this review we will discuss how alterations of these factors or their interaction can affect cancer development and promote or enhance the malignant phenotype of cancer cells. Understanding these altera-tions and their impact on 3'UTR-directed posttran-scriptional gene regulation will uncover promising new targets for therapeutic intervention and diagnostics. We will also discuss emerging new tools in cancer di-agnostics and therapy based on 3'UTR binding factors and approaches to improve them.
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,
基金Supported by Peking Union Medical College Youth Fundthe Fundamental Research Funds for the Central Universities(3332013052)
文摘Objective To screen the proteins associated with four-and-a-half LIM domains 3(FHL3) 3' untranslated region(3'UTR) in glioma cells. Methods Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2(PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry(LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immunoprecipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1(PTBP1), a binding protein identified by LC-MS/MS. Results PCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98 G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein. Conclusion PCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.
