Objective: To amplify HBV-RNase H gene fragment and express RNase H for further use in the studiesof HBV associated liver diseases. Methods: The encoding gene of HBV-RNase H was separately amplified for thefirst half ...Objective: To amplify HBV-RNase H gene fragment and express RNase H for further use in the studiesof HBV associated liver diseases. Methods: The encoding gene of HBV-RNase H was separately amplified for thefirst half and second half (H1 and H2)by PCR from full length HBV gene and cloned into pT7Blue-T vector.Clones were first screened by digestion with Xbal and HindⅢ enzyme for the correct size, and analyzed further byDNA sequencing. The RNase H1 and H2 fragments isolated from XbaⅠ and HindⅢ digestion products of pT7 BlueRNaseH plasmid were ligated to the GSTag expressing vectors separately, and expressed in E. coli BL21. The expressed proteins were checked by PAGE gel and Western blot. Results: Both H1 and H2 nucleotide seqences wereconsisted with the known genes and the proteins, with correct size, were further confirmed by western blot to bethe GST and RNaseHl or H2 fusion proteins. Conclusion: The successful cloning and expression of HBV-RNase Hwill contribute to further research and application in HBV associated diseases.展开更多
HIV-1 reverse transcriptase (RT) RNase H (HIV-RH) is a key target of anti-AIDS drugs. Metal-chelating compounds are an important class of chemicals in pharmacological drug discovery, especially in relation to HIV-RT a...HIV-1 reverse transcriptase (RT) RNase H (HIV-RH) is a key target of anti-AIDS drugs. Metal-chelating compounds are an important class of chemicals in pharmacological drug discovery, especially in relation to HIV-RT and the highly-related HIV-integrase. The correlation between the metal-chelating properties and enzyme activities of the metal chelators is always of high scientific interest, as an understanding of this may accelerate the rational optimization of this class of inhibitors. Our NMR data show that Mg2+ and Ca2+ bind specifically to the active site of the RNase H domain and two Mg2+ ions sequentially bind one molecule of RNase H. We also demonstrate here, using saturated and unsaturated tricyclic N-hydroxypyridones designed to block the active site, that the primary binding sites and affinities of divalent metal ions are correlated with the structures of the chelating motifs. Chemical shift perturbation studies of protein/metal-ion/compound ternary complexes also indicate that divalent metal ions play important roles for the specific interaction of the compounds with the RNase H active site.展开更多
Circular intronic RNAs(ci RNAs) escaping from DBR1 debranching of intron lariats are co-transcriptionally produced from prem RNA splicing, but their turnover and mechanism of action have remained elusive. We report th...Circular intronic RNAs(ci RNAs) escaping from DBR1 debranching of intron lariats are co-transcriptionally produced from prem RNA splicing, but their turnover and mechanism of action have remained elusive. We report that RNase H1 degrades a subgroup of ci RNAs in human cells. Many ci RNAs contain high GC% and tend to form DNA:RNA hybrids(R-loops) for RNase H1 cleavage, a process that appears to promote Pol II transcriptional elongation at ci RNA-producing loci. One ci RNA, ciankrd52, shows a stronger ability of R-loop formation than that of its cognate pre-m RNA by maintaining a locally open RNA structure in vitro. This allows the release of pre-m RNA from R-loops by ci-ankrd52 replacement and subsequent ci RNA removal via RNase H1 for efficient transcriptional elongation. We propose that such an R-loop dependent ci RNA degradation likely represents a mechanism that on one hand limits ci RNA accumulation by recruiting RNase H1 and on the other hand resolves Rloops for transcriptional elongation at some GC-rich ci RNA-producing loci.展开更多
Accurate quantitation of site-specific mRNA mutation in single cells or in peripheral blood is of great significance for both biological and biomedical studies.How to eliminate the false-positive interference from the...Accurate quantitation of site-specific mRNA mutation in single cells or in peripheral blood is of great significance for both biological and biomedical studies.How to eliminate the false-positive interference from the abundant normal mRNA is still a big challenge.Herein,we have proposed an LNA(locked nucleic acid)-assisted high-specificity strategy which can selectively guide the RNase H to cleave only the wildtype mRNA(wtRNA)while the mutant mRNA(mutRNA)will remain intact.The intact mutRNA can be amplified and detected by real-time reverse transcription(RT)-PCR but the disconnected wtRNA will be not replicated at all.Based on the highly selective depletion of wtRNA,this elegant design effectively avoids the false-positive interference from the high background of normal mRNA and thus can guarantee the accurate and reliable detection of rare mutRNA in real biomedical samples.Besides for the excellent specificity,ultrahigh sensitivity is also achieved for this proposed assay,which allows the quantification of mutRNA at single molecule and single cell level.Due to its easy design,high sensitivity and specificity,the established LNA probe-assisted RT-PCR strategy provides a powerful tool for studying the function of mutRNA at the single cell level and for the mutRNA-associated liquid biopsy.展开更多
Badnaviruses are serious plant pararetroviruses affecting banana and causes serious economic losses to banana production worldwide. This study aims to examine the variability of BSV and SCBV nature infecting banana in...Badnaviruses are serious plant pararetroviruses affecting banana and causes serious economic losses to banana production worldwide. This study aims to examine the variability of BSV and SCBV nature infecting banana in Burkina Faso. Polymerase Chain Reaction (PCR) used the Badna FP/RP specific primers for the RT/RNase H regions present in badnaviruses. The PCR yielded about 579 bp amplicons from banana infected by BSV and SCBV. The 38 BSV isolates recorded low nucleotide identity ranging from 58.9% - 98.1%. Based on percentage nucleotide sequence identity and phylogenetic analyse, BSV isolates were categorized into four groups: 1, 2, 3 and 4. Group 4 shared 76.9% - 100% identity with BSOL species. However, Groups 1 and 3 recorded a low identity ranging, from 76.8% - 79.2%, 68.8% - 79.7% with BSCV, and 72.8% - 79.0% between Group 2 and BSOLV. Groups 1, 2 and 3 were assigned to a potentially new BSV species. The two SCBV isolates recorded a low nucleotide identity of 68% among themselves indicating high diversity. In addition, SCBV_Cd and SCBV_CE showed high nucleotide identity 91.3% and 58.7% with SCBV_C and SCBV, when they were compared to all published SCBV genotypes. In addition, phylogenetic analysis revealed the segregation of SCBV isolates into two genotypes, SCBV_Cd in C and SCBV_CE segregated in a new genotype namely Z. Recombination analyses showed weak signatures of recombination among some of the BSV and SCBV sequences.展开更多
文摘Objective: To amplify HBV-RNase H gene fragment and express RNase H for further use in the studiesof HBV associated liver diseases. Methods: The encoding gene of HBV-RNase H was separately amplified for thefirst half and second half (H1 and H2)by PCR from full length HBV gene and cloned into pT7Blue-T vector.Clones were first screened by digestion with Xbal and HindⅢ enzyme for the correct size, and analyzed further byDNA sequencing. The RNase H1 and H2 fragments isolated from XbaⅠ and HindⅢ digestion products of pT7 BlueRNaseH plasmid were ligated to the GSTag expressing vectors separately, and expressed in E. coli BL21. The expressed proteins were checked by PAGE gel and Western blot. Results: Both H1 and H2 nucleotide seqences wereconsisted with the known genes and the proteins, with correct size, were further confirmed by western blot to bethe GST and RNaseHl or H2 fusion proteins. Conclusion: The successful cloning and expression of HBV-RNase Hwill contribute to further research and application in HBV associated diseases.
文摘HIV-1 reverse transcriptase (RT) RNase H (HIV-RH) is a key target of anti-AIDS drugs. Metal-chelating compounds are an important class of chemicals in pharmacological drug discovery, especially in relation to HIV-RT and the highly-related HIV-integrase. The correlation between the metal-chelating properties and enzyme activities of the metal chelators is always of high scientific interest, as an understanding of this may accelerate the rational optimization of this class of inhibitors. Our NMR data show that Mg2+ and Ca2+ bind specifically to the active site of the RNase H domain and two Mg2+ ions sequentially bind one molecule of RNase H. We also demonstrate here, using saturated and unsaturated tricyclic N-hydroxypyridones designed to block the active site, that the primary binding sites and affinities of divalent metal ions are correlated with the structures of the chelating motifs. Chemical shift perturbation studies of protein/metal-ion/compound ternary complexes also indicate that divalent metal ions play important roles for the specific interaction of the compounds with the RNase H active site.
基金This work was supported by the National Natural Science Foundation of China(NSFC)(91940303,31725009)the HHMI International Program(55008728)to L.-L.C.+2 种基金NSFC(31730111,31925011)to L.Y.Young Elite Scientists Sponsorship Program(2020QNRC001)to X.L.L.-L.C.the support from the XPLORER PRIZE.
文摘Circular intronic RNAs(ci RNAs) escaping from DBR1 debranching of intron lariats are co-transcriptionally produced from prem RNA splicing, but their turnover and mechanism of action have remained elusive. We report that RNase H1 degrades a subgroup of ci RNAs in human cells. Many ci RNAs contain high GC% and tend to form DNA:RNA hybrids(R-loops) for RNase H1 cleavage, a process that appears to promote Pol II transcriptional elongation at ci RNA-producing loci. One ci RNA, ciankrd52, shows a stronger ability of R-loop formation than that of its cognate pre-m RNA by maintaining a locally open RNA structure in vitro. This allows the release of pre-m RNA from R-loops by ci-ankrd52 replacement and subsequent ci RNA removal via RNase H1 for efficient transcriptional elongation. We propose that such an R-loop dependent ci RNA degradation likely represents a mechanism that on one hand limits ci RNA accumulation by recruiting RNase H1 and on the other hand resolves Rloops for transcriptional elongation at some GC-rich ci RNA-producing loci.
基金supported by the National Natural Science Foundation of China(Nos.21622507 and 21575086)the Program for Changjiang Scholars and Innovative Re-search Team in University(No.IRT_15R43)the Fundamental Research Funds for the Central Universities(No.GK201802016)。
文摘Accurate quantitation of site-specific mRNA mutation in single cells or in peripheral blood is of great significance for both biological and biomedical studies.How to eliminate the false-positive interference from the abundant normal mRNA is still a big challenge.Herein,we have proposed an LNA(locked nucleic acid)-assisted high-specificity strategy which can selectively guide the RNase H to cleave only the wildtype mRNA(wtRNA)while the mutant mRNA(mutRNA)will remain intact.The intact mutRNA can be amplified and detected by real-time reverse transcription(RT)-PCR but the disconnected wtRNA will be not replicated at all.Based on the highly selective depletion of wtRNA,this elegant design effectively avoids the false-positive interference from the high background of normal mRNA and thus can guarantee the accurate and reliable detection of rare mutRNA in real biomedical samples.Besides for the excellent specificity,ultrahigh sensitivity is also achieved for this proposed assay,which allows the quantification of mutRNA at single molecule and single cell level.Due to its easy design,high sensitivity and specificity,the established LNA probe-assisted RT-PCR strategy provides a powerful tool for studying the function of mutRNA at the single cell level and for the mutRNA-associated liquid biopsy.
文摘Badnaviruses are serious plant pararetroviruses affecting banana and causes serious economic losses to banana production worldwide. This study aims to examine the variability of BSV and SCBV nature infecting banana in Burkina Faso. Polymerase Chain Reaction (PCR) used the Badna FP/RP specific primers for the RT/RNase H regions present in badnaviruses. The PCR yielded about 579 bp amplicons from banana infected by BSV and SCBV. The 38 BSV isolates recorded low nucleotide identity ranging from 58.9% - 98.1%. Based on percentage nucleotide sequence identity and phylogenetic analyse, BSV isolates were categorized into four groups: 1, 2, 3 and 4. Group 4 shared 76.9% - 100% identity with BSOL species. However, Groups 1 and 3 recorded a low identity ranging, from 76.8% - 79.2%, 68.8% - 79.7% with BSCV, and 72.8% - 79.0% between Group 2 and BSOLV. Groups 1, 2 and 3 were assigned to a potentially new BSV species. The two SCBV isolates recorded a low nucleotide identity of 68% among themselves indicating high diversity. In addition, SCBV_Cd and SCBV_CE showed high nucleotide identity 91.3% and 58.7% with SCBV_C and SCBV, when they were compared to all published SCBV genotypes. In addition, phylogenetic analysis revealed the segregation of SCBV isolates into two genotypes, SCBV_Cd in C and SCBV_CE segregated in a new genotype namely Z. Recombination analyses showed weak signatures of recombination among some of the BSV and SCBV sequences.
