AIM: To investigate the subcellular localization and the function of mouse transducin β-like 3(Tbl3).METHODS: The coding sequence of mouse Tbl3 was cloned from the c DNAs of a promyelocyte cell line by reverse transc...AIM: To investigate the subcellular localization and the function of mouse transducin β-like 3(Tbl3).METHODS: The coding sequence of mouse Tbl3 was cloned from the c DNAs of a promyelocyte cell line by reverse transcription-polymerase chain reaction. Fusion constructs of Tbl3 and enhanced green fluorescent protein(EGFP) were transfected into fibroblasts and examined by fluorescence microscopy to reveal the subcellular localization of tbl3. To search for nucleolar targeting sequences, scanning deletions of Tbl3-EGFP were constructed and transfected into fibroblasts. To explore the possible function of Tbl3, small hairpin RNAs(sh RNAs) were used to knock down endogenous Tbl3 in mouse promyelocytes and fibroblasts. The effects of Tbl3 knockdown on ribosomal RNA(r RNAs) synthesis or processing were studied by labeling cells with 5,6-3H-uridine followed by a chase with fresh medium for various periods. Total RNAs were purified from treated cells and subjected to gel electrophoresis and Northern analysis. Ribosome profiling by sucrose gradient centrifugation was used to compare the amounts of 40 S and 60 S ribosome subunits as well as the 80 S monosome. The impact of Tbl3 knockdown on cell growth and proliferation was examined by growth curves and colony assays.RESULTS: The largest open reading frame of mouse Tbl3 encodes a protein of 801 amino acids(AA) with an apparent molecular weight of 89-90 kilodalton. It contains thirteen WD40 repeats(an ancient protein-protein interaction motif) and a carboxyl terminus that is highly homologous to the corresponding region of the yeast nucleolar protein, utp13. Virtually nothing is known about the biological function of Tbl3. All cell lines surveyed expressed Tbl3 and the level of expression correlated roughly with cell proliferation and/or biosynthetic activity. Using Tbl3-EGFP fusion constructs we obtained the first direct evidence that Tbl3 is targeted to the nucleoli in mammalian cells. However, no previously described nucleolar targeting sequences were found in Tbl3, sugge展开更多
RtcB, a highly conserved RNA ligase, is found in all three domains of life, and demonstrated to be an essential tRNA splicing component in archaea and metazoans. However, the biological functions of RtcB in bacteria, ...RtcB, a highly conserved RNA ligase, is found in all three domains of life, and demonstrated to be an essential tRNA splicing component in archaea and metazoans. However, the biological functions of RtcB in bacteria, where there is no splicing, remains to be clarified. We first performed bioinformatics analysis which revealed highly conserved structures and presumably conserved functions of RtcB in bacteria. However, its orthologs only occur in ~0.5% of bacterial species across diverse phyla with significant signals of frequent horizontal transfer, highlighting its non-essential role in bacteria. Next, by constructing an rtcBknockout strain, we find that the removal of antibiotic stress induces a significant impact on rtcB expression in wild-type strain,and furthermore the depletion of RtcB(?RtcB strain) delays the recovery process. Our transcriptomic analysis, comprising the3′-end labeling of RNAs, highlights a significant increase in unmapped reads and cleaved rRNAs in the ?RtcB strain, particularly during recovery. Our observations suggest that the conserved RNA ligase RtcB, repairs damaged r RNAs following stress,which potentially saves energy and accelerates recovery of its host. We propose that acquisition of RtcB by diverse bacterial taxa provides a competitive advantage under stressful conditions.展开更多
Prorocentrum donghaiense is an important harmful algae bloom （HAB） causing creature in China＇s seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects...Prorocentrum donghaiense is an important harmful algae bloom （HAB） causing creature in China＇s seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay （NPA-SH） was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, and the regression equation is y=4× 10^- 6x＋ 0.694 9, in which x is the cell number, and y is the optical density, with r2=0.953 5. These results show that the NPA-SH method has good feasibility in the detection of P. donghaiense. Results of NPA-SH and microscopy are excellent for each sample. The NPA-SH method was a simple way in quantitative detection of P. donghaiense, and the whole process could be finished in about six hours, which provided a new approach in high-throughput sampling and long-term monitoring of P. donghaiense.展开更多
X-ray structures of transfer RNAs (tRNAs) bound to the whole ribosome do not fully explain the mechanism of translation. The cause of the failure seems to come mainly from a high Mg2+ ion concentration compared to tha...X-ray structures of transfer RNAs (tRNAs) bound to the whole ribosome do not fully explain the mechanism of translation. The cause of the failure seems to come mainly from a high Mg2+ ion concentration compared to that in the living cells. There exists a wide range of nucleotide sequence conservation in tRNA and ribosomal RNAs (rRNAs) of small and large subunits as well as sequence complementarities, that seems to explain how high accuracy in translation can be achieved at the decoding site. Conformational transition between U33-folded and U33-extended forms of anticodon loops of tRNAs and G-C pair formation and disruption between C1399 and G1504 of 16S rRNA, etc. play the central role in explaining why E-site tRNA can automatically be expelled when an aminoacyl-tRNA at the A site turns out to be cognate.展开更多
目的以高通量测序技术对1例上呼吸道感染病例的可能病原进行鉴定并对样品前处理方法进行优化。方法以核酸酶和/或核糖体RNA探针杂交预处理患者咽拭子样本，非序列依赖单引物扩增（sequence-independent single primer amplification，SI...目的以高通量测序技术对1例上呼吸道感染病例的可能病原进行鉴定并对样品前处理方法进行优化。方法以核酸酶和/或核糖体RNA探针杂交预处理患者咽拭子样本，非序列依赖单引物扩增（sequence-independent single primer amplification，SISPA）后制备测序文库，利用MiSeq高通量测序，对测序结果进行生物信息分析及Real time RT-PCR验证。结果测序reads经拼接后与病毒数据库比对发现存在乙型流感病毒序列，进化分析该毒株属于Yamagat系一株新的变种，核酸酶结合核糖体RNA探针杂交处理样本可提高高通量测序目标序列数及覆盖率。结论应用核酸酶和/或核糖体RNA探针杂交处理样本结合高通量测序技术可用于快速高效地鉴定不明原因感染的病原。展开更多
基金Supported by In part by a grant from the St.Perres Fund,No.11-02011
文摘AIM: To investigate the subcellular localization and the function of mouse transducin β-like 3(Tbl3).METHODS: The coding sequence of mouse Tbl3 was cloned from the c DNAs of a promyelocyte cell line by reverse transcription-polymerase chain reaction. Fusion constructs of Tbl3 and enhanced green fluorescent protein(EGFP) were transfected into fibroblasts and examined by fluorescence microscopy to reveal the subcellular localization of tbl3. To search for nucleolar targeting sequences, scanning deletions of Tbl3-EGFP were constructed and transfected into fibroblasts. To explore the possible function of Tbl3, small hairpin RNAs(sh RNAs) were used to knock down endogenous Tbl3 in mouse promyelocytes and fibroblasts. The effects of Tbl3 knockdown on ribosomal RNA(r RNAs) synthesis or processing were studied by labeling cells with 5,6-3H-uridine followed by a chase with fresh medium for various periods. Total RNAs were purified from treated cells and subjected to gel electrophoresis and Northern analysis. Ribosome profiling by sucrose gradient centrifugation was used to compare the amounts of 40 S and 60 S ribosome subunits as well as the 80 S monosome. The impact of Tbl3 knockdown on cell growth and proliferation was examined by growth curves and colony assays.RESULTS: The largest open reading frame of mouse Tbl3 encodes a protein of 801 amino acids(AA) with an apparent molecular weight of 89-90 kilodalton. It contains thirteen WD40 repeats(an ancient protein-protein interaction motif) and a carboxyl terminus that is highly homologous to the corresponding region of the yeast nucleolar protein, utp13. Virtually nothing is known about the biological function of Tbl3. All cell lines surveyed expressed Tbl3 and the level of expression correlated roughly with cell proliferation and/or biosynthetic activity. Using Tbl3-EGFP fusion constructs we obtained the first direct evidence that Tbl3 is targeted to the nucleoli in mammalian cells. However, no previously described nucleolar targeting sequences were found in Tbl3, sugge
基金supported by the National Key Research and Development Program of China(2016YFC0903800)the National Scientific Foundation of China(31470180,31471237,31671350)+2 种基金the Programs of Beijing Municipal Science and Technology Project(Z171100001317011)the Scientific Research Project of Public Welfare Industry(2013FY114300,201402018)the Key Research Program of Frontier Sciences,the Chinese Academy of Sciences(QYZDY-SSWSMC017)。
文摘RtcB, a highly conserved RNA ligase, is found in all three domains of life, and demonstrated to be an essential tRNA splicing component in archaea and metazoans. However, the biological functions of RtcB in bacteria, where there is no splicing, remains to be clarified. We first performed bioinformatics analysis which revealed highly conserved structures and presumably conserved functions of RtcB in bacteria. However, its orthologs only occur in ~0.5% of bacterial species across diverse phyla with significant signals of frequent horizontal transfer, highlighting its non-essential role in bacteria. Next, by constructing an rtcBknockout strain, we find that the removal of antibiotic stress induces a significant impact on rtcB expression in wild-type strain,and furthermore the depletion of RtcB(?RtcB strain) delays the recovery process. Our transcriptomic analysis, comprising the3′-end labeling of RNAs, highlights a significant increase in unmapped reads and cleaved rRNAs in the ?RtcB strain, particularly during recovery. Our observations suggest that the conserved RNA ligase RtcB, repairs damaged r RNAs following stress,which potentially saves energy and accelerates recovery of its host. We propose that acquisition of RtcB by diverse bacterial taxa provides a competitive advantage under stressful conditions.
基金The National High Technology Research and Development Program ("863" Program) of China under contract No 2006AA09Z178 the National Natural Science Foundation of China under contract No 40706044
文摘Prorocentrum donghaiense is an important harmful algae bloom （HAB） causing creature in China＇s seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay （NPA-SH） was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, and the regression equation is y=4× 10^- 6x＋ 0.694 9, in which x is the cell number, and y is the optical density, with r2=0.953 5. These results show that the NPA-SH method has good feasibility in the detection of P. donghaiense. Results of NPA-SH and microscopy are excellent for each sample. The NPA-SH method was a simple way in quantitative detection of P. donghaiense, and the whole process could be finished in about six hours, which provided a new approach in high-throughput sampling and long-term monitoring of P. donghaiense.
文摘X-ray structures of transfer RNAs (tRNAs) bound to the whole ribosome do not fully explain the mechanism of translation. The cause of the failure seems to come mainly from a high Mg2+ ion concentration compared to that in the living cells. There exists a wide range of nucleotide sequence conservation in tRNA and ribosomal RNAs (rRNAs) of small and large subunits as well as sequence complementarities, that seems to explain how high accuracy in translation can be achieved at the decoding site. Conformational transition between U33-folded and U33-extended forms of anticodon loops of tRNAs and G-C pair formation and disruption between C1399 and G1504 of 16S rRNA, etc. play the central role in explaining why E-site tRNA can automatically be expelled when an aminoacyl-tRNA at the A site turns out to be cognate.
文摘目的以高通量测序技术对1例上呼吸道感染病例的可能病原进行鉴定并对样品前处理方法进行优化。方法以核酸酶和/或核糖体RNA探针杂交预处理患者咽拭子样本，非序列依赖单引物扩增（sequence-independent single primer amplification，SISPA）后制备测序文库，利用MiSeq高通量测序，对测序结果进行生物信息分析及Real time RT-PCR验证。结果测序reads经拼接后与病毒数据库比对发现存在乙型流感病毒序列，进化分析该毒株属于Yamagat系一株新的变种，核酸酶结合核糖体RNA探针杂交处理样本可提高高通量测序目标序列数及覆盖率。结论应用核酸酶和/或核糖体RNA探针杂交处理样本结合高通量测序技术可用于快速高效地鉴定不明原因感染的病原。
