目的应用SELEX(system evolution of ligands by exponential enrichment)技术筛选与HER2特异性结合的寡聚核酸适配体(aptamer)。方法体外合成了长度为78个碱基并含有35个随机寡核苷酸的ssDNA文库,通过生物素-链亲和素磁珠系统分离法制...目的应用SELEX(system evolution of ligands by exponential enrichment)技术筛选与HER2特异性结合的寡聚核酸适配体(aptamer)。方法体外合成了长度为78个碱基并含有35个随机寡核苷酸的ssDNA文库,通过生物素-链亲和素磁珠系统分离法制备次级ssDNA文库,且经以微孔为介质的SELEX正反筛法获得了与HER2特异性结合的DNA适配体;同时利用生物素-链霉亲和素-辣根过氧化物酶系统检测每轮ssDNA与靶蛋白HER2的亲和力,将亲和力最高的适配体群进行克隆、测序。最后利用生物学软件DNAMAN对适配体群进行了一级结构和二级结构的分析,同时将亲和性最高的适配体做了进一步的特异性的检验。结果经过12轮的筛选,获得了与HER2蛋白具有极高亲和力的适配体9号,亲和力从初始的0.18上升至0.86,提高了约5倍。特异性结果显示,9号适配体只与HER2具有较高的特异性。结论经过多轮的筛选,成功获得了特异性识别骨肉瘤HER2蛋白的单链DNA适配体,为骨肉瘤的诊断和治疗奠定了基础。展开更多
DNA-binding fluorochromes are often used for vital staining of plant cell nuclei. However, it is not always sure whether the cells after staining still remain in living state. We chose several criteria to estimate the...DNA-binding fluorochromes are often used for vital staining of plant cell nuclei. However, it is not always sure whether the cells after staining still remain in living state. We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei. These were: the cytoplasmic streaming in pollen tubes whose nuclei were stained, the simultaneous visualization of fluo-rochromatic reaction and nucleus staining in isolated generative cells, and the capability of isolated, prestained generative or sperm cells to fuse with other protoplasts. The results confirmed that 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33258 and mithramycin could be used as real vital stains, though their efficiency varied from case to case; among them DAPI showed best effect. The fluorescent vital staining technique offered a useful means fori-dentification and selection of heterokaryons in gametoplast manipulation studies.展开更多
Meiosis comprises two rounds of nuclear division following a single phase of DNA replication, leading to the production of haploid gametes and is essential for sexual reproduction in eukaryotes. Unlike mitosis, meiosi...Meiosis comprises two rounds of nuclear division following a single phase of DNA replication, leading to the production of haploid gametes and is essential for sexual reproduction in eukaryotes. Unlike mitosis, meiosis involves homologous chromosome pairing, synapsis, and recombination during prophase I. Meiotic recombination not only ensures the accurate segregation of homologs, but also redistributes alleles among offspring. DNA synthesis is a critical process during meiotic recombination, but our understanding of the proteins that execute and regulate it is limited. This review summarizes the recent advances in defining the role of DNA synthesis in meiotic recombina- tion through analyses of DNA synthesis genes, with specific emphasis on DNA polymerases （e.g., Pole and PolS）, replication processivity factor RFC1 and translesion polymerases （e.g., Pol~）. We also present a new double strand break repair model for meiotic recombination, which includes lagging strand DNA synthesis and leading strand elongation. Finally, we propose that DNA synthesis is one of critical factors for discriminating meiotic recombination pathways and that this differentiation may be conserved among eukaryotes.展开更多
Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks(DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. Howeve...Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks(DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction.Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I.Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans.展开更多
The fast development of next-generation sequencing technology presents a major computational challenge for data processing and analysis.A fast algorithm,de Bruijn graph has been successfully used for genome DNA de nov...The fast development of next-generation sequencing technology presents a major computational challenge for data processing and analysis.A fast algorithm,de Bruijn graph has been successfully used for genome DNA de novo assembly;nevertheless,its performance for transcriptome assembly is unclear.In this study,we used both simulated and real RNA-Seq data,from either artificial RNA templates or human transcripts,to evaluate five de novo assemblers,ABySS,Mira,Trinity,Velvet and Oases.Of these assemblers,ABySS,Trinity,Velvet and Oases are all based on de Bruijn graph,and Mira uses an overlap graph algorithm.Various numbers of RNA short reads were selected from the External RNA Control Consortium(ERCC) data and human chromosome 22.A number of statistics were then calculated for the resulting contigs from each assembler.Each experiment was repeated multiple times to obtain the mean statistics and standard error estimate.Trinity had relative good performance for both ERCC and human data,but it may not consistently generate full length transcripts.ABySS was the fastest method but its assembly quality was low.Mira gave a good rate for mapping its contigs onto human chromosome 22,but its computational speed is not satisfactory.Our results suggest that transcript assembly remains a challenge problem for bioinformatics society.Therefore,a novel assembler is in need for assembling transcriptome data generated by next generation sequencing technique.展开更多
Meiotic recombination is a deeply conserved process within eukaryotes that has a profound effect on patterns of natural genetic variation. During meiosis homologous chromosomes pair and undergo DNA double strand break...Meiotic recombination is a deeply conserved process within eukaryotes that has a profound effect on patterns of natural genetic variation. During meiosis homologous chromosomes pair and undergo DNA double strand breaks generated by the Spo11 endonuclease. These breaks can be repaired as crossovers that result in reciprocal exchange between chromosomes. The frequency of recombination along chromosomes is highly variable, for example, crossovers are rarely observed in heterochromatin and the centromeric regions. Recent work in plants has shown that crossover hotspots occur in gene promoters and are associated with specific chromatin modifications, including H2 A.Z. Meiotic chromosomes are also organized in loop-base arrays connected to an underlying chromosome axis, which likely interacts with chromatin to organize patterns of recombination.Therefore, epigenetic information exerts a major influence on patterns of meiotic recombination along chromosomes, genetic variation within populations and evolution of plant genomes.展开更多
文摘目的应用SELEX(system evolution of ligands by exponential enrichment)技术筛选与HER2特异性结合的寡聚核酸适配体(aptamer)。方法体外合成了长度为78个碱基并含有35个随机寡核苷酸的ssDNA文库,通过生物素-链亲和素磁珠系统分离法制备次级ssDNA文库,且经以微孔为介质的SELEX正反筛法获得了与HER2特异性结合的DNA适配体;同时利用生物素-链霉亲和素-辣根过氧化物酶系统检测每轮ssDNA与靶蛋白HER2的亲和力,将亲和力最高的适配体群进行克隆、测序。最后利用生物学软件DNAMAN对适配体群进行了一级结构和二级结构的分析,同时将亲和性最高的适配体做了进一步的特异性的检验。结果经过12轮的筛选,获得了与HER2蛋白具有极高亲和力的适配体9号,亲和力从初始的0.18上升至0.86,提高了约5倍。特异性结果显示,9号适配体只与HER2具有较高的特异性。结论经过多轮的筛选,成功获得了特异性识别骨肉瘤HER2蛋白的单链DNA适配体,为骨肉瘤的诊断和治疗奠定了基础。
文摘DNA-binding fluorochromes are often used for vital staining of plant cell nuclei. However, it is not always sure whether the cells after staining still remain in living state. We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei. These were: the cytoplasmic streaming in pollen tubes whose nuclei were stained, the simultaneous visualization of fluo-rochromatic reaction and nucleus staining in isolated generative cells, and the capability of isolated, prestained generative or sperm cells to fuse with other protoplasts. The results confirmed that 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33258 and mithramycin could be used as real vital stains, though their efficiency varied from case to case; among them DAPI showed best effect. The fluorescent vital staining technique offered a useful means fori-dentification and selection of heterokaryons in gametoplast manipulation studies.
基金Acknowledgments We apologize to colleagues whose work could not be cited owing to space constraints. J.H., H.M. and Y.W. are supported by the Ministry of Science and Technology of China （2011CB944603）, the National Natural Science Foundation of China （31370347）, and by funds from Fudan University and Rijk Zwaan. G.P.C. is supported by the US National Science Foundation （MCB- 1121563） and Rijk Zwaan.
文摘Meiosis comprises two rounds of nuclear division following a single phase of DNA replication, leading to the production of haploid gametes and is essential for sexual reproduction in eukaryotes. Unlike mitosis, meiosis involves homologous chromosome pairing, synapsis, and recombination during prophase I. Meiotic recombination not only ensures the accurate segregation of homologs, but also redistributes alleles among offspring. DNA synthesis is a critical process during meiotic recombination, but our understanding of the proteins that execute and regulate it is limited. This review summarizes the recent advances in defining the role of DNA synthesis in meiotic recombina- tion through analyses of DNA synthesis genes, with specific emphasis on DNA polymerases （e.g., Pole and PolS）, replication processivity factor RFC1 and translesion polymerases （e.g., Pol~）. We also present a new double strand break repair model for meiotic recombination, which includes lagging strand DNA synthesis and leading strand elongation. Finally, we propose that DNA synthesis is one of critical factors for discriminating meiotic recombination pathways and that this differentiation may be conserved among eukaryotes.
基金supported by the National Key Research and Developmental Program of China (2018YFC1003700, 2016YFC1000600, 2018YFC1003400 and 2018YFC1004700)the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000)the National Natural Science Foundation of China (31890780, 31630050, 31871514 and 31771668)。
文摘Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks(DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction.Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I.Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans.
基金supported by grants from the National Center for Research Resources (5P20RR016471-12)the National Institute of General Medical Sciences (8 P20 GM103442-12) from the National Institutes of Healththe seed collaborative research grant from the Odegard School of Aerospace Sciences and the School of Medicine and Health Sciences at University of North Dakota
文摘The fast development of next-generation sequencing technology presents a major computational challenge for data processing and analysis.A fast algorithm,de Bruijn graph has been successfully used for genome DNA de novo assembly;nevertheless,its performance for transcriptome assembly is unclear.In this study,we used both simulated and real RNA-Seq data,from either artificial RNA templates or human transcripts,to evaluate five de novo assemblers,ABySS,Mira,Trinity,Velvet and Oases.Of these assemblers,ABySS,Trinity,Velvet and Oases are all based on de Bruijn graph,and Mira uses an overlap graph algorithm.Various numbers of RNA short reads were selected from the External RNA Control Consortium(ERCC) data and human chromosome 22.A number of statistics were then calculated for the resulting contigs from each assembler.Each experiment was repeated multiple times to obtain the mean statistics and standard error estimate.Trinity had relative good performance for both ERCC and human data,but it may not consistently generate full length transcripts.ABySS was the fastest method but its assembly quality was low.Mira gave a good rate for mapping its contigs onto human chromosome 22,but its computational speed is not satisfactory.Our results suggest that transcript assembly remains a challenge problem for bioinformatics society.Therefore,a novel assembler is in need for assembling transcriptome data generated by next generation sequencing technique.
文摘Meiotic recombination is a deeply conserved process within eukaryotes that has a profound effect on patterns of natural genetic variation. During meiosis homologous chromosomes pair and undergo DNA double strand breaks generated by the Spo11 endonuclease. These breaks can be repaired as crossovers that result in reciprocal exchange between chromosomes. The frequency of recombination along chromosomes is highly variable, for example, crossovers are rarely observed in heterochromatin and the centromeric regions. Recent work in plants has shown that crossover hotspots occur in gene promoters and are associated with specific chromatin modifications, including H2 A.Z. Meiotic chromosomes are also organized in loop-base arrays connected to an underlying chromosome axis, which likely interacts with chromatin to organize patterns of recombination.Therefore, epigenetic information exerts a major influence on patterns of meiotic recombination along chromosomes, genetic variation within populations and evolution of plant genomes.
