With the advent of rapid automated in silico identification of biosynthetic gene clusters(BGCs),genomics pre-sents vast opportunities to accelerate natural product(NP)discovery.However,prolific NP producers,Strepto-my...With the advent of rapid automated in silico identification of biosynthetic gene clusters(BGCs),genomics pre-sents vast opportunities to accelerate natural product(NP)discovery.However,prolific NP producers,Strepto-myces,are exceptionally GC-rich(>80%)and highly repetitive within BGCs.These pose challenges in sequencing and high-quality genome assembly which are currently circumvented via intensive sequencing.Here,we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes.Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions.We successfully sequenced and assembled 8 GC-rich Streptomyces genomes whose lengths range from 7.1 to 12.1 Mb with a median N50 of 8.2 Mb.Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species,Streptomyces sydneybrenneri.Further comprehensive characterization of their biosynthetic,pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase(PKS)BGCs reflected their potential as alternative NP hosts.Thus,the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery.展开更多
High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein.The stable expression vectors were ...High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein.The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment(including intron) of extremely GC-rich at the 5’ or/and 3’ flanking regions of these protein genes or their gene promoters.This experiment is the first experimental evidence showing that a non-coding extremely GC-rich DNA fragment is a super "chromatin opening element" and plays an important role in mammalian gene expression.This experiment has further indicated that chromatin-based regulation of mammalian gene expression is at least partially embedded in DNA primary structure,namely DNA GC-content.展开更多
目的：探索高GC含量DNA的PCR扩增条件，为扩增达托霉素生物合成基因簇及拼接奠定基础。方法：在PCR扩增体系中，使用高保真的聚合酶及添加不同浓度的DMSO、7-deaza-dGTP等增强剂，并选择合适的PCR循环程序，优化富含GC的DNA的PCR扩增条...目的：探索高GC含量DNA的PCR扩增条件，为扩增达托霉素生物合成基因簇及拼接奠定基础。方法：在PCR扩增体系中，使用高保真的聚合酶及添加不同浓度的DMSO、7-deaza-dGTP等增强剂，并选择合适的PCR循环程序，优化富含GC的DNA的PCR扩增条件。结果：向反应体系中额外添加1%~4%的DMSO可以显著提高富含GC的DNA的PCR扩增产物量，但会降低其特异性；7-deaza-dGTP可以提高扩增产物的特异性及保真度，但产量会有所下降。应用touch down PCR并在体系中添加7-deaza-dGTP能够提高扩增产物的特异性和产率，增加扩增的保真度。结论：应用优化的PCR扩增条件将所有达托霉素生物合成基因簇分段扩增出来，并可扩增出长达6 kb的片段，且序列完全正确，可以进行后续拼接。展开更多
We use single-molecule FRET and newly-developed D-loop techniques to investigate strand displacement activity of Klenow fragment(exo-)of DNA polymerase I in DNA sequences rich in guanine and cytosine(GC)bases.We find ...We use single-molecule FRET and newly-developed D-loop techniques to investigate strand displacement activity of Klenow fragment(exo-)of DNA polymerase I in DNA sequences rich in guanine and cytosine(GC)bases.We find that there exist in the FRET traces numerous ascending jumps,which are induced by the backsliding of Klenow fragment on DNA chains.Our measurements show that the probability of backsliding is closely related to the GC-richness and d NTP concentration:increasing the GC-richness leads to an increase in the backsliding probability,and increasing the d NTP concentration however leads to a decrease in the backsliding probability.These results provide a new insight into the mechanism of DNA polymerase I.展开更多
基金supported by National Research Foundation,Singapore(NRF-CRP19-2017-05-00)Agency for Science,Technology and Research(A*STAR),Singapore(#21719).
文摘With the advent of rapid automated in silico identification of biosynthetic gene clusters(BGCs),genomics pre-sents vast opportunities to accelerate natural product(NP)discovery.However,prolific NP producers,Strepto-myces,are exceptionally GC-rich(>80%)and highly repetitive within BGCs.These pose challenges in sequencing and high-quality genome assembly which are currently circumvented via intensive sequencing.Here,we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes.Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions.We successfully sequenced and assembled 8 GC-rich Streptomyces genomes whose lengths range from 7.1 to 12.1 Mb with a median N50 of 8.2 Mb.Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species,Streptomyces sydneybrenneri.Further comprehensive characterization of their biosynthetic,pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase(PKS)BGCs reflected their potential as alternative NP hosts.Thus,the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery.
文摘High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein.The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment(including intron) of extremely GC-rich at the 5’ or/and 3’ flanking regions of these protein genes or their gene promoters.This experiment is the first experimental evidence showing that a non-coding extremely GC-rich DNA fragment is a super "chromatin opening element" and plays an important role in mammalian gene expression.This experiment has further indicated that chromatin-based regulation of mammalian gene expression is at least partially embedded in DNA primary structure,namely DNA GC-content.
文摘目的：探索高GC含量DNA的PCR扩增条件，为扩增达托霉素生物合成基因簇及拼接奠定基础。方法：在PCR扩增体系中，使用高保真的聚合酶及添加不同浓度的DMSO、7-deaza-dGTP等增强剂，并选择合适的PCR循环程序，优化富含GC的DNA的PCR扩增条件。结果：向反应体系中额外添加1%~4%的DMSO可以显著提高富含GC的DNA的PCR扩增产物量，但会降低其特异性；7-deaza-dGTP可以提高扩增产物的特异性及保真度，但产量会有所下降。应用touch down PCR并在体系中添加7-deaza-dGTP能够提高扩增产物的特异性和产率，增加扩增的保真度。结论：应用优化的PCR扩增条件将所有达托霉素生物合成基因簇分段扩增出来，并可扩增出长达6 kb的片段，且序列完全正确，可以进行后续拼接。
基金Project supported by the National Natural Science Foundation of China(Grant No.12090051)the CAS Key Research Program of Frontier Sciences(Grant Nos.QYZDJSSW-SYS014 and ZDBS-LY-SLH015)the Youth Innovation Promotion Association of CAS(Grant No.2017015)。
文摘We use single-molecule FRET and newly-developed D-loop techniques to investigate strand displacement activity of Klenow fragment(exo-)of DNA polymerase I in DNA sequences rich in guanine and cytosine(GC)bases.We find that there exist in the FRET traces numerous ascending jumps,which are induced by the backsliding of Klenow fragment on DNA chains.Our measurements show that the probability of backsliding is closely related to the GC-richness and d NTP concentration:increasing the GC-richness leads to an increase in the backsliding probability,and increasing the d NTP concentration however leads to a decrease in the backsliding probability.These results provide a new insight into the mechanism of DNA polymerase I.
