Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools,which include efficient genome editing,gene silencing,and robust protein expression systems.However,plasmid v...Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools,which include efficient genome editing,gene silencing,and robust protein expression systems.However,plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid.Although this plasmid coexists with pRN1 in its original host,early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa.islandicus.We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon.We identified a putative target sequence in orf904 encoding a putative replicase on pRN1(target N1).Mutated targets(N1a,N1b,and N1c)were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid inter-ference assay.The results revealed that the original target triggered CRISPR immunity in this archaeon,whereas all three mutated targets did not,indicating that all the designed target mutations evaded host immunity.These mutated targets were then incorporated into orf904 individually,yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed(pN1aSD,pN1bSD,and pN1cSD).Sa.islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors,but pN1cSD lost the capability for replication.These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase.We further showed that pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination,and this yielded a dual plasmid system for genetic study with this important archaeal model.展开更多
Two Escherichia coli Saccharomyces cerevisiae shuttle vectors containing a resistance marker to geneticin (G418) are constructed. Both vectors contain a kanamycin resistant marker (KanMX4) module coding aminoglyc...Two Escherichia coli Saccharomyces cerevisiae shuttle vectors containing a resistance marker to geneticin (G418) are constructed. Both vectors contain a kanamycin resistant marker (KanMX4) module coding aminoglycoside 3' phosphotransferase (APH) that renders E. coli resistant to kanamycin and S. cerevisiae to geneticin. These vectors overcome the shortage of the conventional yeast vectors bearing HIS3, TRP1, LEU2, and URA3 modules as selection markers, which require hosts to be auxotrophic. Green fluorescent protein (GFP) is used as the reporter to examine the functions of the vectors. The vectors are powerful tools for the convenient cloning and controlled expression of genes in most S. cerevisiae strains.展开更多
Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways wi...Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways with organisms of Bacteria, the sister prokaryotic phylum. Archaeal organisms also possess unique features as revealed by genomics and genome comparisons and by biochemical characterization of prominent enzymes. Nevertheless, diverse genetic tools are required for in vivo experiments to verify these interesting discoveries. Considerable efforts have been devoted to the development of genetic tools for archaea ever since their discovery, and great progress has been made in the creation of archaeal genetic tools in the past decade. Versatile genetic toolboxes are now available for several archaeal models, among which Sulfolobus microorganisms are the only genus representing Crenarchaeota because all the remaining genera are from Euryarchaeota. Nevertheless, genetic tools developed for Sulfolobus are probably the most versatile among all archaeal models, and these include viral and plasmid shuttle vectors, conventional and novel genetic manipulation methods, CRISPR-based gene deletion and mutagenesis, and gene silencing, among which CRISPR tools have been reported only for Sulfolobus thus far. In this review, we summarize recent developments in all these useful genetic tools and discuss their possible application to research into archaeal biology by means of Sulfolobus models.展开更多
N^(6)-methyldeoxyadenosine(6 mdA) modification is considered as a new epigenetic mark that may play important roles in various biological processes.However,it remains unclear about the effect of 6 mdA on DNA replicati...N^(6)-methyldeoxyadenosine(6 mdA) modification is considered as a new epigenetic mark that may play important roles in various biological processes.However,it remains unclear about the effect of 6 mdA on DNA replication in human cells.Herein,we combined next-generation sequencing with shuttle vector technology to explore how 6 mdA affects the efficiency and accuracy of DNA replication in human cells.Our results showed that 6 mdA neither blocked DNA replication nor induced mutations in human cells.Moreover,we found that the depletion of translesion synthesis DNA polymerase(Pol) κ,Pol η,Pol ι or Pol ζ did not significantly change the biological consequences of 6 mdA during replication in human cells.The negligible impact of 6 mdA on DNA replication is consistent with its potential role in epigenetic gene expression.展开更多
基金funded by the National Key R&D Program of China(Grant No.2020YFA0906800 to Q.S.)the National Natural Science Foundation of China(Nos.32270040 to Q.S.,32001022 to X.F.,and 32370033 to Y.S.).
文摘Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools,which include efficient genome editing,gene silencing,and robust protein expression systems.However,plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid.Although this plasmid coexists with pRN1 in its original host,early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa.islandicus.We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon.We identified a putative target sequence in orf904 encoding a putative replicase on pRN1(target N1).Mutated targets(N1a,N1b,and N1c)were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid inter-ference assay.The results revealed that the original target triggered CRISPR immunity in this archaeon,whereas all three mutated targets did not,indicating that all the designed target mutations evaded host immunity.These mutated targets were then incorporated into orf904 individually,yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed(pN1aSD,pN1bSD,and pN1cSD).Sa.islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors,but pN1cSD lost the capability for replication.These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase.We further showed that pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination,and this yielded a dual plasmid system for genetic study with this important archaeal model.
文摘Two Escherichia coli Saccharomyces cerevisiae shuttle vectors containing a resistance marker to geneticin (G418) are constructed. Both vectors contain a kanamycin resistant marker (KanMX4) module coding aminoglycoside 3' phosphotransferase (APH) that renders E. coli resistant to kanamycin and S. cerevisiae to geneticin. These vectors overcome the shortage of the conventional yeast vectors bearing HIS3, TRP1, LEU2, and URA3 modules as selection markers, which require hosts to be auxotrophic. Green fluorescent protein (GFP) is used as the reporter to examine the functions of the vectors. The vectors are powerful tools for the convenient cloning and controlled expression of genes in most S. cerevisiae strains.
基金supported by the Danish Council of Independent Research (DFF-0602-02196, DFF-4181-00274, DFF-1323-00330)the Fundamental Research Funds for the Central Universities (2662015PX199)
文摘Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways with organisms of Bacteria, the sister prokaryotic phylum. Archaeal organisms also possess unique features as revealed by genomics and genome comparisons and by biochemical characterization of prominent enzymes. Nevertheless, diverse genetic tools are required for in vivo experiments to verify these interesting discoveries. Considerable efforts have been devoted to the development of genetic tools for archaea ever since their discovery, and great progress has been made in the creation of archaeal genetic tools in the past decade. Versatile genetic toolboxes are now available for several archaeal models, among which Sulfolobus microorganisms are the only genus representing Crenarchaeota because all the remaining genera are from Euryarchaeota. Nevertheless, genetic tools developed for Sulfolobus are probably the most versatile among all archaeal models, and these include viral and plasmid shuttle vectors, conventional and novel genetic manipulation methods, CRISPR-based gene deletion and mutagenesis, and gene silencing, among which CRISPR tools have been reported only for Sulfolobus thus far. In this review, we summarize recent developments in all these useful genetic tools and discuss their possible application to research into archaeal biology by means of Sulfolobus models.
基金supported by the National Natural Science Foundation of China (Nos. 21807030, 21907028)the Science and Technology Innovation Program of Hunan Province(No. 2019RS2020)+1 种基金Natural Science Foundation of Hunan Province(No. 2020JJ5046)the Fundamental Research Funds for the Central Universities (Nos. 531118010061, 531118010259)。
文摘N^(6)-methyldeoxyadenosine(6 mdA) modification is considered as a new epigenetic mark that may play important roles in various biological processes.However,it remains unclear about the effect of 6 mdA on DNA replication in human cells.Herein,we combined next-generation sequencing with shuttle vector technology to explore how 6 mdA affects the efficiency and accuracy of DNA replication in human cells.Our results showed that 6 mdA neither blocked DNA replication nor induced mutations in human cells.Moreover,we found that the depletion of translesion synthesis DNA polymerase(Pol) κ,Pol η,Pol ι or Pol ζ did not significantly change the biological consequences of 6 mdA during replication in human cells.The negligible impact of 6 mdA on DNA replication is consistent with its potential role in epigenetic gene expression.