Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissoc...Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociated from DNA at 1M NaCl. When the salt concentration was slowly reduced to 650 mMand 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic 'beads-on-a-string' structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution ofnucleosome,the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372to -194 bp) DNA sequences in the 5'flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG 14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene expression.展开更多
A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei. Electron microscopy revealed that the rea...A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei. Electron microscopy revealed that the reassembled nucleus had a double-layered nuclear membrane, nuclear pore complexes, and decondensed chromatin etc. Indirect immunofluorescence analysis demonstrated the presence of lamina in newly assembled nuclei. Western-blotting results showed that lamin LII was present in egg extracts and in lamina of the reassembled nuclei which were previously reported to contain only eggderived lamin LIII.展开更多
After lamins A, B and C were isolated and purified from rat liver, their assembly properties were examined by electron microscopy and scanning tunneling microscopy using negative staining and the glycerol coating meth...After lamins A, B and C were isolated and purified from rat liver, their assembly properties were examined by electron microscopy and scanning tunneling microscopy using negative staining and the glycerol coating method,respectively By varying the assembly time or the ionic conditions under which polymerization takes place, we have observed different stages of lamin assembly, which may provide clues on the structure of the 10 nm lamin filaments. At the first level of structural organization, two lamin polypeptides associate laterally into dimers with the two domains being parallel and in register. At the second level of structural organization, two dimers associate in a half-staggered and atiparallel fashion to form a tetramer 75 nm in length. At the third level of structural organization, 4-10 lamin tetramers associate laterally in register to form 75 nm long 10nm filaments, which in turn combine head to head into long, fully assembled lamin filaments.The assembled lamin filaments are nonpolar.展开更多
基金This work was supported by the National Natural Science Foundation of China(Grant No.39893320 and 39870378)the Foundation of the Chinese Academy of Sciences(Grant No.Kj982-j1-618).
文摘Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociated from DNA at 1M NaCl. When the salt concentration was slowly reduced to 650 mMand 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic 'beads-on-a-string' structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution ofnucleosome,the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372to -194 bp) DNA sequences in the 5'flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG 14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene expression.
文摘A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei. Electron microscopy revealed that the reassembled nucleus had a double-layered nuclear membrane, nuclear pore complexes, and decondensed chromatin etc. Indirect immunofluorescence analysis demonstrated the presence of lamina in newly assembled nuclei. Western-blotting results showed that lamin LII was present in egg extracts and in lamina of the reassembled nuclei which were previously reported to contain only eggderived lamin LIII.
文摘After lamins A, B and C were isolated and purified from rat liver, their assembly properties were examined by electron microscopy and scanning tunneling microscopy using negative staining and the glycerol coating method,respectively By varying the assembly time or the ionic conditions under which polymerization takes place, we have observed different stages of lamin assembly, which may provide clues on the structure of the 10 nm lamin filaments. At the first level of structural organization, two lamin polypeptides associate laterally into dimers with the two domains being parallel and in register. At the second level of structural organization, two dimers associate in a half-staggered and atiparallel fashion to form a tetramer 75 nm in length. At the third level of structural organization, 4-10 lamin tetramers associate laterally in register to form 75 nm long 10nm filaments, which in turn combine head to head into long, fully assembled lamin filaments.The assembled lamin filaments are nonpolar.
