The promoters, PTH4 and P-TH270 involved in the regulation of Streptomyces coelicolor differentiation were subcloned into Streptomyces promoter, i.e. probe plasmid pIJ4083, and the recombinant plasmids, pIJ4470 and pI...The promoters, PTH4 and P-TH270 involved in the regulation of Streptomyces coelicolor differentiation were subcloned into Streptomyces promoter, i.e. probe plasmid pIJ4083, and the recombinant plasmids, pIJ4470 and pIJ4471, were constructed. Two promoters could drive the expression of reporter gene encoding catechol dioxygenase when pIJ4470 and pIJ4471 were introduced into some white mutants (C85, C70, C71, C17 and C119). The total RNA was isolated from these strains containing recombinant plasmid. Probes were prepared by labelling 5 -ends of PTH4 AND PTH270 DNA fragments using radioisotope. DNA - RNA hybridization was carried out with the probes and RNAs isolated from different strains. The S1 mapping result showed that all RNAs from strains of C85/pIJ4470, C85/4471, C70/pIJ4470, C70/pIJ4471 and C17/pIJ4470 as well as C17/pIJ4471 gave rise to strong positive hy-bridization signal, whereas RNAs from C71/pIJ4470 and C71/pIJ4471 did not give any positive signal. RNAs from C119/pIJ4470 and C119/pIJ4471 gave weak hybridization signal. The result indicated that the transcription of both PTH4 and PTH270 promoters might depend on whiG, an essential gene in Streptomyces differentiation, and partially de-pend on whiH, but they did not depend on other differentiation genes (whiA, whiB and whiI) .展开更多
The downstream gene controlled by promoter--PTH4 which is related to Streptomycesdifferentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597...The downstream gene controlled by promoter--PTH4 which is related to Streptomycesdifferentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces.展开更多
Six nucleotides located in the region of translation start site of whiG were changed. whiG was amplified by PCR technique. Reformed sequences were determined. This gene was directly subcloned into expression vector pE...Six nucleotides located in the region of translation start site of whiG were changed. whiG was amplified by PCR technique. Reformed sequences were determined. This gene was directly subcloned into expression vector pET11c containing strong T7 promoter, and the recombinant plasmid was introduced into E. coli BL21(DE3), which could be induced by IPTG to produce T7 RNA polymerase. The SDS-PAGE result showed that whiG highly expressed in E. coli BL21(DE3), and the yield of whiG product was about 20% of insoluble proteins in cell. whiG product (σwhiG) was further identified by Western blot hybridization after making its antibody. whiG gene was subcloned into Streptomyces plasmid pIJ6021, and then it was introduced into sporulation deficient mutant C71 from Streptomyces coelicolor. The result showed that C71 could restore sporulation and σwhiG has biological functions.展开更多
基金Proiect supported by the Chinese Academy of Sciences and the Royal Society of UK.
文摘The promoters, PTH4 and P-TH270 involved in the regulation of Streptomyces coelicolor differentiation were subcloned into Streptomyces promoter, i.e. probe plasmid pIJ4083, and the recombinant plasmids, pIJ4470 and pIJ4471, were constructed. Two promoters could drive the expression of reporter gene encoding catechol dioxygenase when pIJ4470 and pIJ4471 were introduced into some white mutants (C85, C70, C71, C17 and C119). The total RNA was isolated from these strains containing recombinant plasmid. Probes were prepared by labelling 5 -ends of PTH4 AND PTH270 DNA fragments using radioisotope. DNA - RNA hybridization was carried out with the probes and RNAs isolated from different strains. The S1 mapping result showed that all RNAs from strains of C85/pIJ4470, C85/4471, C70/pIJ4470, C70/pIJ4471 and C17/pIJ4470 as well as C17/pIJ4471 gave rise to strong positive hy-bridization signal, whereas RNAs from C71/pIJ4470 and C71/pIJ4471 did not give any positive signal. RNAs from C119/pIJ4470 and C119/pIJ4471 gave weak hybridization signal. The result indicated that the transcription of both PTH4 and PTH270 promoters might depend on whiG, an essential gene in Streptomyces differentiation, and partially de-pend on whiH, but they did not depend on other differentiation genes (whiA, whiB and whiI) .
基金Project supported by Chinese Academy of Sciences and the Royal Society of UK.
文摘The downstream gene controlled by promoter--PTH4 which is related to Streptomycesdifferentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces.
基金Project supported by the National Natural Science Foundation of China and the Foundation of the Royal Society of UK.
文摘Six nucleotides located in the region of translation start site of whiG were changed. whiG was amplified by PCR technique. Reformed sequences were determined. This gene was directly subcloned into expression vector pET11c containing strong T7 promoter, and the recombinant plasmid was introduced into E. coli BL21(DE3), which could be induced by IPTG to produce T7 RNA polymerase. The SDS-PAGE result showed that whiG highly expressed in E. coli BL21(DE3), and the yield of whiG product was about 20% of insoluble proteins in cell. whiG product (σwhiG) was further identified by Western blot hybridization after making its antibody. whiG gene was subcloned into Streptomyces plasmid pIJ6021, and then it was introduced into sporulation deficient mutant C71 from Streptomyces coelicolor. The result showed that C71 could restore sporulation and σwhiG has biological functions.
