Zaman, Sara; (1991) Genetics and molecular biology of the Streptomyces lividans plasmid pIJ101. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The 0.5kb Spel(53)-Bcll(49) fragment containing the Streptomyces dividans plasmid pIJ101 cop/korB gene was cloned into the E. coli plasmid pUC8 under the control of the lacZ promoter, creating pQR206. pQR206 produces a 10kDa LacZ-Cop/KorB fusion protein in vitro and in vivo in E. coli. Cop/KorB crude protein is able to retard a 0.5kb Spel(53)-Bcll(49) coplkorB fragment, a 0.5kb Sau3A cop/korB fragment, and a 0.65kb SalGl(19)-Sstll(16) kilB fragment in band-shift assays; however no retardation was observed with a 0.7kb Spel(53)-Bcl(51) sti fragment. DNA footprinting analysis showed that the Cop/KorB protein protects overlapping 33b sequences on the cop/korB coding and non-coding strands respectively. Furthermore, a hypersensitive site present on each strand divides these 33b regions into two equal 16b regions. The kilB protected region covers 60b and 52b on the coding and non-coding strands respectively. Both these defined kilB and cop/korB operator sites for Cop/KorB binding show strong sequence homology and also correspond with the location of their respective promoters. Competition assays suggest that the Cop/KorB protein may have a higher affinity for the kilB operator than for the cop/korB operator. A 0.7kb Bcl(57)-Spel(53) sti fragment inserted in its correct orientation into pIJ702 (a sti-coplkorB-pIJ101 derivative) and into pIJ702::pUC8 shuttle vectors prevented them from accumulating ssDNA and structurally rearranging respectively. Constructs which contained sti in the reverse orientation were found to accumulate ssDNA. Thus, sti is only active as the site for second-strand synthesis in its natural orientation with respect to the basic replicon. The sti determinant was further defined to a 0.53kb 55/II(55)-5/I(53) fragment which contains a potential stem-loop structure with a AG of -64.00 kcal/mol. Cop/KorB does not inhibit the conversion of ssDNA to the double-stranded form and does not significantly alter copy number. The minimal replicon was further defined to a 2.0kb Ball(15)-SstII(63) fragment encompassing only the rep ORF and a non-coding region which may either contain the plus origin or the rep promoter. The 1.4kb NotI(12)-SstII(63) rep ORF was cloned into pBGS19- under the control of the lacZ promoter, creating pQR431a. pQR431a produces a 50kDa LacZ-Rep fusion protein in vitro.

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