Identification Of A Putative Chromosomal Replication Origin From Cyanothece51142

Cyanothece51142, One marine diazotrophic strain, has a robust diurnal cycle inwhich photosynthesis is performed during the day and nitrogen fixation at night. In2008, Welsh et al. reported the complete genome of Cyanothece51142in PNAS. Butthe authors concluded that “The origin of replication could not be determined foreither the circular or linear chromosome by using standard GC skew and DnaA boxanalysis”. However, by utilizing a webbased system Ori-Finder, Feng Gao et al. haveidentified the locations of ori C for both the circular and linear chromosomes inCyanothece51142. In the present study, we identified the interaction of the purifiedDnaA(IV) protein with the ori C by EMSA, and get good results.Firstly, we amplified the circular and linear chromosomal putative replicationorigin from Cyanothece51142, and then cloned into appropriate vectors, and also itscorresponding dnaA gene. dnaA(IV) gene has been cloned into an overexpressingvector for purification. So we get recombinant vectors pEASY-T1-ori C,pEASY-T1-ori L, pGEX-4T-1-DnaAand pET28a-DnaA(IV).Secondly, the expression of genetic engineering strains of pGEX-4T-1-DnaAandpET28a-DnaA(IV) was done and purified the protein by GST-tag or His-tag affinitychromatography.Thirdly, we characterize the interaction of the purified DnaA(IV) protein with thereplication origin by gel retardation assays, and the results are consistent with thepredicted results and certify that the predicted results are correct.