Molbiyokon'22 8th International COngress of the Molecular Biology Association of Turkey, İstanbul, Turkey, 9 - 12 June 2022, pp.1-2
Background/aim: pAnox1 is a small, novel, cryptic and double stranded plasmid which has 1592 bp in long and with a total G + C content of 40,01 from Anoxybacillus sp. 05S15. The aim of this study is to determine the replication mode of pAnox1 plasmid by transmission electron microscopy (TEM) and southern hybridization techniques.
Materials and methods: TEM analysis was performed to determine whether the replication mode of the pAnox1 plasmid was theta type. pAnox1 plasmid DNA was purified from Anoxybacillus sp. 05S15 and then linearized with NdeI and HindIII restriction endonucleases. 5.0 μg of digested and undigested DNAs were prepared for TEM by using 100-200 mesh formvar-coated grid. The samples were stained with Pb citrate and uranyl acetate, and then dried at room temperature for analysis by TEM. In order to determine whether the plasmid has rolling circle (RC) type replication type, the southern hybridization technique was carried out. Total DNA was isolated from the cultures of Anoxybacillus sp. 05S15 grown in medium with and without rifampicin. Purified DNA from medium with rifampicin was digested with S1 nuclease to determine whether ssDNA intermediates of pAnox1 were formed. Both samples were transferred to a nylon membrane without being denatured and then run on electrophoresis with 1% agarose gel. Two spesicific primers were designed from the internal sequence of ORF3 on pAnox1. The amplified DNA was labeled with DIG High Primer DNA Labeling and Detection Starter Kit (Roche Inc.,Manheim,Germany) according to the manufacturer's instructions. The bands formed after hybridization were observed and photographed.
Result: Grids were examined under TEM and it was observed that there were no structures similar to Theta shape and “Y”-shaped structures in the sample prepared using the undigested and digested with NdeI and HindIII restriction endonucleases, respectively. In the Southern blot analysis, DNA products were used without denaturing, so that only the single stranded DNAs could be detected. Digestion of RC replication intermediates (ssDNA) was achieved by applying S1 nuclease to DNA isolated from bacteria grown both in medium with and without rifampicin. It was found that the samples with degraded ssDNA did not hybridize with the probe and there was no signal on the gel. On the other hand, it was determined that DNA samples untreated with S1 nuclease and containing ssDNA hybridized with the probe and formed strong bands.
Conclusion: When the TEM and Southern hybridization results were evaluated together, it was determined that the pAnox1 plasmid contains replication intermediates (ssDNA) formed in the RC mechanism. As a result of the formation of ssDNA intermediates in the replication process
of the pAnox1 plasmid and the hybridization of these products with probe, it was detected that pAnox1 had a rolling circle (RC) replication mode.