| Autor: |
Martel M; Département de microbiologie, infectiologie et immunologie, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, P. Québec, Canada, H3C 3J7., Balleydier A; Département de microbiologie, infectiologie et immunologie, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, P. Québec, Canada, H3C 3J7., Brochu J; Département de microbiologie, infectiologie et immunologie, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, P. Québec, Canada, H3C 3J7., Drolet M; Département de microbiologie, infectiologie et immunologie, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, P. Québec, Canada, H3C 3J7. marc.drolet@umontreal.ca. |
| Abstrakt: |
In bacteria, replication of the chromosome is normally initiated following the binding of DnaA proteins to the oriC region. However, under certain circumstances, replication can also be initiated independent of the oriC/DnaA system. This is the case, for example, in Escherichia coli cells lacking RNase HI (rnha mutants) or type 1A topoisomerase activity (topA topB mutants). Here, we present a protocol in which replication from the oriC/DnaA system is first inhibited by the addition of the protein synthesis inhibitor, spectinomycin, to exponentially growing bacterial cell cultures. The thymidine analog, 5-ethynyl-2'-deoxyurdine (EdU) is then added to the cells, and after 1 h the cells are fixed and the Alexa Fluor ® 488 dye is conjugated to EdU by the click-iT ® reaction. The oriC-independent replication is detected in fixed cells by flow cytometry and can be visualized by fluorescence microscopy. |
