Simple In Vitro and In Vivo Assays to Follow Protelomerase Activity

Deneke et al. (2000) designed an in vitro assay for protelomerases. DNA fragments containing possible substrates for the protelomerase are inserted into a cloning vector. The recombinant plasmids are linearized by a restriction endonuclease and then incubated with purified protelomerase. For TelN, an incubation at 30 °C for 30 min was used. Following this step, products are analysed on agarose gels under non-denaturing and alkaline conditions. Under alkaline (denaturing) conditions, DNA fragments containing a terminal hairpin exhibit twice the length as those observed under neutral conditions.

Hertwig et al. (2003a) developed an assay to investigate protelomerase activity in vivo. Plasmids containing possible substrates for the protelomerase are introduced into an indicator strain. Bacteria are then infected with a high-titre phage lysate. For PY54, a mutant was used that was unable to lyse the bacteria. After different times of infection (e.g. 10 min, 1 h, 6 h), plasmids are isolated and analysed on an agarose gel. Processing of the substrate is visible by linearization and subsequent loss of the plasmid, which can also be determined by plating the bacteria on agar containing the respective antibiotics.

Was this article helpful?

0 0

Post a comment