Summary
We have previously shown that a membranous cell-free system derived from uninfected penicillin spheroplasts of E. coli transcribes early and late messenger RNA's from λ DNA.
This in vitro system will also transcribe and translate the endolysin gene R of λ DNA. The enzyme activity that results from in vitro synthesis corresponds to λ endolysin (a typical late protein) by several criteria.
DNA from λ CI857 sus R5 ts 9B and λ CI857 sus S7 pgal, mutants carrying nonsense mutations in genes involved in the host lysis, are inactive in the synthesis of endolysin with an extract of non permissive cells, although they are fully active with an extract of permissive cells. Furthermore, suppression of these mutations is entirely dependent on addition of supernatant from suppressor strains.
The endolysin synthesis from a thermosensitive λ CI mutant is observed at 40°C and not at 30°C. This suggests that the product of CI gene is formed and acts in the in vitro system at 30°C.
Enzymatic activity is detected after a 15 min lag period.
Membranes and double stranded λ DNA are absolutely required for the enzyme synthesis. Ribosomes and supernatant highly stimulate the in vitro system.
Inhibitors of RNA, DNA and protein synthesis (actinomycine D; cytosine arabinoside; DNA-ase; and chloramphenicol respectively) will prevent endolysin production when added at zero time. If DNA-ase or actinomycine D are added after 20 min of incubation, only partial inhibition of endolysin synthesis occurs. It is therefore concluded, according to our previous observations, that messengers are stable enough to allow enzyme synthesis after delayed addition of the inhibitors in the in vitro system.
It appears that there is a complete regulation in the membranous system like in vivo and which starts with the early and late messenger formation and leads to active late protein synthesis.
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Lévine, A., Lévine, H.J. & Nisman, B. Synthèse in vitro d'endolysine de bactériophage lambda dans un système membranaire d'Escherichia coli . Molec. gen. Genet. 121, 83–98 (1973). https://doi.org/10.1007/BF00353696
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DOI: https://doi.org/10.1007/BF00353696