| Popis: |
The initiator of coliphage λ DNA replication, λO protein, may be detected among other 35S-labeled phage and bacterial proteins by a method based on immunoprecipitation. This method makes it possible to study λO proteolytic degradation in λ plasmid-harboring or λ phage-infected cells; it avoids ultraviolet (u.v.)-irradiation of bacteria, used for depression of host protein synthesis, prior to λ phage infection. We confirm the rapid decay of λO protein (half-time of 80 s), but we demonstrate the existence of a stable λO fraction. In the standard five minute pulse-chase experiments, 20% of synthesized λO is stable. The extension of the [35S]methionine pulse, possible in λ plasmid-harboring cells, leads to a linear increase of this fraction, as if a part of the synthesized λO was constantly made resistant to proteolysis. Less than 5% of λO protein synthesized during one minute is transformed into a stable form. We presume that the stable λO is identical with λO present in the normal replication complex and thus protected from proteases. We cannot find any stable λO inEscherichia coli recA+ cells that were irradiated with u.v. light prior to λ phage infection, but their recA− counterparts behave normally, suggesting that recA function interferes in the assembly of a normal replication complex in u.v.-irradiated bacteria. The stable λO found in λ plasmid-harboring, amino acid-starved relA cells is responsible for the λO-dependent λ plasmid replication that occurs in this system in the absence of λO synthesis. The existence of stable λO raises doubt concerning its role as the limiting initiator protein in the control of replication. Another significance of λO rapid degradation is proposed. |
