JBC, Vol. 252, Issue 8, 2734-2741, Apr, 1977
Intracellular DNA-protein complexes from bacteriophage T4-infected cells isolated by a rapid two-step procedure. Characterization and identification of the protein components
C. Manoil, N. Sinha and B. Alberts
A simple technique has been developed for isolating intracellular DNA and
its bound proteins from uninfected and phage-infected bacteria. This
technique, which utilizes aqueous salt concentrations in the physiological
range, is based upon the fact that DNA exists in normal cell lysates in a
stiff random coil conformation, and has an unusually large excluded volume
to mass ratio. Such stiff coils display a unique combination of low
sedimentation coefficient and large Stokes radius, enabling them to be
separated rapidly from all other cellular components by successive
centrifugal and gel permeation steps. Analysis of this purified
intracellular DNA fraction from bacteriophage T4-infected Escherichia coli
reveals mainly DNA and protein, with a small amount of RNA also present.
Among the major proteins obtained are the DNA-dependent RNA polymerase of
the host and the products of T4 genes rIIA, rIIB, and 32 (DNA-"unwinding"
protein). Small amounts of the proteins coded by T4 genes 43 (DNA
polymerase) and 42 (dCMP hydroxymethylase) have also been identified, in
addition to at least 13 other phage-coded proteins of unidentified genes.
Much of the phage-coded protein in the complex, including the gene 32
protein, does not exchange readily with the same protein exogenously added
in the lysate.
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