TY - JOUR
T1 - Optimization of the in vitro packaging efficiency of bacteriophage T7 DNA
T2 - effects of neutral polymers
AU - Son, Marjatta
AU - Hayes, Shirley J.
AU - Serwer, Philip
N1 - Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 1989/10/30
Y1 - 1989/10/30
N2 - The in vitro DNA packaging of several DNA bacteriophages is stimulated by the presence of neutral polymers. To optimize bacteriophage T7 DNA packaging and to understand the basis for optimization, the efficiency ofT7 DNA packaging has been determined at completion, as a function of the type, molecular mass, and concentration of the polymer added. When the polymer used was polyethylene glycol (PEG) of 0.2, 0.6 or 12.6 kDa, the efficiency of DNA packaging reached maximum at an intermediate concentration of polymer. The osmotic pressure (Pos) at maximum efficiency was either in, or close to, the range of colloid Pos measured for the intact host cell. The optimum Pos increased as the size of the polymer used decreased. PEG-100 (of 0.1 kDa) did not stimulate in vitro T7 DNA packaging. Dextran of 10 kDa also stimulated packaging and produced maximum efficiency at a physiological Pos. The degree of stimulation increases as DNA packaging extract concentration decreases; stimulation by as much as two to three orders of magnitude is observed. The presence of added polymer reduces fluctuations in DNA packaging efficiency caused by variability in the concentration of DNA packaging extracts. For reproducible and high efficiency packaging, the dextran was more reliable than the PEGs, possibly because the Pos of the dextran solutions is less sensitive to polymer concentration than is the Pos of PEG solutions. The optimum concentration of dextran at completion was also the optimum at all times before completion.
AB - The in vitro DNA packaging of several DNA bacteriophages is stimulated by the presence of neutral polymers. To optimize bacteriophage T7 DNA packaging and to understand the basis for optimization, the efficiency ofT7 DNA packaging has been determined at completion, as a function of the type, molecular mass, and concentration of the polymer added. When the polymer used was polyethylene glycol (PEG) of 0.2, 0.6 or 12.6 kDa, the efficiency of DNA packaging reached maximum at an intermediate concentration of polymer. The osmotic pressure (Pos) at maximum efficiency was either in, or close to, the range of colloid Pos measured for the intact host cell. The optimum Pos increased as the size of the polymer used decreased. PEG-100 (of 0.1 kDa) did not stimulate in vitro T7 DNA packaging. Dextran of 10 kDa also stimulated packaging and produced maximum efficiency at a physiological Pos. The degree of stimulation increases as DNA packaging extract concentration decreases; stimulation by as much as two to three orders of magnitude is observed. The presence of added polymer reduces fluctuations in DNA packaging efficiency caused by variability in the concentration of DNA packaging extracts. For reproducible and high efficiency packaging, the dextran was more reliable than the PEGs, possibly because the Pos of the dextran solutions is less sensitive to polymer concentration than is the Pos of PEG solutions. The optimum concentration of dextran at completion was also the optimum at all times before completion.
KW - Recombinant DNA
KW - concatemers
KW - dextran
KW - osmotic pressure
KW - physiological morphogenesis
KW - polyethylene glycol
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U2 - 10.1016/0378-1119(89)90058-9
DO - 10.1016/0378-1119(89)90058-9
M3 - Article
C2 - 2479593
AN - SCOPUS:0024451648
SN - 0378-1119
VL - 82
SP - 321
EP - 325
JO - Gene
JF - Gene
IS - 2
ER -