1. Osmotic shock disrupts particles of phage T2 into material containing nearly all the phage sulfur in a form precipitable by antiphage serum, and capable of specific adsorption to bacteria. It releases into solution nearly all the phage DNA in a form not precipitable by antiserum and not adsorbable to bacteria. The sulfur-containing protein of the phage particle evidently makes up a membrane that protects the phage DNA from DNase, comprises the sole or principal antigenic material, and is responsible for attachment of the virus to bacteria. 2. Adsorption of T2 to heat-killed bacteria, and heating or alternate freezing and thawing of infected cells, sensitize the DNA of the adsorbed phage to DNase. These treatments have little or no sensitizing effect on unadsorbed phage. Neither heating nor freezing and thawing releases the phage DNA from infected cells, although other cell constituents can be extracted by these methods. These facts suggest that the phage DNA forms part of an organized intracellular structure throughout the period of phage growth. 3. Adsorption of phage T2 to bacterial debris causes part of the phage DNA to appear in solution, leaving the phage sulfur attached to the debris. Another part of the phage DNA, corresponding roughly to the remaining half of the DNA of the inactivated phage, remains attached to the debris but can be separated from it by DNase. Phage T4 behaves similarly, although the two phages can be shown to attach to different combining sites. The inactivation of phage by bacterial debris is evidently accompanied by the rupture of the viral membrane. 4. Suspensions of infected cells agitated in a Waring blendor release 75 per cent of the phage sulfur and only 15 per cent of the phage phosphorus to the solution as a result of the applied shearing force. The cells remain capable of yielding phage progeny. 5. The facts stated show that most of the phage sulfur remains at the cell surface and most of the phage DNA enters the cell on infection. Whether sulfur-free material other than DNA enters the cell has not been determined. The properties of the sulfur-containing residue identify it as essentially unchanged membranes of the phage particles. All types of evidence show that the passage of phage DNA into the cell occurs in non-nutrient medium under conditions in which other known steps in viral growth do not occur. 6. The phage progeny yielded by bacteria infected with phage labeled with radioactive sulfur contain less than 1 per cent of the parental radioactivity. The progeny of phage particles labeled with radioactive phosphorus contain 30 per cent or more of the parental phosphorus. 7. Phage inactivated by dilute formaldehyde is capable of adsorbing to bacteria, but does not release its DNA to the cell. This shows that the interaction between phage and bacterium resulting in release of the phage DNA from its protective membrane depends on labile components of the phage particle. By contrast, the components of the bacterium essential to this interaction are remarkably stable. The nature of the interaction is otherwise unknown. 8. The sulfur-containing protein of resting phage particles is confined to a protective coat that is responsible for the adsorption to bacteria, and functions as an instrument for the injection of the phage DNA into the cell. This protein probably has no function in the growth of intracellular phage. The DNA has some function. Further chemical inferences should not be drawn from the experiments presented.
Zone centrifugation of mixtures of two labeled DNA's at low concentrations in density gradients of sucrose permits accurate measurement of relative sedimentation rates. The individual rates are constant during the run. Measurements with DNA's from phages T2, T5, and lambda conform to the relation D(2)/D(1) = (M(2)/M(1))(0.35), where D and M refer to distances sedimented and molecular weights of the DNA pair. The results show that high molecular weight DNA's sediment artificially fast in the optical centrifuge, owing to a hitherto unknown effect of molecular interactions. The molecular weight of lambda DNA is 31 million, measured either from sedimentation rate or from tests of fragility under shear.
Aggregation of DNA is often suspected but seldom studied. In phage lambda we found a DNA that can form characteristic and stable complexes. A first account of them is given here.Materials and Methods.-DNA was extracted from a clear-plaque mutant (genotype cb+) of phage lambda' by rotation2 or shaking' with phenol. Sodium dodecylsulfate, ethylenediaminetetraacetate, citrate, or trichloroacetate was sometimes included in the extraction mixture without effect on the properties of the DNA. Phenol was removed by dialysis, with or without preliminary extraction with ether, against 0.1 or 0.6 M NaCl.Sedimentation coefficients were measured4 at 10 ,ug DNA/ml in 0.1 and 0.6 M NaCl in aluminum cells at 35,600 rpm with consistent results, and are reported as S20.w. Zone sedimentation' of labeled DNA-s6 was observed in 0.1 M NaCl immobilized by a density gradient of sucrose A sample, usually containing less than 0.5 ug of DNA in 0.15 ml of 0.1 M NaCI, was placed on 4.8 ml of sucrose solution, and the tube was spun for 5 or 6 hr at 28,000 rpm in an SW39L rotor of a Spinco Model L centrifuge at 100C.Solutions containing 5-40 ,ug DNA/ml in 0.1 or 0.6 M NaCl were stirred on occasion for 30 min at 50C with a thin steel blade turning in a horizontal plane.' Since we used two stirrers of different capacities, stirring speeds given in this paper are comparable only within a context. Salt solutions were buffered at pH 6.7 with 0.05 M phosphate. Results.-Disaggregation and breakage: Solutions containing 0.5 mg/ml of lambda DNA in 0.1 M NaCl acquire an almost gel-like character on standing for some hours in a refrigerator. Diluted to 10 ug/ml, the DNA exhibits in the optical centrifuge an exceedingly diffuse boundary sedimenting at 40-60 s (Fig. 1A). If the diluted solution is aged for several days, the sedimentation rate may fall somewhat (not below 40 s), but the boundary remains diffuse and often appears double.Stirring the diluted solution at 1,300 to 1,700 rpm yields a single component sedimenting at 32 s (Fig. 1B). The product so obtained is stable for a week or more in the cold in 0.1 M NaCl. We call this process disaggregation by stirring.If samples of the diluted solution are stirred at increasing speeds between 1,800 rpm and 2,100 rpm, one sees a stepwise transition from 32 s to 25.2 s components, each by itself exhibiting a sharply sedimenting boundary (Figs. 1C and iD). We call this phenomenon breakage. Broken DNA can form aggregates, but the characteristic 32 s species cannot be regained.Aggregation: Disaggregation, in contrast to breakage, is reversible, as shown by the following experiment. Lambda DNA at 40 ug/ml in 0.6 M NaCl was 748 VOL. 49, 1963 BIOCHEMISTRY: HERSHEY ET AL. 749
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