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
