Simian adenovirus SA7 DNA: chemical, physical, and biological studies.

Experimental Procedures. -M11aterials: A 13SC-1 cell line was obtained from lDr. RI. N. Hull; CV-1 was provided by Dr. Leroy McLaren. Simian adenovirus type SA7 was obtained from Dr. R. N. Hull. Snake venom phosphodiesterase, papaini (2X crvstallized), and electrophoretically purified pancreatic DNase I were purchased from Worthington Biochemical Corporation. RNase, sniake venom (Crotalus adamanteus), subtilisin, and alpha-chymotrypsin were obtained from Sigma Chemical Company. P'2-orthophosphate was obtained from the New England Nuclear Corporation. Cesium chloride was purchased from the American Potash Corporation and the Gallard Schlesinger Company. SV40 tumor antiserum was provided by Dr. W. Rowe; and SV40 tumor antigen was obtained from Flow Laboratories (Rockville, Maryland). Virus and cell culture: All cells were routinely propagated in Eagle's (MEM) medium with 10% fetal calf serum. The virus pools were produced by infecting monolayer cultures at a multiplicity of infection of 1-10 in MEM with 5% fetal calf serum and harvesting the infected cells at 36-48 hr postinfection. Virus purification: The virus was concentrated and purified by a procedure to be described in detail elsewhere.5 Briefly, the infected cells were disrupted by sonication; and the resulting virus suspension was treated sequentially with a combination of RNase, DNase, and snake venom followed by subtilisin anid alpha-chymotrypsin. The virus was then concentrated by centrifugation onto a CsCl cushion in a Spinco SW25.1 rotor at 25,000 rpm for 2 hr. The collected virus ban-d was centrifuged at least two times to equilibrium in a CsCl gradient. The final viruts bands were stored at 4? C in 35% CsCl or dialyzed against 0.15 411 NaCl containinig 0.01 Ml sodium phosphate, p1,1 7, and 0.001 M1 ethylenediaminietetraacetate (ED)TA) and stored frozen at 90C. Extraction of viral DNA: 1B3eta-mereaptoethanol (20 ,l) and papain (I mg) were added to each milliliter of dialyzed virus containing approximately 10 mg of virus. The mixture was incubated for 10-12 mini at 37? C or until visible clearing was obtained. Twenlty mg/ml of solid sodium dodecyl sulfate (SDS) was then added and incubation continued for 7 mimi at 37? C. This mixture was extracted at room temperature with an equal volume of phenol containing 0.2% 8-hydroxyquinoline. The aqueous phase was reextracted two times with fresh phenol. Traces of phenol were removed either by dialysis against 0.1 X SSC (SSC = 0.15 Ml NaCl + 0.015 M sodium citrate), or by precipitation and spooling of the DNA witb ethanol. The final preparation was stored in SSC with a drop of chloroform at 4? C. Density of DNA: The buoyant densities of native and alkali-treated viral DNA were determined in an equilibrium density gradient of CsCl containing 0.01 M tris(hydroxymethyl)aminomethane (tris) buffer, pH 7.4. Alkali-denatured DNA was prepared by exposing DNA, in 0.1 X SSC, to 0.2 N NaOH for 10 min at room temperature followed by neutralization with HCl. In each experiment, Micrococcus lysodeikticus or E. coli DNA was added as a standard. Centrifugations were performed at 25? C and 44,770 rpm in the Spinco model E ultracentrifuge equipped with ultraviolet absorptionl optics. Photographs were analyzed with a Spinco Analytrol densitometer.