Site on the RNA of an avian sarcoma virus at which primer is bound

J M Taylor, R Illmensee
1975 Journal of Virology  
In vitro transcription of the avian tumor virus RNA by RNA-directed DNA polymerase is initiated on a unique cellular 4S RNA. Previous studies have shown that on the average there is one such RNA primer hydrogen bonded to each viral 35S RNA. The present study confirms that finding and demonstrates that, at least for the majority of 35S RNA molecules, the primer is bound at a site close to the 5'-terminus. The genome of the avian RNA tumor virus is in a 70S RNA aggregate that contains 2 to 4
more » ... ontains 2 to 4 molecules of 35S RNA (12) plus 4S tRNA (8) and ribosomal 5S RNA (8). The 35S RNAs are probably identical (1, 2, 16) . In vitro the transcription of the 70S RNA into DNA by the viral RNA-directed DNA polymerase is initiated on a unique 4S RNA species (9) that is probably identical to the cellular tryptophan tRNA (5, 17). On the average there is approximately one primer binding site per 35S RNA, and in naturally occurring viral RNA at least 70% of these sites are filled (Virology, in press). The present communication describes experiments undertaken to determine the location of the site (or sites) on the 35S RNA at which primer is bound. MATERIALS AND METHODS Virus growth and purification. Secondary cultures of chicken cells (C/O, gs-, Heisdorf and Nelson Laboratories) were infected at low multiplicity with avian sarcoma virus. Either strain B77 clone 9 (obtained from R. Friis) or a nontransforming derivative, td-B77 (obtained from P. Vogt), was used. The cells were grown in a large roller bottle (1585 cm2 of surface area) with 25 ml of Ham F-10 medium containing 4% calf serum, 10% tryptose phosphate broth, and 20 gCi of [H1 Juridine (27.8 Ci/mmol, New England Nuclear) per ml. The radioactive virus was purified (4) from a single harvest (24 h), subsequently pelleted, and resuspended in 0.2 ml of 0.1 M NaCl-0.01 M Tris-hydrochloride (pH 7.4)-0.001 M EDTA. Preparation of 70S complexes between template and tagged primer RNA. To the resuspended virus was added 0.8 ml of a reaction mixture containing: 0.1 M Tris-hydrochloride, pH 8.1; 0.01 M MgCl2; 2% mercaptoethanol; 0.01% Triton X-100; 10-4 M dGTP; 10-' M TTP; 0.86 x 10-6 M [a-82P]dATP (115.1 Ci/mmol, New England Nuclear); and 100 Ag of actinomycin D (Calbiochem) per ml. After incubation for 30 min at 37 C the reaction was stopped by the addition of sodium dodecyl sulfate (0.5%) and selfdigested Pronase (500 ug/ml) and incubated a further 15 min at 37 C. The 70S RNA was isolated by sedimentation (9) and subsequently collected by ethanol precipitation. Fractionation of partially denatured 70S complexes on oligo(dT)-cellulose. The isolated 70S complex was partially denatured by heating at 59 C for 5 min in 0.2 ml of 0.01 M Tris-hydrochloride (pH 7.4)-0.01 M EDTA, and then applied to a 0.5-g column of oligo(dT)-cellulose (Collaborative Research) in 0.5 M NaCl-0.01 M Tris-hydrochloride, pH 7.4. The RNA which failed to bind to the column is designated "poly(A) deficient" and the remainder, which was subsequently eluted with 0.01 M Trishydrochloride, pH 7.4, is designated "poly(A) containing. " For example, in the experiment described in Fig. 1 , the input RNA contained 187,000 counts/min of [3H]uridine-labeled RNA and 10,600 counts/min of bound primer specifically labeled with 32P. Of these 37 and 16%, respectively, bound to the column. The two fractions were subsequently concentrated by ethanol precipitation. Sedimentation analysis. The poly(A)-containing and poly(A)-deficient fraction were separately analyzed by sedimentation into gradients of 15 to 30% sucrose containing 0.01 M Tris-hydrochloride, pH 7.4; 0.01 M EDTA; and 0.01% sodium dodecyl sulfate. Centrifugation was for 15 h at 28,000 rpm and 22 C in the SW40 rotor of a Spinco L5.50. Aliquots of the gradient fractions were precipitated with 10% trichloroacetic acid in the presence of 80 ,gg of calf thymus DNA carrier, collected onto glass fiber filters, and counted in a Beckman scintillation spectrometer. The 3H-labeled L cell rRNA marker was a gift of Dawn Kelley. Polyacrylamide gel analysis. RNA fractions were analyzed by electrophoresis into cylindrical gels (0.6 by 8 cm) of 2.1% acrylamide cross-linked with 0.28% (vol/vol) ethylene diacrylate. Bromophenol blue was added to the sample prior to electrophoresis. The buffer present both in the gel and the electrode 553 on May 9, 2020 by guest
doi:10.1128/jvi.16.3.553-558.1975 fatcat:cp24e6i2uzae5a4mwsj6i5ququ