Vol. 17, No. 3 Printed in U.S.A.

JOURNAL OF VIROLOGY, Mar. 1976, p. 745- 755 Copyright i 1976 American Society for Microbiology

Endogenous Oncornaviral DNA Sequences: Evidence for Two Classes of Viral DNA Sequences in Guinea Pig Cells D. P. NAYAK* AND A. R. DAVIS Department of Microbiology and Immunology, University of California School of Medicine, Los Angeles, California 90024 Received for publication 14 March 1975

The nature of the endogenous viral DNA sequences in guinea pig cells was studied by hybridization. A segment of the viral RNA (r-VRNA) hybridizing to abundant (or reiterated) DNA sequences (R-VDNA) was isolated by recycling to a Cot of 300. The hybridization of the recycled VRNA, as well as the total VRNA, was followed by determining their kinetics and by Wetmur-Davidson analysis. The kinetics of hybridization of total VRNA were complex, did not follow a second-order kinetics, and revealed two slopes by Wetmur-Davidson analysis. The recycled RNA, on the other hand, had a second-order reaction rate expected of the hybridization between a single species of RNA and DNA sequences and yielded a single straight line in a Wetmur-Davidson plot. The Cot½ and slope of the recycled r-VRNA was almost identical to that of the abundant VDNA sequences obtained from the hybridization data of the total VRNA. Guinea pig 28S rRNA with or without recycling was used in monitoring hybridization rate. The kinetics of hybridization of 28S RNA followed a second-order reaction and produced a single straight line by WetmurDavidson plot, with a second-order reassociation rate constant of 9.6 x 10-3 liters/mol * s, a CotC of 104 mol * s/liter, and reiteration frequency of 146. There was no difference in the kinetics of hybridization of 28S RNA before and after recycling. These experiments showed that guinea pig cells contain two classes of VDNA sequences. (i) R-VDNA sequences with a second-order reassociation rate constant of 8.2 x 10-4 liters/mol s, a Cot% of 1,219 mol-s/liter, and a reiteration frequency of 12 represent 37.5% of the viral genome. (ii) Unique VDNA sequences with a second-order reassociation rate constant of 1.2 x 10-4 liters/mol s, a Cot,, of 7,692 mol - s/liter, and a reiteration frequency of 2 represent 62.5% of the viral genome.

Oncornaviruses replicate via DNA. Two classes of intracellular viral DNA (VDNA) sequences have been reported: (i) abundant or reiterated (R-VDNA) and (ii) less abundant or unique (U-VDNA) sequences. However, a wide variation has been reported in the reiteration frequency of these DNA sequences and in the amount of viral genome they represent. For example, reiterated or frequent DNA sequences may be absent completely (24, 29) or may be present at a frequency of 10 to 500 copies and may represent 20 to 50% of the viral genome (11, 12, 17, 23, 25, 29). On the other hand, unique or infrequent DNA sequences may represent 50 to 100% of the genome and may be present at 1 to 10 copies (17, 23-25, 29). These variations have made it difficult to assess the true nature of intracellular viral DNA. It appears that at least three factors have complicated the estimation of VDNA sequences. (I)

The first factor is methods. Two methods used commonly are either DNA/DNA reassociation or DNA/RNA hybridization using labeled VDNA or viral RNA (VRNA) as a probe, respectively. The relative frequency of VDNAs are estimated from the plateau level and the Cot., of hybridization. It is almost impossible to determine Cot½ with any reasonable accuracy from a composite curve representing two or more classes of DNA sequences reassociating or hybridizing at different rates. (ii) The second factor is probe. Either the radiolabeled RNA or in vitro synthesized DNA is used as a probe. When DNA is used as a probe, the kinetics of reaction can be estimated better, because the reassociation rate is the same as that of DNA and DNA is more thermostable. However, VDNA, often used in these reactions, does not represent the entire viral genome (at best it represents 70 to 80%). Furthermore, the relative

745

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NAYAK AND DAVIS

frequency of DNA sequences in the probes cannot be estimated accurately. RNA, on the other hand, represents the entire viral genome but often hybridizes with complex kinetics (depending on the DNA/RNA ratio) and is often more thermolabile. In addition, the source of the probe, i.e., endogenous or exogenous virus, may further complicate the analysis. Depending on the nature of animal species and virus, the homology between exogenous and endogenous viruses may vary widely. (iii) The third factor is cellular DNA. The source of DNA, whether from infected or uninfected cells or from high-, low-, or nonvirus-yielding animals, will affect both the extent of hybridization and the reiteration frequency of VDNA sequences (17,29). In this report we have minimized some of these difficulties. First, we have used endogenous guinea pig VRNA (GP-VRNA) and guinea pig DNA as reactants in the hybridization. Guinea pigs do not have any known exogenous (or infectious) oncornavirus. We and others have previously reported that bromodeoxyuridine (BUdR) or iododeoxyuridine can activate any guinea pig cells to produce oncornavirus particles (14, 20, 22, 27). We have further characterized these particles and shown that they contain a reverse transcriptase, 70S RNA, and structural proteins similar to but antigenically different from those of known oncornaviruses (20, 22). Furthermore, these particles are noninfectious for guinea pig cells. We have also demonstrated that the same amount of VDNA is present in uninduced and BUdRinduced guinea pig cells, indicating that virus production is caused by the activation of preexisting viral genes rather than the facilitation of infection of a few virus particles produced chronically at an undetectable level (21). Second, we have avoided the overlapping of hybridization by the recycling of VRNA probes and the selection of a segment of the VRNA that hybridizes with reiterated sequences. Finally, we have shown that under our experimental condition the hybridization of 28S rRNA follows a second-order kinetics and that there is no difference in the kinetics of hybridization before and after recycling. We have, therefore, calculated the reiteration frequency and the extent of hybridization from Cot½s as well as by the method of Wetmur and Davidson (32).

J. VIROL. guinea pig embryo fibroblasts were treated for 48 h with 10-4 M BUdR in growth medium supplemented with 5% inactivated fetal calf serum. The medium was then replaced with fresh growth medium containing 50gfCi of [5-3H ]uridine per ml (specific activity, 29 Ci/mmol). Culture supernatant was collected at 8- to 12-h intervals for 2 days. Guinea pig virus was concentrated at the interface of 20 and 65% sucrose cushions and purified on linear 20 to 65% sucrose gradients as previously described (20, 22), except that an initial precipitation with polyethylene glycol 6000 was sometimes performed (10). RNA was isolated from purified virions by a phenol-sodium dodecyl sulfate method (28). 70S RNA was purified on a linear 5 to 20% sucrose velocity gradient in NTE buffer (0.01 M Tris-hydrochloride, pH 7.4, 0.1 M NaCl, 0.001 M EDTA) in an SW50.1 rotor (49,000 rpm for 50 min at 4 C). 35S RNA was prepared by heating 70S RNA at 90 C for 3 min, followed by purification on a 5 to 20% sucrose velocity gradient in an SW50.1 rotor (49,000 rpm for 2 h at 4 C). Purification of rRNA. A nonconfluent culture of guinea pig embryo fibroblasts was labeled for 25 h in phosphate-free medium containing 15% dialyzed inactivated fetal calf serum and 100 ;Ci of carrier-free 32PO4 per ml. Cells were then treated for 90 min with 2.5 ug of puromycin per ml. 80S ribosomes were isolated according to the procedure of Hulse and Wettstein (15). 28S rRNA was purified from the 80S ribosomal fraction by extraction with phenol-sodium dodecyl sulfate, followed by fractionation on a linear 5 to 20% sucrose gradient in NTE buffer in an SW50.1 rotor (49,000 rpm for 2 h at 4 C). Liquid hybridization in DNA excess. The hybridization of 3H-labeled GP-VRNA with an excess of fragmented guinea pig DNA has been described (21). DNA was isolated (1, 2) from various sources, treated with 0.3 N KOH for 18 h at 37 C to remove RNA, dialyzed against NTE buffer, fragmented by sonication to a size of 6S, and precipitated with ethanol. Hybridizations were performed at 68 C. Each DNARNA mixture was heated for 5 min at 100 C and cooled quickly. For accuracy, 0.1 M sodium phosphate was used to hybridize at Cot values less than 150, and 0.5 M sodium phosphate was used for higher values. All values have been corrected for standard conditions of 0.12 M sodium phosphate (7). To determine percentage of hybridization, one-half of each sample was treated for 30 min at 37. C with RNase A (20 ;g/ml) and RNase T-1 (10 U/ml), the other half was untreated, and trichloracetic acidprecipitable counts were determined. A background RNase resistance of 1.6 to 6.0% was subtracted from all results. The DNA concentration (which remains constant in each hybridization) and the DNA-RNA ratio are stated in the figure legends.

RESULTS Hybridization of GP-VRNA with guinea MATERIALS AND METHODS DNA. Labeled 70S GP-VRNA was hybridpig BUdR induction of virus and purification of viral 70S RNA. Procedures for the activation of ized to an excess of unlabeled guinea pig DNA. endogenous guinea pig oncornavirus by BUdR, virus The kinetics of hybridization of GP-VRNA to purification, and isolation of labeled GP-VRNA have cellular DNA were normalized and shown in Fig. been previously described (20, 22). Briefly, cultures of 1. As reported previously (21), the kinetics of hy-

VOL. 17, 1976

ENDOGENOUS ONCORNAVIRAL DNA SEQUENCES

bridization were independent of the source of DNA either from normal or leukemic guinea pigs or from methylcholanthrene-induced sarcoma cells. It is also evident that the kinetics were complex and extended over a Cot of four decades. Hybridization kinetics were essentially the same for 35S RNA isolated after denaturation of 70S RNA. Recycling of GP-VRNA and hybridization of recycled RNA to guinea pig DNA. Because of the complex kinetics of hybridization of oncornaviral RNA to cellular DNA, we and others (11, 17, 21, 23, 25, 29) have postulated two classes of DNA sequences: abundant or reiterated DNA (R-VDNA) hybridizing to a segment of RNA at a rate faster than the unique or less abundant DNA (U-VDNA), which hybridizes at a slower rate to a different segment of VRNA. However, it is difficult to determine where the hybridization of R-VDNA ends and U-VDNA begins (Fig. 1). Thus, it is not possible to estimate from such a composite curve the Cot%, frequency rate, or percentage of the viral

747

genome hybridizing to R-VDNA or U-VDNA. We therefore decided to isolate the reiterated VRNA (r-VRNA) segments hybridizing to RVRNA by recycling experiments and to follow the kinetics of hybridization of r-VRNA segments to the total cellular DNA. Accordingly, 70S or 35S RNA was hybridized to a Cot of 300, and hybridized RNA was isolated after RNase treatment (see legend to Fig. 2 for experimental details). Hybrids isolated at a Cot of 300 were greatly enriched in RNA sequences hybridizing to R-VDNA sequences and contained 84% of r-VRNA and only 16% of unique VRNA (u-VRNA) (see legend to Fig. 4). The recycled low Cot hybrids were denatured, and the kinetics of hybridization of r-VRNA to total DNA were monitored. The data from three such independent experiments were normalized and plotted in Fig. 2. The following characteristics of hybridization of recycled VRNA were observed. (i) Maximum hybridization of recycled RNA was 43% at a Cot of 10,000 or more. Longer

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Cot (mole-sec/liter) FIG. 1. Hybridization kinetics of 70S GP- VRNA with guinea pig DNA . 70S GP- VRNA with a specific activity of 101 counts/min per gg was hybridized as described in the text to guinea pig DNA from various sources. The DNAIRNA ratio was 8 x 10'. DNA was from: (-) leukemic guinea pig lymphoblasts; (A) normal guinea pig embryo; (0) methylcholanthrene-induced guinea pig sarcoma. Hybridization values have been normalized using the maximum observed value (84%) as 100%. For comparison the hybridization kinetics of recycled DNA /RNA (from Fig. 2) has been replotted on the same graph (-----).

748

NAVAK AND DAVIS

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FIG. 2. Hybridization kinetics of recycled DNA/RNA hybrids. GP-VRNA with a specific activity of 106 counts/min per 1Ag was hybridized to gross guinea pig DNA in 0.5 M sodium phosphate to a Cot of 300. At this point the reaction was diluted with ice-cold 0.12 M sodium phosphate buffer to a DNA concentration of approximately 150 ,gg/ml and treated with RNase A (20 ,g/ml) and RNase T-1 (10 U/ml) for 1 h at 37 C. Hybrids were recovered by extraction with phenol-sodium dodecyl sulfate (28) at 25 C, exhaustive dialysis against NTE buffer, and ethanol precipitation. The recovered hybrids were dissolved in 0.01 M sodium phosphate and, after adjustment to 0.12 M sodium phosphate, denatured by heating for 5 min at 100 C. Reassociation kinetics of the recycled hybrids were determined as described in Materials and Methods. The results of three independent experiments are shown. Details of the prehybridization are as follows. Experiment 1: A, 70S RNA, hybridization at 300 Cot, 12.1%; initial DNA concentration, 1.5 mg/ml; initial DNAIRNA ratio, 2.0 x 106; final DNAIRNA ratio, 1.6 x 106. Experiment 2: 0, 70S RNA, hybridization at 300 Cot, 20.1%; initial DNA concentration, 3.6 mg/ml; initial DNAIRNA ratio, 2.7 x 106; final DNAIRNA ratio, 3.5 x 101. Experiment 3: 35S RNA, hybridization at 300 Cot, 13.3%; initial DNA concentration, 7.4 mg/ml; initial DNAIRNA ratio, 1.9 x 105; final DNAIRNA ratio, 2.0 x 101. Values have been normalized using the maximum observed value (43%) as 100%. (-----), An ideal second-order reaction with a Cot% of 1,219 was plotted using the equation: 1 C/Co 1 1/(1 + KCot). Cot,, was calculated from the Wetmur-Davidson plot (Fig. 5), i.e., from the abscissa when the (fraction remaining single-standard) -I was 2.0. U,

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hybridization up to a Cot of 30,000 did not increase hybrid formation. Occasionally there was a decrease in RNase resistance at a Cot greater than 20,000. The reason for this decrease is not clear but may be due to increased thermal degradation of recycled RNA. The lower percentage of hybridization of recycled RNA compared to that of RNA without recycling could not be due to a low DNA/RNA ratio. The final DNA/RNA ratio exceeded 106, a ratio at which 75 to 80% of VRNA hybridizes to DNA at a Cot of 15,000. Low hybridization was not due to alteration of DNA during the recycling procedure, because control DNA treated in an identical manner hybridized to 80% of 70S VRNA at a Cot of 12,000. It appears that the RNA became partly non-hybridizable after recycling. RNase treatment generates small RNase-resistant

cores and produces nicks in hybrid RNA during recycling. These small RNA pieces do not rehybridize efficiently after denaturation. Lower hybridization of recycled RNA has been reported previously and is also seen with 28S rRNA (Fig. 6A). (ii) The hybridization kinetics of the recycled RNA follow a second-order reaction (Fig. 2). The ideal curve was drawn with a Cot* of 1,333, which was obtained from the Wetmur-Davidson plot of the recycled data (Fig. 5). (iii) When this ideal curve is superimposed in Fig. 1, it is evident that, unlike the recycled data, the kinetics of hybridization of 70S RNA is more complex and does not follow a secondorder reaction. Recycled RNA thus established the presence of R-VRNA sequences hybridizing

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VOL. 17, 1976

ENDOGENOUS ONCORNAVIRAL DNA SEQUENCES

A curve expected for the hybridization of U-VDNA sequences was constructed in Fig. 3. Values for unique hybridization were calculated by subtracting the value for the R-VDNA hybridization from the total hybridization. The method of computation is described in the legend to Fig. 3. An ideal second-order curve was drawn using a Cot. of 7,692 for U-VDNA sequences. The majority of the points in the high Cot area fit reasonably well on this ideal curve (Fig. 3). Several attempts to isolate u-VRNA segments by hybridization were not successful. We encountered two major problems. (i) In selecting high Cot hybrids, DNA was reassociated to a Cot of 2,000 before the addition of 70 S RNA. Incubation was continued to a Cot of' 15,000 and hybrids (Cot 2,000 to 15,000) were isolated, denatured, and rehybridized. Hybridization after recycling was poor (only 15 to 207c hybridized), and there was no difference from total hybridization presented in Fig. 1. In addition to RNase treatment mentioned above, poor hybridization was primarily due to extensive thermal degradation (70 h at 68 C) in selecting high Cot hybrids. Further, it appears from Fig. 3 that at a Cot between 2,000 to 15,000 both r-VRNA and u-VRNA sequences will hybridize almost equally without any appreciable enrich-

749

ment of unique sequences. (ii) To obtain enrichment for unique sequences similar to that obtained for reiterated sequences (i.e., 84%- of uVRNA), DNA must be reassociated to a Cot of' 30,000 and hybrids must be selected between 30.000 to 100,000 Cot (Fig. 3). Selecting u-VRNA hybrids by such prolonged incubation is not practical with the present method. Besides, although the estimation of number of copies of' U-VDNA sequences has varied, their presence in cellular DNA has not been questioned. Wetmur-Davidson analysis of hybridization data. If hybridization and reassociation follow a second-order kinetics, a single straightline slope will be obtained by plotting the reciprocal of' single-stranded probe against the Cot (32). The second-order reassociation rate constant (32) and the Cot½ can be calculated from the slope of the straight line (see legends to Fig. 4 and 5) and will be proportional to the reiteration frequency. When the data from Fig. 1 was analyzed, two slopes were evident (Fig. 4). A rapidly hybridizing sequence with a slope of 8.2 x 10' liters/mol-s and a Cot½ of 1,219.5 and slowly hybridizing sequences with a slope of 1.3 x 10-4 liters/mol *s and Cot. of 7,692.3. Furthermore, by extrapolating the slope to a Cot of 0, u-VRNA and r-VRNA were found to

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102 104 10 105 Cot (mole-sec/liter) FIG. 3. Hybridization kinetics of unique VRNA with guinea pig DNA. High Cot points in Fig. 1 are replotted after subtraction of the value of the recycled DNA/RNA hybridization at every point. Symbols: ( ), ideal second-order reaction with a Cot% of 7,692; (-----), ideal second-order reaction with a Cots of 1,219. Again the Cots values of the unique sequences were obtained from the Wetmur-Davidson plot in Fig. 4 as described in Fig. 4. The value for unique hybridization (a) at each point was calculated using the following formula: a = [b (0.375) c]/0.625, where b = total hybridization obtained from points in Fig. 1 and c = reiterated value (in percentage) obtained from the ideal curve in Fig. 2. Percentages of unique (62.5) and reiterated (37.5) viral sequences were obtained from the 0 Cot extrapolate of the unique curve in Fig. 4. 10

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Cot (mole-sec/liter) x 10'3 FIG. 4. Analysis of hybridization kinetics of guinea pig DNA and 70S GP-VRNA by the method of Wetmur and Davidson (32). The data used here is from Fig. 1. The maximum observed hybridization (84%) was normalized to 100%. The symbols used are the same as those in Fig. 1. The percentage of the unique and the reiterated sequences represented in the viral genome was calculated as follows. The intercept of the unique curve at 0 Cot is 1.6, or 1/percentage of single-stranded u-VRNA = 1.6, and u-VRNA represents 62.5% of the total virus genome and therefore the r- VRNA is 37.5%. The rate constants (or slopes) were calculated as follows: slope U-VDNA = 1.3 x 10-4 liters/mol sec, Cot% = 1/slope = 7692.3; slope R-VDNA = 8.2 x 10-4 liters/ mol-sec, Cot, = 1,219.5. The percentage of r- and u-RNA in hybrids at Cot 300 were calculated as follows: (i) from the data in Fig. 3, at Cot 300, r- and u-VRNA hybrids represent 19.7 (or 84%) and 3.75 (or 16%) of total hybrids respectively, or (ii) from the following equation: % u-VRNA hybridized I

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ENDOGENOUS ONCORNAVIRAL DNA SEQUENCES

VOL. 17, 1976

represent 62.5 and 37.5% of the viral genome, respectively (see legend to Fig. 4). However, the straight line of the slope depends on the ideal second-order reaction. It was therefore important to see if the break in the slope was not due to an experimental artifact of hybridization. When the data of the recycled RNA (Fig. 2) was plotted (Fig. 5), a straight line was obtained with a slope of 7.5 x 10-4 liters/ mol s and a Cot% of 1,333, which is close to that obtained for reiterated sequences in Fig. 4. Low C ot hybrids obtained by recycling contained predominantly r-VRNA sequences (84%) and only 16% of u-VRNA sequences (see legends to Fig. 4 for calculation). Hybridization of 28S rRNA. Reports comparing the kinetics of hybridization of RNA to DNA (KU) in DNA excess to the reassociation rate (Kd) of DNA are conflicting. Whereas Kb has been reported to be slower than Kd by some (18), others have reported that both are the same (16). In addition, several reports show that hybridization of RNA to DNA does not follow a second-order kinetics (9, 18), whereas others report that it does (13). However, we found that the hybridization of the recycled VRNA closely followed a second-order reaction, although that of total VRNA did not. It therefore became important to determine the kinetics of hybridization of a single species of RNA under the condition of our reaction and also to find out if the recycling of RNA and normalization of the data artificially produced data which appeared as a second-order reaction. We selected 28S RNA because the frequency of rDNA to rRNA has been studied extensively in many species of

751

animals. 28S RNA free from messenger contamination was isolated as described in Materials and Methods. The kinetics of hybridization of 28S RNA with or without recycling were determined. Hybridization of 28S RNA before recycling reached a plateau of 70%, whereas after recycling maximum hybridization was only 45% (Fig. 6A). The reasons for the lower plateau level of recycled RNA has been discussed previously (see recycling of VRNA). However, when hybridization data of both experiments were normalized by using maximum hybridization as 100%, the hybridization of 28S RNA with or without recycling closely followed a secondorder reaction (Fig. 6B) and yielded a single straight line in the Wetmur-Davidson plot (Fig. 6C). There was no difference either in the reaction rate or the Cot% of 28S RNA before and after recycling. The Cot½ was obtained from the Wetmur-Davidson plot and by the best fit of the normalized data to an ideal second-order curve. The reiteration frequency estimated from Cot½ is 146 (the estimated range of rRNA genes varies from 100 to 360 for different vertebrate species [4, 18, 19, 26, 30]. Thus, although our experiments do not permit a detailed analysis of the conflicting results about Kh and Kd, reported previously (9, 13, 16, 18), it is known that many factors such as temperature, cation concentration, and the size of reactants affect the kinetics of hybridization and reassociation. However, the data presented above indicate that under our experimental conditions these conditions are controlled such that the kinetics of hybridization approximate a second-order reaction (16), and therefore the .

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FIG. 6. Hybridization kinetics of guinea pig 28S rRNA and recycled guinea pig 28S rRNA with guinea pig DNA. Guinea pig 28S rRNA with a specific activity of 2.6 x 101 was purified from 80S ribosomes as described in Materials and Methods, and the kinetics of reassociation with guinea pig DNA were measured either with (0) or without (A) recycling of the 28S rRNA. For the recycled hybridization, the 28S rRNA was hybridized to a Ct of 75 in 0.12 M sodium phosphate, hybrids were recovered as described in Fig. 2 and denatured by heating for 5 min at 100 C, and the reassociation kinetics of recycled hybrids were determined. Details of the recycling are as follows: initial DNA concentration, 7.7 mg/ml; final DNA concentration, 4.0 mg/ml; initial DNAIRNA ratio, 4.9 10'; final DNAIRNA ratio, 1.6 x 106; hybridization at Cot 75, 30.8%. Nonrecycled 28S rRNA was hybridized at a DNA concentration of 6.9 mg/ml and a DNA/RNA ratio of 1.5 x 106. Both hybridizations were performed entirely in 0.12 M sodium phosphate. (A) Hybridization kinetics are shown without normalization. (B) Values have been normalized using the maximum observed values (recycled, 45%; and nonrecycled, 70%) as 100%. (---), Ideal second-order reaction with a Cot½s of 104 is shown. (C) The data shown in (B) was analyzed by a Wetmur-Davidson plot in the manner described in Fig. 4. The slope of the line is 9.6 x 10- 3liters/mol -sec. x

Wetmur-Davidson analysis can be used to determine the reaction rate and reiteration frequency. Thermal stability of DNA/RNA hybrids. To determine if true hybrids were formed at high and low Cot values, thermal stability of two classes of hybrids was determined. Hybrids at low Cot were selected by terminating the reaction at 300 Cot. To select high Cot hybrids, DNA was first reassociated to a Cot of 4,000, RNA was added, and incubation was continued to a Cot of 15,000. Both high and low Cot hybrids were isolated by treatment with RNase, phenol extraction, and precipitation in ethanol. Finally, duplicate aliquots of hybrid in 2.5x SSC were sealed in ampoules, treated for 10 min at the specified temperature, and chilled in ice. Onehalf of each sample was treated with RNase, and the percentage of RNase resistance was determined. Both hybrids had a Tm of 91.5 (Fig. 7). However, it should be pointed out that although low Cot hybrids were greatly enriched in r-VRNA sequences, high Cot hybrids were still a mixture for reasons explained earlier (see Fig. 3). DISCUSSION The results presented here demonstrate the presence of two classes of VDNA sequences in guinea pig cells. R-VDNA sequences representing 37.5% of the viral genome are present at a reiteration frequency of 12, and U-VDNA sequences representing 62.5% of the viral genome are present at a reiteration frequency of 2. These results were obtained by isolating VRNA segments hybridizing only to R-VDNA segments. Two procedures were used to analyze the hybridization data of the recycled r-VRNA and the total VRNA. (i) The kinetics of hybridization were determined by plotting the percentage of hybrid formation as a function of log Cot (6). (ii) The reciprocal of single-stranded RNA was also plotted against the linear Cot (WetmurDavidson analysis). These analyses revealed that the hybridization of recycled r-VRNA closely followed a second-order reaction and had a single straight-line slope. The hybridization of total RNA, on the other hand, had a complex kinetics and revealed two straight-line slopes in Wetmur-Davidson analysis. Cot½ and reiteration frequency were best calculated from the Wetmur-Davidson plot. The number of copies calculated for the R-VDNA sequences are fewer than previous estimates (Tables 1 and 2). One major reason is that in the majority of earlier experiments Cot½j was calculated from a composite curve representing hybridization kinetics of total VRNA. As shown

VOL. 17, 1976

ENDOGENOUS ONCORNAVIRAL DNA SEQUENCES I

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earlier, the overlap of the hybridization between R- and U-VDNA sequences would tend to decrease the Cot% of R-VDNA sequences and, consequently, increase the estimate of number of copies. In a report where number of copies was estimated from the Wetmur-Davidson plot of DNA/DNA reassociation, the results are fairly comparable to the copy number obtained here (17). Finally, the presence of two or more classes of DNA sequences can be demonstrated unequivocally only by isolating r- or u-VRNA sequences after recycling and showing that each of these VRNA sequences hybridizes at a rate expected for single species of DNA and RNA sequences. Here again, unless the termination Cot at the first cycle of hybridization is selected carefully, there may not be any appreciable enrichment of RNA sequences hybridizing to one class of DNA (see legend to Fig. 4). The reiteration frequencies reported here are similar to the ones obtained by Lowy et al. (17), who noted that DNA from virus-yielding mice contained both classes of DNA sequences whereas DNA from non-virus-yielding murine cells contained only R-VDNA and not U-VDNA sequences.

Contrary to the mammalian system, the VDNA sequences in avian cells appear differ-

753

ent. Although both classes of DNA sequences have been reported, using exogenous VRNA as probes, DNA sequences comparable to RVDNA sequences appear to be absent when an avian endogenous oncornaviral RNA (RAV-O) was used (24). Furthermore, Evans, Drohan, and Baluda (personal communications), using a different approach, have not detected any appreciable enrichment of the VRNA complementary to reiterated VDNA by recycling. Thus, in the avian system it is still not clear why one often observes an increased rate of hybridization at a lower Cot with an exogenous oncornaviral RNA probe. The functional role of R-VDNA sequences is still unknown. Preliminary data indicated that viral RNA sequences do not contain the same amount of reiteration found in cellular R-VDNA sequences (21, 31). Therefore, these R-VDNA sequences are not transcribed per se into the structural RNA (70S) of the viral genome. The most likely function of these R-VDNA sequences is in the regulation of the transcription of VDNA and subsequent activation of VRNA. It may be likely that these abundant sequences are tandem and located contiguous to the unique sequences in a manner analogous to the repeated sequences present in heterogeneous nuclear RNA. It is possible that the entire RNA transcribed in the nucleus may contain all of the sequences present in both R-VDNA and U-VDNA. However, during transcriptional processing part of the reiterated sequences may be removed, completing the maturation of TABLE 1. Rate constant, Cot*, and reiteration frequencies of U- and R-VDNA and rDNA sequences RNA used for K a(liters/mol RNAiusedifor

)

tion freCot4 ~~~~~~Reitera-

quencyb

rRNA (28S) r-VRNA

u-VRNA

9.6 x 8.2 x 7.5 x 1.3 x

10-3 10-4c 10-d 10-4

104 1,219 1,333 7,692

146 12 12 2

a Kh, the second-order rate constant was obtained from the slope of the Wetmur-Davidson plots (Fig. 4, 5, and 6C). ° Obtained using rRNA as a standard. The reiteration frequency of rRNA was 146 and was calculated using the equation described by Melli et al. (18) using 1.86 x 1012 (5, 8) and 2.7 x 10' (18, 29) daltons as the respective analytical complexities of guinea pig and Escherichia coli DNA and a Cot½s of E. coli complementary RNA of 22 under identical conditions (29). e Obtained from the initial slope of the kinetics of reassociation of guinea pig DNA and 70S VRNA (Fig. 4). d Obtained from the slope of the kinetics of reassociation of recycled DNA/RNA hybrids (Fig. 5).

754

NAYAK AND DAVIS

J. VIROL.

TABLE 2. Reiteration frequency of cellular DNA complementary to the RNA or DNA of endogenous and exogenous oncornaviruses Source of VRNA or VDNA

PR-RSV-Ca RAV-Oa AMVa AMVa AMVa AMVa AKR-L1 AKR-L1*

AKR-L15 RD-114a RD-114a S-tropic murine5 S-tropic murineh M-7 baboon" M-7 baboon

RT21C rat" PK15 porcine"

Soreo elDAUnique VDNA Source of cell DNA copy

Reiterated

no.uVDNAno.copy

Chicken sarcoma Chicken, gs+ and gsChicken, nonproducing, gs+ Chicken, nonproducing, gsChicken myeloblasts, leukemic Rat cells transformed by B-77 virus Mouse, high virus yielding Mouse, low virus yielding Mouse, non-virus yielding Cat brain or spleen Cat (specific pathogen free) Mouse liver Rabbit cells infected with murine S-tropic virus Baboon lung or liver Canine thymus cells infected with M-7 baboon virus Rat cells Pig kidney cells

Reference

1.5 2 2-6 2-6 9 2 3-4 1-2 None 1-2 -c -c 1-2

50-100 None 40-100 40-100 200 None 7-8 7-8 10 100-200 50-500c 5-15c

_C 1-2

5-15c

3 3

-c -c

5-15c 5-15c

3 3

23 24 29 29 29 29 17 17 17 25 12 3 3

Values obtained using liquid hybridization of 60-70S VRNA with excess cellular DNA. "Values obtained using DNA-DNA reassociation kinetics of VDNA synthesized in endogenous reverse transcriptase reactions. C Cots was determined from the total curve, and no distinction was made for the unique and the reiterated area of the curve. a

VRNA by a process similar to that described by Gillespie and Gallo (11). It is also possible that there may be more than one class of endogenous VDNA sequences containing similar R-VDNA and BUdR is activating only one of these endogenous oncornaviruses. This work was supported by Public Health Service grant CA 16880 from the National Cancer Institute and a grant from the California Institute of Cancer Research. A. R. Davis is a fellow of the Leukemia Society of America, Inc.

1.

2.

3. 4.

5.

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Lowy, D. G., S. K. Chattopadhyay, N. M. Teich, W. P. Rowe, and A. S. Levine. 1974. AKR murine leukemia virus genome: frequency of sequences in DNA of high-, low-, and non-virus yielding mouse strains. Proc. Nati. Acad. Sci. U.S.A. 71:3555- 3559. Melli, M., C. Whitfield, K. V. Rao, M. Richardson, and J. 0. Bishop. 1971. DNA-RNA hybridization in vast DNA excess. Nature (London) New Biol. 231:8-12. Mohan, J., A. Dunn, and L. Casola. 1969. Ribosomal DNA in the rat. Nature (London) 233:295-296. Murray, P. R., and D. P. Nayak. 1974. Characterization of bromodeoxyuridine-induced endogenous guinea pig virus. J. Viol. 14:679-688. Nayak, D. P. 1974. Endogenous guinea pig virus: equability of virus-specific DNA in normal, leukemic, and virus-producing cells. Proc. Natl. Acad. Sci. U.S.A. 71:1164-1168. Nayak, D. P., and P. R. Murray. 1973. Induction of type C viruses in cultured guinea pig cells. J. Virol. 12:-177-187. Neiman, P. E. 1972. Rous sarcoma virus nucleotide sequences in cellular DNA: measurement by RNADNA hybridization. Science 178:750-752. Neiman, P. E. 197:3. Measurement of endogenous leukosis virus nucleotide sequences in the DNA of normal avian embryo by RNA-DNA hybridization. Virology 53:196-204. Neiman, P. E. 1973. Measurement of RD-114 virus nucleotide sequences in feline cellular DNA. Nature

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