Proc. Nati. Acad. Sci. USA Vol. 73, No. 10, pp. 3408-3412, October 1976 Biochemistry

Anticomplementary nature of smaller DNA produced during synthesis of extensive DNA copies of poliovirus RNA (RNA-directed DNA nucleotidyltransferase/comvlementary DNA/avian myeloblastosis virus/nucleic acid hybridization) D. L. KACIAN AND JEANNE C. MYERS Institute of Cancer Research and Department of Human Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032 Communicated by S. Spiegelman, July 9, 1976

ABSTRACT The reverse transcriptase (RNA-directed DNA of '25I-labeled poliovirus RNA have been detailed (1). Char- nucleotidyltransferase) from avian myeloblastosis virus is able acteristics of the specific lots of poliovirus RNA, 125I-labeled to make an extensive, possibly complete, complementary DNA copy of intact poliovirus RNA. In the presence of high concen- poliovirus RNA, and 3H-labeled 18S cDNA made in the pres- trations of deoxyribonucleoside triphosphates, ribonucleoside ence of 4 mM sodium pyrophosphate that were used in these triphosphates, or sodium pyrophosphate, this DNA is the only studies have been described (1). species produced. Without these additives, however, a second Hydroxylapatite Column Chromatography. A bed of hy- size class of DNA is also synthesized. This material has a sedi- droxylapatite (0.5 g dry weight) was prepared inside a 1-cm mentation coefficient between roughly 4 and 10 S and is pro- diameter jacketed column. The bed was equilibrated with 0.12 duced later in the reaction, largely after synthesis of the larger complementary DNA has ceased. The smaller DNA consists M sodium phosphate (pH 6.8), 0.4% (wt/vol) sodium dodecyl primarily of material anticomplementary to the RNA template sulfate at 600. Samples were diluted into 1 ml of the same buffer and contains a faithful and uniform representation of the viral and applied to the column under gentle air pressure. The sequences. It most likely arises by transcription of the larger temperature was raised in 5° steps, and at each temperature DNA species. the column was eluted with five 1-ml portions of the buffer. At 1000, the column was washed in addition with five 1-ml por- In a previous report (1), we showed that the reverse transcrip- tions of 0.4 M sodium phosphate (pH 6.8), 0.4% (wt/vol) sodium tase (RNA-directed DNA nucleotidyltransferase) from avian dodecyl sulfate. Each fraction was then precipitated with tri- myeloblastosis virus is able to make an extensive, possibly chloroacetic acid, and the radioactive samples were collected complete, complementary DNA (cDNA) copy of intact polio- on nitrocellulose membrane filters. virus RNA. The DNA sedimented at approximately 18 S in alkaline sucrose gradients, indicating a chain length of about 7000 nucleotides. It hybridized 96% back to the template RNA RESULTS and was able to protect the RNA from digestion Time course of appearance of smaller DNA when approximately equivalent amounts were annealed to it; thus, it is a uniform representation of the sequences in the The size distribution of the DNA synthesized using reverse template. The material was produced in high yields (50-75% transcriptase of avian myeloblastosis virus and intact poliovirus of the input RNA) and could be made at high specific activities RNA template at various times is shown in Fig. 1. Polymer- without using prohibitively large amounts of isotope. ization is slow (less than 10 nucleotides per sec). The material In the presence of high concentrations (4 mM) of deoxyri- increases in size up to about 30 min, after which time very little bonucleoside triphosphates, ribonucleoside triphosphates, or of the larger species is produced. The smaller DNA lags behind sodium pyrophosphate, this DNA was by far the predominant until this point, but then the ratio of the two size classes changes species produced. Without these additives, however, a second greatly. At 60 min, the smaller species is about one-fourth of size class of DNA was also synthesized. This material had a the total DNA; at 2 hr, the two are approximately equal (ref. sedimentation coefficient between roughly 4 and 10 S and was 1 and Fig. 4). At still later times, the smaller species predomi- produced later in the reaction, largely after synthesis of the 18S nates (data not shown). cDNA had ceased. In the presence of phosphodiester bond-containing additives, In this communication we describe the properties of the the smaller cDNA synthesis can be completely suppressed (1). smaller size DNA and demonstrate that it consists primarily of Fig. 2 shows the size distribution at 2 and 7 hr of poliovirus DNA with faithful sequence representation that is anticom- cDNA synthesized in the presence of optimum levels of deox- plementary to the RNA template. yribonucleoside triphosphates. The profiles are virtually in- distinguishable and illustrate the effectiveness of the additives MATERIALS AND METHODS in preventing the appearance of the smaller species. Actinomycin D was obtained from Sigma, and [32P]dCTP was Origin of the smaller DNA obtained from New England Nuclear. Sources of other mate- On the basis of the temporal sequence in which the 18S cDNA rials have been described (1). and the smaller DNA were synthesized, it was postulated (1) Procedures for preparation of reverse transcriptase, isolation that the smaller DNA might result from the operation of de- of poliovirus RNA, nucleic acid hybridization and analysis using gradative activities during the reaction. These could be frag- micrococcal and A and T1, synthesis of menting the 18S species itself or providing broken template or DNA, alkaline sucrose gradient centrifugation, and preparation primer molecules. In the first two cases, the smaller species in Abbreviations: cDNA, complementary DNA; CDNA-t, product of would be complementary to the poliovirus RNA template; the nucleic acid concentration (mol-liter-l) and time of incubation the third case, the smaller DNA would be anticomplementary (sec). to the viral RNA. 3408 Downloaded by guest on September 27, 2021 Biochemistry: kacian and Myers Proc. Natl. Acad. Sci. USA 73 (1976) 3409

5 Min 15 Min 8.0K 4.0 0X174 0X174 DNA DNA

3.2 6.4-

ff 0 2.4_ 4.8k

'C 1.6_ 3.2F

0.8_ 1.6F

I I I 1 1 5 10 15 20 25 30 35 5 10 15 20 25 30 35

I 0 0

0

20 25 30 35 5 10 15 20 25 30 35 FRACTION NUMBER FIG. 1. Time course of poliovirus DNA synthesis. A 100-,M1 standard reaction mixture was prepared as described in Materials and Methods and contained [3H]dCTP at 10.2 Ci/mmol. Samples (2 ,l) were removed at the times shown, added to 75 gl of 1 mM EDTA, and frozen. When the reaction was complete, they were thawed and analyzed by alkaline sucrose density gradient centrifugation. kX174 [3H]DNA was run on a parallel gradient.

In order to distinguish among these alternatives, the ability resistant material with an increase in CDNA-t in the reaction with of the smaller DNA to hybridize to the template RNA and to E. coli RNA is additional evidence for self-annealing and in- protect that RNA from digestion by ribonuclease was tested. dicates that at least part of the material consists of separate A reaction mixture was prepared and fractionated on an alka- positive and negative strands rather than hairpin structures. It line sucrose gradient. The fractions containing the two size was, however, possible that the observed difference was for- classes of DNA were pooled separately, and the DNA was freed tuitous and merely represented different background levels of small-molecular-weight substances by gel filtration. The with the different preparations. ability of the two species to anneal to poliovirus RNA was then Annealing the 18S cDNA to the RNA rendered it 90-95% examined, and the results shown in Table 1 were obtained. RNase-insensitive (1). The smaller DNA, however, was unable Only about 25% of the smaller cDNA hybridized to the to protect most of the template from the (Table 2), template RNA compared with the larger material, which an- suggesting that only a limited portion of the RNA had been nealed essentially completely. In addition, the smaller material transcribed. Were this the case, several of the hybridizations showed a much higher nuclease resistance when incubated with described in Table 1 may not have contained sufficient RNA Escherichia coli RNA than did the 18S cDNA. These results to permit the reactions to go to completion. To check this pos- were obtained with three different preparations of cDNA, and sibility and to verify by another method of analysis that the since it is unlikely that the DNA hybridizes to the bacterial annealing being observed was real, we repeated the hybrid- nucleic acid, self-hybridization of the DNA was probably oc- ization experiments using larger amounts of RNA, and the ex- curring. The fact that there was an increase in the nuclease- tent of annealing and the melting temperatures of the hybrids Downloaded by guest on September 27, 2021 3410 Biochemistry: Kacian and Myers Proc. Natl. Acad. Sci. USA 73 (1976) Table 1. Hybridization specificity of poliovirus DNAs DNA prepa- Larger Smaller ration poliovirus poliovirus Poliovirus E. coli Time % Nuclease no. DNA (ng) DNA (ng) RNA (ng) RNA (ng) (hr) CRNA t CDNA t resistant 1 1.7 - 200.0 2 0.2 0.002 100.0 ± 2.1 1 1.7 - 200.0 2 0.2 0.002 3.2 ± 0.7 1 2.2 200.0 2 0.2 0.002 24.7 ± 0.4 1 2.2 - 200.0 2 0.2 0.002 24.5 ± 0.6 2 - 1.4 17.3 23 0.2 0.016 20.7 ± 0.9 2 1.4 17.3 23 0.2 0.016 32.1 ± 1.5 3 1.1 10.0 46 0.23 0.025 25.3 ± 0.7 3 1.1 10.0 46 0.23 0.025 36.2 ± 2.7 Hybridizations were performed as described in Materials and Methods in a final volume of 0.02 ml and assayed with . E. coli RNA is total cell ribonucleic acid. The percent nuclease-resistant is given as the average of two determinations followed by the range of values obtained. The DNAs were fractionated on alkaline sucrose gradients to obtain the larger and smaller species. Results are shown for three different DNA preparations as identified in the first column. CAt values are the product of the concentration of the subscripted nucleic acid and the time of incubation expressed in moles-nucleotide-liter-'-sec.

were measured on hydroxylapatite. The results, shown in Fig. The most probable template for the smaller DNA would then 3 and Table 3, indicate that stable, well-matched duplexes were be the 18S cDNA, although it was conceivable that the material formed in both reactions containing poliovirus RNA and E. coli arose by random end addition or some other abortive process. RNA (Te = 870 in both). In addition, there was a CDNA-t-de- To determine which of these alternatives was correct, the ability pendent increase in the amount of duplex formed in the pres- of the smaller DNA to hybridize to the larger species was ence of the bacterial nucleic acid, strongly suggesting that measured. A reaction mixture was prepared containing [32P] self-annealing of positive-and negative-strand DNA was taking dCTP, and the DNA was fractionated as above into the two size place. The amount of hybrid formed between the DNA and the classes (Fig. 4). The smaller [32P]DNA was then annealed to 18S poliovirus RNA was not, however, substantially greater than [3H]cDNA made in the presence of sodium pyrophosphate. This was obtained in the previous experiments; therefore, it appeared latter material had previously been shown to hybridize 96% that the poliovirus RNA itself was not serving as template for back to the template RNA and to be able to protect at least 90% the synthesis of the major portion of the smaller DNA and that of the poliovirus sequences from ribonuclease digestion (1). most likely it was predominantly plus-strand DNA. When this experiment was performed, the results shown in Table 4 were obtained. The smaller DNA hybridized exten- sively to the 18S species, showing that the major part of it is 1.6- - JX174 complementary to the larger cDNA. The remainder of the DNA material (22-30%) was equivalent in amount to that which hybridized to the poliovirus RNA itself; therefore, the smaller 1.4k species is composed of about 25% plus-strand DNA and about 75% minus-strand DNA. 7 Hours 1 .2I Sensitivity of smaller DNA synthesis t actinomycin D Several groups of investigators have shown that DNA-in- DNA reverse 1.0 - structed, but not RNA-instructed, synthesis by N) .p 2 Hours transcriptase is inhibited by the presence of actinomycin D (2-4). As an additional test of the origin of the smaller DNA, 0.8 Table 2. RNase resistance of poliovirus 12'I-labeled tu 0.61 RNA- DNA hybrids - N Poliovirus Poliovirus ng DNA Nuclease 0.41 RNA (ng) DNA (ng) ng RNA resistant 5.75 0.0 0.00 1.7 0.21 5.75 3.0 0.52 3.8 5.75 6.0 1.04 9.4 vi I 5.75 9.0 1.56 9.0 5 10 15 20 25 30 5.75 12.0 2.08 10.6 FRACTION NUMBER 5.75 18.0 3.13 9.1 FIG. 2. Suppression of smaller DNA synthesis. Poliovirus cDNA Reactions were performed as described in Materials and Meth- was synthesized as described in Materials and Methods except that ods in a final volume of 0.02 ml and were assayed with both ribo- each deoxyribonucleoside triphosphate was present at 2 mM. Samples nuclease A and ribonuclease T1. Hybridization reactions were of 0.5 ,l were removed at the indicated times and analyzed by alkaline incubated for 23 hr to attain CRNA*CDNA*t 2 0.1 mol of nucleotide- sucrose density gradient centrifugation. liter-'sec. Downloaded by guest on September 27, 2021 Biochemistry: Kacian and Myers Proc. Natl. Acad. Sci. USA 73 (1976) 3411

I 0 ( ) 1, f

0 0D

> 1,0 0 m - Z 0.83. 0~~~~ ,Ij.

0 Z 0.651 x) 0 ) I I I X 0,43 LL~ I uJ I < 0.2 /Cog?/ -j !I

I I- -I -I -I I I 65 70 75 80 85 90 95 100 100 (,12M)( 4M) 5 10 15 20 25 30 TEMPERATURE (0C) FRACTION NUMBER FIG. 4. Fractionation of poliovirus [32P]DNA into larger and FIG. 3. Thermal elution hydroxylapatite column chromatography smaller species. Poliovirus DNA was synthesized as described in of hybrids formed with smaller poliovirus DNA. Hybridization re- Materials and Methods. [32P]dCTP was present at 6 Ci/mmol, and actions containing smaller poliovirus [3H]DNA (1.7 ng) and poliovirus the reaction mixture was incubated at 370 for 2 hr. The reaction RNA (2.0 ,g) or E. coli RNA (2.0 ,g) were incubated for 2 hr at 680 mixture was extracted with sodium lauroyl sarcosinate and phenol- to attain CRNA-t = 2.0 and CDNA-t = 1.7 X 10-3 mol of nucleotide- cresol-chloroform as detailed (1) and fractionated by alkaline sucrose liter-'-sec. The reaction mixtures were analyzed by hydroxylapatite density gradient centrifugation. Fractions 8-13 and 18-23 were column chromatography as described in Materials and Methods. pooled, neutralized, and diluted 3-fold with water. Each sample was When the reaction mixture with poliovirus RNA was applied, 75.6% passed through a column of Sephadex G-50; the peak fractions were of the radioactivity eluted at 600, whereas when that containing E. pooled and lyophilized to dryness. The DNA was taken up in 0.3 M at same The coli RNA was tested, 70.6% was eluted the temperature. NaOH, 3 mM EDTA and incubated at 370 for 16 hr to ensure com- were then thermal elution profiles of the remaining radioactivity plete removal of the template RNA. Tris-HCl (pH 7.4) was added to the same determined. When a control hybridization containing 0.05 M,,and the pH was adjusted to 7.4 by addition of HCl. amounts of E. coli RNA and poliovirus DNA was applied to the col- umn immediately after being melted at 1000 for 2 min, 94.5% of the radioactivity eluted at 600. The temperature at which 50% of the Sequence representation of the smaller DNA double-strand-containing nucleic acid was eluted (Te) was 870 for both reactions. Additional insight into the possible mechanism of smaller DNA synthesis would result from a determination of the sequence representation of the plus-strand material. To this end, in- the ability of the antibiotic to affect its production was deter- creasing amounts of 32P-labeled smaller DNA were annealed mined. As shown in Fig. 5, addition of 100 Ag/ml of actino- to 18S [3H]cDNA made in the presence of sodium pyrophos- mycin D to the reaction strongly inhibited the synthesis of the phate, and the resistance of the latter to digestion by micro- smaller species. This finding supports the hybridization data, coccal nuclease was measured. As can be seen in Table 5, vir- showing that the material was copied from a DNA template. tually all of the larger material can be covered by sequences present in three weight equivalents of the smaller species; Table 3. Hybridizability of smaller poliovirus DNA Table 4. Hybridization of smaller DNA to 18S Polio- S poliovirus cDNA virus E. coli Eluting DNA RNA RNA Time above 18S % (ng) (ng) (ng) (min) CRNA- t CDNA- t 600 DNA Polio- Nuclease- prepa- Smaller virus resistant 1.7 2000 12 0.2 0.00017 19.0 ration DNA cDNA Time 32P-labeled 1.7 2000 12 0.2 0.00017 16.6 no. (ng) (ng) (hr) * t material 1.7 2000 120 2.0 0.00170 24.4 CDNA(-) 1.7 - 2000 120 2.0 0.00170 29.4 2 1.4 17.3 23 0.2 77.6 ± 0.2 1.7 200 1200 2.0 0.01700 26.7 3 1.1 10.4 46 0.2 69.4± 3.6 1.7 - 200 1200 2.0 0.01700 40.0 1.7 200 0 - 5.5 Hybridizations were performed as described in the legend of Table 1. As seen in that table, under the same conditions in the Reactions were performed as described in Materials and Meth- presence of equivalent amounts of E. coli RNA instead of 18S ods in a final volume of 0.02 ml. After incubation, mixtures were cDNA, the percent nuclease-resistant material was 32.1 and 36.2%, analyzed by hydroxylapatite column chromatography. respectively, for DNA preparations 2 and 3. Downloaded by guest on September 27, 2021 3412 Biochemistry: Kacian and Myers Proc. Natl. Acad. Sci. USA 73 (1976) 20r Table 5. Nuclease resistance of 18S poliovirus cDNA annealed to smaller DNA 0X 174 DNA 18S Polio- % Smaller virus Nuclease- Expected 1.61 [32P]- [3H]- resistant % DNA cDNA ng smaller DNA 3H-labeled nuclease (ng) (ng) ng 18S DNA material resistant 0.00 6.92 0.00 4.0 ± 0.0 4.0 I- ,11.2O. ,_ 3.27 6.92 0.47 37.8 ± 0.2 33.0 01 6.54 6.92 0.95 55.1 ± 3.5 56.0 14.17 6.92 2.05 82.8 ± 5.7 93.0 x 20.70 6.92 2.99 96.4 ± 2.6 100.0 27.25 6.92 3.94 93.6 ± 3.4 100.0 X 0.81 Reaction mixtures were prepared as described in Materials and Methods and incubated for 46 hr to attain CDNA-t > 0.2 mol of nucleotide-liter-'-sec. Assays were performed with micrococcal nuclease. The expected percent nuclease resistant is calculated assuming that the smaller DNA contains 30% minus-strand cDNA and 70% plus-strand DNA, each with full sequence representation. 0.4_ The 4% background level is added to each of the calculated per- centages.

/ ^'V' is anticomplementary to the poliovirus RNA, strongly suggesting that it is transcribed from the larger cDNA. In ad- I I I I dition, the appearance of the smaller DNA is inhibited by ac- 5 10 15 20 25 30 tinomycin D, providing additional evidence that the major FRACTION NUMBER fraction of the material is copied from a DNA template. The FIG. 5. Synthesis of poliovirus DNA in the presence of actino- smaller DNA complementary to the RNA may arise by limited mycin D. A standard reaction mixture was prepared containing ac- degradation of the 18S species, by abortive synthesis, by tran- tinomycin r at a final concentration of 100,g/ml, and incubated at scription from fragmented template, or as copies of the smaller, 370 for 2 hr. The yield of DNA in this reaction was 27% of the input anticomplementary material. The ability of actinomycin D to RNA template compared with 63% in the control reaction without antibiotic (not shown). A sample ofthe reaction mixture was analyzed effectively suppress synthesis of the smaller complementary by alkaline sucrose density gradient centrifugation as described in material favors the last possibility. Materials ajnd Methods. Several possible schemes for synthesis of anticomplementary DNA consistent with these results can be imagined. That ac- therefore,,all of the sequence information present in the 18S counting best for the production of both the necessary template cDNA is represented in the smaller plus-strand material. In and primers would be that degradation of the RNA template, addition, if a correction is made for the amount of minus-strand possibly by RNase H activity of the , affords primer DNA presment in the smaller species as shown in Table 5, the molecules that serve to initiate synthesis at various points along results indiicate that the amount of actual plus strand needed the 18S cDNA. The ability of phosphodiester bond-containing to protect aall of the 18S species is only slightly over one weight additives to inhibit smaller DNA synthesis could then be ex- equivalent Thus, it is clear that the plus-strand DNA is a plained if these alter or suppress the RNase H activity. faithful, bLat fragmented, copy of the sequences present in the 18S specieis. We thank Dr. S. Spiegelman for his support. This work was funded It is important to recognize that because of the presence of by National Institutes of Health Contract N01-6-1010 and National excess plus-strand DNA in the smaller material, the protection Institutes of Health Grant CA-02332-21. experimenit described in Table 2 is not informative regarding ce of the smaller minus-strand DNA. the sequeni representation 1. D. L. & Proc. Acad. Sci. USA Isolation olf this material will be required to determine the ex- 2191-2195.Kacian, Myers, J. (1976) Nati. 73, tent to whi4ch the sequences complementary to the template are 2. McDonnell, J. P., Garapin, A. C., Levinson, W. E., Quintrell, N., present. Fanshier, L. & Bishop, J. M. (1970) Nature 228, 433-435. 3. Manly, K. F., Smoler, D. F., Bromfeld, E. & Baltimore, D. (1971) DISCUSSION J. Virol. 7,106-111. The results presented above show that the smaller DNA is not 4. Ruprecht, R. M., Goodman, N. C. & Spiegelman, S. (1973) Bio- the Most of it chim. Acta produced byI simple degradation of 18S species. Biophys. 294,192-203. Downloaded by guest on September 27, 2021