Cell, Vol. 7. 361-371,
Ft. Kamen and H. Shure* Department of Molecular Virology Imperial Cancer Research Fund Lincoln’s Inn Fields London WC2A 3PX, England
Summary The different species of poiyoma virus-specific RNA molecules present in the cytoplasm of 3T6 ceils 30 hr after viral infection have been characterized by molecular hybridization between nonradioactive poiyadenyiated RNA, fractionated by sedimentation through sucrose-formamide density gradients, and the 32P-labeled separated strands of restriction endonuciease fragments of poiyoma DNA. Two relatively abundant RNA molecules, sedimenting at 16s and at 19S, transcribed from the L strand of the viral DNA, as well as a minor 20s species transcribed from the E strand of the DNA, were detected. The most abundant viral transcript, the 16s RNA molecule, was estimated to be complementary to the 22% of the L-strand DNA extending from 47 to 25 map units. The less abundant 19s L DNA strand transcript included ail the sequences present in the 16s RNA and mapped between 66 and 25 map units. The mtnor 20s RNA molecule was tentatively identified as a transcript of the E-strand DNA from the entire early region of the poiyoma genome. These three viral RNA moiecuies together exhaust >95% of the coding capacity of the viral DNA. A small region of the DNA (4-S%), including the origin of DNA replication, does not appear to determine sequences present among the major stable species of viral mRNA. introduction Virus-specific RNA in the cytoplasm of permissive cells infected by the very similar papovaviruses SV40 or polyoma has been characterized by two different experimental approaches (reviewed by Salzman and Khoury, 1974; Sambrook, 1975). Since these viruses do not block the transcription of the host genome, viral RNA can only be studied in the presence of a high background of cellular RNA sequences. In the earlier approach, viral RNA was assayed or purified by hybridizing radioactive RNA to viral DNA immobilized on nitrocellulose filters. The more recent approach has alternatively used the hybridization of unlabeled RNA extracted from infected cells to radioactive separated strands of specific DNA fragments produced by cleavage of the viral DNAs with various restriction endonucleases. The former approach has yielded informa“Present address: Department of Science, Rehovof, Israel.
tion on the number and size of viral mRNA molecules present during the early and late phases of infection, while the latter approach has been used to determine the DNA strand from which unfractionated viral RNA is transcribed and to position the total viral RNA sequences present at different stages of infection on the physical maps of the SV40 and polyoma genomes. The conclusions of these studies, all virtually identical for the two viruses, are the following. First, before viral DNA replication, the cytoplasm of infected cells contains a small quantity of a single viral RNA sedimenting at 19s (Weinberg, Warnaar, and Winocour, 1972a; Weinberg, Ben-lshai, and Newbold, 1974; Weil et al., 1974). Viral cytoplasmic RNA present during this early phase of infection is complementary to only one of the two strands (called the “E” strand) of the viral DNA (Khoury, Byrne, and Martin, 1972; Sambrook, Sharp, and Keller, 1972; Lindstrom and Dulbecco, 1972), hybridizing to about 50% of the sequences of this strand (Khoury et al., 1975; Kamen et al., 1974). This early cytoplasmic RNA is transcribed from a continuous portion of the E strand, extending in the case of polyoma virus clockwise from 72 to 25 map units (Kamen et al., 1974) around the Griffin, Fried, and Cowie (1974) physical map of polyoma DNA, as indicated in Figure 1. Second, after the initiation of viral DNA replication, two relatively abundant species of cytoplasmic viral RNA are present. The more abundant sediments at 16S while the less abundant sediments at 19s (Tonegawa et al., 1970; Weinberg et al., 1972a, 1974; Buetti, 1974). Unfractionated cytoplasmic viral RNA late during infection hybridizes to the same 50% of the E DNA strand as early RNA and, in addition, hybridizes to about 50% of the complementary (or “L”) strand of the DNA (Khoury et al., 1975; Kamen et al., 1974). The L-strand transcripts are far more abundant than those from the E strand and in the case of polyoma virus, are complementary to the region of the L strand extending on the physical map counterclockwise from 72 to 25 map units, as shown in Figure 1. Third, the 5’ ends of the E- and L-strand transcripts are proximal to the point on the genome where bidirectional viral DNA replication initiates (Khoury et al., 1973; Sambrook et al., 1974; Kamen et al., 1974; Kamen, Sedat, and Ziff, 1976). Fourth, viral cytoplasmic RNA is polyadenylated (Weinberg, Ben-lshai, and Newbold, 1972b; Rutherford and Hare, 1974). Fifth, the late SV40-specific 16s and 19s RNA molecules share common nucleotide sequences (Weinberg et al., 1974). The 19s component may be the cytoplasmic precursor of the 16s (Aloni, Shani, and Reuveni, 1975).
In the experiments described here, we applied the approach which uses the annealing of nonradioactive RNA to 3zP-labeled separated strands of polyoma DNA fragments to determine the number, size, and map position of the discrete polyoma RNA molecules present in the cytoplasm of 3T6 cells at a late time after productive infection. We have mapped three different viral RNA molecules, which together account for over 95% of the coding capacity of the viral DNA. The “19s” RNA previously shown to be present at late times by experiments using radioactive RNA (Buetti, 1974) proved to be a mixture of a minor 20s species transcribed from the E DNA strand (thus probably identical to the early “19s” RNA) and a 19s transcript of the late region of the L DNA strand. The late 16s RNA, as suggested for SV40 virus by Weinberg et al. (1974), is a specific fragment of the 19s molecule. Since the proteins encoded by the two late RNA molecules have been determined through in vitro translation studies (Smith et al., 1975), the present data on the map positions of these molecules allow us
to deduce sequences
the location on the genome of the DNA encoding the late viral proteins.
Results Mapping Total Cytoplasmic Polyoma RNA
Cytoplasmic RNA was extracted from 3T6 cells 30 hr after infection at a high multiplicity with plaquepurified polyoma virus, as described in Experimental Procedures. Polyadenylated RNA was then selected by chromatography on a poly(U)Sepharose column. We chose to use polyadenylated RNA to reduce the mass of RNA to a quantity small enough for high resolution fractionation of viral molecular species on sucrose gradients, as described in the subsequent section. Although Rutherford and Hare (1974) have shown that the polyoma-specific cytoplasmic RNA contains poly(A) sequences, preliminary experiments were undertaken to verify that there was no difference between viral mRNA prepared by poly(A) selection and the total polysome-associated mRNA mapped in our previous studies (Kamen et al., 1974). Figure 2 shows the results of a hybridization saturation experiment in which the nonradioactive polyadenylated cytoplasmic RNA (which we shall refer to as “mRNA”) was annealed with the 3zP-labeled E or
Figure 1. Restriction Endonuclease Map of Polyoma DNA Showing the Positions of Polyadenylated Cytoplasmic RNA Molecules The physical map of polyoma DNA (Griffin et al., 1974) is shown with the unique site of cleavage by restriction endonuclease EcoRl at the top. Reading outward from the innermost concentric circle, we indicate: (1) the regions of the DNA which are the templates for transcription of early and late cytoplasmic RNA (Kamen et al., 1974); (2) standard polyoma map units measured in percentage of the total DNA length with the EcoRl site as the reference point; (3) restriction endonuclease Hpall cleavage map (Griffin et al., 1974); (4) map of the four fragments generated by cleavage with EcoRl plus Hhal (Griffin and Fried, 1975); OR indicates the location of the origin of bidirectional viral DNA replication (Griffin et al., 1974; Crawford et al., 1974); (5) orientations of the three polyadenylated cytoplasmic RNA molecules mapped in this paper. The arrows indicate the 5’ to 3’ RNA polarities (Kamen et al., 1976).
w RNA Figure 2. Saturation of Total L- or E-Strand Polyadenylated Cytoplasmic RNA
Increasing quantities of cytoplasmic polyadenylated RNA purified from 3T6 cells 30 hr after polyoma infection were annealed with 470 cpm of total L-strand DNA (O-o) or 590 cpm of total Estrand DNA (Od) in 100 pl of hybridization buffer (see Experimental Procedures) for 2830 min. The fraction of ‘ZP-labeled DNA hybridized was determined by digestion with endonuclease Si as described in Experimental Procedures.
L strand of polyoma DNA. The fraction of the radioactive DNA strand which hybridized to the RNA was determined here, as in all subsequent experiments, by digestion of the unhybridized DNA with the fungal single-strand specific endonuclease Sl (Leong et al., 1972). The late viral mRNA was complementary to one half of each of the complementary viral DNA strands. We estimated (data not shown) that 0.5-l % of the total mRNA was complementary to the E DNA strand and lo-15% was complementary to the L DNA strand. The viral mRNA sequences were next positioned on the DNA physical map by hybridization saturation experiments using J*P-labeled separated strands of the four DNA fragments resulting from digestion of polyoma DNA with restriction endonucleases EcoRl and Hhal. The locations of the four fragments on the Griffin, Fried, and Cowie (1974) physical map are shown in Figure 1. Figure 3 shows that the mRNA annealed to all of the L strand of 0.4
Figure 3. Mapping Separated Strands
Late Polyadenylated Cytoplasmic of EcoRI/Hhal Fragments
RNA with the
The indicated amounts of late polyadenylated cytoplasmic RNA (bottom abscissa-annealings with L strands; top abscissa-annealings with E strands) were annealed with fixed quantities of the separated strands of the four EcoRI/Hhal fragments of polyoma DNA (see map in Figure 1) in 100 ~1 of hybridization buffer for 2400 min. The input 3*P radioactivities used were: fragment 1 L: 1150 cpm; 1 E: 1190 cpm; 2L: 600 cpm; 2E: 320 cpm; 3L: 145 cpm; 3E: 205 cpm; 4L: 370 cpm; 4E: 380 cpm. The hybridizabilities of the L-strand probes were measured by parallel annealings containing 0.5 pg of asymmetric polyoma cRNA. The values obtained were 69%, 66%, 91%, and 78%, for fragments 1 L-4L, respectively. The fraction of the DNA probe hybridized in polyadenylated RNA was calculated as the fraction of the DNA resistant to Sl divided by the fraction of the DNA resistant after annealings to cRNA. In the case of E-strand probes, it was assumed that the hybridizability of both strands of a particular fragment were identical.
fragment EcoRI/Hhal-1, as well as to about 20% of the L strand of EcoRI/Hhal-4. Annealing at higher input in RNA levels was obtained with all the E strand of fragments EcoRI/Hhal-2 and 3, and with at least 72% of that of EcoRI/Hhal-4. The data shown in Figures 2 and 3 agree with our previous mapping results for total polysome-associated RNA, which are summarized in Figure 1. We therefore conclude that all species of viral mRNA have poly(A) tracts. The data presented thus far confirm the positioning of one of the boundaries between E DNA strandspecific and L DNA strand-specific transcriptional regions very near the origin of viral DNA replication, which is at 71 f2 units on the physical map (Crawford, Robbins, and Nicklin, 1974; Griffin et al., (1974). This boundary corresponds to the 5’ ends of E- and L-strand transcripts (Kamen et al., 1976). The control possibilities suggested by the proximity of the mRNA 5’ ends to the origin of DNA replication made it interesting to attempt to locate these ends with greater precision. As shown in Figure 1, the DNA replication origin lies within 2 map units of the juncture between restriction fragments Hpall-3 and Hpalld. We thus chose these two fragments for more detailed hybridization saturation experiments. A constant amount of the complementary strands of Hpall-3 and Hpall-5 was separately annealed with increasing amounts of late mRNA under exhaustive hybridization conditions. By “exhaustive hybridization conditions” we imply that the DNA concentration and the time of annealing were such that virtually all the DNA would have reannealed if both complementary strands were present in the same reaction (~8 times the Cot% of polyoma DNA). Figure 4A shows that the mRNA annealed to 95-100% of the L-strand DNA of Hpall-3 (Hpall9L), with no significant hybridization to the E-strand DNA (Hpall3E) of this fragment. The same mRNA preparation, as indicated in Figure 48, saturated 80-85% of HpalldE and also annealed to at least 40% of Hpall5L; the curve obtained with DNA from Hpall6L, however, is complex and a saturation plateau was not reached. This result means that the RNAs transcribed from the E and L DNA strands of Hpalld overlap for at least 20% (approximately 80 nucleotides) of the fragment. The data shown in Figure 4 can be quantitatively interpreted in the following manner: since the annealing conditions were exhaustive, we can assume that when 50% of the probe DNA was hybridized, the quantity of complementary viral RNA in the annealing was equal to one half the amount of probe DNA added. To compare the data shown in Figures 4A and 48, correction must be made for the DNA fragment size and for the amount of probe DNA strand used in each annealing. We therefore esti-
w RNA Figure 4. Hybridization of Late Cytoplasmic Polyadenylated RNA to the Separated Strands of Restriction Fragments Spanning the Origin of DNA Replication (A) Annealings with the separated strands of fragment Hpall-3. Each 50 ~1 hybridization contained either 550 cpm of Hpall-3L or 460 cpm of Hpall-SE and was incubated for 2860 min. (O-0) Hpall-BE DNA annealed with the quantities of polyadenylated RNA shown on the upper abscissa; (-0) Hpall9L DNA annealing with the quantities of RNA shown on the lower abscissa; (I-A) Hpall3L DNA, upper abscissa. The data are corrected for the hybridizability of the L-strand probe, which became 90% Sl -resistant after annealing with 0.12 1-19of cRNA. (B) Annealing with the separated strands of fragment Hpall-5. Each 50 pl hybridization contained either 420 cpm of Hpall-5L or 390 cpm of Hpall-5E. Annealing conditions were as in (A). (0-O) Hpall-5E DNA: (-0) Hpall-5L DNA. The data are corrected for hybridizability of the L-strand probe, which became 69% Siresistant after annealing with 0.12 pg of cRNA.
mate the relative abundance of RNA complementary to the different strands of the two fragments by calculating the mass of RNA necessary to half-saturate an amount of probe DNA equivalent to 1000 cpm of full length polyoma DNA. DNA quantities are unavoidably expressed in cpm because the DNA specific activity could not be accurately determined (see Experimental Procedures). Such calculation showed that annealing to Hpall-3L DNA is half-maximal at 5ng RNA per 1000 cpm DNA,
while that to HpalME DNA is half-maximal at 75 ng RNA per 1000 cpm DNA. Thus, as expected from the overall distribution of L- and E-strand transcripts mentioned above, the RNA transcribed from Hpall3L is much more abundant than that from HpalldE. Quantitative analysis of the annealing between viral RNA and HpalldL DNA is difficult because of the competing RNA-RNA annealing reaction between the overlapping E- and L-strand transcripts of this fragment. We have previously discussed an equivalent problem in relation to analysis of polyoma nuclear transcripts (Kamen et al., 1974). The present data, however, allow us to conclude that the transcript of HpalldL DNA is no more abundant than the transcript HpallBE DNA and is therefore less abundant than the transcript of Hpall-3L DNA. We thus further conclude that only a minor fraction of the abundant RNA molecules complementary to Hpall3L extend into Hpall-5. Because of possible RNA-RNA annealing competition, it cannot be determined whether the overlap between this minor class of L-strand transcripts and the E-strand transcript is necessarily restricted to the region of the genome containing the origin of viral DNA replication. We return to this point in the Discussion. Mapping Discrete Viral mRNA Molecules Polyadenylated RNA extracted from the cytoplasm of 3T6 cells 30 hr after polyoma infection, equivalent to the RNA analyzed in the preceding section, was fractionated into its component molecular species by sedimentation through sucrose gradients containing 50% formamide. The distribution of viral RNA molecules across the gradients was determined by hybridizing the RNA in the sucrose gradient fractions with the 3*P-labeled separated strands of either total viral DNA or of particular restriction endonuclease fragments. The profile of the endonuclease Sl resistance of the probe DNA will be proportional to the amount of viral RNA present in each fraction only if none of the various resolved RNA species approaches saturation of the available complementary probe sequences under the annealing conditions used. For this reason, peak fractions of different viral mRNAs were subsequently analyzed further in more detail to define both their relative abundance and their sequence composition by hybridization saturation experiments using the appropriate strand of selected restriction endonuclease fragments. Figure 5 shows the profile of Sl resistance obtained when late mRNA fractionated on a sucroseformamide gradient was assayed by hybridization with the total E or L strand of polyoma DNA. A single RNA species, sedimenting at 2OS, was detected which hybridized to E-strand DNA. We have previously shown (Figure 2) that the E-strand transcript
present in the mRNA preparation before sucrose gradient fractionation saturates 50% of the total Estrand DNA probe. Since the maximum amount of hybridization found in the 20s peak fraction (13%) exhausts only about 25% of available probe sequences, we can conclude that the hybridization profile shown is proportional to the distribution across the gradient of RNA transcribed from the DNA E strand. In contrast, when the RNA in the different gradient fractions was annealed to the L strand of polyoma DNA (Figure 5), two major peaks, at 19s and at about 16S, were observed; this profile, however, cannot be proportional to the distribution of viral RNA across the gradient, since the RNA in the 19s peak fraction annealed to all the available probe sequences (50% of the L-strand DNA) previously shown in Figure 2 to be complementary to unfractionated late mRNA. The Sl resistance profile shown in this case probably represents the hybridization saturation values attainable with the 19s and 16s species rather than their relative abundance. This supposition was confirmed by reassay of the
Sucrose-Formamide Gradient Polyadenylated RNA
of Late Cyto-
Cytoplasmic polyadenylated RNA (8 pg) purified from 3T6 cells 30 hr after polyoma infection was denatured at 37°C in 200 ~1 of a buffer containing 0.05 M Tris-HCI (pH 79, 0.05% SDS, and 50% v/v formamide (Analar). After addition of 200 pl of 0.1% SDS, the RNA was loaded onto a 5-20% sucrose gradient containing 50% v/v formamide (see Experimental Procedures). Centrifugation was for 17.5 hr at 35,000 rpm and 20°C in the Spinco SW40 rotor. 15 drop fractions were collected and then diluted with 200 ~1 of 0.1% SDS. An internal marker (2000 Cerenkov cpm) of ‘IP-labeled cytoplasmic RNA extracted from uninfected 3T6 cells was included: the 28s and 18s positions are indicated by the arrows. Aliquots of gradient fractions were assayed for viral RNA by hybridization to QP-labeled separated strands of polyoma DNA as follows: (O-O). 5 pl of aliquots of gradient fractions annealed with 765 cpm total E-strand DNA for 2700 min in 50 pl of hybridization buffer; (O----O) 5 ~1 aliquots of gradient fractions annealed with 440 cpm of total L-strand DNA for 550 min in 50 pl of hybridization buffer; (O--O) 4 ~1 aliquots of gradient fractions annealed with 880 cpm of total L strand for 420 min in 100 pl of hybridization buffer.
gradient fractions using more probe DNA, less RNA, and a shorter annealing time. As shown in Figure 5, under these conditions hybridization at the 19s position decreased to approximately 10% of the added L-strand DNA, whereas that detected at the 16s position remained at its apparent saturation level. Therefore the 19s viral transcript is complementary to a larger fraction of the DNA L strand, but the smaller 16s RNA is more abundant. This interpretation was confirmed by hybridization across a second similar sucrose gradient, as shown in Figure 6, with the L-strand DNA of restriction endonuclease fragments which span the late region of the viral genome, EcoRI/Hha-l-l, Hpall-1, and Hpall-3. Analysis using these fragments provides direct information on the map positions of DNA sequences complementary to the late viral mRNAs. Both 19s and 16s RNA molecules hybridized to EcoRI/Hhal-1 L and to Hpall-1L DNA, but only the 19s RNA annealed to Hpall-3L DNA. The profiles obtained clearly show that the 16s molecule is the more abundant component. It should also be noted that a minor RNA sedimenting at 18s and low concentrations of viral RNA sedimenting faster than 19s were also detected. RNA in the fractions of the sucrose gradient shown in Figure 6 was also annealed to the E-strand DNA of fragment EcoRI/Hhal-2. Once again a single 20s (as well as a small amount of heterogeneous, faster sedimenting RNA) peak was detected. This demonstrated that the 20s RNA is transcribed from the E strand of the early region of the polyoma genome (see map in Figure 1). To position the 19s and 16s late polyoma messengers more accurately on the viral genome and to obtain a more precise estimate of their relative abundance, hybridization saturation experiments were performed between aliquots of the respective peak fractions and the L strands of late region restriction endonuclease fragments of polyoma DNA. Figure 7 shows the result of exhaustive annealings between 19s or 16s RNA and the L strand of a fragment (EcoRI/Hhal-1 L) which covers virtually the entire late region. The 19s RNA saturated at least 90% of this probe. The curve obtained with 16s RNA is clearly biphasic; the RNA contains a major component complementary to about 50% of EcoRI/Hhal1L DNA, as well as a component present at much lower concentration complementary to some of the remaining sequences of the probe. The 16s region of a gradient similar to that shown in Figure 6 was pooled and sedimented through a second sucrose gradient in an effort to reduce cross-contamination with 19s RNA sequences. Figure 8 shows that when increasing amounts of the repurified 16s RNA were annealed to EcoRI/Hhal-1 L, only the major component saturating 45-50% of the probe was detected;
a parallel annealing experiment between the same 3zP-labeled DNA probe and polyoma cRNA is included in Figure 8 to demonstrate that 100% of the added DNA was hybridizable. The results of hybridization saturation experiments between 19s and 16s peak fractions from two different mRNA preparations and Hpall-1L or
Figure 7. Saturation with 19s and 16s
Increasing amounts of 19s RNA (A, fraction 25 of the gradient shown in Figure 6) or 16s RNA (6, fraction 33) were annealed for 1500 min in 100 ~1 of hybridization buffer to either 915 cpm (first four data points in A and 6) or to 235 cpm (last five data points in A and 6) of the L-strand DNA of fragment EcoRI/Hhal-1. The data are plotted as uncorrected percentage of Sl resistance (the probe was totally hybridizable) against the ratio of RNA: DNA in units of ~1 cpm-r x 104. To fit all the data on a linear scale, the curves are plotted against two different abscissa. The lower curves refer to the lower abscissa, while the upper curves refer to the upper abscissa.
FRACTION Figure 6. Cytoplasmic
Cytoplasmic polyadenylated RNA (11 Pg) was sedimented through a sucrose-formamide gradient as described in Figure 5. A parallel gradient containing IH-uridine-labeled cytoplasmic RNA was used to estimate the 28s and 16s positions (arrows). (A) Polyoma-specific RNA was measured as follows: (Od) 0.5 pl aliquots of gradient fractions were annealed with 490 cpm of the L strand of fragment EcoRI/Hhal-1 for 1445 min in 100 ~1 of hybridization buffer. A parallel annealing with excess cRNA demonstrated that the probe used could be rendered 97% Sl-resistant: (0-O) 5 gl aliquots annealed with 570 cpm of the L strand of fragment Hpall-3 for 1400 min in 100 Al of hybridization buffer. The probe used was 92% Sl-resistant after parallel annealing with cRNA. (6) (O--O) 1 ~1 aliquots of gradient fractions were annealed with 600 cpm of the L strand of fragment Hpall-1 for 630 min in 200 pl of hybridization buffer. The probe used was 100% Sl-resistant after parallel annealing with cRNA; (0-O) 5 ~1 aliquots were annealed with 155 cpm of the E strand of fragment EcoRI/Hhal-2 for 4440 min in 50 ~1 of hybridization buffer.
RNA/DNA Figure 6. Saturation Hybridization Repurified 16s RNA
Late cytoplasmic polyadenylated RNA (a preparation provided by A. Smith and T. Wheeler) was sedimented through a sucroseformamide gradient as described in Figure 5. The 16s region was pooled, concentrated by ethanol precipitation, and rerun on a second sucrose-formamide gradient. Aliquots of the 16s peak region from the second gradient (localized by in vitro translation as well as by hybridization across the gradient) were annealed with 304 cpm of the L-strand DNA of fragment EcoRI/Hhal-1 for 1440 min in 50 gl of hybridization buffer. A parallel annealing series using different amounts of polyoma cRNA served to measure the hybridizability of the probe. The 16s RNA data are plotted as uncorrected percentage of Sl resistance against the ratio of RNA to DNA in units of gl cpm-’ x 104. (e-0) 16s RNA; (H ) cRNA.
Hpall-3L DNA are shown in Figures 9 and 10. The 19s RNA peak fraction from the sucrose gradient shown in Figure 6 saturated 90-100% of Hpall-1L and 80% of Hpall9L DNA (Figure 9). The 16s peak fraction from the same sucrose gradient contained a major component complementary to approximately 70% of Hpall-1L DNA and a minor component complementary to some fraction of Hpall3L DNA. While the quantity of 16s RNA sequences which annealed to Hpall3L DNA was so small that saturation was not approached at the highest RNA/ DNA ratio used, extrapolation of the data obtained indicates that the amount of RNA required to saturate one half of the Hpall-1L probe is 34% of the quantity that would be required to saturate one half of the Hpall-3L DNA probe. Results of a similar experiment with a different polyadenylated mRNA preparation are shown in Figure 10. In this case, pairs of sucrose gradient fractions were pooled and concentrated by ethanol precipitation prior to hybridization analysis; one would thus expect somewhat lower resolution between the 19s and 16s RNAs. The 19s peak fraction from this RNA preparation annealed to >90% of Hpall-1L DNA and to at least 70% of Hpall-3L DNA; sufficient RNA was not available in this experiment to obtain saturation plateaus. The pooled 16s peak fraction yielded a result qualitatively similar to that shown in Figure 9; the RNA contained a major component complementary to about 70-75% of Hpall-1L DNA, as well as a minor RNA complementary to Hpall9L. From a comparison of amounts of RNA required for half-maximal satura-
RNA/DNA Figure 10. Saturation Hybridization HpallJL with 19s and 16s RNA
9. Saturation Hybridization with 19s and 16s RNA
(A) increasing amounts of 19s RNA (Figure 6, fraction 25) were annealed with either 390 cpm of the L-strand DNA of fragment Hpall-1 or with 155 cpm of the L-strand DNA of Hpall-3 for 1410 min in 100 pl of hybridization buffer. Both probes were completely hybridized in parallel annealings containing excess cRNA. The data are plotted as in Figure 8, except that DNA* was used instead of the actual cpm DNA, where DNA* = cpm DNA strand per fractional length of the restriction fragment relative to total polyoma DNA. (-0) Hpall-I L; ( H) Hpall3L. (B) Increasing amounts of 16s RNA (Figure 6, fraction 33) were annealed with 370 cpm of Hpall-1L or with 240 cpm of Hpall3L for 810 min in 200 ~1 of hybridization buffer. The data are plotted as in (A).
19s and 16s RNA was prepared (in collaboration with A. Smith and T. Wheeler) essentially as described in Figure 6, except that the sucrose gradient fractions were pooled in pairs and concentrated by ethanol precipitation prior to analysis. The peak fractions of polyoma mRNA were localized by hybridizing across the gradients with QP single-stranded restriction fragments (data not shown) and by translating each fraction in the wheat germ in vitro system (A. Smith, S. T. Bayley, and T. Wheeler, manuscript in preparation). The data are plotted as in Figure 9. (A) 19s peak fraction, annealed to fragment Hpall-1 L or to Hpall3L DNA. Incubations (100 ~1) for 1440 min contained 285 cpm of Hpall1 L (0) or 210 cpm of Hpall9L (B). Incubations (100 pl) for 2760 min contained 215 cpm of Hpall-1L (0) or 130 cpm of Hpall-3L 0. (B) 16s Hpall-3L (A).
peak fraction, annealed to fragment Hpall-1L DNA or to DNA. Annealing conditions and data presentation as in
tion, we estimate that this RNA preparation contained 2.5 times more 16s viral RNA than 19s RNA, and that the ratio of the major and minor 16s RNA components was approximately 1O:l. Table 1 summarizes the results of the hybridization saturation experiments described. They are consistent with the positioning of the 19s and 16s RNAs on the restriction endonuclease map shown in Figure 1, in which the 16s is represented as the 3’ terminal half of the 19s mRNA molecule. We conclude here that there are only two polyadenylated mRNAs. The minor component detected in the 16s fractions, which has a sequence composition similar to 19s RNA, we attribute to the expected level of contamination of the 16s region of a sucrose gradient with 19s RNA and its fragmentation products. If, as Aloni et al. (1975) have demonstrated for SV40, the polyoma 19s RNA is the cytoplasmic precursor to the 16s RNA, it is further possible that the minor 16s RNA results from the in vitro dissociation of 19s molecules nicked in vivo, which might produce a 16s polyadenylated mRNA as well as a large 5’ terminal fragment.
Discussion The map shown in Figure 1 summarizes our conclusions on the number and map positions of polyadenylated viral RNA molecules present in the cytoplasm of 3T6 cells 30 hr after polyoma infection. We have shown that the late 16s and 19s RNA molecules previously identified by Buetti (1974) are transcripts of the L strand of polyoma DNA and share common sequences, the 16s RNA constituting the 3’ terminal one half of the 19s RNA. P. Beard and N. Acheson (personal communication) have reached similar conclusions. The third viral RNA, which is far less abundant than the other two components, sediments at 20s and is transcribed from the E strand of polyoma DNA. While insufficient quantities of this RNA were available to determine the sequence composition by detailed hybridiTable
Gel Electrophoresis with Restriction
of s*P-Labeled Polyoma Endonucleases EcoRl and
32P-labeled polyoma DNA was prepared, digested with endonucleases EcoRl and Hha 1, and electrophoresed on a 20 x 40 cm gel containing 1.4% agarose as described in Experimental Procedures.
Figure 11. Agarose DNA after Digestion Hhal
of Py DNA0
OThis estimate includes the fraction of fragment Hpall-6 (equivalent to about 2% of genome) complementary to the 3’ terminal region of the viral messengers (Figure 3 and Kamen et al., 1974).
zation saturation experiments, we suggest that it is a transcript of the entire polyoma early region because it is the only detectable E-strand transcript and because its sedimentation rate, with respect to 19s late RNA molecule, is consistent with that of an RNA of this size. We also suggest that the 20s RNA is identical to the “19s” species detected by Weil et al. (1974) during the early phase of infection. The proposal that early and late “19s” RNAs are the same (Buetti, 1974) cannot be true, since they are transcripts of different DNA strands; the two species fortuitously have very similar sedimentation rates, and the 20s RNA represents too small a fraction of the viral RNA to be detected in hybridizations between radioactive late RNA and total polyoma DNA. The specification of the total number of viral RNA molecular species as only three should be regarded as a minimal estimate. The resolution obtained on sucrose gradients is not fine enough to decide this issue definitively. From the data shown in Figures 5 and 6, it is probable that minor components sedimenting between 19s and 16s are also present. Whether these represent true mRNA molecules or simply degradation products of the major species is not known. The observed heterogeneity may also simply reflect the distribution of molecules with different lengths of terminal poly(A). We are currently attempting to fractionate labeled viral mRNA on denaturing gels after enzymatic removal of poly(A) tracts in the hope of directly visualizing discrete species by autoradiography. Our identification of at least the two major polyadenylated L-strand transcripts as true mRNAs is based on the stringent criterion of in vitro translation. Smith et al. (1975) showed that the 16s RNA encodes the major virion capsid protein of molecular weight 45,000 daltons, VP1 . In Table 1, we estimate that the 16s RNA could determine 44,000 daltons of protein and thus is within the error inherent in such calculations, just large enough to encode VPl. L. K. Miller and M. Fried (personal communication) have recently shown that polyoma late temperature-sensitive mutants tsl0, tsl260, and tsC map within the region of the polyoma genome determining the 16s RNA molecule. Since this RNA encodes VP1 in vitro, these mutations occur in the gene for the major capsid protein. The 19s late RNA codes for the two minor capsid proteins, VP2 and VP3 (Smith et al., 1975; A. Smith and T. Wheeler, personal communication). The combined molecular weights of these two proteins (35,000 and 23,000 daltons for VP2 and VP3, respectively) is greater than the coding capacity of the additional sequences present in the 19s molecule but not in the 16S, as calculated in Table 1. This is consistent with the data of Fey and Hirt
(1974) Gibson (1974) and Hewick, Fried, and Waterfield (1975) showing that VP3 contains most of the tryptic peptides present in VP2. It is interesting that although the 19s RNA contains all of the viral sequences necessary to encode VPl, highly purified 19s RNA does not direct the synthesis of significant amounts of this protein in the wheat germ in vitro system (A. Smith and T. Wheeler, personal communication). This suggests that cleavage and perhaps 5’ terminal modification is essential for translation of the VP1 coding sequences. The remarkable identity in molecular organization between polyoma and SV40 viruses which has emerged in recent years was described in the Introduction. Two groups (May, Kopecka, and May, 1975; G. Khoury, B. J. Carter, F.-J. Ferdinand, P. Howley, M. Brown, and M. A. Martin, personal communication) have now shown that the late SV40 mRNA molecules have map positions almost precisely equivalent to those described here for polyoma virus. Moreover, Prives et al. (1974) have translated SV40 messengers in vitro and have made the same protein assignments as those reported subsequently for polyoma virus. In our positioning of the polyoma 19s mRNA molecule shown in Figure 1, we have used the results of hybridization saturation experiments with purified 19s material to locate the 5’ end of the molecule within fragment Hpall-1 at 68 map units. This leaves a small “gap” in the mRNA map, indicating that the region between 68 and 72 map units, which includes the origin of viral DNA replication, does not determine a stable messenger molecule. Our efforts to position the origin proximal 5’ end of unfractionated polyadenylated RNA, which were presented in Figure 4, however, showed that at least a minor fraction of the RNA molecules transcribed from the L strand extended through fragment Hpall-3 into some portion of Hpall-5; they thus overlapped the 5’ terminal region of the RNA molecules transcribed from the E strand. A similar conclusion, based on three lines of evidence, can be reached for RNA transcribed from the equivalent region of SV40 DNA. G. Khoury et al. (personal communication), in hybridization experiments with SV40 RNA analogous to those described here, had similar difficulty in positioning the 5’ end of the largest L-strand transcript. The RNA fingerprinting approach used by R. Dhar, S. Weissman, and their co-workers (personal communication) suggested that the 5’ end of a major L-strand transcript lies about 9% of the SV40 map away from the origin of DNA replication, whereas a less abundant L-strand transcript continues across the origin into a DNA region also determining the 5’ terminal portion of an E-strand transcript. Furthermore, the genetic experiments of Mertz and Berg (1974) showed that a small portion
of the SV40 genome on the late region side of the origin of DNA replication can be deleted without eliminating viability, suggesting that this DNA contains neither critical control signals nor protein-encoding sequences. Clearly, the significance of these results with both viruses cannot be ascertained until future experiments answer two critical questions: where does transcription of viral RNA initiate, and how are the nascent transcripts processed to produce the final mRNA molecules? Experlmental
Growth of Infected Cells and RNA Extraction Plaque-purified polyoma virus of the A2 strain (Griffin et al., 1974) was grown at low multiplicity in secondary mouse embryo cells and checked for the absence of defective virus as described previously (Kamen et al., 1974). RNA was purified from infected cells as follows: 3T6 cells grown in 60 oz roller bottles were infected (50 PFU per cell) in 10 ml of phosphate-buffered saline (PBS) for 2 hr at 37°C. Dulbecco’s modified Eagle’s Medium (E4) containing 5% fetal calf serum (200 ml per roller bottle) was then added. 30 hr after medium addition, the infected cells were removed after rinsing the bottles with 1 O-20 ml of versene buffer followed by swirling with 25-50 ml of PBS, and were collected by low speed centrifugation. The cells were washed twice in PBS and then resuspended in 7.5 ml per roller bottle of iso-Hi-pH (0.14 M NaCI, 1.5 mM MgCl?, 0.01 M Tris-HCI (pH 8.5) 0.5% NP 40; Lindberg and Darnell, 1970) at 0°C. After approximately 10 min at O”C, the nuclei were sedimented by centrifugation at 2500 x g for 10 min. The cytoplasmic supernatant fraction was further centrifuged at 12,000 x g for 10 min, urea (to 7 M; Mann Ultra Pure) and SDS (to 1% w/v) were added, and protein was removed by extraction with an equal volume of phenol mixture (phenol:CHC13:isoamyI alcohol, 50:50:1). RNA was precipitated from the aqueous phase with 2 vol of ethanol and dissolved in 0.5 ml per roller bottle of buffer A [O.Ol M Tris-HCI (pH 7.5), 1 mM EDTA, 0.1% SDS]. Residual protein was removed by a further extraction with phenol mixture and the RNA was reprecipitated with ethanol. The pelleted nuclei were dissolved in 5 ml per roller bottle of lysis mix [7 M urea, 0.35 M NaCI, 0.01 M Tris-HCI (pH 8.5), 2% SDS; Holmes and Bonner, 19731 by blending at low speed in an Omni-mixer (Sorvall) for l-2 min. An equal volume of phenol mixture was then added and blending continued for a further l-2 min. The phases were separated by centrifugation at 10,000 x g for 30 min in the GSA head of the Sorvall RB-4 centrifuge. Nucleic acid was precipitated from the aqueous phase with 2 vol of ethanol, redissolved in buffer A, reextracted once with phenol mixture, reprecipitated from ethanol, and dissolved in buffer A. An equal volume of 4 M LiCl was then added, and the nuclear RNA was allowed to precipitate at 0°C for at least 12 hr. The RNA was recovered by centrifugation, dissolved in 10 mM Tris-HCI (pH 7.5), 5 mM MgCb, and incubated for 20 min at 37°C with 5 pg/ml of RNAase-free DNAase (Worthington RNAase-free, further purified as described by Holmes and Bonner, 1973). The DNAase was removed by extraction with phenol mixture and the nuclear RNA was recovered by ethanol precipitation. Polyadenylated RNA was purified from the cytoplasmic and nuclear RNA fractions by poly(U)-Sepharose chromatography on 5 ml columns (for the RNA from 10 roller bottles) as described by Lindberg and Persson (1974). Preparation of 12P-Labeled Polyoma DNA Fragments Sub-confluent monolayers (6-7 x 106 cells per 90 mm dish) of 3T6 cells grown in E4 medium containing 5% fetal calf serum were washed twice with 10 ml of phosphate-free E4 medium and infected at lo-20 PFU per cell with polyoma virus in 1 ml of phosphate-free medium for 2 hr. Phosphate-free E4 medium containing 3% horse
serum (10 ml) was then added and the cells incubated at 37°C for 24-27 hr, at which time the medium was replaced with 10 ml of fresh phosphate-free medium containing 2.5 mCi per dish ‘2P04 (Amersham high specific activity). After a further 24 hr at 37”C, the cells were lysed and fractionated by selective SDS extraction (Hirt, 1967). The resulting supernatant was extracted with an equal volume of phenol mixture, and the nucleic acids were precipitated from the aqueous phase with ethanol. After redissolving in 0.05 ml per dish of 1 mM EDTA, the contaminating RNA was digested with 80 @g/ml of pancreatic ribonuclease A (Sigma X-A grade) for 60 min at room temperature. The DNA was separated from ribonucleotides by chromatography on a 4 ml column of Sephadex G-75 (in a disposable 5 ml plastic syringe) equilibrated in a buffer containing 10 mM Tris-HCI (pH 7.5) 10 mM MgCI?, and 1 mM dithiothreitol. Portions of the DNA were then digested to completion with restriction and endonucleases EcoRl (after addition of NaCl to 0.1 M) or with Hhal followed by EcoRI. After concentration by ethanol precipitation, the digested DNA was fractionated on 20 x 40 cm gels containing 1.4% agarose (SeaKern) in E buffer (Sharp, Sugden, and Sambrook, 1973). As shown in Figure 11, this procedure yields polyoma DNA fragments of high purity even though the viral DNA is not purified by CsCI-ethidium bromide centrifugation. The majority of the labeled DNA extracted by the Hirt procedure appears to be viral, and further purification is achieved by gel electrophoresis of the restriction fragments. Preliminary experiments showed that very little superhelical DNA is recovered from cells labeled for 24 hr with 250 &i/ml of 32P, and thus ethidium bromide equilibrium centrifugation would not be useful in separating viral from contaminating host DNA. One limitation of the procedure is that the DNA-specific activity cannot be accurately determined. From the rate of reannealing of EcoRl linear DNA prepared by this method, we estimated that the initial specific activity was l-2 x 107 cpm/pg. DNA fragments were eluted from the agarose gel by a modification of the procedure described by Lewis et al. (1975): gel bands were passed through a 5 ml disposable plastic syringe to fragment the agarose which was then dissolved in 5 M NaC104 containing 10 mM Tris-HCI (pH 7.5) at 5O’C. Calf thymus DNA (25 pg) was added, and the DNA fragments were absorbed to 0.5 ml columns of hydroxylapatite (Biorad HTP, DNA grade) at 60°C. The columns were washed with about 5 ml of 5 M NaCIO, before elution of the DNA with 2.5 ml of 0.4 M Na phosphate buffer. The phosphate was removed by dialysis against a buffer containing 0.05 M TrisHCI (pH 7.5), 0.1 M NaCI, 1 mM EDTA, and 0.1% SDS before concentration of the DNA by ethanol precipitation. DNA strand separation was achieved by hybridization to asymmetric polyoma cRNA (prepared as described by Kamen et al., 1974) followed by hydroxylapatite chromatography essentially as described by Sambrook et al. (1974). DNA-RNA Hybridization 32P-labeled single-stranded polyoma DNA fragments were annealed to unlabeled cellular RNA in 50 or 100 ~1 of hybridization buffer [l M NaCI, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.1% SDS, 100 ag/ml yeast RNA] at 70°C in Beckman plastic microfuge tubes. The details of each experiment are described in the figure legends. After the stipulated annealing time, the samples were stored frozen until further processing. To determine the percentage of the DNA in hybrid, the thawed incubation mixtures were diluted with 2 ml of Sl buffer [0.05 M KCHIC02 (pH 4.5), 0.2 M NaCI, 0.0035 M ZnSO+ 25 pg/ml native calf thymus DNA, and 7 mM P-mercaptoethanol] and were then incubated for 1 hr at 50°C with sufficient Sl endonuclease to digest >97% of the single-stranded DNA. After chilling on ice, trichloroacetic acid (TCA) was added to a final concentration of 10% (w/v), and the acid-insoluble material was collected on Whatman GF/C filters which were then washed with 5% TCA followed by 80% ethanol. Radioactivity was determined by liquid scintillation counting in a toluene-based scintillation fluid. In each experiment, two annealings processed in parallel but
incubated without Sl nuclease were used to determine the total input acid-insoluble radioactivity; one annealing without viral RNA was used to determine the Sl-resistant background (l-3%). and in the case of L strand DNA probe, a further tube containing excess asymmetric cRNA served to measure the hybridizability of the probe. When indicated in the figure legends, the percentage of DNA hybridized was corrected by dividing the measured percentage of Sl resistance by the fraction of the probe hybridizable to cRNA.
Lindberg, 65, 1089.
U., and Darnell,
J. F. (1970).
U.. and Persson,
Sucrose Gradtent Centrifugation RNA was sedimented through 5-20% sucrose gradients containing 50% formamide prepared as described by Lewis et al. (1975). The details of the particular experiments are given in the figure legends,
Enzyme Purification E. coli RNA polymerase was the gift of W. Mangel. Restriction endonuclease Hpall was prepared as described by Sharp et al. (1973); EcoRl by the unpublished procedure of W. Sugden and J. Sambrook (personal communication), and Hhal by the unpublished procedure of R. Roberts and P. Myers (personal communication). Endonuclease Sl was extracted from a-amylase powder (Sigma) by a modification of the methods of Vogt (1973) and Sutton (1971).
Lindstrom, D. M., and Dulbecco, USA 69, 1517. May, E., Kopecka, 2, 1995.
Mertz. J. E., and 71. 4879.
C. L., Aviv, Cold Spring
We thank Clark Tibbetts, Lionel Crawford, and Brad Ozanne for their help in the preparation of this manuscript, and are grateful to Cilla Conway for her editorial assistance. We also thank Drs. E. May, H. Kopecka, and P. May, as well as Dr. G. Khoury, for providing us with their data prior to publication. 8, 1975
M., and Reuveni, J. Virol.
Griffin, B., Fried, USA 71, 2077. Hewick, 66, 408. Hirt,
M., and Cowie,
P. M. (1974).
M., and Waterfield,
M. D. (1975).
J. Mol. Biol. 26, 365.
D. S., and Bonner,
J., and Ziff,
Kamen, R., Lindstrom, Spring Harbor Symp.
M. A. (1972).
Khoury, G., Martin, M. A., Lee, T. N. H., Danna. D. (1973). J. Mol. Biol. 78, 377. G., Howley, 15, 433.
Leong, J. A., Garapin. A. C., Jackson, W., and Bishop, J. M. (1972). J. Virol. Lewis, J. B., Atkins, land, R. F. (1975).
12, 2330. in press.
D. M., Shure, H., and Old, R. (1974). Quant. Biol. 39, 187.
Khoury, G.. Byrne, J. C., and Martin, Sci. USA 69, 1925.
Khoury, J. Virol.
Nat. Acad. Nucl.
K. J., and Nathans,
N. Fanshier. 9, 891.
M. A. (1975). L.. Levinson,
J. F., Anderson, C. W., Baum. P. R., and GesteProc. Nat. Acad. Sci. USA 72, 1344.
H., Gilboa, E., Revel, M., and Winocour, Harbor Symp. Quant. Biol. 39, 309.
R. B., and Hare, 56, 839.
Sambrook, 72, 57.
Sharp, P. A., Sugden, 12, 3055.
J. D. (1974).
G. (1974). In Comprehensive Virology, R. Wagner, eds. (New York: Plenum
P. A., and
B., and Sambrook,
P. A. (1974).
Smith, A. E., Bayley, S. T., Mangel. W. F., Shure, H., Wheeler, T., and Kamen, R. I. (1975). Proceedings Tenth FEBS Symposium (Amsterdam: North-Holland), p. 151. W. D. (1971).
V. M. (1973).
Eur. J. Biochem.
R. A., Warnaar,
Weil, R., Salomon, C., May, E., and May, Harbor Symp. Quant. Biol. 39, 381-395. Weinberg, 10. 193.
R. W., Fried,
A. K., and Nicklin,
Fey, G., and Hirt, 8. (1974). 39, 235. B. E., and Fried,
Crawford, L. V., Robbins, Virol. 25, 133.
Sambrook, J. F. (1975). In Control in Virus Multiplication, D. C. Burke and W. C. Russell, eds. (London and New York: Cambridge University Press), p. 153.
Tonegawa, S., Walter, G., Bernardini, A., and Dulbecco, Cold Spring Harbor Symp. Quant. Biol. 35, 823.
Salzman, N. P., and Khoury, 3, H. Fraenkel-Conrat and Press), p. 63.
Aloni, Y., Shani, USA, in press.
Sambrook, J., Sugden, B., Keller, W., and Proc. Nat. Acad. Sci. USA 70, 3711.
S. O., and Winocour,
Spring J. Viral.
Weinberg, R. A., Ben-lshai, New Biol. 238, 111.
Z., and Newbold,
J. E. (1972b).
Weinberg, 13, 1263.
Z., and Newbold,
J. E. (1974).
R. A., Ben-lshai,