Proc. Nati. Acad. Sci. USA Vol. 75, No. 6, pp. 2964-2968, June 1978

Microbiology

Large T1 oligonucleotides of Moloney leukemia virus missing in an env gene recombinant, HIX, are present on an intracellular 21S Moloney viral RNA species (spliced gp7O mRNA)

DOUGLAS V. FALLER, JEAN ROMMELAERE, AND NANCY HOPKINS Biology Department and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts

Communicated by Wallace P. Rowe, March 10, 1978

HIX, a recombinant derived from Moloney ABSTRACT leukemia virus, has an envelope glycoprotein different from that of the Moloney virus. HIX and Moloney viruses share the majority of the large Ti oligonucleotides derived from their genomes but each possesses a set of distinctive oligonucleotides that lie clustered in corresponding regions in the 3' halves of their oligonucleotide maps. These regions presumably contain envelope glycoprotein coding sequences. The type C viral envelope glycoprotein is believed to be translated from a 21S RNA. Thus, at least part of the region of the Moloney virus genome that is altered relative to HIX was expected to be present on such a species. To test this prediction, we purified an intracellular 21S Moloney viral RNA species and analyzed its large Ti oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. This RNA contains one Ti oligonucleotide that is probably derived from the 5' end of the Moloney virus genome, the Molone virus Ti oligonucleotides that are missing in HIX, and those gat lie to their 3' side on the Moloney virus Ti oligonucleotide map.

An unusual class of murine leukemia viruses (MuLVs) appears to arise by recombination between ecotropic and as yet unidentified xenotropic viruses. The viruses are designated dual tropic viruses or env gene recombinants because their envelope glycoproteins (gp7Os) appear to be specified partially by their ecotropic parent and partially by their putative xenotropic parent (1, 2, 3). All the other virion proteins of the dual tropic viruses that have been resolved on sodium dodecyl sulfate/ polyacrylamide gels are indistinguishable from those of their ecotropic parent by tryptic peptide mapping (ref. 3; P. Fis-

chinger, personal communication). The genetic organization of the MuLV genome is probably similar to that of avian leukosis viruses. Thus, three major genes, gag, pol, and env, lying from 5' to 3' on the genome, give rise, after cleavage of polyproteins, to all the known virion proteins (4). By using T1 oligonucleotide mapping, we analyzed the genomes of four dual tropic viruses designated MCF and their putative ecotropic parent, Akv (2, 5, 6), and also those of a dual tropic virus designated HIX and its ecotropic parent, Moloney MuLV (M-MuLV) (ref. 1; D. V. Faller and N. Hopkins, unpublished data). The dual tropic viruses share the 5' half and a portion of the extreme 3' end of their oligonucleotide maps with Akv or M-MuLV. However, each isolate has lost a cluster of ecotropic-specific oligonucleotides in the 3' half of its map and replaced the missing oligonucleotides with a cluster of MCF- or HIX-specific sequences. In conjunction with the other known properties of dual tropic viruses, it seems probable that these sequences contain information specifying gp7O. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

The major envelope glycoprotein of avian and murine type C viruses appears to be translated from an RNA species approximately one-third of the genome length (7, 8). Recent evidence has suggested that the glycoprotein mRNA is a spliced (9-12) 21S RNA in which sequences from the 5' terminus of the viral genome are contiguous with sequences comprising the 3' one-third of the genome (13-15). Together these observations suggested that regions of the Akv or M-MuLV genomes that are altered in MCF or HIX viruses will be present on an intracellular 21S spliced viral RNA species. To test this prediction, we isolated a 21S intracellular M-MuLV RNA, the putative gp7O mRNA, from M-MuLV infected cells and analyzed its large Tl-resistant oligonucleotides by two dimensional polyacrylamide gel electrophoresis. DISCUSSION We have presented evidence that an intracellular, poly(A)containing, 21S M-MuLV RNA species contains one T1 oligonucleotide probably derived from the 5' end of the M-MuLV genome, the T1 oligonucleotides that are missing in HIX, and those to their 3' side (Fig. 1). Because it has been reported that the envelope glycoprotein mRNA of type C viruses is a 21S species (7, 8), because HIX and M-MuLV possess different gp7Os (P. Fischinger, personal communication), and because the T1 oligonucleotides of M-MuLV that are missing in HIX are present on the 21S species we analyzed, it is not unlikely that this species is the M-MuLV gp7O mRNA. We did not determine the relative order of the M-MuLV oligonucleotides present on the 21S species. However, the 3' portions of full length M-MuLV cDNA and of an intracellular 21S M-MuLV RNA similar to the species analyzed here form an apparently uninterrupted heteroduplex by electron microscopy (15). Thus, it is simplest to imagine that the M-MuLV oligonucleotides present on the 21S RNA that we studied retain their relative genomic order. Proximity of a junction of M-MuLV- and HIX-Specific Sequences to a Region of the Genome Presumably Involved in RNA Splicing. The M-MuLV Ti oligonucleotide at position 21 in Fig. 1 appears to define two regions of interest: (i) a junction of M-MuLV- and HIX-specific T1 oligonucleotides in HIX genomic RNA and (ii) the junction of Ti oligonucleotides presumably located in the leader and body sequences of a M-MuLV 21S intracellular RNA. Thus, the 5'-end point of the region of nonidentity between the M-MuLV and HIX genomes appears to lie near, and might coincide with, a point at which splicing generates the 21S RNA. Ti oligonucleotide Abbreviations: M-MuLV, Moloney murine leukemia virus; HIX, dual tropic virus whose ecotropic parent is M-MuLV; MCF, dual tropic virus whose ecotropic parent is Akv.

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Microbiology: Faller et al. mapping does not allow us to determine whether or not points (i) and (ii) coincide for the following reasons. If the M-MuLV T1 oligonucleotides that were ordered (Fig. 1) were evenly distributed along the genome, then they would be separated by approximately 225 nucleotides, and this would be the largest distance that could exist between points (i) and (ii) defined above. These points could lie more than 225 bases apart because deviations from an even distribution of the mapped oligonucleotides occur. For example, the 5' halves of the oligonucleotide maps in Fig. 1 appear to be significantly underrepresented by T1 oligonucleotides, and thus the average distance between adjacent oligonucleotides in this region would be greater than 225 nucleotides. Determining precisely how near the 5'-end junction of MMuLV- and HIX-specific sequences is to the splice point in a viral 21S RNA species will require analysis of this region of the appropriate RNAs by additional methods, optimally by nucleotide sequencing. The answer to this question seems to be of interest for the following reasons: (i) ultimately, in conjunction with sequence analysis of gp70s, it may help in determining where gp7O coding sequences begin on the genome; (ii) it is possible that the junction of M-MuLV- and HIX-specific sequences corresponds to a site at which recombination generated HIX virus (see below). In this case, a coincidence of this position with a splice point might raise the interesting speculation that dual tropic viruses could conceivably be generated by mechanisms similar to those that generate spliced RNAs-in other words, by RNA recombination. Do the Junctions of M-MuLV- and HIX-Specific Ti Oligonucleotides Define Regions in which Recombination Events Generated HIX? To know if a putative recombination event that generated HIX occurred near position 21 in Fig. 1 we would have to know whether or not the putative xenotropic parent of HIX shares T1 oligonucleotides with M-MuLV to the 5' side of this position. (If this were the case, then recombination events occurring to the 5' side of position 21 would not be detected.) Of course, we will not be able to answer this question until the putative xenotropic parent of HIX has been identified and its RNase Tl-resistant oligonucleotides have been analyzed. However, the following considerations suggest that a recombination event generating HIX may have occurred near position 21 in Fig. 1. (i) HIX was isolated from stocks of M-MuLV-IC (1) and the 5' half of its oligonucleotide map is indistinguishable from that of M-MuLV-IC but distinct from that of another clonal isolate of M-MuLV, M-MuLV-Cl-i (see Fig. 1A). (ii) The Ti oligonucleotides in the 5' half of the M-MuLV oligonucleotide map are distinct from those of endogenous ecotropic viruses of inbred mice that we have analyzed (6, 18). To explain our failure to detect recombination events in the 5' half of the HIX genome relative to M-MuLV we would have to postulate that there exists a xenotropic virus that shares Ti oligonucleotides in the 5' half of its genome with a particular clonal isolate of M-MuLV, and this seems somewhat unlikely. Therefore, it seems quite possible that a recombination event that generated this particular HIX isolate occurred near position 21 in Fig. 1.

MATERIALS AND METHODS Cells and Viruses. NIH/3T3 cells chronically infected with clone 1 M-MuLV (M-MuLV-Cl-l) (16) and grown in roller bottles were used to prepare virion or intracellular viral RNA. RNase Ti Fingerprints and Analysis of Oligonucleotides

by Pancreatic RNase Digestion. These methods have been described in detail (17).

Proc. Nati. Acad. Sci. USA 75 (1978)

2965

Synthesis of Viral cDNAs. cDNA representative of the entire M-MuLV genome was prepared in the presence of actinomycin D essentially as described (18) using calf thymus DNA primers (19). Hybridization of the resulting cDNA to M-MuLV 70S virion RNA at a cDNA/RNA ratio of 5:1 rendered the RNA 90% resistant to digestion by RNase A plus RNase T1. A cDNA probe specific for the 5' end of the M-MuLV genome was synthesized as described (15). One preparation of this cDNA was a generous gift from E. Rothenberg. Purification of 32P-Labeled (and Unlabeled) Intracellular M-MuLV RNAs. Ten roller bottles of confluent M-MuLV-Cl-1infected NIH/3T3 cells were incubated for 10-12 hr with 20 mCi of [32P]phosphate (100 ,uCi/ml of medium) as described (17). The cells were washed twice with cold phosphate-buffered saline and the total cellular RNA was extracted by a modification (20) of the guanidine extraction method (21). Poly(A)containing RNA was selected by affinity chromatography on an oligo(dT)-cellulose (Collaborative Research, T3) column (22). For some experiments, this RNA was fractionated by sedimentation on 15-30% sucrose gradients (4 hr, 40,000 rpm, 200 in a Beckman SW-41 rotor). Poly(A)-containing RNA was dissolved in buffer containing 80% (vol/vol) deionized formamide/0.4 M NaCl/60 mM 1,4-piperazinediethanesulfonic acid (Pipes) (Calbiochem), pH 6.4/1 mM EDTA at a concentration of 10 ,ug of RNA per ml. M-MuLV cDNA was then added to achieve a cDNA/RNA ratio of 1:4 (wt/wt). Hybridization was carried out for 5 hr at 450. The sample was then adjusted to 10 mM sodium pyrosulfite and 100 mM Tris (pH 8), the volume was brought to 2 ml with water, and 1.3 g of KI was added. The sample was layered on 5 ml of a solution of KI in 15 mM sodium citrate/10 mM sodium pyrosulfite, refractive index 1.4330, and centrifuged for 17 hr at 50,000 rpm using a type 65 rotor (Beckman). Fractions (0.3 ml) were analyzed for radioactivity by Cerenkov counting. 32P-Labeled cellular poly(A)-containing RNA and 70S virion M-MuLV RNA-cDNA hybrids that were run in parallel gradients marked the density positions of RNA or RNA-DNA hybrids. Cellular RNA banding at the hybrid position was 70% resistant to RNases A plus T1 digestion; the material at the position of single-strand RNA was less than 1% resistant. Electrophoresis of RNA on Denaturing Agarose Gels. This was performed essentially as described (23). Hybridization across Gels with a 5'-End-Specific M-MuLV cDNA Probe. Gel slices 2 mm thick were each dissolved at 100° in 75 pJ containing 5'-end-specific [32P]cDNA probe (15), 400 jig of calf thymus DNA (Sigma) per ml, and 2 M sodium perchlorate (13); these solutions were incubated at 68° under mineral oil. They were then digested by addition of 0.4 ml of prewarmed (500) buffer containing nuclease S1 at 2000 units/ml (24). After incubation at 500 for 30 min the reaction was stopped, the mixtures were filtered, the filters were washed with ethanol (1:1), and the radioactivity was quantitated.

RESULTS

Background The particular clonal isolate of M-MuLV from which HIX was derived is designated Moloney IC (M-MuLV-IC) (1). RNase T1 oligonucleotide maps of the genomes of HIX and M-MuLV-IC are shown in Fig. 1. Because M-MuLV and HIX possess different gp70s but have other virion proteins that are indistinguishable, the maps in Fig. 1 suggest that a region of the MMuLV genome corresponding to oligonucleotide map positions 21-30 contains gp7O coding sequences. The oligonucleotide map of another clonal isolate of M-MuLV, designated M-

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Proc. Natl. Acad. Sci. USA 75 (1978)

Faller et at.

U 213145 617 122122232425262728293312333435337389404142 W POSM 5D 1 2 3 4 5 6 7 8 93 IC

A

IC IC

IC

IC

4)36 30 20224 KEUCY IC0(42l 2D40( 4 15)1 255L6(1299 34(48 4729241427D77662( 2 .18 23141177D22 2633 39 19 135 IC IC IC IC IC HIX 5)(42 2140)(4 15) 25(46 12) 9 45( 5 47iXl 34 48) + 4127 H18 Hi HR H3 ( Hi 22)2633 38 3 19 B 315 3 20 + 50 MUU CL-1 50 21 40 42 4 16 25 fl223338 39193532024 9 315 34 47294127 7 632 2 3182314 F L i JmEY IC _HIX

C 21S

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FIG. 1. RNase T1 oligonucleotide maps of M-MuLV-IC, M-MuLV-Cl-1, and HIX, and T1 oligonucleotides present in M-MuLV-Cl-1 21S RNA. (A) Relative order from 5' to 3' of large RNase Ti-resistant oligonucleotides derived from the genomes of M-MuLV and HIX (D. V. Faller and N. Hopkins, unpublished data). "IC" indicates T1 oligonucleotides of M-MuLV-IC not found in M-MuLV-Cl-1, "H" indicates oligonucleotides found in HIX but not in either M-MuLV isolate. Oligonucleotides within parentheses could not be unambiguously ordered with respect to one another. +, Corresponding oligonucleotide was not "mapped" but is present in the virus. (B) Schematic comparison of oligonucleotide maps of M-MuLV-IC and HIX. Triangles represent oligonucleotides found in one virus but not in the other. Solid line indicates oligonucleotides shared by HIX and M-MuLV-IC. (C) Triangles indicate oligonucleotides of M-MuLV-Cl-i that are contained in the 21S intracellular RNA. Oligonucleotide maps are only approximate physical maps and consideration of the data in this report indicates that the 5' half ofthese maps is underrepresented by T1 oligonucleotides and that position 21 lies approximately within the 3' third of the genome.

MuLV-Cl-i is also shown in Fig. 1A (16). To determine if the region of the M-MuLV genome that differs from HIX is present on an intracellular 21S RNA, we analyzed this species from M-MuLV-Cl-i-infected cells rather than from M-MuLV-ICinfected cells, because the former produce larger amounts of virus and thus might be expected to contain higher levels of intracellular viral RNA. M-MuLV-IC and M-MuLV-Cl-I differ by the presence of eight T1 oligonucleotides. Five of these could be mapped and they lie to the 5' side of the region that differs between M-MuLV and HIX viruses (positions 21-0 in Fig. 1). It should be noted that oligonucleotide 50 is present in twice molar amount relative to the other (unique) large T1 oligonucleotides derived from the M-MuLV and HIX genomes. Coffin et al. (25) have shown that this oligonucleotide resides in the redundant sequences located at the 5' and 3' ends of the genome. Isolation and Ti oligonucleotide analysis of a 21S viral RNA species from M-MuLV-infected cells To partially purify 32P-labeled 21S intracellular M-MuLV RNA, poly(A)-containing intracellular RNA was first selected from 32P-labeled total cellular RNA of M-MuLV-Cl-l-infected cells by oligo(dT)-affinity chromatography. To reduce potential contamination of the 21S RNA species with degradation products of intracellular 35S RNA, the majority of the poly(A)-selected RNA was subjected to sucrose gradient sedimentation and the material sedimenting in the 1i-28S size range was pooled. Both unselected and size-selected poly(A)containing cellular RNAs were then hybridized to an excess of viral cDNA and the hybridized material [1% or less of the total poly(A)-containing RNA] was separated by density banding sedimentation on KI equilibrium gradients. To ascertain if this procedure resulted in the selection of discrete intracellular viral RNA species, the various selected and unselected preparations of 32P-labeled cellular RNA were analyzed by electrophoresis on a 1% agarose gel containing 5 mM methylmercuric hydroxide. Fig. 2 is an autoradiogram of such a gel. It is apparent that hybridization to viral cDNA and subsequent purification of the hybridized material by density banding results in the selection of two discrete RNA species: one comigrates with 32P-labeled virion 35S RNA and presumably represents intracellular, or cell-associated, full length viral RNA (lanes 3 and 4); the other has an apparent molecular weight of 1.2 X 106 and

thus tentatively corresponds to the previously described gp7O mRNA (lane 4). To show that the 1.2 X 10i' molecular weight band in lanes 3 and 4 of Fig. 2 possessed sequences from the 5' end of the genome, we used a 5S-nucleotide-long cDNA probe specific for the 5' end of M-MuLV-Cl-i genomic RNA (15). Poly(A)containing RNA from unlabeled M-MuLV-Cl-l-infected cells was electrophoresed on a gel (Fig. 2 left), and hybridization across the gel using the 5'-end-specific probe showed two peaks of 5' sequences, comigrating with the 35S and the 1.2 X 106 viral RNAs (Fig. 2 right). The 1.2 X 106 band from two lanes of a gel loaded with most of the material from lane 4 of Fig. 2 left was eluted and digested Origin-

35S-

28S-

18S- _ 20

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2 3 4

40

60

80

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FIG. 2. Electrophoresis of M-MuLV-specific intracellular RNA on denaturing agarose gels. (Left) Autoradiogram of a 1% agarose gel containing 5 mM methylmercuric hydroxide after electrophoresis of: lane 1, 32P-labeled, non-oligo(dT)-cellulose-bound RNA from MMuLV-infected cells with 32P-labeled virion RNA added as a 35S marker; lane 2,,poly(A)-containing RNA from M-MuLV-infected cells; lane 3, poly(A)-containing RNA that banded at the position of RNA-DNA hybrids on KI gradients after hybridization to M-MuLV cDNA; lane 4, size-selected (1i-28S) poly(A)-containing RNA after hybridization to viral cDNA and selection of hybridized RNA on KI gradients. (Right) Localization in the denaturing gel shown in Left of RNA species containing the 5' end of the M-MuLV genome. Twenty micrograms of unlabeled, poly(A)-containing RNA from M-MuLV-Cl-1-infected cells [obtained in parallel with the 32P-labeled poly(A) RNA shown in Left, lane 21 was run on the gel shown in Left. The presence of species containing the 5' end of the M-MuLV genome was determined by hybridization to a 32P-labeled 5'-end-specific M-MuLV cDNA fragment.

Microbiology: Faller et al.

Proc. Nati. Acad. Sci. USA 75 (1978)

2967

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FIG. 3. Autoradiograms (fingerprints) of RNase Ti-resistant products from 32P-labeled M-MuLV-Cl-1 70S virion RNA and M-MuLV-Cl-1 35S and 21S intracellular RNAs. (Upper Left) M-MuLV 70S virion RNA. (Upper Middle and Right) 32P-Labeled intracellular RNA obtained after hybridization of a 29-38S pool (Middle) and a 17-23S pool (Right) of poly(A)-containing M-MuLV-Cl-1-infected cellular RNA to M-MuLV cDNA, selection of RNA banding at RNA-DNA hybrid position on KI gradients, and RNase T1 digestion of the denatured hybrids. (Lower Left and Right) Diagrams of autoradiograms. Numbered open circles represent unique oligonucleotides present in equimolar amount (D. V. Faller and N. Hopkins, unpublished-data). Spot 50 represents two identical oligonucleotides (see text; ref. 25) and thus is present in twice the molar amount relative to other numbered oligonucleotides. Crosshatched areas without numbers represent multiple oligonucleotides with similar or identical electrophoretic mobilities. Arrows indicate directions of migration in first and second dimensions of the gel electrophoresis. "XC" and "B" indicate positions of dye markers xylene cyanol FF and bromphenol blue, respectively.

with RNase Ti, and the digestion products were separated by two-dimensional gel electrophoresis. The resulting autoradiogram was similar to that shown in Fig. 3 upper right which was obtained as described below. This species appeared to possess some but not all of the M-MuLV genomic T1 oligonucleotides. The M-MuLV oligonucleotides present in this RNA (see below) are indicated in Fig. 1C; they include one T1 oligonucleotide derived from the 5' end of the viral genome, the oligonucleotides present in M-MuLV but not HIX (positions 21-S0), and those to the 3' side of this region (positions 31-42). To confirm which M-MuLV oligonucleotides are present in the 21S intracellular RNA species, it was necessary to obtain sufficient quantities of the 21S viral RNA to allow analysis of the secondary digestion products of its large Ti oligonucleotides and quantitation of their relative molarities. Since the procedure employing gels incurred considerable losses, we prepared intracellular viral 21S material for further analysis by sucrose gradient fractionation. Poly(A)-containing cellular RNA of 29-38 S and 17-23 S was hybridized to viral cDNA, the hybrids were selected on KI equilibrium gradients and denatured, and the RNA was fingerprinted (Fig. 3 upper middle and right). The fingerprint in Fig. 3 upper middle looks like that of MMuLV-Cl-I 70S RNA (Fig. 3 upper left) except for a faint background of oligonucleotides, presumably from contaminating cellular RNAs. The fingerprint in Fig. 3 upper right possesses some but not all of the M-MuLV Ti oligonucleotides as well as a higher background of contamination. The putative M-MuLV Ti oligonucleotides in these fingerprints, and any RNA migrating in the regions of the fingerprint in Fig. 3 upper right that should have contained the missing M-MuLV oligonucleotides, were removed, the amount of radioactivity in each was quantitated, and -their secondary digestion products were compared with the corresponding oligonucleotides derived from M-MuLV-CI-i 70S virion RNA. The results of this analysis

indicate that the 35S species contained all the T1 oligonucleotides present in 70S virion RNA in equal molar amount (±15%), whereas the 21S species contains only those M-MuLV Ti oligonucleotides indicated in Figs. 3 lower right and IC. Quantitation of the radioactivity present in spot 50 of the 21S intracellular species suggests that it is present in approximately twice the molar amount (2.1 + 0.3 and 1.9 ± 0.3 in two experiments) relative to the other oligonucleotides. Because this spot resides in the redundant sequences located at the 5' and 3' termini of the M-MuLV genome and because the 21S RNA we analyzed hybridizes to a cDNA fragment homologous to the 5' end of the M-MuLV genome, we assume that both the 5'- and 3'-end copies of oligonucleotide 50 are present in the 21S species.-No other Ti oligonucleotides derived from the 5' end of the M-MuLV oligonucleotide map appear to be present in this RNA. To obtain further evidence that Fig. 3 upper right is not merely a fingerprint of degraded RNA from the intracellular 35S RNA, 32P-labeled virion RNA was fragmented, the poly(A)-containing RNA was selected, and a size class (17-23 S) like that used to fingerprint the 21S material was processed in parallel with the intracellular species. The T1 fingerprint of the resulting material-showed a gradient of relative molar yields of M-MuLV T1 oligonucleotides, and oligonucleotide 50 was present in only equimolar amounts relative to other oligonucleotides mapping near the 3' terminus. It should also be noted that a Ti fingerprint of intracellular 24-28S poly(A)-containing RNA from the gradients employed above revealed the presence of a smaller amount of M-MuLV RNA than was found in the 17-23S fraction, and analysis of this RNA showed a gradient of molar yield of M-MuLV Tl-resistant oligonucleotides as expected for degraded 35S RNA of this size class. These studies would seem to provide strong motivation both for identifying the missing putative xenotropic parent of dual

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tropic viruses and for further investigating the physical relationship between the region of dual tropic virus genomes that is altered relative to their ecotropic parents and a region of the viral genome involved in generating a spliced 21S putative gp70

mRNA. We thank Phillip Sharp for many stimulating discussions. This work was supported by National Cancer Institute Grant CA-19308 to N.H. and National Institutes of Health Grant CA-14051 to S. E. Luria. J.R. is a Charge de Recherches du Fonds National de la Recherche Scientifique de Belgique and Fellow of the Fondation Rose et Jean Hoguet. 1. Fischinger, P. J., Nomura, S. & Bolognesi, D. P. (1975) Proc. Natl. Acad. Sci. USA 72,5150-5155. 2. Hartley, J. W., Wolford, N. K., Old, L. J. & Rowe, W. P. (1977) Proc. Nati. Acad. Sci. USA 74,789-792. 3. Elder, J., Jensen, F., Lerner, R., Hartley, J. W. & Rowe, W. P. (1977) Proc. Natl. Acad. Sci. USA 74,4676-4680. 4. Baltimore, D. (1974) Cold Spring Harbor Symp. Quant. Biol. 34, 1187-1200. 5. Rommelaere, J., Faller, D. V. & Hopkins, N. (1978) Proc. Natl. Acad. Sci. USA 75,495-499. 6. Rommelaere, J., Faller, D. V. & Hopkins, N. (1977) J. Virol. 24, 690-694. 7. Stacey, D. W., Allfrey, V. G. & Hanafusa, H. (1977) Proc. Natl. Acad. Sc. USA 74, 1614-1618. 8. Gielkens, A. L. J., Van Zaane, D., Bloemers, H. P. J. & Bloemendal, H. (1976) Proc. Natl. Acad. Sci. USA 73,356-360.

Proc. Nati. Acad. Sci. USA 75 (1978) 9. Berget, S. M., Moore, C. & Sharp, P. A. (1977) Proc. Natl. Acad. Sci. USA 74,3171-3175. 10. Chow, L. T., Gelinas, R. E., Broker, T. R. & Roberts, R. J. (1977) Cell 12, 1-8. 11. Klessig, D. F. (1977) Cell 12,9-21. 12. Gelinas, R. E. & Roberts, R. J. (1977) Cell 11, 533-544. 13. Weiss, S. R., Varmus, H. E. & Bis'hop, J. M. (1977) Cell 12, 983-992. 14. Mellon, P. & Duesberg, P. H. (1977) Nature 270,631-634. 15. Rothenberg, E., Donoghue, D. J. & Baltimore, D. (1978) Cell 13, 435-451. 16. Fan, H. & Paskind, M. (1974) J. Virol. 14,421-429. 17. Faller, D. V. & Hopkins, N. (1977) J. Virol. 23, 188-195. 18. Rothenberg, E. & Baltimore, D. (1977) J. Virol. 21,168-178. 19. Taylor, J. M., Illmansee, R. & Summers, J. (1976) Btochtm. Btophys. Acta 442,324-0. 20. Adams, S. L., Sobel, M. E., Howard, B. H., Olden, K., Yamada, K. M., de Crombrugghe, B. & Pastan, I. (1977) Proc. Natl. Acad. Sci. USA 74,3399-3403. 21. Cox, R. A. (1967) in Methods in Enzymology, eds. Grossman, L. & Moldave, K. (Academic Press, New York), Vol. 12B, pp. 120-129. 22. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, 1408-1412. 23. Bailey, J. M. & Davidson, N. (1976) Anal. Biochem. 70, 7585. 24. Vogt, V. M. (1973) Eur. J. Biochem. 33,192-200. 25. Coffin, J. M., Hageman, T., Maxam, A. & Haseltine, W. A. (1978) Cell, in press.