The integrated genome of murine leukemia virus

Cell, Vol. 15, 1003-1010,

November

The Integrated

1978, Copyright

8 1978 by MIT

Genome

David Steffen and Robert A. Weinberg Department of Biology and Center for Cancer Research Massachusetts Institute of Technology Cambridge, Massachusetts 02139

Summary The Southern gel filter transfer technique has been used to characterize the integrated genome of Moloney murine leukemia virus (M-MuLV) and the genomes of the endogenous viruses of the mouse. Study of 10 clones of rat cell independently infected by M-MuLV indicates a minimum of 15 integration sites into which the M-MuLV provirus can be inserted. No common integration site is observed among these clones. Clones productively infected by M-MuLV acquire multiple proviruses, whereas infected cells unable to produce virus contain only one M-MuLV provirus. Once established, the integrated genomes are stable for at least two years after initial infection. The use of M-MuLV probe allows detection of a spectrum of Eco RI-cleaved mouse DNA fragments containing endogenous MuLV genomes. DNAs of different inbred laboratory mouse strains yield similar patterns of provirus with each strain showing minor characteristic differences. In some instances, mouse cells infected by M-MuLV reveal additional proviruses beyond those seen in the uninfected cell. DNAs from three different M-MuLV-induced thymomas indicate, as in rat cells, multiple possible integration sites. Introduction Cells which have been recently infected by retroviruses yield a series of viral DNAs that represent the products of the initial reverse transcription of the virion RNA (Gianni, Smotkin and Weinberg, 1975; Guntaka et al., 1976; Weinberg, 1977; Ringold et al., 1977). A linear double-stranded viral DNA is found in the cytoplasm while closed circular viral DNA can be detected in the nucleus. Kinetic evidence suggests that the linear cytoplasmic DNA is precursor to the closed circular, nuclear viral DNA, and that this closed circular DNA is the immediate precursor of the integrated viral genome, sometimes termed the provirus (Shank and Varmus, 1978). The present investigation was undertaken to characterize the integrated form of murine leukemia virus DNA, with emphasis placed on the genome of Moloney MuLV. The study of integrated viral genomes has been greatly facilitated by the development of the Southern gel filter transfer technique (Southern, 1975).

of Murine

Leukemia

Virus

This technique allows the detection of several picograms of discretely sized DNA which has been resolved by agarose gel electrophoresis and then adsorbed to a cellulose nitrate filter. Incubation of the filter with a high specific activity viral RNA or DNA probe and subsequent autoradiography permit detection of these small amounts of viral DNA among a million fold excess of cellular DNA fragments. This technology was initially exploited by Botchan, Topp and Sambrook (1976) and Ketner and Kelly (1976) to characterize the integrated genome of the papovavirus SV40. Examination of the DNA fragments produced by site-specific endonucleases from SV40-infected cell DNA showed that the size of fragments generated at the junction between viral and cellular DNA is not constant in independently derived clones of SV40-infected cells. Thus SV40 integrates its DNA at many sites in the host cell genome. We report here the use of a similar experimental design to investigate integrated proviruses of murine retroviruses. We investigated integrated Moloney MuLV (M-MuLV) proviruses in rat cells, which lack genetically acquired murine-like proviruses, to study in detail the site in the host genome at which M-MuLV integrates its provirus. We also examined genetically acquired murine proviruses as well as proviruses introduced by infection in mouse cells.

Results Moloney MuLV (M-MuLV) is a potent leukemogenic virus whose ease of growth and passage made it a useful agent for studies reported here. The experiments described below were designed to determine whether M-MuLV integrates its DNA into a small number of sites in the host cell genome. Unfortunately, the genomes of M-MuLV and of a variety of other related MuLVs show extensive sequence homology with the DNA of uninfected mouse cells. This homology derives from the presence of a large number of endogenous MuLV proviruses present in the DNAs of all mice. Although these endogenous proviruses are interesting in themselves, their presence in mouse DNA complicates any attempts at analysis of M-MuLV integration into the mouse genome. We considered it improbable that we would always be able to detect clearly an M-MuLV provirus among the large number of endogenous proviruses found in the mouse cell DNA. Thus we began by introducing M-MuLV into rat cells. Rat DNA contains no endogenous proviruses having sequence homology with M-MuLV. Rat cells, however, have sufficient affinities with mouse cells that their infection by the ecotropic M-MuLV took place readily. Such infected cells yield high

Cell 1004

titers of M-MuLV and thus represent reasonable models of M-MuLV infected mouse cells. We were also able to induce rapid leukemias in rats with MMuLV, further strengthening the biological relevance of the M-MuLV/rat model. Thus rat cells represent a permissive and quasi-normal host for M-MuLV while containing no complicating endogenous mouse proviruses. Previous work with the unintegrated forms of MMuLV indicated that the viral genome is not cleaved by Eco RI endonuclease (Gianni et al., 1976). Treatment of cellular DNA containing an integrated M-MuLV genome with this enzyme would yield -lo6 different DNA fragments, one of which would contain the entire provirus. Since Eco RI cuts cellular but not viral sequences, this fragment will contain cellular sequences at both ends and the viral genome in the middle. It is significant that the size of this fragment is dependent upon the distances between the ends of the viral genome and the locations of the nearest Eco RI sites in the cellular DNA. Proviruses integrated in different chromosomal sites will in most cases be found in differently sized DNA fragments, each fragment reflecting the distances between the integration site and the two nearest Eco RI endonuclease cleavage sites. To compare the site of provirus integration in different clones of M-MuLV infected normal rat kidney (NRK) cells, we fragmented the cellular DNA with Eco RI and fractionated the fragments by size using electrophoresis in 1% agarose gels. Those fragments containing viral sequences were detected by transferring the DNA from the gel to a nitrocellulose filter and hybridizing the DNA on the filter to radioactive M-MuLV RNA. The hybridized viral DNA was detected by autoradiography. The results of this analysis are shown in Figure 1. NRK, the uninfected rat cell line, displays only one faint band of hybridization (Figure 1, lane A). This band, detected among DNA fragments having molecular weights of approximately 4 x lo6 daltons, is consistently seen in all rat DNAs. Furthermore, its location on the filter is the same as that of a prominent band seen after hybridization with radioactive ribosomal 28s RNA. We therefore conclude that detection of this band of hybridization is a consequence of a low level of ribosomal RNA which contaminates our viral RNA probe despite our extensive efforts to assure its purity (see Experimental Procedures). NRK cells A mass population of M-MuLV-infected (lane B) displays a continuum of hybridization, ranging from DNA fragments of >15 x lo6 to about 6 x lo6 daltons molecular weight. Since the unintegrated provirus has a molecular weight of 5.7 x lo6 daltons (F. K. Yoshimura, personal communication) almost all the DNA detected probably rep-

Figure

1. M-MuLV

Proviruses

Integrated

into the DNA of

Rat Cells

DNA extracted from the cell line indicated below each lane was cleaved with Eco RI, fractionated by electrophoresis for 26 hr at 1 V per cm in 1% agarose, transferred to a nitrocellulose sheet and hybridized to 32P-M-M~LV RNA as described in Experimental Procedures. The hybrids were detected by autoradiography. All lanes are from the same gel; different lanes were exposed for different times to best resolve bands. DNA run in lane B. labeled uncloned, was extracted from NRK cells infected with M-MuLV at high multiplicity and grown as a mass culture for several weeks. The arrows to the left of the figure indicate the positions of fragments of the genome of bacteriophage lambda generated by cleavage with the endonuclease Hind Ill (Murray and Murray, 1975) run in an adjacent channel. The sizes of these fragments, indicated in units of daltons mass. were used as standards.

resents integrated M-MuLV DNA covalently linked to cellular sequences of different sizes. The heterogeneity of these sizes indicates a very large number of possible adjacent cellular sequences and therefore a large number of possible M-MuLV integration sites. This analysis is more directly pursued by studying the DNAs of individual subclones of such a mass-infected population (lanes C-L). We derived single-cell clones (NRK-1, NRK-3, NRK-4 and NRK5) which produce high titers of M-MuLV. Additional clones producing a high titer of M-MuLV (CP-l), low titers of M-MuLV (LX-l and LX-2) or no virus (NX-1, NX-3 and NX-4) have been described (Rothenberg, Donoghue and Baltimore, 1978; Shields et al., 1978) and were provided by E. Rothenberg. Analysis of these clones as described above reveals several facts. First, each clone shows a characteristic pattern of endogenous proviruses. Thus the sites of provirus integration, once established, are relatively stable. Second, many of the clones (including some clones which produce a

The Integrated 1005

Genome

of Murine

Leukemia

Virus

higher titer of M-MuLV) do not contain unintegrated viral DNA (molecular weight of 5.7 x lo6 daltons). The stabilization of the infection and synthesis of progeny viruses can therefore be specified solely by integrated viral genomes. Third, among the 10 clones analyzed, about 15 distinguishable sizes of Eco RI fragments are present. Thus M-MuLV proviruses can be integrated at more than 15 sites in the host cell genome. Furthermore, since there is no integration site common to all virus-producing clones, there is not one site in the host cell genome into which M-MuLV must integrate its provirus to establish a productive infection. Finally, since all clones that produce virus contain a number of proviruses, a single initial infection can result in a multiplicity of integrated genomes. On the other hand, all the clones infected with M-MuLV but not producing virus contain only a single size of hybridizing DNA fragment which we presume represents a single provirus. An explanation for this last observation will be proposed in the Discussion. The observation of a characteristic spectrum of proviruses in each of the M-MuLV-infected NRK clones argues that the integration of these proviruses must be sufficiently stable to prevent this integration pattern from becoming random. Nevertheless, if proviruses were acquired or lost at a low probability per generation, the results of such events would be present in only a minority of the cell population. Many cells in the population may exhibit one or more of these alterations, yet analysis of the bulk population would reveal only an average pattern equivalent to that seen in the clonal ancestor. In Figure 2, we compare the proviruses of the NRK-5 clone to three subclones of NRK-5 which were isolated by single-cell recloning of an NRK-5 population. These subclones were isolated from a culture which has been passaged for two years since the original cloning. It is apparent that NRK-5 and its subclones contain virtually identical sets of proviruses. Thus all three lineages represented by these three randomly cloned subclones suffered no alterations in their pattern of proviruses during the intervening two years. Perhaps the most striking indication of provirus stability is the readily demonstrable existence of the multiple endogenous proviruses in the mouse genome. These proviruses may have been acquired by infection of germ line tissue with a series of retroviruses at different times during the evolution of the mouse species. They have been perpetuated by genetic transmission since their introduction into the mouse germ line. Analysis of uninfected mouse DNA by Eco RI cleavage and hybridization to M-MuLV RNA reveals about 10 distinct bands of intense hybridization present among DNAs in the molecular weight range

Figure 2. M-MuLV Proviruses Integrated and Three Sub-Clones of NRK-5 As described in the legend subclones of NRK-5.

to Figure

into the DNA of NRK-5

1. NRK-Sa,

5b and 5c are the

~4 x 10s->15 x lo6 daltons (Figure 3). This hybridization is caused by the endogenous proviruses described above. Interpretation of the bands of hybridization seen in Eco RI-cleaved mouse DNA is more difficult than interpretation of the bands seen in M-MuLV-infected rat cell DNA. First, the sensitivities of these proviruses to Eco RI cleavage are unknown; cleavage by Eco RI within a proviral sequence might result in more than one detected fragment. Second, the different endogenous proviruses of mouse have varying degrees of homology to the M-MuLV RNA used here as probe. Those with more homology will be easily detected and those with less homology will be detected with difficulty or not at all. Finally, the large number of provirus-containing fragments cannot be well-resolved by the present gel electrophoresis. Multiple unresolved fragments are almost certainly the cause of the darkest bands of hybridization seen in Figure 3. Despite these reservations, however, a given spectrum of integrated proviruses will produce a characteristic pattern of provirus-containing Eco RI fragments. Thus the pattern of hybridizing Eco RI fragments is a fingerprint of the endogenous proviruses in the DNA examined. The five strains of laboratory mice we examined exhibit a great similarity in their endogenous proviruses (Figure 3, lanes B-F). Despite the similarity of hybridizing Eco RI fragments observed in the strains of mice, there are a small number of strainspecific differences. For example, DNA from a BALB/c mouse (lane B) exhibits a unique proviral

Cell 1006

ABCDEF

6

Figure

3. Endogenous

Proviruses

As described in the legend to figure indicate the position of Figure 1. Arrows to the right hybridization referred to in the adult mouse livers, except the from embryos.

in Uninfected

Mouse

DNA

Figure 1. Arrows to the left of the molecular weight standards as in of the figure point to bands of text. All DNAs were extracted from AKWJ DNA, which was extracted

band of 13 x lo6 daltons and lacks a proviral band of 9.5 x lo6 daltons present in DNAs from the other mice. Other differences between strains of mice are present among DNA fragments of molecular weight near 5 x 10s daltons. The relative intensity of common bands varies from strain to strain but we feel that at least some of this variation may result from technical factors. The cleavage patterns of proviruses appear quite similar among the DNAs of the laboratory mice. In contrast, the cleavage pattern of proviruses in DNA from a feral mouse cell line appears quite different (lane A). The multiplicity of endogenous proviruses in mouse DNA complicates detection of M-MuLV proviruses introduced by exogenous infection. Nevertheless, we have been able to locate at least some of the proviruses present in M-MuLV-infected mouse cells which are not discernible in their uninfected parents. Figure 4 shows a comparison of the Eco RI cleavage pattern of infected and uninfected mouse DNAs. M-MuLV clone 2 is a line of NIH/swiss mouse fibroblasts (NIH/3T3) infected with M-MuLV and then cloned. DNA from this cell line exhibits bands of hybridization among DNA fragments of -14.5 x IO6 and 6.2 x lo6 daltons (lane B) which are absent

Figure 4. Comparison Infected Mouse DNAs

of Proviruses

in Uninfected

and M-MuLV-

As described in the legend to Figure 1, except that the filter was incubated with l~SI-M-M~LV RNA. Mice containing the thymomas were supplied by N. Rosenberg. Neonatal NIH/swiss mice were injected with IO’-lo6 pfu of M-MuLV clone 2 virus, a recloned stock of clone 1 M-MuLV. 2-4 months later, lymphoid tumors developed in the thymus, spleen and lymph nodes. Tumors of the lymph nodes in each case were used as a source of DNA.

from the DNA of an uninfected NIH/swiss mouse (lane A). Comparison of an M-MuLV infected BALB/c mouse cell line JLS-Vll (lane G) with its uninfected parent JLS-V9 (lane F) produces a more ambiguous result. There appears to be a new band of hybridization among DNA fragments of molecular weight slightly greater than 6.2 x IO6 daltons in the infected cell DNA which is absent from the uninfected cell DNA. The ambiguity of this finding points out the difficulty of pursuing this kind of experiment in mouse cells. Thymomas 1, 2 and 3 are independent tumors induced by M-MuLV in three NIH/swiss mice. The proviruses detected in DNAs from these tumors are displayed in Figure 4 (lanes C, D and E). All three

The Integrated 1007

Genome

of Murine

Leukemia

Virus

tumors display bands of hybridization among DNAs of about 14.5 x 10s daltons which are absent from uninfected NIH/swiss mouse liver DNA (lane A). The pattern of these extra bands is different in each tumor, and the variability is analogous to that seen in the in vitro infected rat cell clones. Thus the infection of the natural lymphoid target cell of the natural host species results in integration of new proviruses at several sites, different sites being used in different infected cells. Since new proviruses are introduced into different sites in different tumors, and yet each tumor displays a discrete pattern of new bands of hybridization (as opposed to a continuum of hybridization), a large portion of cells in the tumor must derive from one initially infected cell. A comparison of proviruses in DNA from NIH/ Swiss and BALB/c mouse livers and DNA from cell lines derived from these mice is shown in Figure 5. The pattern of proviruses in the cell lines is identical to that of the mouse from which they were derived. This correspondence assures the identity of the in vitro cell lines and demonstrates that the spectrum of proviruses did not change during their derivation and extensive passaging.

ABCDE

6.2 ~106

Discussion The integrated DNA genome appears to be a central intermediate in the replication of retroviruses. In many of the infected c,ell lines we studied, this proviral DNA represents the only detectable DNA in the cell and is therefore the sole template which specifies the synthesis of progeny viral RNA. Such observations diminish the possibility that unintegrated viral DNA is a template for viral RNA synthesis and a repository of the viral genetic information in chronically infected cells. Although unintegrated DNA synthesis has been observed in certain cells chronically infected by other retroviruses (Varmus and Shank, 1976) and occasionally seen here (Figure I), there is no evidence that such DNAs are involved in the maintenance of the infected state in the cells studied. This potentially obligatory role of the integrated retrovirus genomes contrasts with the function of similar genomic forms observed in other tumor viruses. The integrated DNAs of SV40 and adenoviruses have been extensively studied, yet there is little evidence that integration plays any role in the normal replicative cycle of these viruses. Rather, these viruses appear able to proceed through their entire growth cycle utilizing only unintegrated forms of viral DNAs. Integration of their genomes does occur at low frequency and in a fragmentary fashion upon infection of heterologous, nonpermissive cells, and the resulting rare event can occasionally be identified by a transformed pheno-

Figure Mouse

5. Comparison of Endogenous Proviruses in DNAs from Cell Lines and the Mice from Which They Were Derived

As described

in the legend

to Figure

1.

type. Perhaps reflecting the almost accidental nature of these events, the Southern gel filter analysis has indicated that a large number of integration sites are used by these viruses and that fragmentary and permuted representations of the viral genome are present. Detailed analysis of the SV40 integration sites reveals no recurring pattern of surrounding cellular sequences (Botchan et al., 1976). The large number of retrovirus integration sites (~15) found here would appear to conform to the nonspecific pattern of integration observed with the adeno- and papovaviruses. In reality, however, the techniques we used shed little light on the nature of the integration sites used by M-MuLV. Our evidence is compatible either with random retrovirus integration in the cellular chromosome, or with virus integration in a discrete and specific nucleotide sequence that is repeated many times in the cellular genome. The existence of such a specific integration sequence, which might be relatively short in length, would not be revealed by the Eco RI endonuclease used here. This enzyme

Cell lQQl3

cuts cellular DNA at sites occurring every 4-6 x lo6 daltons. Detection of any specific integration sequences would require the use of endonucleases which cut within such sequences. Since such sequences may be relatively short, it may be necessary to experiment with a series of frequently cutting endonucleases. The presence of multiple integration sites allows an infected cell to acquire a large number of proviruses. In fact, all the infected virus-producing NRK clones we studied do contain multiple proviruses. This multiplicity of proviruses is apparently acquired soon after infection, and the number of proviruses then becomes stabilized. Evidence for this stabilization comes from the isolation and analysis of subclones of one virus-producing clone, NRK-5, which have retained an unchanged pattern of proviruses during two years of cultivation. The stability of the provirus pattern in virusproducing cells is due, at least in part, to the resistance of MuLV-infected cells to reinfection by MuLV. The three infected clones we studied which do not produce virus each contain only a single size of hybridizing DNA fragment. We presume that each of these fragments represents a single provirus, although other possible structures (which we consider improbable) will only be ruled out by detailed structural analysis of these sequences. Although these single proviruses are defective and do not specify all the components required for synthesis of functional virions, they do establish an effective interference barrier preventing reinfection of these clones by additional M-MuLV virus. The presence of multiple proviruses in producer clones and single proviruses in nonproducer clones is not a consequence of the initial multiplicity of infection, as all clones with one provirus were infected with a multiplicity of much greater than one (Shields et al., 1978), and all virus-producing clones infected with a multiplicity of less than one acquired multiple proviruses. Nor is the association between the single provirus and the nonproducer phenotype likely to be a chance occurrence. The probability of such a distribution occurring by chance is about lo++. We therefore propose two explanations for the association of single provirus with the nonproducer phenotype. First, it is possible that at least two proviruses are required for the maintenance of a productive infection, perhaps complementing one another in essential functions. Other work (Cooper and Temin, 1974) which demonstrates the linear dose response of infectious centers after transfection of integrated proviruses would argue against such a possibility, although not conclusively eliminate it. An alternative explanation, which we favor, is that most retrovirus

infections initially yield only one integrated provirus capable of specifying an initial wave of progeny particles. These particles begin to emerge from the infected cell at a time when an effective interference barrier to reinfection has not yet been established. Some of these particles are able to reinfect the cell from which they have just emerged, resulting in the acquisition of a number of additional proviruses. This reinfection process is transient and is blocked after a short time by the establishment of the interference barrier. In cells initially infected with a defective provirus, no additional proviruses are acquired during this period because no progenyvirions are produced at the cell surface. The interference barrier is then established because the provirus, although defective, is still able to specify the viral glycoprotein that seems to be required for interference (Shields et al., 1978). This paper shows a visualization of the endogenous MuLV proviruses of different strains of mice. The analysis of these proviruses is complicated by two factors. First, we are ignorant of the endonuclease cleavage sites, if any indeed exist, within the different endogenous viral genomes. For instance, an endogenous provirus may be dissected into two pieces by Eco RI, resulting in its visualization as two differently sized DNA fragments in the Southern analysis used here. Second, each of these endogenous proviruses has a different degree of sequence homology with our M-MuLV probe. In fact, any probe generated from the genomic RNA of an exogenously transmitted MuLV represents an equally arbitrary choice whose usefulness in detecting all MuLV proviruses is limited. We believe that many endogenous MuLV proviruses have therefore escaped detect ion. The multitude of endogenous genomes complicates any analysis of the exogenously introduced MuLV proviruses of mouse cells. In some cases, however, we have been able to discover new viral bands above the endogneous background. In the case of three MuLV-induced thymomas, the different patterns of exogenous proviral integration allowed us to conclude that multiple integration sites for provirus integration exist in mouse as well as in rat cells. The endogenous genomes detected in the present study allow us to establish a new typing of different mouse strains and their derivative cell lines. We show (Figure 5) that DNA from a cell line derived from NIH/swiss mice has endogenous proviruses with an Eco RI cleavage pattern identical to that observed in DNA from an NIH/swiss mouse liver. Similarly, the cleavage pattern displayed by two cell lines derived from BALB/c mice is identical to that of BALB/c mouse liver. The BALB/c and NIH/swiss patterns are clearly distinguishable,

The Integrated 1009

Genome

of Murine

Leukemia

Virus

however, confirming the origins of the cell lines. The DNAs of five strains of laboratory mice appear to contain basically similar proviruses. Although the origin of these mice is obscure, they are known to constitute distinct lines since at least 1920. Although all laboratory mice exhibit a similar spectrum of proviruses, DNA from a feral mouse cell line (SC-l) has endogenous proviruses with an Eco RI cleavage pattern very different from that of the laboratory mice. Consequently, the constancy of proviruses observed in laboratory mice is not a property of Mus musculus, but rather of some subpopulation of mice from which laboratory mice were derived. We are currently analyzing DNAs from various subspecies of Mus musculus in an attempt to define this subspecies. Experlmental

Procedures

Mice AKRlJ strains animal

mice were obtained from Jackson Laboratories. All other of mice were obtained from colonies maintained in the care center of the Center for Cancer Research. MIT.

Cell Lines NRK cells (a continuous line of normal rat kidney cells), NIH/3T3 (a continuous line of NIH/swiss mouse fibroblasts), BALB/3T3 (a continuous line of BALB/c mouse fibroblasts), JLS-V9 and JLSVi1 (described by Wright et al., 1967; cloned in our laboratory before use), and SC-1 (a continuous line of feral mouse fibroblasts, cloned in our laboratory before use) were obtained from stocks maintained at the Center for Cancer Research, MIT. LX-l, LX-2, NX-1, NX-3, NX-4 and CP-1 have been described (Rothenberg et al., 1978; Shieldset al., 1978). NRK-1, NRK-3. NRK-4 and NRK-5 were derived by infection of NRK cells with Maloney clone 1 virus (Fan and Paskind, 1974). Within a few hours after infection, cells were trypsinized and transferred to cloning wells at less than one cell per well. Since only a minority of the single-cell clones were found to have been infected, the effective multiplicity of infection was less than one. All cells were grown in Dulbecco’s modified Eagle’s medium containing 10% calf serum, except NRK cells and derivatives of NRK cells. which received 10% heat-inactivated fetal calf serum.

Extraction

of DNA

Mouse livers were minced with a razor blade, homogenized in RSB [lo mM Tris-HCI (pH 7.5), 10 mM NaCl, 1.5 mM MgCI,] using IO strokes with a Dounce homogenizer, and the resulting nuclei were removed by centrifugation at 2ooO x g for 10 min. The nuclei were resuspended in 25 ml of STE [lo0 mM NaCI, 10 mM Tris-HCI (pH 8) 1 mM Na EDTA] and slowly poured into STE containing 2% sodium dodecylsulfate and 0.4 mglml proteinase K (obtained from Boehringer-Mannheim). Thymomas were dispersed by passage through an 80 mesh stainless steel screen into 25 ml of STE. The suspension was then slowly poured into STE containing 2% sodium dodecylsulfate and 0.4 mg/ml of proteinase K. Seventeen day mouse embryos had heads and limbs removed, were minced with a razor blade, were passed through an 86 mesh stainless steel screen and then processed as described for mouse livers. Cells had medium removed, were washed with phosphatebuffered saline and then STE containing 0.5% SDS, and 0.2 mg/ ml proteinase K was applied directly to the monolayer (10 ml per 1 O* cells). In all cases, extracted DNA was incubated at 37°C for 2 hr,

extracted twice with one vol of redistilled phenol (pH 8 to 9) and dialyzed overnight at room temperature against 2 x SSC [0.3 M NaCl. 0.03 M Na citrate (pH 7.5)]. Pancreatic RNAase (recrystalized, obtained from Worthington Laboratories) was added to 50 pg/ml; the sample was incubated at 37°C for 1 hr; SDS was added to 0.5% and proteinase K to 0.2 mg/ml; the sample was incubated at 37°C for 1 hr and extracted twice with one vol of redistilled phenol (pH 8 to 9) and twice with one vol of 96% CHCl,/4% isoamyl alcohol: and was dialyzed three times for 24 hr each at 4°C against 2 x SSC, STE and 10 mm Tris-HCI (pH 8), 1 mM Na EDTA, respectively. DNA was stored at 4°C.

Endonuclease

Eco RI

Eco RI was prepared as described (Greene et al., 1975) through the phosphocellulose column step from the RY23 strain of Escherichia coli supplied by New England Biolabs. Both the endonuclease and methylase activities were recovered. DNA was cleaved at 25 pg/ml in 50 mM NaCl, 100 mM Tris-HCI (pH 7.3), 10 mM MgCI, for 1 hr at 37°C. The activity of the enzyme was determined by digestion of bacteriophage lambda DNA. Five times the amount of enzyme needed to completely digest the lambda DNA was used to cleave rat and mouse cell DNAs. Completeness of cleavage of the rat and mouse DNAs was monitored by testing the ability of the DNA to serve as a substrate for the Eco RI methylase. DNA that is completely cleaved by the Eco RI nuclease is not a substrate for the methylase. Reaction of the methylase has been described (Greene et al., 1975). After digestion with Eco RI the DNA was adjusted to 250 mM NaCl, 10 mM Na EDTA; extracted with onevol of redistilled phenol (pH 8 to 9); and extracted with one vol of 98% CHCI,M% isoamyl alcohol. 2 vol of ethanol were added and the DNA was incubated at -20°C overnight. The DNA was precipitated by centrifugation at 72.000 x g for 30 min and resuspended at 1 mg/ml in 10 mM Tris-HCI (pH 8), 1 mM Na EDTA.

Electrophoresis,

Transfer

and Hybridization

of the DNA

Electrophoresis of the DNA in 1% agarose gels, transfer of the DNA to nitrocellulose filters and hybridization of the DNA to radioactive M-MuLV RNA have been described (Smotkin, Yoshimura and Weinberg, 1976). For work reported here, ~2SI-labeled M-MuLV RNA was purified by chromatography on cellulose powder as described (Franklin, 1966). RNA from purified virions was purified first as a 70s dimer and then as a 355 monomer before iodination. 32P-labeled M-MuLV RNA was provided by F. Yoshimura. For hybridization of DNA on nitrocellulose filters to 52P-labeled RNA, the procedure used for soaking the filter before hybridization was modified as follows: 10 mM NaPO, replaced the 1 mM Nal in the soaking solution and the soaking was performed at 67°C rather than at room temperature. After hybridization, filters were briefly rinsed several times with 10-20 ml of 20°C 2 x SSC washed twice for 1 hr each time with 20 ml of 5 x SSC containing 10 mM Na EDTA, 0.2% SDS and adjusted to pH 5.5. The filter was incubated at 37°C in 2 x SSC containing 59 pg/ml RNAase and washed a final 30 min at 6PC versus the 5 x SSC buffer described above. Filters were exposed to Kodak XR-5 high speed film using DuPont Cronex Lightening Plus intensifying screens at -70°C for 1-14 days.

Acknowledgments We thank Dr. Fayth Yoshimura for many useful discussions and Cliff Tabin, Stephanie Bird and Julie Sexton for help in preparation of DNAs. This work was supported by grants from the American Cancer Society and the NIH. D. S. was a postdoctoral fellow of the American Cancer Society. R. A. W. was a research scholar of the American Cancer Society, Massachusetts Division, and subsequently a fellow of the Rita Allen Foundation.

Cell 1010

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. Section 1734 solely to indicate this fact. Received

July 18,1978;

revised

August

10.1978

References Botchan, Cooper,

M., Topp,

W. and Sambrook.

G. M. and Temin.

Fan, H. and Paskind, Franklin,

M. (1974).

R. M. (1966).

Proc.

J. (1976).

H. M. (1974).

J. Virol.

J. Virol.

A. M., Science

269-267.

74,421-429.

Nat. Acad.

Sci. USA55,

Gianni, A. M., Smotkin, D. and Weinberg, Acad. Sci. USA 76,447-451. Gianni. (1976).

Cell9,

74, 1132-1141. 1504-1511.

R. A. (1975).

Hutton, J. R., Smotkin, 791, 569-571.

D. and

Greene, P. J., Poonian, M. S.. Nussbaum. Garfin, D. E., Boyer, H. W. and Goodman, Biol. 99, 237-261.

Proc.

Weinberg,

Nat. R. A.

A. L., Tobias, L., H. M. (1975). J. Mol.

Guntaka, R. V., Richards, 0. C., Shank, P. R., Davidson, N., Fritsch, E., Bishop, J. M. and Varmus, J. Mol. Biol. 706, 337357.

Kung, H. J., H. E. (1976).

Ketner, G., and Kelley, 1102-1106.

Sci. USA 73,

Murray,

T. J. (1976).

K. and Murray,

N. E. (1975).

Ringold, G. M., Yamamoto, (1977). Cell 70, 19-26. Rothenberg, 73, 435-451. Shank,

E., Donoghue,

P. R. and Varmus,

Shields, A., Witte, Cell 74,601-609.

Southern,

E. M. (1975).

Varmus,

H. E. and Shank, R. A. (1977).

Nat. Acad.

J. Mol.

K. R., Shank, D. J. and H. E. (1978).

0. N., Rothenberg,

Smotkin, D., Yoshimura, Virol. 20, 621-626.

Weinberg,

Proc.

D. (1978).

J. Virol.25,

104-114.

Weinberg,

H. E. Cell

D. (1978).

R. A. (1976).

J.

Biol. 98, 503-517.

P. R. (1976). Biochim.

Baltimore,

E. and Baltimore,

F. K. and J. Mol.

Biol. 98, 551-564.

P. R. and Varmus,

J. Virol.

Biophys.

78, 567-573.

Acta473,

Wright, B. S.. O’Brien, P. A., Shibley, G. P., Mayyasi, Lasfargues, J. C. (1967). Cancer Res. 27, 1672-1675.

39-55. S. A. and