Cell, Vol. 11, 321-329,

June

1977, Copyright

0 1977 by MIT

Organization of Shared and Unshared the Genomes of Chicken Endogenous Viruses Paul E. Neiman, Sarojini Das, Don Macdonnell and Christine McMillin-Helsel Department of Medicine Division of Oncology University of Washington School of Medicine Seattle, Washington 98195 and The Fred Hutchinson Cancer Research Center Seattle, Washington 98104

Summary The genome of the genetically transmitted endogenous C type virus of chickens, RAV-0, is closely related to that of Rous sarcoma virus (RSV). Nevertheless, these viruses differ widely in oncogenicity and regulation by the host cell. Competitive hybridization analysis of 1*51-labeled genomic RNA demonstrated that the genome of RAV-0 lacks about 35% of the sequences of nondefective RSV which formed hybrids with proviral DNA from RSV-infected cells, and that the genome of transformation-defective deletion mutants of RSV (fd RSV) lacks about 15% of these sequences. Conversely, about 12% of the RAV-0 sequences forming hybrids with normal chicken cell DNA were not detected in the sarcoma virus. A technique was developed to map the location of these unshared sequences by competitive hybridization. The deletion in the genome of td RSV was seen to begin at about 0.2 and to end at about 0.05 of the genome length from the 3’ end of sarcoma virus RNA, confirming the results of other laboratories using the method of mapping RNAase TI resistance of oligonucleotides. The 35% of RSV sequences missing and/or diverged in the genome of RAV-0 were concentrated within 40% of the sarcoma virus genome from the 3’ end, and most of this large section did not appear to form hybrids with chicken DNA under the conditions of these experiments. A low level of hybrid formation was, however, detected between uninfected chicken cellular DNA and a small fraction of the nucleotides in the region of the td deletion. Analysis of RAV-0 3’ end fragments demonstrated that the genomic sequences of RAV-0 missing in RSV were concentrated at the 3’ end of the endogenous viral genome. We conclude that the sequence differences between endogenous and sarcoma viruses are largely concentrated in specific regions of the viral genome. Introduction Rous-associated virus type 0 (RAV-0) is an endogenous RNA tumor virus of chickens (Vogt and Friis,

Sequences in and Sarcoma

1971; Weiss et al., 1971). Complete or nearly complete sets of endogenous viral sequences have been detected in the DNA of normal chicken embryo cells by molecular hybridization techniques (Neiman, 1973; Tereba, Skoog and Vogt, 1975; Shoyab and Baluda, 1975), including direct reactions of viral genomic RNA with an excess of cellular DNA fragments. RAV-0 is nontransforming in vitro, and either poorly oncogenic or nononcogenie in chickens (Neiman, 1973; Motta et al., 1975). Furthermore, expression of the genome of this virus, both in the form of genetically transmitted viral genes and following exogenous introduction of RAV-0 into chicken cells (Robinson, 1976; Linial and Neiman, 1976), appears to be regulated by the host cell. Rous sarcoma virus (RSV) differs from RAV-0 in a number of significant biological properties. Nondefective strains of this prototype exogenous virus replicate in and transform chick embryo fibroblasts in culture apparently independent of the intracellular regulation affecting the expression of endogenous viral genes. RSV also induces rapidly growing sarcomas when injected into chickens. Spontaneously arising nonconditional mutants of RSV (td RSV) containing an approximately 15% deletion in the viral genome (Vogt, 1971; Martin and Duesberg, 1972; Duesberg and Vogt, 1973; Neiman et al., 1974b) are transformation-defective in vitro and with respect to fibrosarcoma formation in vivo, but retain the ability to induce malignant lymphomas of the Bursa of Fabricius in chickens (Biggs et al., 1973). While a complete provirus is found in the DNA of RSV-infected cells (Cooper and Temin, 1974), only a portion of sarcoma virus sequences are detected in the DNA of uninfected chicken cells (Neiman, 1972; Varmus et al., 1973; Wright and Neiman, 1974; Neiman et al., 1974b; Varmus, Heasley and Bishop, 1974; Tereba et al., 1975). Despite the differences between RSV and RAV-0, the two viruses are genetically highly related. Studies which measured sequence relationships by competition in hybridization reactions between viral RNA and proviral DNA determined that 6585% of the genome of the two viruses were shared (Wright and Neiman, 1974; Neiman et al., 1974b). In the competition analysis, genomic RNA from RAV0 appeared to lack about 35% of the sarcoma virus genome. Subunit RNA of RAV-0 is the same size as that of td RSV, about 15% smaller than that of RSV (Duesberg and Vogt, 1973). Sarcoma virus (as well as td RSV) appeared to lack about lo-15% of the genome of the endogenous virus. This paper reports an attempt to determine the organization of shared and unshared sequences in the genome of these viruses. The results of this efford show that

Cell 322

the missing (or divergent) each virus are concentrated the genomic RNA molecule.

sets of sequences in specific regions

in of

Results Hybridization of 1251-Labeled Viral RNA to Cellular DNA Fragments in either the Presence or Absence of Competing RNA As in previous experiments, genomic Is51 RNA sequences from Prague strain of RSV subgroup C (PR-RSV-C) formed hybrids with an excess of fragments of DNA from virus-induced sarcomas at a rate suggesting about 2 complete copies per haploid genome. About 75% of the input viral RNA entered RNAase-resistant hybrid at values of Cot near 105. Hybridization of this same RNA to DNA from normal uninfected chicken embryo cells was considerably less extensive. At values of Cot near 105, about 45% of the RSV RNA entered hybrids with normal DNA. This was about two thirds the extent of the reaction seen with RAV-0 RNA forming hybrids with normal DNA under the same conditions. The kinetics of hybridization of this endogenous virus RNA corresponded closely to those expected for about 1 copy per haploid genome. These hybridization data as well as the method of estimating copy numbers have been described in detail (Neiman, 1972; 1973; Neiman, Wright and Purchase, 1974a; Neiman et al., 1974b). The relationships between these viral sequences and the apparent absence of some sequences of RSV from the DNA of uninfected chicken cells are demonstrated in Figure 1, which depicts the competitive effects of unlabeled RNA from RAV-0, PR-RSV-C and the corresponding deletion mutant to-PR-C in these reactions. RNA from to-PR-C competed for all but 15%, and RAV-0 for all but 35% of the sequences of the nondefective sarcoma virus which formed hybrids with sarcoma DNA. When DNA from normal chickens was used in the reaction with the same RNA preparations, these differences were nearly obliterated. This apparent identity of nearly all the sequences of RSV endogenous to the normal chicken genome with those of RAV-0 led us to conclude previously that the remaining 35% of the RSV genome was not present in the normal cell including, presumably, the majority of the sequences deleted from td-PR-C (Neiman et al., 197413). This is demonstrated more directly by the present analysis in which very little hybrid was formed between labeled RSV RNA and normal DNA in the presence of an excess of competing RNA from the deletion mutant. The competitive effect of RSV RNA in the reaction between labeled RAV-0 RNA and normal DNA, demonstrating that lo-15% of the RAV-0 genome detected in this manner is

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Analysis

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Gen-

(A) ‘**l-labeled RNA from PR-RSV-C was incubated with sarcoma cell DNA to a Cot level of about 1.5 x 104. The extent of hybridization of radioactive RNA in the presence of varying quantities of unlabeled 70s RNA competitive from RAV-0 (m), M-P&C (0) and PR-RSV-C (0) was compared with the 61% achieved in control hybridization reactions in the absence of competitor. Percentage of control hybridization was plotted as a function of the calculated ratio of proviral DNA:total viral RNA in the reaction mixture (assuming a single-stranded viral genomic molecular weight of 3.3 x lo6 daltons present in 2 complete copies per haploid genome). The actual quantity of viral RNA added to each reaction mixture is also shown on the abcissa. The dashed line is a theoretical curve for complete competition by homologous RNA and follows the equation y = x/(x + I), where y is the fraction of control hybridization and x is the viral DNA:RNA ratio in the reaction mixture (Wright and Neiman, 1974). (B) The same reactions carried out with DNA from normal uninfected chicken embryo cells. Because of the partial representation of RSV sequences in normal DNA, the extent of hybridization of the ordinate was not normalized. The calculated proviral DNA:viral RNA ratio was adjusted for 1 proviral copy per haploid genome.

absent from the genome of the sarcoma virus (as well as a number of other exogenous chicken leukosis-sarcoma viruses), has been described elsewhere (Wright and Neiman, 1974; Neiman et al., 1974b; Neiman, Purchase and Okazaki, 1975). Preparation of 3’ End Fragments of PR-RSV-C The principal strategy for determining the organization of the sets of sequences discriminated in the genome of RSV and RAV-0 by competitive hybridization was to prepare fragments of genomic RNA of various lengths from the poly(A) tract at the 3’ end of the molecule (Keith, Gleason and Fraenkel-Conrat, 1974; Wang and Duesberg, 1974) by affinity chromatography on oligo(dT)-cellulose as described in Experimental Procedures. These fragments could then be analyzed by competitive hybridization for their content of the sequences of interest. Figure 2 shows the distribution on mixed agarose-acrylamide gels of 3’ end fragments of 1251-labeled 355 RNA from PR-RSV-C which varied from full-length molecules to very small sizes. An

Organization 323

of the Genome

1

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2. Electrophoresis ‘251-Labeled 35s Gels

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of 1251-Labeled 3’ End Fragments of PRRNA Fragments on Mixed Agarose-Acryl-

The upper panel demonstrates the distribution of radioactivity in 1 mm gel slices of PR-RSV-C RNA following the iodination and processing as described in Experimental Procedures. Migration was from left to right. Background was 25-50 cpm. The position of E. coli RNA markers is shown. The arrows are the position of slices corresponding to RNA of about 16s and 225 in size. RNA extracted from these slices was subjected to simultaneous electrophoresis on a second set of gels (which also included the E. coli markers running in about the same positions shown in the upper panel), and the results are superimposed in the lower panel [(O-O) 16s RNA; (O- - -0) 11s RNA].

aliquot of the radioactivity eluted from two separate gel slices was analyzed for size distribution by reelectrophoresis on the same type of gel. The band width and degree of separation of fragments presumed to represent 0.16 and 0.08 of the genome length from the 3’ end are shown in the same figure. One hazard to the quantitative interpretation of hybridization carried out with such presumptive 3’ end fragments is aggregation of RNA fragments in the gel, which might distort the correlation of specific regions of the genome with fragment length. Coelectrophoresis of labeled fragments with unlabeled viral RNA of varying sizes failed to demonstrate evidence of aggregation (data not shown). Analysis of Sequence Representation in PR-RSVC Fragments Figure 3 describes an analysis of the distribution of

sequences in the genome of PR-RSV-C which are unshared with RAV-0 and td-PR-C using the 3’ end fragments from the subgroup C sarcoma virus 35s RNA and, for purposes of comparison, from identically prepared fragments from the 705 RNA complex of PR-RSV subgroup B. The data were obtained by reacting ‘251-labeled RSV-RNA fragments of various sizes with an excess of proviral sequences in sarcoma DNA to a Cot value of about 1.5 X 104. The extent of hybridization of fragments representing various lengths of the genome from the 3’ end did not vary significantly, consistent with a complete and nearly uniform representation of proviral sequences in the cellular DNA. There was no apparent major effect of size of the different fragments on the extent of hybridization. This observation was expected, since the length of the DNA fragments was shorter than all but the smallest initial sizes of RNA fragments (Wetmur, 1971; Hutton and Wetmur, 1973). The extent of hybridization of the fragments was normalized on the ordinate as a control for comparison with hybridization in the presence of an excess of unlabeled competitor RNA from td RS\i and RAV-0. As shown in Figure 3, RNA from the td deletion mutant failed to compete for about 15% of the sequences of fulllength molecules from both sarcoma viruses (as expected from the standard competition analysis shown in Figure 1). This competitive effect progressively diminished with shorter genome lengths, with maximum hybridization being achieved with fragments representing about 0.2 of the genome from the 3’ end. Competition then progressively increased further toward the 3’ end. The data are compared and fit fairly closely with a theoretical model (Figure 3, model l), in which a 15% deletion begins at 0.2 and ends at 0.05 of the genome from the 3’ poly(A) tract. The theoretical curve for hybridization in the presence of excess RNA from td-PR-C was obtained by calculating the fraction of sequences in each fragment length missing (according to the model) from the competitor. Experimental data demonstrated a slight deviation from the model for very small (500 nucleotides and less) end fragments. Whether this alteration reflects a degree of error in the model or background noise (for example, low level contamination with small nonspecific fragments generated during preparation) could not be ascertained. Unlabeled genomic RNA from RAV-0 gave a different competition pattern. As expected, competition with full-length molecules left about 35% of the level of hybridization in the absence of a competitor and rose to a peak level near 85% with 3’ end fragments representing about 0.4 of the genome. Thus the data indicated that most of the sequences of RSV missing in the endogenous virus are con-

Cell 324

This issue was more directly addressed in subsequent experiments. Finally, these results were not altered by the use of fragments of 70s complex RNA from PR-RSV-6.

s

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Figure 3. Competition of RAW0 and td-PR-C RNA in Hybridization Reactions between 3’ End Fragments of PR-RSV RNA and Sarcoma Cell DNA Standard reaction mixtures containing a constant amount of radioactivity, about 2000 cpm representing 3’ end fragments of varying lengths from the 3’ poly(A) tract, shown as fractions of the genome on the abcissa, were incubated to a Cot value of about 1.5 x 104. The extent of hybridization obtained in the absence of competitor (A) was normalized on the ordinate. The extent of hybridization of sequences of 3’ end fragments prepared from PRRSV-C 355 RNA (0, 0) and PR-RSV-8 70s RNA (m, 0) was measured in the presence of 0.15 pg of unlabled 70s RNA sequences from RAV-0 (closed symbols) and td-PR-C (open symbols), and plotted as a function of control hybridization reactions in the absence of competitor. The data are compared to theoretical curves calculated from model I(-) and model 2 (. . ) shown above in which the sequences missing from the competitor RNA are represented by an open box.

centrated from about 0.4 of the genome to a short segment just proximal to the 3’ poly(A) tract as seen by comparison with the suggested model (Figure 3, model 2). The data, however, deviated from the model more substantially than was the case tiith td RSV RNA with respect to the very short 3’ end fragments, an observation which suggests more sequence divergence between RAV-0 and RSV within this region than was the case between RSV and the td deletion mutant. If so, one might have expected an even higher “peak” of hybridization than was detected and suggested by the model. It is therefore possible that some low level sequence sharing could have been detected within the region shown as totally divergent by the model.

Analysis of RSV-Related Sequences in DNA from Uninfected Chicken Embryo Cells The same sets of 3’ end fragments and competitor RNAs were used in hybridization reactions with normal uninfected chicken cell DNA, as shown in Figure 4. As in standard competition analysis, a very small fraction of full-length molecules of RSV RNA formed hybrids with normal DNA in the presence of either competitor near the background noise level with this technique (O-5%). However, fragments composed of sequences representing a little less than 0.2 of the genome from the 3’ end did show some increase in hybridization. The data were compared with an alteration of model 1 (Figure 3), which predicted the hybridization pattern if about one third of the approximately 1500 nucleotides deleted from the genome of td PR-C, distributed within the full region of the deletion, were detected in normal DNA by this technique. These sequences comprise only about 5% of the viral genome, which presumably explains our difficulties in detecting them in competition experiments involving the entire viral genome (for example, Figure 1). In contrast to the observations made with DNA from RSV-infected cells, the pattern of competition by RAV-0 RNA with PR-RSV-C fragments for normal DNA did not differ from that seen with td RSV RNA. Thus we did not detect a segment of the RSV genome represented in DNA from uninfected chicken cells which was not shared with RAV-0, except within the region of the td deletion. This observation suggested that the fraction between 0.4 and 0.2 of the RSV genome from the 3’ end, which is largely unshared with RAV-0, is more completely divergent from the normal chicken genome than is the region of the deletion in the td mutant. The distribution of sequences in RSV-RNA molecules exogenous to the normal chicken genome were also analyzed by simply measuring the extent of hybridization of the 3’ end fragments with normal DNA in the absence of competitor (Figure 4). The full-length fragments hybridized about 65% as well as expected if they were fully or nearly fully represented at 1 copy per haploid genome (that is, as well as RAV-0 RNA under these conditions). Hybridization decreased to a low value of about 20% with fragments composed of sequences beginning 0.4 of the genome length from the poly(A) tract and then increased slightly with shorter genome lengths. A model was calculated for the rather complex arrangement suggested by the pre-

Organization 325

of the Genome

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Figure 4. Competition of RAV-0 and td-PR-C RNA in Hybridization Reactions between 3’ End Fragments of PR-RSV RNA and Normal Chicken DNA The extent of reaction of the radioactive fragments was plotted on the same scale used for reactions with sarcoma DNA shown in Figure 3. The conditions and symbols are identical to those in Figure 3. Model 1 from Figure 3 was modified such that about one third of the genome within the deleted region formed hybrids with normal DNA; this modification is represented by the interrupted line (-). Model 3 (. ) proposes an arrangement for exogenous sequences in the genome of RSV. This theoretical curve was obtained simply by calculating the fraction of sequences in each fragment length represented in uninfected chicken DNA. The open boxes mark the location of RSV sequences not detected in normal chicken DNA. The spaced open boxes beginning at 0.2 from the 3’ end are meant to represent a situation in which a random two thirds of the sequences in the 3’ terminal 20% of the genome were exogenous.

ceding data in which sequences in the region of the WV genome from 0.4-0.2 from the 3’ end did not enter hybrids and only about one third of the sequences distributed in the remainder of the 3’ end of the genome were detected in normal DNA. The data points obtained were in rough agreement with the complex curve predicted by this model, although no attempt was made to determine whether all the deflections and contours could be demonstrated. Furthermore, the segmental nature of the unreactive sequences in the terminal 0.2 fraction suggested by the drawing of model 3 (Figure 4) was shown in that fashion for graphic convenience and is neither supported nor ruled out by the data (although stretches long enough to form and be scored as stable hybrids must exist).

Analysis of the Sequences in 3’ End Fragments of RAV-0 RNA Fragments of 1Z51-labeled 70s complex RNA from RAV-0 were prepared in the same manner as the 3’ end fragments of the sarcoma virus and analyzed for the localization of sequences unshared with RSV. As shown in Figure 5, sarcoma virus RNA failed to compete for about 12% of the sequences in full-length (35s) molecules of radioactive RAV-0 genomic RNA which formed hybrids with normal chicken DNA. This percentage progressively increased with shorter fragment lengths to a level of about 67% with sequences from fragments beginning between 0.2 and 0.15 from the 3’ end of the genomic RNA molecule and remained at about that level, albeit with some variation, for the remainder of the fragment sizes studied. The data fit reasonably well with a model in which the 12% of the RAV0 sequences not detected in RSV were distributed in about 18% of the RNA molecule from the 3’ end. This mode of organization, if correct, requires some interspersion of shared and unshared sequences within this largely divergent terminal segment of RAV-0 RNA. Analysis of RAV-0 Sequences in a Recombinant between RAW0 and PR-RSV-C Cloned recombinant sarcoma viruses formed following mixed infection of quail cells with PR-RSVC and RAV-0, which acquired the envelope properties of the endogenous virus (PR-RSV subgroup E), were recently characterized for their content of specific parental sequences by competitive hybridization (Linial and Neiman, 1976). One such clone of PR-RSV-E showed little loss (O-4%) of parental PRRSV genomic sequences, but had acquired RAV-Ospecific sequences not present in the sarcoma virus parent which comprised about 8% of the recombinant genome. An attempt was therefore made to locate these endogenous virus-specific sequences within the genome of such a recombinant. The virus selected was PR-RSV-E clone 95b, a different clone from that studied previously. Fragments beginning at the 3’ end were prepared as before from isolated 35s subunits from this virus and hybridized to DNA from sarcomas (induced in Japanese quails by this recombinant) in either the presence or absence of RNA from the parental sarcoma virus. The results are described in Figure 6. As with the previously studied clone, parental sarcoma virus RNA failed to compete with about 8% of the sequences of full-length subunits from PRRSV-E clone 95b forming hybrids in this reaction. Two regions of decreased competition were detected, one beginning at about 0.4 and ending about 0.25 of the genome length from the 3’ end, and another very small segment within 300-400

Cell 326

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Figure 5. Competition by RNA from RSV in the Reaction between 3’ End Fragments of RAV-0 RNA and Normal Chicken DNA The conditions of reaction were the same as described in Figure 6, except that the radioactive fragments were prepared from 70s complex RNA from RAV-0. The extent of hybridization in the presence of 0.15 pg of unlabeled 705 RNA from RSV (0) was again plotted as the fraction of hybridization achieved in the absence of competitor. The data were compared to a curve calculated from a model (. ) in which 12% of the RAV-0 sequences missing from the competitor were distributed in the 3’ terminal 16% of the genome [excluding the poly(A) tract].

nucleotides of the poly(A) tract. Again a reasonable model could be constructed in which the RAV-0 sequences were located between 0.4 and 0.25 with a very short stretch of 100-200 nucleotides very close to the 3’ end. This slightly extended region with respect to the total fraction of RAV-O-specific sequences in PR-RSV-E RNA seemed consistent with the evidence of some interspersion of these nucleotides in the endogenous viral genome suggested by the previous experiments. Discussion The principal conclusion we have drawn from this series of experiments is that the large majority of sequences which appear unshared between RAV-0 and nondefective RSV are concentrated in specific regions of the genome of these two types of viruses. A reasonable speculation based on this observation is that the biological differences between chicken endogenous and sarcoma viruses, including the properties of oncogenicity and regulation by the host cell, may be influenced by viral func-

,tions located in these divergent regions of the genome. The theoretical models suggesting a precise location and distribution for the various sets of sequences studied were generated to provide a basis of comparison with other mapping data and to establish working hypotheses for future studies. There are, however, technical limitations to the method which dictate a degree of caution in the quantitative interpretation of these experiments. First, the technique is, of necessity, more sensitive to alterations at the 3’ end than at the 5’ end of the genome. Second, the extent of hybridization of any of the putative genome segments we prepared may be modified by the content of contaminating random fragments generated by breakage occurring after selection of the 3’ ends on oligo(dT)-cellulose (a factor which might vary somewhat from one preparation to the next and from one size class of fragments to the next) and by the degree of actual separation between the various size classes during gel electrophoresis. These factors are difficult and impractical to measure and their effects hard to predict. Third, identification of sequences as either shared or unshared by hybridization techniques very much depends upon the conditions used for formation and assay of hybrid structures. Thus the degree of divergence in the sets of sequences which differ between RAV-0 and RSV was not assessed in this study. Shared sequences have been analyzed previously by thermal chromatography (Neiman, 1972, 1973; Neiman et al., 1974b) which suggested a high level of fidelity of base pairing (no more than about l-2% mismatching) In this connection, the experiments in which the localization of the deletion in td PR-C was determined provided a basis of comparison for this method of mapping by competitive hybridization. The size of the deletion has been established by a number of techniques and laboratories, and its localization was previously determined by mapping of ribonuclease TI-resistant oligonucleotides (Lai et al., 1973; Want and Duesberg, 1974; Wang et al., 1975; Coffin and Billiter, 1976; Wang et al., 1975). Both the data and the model (Figure 3, model 1) agree well with these preceding studies. In this case, the rather minor variation of data from the model provides reasonable assurance that the degree of error due to technical hazards was not very great. Another level of complexity was introduced in examination of RSV-related sequences in uninfected chicken cells (Figure 4), where we have suggested an interpretation involving partial sequence sharing between the normal chicken genome and the region of the RSV genome containing the sequences deleted from td RSV. Stehelin et al. (197613) have prepared cDNA from these sequences (cDNA&, and have demonstrated extensive reas-

Organization 327

of the Genome

of WV

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Sarcoma Virus RNA in the of RNA and the Recombinant Quail Sarcoma induced by the

Genomic 3% RNA from PR-RSV-E clone 95b was used to prepare 3’ end fragments which were reacted with DNA from quail sarcomas either in the absence (A) or presence (0) of 70s complex RNA from parental sarcoma virus. The model at the top of the figure depicts the proposed localization of sequences within the genome of the recombinant which was contributed solely by the RAV-0 parent (and thus not competed for by sarcoma virus RNA) and is represented by a dotted line.

sociation reactions between cDNA,,,, and low frequency sequences in normal chicken DNA as well as in DNA from other avian species (Stehelin, Varmus and Bishop, 1976a). A decreased thermal stability of these duplex structures also suggested some sequence divergence between viral and cellular sequences. While the extent of hybridization between viral and endogenous “sarc” sequences in this RNA-DNA system is less extensive than that reported by Stehelin and his co-workers, both observations are at least qualitatively in agreement that some sequences appear to be present in the DNA of normal chickens which are partially homologous to those deleted from the td RSV mutants. It is reasonable to speculate on this basis that the cellular sequences detected share a common ancestry with the present src gene (Baltimore, 1974) of RSV. Their function in the chicken genome, and in particular their role in oncogenesis, is more problematic. The results of a number of experiments using both RNA-DNA and DNA-DNA hybridization tech-

niques have supported the conclusion that a substantial fraction of the RSV genome cannot be detected in the normal chicken genome (Neiman, 1972; Varmus et al., 1973, 1974; Wright and Neiman, 1974; Neiman et al., 1974b; Tereba et al., 1975). The present experiments suggest that the majority of these more exogenous sequences are concentrated between 0.4 and 0.2 of the viral genome from the 3’ poly(A) tract. Furthermore, since RAV-0 lacks src, this segment corresponds with the 3’ terminal segment of RAV-0 subunit RNA which contains the bulk of endogenous viral sequences not detected in the genome of RSV. It is not possible to determine from these data whether such sequence differences reflect either more extensive divergence or an origin completely separate from that of the more conserved regions of the genome. While the rather sharply segmental arrangement of these distinct sets of sequences makes attractive a recombinational theory of origin for the present genome of nondefective RSV, nothing in this study proves that such a concept is correct. Another important issue is the identity of the viral gene(s) at the 3’ end of the genome of RAV-0 and the corresponding exogenous segment of RSV which appear so strikingly different in sequence content. On the basis of TI-resistant oligonucleotide mapping of the deletion in replication-defective RSV, the gene for envelope glycoprotein, gp 85 (env), should lie next on the 5’ side of src in the genome of RSV (Joho, Billeter and Weissmann, 1975; Wang et al., 1975, 1976). We have not yet placed the envelope deletion by this competitive hybridization technique, but the characteristic TI oligonucleotides for this region have been reported to begin at about the middle of the RSV subunit RNA molecule. Thus the genome segment in question may extend into, but not encompass, the env region located by deletion mapping. Most, but not necessarily all, of the RAV-O-specific segment was found in the recombinant studied. The largest fraction of these sequences was located on the 5’ side of the region of the recombinant genome presumed to contain src from the RSV parent [although a very short RAV-O-specific segment was also detected very near the 3’ poly(A) tract of the recombinant]. A minimum of three crossovers would be required to create this rather complex pattern of recombination. Since this recombinant was selected for the envelope properties of the endogenous virus, the largest segment of RAV-Ospecific information between 0.4 and 0.25 from the 3’ end of the PR-RSV-E genome may well specify host range and related characteristics of subgroup E viruses. Again the evidence is only circumstantial. Furthermore, clone 95b is a subclone of a PR-

Cell 320

RSV-E isolate which appears to be subject to the intracellular growth restriction on chicken embryo cells characteristic of chicken endogenous viruses, but not of exogenous viruses and most other recombinants between RAV-0 and exogenous viruses (Linial and Neiman, 1976). Study of more such recombinants which vary in their expression of these properties may help to determine the function of the RAV-O-specific sequences. Experimental

Procedures

Cells and Viruses Viruses were propagated on secondary chicken embryo cells cultured by previously described methods (Rubin, 1960). RAV-0 was either spontaneously released by such cultures from line 7 x 15 embryos contributed by Lyman Crittenden or obtained by propagation in cultured line 15 cells which were a gift from Harriet Robinson. PR-RSV-B and PR-RSV-C were recently cloned and propagated on susceptible cultures of embryos negative for chicken helper factor (chf) obtained from H and N, Inc. (Redmond, Washington). PR-RSV-E clone 95b was a recombinant virus isolated by Maxine Llnial following mixed infection of quail embryos with PR-RSV-C and RAV-0. This virus was selected for host range properties mediated by the transforming function(s) of RSV. Isolation and characterization of this class of recombinants have recently been described in detail (Linial and Neiman, 1976). Isolation of Viral Nucleic Acids Viruses were isolated from 2-4 liters of supernatant tissue culture fluids from cells grown either on 75 mm plastic tissue culture plates or in roller bottles on a Bellco perfusion apparatus (Smith and Kozoman, 1973). For preparing viral 70s RNA complexes, harvests were made at 18-24 hr intervals, and for preparation of 35s genomic subunit RNA at 2-3 hr intervals. Purification of virus by equilibrium density centrifugation, extraction of total viral nucleic acids by the phenol-SDS method and separation of the 70s complex containing the viral genome from lower molecular weight viral RNA by velocity sedimentation in 1530% glycerol gradients have been described previously (Robinson, Pitkanen and Rubin, 1965; Wright and Neiman, 1974). For preparation of dissociated subunits, ethanol-precipitated total virion nucleic acid was dissolved in 0.01 M LiCI, 0.001 M EDTA, 0.05 M Tris (pH 7.4) containing 80% dimethyl sulfoxide (DMSO) to dissociate the 70s complex and layered over 5 ml preformed 5-20% sucrose gradients in the same DMSO-buffer solution (Leis et al., 1975). Gradients were centrifuged in polyallomer tubes for 18 hr at 190,000 x g, and fractions containing 35s RNA were pooled, diluted to 5 ml in 0.15 M NaCl, 0.001 EDTA, 0.05 M Tris (pH 7.4) and precipitated at -20°C with 2 vol of ethanol. The RNA pellet was dissolved in water and stored in a liquid nitrogen freezer until use. Labeling and Fragmentation of RNA RNA (4 pg at a time) was labeled with carrier-free ‘*Y (Amersham) to specific activities of 2-6 x 10’ cpmlpg by a previously described technique (Tereba and McCarthy, 1973; Neiman et al., 1974b). This process, which involved heating to 67°C for 15 min in 0.15 M sodium acetate buffer (pH 5.1) containing 0.2 mM Na lZ51 and 0.1 mM TICI,, and again at 60°C for 45 min in 0.5 M phosphate buffer (pH 7.2), resulted in partial fragmentation of 355 subunit RNA to lengths ranging from full-length molecules to pieces of about 4-55 in size. Isolation and Sizing of 3’ End Fragments Labeled viral RNA fragments in 2.0 ml, 0.5 M NaCI, 0.01 M Tris (pH 7.5) were mixed for 5 min at room temperature with 75 mg oligo(dT)-cellulose (Collaborative Research) to bind fragments

containing 3’ poly(A) tracts. The cellulose beads were then sedimented at 100 g, washed twice in the same buffer to remove unbound RNA and loaded on a small glass column. The column was washed with the Tris buffer containing 0.15 M NaCl and then with Tris buffer alone. The 3’ end fragments were sharply eluted in the first 0.6-0.8 ml of the final wash. This fraction was made 0.5 M in NaCl and the procedure was repeated. Recovery of initial radioactivity (0.8-2.4 x 108cpm, 4 fig of RNA) in the final eluate ranged from 3-6%, and fragments so prepared bound oligo(dT)-cellulose with about 95% efficiency under these conditions. Freshly prepared 3’ end fragments were passed over Sephadex G-100 in water and concentrated by lyophilization in polypropylene tubes. Sizing was carried out within 15 hr of preparation of fragments by electrophoresis on 5 x 60 mm mixed gel columns of 2% acrylamide, 0.1% bis-acrylamide, 0.25% agarose prepared as previously described (Neiman and Henry, 1971). Lyophilized fragments were dissolved in 15 ~1 of 0.001 M EDTA, 0.004 M Tris-acetate buffer (pH 7.2), mixed with an equal volume of 50% sucrose dissolved in formamide and distributed on the gel column by electrophoresis at 1 mA per gel in the Tris-acetate buffer for 1.5 hr in a temperature-controlled apparatus (Buchler Instruments) at 20°C. The gel columns were then divided into 1 mm slices which were kept at 4°C. Labeled RNA could not be efficiently eluted directly from the slices, but was recovered by one of two methods with about equal efficiency (30-60%). In method 1 slices were placed on 5 x 40 mm columns of 0.8% agarose, and RNA was transferred to the upper 5 mm of the agarose column by electrophoresis for 15 min under the same conditions. The upper 5 mm of the agarose column were then sliced off and RNA was directly eluted in water. (Electrophoresis of RNA from slices directly into buffer rendered radioactivity acid-soluble.) In method 2, the slices were rapidly frozen in liquid nitrogen and thawed in 0.4 ml of water at room temperature, and the mixture was refrozen and thawed again. The solution Of released RNA was stored in liquid nitrogen until use. Preparation of Cellular DNA DNA containing endogenous viral sequences was obtained from normal &f-negative chick embryos from the H and N flock. Proviral DNA sequences for PR-RSV-C were contained in DNA from IO day wing web sarcomas induced by this virus in newly hatched chickens, and for PR-RSV-E clone 95b from tumors induced in analogous fashion in Japanese quail as previously described (Neiman, 1972: Linial and Neiman, 1976). DNA was extracted by the method of Marmur (1961) and fragmented to average singlestrand length of 300 nucleotides by the method of limited depurination and alkaline hydrolysis (McConaughy and McCarthy, 1967). RNA-DNA Hybridization Hybridization reaction mixtures consisted of a maximum of 0.1 ng (2-6 x IO3 cpm) of labeled viral RNA and 250 pg of cellular DNA fragments in 25 ~1 of 0.75 M NaCI, 0.075 M sodium citrate, 50% formamide. Proviral DNA was present in all reactions at a calculated lo-20 fold excess over input viral RNA. To maintain an adequate DNA excess for reactions with 3’ end fragments representing 10% of the genome, input ‘251-labeled RNA was decreased to 0.03 ng. Fragments representing ~3% of the genome were not studied. Reaction mixtures were incubated at 49°C for varying periods of time. Hybridization of labeled RNA was assayed as percentage of resistance to ribonuclease as previously described (Neiman, 1972, 1973). Data were adjusted for background measurements made using unincubated reaction mixtures which varied from 2-10% with different preparations of ‘251-labeled viral RNA and were generally lower (l-5%) following oligo(dT)-cellulose chromatography. For competitive hybridization experiments, appropriate unlabeled competitor RNA was added to the reaction mixture in sufficient quantity to produce an RNA excess. The characteristics of this technique for determining sequence relationships of viral RNA have been described in detail (Wright and Neiman, 1974; Neiman et al., 1974a, 197413). For mapping experi-

Organization 329

of the Genome

of RSV

ments with labeled 3’ end fragments, 0.15 pg of competitor were added corresponding to a calculated 40 fold RNA excess for 1 proviral copy per haploid genome. In these experiments, background controls included hybridization in the presence of 0.15 pg of homologous competitor RNA. Asexpected, these controlswere not significantly above background. Acknowledgments This investigation was supported by research grants awarded by the National Cancer Institute. P.E.N. is a scholar of the Leukemia Society of America. The authors thank M. Linial and Ft. Eisenman for helpful comments in the preparation of this manuscript. Received

January

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